CD8 binding polypeptides and methods of use

Abstract

The present disclosure relates to novel CD8 binding polypeptides, conjugates thereof, and methods of use thereof.

Description

Attorney Docket No. OTPC-050 / 02WO CD8-BINDING POLYPEPTIDES AND METHODS OF USE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 670,723 filed on July 12, 2024, and U.S. Provisional Application No.63 / 774,042 filed on March 18, 2025, the contents of all of which are incorporated herein by reference in their entirety. BACKGROUND

[0002] CD8 receptors are transmembrane glycoproteins found primarily on cytotoxic T cells and typically exist as heterodimers of CD8α and CD8β chains. Both the α and β chains consist of an extracellular immunoglobulin-like domain that binds MHC class I molecules, a transmembrane region anchoring the protein to the cell membrane, and a cytoplasmic tail that interacts with Lck (Lymphocyte-specific protein tyrosine kinase) to relay activation signals to the T cell. CD8 chains can also exist as homodimers, e.g., CD8α homodimers, especially on some specialized T cell subsets.

[0003] The CD8 receptor plays a critical role in the immune system by enabling cytotoxic T cells (also known as CD8+ T cells) to recognize and eliminate infected or malignant cells. Specifically, CD8 binds to the major histocompatibility complex class I (MHC I) molecules that present antigenic peptides on the surface of virtually all nucleated cells. This interaction gives rise to the activation and targeted response of CD8+ T cells against, for example, cells infected with viruses, intracellular bacteria, or against transformed cancer cells. Without functional CD8 receptors, the immune system would be significantly impaired in its ability to detect and destroy these abnormal cells, making CD8 indispensable for effective adaptive immunity and immune surveillance.

[0004] On the other hand, molecules that inhibit the CD8 receptor are important tools in both research and therapeutic contexts because they can selectively modulate the activity of CD8+ cytotoxic T cells. These T cells, while essential for clearing infected or cancerous cells, can also contribute to tissue damage and disease when their activity becomes excessive or misdirected such as in autoimmune disorders, transplant rejection, or certain inflammatory conditions. By blocking the interaction between CD8 and MHC class I molecules, CD8 inhibitory molecules can reduce T cell activation and cytotoxic function and can be harnessed to clinically dampen overactive immune reactions. In contexts like cancer immunotherapy, 1 321490133Attorney Docket No. OTPC-050 / 02WO temporary inhibition of CD8 may help reduce immune-related adverse events caused by overly aggressive T cell responses or allow fine-tuned control in combination treatments.

[0005] New CD8 binding molecules are needed to provide optimal immune regulation for cancer and autoimmune treatments as well as to improve the safety of gene and cell therapies for patients. The present invention addresses these needs and relates to the development of improved CD8-binding polypeptides and their use. SUMMARY

[0006] In one aspect, the disclosure provides a CD8-binding polypeptide comprising a CD8-specific single domain antibody (VHH), where the VHH comprises at least one modification with respect to SEQ ID NO: 314. In some embodiments, the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, V79, K87, P88, A91, K124, and a combination thereof. In some embodiments, the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, P88, A91, K124, and a combination thereof. In some embodiments, the at least one modification is a substitution, deletion, or insertion. In some embodiments, the at least one modification is selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, E44G, S72R, A75S, V79L, K87R, P88A, A91T, K124Q, and a combination thereof, each with respect to SEQ ID NO: 314.

[0007] In some embodiments of the foregoing or related aspects, the at least one modification is in the VHH complementarity-determining region (CDR) 1, CDR2, and / or CDR3. In certain embodiments, the at least one modification is in the VHH framework region 1 (FR), FR2, FR3, and / or FR4. In some embodiments, the VHH CDR1 sequence comprises GFTFDDYAX1G (SEQ ID NO: 373), the VHH CDR2 sequence comprises X2IRVSDGSTYYADX3VKG (SEQ ID NO: 374), and the VHH CDR3 sequence comprises GSLYTX4VQSIVVVPARPYYDMDY (SEQ ID NO: 375), wherein X1is M or I, X2is C, S, or A, X3 is P or S, and X4 is C, S, or A. In certain embodiments, (i) if X1 is I, then X2 is S or A, X3is S, and / or X4is S or A; (ii) if X2is C, then X1is M, X3is S, and / or X4is S or A; (iii) if X3is P, then X1 is M, X2 is S or A, and / or X4 is S or A; and (iv) if X4 is C, then X1 is M, X2 is S or A, and / or X3is S.

[0008] In some embodiments of the foregoing or related aspects, the VHH CDR1, CDR2, and CDR3 respectively comprise: GFTFDDYAMG (SEQ ID NO: 14); 2 321490133Attorney Docket No. OTPC-050 / 02WO CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); GFTFDDYAMG (SEQ ID NO: 14); CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494); GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494); GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and AGSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 8);GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and GSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 495); GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and AGSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 12); or GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and GSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 496).

[0009] In some embodiments of the foregoing or related aspects, the at least one modification comprises: (i) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, A91T, and K124Q; (ii) Q1E, Q5V, A14P, A75S, A91T, and K124Q; (iii) Q1E, Q5V, A14P, S72R, A75S, V79L, K87R, A91T, and K124Q; (iv) Q1E, Q5V, A14P, S72R, A75S, A91T, and K124Q; (v) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, A91T, and K124Q; (vi) Q1E, Q5V, A14P, A75S, V79L, K87R, A91T, and K124Q; (vii) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; (viii) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; (ix) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, V79L, A91T, and K124Q; (x) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, A91T, and K124Q; or (xi) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, K87R, P88A, A91T, and K124Q. In some embodiments, the at least one modification comprises Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, A91T, and K124Q.

[0010] In some embodiments of the foregoing or related aspects, the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 17, 303, 1, 5, 9, 13, 21, 25, 29, 33, 37, and 315-323, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, the VHH comprises SEQ ID NO: 17, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0011] In some embodiments of the foregoing or related aspects, the VHH CDR1 sequence comprises DYAX5G (SEQ ID NO: 376), the VHH CDR2 sequence comprises CIRX6X7DX8X9X10YYADPX11KG (SEQ ID NO: 377), the VHH CDR3 sequence comprises 3 321490133Attorney Docket No. OTPC-050 / 02WO GSLX12X13CVQX14X15X16X17X18X19X20X21X22X23YDMDY (SEQ ID NO: 378), X5 is I or L, X6is A, C, F, I, L, N, S, T, V, W, or Y, X7is F, S, W, or Y, X8is G, R, or V, X9is A, M, R, S, or W, X10 is D, F, S, or T, X11 is L or V, X12 is F, W, or Y, X13 is A, S, or T, X14 is G, S, W, or Y, X15is F, H, I, L, or W, X16is A, E, G, I, L, N, P, R, S, or V, X17is A, G, P, S, or V, X18is D, E, or V, X19 is P or S, X20 is A, D, E, N, S, or T, X21 is A, D, E, G, H, L, Q, R, S, or V, X22 is A, D, E, N, or P and X23is R, V, or Y.

[0012] In some embodiments of the foregoing or related aspects, (i) the VHH CDR1 comprises DYAX5G (SEQ ID NO: 376); (ii) the VHH CDR2 sequence comprises CIRX24X25DX26X27X28YYADPX29KG (SEQ ID NO: 379); and (iii) the VHH CDR3 sequence comprises: GSLYTCVQSX30X31X32X33PYYDMDY (SEQ ID NO: 380), GSLYTCVQX34X35X36X37X38X39PX40YDMDY (SEQ ID NO: 381), GSLYTCVQX41X42X43X44X45X46X47X48YYDMDY (SEQ ID NO: 382), GSLYTCVQSX49X50X51X52X53X54X55X56X57 X58DMDY (SEQ ID NO: 383), GSLYTCVQX59X60X61X62VX63X64X65X66X67X68YDMDY (SEQ ID NO: 384), GSLYTCVLX69X70X71X72X73X74X75X76X77X78X79X80YDMDY (SEQ ID NO: 385), or GSLYTCVQX81X82X83X84X85X86 X87X88X89X90X91X92X93YDMDY (SEQ ID NO: 386); wherein: X5 is I or L, X24 is A, C, F, I, L, N, S, T, V, W, or Y; X25 is F, S, W, or Y; X26 is A, G, I, R, or V; X27 is A, M, R, S, or W; X28 is D, F, S, or T; X29 is V or L; X30 is F, H, I, or L; X31 is A, D, G, H, I, K, L, N, Q, R, S, T, V, or Y; X32is A, D, or G; X33is D, E, I, K, or R; X34is F, H, L, S, W, or Y; X35 is F, H, I, L, or W; X36 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W; X37is D, P, or V; X38is A, E, or V; X39is P or R; X40is E or Y; X41is F, S, or Y; X42is F, I, L, W, or Y; X43 is A, G, H, K, L, M, P, R, S, V, or W; X44 is P, V, or Y; X45 is D, E, H, P, or V; X46is A, E, G, or P; X47is A, E, G, R, or S; X48is D, E, P, or V; X49is F, H, I, or L; X50is I, L, N, or V; X51 is A, I, K, M, P, S, T, or V; X52 is E, N, P, R, T, or V; X53 is G or P; X54 is A, G, H, T, or Y; X55is E, G, L, R, or Y; X56is D, E, or P; X57is S or Y; X58is D or Y; X59is S or Y; X60is F, I, L, or W; X61 is A, E, F, G, K, L, Q, S, T, or V; X62 is A, D, E, K, M, P, S, T, or V; X63 is P or V; X64is A, D, E, F, G, I, N, P, R, or V; X65is A, D, E, G, I, L, N, P, Q, R, S, T, V, W, or Y; X66 is A, D, E, G, R, S, T, V, or Y; X67 is A, D, E, G, P, or V; X68 is L, S, or Y; X69 is H, K, L, Q, R, S, or Y; X70is F, I, L, Q, or W; X71is G, K, L, P, Q, R, S, or V; X72is D, I, S, or V; X73 is D, E, N, P, or V; X74 is A, D, E, G, P, or V; X75 is A, E, R, T, or V; X76 is P, R, or V; X77 is A, D, E, K, N, P, S, T, or V; X78is E, G, Q, R, S, V, or Y; X79is D, E, P, S, or T; X80is R or Y; X81 is H, R, S, or W; X82 is F, I, or L; X83 is I, N, R, V, or W; X84 is P, R, S, or V; X85 is A, D, E, I, R, V, or Y; X86is D, G, N, P, Q, or V; X87is A, D, E, G, L, R, or V; X88is R or V; X894 321490133Attorney Docket No. OTPC-050 / 02WO is D, G, or P; X90 is A, D, E, G, H, L, M, P, Q, R, S, T, V, or Y; X91 is A, D, E, G, L, R, S, V, or Y; X92is D, E, F, L, P, Q, or V; and X93is E, I, M, R, T, V, or Y.

[0013] In some embodiments of the foregoing or related aspects, the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352 and 394-493, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0014] In some embodiments of the foregoing or related aspects, the VHH specifically binds to CD8α. In certain embodiments, the VHH specifically binds to human CD8α.

[0015] In some embodiments of the foregoing or related aspects, the VHH comprises one or more characteristics relative to a VHH set forth in SEQ ID NO: 314, where the one or more characteristics is selected from the group consisting of: an increased binding affinity (KD) to human CD8α; a decreased off-rate (koff) for binding to human CD8α; an increased melting temperature (Tm); an increased expression yield; and an increased purity following a single-step purification.

[0016] In some embodiments of the foregoing or related aspects, the polypeptide is operably linked to one or more of a second polypeptide, a nucleic acid, an inorganic molecule, an organic molecule or another moiety. In some embodiments, the polypeptide selectively targets a CD8-expressing (CD8+) cell. In particular embodiments, the CD8+ cell is a T cell.

[0017] In some embodiments of the foregoing or related aspects, the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising a cytokine. In certain embodiments, the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 5 321490133Attorney Docket No. OTPC-050 / 02WO polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

[0018] In some embodiments of the foregoing or related aspects, the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising an interferon. In certain embodiments, the interferon comprises one or more of an interferon type I, an interferon type II, and an interferon type III. In certain embodiments, the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN-ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN-γ).

[0019] In some embodiments of the foregoing or related aspects, the polypeptide is operably linked to a second polypeptide comprising a designed cytokine.

[0020] In another aspect, provided herein is a CD8-targeted conjugate, comprising a CD8- binding polypeptide of the disclosure and at least one second moiety, where the CD8-binding polypeptide is operably linked to the second moiety. In some embodiments, the CD8-binding polypeptide is operably linked to the second moiety by a linker. In certain embodiments, the linker comprises one or more of an amino acid sequence, a nucleic acid sequence, an amino acid sequence, a small molecule, and any combination thereof.

[0021] In some embodiments of the foregoing or related aspects, the at least one second moiety comprises a detection label, a cytotoxic agent, an antigen-targeting domain, a signaling domain, and / or a second polypeptide. In some embodiments, the at least one second moiety comprises a detection label. In certain embodiments, the detection label comprises a radioactive isotope, an affinity tag, or a fluorescent tag. In some embodiments, the at least one second moiety comprises a cytotoxic agent, where the cytotoxic agent comprises a chemotherapeutic drug or a toxin. In some embodiments, the at least one second moiety comprises an antigen-targeting domain. In certain embodiments, the antigen-targeting domain specifically binds to an antigen expressed by an immune cell. In certain embodiments, the antigen-targeting domain specifically binds to a tumor associated antigen. In some embodiments, the CD8-targeted conjugate is a multi-specific antibody. In some embodiments, the CD8-targeted conjugate is an antibody drug conjugate. 6 321490133Attorney Docket No. OTPC-050 / 02WO

[0022] In some embodiments of the foregoing or related aspects, the at least one second moiety comprises a second polypeptide. In some embodiments, the second polypeptide comprises a cytokine, an interferon, a chemokine, a growth factor, and / or a tumor necrosis factor. In certain embodiments, the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL- 36). In certain embodiments, the interferon comprises one or more of an interferon type I, an interferon type II, and an interferon type III. In certain embodiments, the interferon comprises one or more of an interferon alpha (IFN-a), an interferon beta (IFN-b), and interferon epsilon (IFN-e), an interferon kappa (IFN-k), an interferon omega (IFN-w), and an interferon gamma (IFN-g). In some embodiments, the second polypeptide comprises a designed cytokine. In certain embodiments, the designed cytokine comprises a sequence set forth in any one of SEQ ID NOs: 69-300.

[0023] In another aspect, provided herein is a chimeric antigen receptor comprising a CD8- binding polypeptide of the disclosure.

[0024] In another aspect, provided herein is a composition comprising a CD8-binding polypeptide of the disclosure and a delivery vector, where the CD8-binding polypeptide is operably linked to the delivery vector. In some embodiments, the delivery vector comprises a viral particle, a virus-like particle, a synthetic particle, a liposome, or a lipid nanoparticle.

[0025] In another aspect, provided herein is a nucleic acid sequence encoding a CD8- binding polypeptide of the disclosure. In some embodiments, the nucleic acid sequences of the disclosure comprise a regulatory sequence capable of driving expression of the nucleic 7 321490133Attorney Docket No. OTPC-050 / 02WO acid sequence in a mammalian cell. In certain embodiments, the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof. In certain embodiments, the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem. In certain embodiments, the regulatory sequence comprises a constitutive promoter. In certain embodiments, the regulatory sequence comprises an inducible promoter. In certain embodiments, the regulatory sequence comprises a minimal promoter. In certain embodiments, the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs. In certain embodiments, the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

[0026] In another aspect, provided herein is a vector comprising a nucleic acid sequence of the disclosure. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a delivery vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a viral vector.

[0027] In another aspect, provided herein is a cell comprising one or more of: a CD8- binding polypeptide of the disclosure; a CD8-targeted conjugate of the disclosure; a chimeric antigen receptor of the disclosure; a composition of the disclosure; a nucleic acid of the disclosure; and a vector of the disclosure.

[0028] In some embodiments of the foregoing or related aspects, the cell is an immune cell. In some embodiments, the cell is a stem cell or a T-cell precursor cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell. In some embodiments, the cell is a somatic cell or a differentiated cell. In some embodiments, the cell is a T cell, a Natural Killer (NK) cell, or a macrophage.

[0029] In some embodiments of the foregoing or related aspects, the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood. In certain embodiments, the cell has been cryopreserved. In certain embodiments, the cell has been modified to become an allogeneic cell. In certain embodiments, the cell has been modified to produce an allogeneic cell.

[0030] In some embodiments of the foregoing or related aspects, the cell is capable of expressing or is modified to express an exogenous protein. In some embodiments, the exogenous protein comprises an antigen receptor. In certain embodiments, the antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the cell is capable of expressing or modified to express a safety switch. In some embodiments, 8 321490133Attorney Docket No. OTPC-050 / 02WO the cell is capable of expressing or modified to express a cell marker. In some embodiments, the cell is capable of expressing or modified to express a truncated EGFR (EGFRt), or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, the EGFRt comprises a sequence of SEQ ID NO: 53. In some embodiments, the cell is capable of expressing or modified to express a truncated and optimized EGFR (EGFRopt). In certain embodiments, the EGFRopt sequence comprises a sequence of one or more of SEQ ID NOs: 54-68, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0031] In another aspect, provided herein is a composition comprising: a CD8-binding polypeptide of the disclosure; a CD8-targeted conjugate of the disclosure; a chimeric antigen receptor of the disclosure; a nucleic acid sequence of the disclosure; a vector of the disclosure; or a cell of the disclosure.

[0032] In another aspect, provided herein is a pharmaceutical composition comprising: a CD8-binding polypeptide of the disclosure; a CD8-targeted conjugate of the disclosure; a chimeric antigen receptor of the disclosure; a composition of the disclosure; a nucleic acid sequence of the disclosure; a vector of the disclosure, or a cell of the disclosure, and a pharmaceutically acceptable carrier.

[0033] In some embodiments of the foregoing or related aspects, the pharmaceutical composition is formulated for systemic administration. In certain embodiments, the systemic administration comprises an intravenous injection or an intravenous infusion. In some embodiments, the pharmaceutical composition is formulated for local administration. In certain embodiments, the local administration comprises an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

[0034] In another aspect, provided herein is a use of one or more of a CD8-binding polypeptide of the disclosure, a CD8-targeted conjugate of the disclosure, a chimeric antigen receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the disclosure, a cell of the disclosure, a composition of the disclosure, or a pharmaceutical composition of the disclosure in the manufacture of a medicament for treating a disease or disorder.

[0035] In another aspect, provided herein is a use of one or more of a CD8-binding polypeptide of the disclosure, a CD8-targeted conjugate of the disclosure, a chimeric antigen receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the 9 321490133Attorney Docket No. OTPC-050 / 02WO disclosure, a cell of the disclosure, a composition of the disclosure, or a pharmaceutical composition of the disclosure for treating a disease or disorder.

[0036] In some embodiments of the foregoing or related aspects, the disease or disorder comprises a proliferative disorder. In some embodiments, the proliferative disorder is a metastatic disorder. In certain embodiments, the metastatic disorder comprises a solid tumor.

[0037] In another aspect, provided herein is a method of treating a disease or disorder comprising administering an effective amount of one or more of a CD8-binding polypeptide of the disclosure, a CD8-targeted conjugate of the disclosure, a chimeric antigen receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the disclosure, a cell of the disclosure, a composition of the disclosure, or a pharmaceutical composition of the disclosure, to a subject in need thereof, thereby treating the disease or disorder. In some embodiments, the subject is at risk of developing the disease or disorder. In some embodiments, the subject has been diagnosed with the disease or disorder. In some embodiments, the disease or disorder comprises a proliferative disorder. In certain embodiments, the proliferative disorder is a metastatic disorder. In particular embodiments, the metastatic disorder comprises a solid tumor. In some embodiments, treating the disease or disorder comprises one or more of: decreasing the severity of a sign or symptom of the disease or disorder; improving a function negatively affected by the disease or disorder; improving the subject’s prognosis; increasing the subject’s duration of progression-free survival (PFS); reducing a quantity of cells affected by the disease or disorder; inducing a remission form the disease or disorder; delaying or preventing a relapse of the disease or disorder; delaying or preventing a second presentation of the disease or disorder; and / or increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG.1 illustrates the alignment of parental aCD8 VHH, humanized VHH and related human germline gene segments IGHG3-23*04 and IGHJ4*01. Highlighted CDRs are an inclusive combination of Kabat + Chothia CDR definitions.

[0039] FIG.2 provides a summary table of biophysical data on the first round of humanization variants. The data demonstrates that relatively conserved V79L and K87R mutations have an unexpected negative impact on yield and binding. An exemplary intermediate variant (VHH 1.4.0; SEQ ID NO: 1) displayed higher yield and increased 10 321490133Attorney Docket No. OTPC-050 / 02WO thermostability in the presence of TCEP compared to the parent (VHH 1, SEQ ID NO: 314), but it also showed slightly weaker binding to CD8a by Octet™ BLI based on a reduced response at the end of association and ~1.5 fold faster apparent off-rate compared to the parent.

[0040] FIGS.3A-3B provides a summary of biophysical data on second round of humanization variants in the context of VHH only (FIG.3A) and cytokine-VHH fusions (FIG.3B). These data result from additional humanization mutations in the context of the exemplary intermediate variant (VHH 1.4.0, SEQ ID NO: 1) and from a separate round of expression, purification and testing compared to the first round. The second round identified a combination of mutations (I34M, E44G, and P63S) that maintained the increased thermostability of VHH 1.4.0 (SEQ ID NO: 1) relative to the parental VHH 1 (SEQ ID NO: 314), while restoring some of the favorable binding properties of the parental VHH 1 (SEQ ID NO: 314) to CD8a (e.g. a slower off-rate and higher response at the end of the association phase compared to VHH 1.4.0, SEQ ID NO: 1). The favorable properties of a selected humanized VHH (VHH 1.4.0+, SEQ ID NO: 17) compared to the parental VHH (increased thermostability without loss of binding to CD8a) are demonstrated in the VHH binder only (SEQ ID NO: 17) and in the cytokine-VHH fusion (SEQ ID NO: 327) data.

[0041] FIGS.4A-4D illustrates Octet™ BLI affinity data of variants to human CD8a in the following plots: FIG.4A shows humanized Cytokine-VHH 1.4.0+ (SEQ ID NO: 327), FIG. 4B shows humanized VHH 1.4.0+ (SEQ ID NO: 17), FIG.4C shows non-humanized Cytokine-VHH 1 (SEQ ID NO: 324) and FIG.4D shows non-humanized parental VHH 1 (SEQ ID NO: 314). The selected humanized VHH 1.4.0+ data illustrates high affinity despite a weaker affinity for human CD8a compared to the parental VHH in the context of both the VHH alone or a cytokine-VHH fusion.

[0042] FIGS.5A-5L represents CDR sequences of yeast-displayed VHH variants enriched from hCD8a binding. Original CDR sequences are bolded and >1% frequency is highlighted in gray. FIG.5A illustrates the top hits for CDR1 from fixed length library; FIG.5B illustrates the top hits for CDR2 from fixed length library; FIG.5C illustrates the top hits for CDR3 from fixed length library; FIG.5D illustrates the top hits for CDR1 from variable length library; FIG.5E illustrates the top hits for CDR2 from variable length library; FIG. 5F illustrates the top hits for CDR3 at length of 20 amino acids from variable length library; FIG.5G illustrates the top hits for CDR3 at length of 21 amino acids from variable length library; FIG.5H illustrates the top hits for CDR3 at length of 22 amino acids from variable length library; FIG.5I illustrates the top hits for CDR3 at length of 23 amino acids from 11 321490133Attorney Docket No. OTPC-050 / 02WO variable length library; FIG.5J illustrates the top hits for CDR3 at length of 24 amino acids from variable length library; FIG.5K illustrates the top hits for CDR3 at length of 25 amino acids from variable length library; FIG.5L illustrates the top hits for CDR3 at length of 26 amino acids from variable length library.

[0043] FIGS.6A-6B provides a summary of biophysical characterization of cytokine-VHH affinity variants. Based on CDR3 clustering and frequency in the sequenced sorted combinatorial affinity maturation library, 15 VHH 1.4.0 scaffolds (FIG.6A) and 4 VHH 1.4.0+ scaffolds (FIG.6B) with affinity matured CDR grafts were selected for further characterization as cytokine-VHH-STII fusions, including their binding to hCD8a (Octet response, koff), thermal stability (Tm), purity from single step purification (% monomer on HPLC-SEC), and yield from mammalian expression. All affinity variants showed slower koff and maintained reasonable biophysical characteristics.

[0044] FIG.7 illustrates the activation of STAT5 in CD8+ T cells by cytokine-VHH fusion proteins. Efficiency of Cytokine-VHH fusions to activate the IL-2 receptor was assessed in CD8+ T cells using a 7-point dilution series. Percent pSTAT5 positive cells was determined by flow cytometry. When fused with designed cytokine, anti-CD8a VHH affinity variants increased activation of CD8+ T cells, as indicated by increased percentage of pSTAT5+ CD8+ T cells, compared to the untargeted IL-2 cytokine.

[0045] FIG.8 shows data representing tumor spheroid killing by chronically stimulated anti-MSLN CAR-T cells armored with cytokine-VHH molecules. Compared to CAR-T cells alone, CAR-T cells armored with cytokine-VHH molecule show superior spheroid tumor clearance. DETAILED DESCRIPTION

[0046] The disclosure provides CD8-binding polypeptides, nucleic acids encoding same, vectors comprising the nucleic acid sequences and cells modified to contain or express one or more of these components. In some aspects, the disclosure provides CD8-binding polypeptides comprising a CD8-specific single domain antibody that is a variable domain of a heavy-chain-only antibody (VHH) as well as conjugates thereof (e.g., cytokine conjugates, multi-specific conjugates, targeted delivery compositions, antigen receptors). The present disclosure also relates to methods of use.

[0047] In some aspects, the CD8-binding polypeptides of the disclosure comprise a humanized CD8-specific single domain antibody (VHH). As further described herein, a non- 12 321490133Attorney Docket No. OTPC-050 / 02WO human parental CD8-specific VHH was compared to a germline human immunoglobulin sequence to identify one or more modifications that would be expected to increase homology of the non-human parental sequence to the human immunoglobulin sequence (“humanizing modifications”). The humanizing modification(s) were introduced into the parental CD8- specific VHH to generate a set of humanized CD8-specific VHHs, which were experimentally evaluated to identify the humanizing modification(s) that maintain or improve one or more functional properties relative to the non-human parental CD8-specific VHH (e.g., thermostability, CD8-binding, expression yield, and / or purity following a single- purification step). Without being bound by theory, by increasing homology of the parental CD8-specific VHHs to a germline human immunoglobulin sequence, the resulting humanized CD8-specific VHHs would be expected to exhibit reduced immunogenicity when introduced to a human subject compared to the parental CD8-specific VHH (e.g., as measured by an anti-drug antibody response).

[0048] As further described herein, exemplary humanized CD8-specific VHHs of the disclosure were affinity matured using a combinatorial library approach. Affinity maturation libraries were generated having variants with a modification in at least one of the heavy chain CDR1, CDR2, and CDR3 with respect to the humanized CD8-specific VHH parent. The variants were selected based on CD8-antigen binding and sequenced to identify the CDR modification(s) that increase binding affinity to CD8 relative to the humanized CD8-specific VHH parent. It was demonstrated that affinity matured CD8-specific VHHs selected from the library maintain the desirable biophysical properties of the humanized CD8-specific VHH parent (e.g., thermostability, expression yield, and / or purity following a single-purification step). Furthermore, it was demonstrated that the improved antigen binding properties of the affinity matured CD8-specific VHHs provide improved targeting capability. For example, as described herein, a cytokine conjugate comprising an exemplary affinity matured CD8- specific VHH of the disclosure and an engineered IL-2 cytokine has improved potency for activation of CD8+ T cells compared to a control cytokine conjugate comprising the humanized CD8-specific VHH parent.

[0049] Accordingly, in some embodiments, the present disclosure provides CD8-binding polypeptides comprising a CD8-specific VHH, wherein the CD8-specific VHH comprises at least one humanizing modification described herein with respect to a parental CD8-specific VHH, and optionally, one or more modifications described herein for increasing binding affinity to a CD8 antigen (e.g., a human CD8 antigen). The CD8-specific VHH of the disclosure are characterized by one or more functional properties that are comparable to or 13 321490133Attorney Docket No. OTPC-050 / 02WO improved with respect to the parental CD8-specific VHH (e.g., thermostability, CD8-binding, expression yield, and / or purity following a single-purification step). In some embodiments, the CD8-specific VHH has reduced immunogenicity as compared to the parental CD8- specific VHH. In some embodiments, the CD8-specific VHH has increased binding affinity to a CD8 antigen (e.g., a human CD8 antigen) as compared to the parental CD8-specific VHH.

[0050] The disclosure further provides CD8-targeted conjugates comprising a CD8-binding polypeptide described herein and at least one second moiety. In some embodiments, the CD8- binding polypeptide is operably linked to the at least one second moiety. In some embodiments, the CD8-targeted conjugate is a conjugate comprising the CD8-binding polypeptide operably linked to a cytokine described herein. In some embodiments, the CD8- targeted conjugate is a multi-specific antibody comprising the CD8-binding polypeptide operably linked to at least one second antibody or antigen binding fragment thereof. In some embodiments, the CD8-targeted conjugate comprises a CD8-binding polypeptide operably linked to a delivery vehicle (e.g., a viral vector or a lipid nanoparticle). In some embodiments, the CD8-targeted conjugate is a chimeric antigen receptor (CAR) comprising the CD8-binding polypeptide operably linked to at least one domain, e.g. at least one signaling domain, or a scFv.

[0051] The disclosure further provides nucleic acid sequences encoding the CD8-encoding polypeptides described herein, vectors comprising the nucleic acid sequences, and host cells for expression of the CD8-encoding polypeptides.

[0052] The disclosure also provides cells comprising the CD8-binding polypeptides or CD8-targeted conjugates described herein. In some embodiments, the cell is engineered to express a T cell receptor (TCR) or CAR. In some embodiments, the cell further comprises a safety switch. In some embodiments, the cell is engineered to express a soluble factor described herein.

[0053] In related aspects, the disclosure provides methods for treating a disease or disorder in a subject comprising administering to the subject a CD8-binding polypeptide, CD8- targeted conjugate, cell, or pharmaceutical compositions thereof. 14 321490133Attorney Docket No. OTPC-050 / 02WO CD8-binding polypeptides

[0054] In some embodiments, a CD8-binding polypeptide of the disclosure comprises one or more of an antibody or a functional fragment thereof. The antibody or functional fragment thereof specifically binds to a CD8 antigen described herein.

[0055] In some embodiments, a CD8-binding polypeptide of the disclosure comprises a sequence, which may be isolated or derived from any species, including but not limited to, human, non-human primate, rodent (including, but not limited to, mouse), and camelid species. In some embodiments, a targeting moiety comprises a sequence, which may be humanized, chimeric, recombinant, non-naturally occurring, modified (for example, to include synthetic nucleic acids or synthetic amino acids), optimized (for example, to reduce immunogenicity and / or aggregation during manufacturing).

[0056] In some embodiments, described herein are anti-CD8α antibodies that can comprise regions such as but not limited to a complementarity-determining region (CDR), a variable domain, or a constant domain. In some embodiments, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some embodiments, the CDR is CDR1, CDR2, or CDR3. In some embodiments, the CDR is a heavy domain including, but not limited to, CDRH1, CDRH2, and CDRH3. In some embodiments, the CDR is a light domain including, but not limited to, CDRL1, CDRL2, and CDRL3. For example, Table 4 provides exemplary CDRs.

[0057] In some embodiments of the disclosure, a CD8-binding polypeptide of the disclosure comprises an antibody, including, but not limited to, a heavy-chain antibody (a VH or a VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific single domain antibody. A “single-domain antibody” or “sdAb” refers to a single antigen-binding domain having three complementarity determining regions (CDRs). A sdAb is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. A “VHH” refers to a single domain antibody consisting of a variable region of a heavy chain. VHH molecules can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Generally, a basic VHH has the following structure from the N-terminus to the C- terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively. 15 321490133Attorney Docket No. OTPC-050 / 02WO

[0058] In some embodiments, a CD8-binding polypeptide of the disclosure comprises a VH or a VHH. In some embodiments, a CD8-binding polypeptide of the disclosure comprises a VHH. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific VHH. Functional Characteristics

[0059] The CD8-binding polypeptides of the disclosure comprise one or more functional characteristics described herein.

[0060] In some embodiments, the CD8-binding polypeptide binds to CD8. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific antibody or antigen binding fragment thereof that binds to CD8. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific VHH that binds to CD8. In some embodiments, the CD8-binding polypeptide binds to mouse, non-human primate, or human CD8. In some embodiments, the CD8-binding polypeptide binds to human CD8 and non-human primate CD8. In some embodiments, the non-human primate CD8 is rhesus CD8 or cynomolgus CD8. In some embodiments, the CD8-binding polypeptide binds to human CD8, rhesus CD8, and / or cynomolgus CD8. In some embodiments, the CD8-binding polypeptide binds to human CD8. In some embodiments, the CD8-binding polypeptide binds to rhesus CD8. In some embodiments, the CD8-binding polypeptide binds to cynomolgus CD8. In some embodiments, the CD8-binding polypeptide binds to rhesus CD8 and cynomolgus CD8.

[0061] In some embodiments, the CD8-binding polypeptide specifically binds to CD8. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific antibody or antigen binding fragment thereof that specifically binds to CD8. In some embodiments, the CD8-binding polypeptide comprises a CD8-specific VHH that specifically binds to CD8. In some embodiments, the CD8-binding polypeptide specifically binds to mouse, non-human primate, or human CD8. In some embodiments, the CD8-binding polypeptide specifically binds to human CD8 and non-human primate CD8. In some embodiments, the non-human primate CD8 is rhesus CD8 or cynomolgus CD8. In some embodiments, the CD8-binding polypeptide specifically binds to human CD8, rhesus CD8, and / or cynomolgus CD8. In some embodiments, the CD8-binding polypeptide specifically binds to human CD8. In some embodiments, the CD8-binding polypeptide specifically binds to rhesus CD8. In some embodiments, the CD8-binding polypeptide specifically binds to cynomolgus CD8. In some embodiments, the CD8-binding polypeptide specifically binds to rhesus CD8 and cynomolgus CD8. 16 321490133Attorney Docket No. OTPC-050 / 02WO

[0062] In some embodiments, the CD8-binding polypeptide binds to human CD8α. In some embodiments, the CD8-binding polypeptide binds to a CD8α isoform comprising an amino acid sequence in Table 1, or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In some embodiments, the CD8-binding polypeptide does not substantially bind to human CD8β.

[0063] In some embodiments, the CD8-binding polypeptide specifically binds to human CD8α. In some embodiments, the CD8-binding polypeptide specifically binds to a CD8α isoform comprising an amino acid sequence in Table 1, or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In some embodiments, the CD8-binding polypeptide does not substantially bind to human CD8β.

[0064] In some embodiments, the CD8-binding polypeptide does not bind to murine CD8. In some embodiments, the CD8-binding polypeptide does not bind to murine CD8α. In some embodiments, the CD8-binding polypeptide does not bind to murine CD8β. Table 1: Human CD8 Domains SEQ Name Exemplary Protein Sequence ID17 321490133Attorney Docket No. OTPC-050 / 02WO SEQ Name Exemplary Protein Sequence ID NO:

[0065] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KD that is substantially similar to the KD of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314 (an exemplary parental CD8 VHH). In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KDthat is at least about 150% lower than the KDof an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KD that is lower relative to the KD of an antibody or antigen binding 18 321490133Attorney Docket No. OTPC-050 / 02WO fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KDthat is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% lower relative to the KD of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KDthat is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold lower relative to the KD of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314.

[0066] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KDthat is lower relative to the KDof a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KD that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% lower relative to the KD of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8- binding polypeptide binds to CD8α (e.g., human CD8α) with a KD that is at least 1 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold lower relative to the KDof a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314.

[0067] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a KD of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 nM or less, about 0.05 nM or less, about 0.01 nM or less, about 0.005 nM or less, or about 0.001 nM or less. In some embodiments, the KDis about 0.01nM to about 100nM. In some embodiments, the KD is between about 0.01 nM and about 0.05 nM, between about 0.01 nM and about 0.1 nM, between about 0.05 nM and about 2 nM, between about 0.1 nM and about 1 nM, between about 0.5 nM and about 5 nM, between about 1 nM and about 5 nM, between about 19 321490133Attorney Docket No. OTPC-050 / 02WO 1 nM and about 10 nM, between about 5 nM and about 10 nM, between about 5 nM and about 20 nM, between about 10 nM and about 25 nM, between about 10 nM and about 50 nM, between about 25 nM and about 50 nM, between about 30 nM and about 60 nM, between about 50 nM and about 70 nM, between about 60 nM and about 80 nM, between about 70 nM and about 90 nM, between about 80 nM and about 100 nM, or between about 50 nM and about 100 nM.

[0068] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koffthat is similar to the koffof an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koffthat is less than about 150% of the koff of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is lower relative to the koff of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% lower relative to the koff of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is at least 1 fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold lower relative to the koffof an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314.

[0069] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is lower relative to the koff of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% lower relative to the koff of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8- binding polypeptide binds to CD8α (e.g., human CD8α) with a koff that is at least 1 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, 20 321490133Attorney Docket No. OTPC-050 / 02WO at least 50-fold, or at least 100-fold lower relative to the koff of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314.

[0070] In some embodiments, the CD8-binding polypeptide binds to CD8α (e.g., human CD8α) with a koffof about 0.01 / s or less, about 0.005 / s or less, about 0.004 / s or less, about 0.003 / s or less, about 0.002 / s or less, about 0.0015 / s or less, about 0.001 / s or less, about 0.0009 / s or less, about 0.0008 / s or less, about 0.0007 / s or less, about 0.0006 / s or less, about 0.0005 / s or less, about 0.0004 / s or less, about 0.0003 / s or less, about 0.0002 / s or less, or about 0.0001 / s or less. In some embodiments, the koffis between about 0.00001 / s to about 0.1 / s. In some embodiments, the Koff is between about 0.00001 / s and about 0.00005 / s, between about 0.00002 / s and about 0.00005 / s, between about 0.000025 / s and about 0.00005 / s, between about 0.000025 / s and about 0.000075 / s, between about 0.00005 / s and about 0.0001 / s, between about 0.00005 / s and about 0.00025 / s, between about 0.00075 / s and about 0.00025 / s, between about 0.00075 / s and about 0.001 / s, between about 0.001 / s and about 0.005 / s, between about 0.001 / s and about 0.01 / s, between about 0.005 / s and about 0.01 / s, between about 0.001 / s and about 0.002 / s, between about 0.001 / s and about 0.005 / s, between about 0.0025 / s and about 0.005 / s, between about 0.0025 / s and about 0.0075 / s, between about 0.005 / s and about 0.01 / s, between about 0.005 / s and about 0.02 / s, between about 0.01 / s and about 0.025 / s, between about 0.01 / s and about 0.05 / s, between about 0.025 / s and about 0.05 / s, between about 0.025 / s and about 0.075 / s, between about 0.05 / s and about 0.075 / s, between about 0.05 / s and about 0.1 / s, between about 0.075 / s and about 0.1 / s, or between about 0.09 / s and about 0.1 / s.

[0071] In some embodiments, the koff is between 1.0x10-8 / s and about 1.0x10-5 / s. In some embodiments, the K koffis between about 1.0x10-8 / s and about 2.5x10-8 / s, between about 1.0x10-8 / s and about 5.0x10-8 / s, between about 2.5x10-8 / s and about 7.5x10-8 / s, between about 5.0x10-8 / s and about 1.0x10-7 / s, between about 5.0x10-8 / s and about 2.0x10-7 / s, between about 1.0x10-8 / s and about 1.0x10-7 / s, between about 1.0x10-7 / s and about 5.0x10-7 / s, between about 2.5x10-7 / s and about 5.0x10-7 / s, between about 5.0x10-7 / s and about 1.0x10-6 / s, between about 7.5x10-7 / s and about 1.0x10-6 / s, between about 7.5x10-7 / s and about 2.5x10-6 / s, between about 1.0x10-6 / s and about 5.0x10-6 / s, between about 2.5x10-6 / s and about 7.5x10-6 / s, between about 5.0x10-6 / s and about 1.0x10-5 / s, between about 7.5x10-6 / s and about 1.0x10-5 / s, between about 8.0x10-6 / s and about 1.0x10-5 / s, or between about 9.0x10-6 / s and about 1.0x10-5 / s.

[0072] The KD of the CD8-binding polypeptide can be determined by any method known in the art, including, but not limited to surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, biolayer interferometry (BLI), and 21 321490133Attorney Docket No. OTPC-050 / 02WO radioimmunoassay (RIA). In some embodiments, the KD of a CD8-binding polypeptide described herein is determined via SPR. In some embodiments, the KDand / or koffof a CD8- binding polypeptide described herein is determined via BLI. In some embodiments, methods for measuring the KDfor a CD8-binding polypeptide described herein comprise detecting a binding signal at different concentrations of the CD8-binding polypeptide and generating a binding curve.

[0073] In some embodiments, an exact determination of KD is unnecessary, as it is sufficient to obtain a qualitative measurement of binding affinity. For example, by comparing binding of a CD8-binding polypeptide described herein to CD8 (e.g., CD8α) as compared to an antibody (e.g., a single-domain antibody or VHH) or antigen binding fragment thereof comprising the sequence set forth in SEQ ID NO: 314, e.g., it is determined whether affinity of the CD8-binding polypeptide for CD8 (e.g., CD8α) is higher (e.g., at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, etc.) than that of the antibody or antigen binding fragment thereof comprising the sequence set forth in SEQ ID NO: 314. Melting temperature

[0074] In some embodiments, the CD8-binding polypeptide has a melting temperature (Tm) similar to that of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has an increased melting temperature (Tm) relative to that of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has an increased Tm relative to that of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has a Tm that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 70%, at least 19%, or at least 20% higher relative to the Tm of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has a Tm that is at least 0.5°C, at least 0.6°C, at least 0.7°C, at least 0.8°C, at least 0.9°C, at least 1.0°C, at least 1.5°C, at least 2.0°C, at least 2.5°C, at least 3.0°C, at least 3.5°C, at least 4.0°C, at least 4.5°C, at least 5.0°C, at least 5.5°C, at least 6.0°C, at least 6.5°C, at least 7.0°C, at least 7.5°C, at least 8.0°C, at least 8.5°C, at least 22 321490133Attorney Docket No. OTPC-050 / 02WO 9.0°C, at least 9.5°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, or at least 15°C higher than the Tm of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314.

[0075] In some embodiments, the CD8-binding polypeptide has a melting temperature (Tm) similar to that of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has an increased melting temperature (Tm) relative to that of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has an increased Tm relative to that of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has a Tm that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 70%, at least 19%, or at least 20% higher relative to the Tm of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. In some embodiments, the CD8-binding polypeptide has a Tm that is at least 0.5°C, at least 0.6°C, at least 0.7°C, at least 0.8°C, at least 0.9°C, at least 1.0°C, at least 1.5°C, at least 2.0°C, at least 2.5°C, at least 3.0°C, at least 3.5°C, at least 4.0°C, at least 4.5°C, at least 5.0°C, at least 5.5°C, at least 6.0°C, at least 6.5°C, at least 7.0°C, at least 7.5°C, at least 8.0°C, at least 8.5°C, at least 9.0°C, at least 9.5°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, or at least 15°C higher than the Tm of a VHH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 314. Binding to CD8α expressing cells

[0076] In some embodiments, the CD8-binding polypeptide binds to a cell expressing CD8 (e.g., human CD8). In some embodiments, the CD8-binding polypeptide binds to a cell expressing CD8α (e.g. human CD8α). In some embodiments, the CD8 expressing cell is a mammalian cell. In some embodiments, the CD8 expressing cell is a human cell. In some embodiments, the CD8 expressing cell is a non-human primate cell. In some embodiments, the cell is a naturally occurring cell. In other embodiments, the cell is an engineered cell, including but not limited to a genetically engineered cell.

[0077] Examples of CD8α expressing cells include, but are not limited to, T cells, natural killer (NK) cells, natural killer T (NKT) cells, monocytes, and dendritic cells. In some 23 321490133Attorney Docket No. OTPC-050 / 02WO embodiments, the CD8-binding polypeptide binds to a T cell, an NK cell, and / or a NKT cell. In certain embodiments, the CD8-binding polypeptide binds to an NK cell. In certain embodiments, the CD8-binding polypeptide binds to a NKT cell. In certain embodiments, the CD8-binding polypeptide binds to a T cell. In some embodiments, the T cell is a CD8+ T cell, a naïve cytotoxic T cell, a mature cytotoxic T cell, a memory T cell, a mucosal associated invariant T cells, a γδ T cells, an intraepithelial T cell, or a thymocyte.

[0078] The binding of the CD8-binding polypeptide to a CD8α expressing cell can be determined by any method known in the art, including, but not limited to flow cytometry and fluorescence microscopy. Expression yield

[0079] In some embodiments, the CD8-binding polypeptide has a similar yield relative to that of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314 when expressed in a yeast cell, an insect cell, a bacterium, a mammalian cell, or a plant cell using the same protein expression method. In some embodiments, the CD8-binding polypeptide has an increased yield relative to that of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314 when expressed in a yeast cell, an insect cell, a bacterium, a mammalian cell, or a plant cell using the same protein expression method. In some embodiments, the CD8-binding polypeptide has a yield that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 70%, at least 19%, or at least 20% higher relative to the yield of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314 when expressed in a yeast cell, an insect cell, a bacterium, a mammalian cell, or a plant cell using the same protein expression method. In some embodiments, the CD8-binding polypeptide has a yield that is at least 1.1-fold, at least 1.15-fold, at least 1.2-fold, at least 1.25-fold, at least 1.13-fold, at least 1.35-fold, at least 1.4-fold, at least 1.45-fold, at least 1.5- fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.8-fold, or at least 2-fold higher relative to the yield of an antibody or antigen binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 314 when expressed in a yeast cell, an insect cell, a bacterium, a mammalian cell, or a plant cell using the same protein expression method. The CD8-binding polypeptides of the disclosure can be produced using any method known in 24 321490133Attorney Docket No. OTPC-050 / 02WO the art for protein expression, including expression in a yeast cell, an insect cell, a bacterium, a plant cell, or a mammalian cell. Examples of mammalian protein expression systems include, but are not limited, to a CHO cell expression system and a HEK293 expression system. Examples of insect protein expression systems include, but are not limited to, a Sf9 expression system and a High Five expression system. Bacterial protein expression systems are known in the art and described, for example, in Terpe et al, Appl Microbiol Biotechnol. 2006; 72(2):211-22. Examples of yeast protein expression systems include protein expression in Saccharomyces cerevisiae or Pichia pastoris. CD8-Specific Single Domain Antibody (VHH) Sequences

[0080] In some embodiments, the CD8-binding polypeptide comprises a VHH that binds to CD8. In some embodiments, the VHH specifically binds to CD8 relative to a non-CD8 molecule. In some embodiments, the VHH specifically binds to human CD8 relative to a non- CD8 molecule or a non-human CD8 molecule. In some embodiments, the VHH specifically binds to human CD8α relative to a non-CD8 molecule, a non-human CD8 molecule, a non- human CD8α molecule, or a CD8β domain. In some embodiments, the VHH does not substantially bind to an epitope on CD8β. In some embodiments, the VHH does not substantially bind to an epitope on human CD8β.

[0081] In some embodiments, the CD8-binding polypeptide comprises a VHH that binds to CD8, and the VHH comprises a single amino acid chain comprising four framework regions (FRs) and three complementarity determining regions (CDRs). The term “FR” with respect to a VHH described herein refers to the region in the variable domain located between the CDRs. The term “CDR” with respect to a VHH described herein refers to the amino acid sequences capable of specifically binding to an antigen described herein.

[0082] The sequence boundaries for a given CDR and FR are determined using numbering schemes known in the art, and further described herein. As appreciated by the skilled artisan, the boundaries of a given CDR or FR vary depending on the numbering scheme used for identification. In some embodiments, the CDRs provided herein are defined according to a numbering scheme described herein or known in the art, e.g., the Kabat numbering scheme, the Chothia numbering scheme, the Kabat + Chothia numbering scheme, or the alternate CDR definition scheme. The FRs are located between the CDRs, for example, with FR1 located at the N- terminus of CDR1, FR2 located between CDR1 and CDR2, FR3 located between CDR2 and 25 321490133Attorney Docket No. OTPC-050 / 02WO CDR3, and FR4 located at the C-terminus of CDR3. Unless otherwise specified, a "CDR" or "complementary determining region," or individual specified CDRs (e.g., CDR1, CDR2, CDR3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a VHH comprises a CDR (e.g., a CDR3) comprising a particular amino acid sequence, it is understood that the CDR comprises the sequence when defined by any of numbering schemes described herein. It is understood that any antibody, such as a VHH, includes CDRs and such can be identified according to any of the numbering schemes described herein or other numbering schemes known to a skilled artisan. (i) Humanized CD8-Specific Single Domain Antibody (VHH)

[0083] In some embodiments, the VHH is a humanized variant with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least one modification described herein with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH has less than 95% sequence identity to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH has less than 90% sequence identity to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH has less than 85% sequence identity to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH has less than 80% sequence identity to the VHH sequence set forth in SEQ ID NO: 314.

[0084] In some embodiments, the at least one modification increases homology of the VHH sequence set forth in SEQ ID NO: 314 to a human immunoglobulin sequence set forth in SEQ ID NO: 387 and / or SEQ ID NO: 388. In some embodiments, at least one residue in the VHH sequence set forth in SEQ ID NO: 314 is substituted with a corresponding residue in the human immunoglobulin sequence set forth in SEQ ID NO: 387 and / or SEQ ID NO: 388, wherein the corresponding residue in the human immunoglobulin sequence is identified from a sequence alignment of SEQ ID NO: 314 and SEQ ID NO: 387 and / or SEQ ID NO: 388. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 residues in the VHH sequence set forth in SEQ ID NO: 314 are substituted with a corresponding residue in the human immunoglobulin sequence set forth in SEQ ID NO: 387 and / or SEQ ID NO: 388. In some embodiments, 10 residues are substituted with a corresponding residue in the human immunoglobulin sequence set forth in SEQ ID NO: 387 and / or SEQ ID NO: 388. 26 321490133Attorney Docket No. OTPC-050 / 02WO In some embodiments, the at least one residue in the VHH sequence set forth in SEQ ID NO: 314 is substituted with a conservative alternate to the corresponding residue in the human immunoglobulin sequence set forth in SEQ ID NO: 387 and / or SEQ ID NO: 388. For example, wherein the corresponding residue is alanine, the residue is substituted with alanine or with a conservative alternate residue to alanine (e.g., valine, leucine, or isoleucine).

[0085] In some embodiments, the VHH comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 5 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 10 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 11 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 12 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 13 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 14 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises at least 15 modifications with respect to the VHH sequence set forth in SEQ ID NO: 314.

[0086] In some embodiments, the at least one modification is in a CDR. In some embodiments, the CDR is defined according to a Kabat numbering scheme. In some embodiments, the CDR is defined according to a Chothia numbering scheme. In some embodiments, the CDR is defined according to an IMGT numbering scheme. In some embodiments, the CDR is defined according to a Kabat+Chothia numbering scheme. In some embodiments, the CDR is defined according to an alternative CDR numbering scheme. In some embodiments, CDR1, CDR2, and CDR3 are defined by different numbering schemes. For example, in some embodiments, CDR1 is defined according to a Kabat+Chothia numbering scheme, CDR2 is defined according to a Kabat numbering scheme, and CDR3 is defined according to a Kabat numbering scheme. In another embodiment, CDR1 is defined according to a Kabat+Chothia numbering scheme, CDR2 is defined according to a Kabat numbering scheme, and CDR3 is defined according to the alternative CDR definition.

[0087] In some embodiments, the at least one modification is in a CDR1, CDR2, and / or CDR3. In some embodiments, the at least one modification is in a CDR1. In some embodiments, the at least one modification is in a CDR2. In some embodiments, the at least one modification is in a CDR3. 27 321490133Attorney Docket No. OTPC-050 / 02WO

[0088] In some embodiments, the at least one modification is in a framework region (FR). In some embodiments, the FR is defined according to a Kabat numbering scheme. In some embodiments, the FR is defined according to a Chothia numbering scheme. In some embodiments, the FR is defined according to an IMGT numbering scheme. In some embodiments, the FR is defined according to a Kabat+Chothia numbering scheme. In some embodiments, the FR is defined according to an alternative CDR numbering scheme.

[0089] In some embodiments, the at least one modification is in an FR1, FR2, FR3, and / or FR4. In some embodiments, the at least one modification is in an FR1. In some embodiments, the at least one modification is in an FR2. In some embodiments, the at least one modification is in an FR3. In some embodiments, the at least one modification is in an FR4.

[0090] In some embodiments, the at least one modification comprises a deletion. In some embodiments, the at least one modification comprises an insertion. In some embodiments, the at least one modification comprises a substitution. In some embodiments, the at least one modification comprises a conservative substitution. In some embodiments, the at least one modification comprises a substitution to increase homology to a human germline immunoglobulin sequence (e.g., a human germline immunoglobulin sequence set forth in SEQ ID NO: 387 or SEQ ID NO: 388).

[0091] In some embodiments, the at least one modification with respect to the VHH sequence set forth in SEQ ID NO: 314 is at a position selected from the group consisting of 1, 5, 14, 34, 37, 44, 45, 47, 63, 72, 75, 79, 87, 88, 91, 124, 127, and a combination thereof. In some embodiments, the at least one modification with respect to the VHH sequence set forth in SEQ ID NO: 314 is at a position selected from the group consisting of 1, 5, 14, 34, 44, 63, 72, 75, 79, 87, 88, 91, 124 and a combination thereof. In some embodiments, the at least one modification with respect to the VHH sequence set forth in SEQ ID NO: 314 is at a position selected from the group consisting of 1, 5, 14, 34, 44, 63, 72, 75, 88, 91, 124 and a combination thereof. In some embodiments, the VHH comprises a modification at positions 1, 5, 14, 72, 75, 91, and 124 with respect to the VHH sequence set forth in SEQ ID NO: 314. In some embodiments, the VHH comprises a modification at positions 1, 5, 14, 72, 75, 91, and 124 with respect to the VHH sequence set forth in SEQ ID NO: 314.

[0092] In some embodiments, the VHH comprises at least one modification selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, F37V, E44G, R45L, G47W, S72R, A75S, V79L, K87R, P88A, A91T, K124Q, Q127L, and a combination thereof. In some embodiments, the VHH comprises at least one modification is selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, E44G, S72R, A75S, V79L, K87R, P88A, A91T, 28 321490133Attorney Docket No. OTPC-050 / 02WO K124Q, and a combination thereof. In some embodiments, the VHH comprises at least one modification is selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, E44G, S72R, A75S, P88A, A91T, K124Q, and a combination thereof.

[0093] In some embodiments, the VHH comprises modifications with respect to the VHH sequence set forth in SEQ ID NO: 314, wherein the modifications comprise or consist of: Q1E, Q5V, A14P, A75S, V79L, K87R, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, A75S, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, S72R, A75S, V79L, K87R, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, S72R, A75S, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, V79L, A91T, and K124Q. In some embodiments, the modifications comprise or consist of: Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, A91T, and K124Q. In some embodiments, the VHH comprises a modification at positions Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, K87R, P88A, A91T, and K124Q.

[0094] In some embodiments, the VHH comprises a CDR1, CDR2, and CDR3, each defined according to a Kabat+Chothia numbering scheme. In some embodiments, CDR1 according to the Kabat+Chothia numbering scheme consists of GFTFDDYAX1G (SEQ ID NO: 373), wherein X1is I or M. In some embodiments, the CDR2 according to the Kabat+Chothia numbering scheme consists of X2IRVSDGSTYYADX3VKG (SEQ ID NO: 374), wherein X2is C, S, or A, and wherein X3is P or S. In some embodiments, the CDR3 according to the Kabat+Chothia numbering scheme consists of GSLYTX4VQSIVVVPARPYYDMDY (SEQ ID NO: 375), wherein X4is C, S, or A.

[0095] In some embodiments, the VHH comprises a CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of GFTFDDYAX1G (SEQ ID NO: 373), wherein the CDR2 comprises or consists of X2IRVSDGSTYYADX3VKG (SEQ ID NO: 374), wherein the CDR3 comprises or consists of GSLYTX4VQSIVVVPARPYYDMDY (SEQ ID NO: 375), wherein X1 is I or M, wherein X2 is C, S, or A, wherein X3 is P or S, and wherein X4 is C, S, or A. In 29 321490133Attorney Docket No. OTPC-050 / 02WO some embodiments, if X1 is I, then X2 is S or A, X3 is S, and / or X4 is S or A. In some embodiments, if X2is C, then X1is M, X3is S, and / or X4is S or A. In some embodiments, if X3 is P, then X1 is M, X2 is S or A, and / or X4 is S or A. In some embodiments, if X4 is C, then X1is M, X2is S or A, and / or X3is S.

[0096] In some embodiments, the VHH comprises a CDR1, CDR2, and CDR3 identified in any one of Tables 2-4. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to a Kabat numbering scheme. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to a Chothia numbering scheme. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to a IMGT numbering scheme. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to a Kabat+Chothia numbering scheme. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to an alternative CDR numbering scheme. In some embodiments, the CDR1 is defined according to a Kabat+Chothia numbering scheme, the CDR2 is defined according to a Kabat numbering scheme, and the CDR3 are defined according to an alternative CDR numbering scheme. In some embodiments, the CDR1 is defined according to a Kabat+Chothia numbering scheme, the CDR2 is defined according to a Kabat numbering scheme, and the CDR3 are defined according to a Kabat numbering scheme.

[0097] In some embodiments, the CDR1 comprises or consists of SEQ ID NO: 2, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR1 comprises or consists of SEQ ID NO: 14, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR2 comprises or consists of SEQ ID NO: 3, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR2 comprises or consists of SEQ ID NO: 7, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR2 comprises or consists of SEQ ID NO: 11, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR2 comprises or consists of SEQ ID NO: 15, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR3 comprises or consists of SEQ ID NO: 4, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR3 comprises or consists of SEQ ID NO: 494, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR3 comprises or consists of SEQ ID NO: 8, or a sequence having 1, 2, or 3 modifications thereto. n some embodiments, the CDR3 comprises or consists of SEQ ID NO: 495, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the CDR3 comprises or consists of SEQ ID NO: 12, or a sequence having 1, 2, or 3 modifications thereto. In some embodiments, the 30 321490133Attorney Docket No. OTPC-050 / 02WO CDR3 comprises or consists of SEQ ID NO: 496, or a sequence having 1, 2, or 3 modifications thereto.

[0098] In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 494. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 494.

[0099] In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 495. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 495.

[0100] In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 496. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID 31 321490133Attorney Docket No. OTPC-050 / 02WO NO: 11, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 496.

[0101] In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the VHH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 494. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the VHH comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 494.

[0102] In some embodiments, an antibody or antibody fragment described herein comprises a CDRH1 sequence of any one of SEQ ID NOs: 304-307. In some embodiments, an antibody or antibody fragment described herein comprises at least about 1, at least about 2, at least about 3, at least about 4, or at least about 5 amino acid substitutions from the sequence of any one of SEQ ID NOs: 304-307. In some embodiments, the antibody or antibody fragment is the VHH that binds to CD8.

[0103] In some embodiments, an antibody or antibody fragment described herein comprises a CDRH2 sequence of any one of SEQ ID NOs: 308-310. In some embodiments, an antibody or antibody fragment described herein comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, or at least about 8 amino acid substitutions from the sequence of any one of SEQ ID NOs: 308-310. In some embodiments the antibody or antibody fragment is the VHH that binds to CD8.

[0104] In some embodiments, an antibody or antibody fragment described herein comprises a CDRH3 sequence of any one of SEQ ID NOs: 311-313. In some embodiments, an antibody or antibody fragment described herein comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 amino acid 32 321490133Attorney Docket No. OTPC-050 / 02WO substitutions from the sequence of any one of SEQ ID NOs: 311-313. In some embodiments the antibody or antibody fragment is the VHH that binds to CD8.

[0105] In some embodiments, the VHH is a sequence provided in Table 2 or Table, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. Tables 2 and 3 provides exemplary CD8-specific VHH of the disclosure and CDRs thereof.

[0106] In some embodiments, the VHH comprises SEQ ID NO: 1. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

[0107] In some embodiments, the VHH comprises SEQ ID NO: 5. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

[0108] In some embodiments, the VHH comprises SEQ ID NO: 9. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.

[0109] In some embodiments, the VHH comprises SEQ ID NO: 13. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.

[0110] In some embodiments, the VHH comprises SEQ ID NO: 17. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17.

[0111] In some embodiments, the VHH comprises SEQ ID NO: 21. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0112] In some embodiments, the VHH comprises SEQ ID NO: 25. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25.

[0113] In some embodiments, the VHH comprises SEQ ID NO: 29. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 29.

[0114] In some embodiments, the VHH comprises SEQ ID NO: 33. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33. 33 321490133Attorney Docket No. OTPC-050 / 02WO

[0115] In some embodiments, the VHH comprises SEQ ID NO: 37. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37.

[0116] In some embodiments, the VHH comprises SEQ ID NO: 315.In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 315.

[0117] In some embodiments, the VHH comprises SEQ ID NO: 316. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 316.

[0118] In some embodiments, the VHH comprises SEQ ID NO: 317. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 317.

[0119] In some embodiments, the VHH comprises SEQ ID NO: 318. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 318.

[0120] In some embodiments, the VHH comprises SEQ ID NO: 319. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 319.

[0121] In some embodiments, the VHH comprises SEQ ID NO: 320. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 320.

[0122] In some embodiments, the VHH comprises SEQ ID NO: 321. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 321.

[0123] In some embodiments, the VHH comprises SEQ ID NO: 322. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 322. 34 321490133Attorney Docket No. OTPC-050 / 02WO

[0124] In some embodiments, the VHH comprises SEQ ID NO: 323. In some embodiments, the VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 323. Table 2: Exemplary CD8-Specific VHH Sequences of the Disclosure Name SEQ ID Sequence (changes compared to VHH No. 1 shown NO: in bold, CDRs are underlined) VHH 140 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKE P S E P S E P S E P S35 321490133Attorney Docket No. OTPC-050 / 02WO Name SEQ ID Sequence (changes compared to VHH No. 1 shown NO: in bold, CDRs are underlined) (Kabat) G P S E A S G A S E P S G P S36 321490133Attorney Docket No. OTPC-050 / 02WO Name SEQ ID Sequence (changes compared to VHH No. 1 shown NO: in bold, CDRs are underlined) VHH 1.4.0.e CDR3 4 AGSLYTCVQSIVVVPARPYYDMDY G A SName SEQ ID Sequence NO: E P S E P S E P S E P S37 321490133Attorney Docket No. OTPC-050 / 02WO Name SEQ ID Sequence NO: VHH 1.1.2 CDR1 2 GFTFDDYAIG E P S E P S E P S E P S E P S38 321490133Attorney Docket No. OTPC-050 / 02WO Name SEQ ID Sequence NO: VHH 1.3.2 323 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKE P SSEQ SEQ SEQ Descriptio ID CDRH1 ID CDRH2 ID CDRH3 n NO NO NO I D I D Q Y I D I D , n S Y

[0125] In some embodiments, the VHH is affinity matured with respect to a CD8-specific VHH described herein (e.g., a humanized CD8-specific VHH described herein). In some embodiments, the VHH is affinity matured with respect to a CD8-specific VHH sequence set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, and 315-323. In some embodiments, the VHH is affinity matured with respect to the CD8-specific VHH sequence of SEQ ID NO: 17. In some embodiments, the VHH is affinity matured with respect to the CD8-specific VHH sequence of SEQ ID NO: 1. 39 321490133Attorney Docket No. OTPC-050 / 02WO

[0126] In some embodiments, the affinity matured VHH comprises at least one modification in a CDR with respect to the CD8-specific VHH described herein (e.g., the humanized CD8-specific VHH described herein). In some embodiments, the CDR is defined according to a numbering scheme described herein. In some embodiments, the CDR is defined according to Kabat. In some embodiments, the affinity matured VHH comprises at least one modification in a FR with respect to the CD8-specific VHH described herein (e.g., the humanized CD8-specific VHH described herein). In some embodiments, the FR is defined according to a numbering scheme described herein. In some embodiments, the FR is defined according to Kabat. In some embodiments, the affinity matured VHH comprises a FR1, FR2, FR3, and / or FR4 that are identical relative to the respective sequences in the CD8- specific VHH described herein (e.g., the humanized CD8-specific VHH described herein). In some embodiments, the CD8-specific VHH is set forth in SEQ ID NO: 17. In some embodiments, the CD8-specific VHH is set forth in SEQ ID NO: 1.

[0127] In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and / or CDR3 comprising a sequence provided in Table 5. Table 5 provides CDR sequences for exemplary affinity matured CD8-specific VHH of the disclosure.

[0128] In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 defined according to Kabat. In some embodiments, the CDR1 defined according to Kabat comprises or consists of DYAX5G (SEQ ID NO: 376), wherein X5is I or L. In some embodiments, CDR2 defined according to Kabat comprises or consists of CIRX6X7DX8X9X10YYADPX11KG (SEQ ID NO: 377), wherein X6is A, C, F, I, L, N, S, T, V, W, or Y, X7 is F, S, W, or Y, X8 is G, R, or V, X9 is A, M, R, S, or W, X10 is D, F, S, or T, and X11is L or V. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLX12X13CVQX14X15X16X17X18X19X20X21X22X23YDMDY (SEQ ID NO: 378), wherein X12is F, W, or Y, X13is A, S, or T, X14is G, S, W, or Y, X15is F, H, I, L, or W, X16is A, E, G, I, L, N, P, R, S, or V, X17 is A, G, P, S, or V, X18 is D, E, or V, X19 is P or S, X20 is A, D, E, N, S, or T, X21is A, D, E, G, H, L, Q, R, S, or V, X22is A, D, E, N, or P and X23is R, V, or Y.

[0129] In some embodiments, the CDR1 defined according to Kabat comprises or consists of the VHH CDR1 comprises DYAX5G (SEQ ID NO: 376), wherein X5 is I or L. In some embodiments, the CDR2 defined according to Kabat comprises or consists of CIRX24X25DX26X27X28YYADPX29KG (SEQ ID NO: 379), wherein X24 is A, C, F, I, L, N, S, T, V, W, or Y; X25is F, S, W, or Y; X26is A, G, I, R, or V; X27is A, M, R, S, or W; X28is D, F, S, or T; and X29 is V or L. In some embodiments, the CDR3 defined according to Kabat 40 321490133Attorney Docket No. OTPC-050 / 02WO comprises or consists of GSLYTCVQSX30X31X32X33PYYDMDY (SEQ ID NO: 380), wherein X30is F, H, I, or L; X31is A, D, G, H, I, K, L, N, Q, R, S, T, V, or Y; X32is A, D, or G; X33 is D, E, I, K, or R. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVQX34X35X36X37X38X39PX40YDMDY (SEQ ID NO: 381), wherein X34 is F, H, L, S, W, or Y; X35 is F, H, I, L, or W; X36 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W; X37is D, P, or V; X38is A, E, or V; X39is P or R; X40is E or Y. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVQX41X42X43X44X45X46X47X48YYDMDY (SEQ ID NO: 382), wherein X41is F, S, or Y; X42 is F, I, L, W, or Y; X43 is A, G, H, K, L, M, P, R, S, V, or W; X44 is P, V, or Y; X45 is D, E, H, P, or V; X46is A, E, G, or P; X47is A, E, G, R, or S; X48is D, E, P, or V. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVQSX49X50X51X52X53X54X55X56X57X58DMDY (SEQ ID NO: 383), wherein X49is F, H, I, or L; X50 is I, L, N, or V; X51 is A, I, K, M, P, S, T, or V; X52 is E, N, P, R, T, or V; X53 is G or P; X54is A, G, H, T, or Y; X55is E, G, L, R, or Y; X56is D, E, or P; X57is S or Y; X58is D or Y. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVQX59X60X61X62VX63X64X65X66X67X68YDMDY (SEQ ID NO: 384), wherein X59 is S or Y; X60 is F, I, L, or W; X61 is A, E, F, G, K, L, Q, S, T, or V; X62 is A, D, E, K, M, P, S, T, or V; X63 is P or V; X64 is A, D, E, F, G, I, N, P, R, or V; X65 is A, D, E, G, I, L, N, P, Q, R, S, T, V, W, or Y; X66is A, D, E, G, R, S, T, V, or Y; X67is A, D, E, G, P, or V; X68is L, S, or Y. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVLX69X70X71X72X73X74X75X76X77X78X79X80YDMDY (SEQ ID NO: 385), X69is H, K, L, Q, R, S, or Y; X70 is F, I, L, Q, or W; X71 is G, K, L, P, Q, R, S, or V; X72 is D, I, S, or V; X73is D, E, N, P, or V; X74is A, D, E, G, P, or V; X75is A, E, R, T, or V; X76is P, R, or V; X77is A, D, E, K, N, P, S, T, or V; X78 is E, G, Q, R, S, V, or Y; X79 is D, E, P, S, or T; X80 is R or Y. In some embodiments, the CDR3 defined according to Kabat comprises or consists of GSLYTCVQX81X82X83X84X85X86 X87X88X89X90X91X92X93YDMDY (SEQ ID NO: 386), wherein X81is H, R, S, or W; X82is F, I, or L; X83is I, N, R, V, or W; X84is P, R, S, or V; X85is A, D, E, I, R, V, or Y; X86 is D, G, N, P, Q, or V; X87 is A, D, E, G, L, R, or V; X88 is R or V; X89is D, G, or P; X90is A, D, E, G, H, L, M, P, Q, R, S, T, V, or Y; X91is A, D, E, G, L, R, S, V, or Y; X92 is D, E, F, L, P, Q, or V; and X93 is E, I, M, R, T, V, or Y.

[0130] In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 in a VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493 according to a Kabat numbering scheme. In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 in a VHH sequence set forth in any one of SEQ ID 41 321490133Attorney Docket No. OTPC-050 / 02WO NOs: 333-372 and 394-493 according to a Chothia numbering scheme. In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 in a VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493 according to a Kabat+Chothia numbering scheme. In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 in a VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493 according to a IMGT numbering scheme. In some embodiments, the affinity matured VHH comprises a CDR1, CDR2, and CDR3 in a VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493 according to an alternative CDR definition numbering scheme. In some embodiments, the affinity matured VHH comprises a CDR1 in a VHH sequence according to a Kabat+Chothia numbering scheme, a CDR2 in the VHH sequence according to a Kabat numbering scheme, and a CDR3 in the VHH sequence according to a Kabat numbering scheme, wherein the VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493. In some embodiments, the affinity matured VHH comprises a CDR1 in a VHH sequence according to a Kabat+Chothia numbering scheme, a CDR2 in the VHH sequence according to a Kabat numbering scheme, and a CDR3 in the VHH sequence according to an alternative CDR definition numbering scheme, wherein the VHH sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493.

[0131] In some embodiments, the affinity matured VHH comprises a sequence provided in Table 6, or an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Table 6 provides exemplary affinity matured CD8-specific VHH of the disclosure.

[0132] In some embodiments, the affinity matured VHH comprises a sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence set forth in any one of SEQ ID NOs: 333-372 and 394-493.

[0133] In some embodiments, the affinity matured VHH comprises SEQ ID NO: 333. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 333. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 334. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 334. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 335. In some embodiments, the affinity matured VHH 42 321490133Attorney Docket No. OTPC-050 / 02WO comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 335. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 336. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 336. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 337. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 337. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 338. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 338. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 339 In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 339. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 340. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 340. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 341. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 341. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 342. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 342. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 343. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 343. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 344. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 344. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 345. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 43 321490133Attorney Docket No. OTPC-050 / 02WO to SEQ ID NO: 345. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 346. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 346. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 347. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 347. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 348. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 348. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 349. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 349. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 350. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 350. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 351. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 351. In some embodiments, the affinity matured VHH comprises SEQ ID NO: 352. In some embodiments, the affinity matured VHH comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 352. Table 5: CDR Sequences for Exemplary Affinity Matured CD8-specific VHH Name / Identifier Sequence SEQ ID NO44 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO is A, D, E, N, S, or T, X is A, D, E, G, H, L R V X i A D E N Pp y y p Name / Identifier Sequence SEQ ID NO5 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO Substitutions relative to SEQ ID NO: 314 are shown in bold / underline46 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO VHH 1.4.0_aff.14 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGV47 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGV48 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGV49 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGV50 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGV51 321490133Attorney Docket No. OTPC-050 / 02WO Name / Identifier Sequence SEQ ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVCD8-Targeted Conjugates

[0134] The present disclosure provides CD8-targeted conjugates comprising a CD8- binding polypeptide described herein. CD8-targeted conjugates of the disclosure comprise a CD8-binding polypeptide operably linked to a second moiety.

[0135] In some embodiments, the second moiety is comprises a detection label, a cytotoxic agent, an antigen-targeting domain, a signaling domain, and / or a second polypeptide. 52 321490133Attorney Docket No. OTPC-050 / 02WO

[0136] In some embodiments, the CD8-binding polypeptides of the disclosure is operably linked to a nucleic acid. In some embodiments, the nucleic acid comprises an antisense oligonucleotides (ASOs), a small interfering RNA (siRNA), a microRNA (miRNA), a messenger RNA (mRNA), an aptamer, plasmid DNA, a guide RNA (gRNA), or a circular DNA.

[0137] In some embodiments, the CD8-specific VHH domain does not comprise an Fc domain. In some embodiments, the CD8-specific VHH domain is fused to an Fc domain. In some embodiments, the CD8-binding polypeptide is fused to an Fc domain. In some embodiment, the Fc domain comprises a hinge, a CH1, a CH2, and / or a CH3 domains. In some embodiments, the Fc domain is an IgG, IgD, IgE, IgM, or IgA isotype. In some embodiments, the Fc domain is an IgG subtype (e.g., IgG1).

[0138] In some embodiments, the VHH domain is operably linked to one or more chemical moieties. In some embodiments, the one or more chemical moieties comprise a polymer, an affinity label, a radiolabel, or a fluorophore. In some embodiments, the one or more chemical moieties comprise a polymer. In certain embodiments, the polymer is PEG. In some embodiments, the one or more chemical moieties comprise an affinity label. In certain embodiments, the affinity label is biotin. In some embodiments, the one or more chemical moieties comprise a radiolabel. In certain embodiments, the radiolabel comprises a gamma- emitting radionuclide. Examples of gamma-emitting radionuclides include, but are not limited to,67Ga,99mTc,luIn,123I,131I,153Sm, or186Re. In some embodiments, the one or more chemical moieties comprise or a fluorophore. In some embodiments, the one or more chemical moieties comprises a chelating agent. In some embodiments, the one or more chemical moieties comprises a carbohydrate. In some embodiments, the one or more chemical moieties comprises a cytotoxic agent (e.g., a chemotherapeutic drug).

[0139] In some embodiments, the CD8-binding polypeptide is operably linked to a half-life extension domain. In some embodiments, the half-life extension domain is selected from a polymer (e.g., PEG), albumin (e.g., human serum albumin), an elastin-like protein, transferrin, and an Fc fragment. In some embodiments, the polymer is a poly(ethylene glycol) (PEG) moiety or a derivative thereof. In some embodiments, the PEG moiety increases half- life and / or reduces immunogenicity of the CD8-binding polypeptide.

[0140] In some embodiments, the CD8-binding polypeptide is operably linked to a detectable moiety. In some embodiments, the detectable moiety comprises a fluorescent label, a phosphorescent label, a chemiluminescent label, a radio-isotope, a bioluminescent label, a chromophore, and / or an enzyme. In some embodiments, the detectable moiety is detectable in 53 321490133Attorney Docket No. OTPC-050 / 02WO an immunoassay (e.g., ELISA). In some embodiments, the detectable moiety is detectable by imaging (e.g., flow cytometry or microscopy). In some embodiments, the detectable moiety is detectable by spectroscopy (e.g., NMR).

[0141] In some embodiments, the CD8-binding polypeptide is operably linked to a cytotoxic agent (i.e., a substance that inhibits or prevents proliferation, function, expression of cells and / or promotes cell killing). In some embodiments, the cytotoxic agent is a chemotherapeutic drug (e.g., an anthracycline, a platinum-based agent, a taxane, an alkylating agent, or exatecan or a derivative thereof). In some embodiments, the cytotoxic agent is a toxin. In some embodiments, the toxin is a small molecule. In some embodiments, the toxin is an enzyme.

[0142] In some embodiments, the CD8-binding polypeptide operably linked to the second moiety via a linker. In some embodiments, the linker comprises a polypeptide, a sugar, a nucleic acid, or a small molecule. In some embodiments, the linker is any that does not affect or reduce the stability, orientation, function, and / or binding properties of the CD8-binding polypeptide and second moiety. In some embodiments, the linker is cleavable. For example, in some embodiments, the linker is acid-labile, photosensitive, or susceptible to enzymatic cleavage.

[0143] In some embodiments, the linker is a linking segment present in a naturally occurring multi-domain protein. In some embodiments, the linker is a hinge region of an antibody (e.g., an IgG, IgA, IgD, IgE antibody).

[0144] In some embodiments, the linker is a polypeptide of less than about 100, 90, 80, 70, 60, 50, 40, 30, or 20 amino acid residues in length. In some embodiments, the polypeptide comprises a sequence that is flexible. In some embodiments, the polypeptide comprises a sequence that is rigid. In some embodiments, the polypeptide is substantially comprises of glycine and serine residues (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more glycine and serine).

[0145] In some embodiments, the linker is a synthetic polymer (e.g., a PEG). Cytokine Conjugates

[0146] In some embodiments, the CD8-binding polypeptides of the disclosure is operably linked to a second polypeptide, wherein the second polypeptide is a cytokine. In some embodiments, the second polypeptide comprises a naturally occurring cytokine. In some embodiments, the second polypeptide comprises a fragment of a naturally occurring cytokine. 54 321490133Attorney Docket No. OTPC-050 / 02WO In some embodiments, the second polypeptide comprises a variant of a naturally occurring cytokine or a fragment thereof.

[0147] In some embodiments, a cytokine can comprise an interleukin (IL), an interferon (IFN), a tumor necrosis factor (TNF), a growth factor, a chemokine (CXCL), a fragment thereof, an engineered version thereof, or a combination thereof.

[0148] In some embodiments, a cytokine can comprise an interleukin. In some embodiments, the cytokine comprises a naturally occurring interleukin. In some embodiments, the cytokine comprises a fragment of a naturally occurring interleukin. In some embodiments, the cytokine comprises a variant of a naturally occurring interleukin or a fragment thereof.

[0149] In some embodiments, the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), or interleukin 36 polypeptide (IL- 36). In some embodiments, a cytokine can comprise an IL-2 cytokine. In certain embodiments, the cytokine comprises an IL-2 polypeptide or a variant thereof. In certain embodiments, the cytokine comprises an IL-15 polypeptide or a variant thereof.

[0150] In some embodiments, a cytokine can comprise an interferon. Non-limiting examples of interferons can include IFN-α, IFN-β, IFN-γ, or any combination thereof. In some embodiments, the cytokine comprises an interferon. In some embodiments, the cytokine comprises a naturally occurring interferon. In some embodiments, the cytokine comprises a fragment of a naturally occurring interferon. In some embodiments, the cytokine comprises a variant of a naturally occurring interferon or a fragment thereof. In some embodiments, the 55 321490133Attorney Docket No. OTPC-050 / 02WO interferon comprises one or more of an interferon type I, an interferon type II, or an interferon type III. In some embodiments, the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN-ε), an interferon kappa (IFN- κ), an interferon omega (IFN-ω), or an interferon gamma (IFN-γ).

[0151] In some embodiments, a cytokine can comprise a tumor necrosis factor. Non- limiting examples of tumor necrosis factors can be TNF-α, TNF-β, or a combination of TNF- α and TNF-β. In some embodiments, the cytokine comprises a tumor necrosis factor (TNF). In some embodiments, the cytokine comprises a naturally occurring TNF. In some embodiments, the cytokine comprises a fragment of a naturally occurring TNF. In some embodiments, the cytokine comprises a variant of a naturally occurring TNF or a fragment thereof. In some embodiments, the cytokine comprises TNF-α. In some embodiments, the cytokine comprises TNF-β. In some embodiments, the cytokine comprises TNF-α and TNF- β.

[0152] In some embodiments, a cytokine can be a growth factor. Non-limiting examples of growth factors can include GM-CSF, G-CSF, M-CSF, EGF, PDGF, VEGF, or any combination thereof. In some embodiments, the cytokine comprises a growth factor.

[0153] In some embodiments, a cytokine can comprise a chemokine. Non-limiting examples of chemokines can include CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, or any combination thereof. In some embodiments, the cytokine comprises a chemokine. In some embodiments, the cytokine comprises a naturally occurring chemokine. In some embodiments, the cytokine comprises a fragment of a naturally occurring chemokine. In some embodiments, the cytokine comprises a variant of a naturally occurring chemokine or a fragment thereof.

[0154] In some embodiments, the second polypeptide comprises a designed cytokine. Cells of the disclosure may be modified to express a cytokine, including but not limited to a de novo polypeptide (also referred to herein as a “designed cytokine” of the disclosure). In some embodiments, the engineered cytokine construct of the disclosure can comprise an antibody (e.g., anti-CD8α antibody), which can assist in directing T cells to target cells. A cytokine (e.g., a designed cytokine) operably linked to an antibody is referred to as an “engineered cytokine.” An engineered cytokine of the disclosure comprises a cytokine (e.g., a designed cytokine described herein) operably linked to a CD8-binding polypeptide described herein.

[0155] A cytokine can comprise a naturally occurring sequence. Alternatively, a cytokine can comprise an engineered cytokine sequence. In some embodiments, a cytokine of the disclosure can reduce preferential Treg stimulation, potentiates T cell subtype targeting, 56 321490133Attorney Docket No. OTPC-050 / 02WO stabilizes protein folding, and reduces immunogenicity. In some embodiments, engineered cytokines eliminate preferential Treg stimulation. In some embodiments, engineered cytokines eliminate immunogenicity. In some embodiments, engineered cytokines can provide enhanced function and improved stability without post-translational modifications. In some embodiments, an engineered cytokine of the disclosure can comprise an extracellular domain, a transmembrane domain, an intercellular domain, or any combination thereof. Exemplary designed cytokines of the disclosure comprise one or more of sequences provided in Table 7 or Table 8 or a sequence having at least 70% identity to a sequence of provided in Table 7. Exemplary designed cytokines of the disclosure include, but are not limited to, those designed cytokines disclosed in PCT Publication No. WO 2023 / 137346, the contents of which are incorporated herein by reference in their entirety.

[0156] In some embodiments of the designed cytokine of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 283.

[0157] In some embodiments of the designed cytokine of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 177.

[0158] In some embodiments of the designed cytokine of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 242.

[0159] In some embodiments of the designed cytokine of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 220.

[0160] In some embodiments, the engineered cytokine construct of the disclosure can reduce or ablate binding of the designed cytokine to the alpha subunit of the IL-2 Receptor (IL-2Rɑ). Alternatively or in addition to, the engineered cytokine construct of the disclosure can bind to the beta and / or gamma subunit of the IL-2 Receptor (IL-2Rβ / γ). Alternatively or in addition to, the engineered cytokine construct of the disclosure can bind to the IL-2 receptor β / γ heterodimer (IL-2Rβ / γ) as a heterodimeric receptor.

[0161] In some embodiments, the engineered cytokine construct of the disclosure can comprise alpha helices H1, H2, H3, and H4. In some embodiments, from an amino terminus to a carboxy terminus, a first loop (L1) connects H1 and H4; a second loop (L2) connects H4 and H2; a third loop (L3) connects H2 and H3.

[0162] In some embodiments, an engineered cytokine of the disclosure can further comprise a signal peptide, which can be used to direct the insertion of the cytokine into a membrane of a cell or intracellular component. A signal peptide can be located on the 5’ end of the engineered cytokine. Alternatively, a signal peptide can be located on the 3’ end of the engineered cytokine or in the middle of the engineered cytokine. 57 321490133Attorney Docket No. OTPC-050 / 02WO

[0163] In some embodiments, an engineered cytokine of the disclosure can further comprise a linker sequence to connect portions of the engineered cytokine construct. For example, an engineered cytokine can comprise an engineered IL-2 cytokine coupled to an anti-CD8a humanized VHH using a linker sequence. Table 7: Designed Cytokines Designed Sequence SEQ ID Cytokine NO:58 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:59 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:60 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:61 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:62 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:63 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:64 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:65 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:66 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:67 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:68 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:69 321490133Attorney Docket No. OTPC-050 / 02WO Designed Sequence SEQ ID Cytokine NO:

[0164] The present disclosure provides exemplary CD8-targeted conjugate comprising a CD8-binding polypeptide described herein and a designed cytokine described herein. The 70 321490133Attorney Docket No. OTPC-050 / 02WO exemplary CD8-targeted conjugate is also referred to as an “engineered cytokine” in this section.

[0165] In some embodiments, an engineered cytokine described herein comprises a CD8- binding polypeptide and a designed cytokine, wherein the CD8-binding polypeptide comprises a CD8-specific VHH comprising a sequence set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, and 315-323 or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the designed cytokine comprises a sequence set forth in Table 7 or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0166] In some embodiments, an engineered cytokine described herein comprises a CD8- binding polypeptide and a designed cytokine, wherein the CD8-binding polypeptide comprises a CD8-specific VHH comprising a sequence set forth in any one of SEQ ID NOs: 333-352 or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the designed cytokine comprises a sequence set forth in Table 7 or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0167] In some embodiments, the designed cytokine comprises SEQ ID NO: 302 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the designed cytokine comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acid substitutions relative to SEQ ID NO: 302.

[0168] In some embodiments, an engineered cytokine as described herein comprises the sequence of SEQ ID NO: 301 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered cytokine as described herein comprises a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 80% identical to a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 85% identical to a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 90% a sequence 71 321490133Attorney Docket No. OTPC-050 / 02WO of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 95% identical to a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 96% a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 97% a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 98% a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that is at least 99% a sequence of SEQ ID NO: 301. In some embodiments, an engineered cytokine as described herein comprises a sequence that comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acid substitutions from the sequence of SEQ ID NO: 301.

[0169] In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 80% identical to a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 85% identical to a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 90% a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 95% identical to a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 96% a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 97% a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 98% a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that is at least 99% a sequence of SEQ ID NO: 303. In some embodiments, an engineered cytokine as described herein comprises an anti-CD8a VHH sequence that comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 72 321490133Attorney Docket No. OTPC-050 / 02WO 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acid substitutions from the sequence of SEQ ID NO: 303.

[0170] In some embodiments, an engineered cytokine as described herein comprises the sequence of any one of SEQ ID NOs: 324-372 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Table 8: Engineered Cytokines SEQ ID Description Sequence NO: MVLQTQVFISLLLWISGAYGAPTSSSTKKTQLQLEHLLLDLQMILNGINNM S P K Y I E I Y I E I S I E I S I E M S I E M S I E M S I E M S I E73 321490133Attorney Docket No. OTPC-050 / 02WO SEQ ID Description Sequence NO: LKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAM S I E M S I E M S I E I S I E I S I E I S I E I S I E I S I E I S I E I S I E I S I E I S74 321490133Attorney Docket No. OTPC-050 / 02WO SEQ ID Description Sequence NO: APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSI E I S I E I S I E I S I E I S I E I S I E I S I E I S I E M S I E M S I E M S I E M Su -spec c onjugaes 75 321490133Attorney Docket No. OTPC-050 / 02WO

[0171] In some embodiments, the CD8-targeted conjugate is multi-specific, i.e., the CD8- targeted conjugate comprises two or more targeting moieties comprising antigen binding domains that bind different targets, e.g., different antigens or different epitopes on a same target. In some embodiments, at least one of the two or more targeting moieties comprise a CD8-binding polypeptide described herein and at least one of the two or more targeting moieties comprises an antibody or antigen binding fragment thereof that recognizes an epitope that is different from the epitope recognized by the CD8-binding polypeptide.

[0172] In some embodiments, the multi-specific conjugate comprises a CD8-binding polypeptide described herein and at least one second polypeptide, wherein the second polypeptide is an antibody or antigen binding fragment thereof that recognizes an epitope on an antigen that is not CD8. In some embodiments, the multi-specific conjugate comprises a CD8-binding polypeptide described herein and a second polypeptide, a third polypeptide, and / or a fourth polypeptide, wherein the second, third, and / or fourth polypeptide is an antibody or antigen binding fragment thereof that recognizes an epitope on an antigen that is not CD8.

[0173] In some embodiments, the multi-specific conjugate comprises at least two CD8- binding polypeptides described herein. In some embodiments, the multi-specific conjugate has increased binding avidity to CD8 relative to a conjugate comprising a single CD8-binding polypeptide.

[0174] In some embodiments, the multi-specific conjugate comprises a CD8-binding polypeptide described herein and an antibody or antigen binding fragment thereof that binds to a tumor antigen. In some embodiments, the multi-specific conjugate binds to a tumor cell via the antibody or antigen binding fragment thereof that binds to a tumor antigen and recruits a CD8-expressing cell (e.g., a CD8-expressing T cell or NK cell) via the CD8- binding polypeptide. In some embodiments, the CD8-expressing cell is an effector cell capable of killing and / or inhibiting proliferation of the tumor cell.

[0175] In some embodiments, the multi-specific conjugate comprises a CD8-binding polypeptide described herein and an antibody or antigen binding fragment thereof that binds to a viral antigen.

[0176] In some embodiments, the multi-specific conjugate comprises a CD8-binding polypeptide described herein and an antibody or antigen binding fragment thereof that binds to an antigen present on an immune cell (e.g., a T cell, B cell, dendritic cell, macrophage, monocyte, NK cell). In some embodiments, the multi-specific conjugate binds to a CD8 antigen on an immune cell (e.g., a CD8-expressing T cell or NK cell) via the CD8-binding 76 321490133Attorney Docket No. OTPC-050 / 02WO polypeptide and recruits a second immune cell via the antibody or antigen binding fragment thereof that binds to an antigen present on the second immune cell.

[0177] In some embodiments, the multi-specific conjugate is an antibody drug conjugate. In some embodiments, the multi-specific conjugate further comprises a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic drug (e.g., exatecan or a derivative thereof). In some embodiments, the cytotoxic agent is a toxin (e.g., auristatin E, auristatin F, MMAE, or MMAF). In some embodiments, the cytotoxic agent is operably linked to a targeting moiety or an Fc domain of the multi-specific conjugate via a linker. In some embodiments, the linker is cleavable.

[0178] The multi-specific conjugates of the disclosure are constructed using methods known in the art. For example, in some embodiments, the multi-specific conjugate is constructed by chemically crosslinking a CD8-binding polypeptide described herein to a second targeting moiety. In another embodiment, the multi-specific conjugate is constructed by genetic fusion, i.e., constructing a gene encoding a fusion polypeptide comprising a CD8- binding polypeptide described herein to a second targeting moiety. In some embodiments, the encoded fusion polypeptide comprises a linker between the CD8-binding polypeptide and the second targeting moiety. Targeted Delivery

[0179] In some embodiments, the CD8-binding polypeptides of the disclosure is operably linked to a delivery vehicle. In some embodiments, the delivery vehicle is a viral vector. In some embodiments, the viral vector is derived from a lentivirus, a retro virus, a vaccinia virus, herpes simplex virus, an adenovirus, an oncolytic virus, or an adeno-associated virus (AAV). In some embodiments, the CD8-binding polypeptide is encoded by the viral vector. In some embodiments, the CD8-binding polypeptide is encoded as a fusion protein to envelope protein encoded by the viral vector.

[0180] some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the non-viral vector comprises a particle. In some embodiments, the CD8- binding polypeptide is linked to a surface of the particle.

[0181] In some embodiments, the particle is a virus-like particle. In some embodiments, the particle is a protein nanoparticle. In some embodiments, the particle is a lipid-based particle. In some embodiments, the lipid-based particle comprises a phospholipid bilayer. In some embodiments, the lipid-based particle is a liposome. In some embodiments, the lipid-based 77 321490133Attorney Docket No. OTPC-050 / 02WO particle is a lipid nanoparticle comprising a cationic lipid, a phospholipid, and optionally a pegylated lipid and / or a structural lipid (e.g., cholesterol). In some embodiments, the particle is a polymer-based particle (e.g., a hydrogel particle). In some embodiments, the particle is a metal nanoparticle. Chimeric Antigen Receptors

[0182] Chimeric antigen receptors (CARs) are synthetic receptors that can be used to provide immune cells with engineered specificity. Provided herein is a chimeric antigen receptor (CAR) comprising a CD8-binding polypeptide of the disclosure. In some embodiments, a CAR of the disclosure comprises (a) an extracellular domain comprising a CD8-binding polypeptide described herein, (b) a transmembrane domain, and (c) an intracellular domain. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a human endogenous transmembrane protein. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a sequence encoding human CD3-zeta, human CD4, human CD8, human CD28 and / or human ICOS. In some embodiments, the intracellular domain comprises a sequence isolated or derived from a human CD3-zeta sequence. In some embodiments, the intracellular domain further comprises at least one co-stimulatory domain. In some embodiments, the at least one co-stimulatory domain comprises a sequence isolated or derived from a human CD28 sequence. In some embodiments, the at least one co-stimulatory domain comprises a sequence isolated or derived from a human 41BB sequence or an OX40 sequence. In some embodiments, the intracellular domain comprises a sequence isolated or derived from a cytokine inducer. In some embodiments, the cytokine inducer comprises an IL-12 cytokine inducer. In some embodiments, the cytokine inducer binds to a sequence isolated or derived from a Nuclear factor of activated T cells (NFAT) sequence. In some embodiments, the intracellular domain comprises a sequence isolated or derived from an IL-2 receptor (IL-2R). In some embodiments, the IL-2R comprises IL-2R beta. Production of CD8-Binding Polypeptides and Conjugates Thereof

[0183] The disclosure provides a nucleic acid sequence encoding a CD8-binding polypeptide or a conjugate thereof described herein, a vector comprising the nucleic acid sequence, and a cell comprising the CD8-binding polypeptides described herein or conjugate thereof, the nucleic acid sequence, and / or the vector. 78 321490133Attorney Docket No. OTPC-050 / 02WO

[0184] In some embodiments, the disclosure provides a cell for expression of the CD8- binding polypeptide or the conjugate thereof. In some embodiments, the cell comprises a nucleic acid sequence encoding the CD8-binding polypeptide or the conjugate thereof described herein or a vector comprising the nucleic acid. In some embodiments, the vector is an expression vector (e.g., a plasmid or an mRNA). In some embodiments, the expression occurs in vitro.

[0185] In some embodiments, the disclosure provides a therapeutic cell for expression of the CD8-binding polypeptide or the conjugate thereof. In some embodiments, the therapeutic cell is a stem cell. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a differentiated cell (e.g., a fully differentiated cell). In some embodiments, the therapeutic cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell. In some embodiments, the cell is a hematopoietic precursor cell. In some embodiments, the cell is a T-cell precursor cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell, a Natural Killer (NK) cell, or a macrophage. In some embodiments, the cell is differentiated in vitro or ex vivo from a stem cell (e.g., an embryonic stem cell, an HSC) or a precursor cell (e.g., a hematopoietic precursor cell). In some embodiments, the cell is isolated or harvested from a subject. In some embodiments, the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood obtained from a subject. In some embodiments, the cell has been cryopreserved. In some embodiments, the cell has been modified to become an allogeneic cell. In some embodiments, the cell has been modified to produce an allogeneic cell. In some embodiments, the cell is capable of expressing or is modified to express an exogenous protein. In some embodiments, the exogenous protein comprises an antigen receptor. In some embodiments, the antigen receptor is a T cell receptor or a chimeric antigen receptor. In some embodiments, the cell is capable of expressing or is modified to express a safety switch. In some embodiments, the cell is capable of expressing or is modified to express a cell marker. In some embodiments, the cell is capable of expressing or is modified to express a truncated EGFR (EGFRt). In some embodiments, the truncated EGFR (EGFRt) comprises a sequence set forth in Table 9. In some embodiments, the EGFRt comprises SEQ ID NO: 53 or a sequence having at least about 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the truncated and optimized EGFR (EGFRopt) comprises a sequence set forth in Table 9. In some embodiments, the EGFRopt comprises a sequence selected from SEQ ID NOs: 4-68, or a sequence having at least about 80%, 85%, 90%, or 95% identity thereto. 79 321490133Attorney Docket No. OTPC-050 / 02WO Table 9: EGFR Safety Switch Sequences SEQ ID Name Exemplary EGFR Safety Switch Sequences NO: MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTS80 321490133Attorney Docket No. OTPC-050 / 02WO SEQ ID Name Exemplary EGFR Safety Switch Sequences NO: MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTS

[0186] In some embodiments, the disclosure provides a delivery vector described herein (e.g., a viral vector or a particle) for expression of the CD8-binding polypeptide or conjugate thereof in vivo. In some embodiments, the delivery vector comprises a nucleic acid sequence comprising the CD8-binding polypeptide or conjugate thereof. In some embodiments, the delivery vector is a viral vector encoding the CD8-binding polypeptide or conjugate thereof. 81 321490133Attorney Docket No. OTPC-050 / 02WO In some embodiments, the delivery vector is a nucleic acid (e.g., a plasmid or mRNA) encoding the CD8-binding polypeptide or conjugate thereof. In some embodiments, the delivery vector is a lipid particle comprising the CD8-binding polypeptide or conjugate thereof or the nucleic acid. In some embodiments, the delivery vector comprises a gene expression vector for expression of the CD8-binding polypeptide or conjugate thereof in vivo. Nucleic Acids

[0187] In some embodiments, the disclosure provides a nucleic acid sequence comprising a CD8-binding polypeptide described herein or a conjugate described herein comprising the CD8-binding polypeptide and at least one second polypeptide. The term “nucleic acid sequence” or “nucleic acid” is used interchangeably with “polynucleotide” herein.

[0188] In some embodiments of the disclosure, a polynucleotide comprises a DNA sequence. In some embodiments of the disclosure, a polynucleotide comprises a DNA sequence inserted in a vector or a vector comprising a DNA sequence.

[0189] In some embodiments of the disclosure, a polynucleotide comprises an mRNA. In some embodiments, the mRNA is a synthetic mRNA, or the mRNA comprises a synthetic nucleotide.

[0190] In some embodiments of the disclosure, a polynucleotide comprises at least one unnatural, non-naturally occurring or modified nucleic acid. In some embodiments, the polynucleotide comprises a plurality of unnatural, non-naturally occurring or modified nucleic acids. In some embodiments, all nucleic acids of a certain class are unnatural, non- naturally occurring or modified nucleic acids (e.g., all uridines in a polynucleotide can be replaced with an unnatural nucleobase, e.g., 5-methoxy uridine).

[0191] Methods for producing polynucleotides are known in the art. In some embodiments, a polynucleotide encoding a CD8-binding polypeptide described herein is fused to one or more regulatory sequences capable of driving expression of the nucleic acid sequence in a cell (e.g., a mammalian cell). In some embodiments, the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof. In some embodiments, the regulatory sequence comprises two or more response elements or repeated response elements. In some embodiments, the two or more response elements or the repeated response elements are arranged in tandem. In some embodiments, the regulatory sequence comprises a constitutive promoter. In some embodiments, the regulatory sequence 82 321490133Attorney Docket No. OTPC-050 / 02WO comprises an inducible promoter. In some embodiments, the regulatory sequence comprises a minimal promoter. In some embodiments, the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs. In some embodiments, the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

[0192] In some embodiments, the polynucleotides encoding the CD8-binding polypeptide or conjugate thereof is ligated into an expression vector. In some embodiments, the expression vector is introduced to a host cell to produce the CD8-binding polypeptide or conjugate thereof. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the expression vector is introduced to the host cell under conditions suitable for expression of the CD8-binding polypeptide or conjugate thereof. In some embodiments, the CD8-binding polypeptide or conjugate thereof is purified using a method known in the art (e.g., chromatography). Polypeptides

[0193] Polypeptides of the disclosure may comprise naturally or synthetically created or modified amino acids, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides in which one or more amino acid residues are artificial chemical analogs of a corresponding naturally occurring amino acid (including, for example, synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides also include gene products, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may comprise a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides of the disclosure may comprise single-chain or multi-chain polypeptides. Most commonly disulfide linkages are found in multi-chain polypeptides.

[0194] The polypeptides of the disclosure may comprise L-amino acids + glycine, D-amino acids + glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids + glycine. Polypeptides described may be chemically synthesized or recombinantly expressed. 83 321490133Attorney Docket No. OTPC-050 / 02WO

[0195] The polypeptides of the disclosure can include additional residues at the N- terminus, C-terminus, internal to the polypeptide, or a combination thereof; these additional residues are not included in determining the percent identity of the polypeptides of the disclosure relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to tags. Therapeutic Methods

[0196] The disclosure provides methods for treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a CD8- binding polypeptide or conjugate thereof described herein, a nucleic acid sequence encoding the CD8-binding polypeptide or conjugate thereof, a vector comprising the nucleic acid sequence or the CD8-binding polypeptide or conjugate thereof, a pharmaceutical composition comprising the vector, the nucleic acid sequence, or the CD8-binding polypeptide or conjugate thereof, or a cell comprising the vector, the nucleic acid sequence, or the CD8- binding polypeptide or conjugate thereof. In some embodiments, the administration is local or systemic. In some embodiments, the administration is oral. In some embodiments, the administration is by parenteral injection (e.g., subcutaneous, intravenous, or intramuscular). In some embodiments, the administration is by inhalation. In some embodiments, the administration is topical.

[0197] In some embodiments of the disclosure, the term “therapeutically effective” may refer to imparting a beneficial effect on the recipient, e.g., providing some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Therapeutic effects of the disclosure need not be complete or curative, as long as some benefit is provided to the subject. For example, a therapeutic regimen that incorporates the polynucleotides, gene therapy vectors or cells of the disclosure with the small molecules of the disclosure may be structured such that the regimen is therapeutically effective as a whole.

[0198] In some embodiments of the disclosure, the term “therapeutically effective amount” refers to a dose or an amount of a nucleic acid, vector, polypeptide, composition, pharmaceutical composition or cell of the disclosure sufficient to impart a therapeutically effective benefit on the recipient. For example, polynucleotides, gene therapy vectors or cells of the disclosure may be administered in a therapeutically effective amount. A subject who has been administered polynucleotides, gene therapy vectors or cells of the disclosure may subsequently be administered a therapeutically effective amount of a small molecule of the 84 321490133Attorney Docket No. OTPC-050 / 02WO disclosure, i.e., an amount sufficient to impart a beneficial effect on the recipient given the previous administration of polynucleotides, gene therapy vectors or cells.

[0199] In some embodiments, the CD8-binding polypeptide or conjugate thereof, the nucleic acid sequence, the vector, the pharmaceutical composition, or the cell is administered as a single agent. In some embodiments, the CD8-binding polypeptide or conjugate thereof, the nucleic acid sequence, the vector, the pharmaceutical composition, or the cell is administered in combination with at least one second therapeutic agent. In some embodiments, the at least one second therapeutic agent is an immunomodulatory agent.

[0200] In some embodiments, the subject has cancer, wherein the administering is performed to reduce or ameliorate one or more clinical symptoms of cancer. In some embodiments, the administering is performed to reduce tumor burden in the subject. In some embodiments, the administering is performed to reduce the risk of metastases in the subject.

[0201] In some embodiments, the subject has an infectious disease, wherein the administering is performed to reduce or ameliorate one or more clinical symptoms of infectious disease. In some embodiments, the infectious disease is a viral infection. In some embodiments, the infectious disease is a bacterial infection.

[0202] In some embodiments, the subject has an autoimmune disease, wherein the administering is performed to reduce or ameliorate one or more clinical symptoms of autoimmune disease. Formulations

[0203] The present disclosure provides compositions comprising a CD8-binding polypeptide or conjugate thereof described herein, a nucleic acid sequence encoding the CD8-binding polypeptide or conjugate thereof, a vector comprising the nucleic acid sequence or the CD8-binding polypeptide or conjugate thereof, or a cell comprising the vector, the nucleic acid sequence, or the CD8-binding polypeptide or conjugate thereof.

[0204] The present disclosure also provides pharmaceutical compositions comprising the CD8-binding polypeptide or conjugate thereof described herein, the nucleic acid sequence, the vector, or the cell, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration (e.g., in a solid or liquid dosage form). In some embodiments, the pharmaceutical composition is formulated for topical administration (e.g., in a lotion or gel). In some embodiments, the pharmaceutical composition is formulated for parenteral administration (e.g., in a liquid suspension). 85 321490133Attorney Docket No. OTPC-050 / 02WO

[0205] In some embodiments of the disclosure, “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the disclosure may be administered as part of a composition together with other pharmaceutically acceptable components, including pharmaceutically acceptable carriers.

[0206] In some embodiments, pharmaceutically acceptable carrier comprises a pharmaceutically acceptable salt of a CD8-binding polypeptide described herein or a conjugate thereof. In some embodiments of the disclosure, the term “pharmaceutically acceptable salts” refers to derivatives of the small molecules of the disclosure wherein the specified compound is converted to an acid or base salt thereof. Such pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluensulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like. For example, the small molecules of the disclosure may be provided as pharmaceutically acceptable salts.

[0207] In some embodiments, the pharmaceutical composition comprises an excipient. In some embodiments of the disclosure, the term “excipients” refer to pharmacologically inert ingredients that are not active in the body. See, for example, Hancock, B. C., Moss, G. P., & Goldfarb, D. J. (2020). Handbook of pharmaceutical excipients. London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. The small molecules of the disclosure may be mixed with pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, lubricating agents, acidifying agents, 86 321490133Attorney Docket No. OTPC-050 / 02WO and dispensing agents, depending on the nature of the mode of administration and dosage forms. Such ingredients, including pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms. Pharmaceutically acceptable carriers include water, ethanol, polyols, vegetable oils, fats, waxes polymers, including gel forming and non- gel forming polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and lake blend. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, as well as high molecular weight polyethylene glycols. For example, the small molecules, polynucleotides, gene therapy vectors or cells of the disclosure may be provided and administered in compositions that include pharmaceutically acceptable excipients. Definitions

[0208] In some embodiments of the disclosure, the term “subject” refers to any mammal, including without limitation, humans.

[0209] The terms "a", "an" and "the" include their plural forms unless the context clearly dictates otherwise.

[0210] The term “and” is used interchangeably with “or” unless expressly stated otherwise.

[0211] The term "And / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, "and / or" as used in a phrase such as "A and / or B," includes "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, "and / or," as used in a phrase such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0212] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is stated, each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, as is each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For 87 321490133Attorney Docket No. OTPC-050 / 02WO example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0213] Where a value is explicitly stated, it is to be understood that values which are about the same quantity or amount as the stated value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0214] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0215] Singular or plural words also include the plural and singular number, respectively. Thus, for example, where the specification describes a gene of interest, the disclosure includes polynucleotides with a single gene of interest or multiple genes of interest.

[0216] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form.

[0217] Headings are included herein for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described with respect to the headings. Such concepts may have applicability throughout the present specification.

[0218] The term “CD8” as used herein refers to a protein, polypeptide, or portions thereof that corresponds to a cluster of differentiation 8 molecule. In humans, the full-length CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor. CD8 protein may be a dimer, consisting of a pair of CD8 chains, including CD8α and CD8β chains. The term “CD8” encompasses CD8α / CD8α homodimer, CD8α / CD8β heterodimer, CD8α chain, CD8β chain, or portions thereof, such as extracellular domain(s).

[0219] The term “CD8-binding polypeptide” as used herein refers to a polypeptide that binds to a CD8 antigen. The CD8-binding polypeptide may be a protein, an antibody 88 321490133Attorney Docket No. OTPC-050 / 02WO (including a 4-chain antibody, or a heavy chain antibody), an antibody fragment (e.g., VHH), or an immunoconjugate that binds to human CD8, cynomolgus CD8, and / or other non-human CD8 proteins or peptides.

[0220] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, monovalent antibodies (e.g., one-armed antibodies), 4-chain antibodies (such as IgG antibodies), single domain antibodies, and antibody fragments thereof so long as they exhibit the desired antigen-binding activity, i.e., binding to CD8 (such as a human CD8, a cynomolgus CD8, and / or a rhesus CD8). The terms “4- chain antibody” are used herein interchangeably to refer to an antibody or antigen-binding fragments having two heavy chains and two light chains.

[0221] The terms “antibody functional fragment,” “antibody fragment,” and “antigen binding fragment” are used to describe a fragment comprising a portion of an antibody. In some embodiments, the fragment comprises the antigen binding or variable region of the antibody. Examples of such fragments include VHHs, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No.5,641,870, Example 2; Zapata et al, Protein Eng.8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from such fragments.

[0222] The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non- contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1 (also called HC CDR1), CDR-H2 (also called HC CDR2), and CDR-H3 (also called HC CDR3)) and three CDRs in each light chain variable region (CDR- L1 (also called LC CDR1), CDR-L2 (also called LC CDR2), and CDR-L3 (also called LC CDR3)). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by 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); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site 89 321490133Attorney Docket No. OTPC-050 / 02WO topography,” J. Mol. Biol.262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng.2000 Dec;13(12):819-24 (“AbM” numbering scheme. In the "Chothia + Kabat" scheme, also herein called the “Kabat + Chothia” scheme, the CDR range definition is based on Kabat scheme, with CDR H1 region extended by five additional residues in the N-terminal direction. In some embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0223] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0224] The term “affinity” is intended to have a conventional meaning in the art, e.g., the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., antigen). The term as it applies to a molecule-binding partner interaction refers to the binding energy between the binding site and the bound binding partner. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD) which is used to evaluate and rank strengths of bimolecular interactions. The terms “KD”, “KD”, and “Kd” are used interchangeably to refer to the equilibrium dissociation constant. The value of KDis a numeric representation of the ratio of the off-rate constant (koff) for the dissociation of a binding partner from a molecule / binding partner complex to the on-rate constant (kon) for the association of the molecule and binding partner. The terms “koff”, “Koff”, “Koff”, “koff”, “K_off” and “k_off” are used interchangeably to refer to the off-rate constant. The value of koffis a numeric representation of the fraction of complexes that decay or dissociate per second, and is 90 321490133Attorney Docket No. OTPC-050 / 02WO expressed in units per sec. The value of kon is a numeric representation of the number of complexes formed per second in a 1 molar (1M) solution of molecule and binding partner, and is expressed in units M-1sec-1. The value of KD is inversely proportional to the binding affinity of a molecule and binding partner. The smaller the KDvalue, the greater the affinity of the molecule and the binding partner.

[0225] As used herein, the terms “specific binding,” “specifically binds,” or “is specific for” refer to a polypeptide (e.g., a CD8-binding polypeptide) binding to an epitope of a target molecule (e.g., CD8), where the polypeptide reacts or associates more frequently, more rapidly, with greater duration, and / or with greater affinity with the target molecule than it does with alternative molecules. A polypeptide (e.g., a CD8-binding polypeptide), such as a single domain antibody (e.g., VHH), “specifically binds” to a target molecule (e.g., CD8) if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other molecules. The polypeptide (e.g., a CD8-binding polypeptide) binds to a target molecule (e.g., human CD8α) with an equilibrium dissociation constant (KD) of less than about 10-6M, 10-7M, and more preferably less than about 10-8M or 10-9M when determined by a method described herein and / or known in the art. The polypeptide (e.g., a CD8-binding polypeptide) may further bind to a target molecule (e.g., CD8α) with a (KD) that is at least 10- fold, at least 100-fold, at least 103-fold, or at least 104-fold lower than its KD for a non-target molecule (e.g., a molecule that is not CD8). It is understood that a binding polypeptide, such as a single domain antibody (e.g., VHH), that specifically binds to a first target may or may not specifically bind to a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding.

[0226] In some embodiments of the disclosure, the terms “Nucleic acid,” “nucleic acid molecule,” “nucleotide,” “nucleotide sequence,” “polynucleotide,” and grammatical variants thereof are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.

[0227] In some embodiments of the disclosure, “Nucleic acid,” and in particular a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. In some embodiments of the disclosure, “Nucleic 91 321490133Attorney Docket No. OTPC-050 / 02WO acid,” includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences are provided according to the normal convention of writing the sequence left to right in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the messenger RNA or mRNA). Unless otherwise indicated, all nucleic acid and nucleotide sequences are written left to right in 5’ to 3’ orientation.

[0228] Nucleotides are referred to by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, ‘A’ represents adenine, ‘C’ represents cytosine, ‘G’ represents guanine, ‘T’ represents thymine, and ‘U’ represents uracil.

[0229] In some embodiments of the disclosure, the term “Polynucleotide” refers to polymers of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”). It also includes modified, for example by alkylation and / or by capping, and unmodified forms of the polynucleotide. More particularly, “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D- ribose) and polyribonucleotides (containing D-ribose), including mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nucleotide backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0230] In some embodiments of the disclosure, “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0231] In some embodiments of the disclosure, “expression vector” refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression in a cell. The vector or construct is used to introduce a gene into a host cell whereby the vector will interact with polymerases in the cell to express the protein encoded in the vector / construct. The expression vector may exist in the cell extrachromosomally or may be integrated into the chromosome. Expression vectors may include additional sequences which render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle 92 321490133Attorney Docket No. OTPC-050 / 02WO vectors). The polynucleotides of the disclosure may be provided as components of expression vectors.

[0232] In some embodiments of the disclosure, “cloning vector” refers to a plasmid, virus, or other nucleic acid designed for producing copies of a polynucleotide. Cloning vectors may contain transcription and translation initiation sequences, transcription and translation termination sequences and a polyadenylation signal. Such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof. The polynucleotides of the disclosure may be provided as components of cloning vectors, which may be used to produce the polynucleotides of the disclosure.

[0233] In some embodiments of the disclosure, “encoding” or the like refers to the capacity of specific sequences of nucleotides in a polynucleotide (e.g. a gene, cDNA, or mRNA) to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non- coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0234] Unless otherwise specified, a nucleotide sequence “encoding an amino acid sequence,” e.g., a polynucleotide “encoding” a chimeric polypeptide, defined below of the present disclosure, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0235] Amino acids are referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The amino acid residues are abbreviated as follows, where the abbreviations are shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0236] Amino acid sequences are written left to right in amino to carboxy orientation. 93 321490133Attorney Docket No. OTPC-050 / 02WO

[0237] In some embodiments of the disclosure, “Polypeptide” may refer to a sequence of amino acid subunits. In some embodiments, a “peptide” can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. “Polypeptide,” refers to proteins, polypeptides, and peptides of any length, size, structure, or function. “Polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length.

[0238] In some embodiments of the disclosure, “chimeric polypeptide” may refer to any polypeptide comprised of a first amino acid sequence derived from a first source, bonded, covalently or noncovalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same. In some embodiments, a first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein can include for example, a protein derived from at least 2 different biological sources. In some embodiments, the chimeric polypeptide may include sequences from similar proteins derived from two distinct species. In some embodiments, the chimeric polypeptide may include sequences from dissimilar proteins derived from the same species. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g., solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or noncovalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein can comprise a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0239] A polypeptide "derived from" a reference polypeptide comprises an amino acid sequence having at least about 60% identity, about 70%, about 80%, or about 90% to the sequence of the reference polypeptide or a portion thereof (e.g., a portion that is at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids in length). Polypeptides derived from a reference polypeptide may have one or more modifications relative to the sequence of the reference polypeptide or the portion thereof, e.g., one or more amino acid 94 321490133Attorney Docket No. OTPC-050 / 02WO residues which have been substituted with another amino acid residue, an insertion, and / or a deletion. In some embodiments, the polypeptides derived from a reference polypeptide is a recombinant or synthetic polypeptide.

[0240] In some embodiments of the disclosure, a “fragment” of a polypeptide, or a “truncated polypeptide” may refer to an amino acid sequence of a polypeptide that is shorter than the sequence of a reference polypeptide (which may be a naturally-occurring sequence). In comparison to the reference polypeptide, the fragment may comprise an N- and / or C- terminal deletion. In comparison to the reference polypeptide, the fragment may comprise a deletion of any part of the sequence, whether or not the deletion is contiguous. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment. The various polypeptide components of the disclosure may be provided as fragments or truncated versions of a reference protein.

[0241] In some embodiments of the disclosure, a “functional fragment” may refer to a polypeptide fragment that retains a function of the polypeptide. In some embodiments, a functional fragment of a bioactive peptide (e.g., an enzyme), retains the ability to catalyze a biological action because the functional fragment comprises a catalytic domain of the enzyme. Polypeptides of the disclosure may be provided as functional fragments or truncated versions.

[0242] In some embodiments of the disclosure, “amino acid substitution” may refer to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. In some embodiments, the parent or reference sequence comprises a wildtype sequence. An amino acid can be substituted, for example, via chemical peptide synthesis or through recombinant methods known in the art. For example, substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid. Polypeptides of the disclosure may be provided with one or more amino acid substitutions.

[0243] In some embodiments of the disclosure, a “conservative amino acid substitution” is one in which one amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar 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., 95 321490133Attorney Docket No. OTPC-050 / 02WO tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In some embodiments, a string of amino acids can be conservatively replaced with a chemically similar string that differs in order and / or composition of side chain family members. The various polypeptide components of the disclosure may be provided with conservative amino acid substitutions.

[0244] In some embodiments of the disclosure, non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). The various polypeptide components of the disclosure may be provided with non- conservative amino acid substitutions. The likelihood that one of the foregoing non- conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties. The various polypeptide components of the disclosure may be provided with non- conservative amino acid substitutions that do not significantly alter the functionality of the altered components.

[0245] In some embodiments of the disclosure, “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules. “Identical” without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., “70% identical,” is equivalent to describing them as having, e.g., “70% sequence identity.”

[0246] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the 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, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. 96 321490133Attorney Docket No. OTPC-050 / 02WO

[0247] In certain embodiments, the percentage identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID = 100 (Y / Z), where Y is the number of amino acid (or nucleobase) residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0248] Calculation of the percent identity of two polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes. For example, gaps can be introduced in one or both of a first and a second polypeptide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes. In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0249] Generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the European Bioinformatics Institute (EBI) at website ebi.ac.uk / Tools / psa. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0250] Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available 97 321490133Attorney Docket No. OTPC-050 / 02WO from the EBI. Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc. Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, values from 80.11 to 80.14 are rounded down to 80.1, while values from 80.15 to 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0251] In some embodiments of the disclosure, “non-naturally occurring” means a polypeptide or a polynucleotide sequence that does not exist in nature. In some embodiments, the non-naturally occurring sequence does not exist in nature because the sequence is altered relative to a naturally occurring sequence. In some embodiments, the non-naturally occurring sequence does not exist in nature because it is a combination of two known, naturally- occurring, sequences (e.g., chimeric polypeptide) that do not occur together in nature. In some embodiments, a non-naturally occurring polypeptide is a chimeric polypeptide. In some embodiments, a polypeptide or a polynucleotide is not naturally occurring because the sequence contains a portion (e.g., a fragment) that cannot be found in nature, i.e., a novel sequence. Any of the polynucleotides described herein may be provided as non-naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polynucleotides which are linked to other polynucleotides in a manner that does not exist in nature. Any of the polypeptides described herein may be provided as non- naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polypeptides which are linked to other polypeptides in a manner that does not exist in nature.

[0252] In some embodiments of the disclosure, the term “stem cell” may refer to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell.

[0253] In some embodiments of the disclosure, the term “Pluripotent stem cell” (PSC) may refer to a cell that can maintain an undifferentiated state indefinitely and can differentiate into most, if not all cells of the body.

[0254] In some embodiments of the disclosure, the term “Induced pluripotent stem cell” (iPS or iPSC) may refer to a pluripotent stem cell that can be generated directly from a somatic cell. This includes, but is not limited to, specialized cells such as skin or blood cells derived from an adult. 98 321490133Attorney Docket No. OTPC-050 / 02WO

[0255] In some embodiments of the disclosure, the term “multipotent” may refer to a cell that can develop into more than one cell type but is more limited than a pluripotent cell. For example, adult stem cells and cord blood stem cells may be considered as multipotent.

[0256] In some embodiments of the disclosure, the term “hematopoietic cell” may refer to a cell that arises from a hematopoietic stem cell (HSC). Hematopoietic cells of the disclosure include, but is not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, thrombocytes, monocytes, natural killer cells, T lymphocytes, B lymphocytes and plasma cells.

[0257] In some embodiments of the disclosure, the term “T-lymphocyte” or “T-cell” may refer to a hematopoietic cell that normally develops in the thymus. T-lymphocytes or T-cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0258] In some embodiments of the disclosure, the term “mesenchyme” may refer to a type of animal tissue comprising loose cells embedded in a mesh of proteins and fluid, i.e., the extracellular matrix. Mesenchyme directly gives rise to most of the body’s connective tissues including bones, cartilage, lymphatic system, and circulatory system.

[0259] In some embodiments of the disclosure, the term “mesenchymal cell” may refer to a cell that is derived from a mesenchymal tissue. In some embodiments, cells of the disclosure may be mesenchymal cells.

[0260] In some embodiments of the disclosure, the term “mesenchymal stromal cell” (MSC) may refer to a spindle shaped plastic-adherent cell isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity in vitro. For example, a mesenchymal stromal cell can differentiate into osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells which give rise to marrow adipose tissue). The term mesenchymal stromal cell is suggested in the scientific literature to replace the term “mesenchymal stem cell”. In some cases, cells of the disclosure may be mesenchymal stromal cells.

[0261] In some embodiments of the disclosure, an “autologous cell” is a cell obtained from the same individual to whom it may be administered as a therapy (the cell is autologous to the subject). Autologous cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0262] In some embodiments of the disclosure, an allogeneic cell is a cell obtained from an individual who is not the intended recipient of the cell as a therapy (the cell is allogeneic to 99 321490133Attorney Docket No. OTPC-050 / 02WO the subject). Allogeneic cells of the disclosure may be selected from immunologically compatible donors with respect to the subject of the methods of the disclosure. Allogeneic cells of the disclosure may be modified to produce “universal” allogeneic cells, suitable for administration to any subject without unintended immunogenicity. Allogeneic cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0263] In some embodiments of the disclosure, the term “Transfect” or “transform” or “transduce” may refer to a process by which exogenous nucleic acid is transferred or introduced into a host cell. In some embodiments, a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid or progeny of the cell.

[0264] In some embodiments of the disclosure, the term “Cell therapy” may refer to the provision or delivery of cells into a recipient for therapeutic purposes. OTHER EMBODIMENTS

[0265] The disclosure further provides the following enumerated embodiments.

[0266] Embodiment I-1. A polypeptide comprising a sequence of one or more of SEQ ID NOs: 1-3.

[0267] Embodiment I-2. A polypeptide comprising a complementarity-determining region 1 (CDR1) sequence comprising GFTFDDYAMG, a complementarity-determining region 2 (CDR2) sequence comprising CIRVSDGSTYYADSVKG and a complementarity- determining region 3 (CDR3) sequence comprising AGSLYTCVQSIVVVPARPYYDMDY.

[0268] Embodiment I-3. The polypeptide of embodiment I-2, wherein the polypeptide comprises a sequence of one or more of SEQ ID NOs: 4-10.

[0269] Embodiment I-4. The polypeptide of any one of embodiments I-1 to I-3, wherein the polypeptide comprises a linker.

[0270] Embodiment I-5. The polypeptide of embodiment I-4, wherein the linker comprises one or more of an amino acid sequence, a nucleic acid sequence, an amino acid sequence, a small molecule, and any combination thereof.

[0271] Embodiment I-6. The polypeptide of any one of embodiments I-1 to I-5, wherein the polypeptide is operably linked to one or more of a second polypeptide, a nucleic acid, an inorganic molecule, an organic molecule or another moiety. 100 321490133Attorney Docket No. OTPC-050 / 02WO

[0272] Embodiment I-7. The polypeptide of any one of embodiments I-1 to I-6, wherein the polypeptide selectively targets a CD8-expressing (CD8+) cell.

[0273] Embodiment I-8. The polypeptide of embodiment I-7, wherein the CD8+ cell is a T cell.

[0274] Embodiment I-9. The polypeptide of any one of embodiments I-6 to I-8, wherein the second polypeptide comprises a sequence isolated or derived from a sequence encoding or comprising a cytokine.

[0275] Embodiment I-10. The polypeptide of embodiment I-9, wherein the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL- 2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

[0276] Embodiment I-11. The polypeptide of any one of embodiments I-6 to I-10, wherein the second polypeptide comprises a sequence isolated or derived from a sequence encoding or comprising an interferon.

[0277] Embodiment I-12. The polypeptide of embodiment I-11, wherein the interferon comprises one or more of an interferon type I, an interferon type II and an interferon type III.

[0278] Embodiment I-13. The polypeptide of embodiment I-11, wherein the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN-ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN-γ). 101 321490133Attorney Docket No. OTPC-050 / 02WO

[0279] Embodiment I-14. The polypeptide of any one of embodiments I-6 to I-10, wherein the second polypeptide comprises a designed cytokine.

[0280] Embodiment I-15. A nucleic acid sequence encoding the polypeptide of any one of embodiments I-1 to I-14.

[0281] Embodiment I-16. The nucleic acid sequence of embodiment I-15 comprising a regulatory sequence capable of driving expression of the nucleic acid sequence in a mammalian cell.

[0282] Embodiment I-17. The nucleic acid sequence of embodiment I-16, wherein the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof.

[0283] Embodiment I-18. The nucleic acid sequence of embodiment I-16 or I-17, wherein the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem.

[0284] Embodiment I-19. The nucleic acid sequence of any one of embodiments I-16 to I- 18, wherein the regulatory sequence comprises a constitutive promoter.

[0285] Embodiment I-20. The nucleic acid sequence of any one of embodiments I-16 to I- 18, wherein the regulatory sequence comprises an inducible promoter.

[0286] Embodiment I-21. The nucleic acid sequence of any one of embodiments I-16 to I- 20, wherein the regulatory sequence comprises a minimal promoter.

[0287] Embodiment I-22. The nucleic acid sequence of any one of embodiments I-16 to I- 21, wherein the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs.

[0288] Embodiment I-23. The nucleic acid sequence of any one of embodiments I-16 to I- 22, wherein the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

[0289] Embodiment I-24. A vector comprising the nucleic acid of any one of embodiments I-15 to I-23.

[0290] Embodiment I-25. The vector of embodiment I-24, wherein the vector is an expression vector.

[0291] Embodiment I-26. The vector of embodiment I-24, wherein the vector is a delivery vector.

[0292] Embodiment I-27. The vector of any one of embodiments I-24 to I-26, wherein the vector is a non- to I-viral vector. 102 321490133Attorney Docket No. OTPC-050 / 02WO

[0293] Embodiment I-28. The vector of any one of embodiments I-24 to I-26, wherein the vector is a viral vector.

[0294] Embodiment I-29. A cell comprising one or more of (a) a polypeptide of any one of embodiments I-1 to I-14; (b) a nucleic acid sequence of any one of embodiments I-15 to I-23; and (c) a vector of any one of embodiments I-24 to I-28.

[0295] Embodiment I-30. The cell of embodiment I-29, wherein the cell is an immune cell.

[0296] Embodiment I-31. The cell of embodiment I-30, wherein the cell is a stem cell or a T-cell precursor cell.

[0297] Embodiment I-32. The cell of embodiment I-30, wherein the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell.

[0298] Embodiment I-33. The cell of embodiment I-30, wherein the cell is a somatic cell or a differentiated cell.

[0299] Embodiment I-34. The cell of embodiment I-30, wherein the cell is a T cell, a Natural Killer (NK) cell, or a macrophage.

[0300] Embodiment I-35. The cell of any one of embodiments I-29 to I-34, wherein the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood.

[0301] Embodiment I-36. The cell of any one of embodiments I-29 to I-35, wherein the cell has been cryopreserved.

[0302] Embodiment I-37. The cell of any one of embodiments I-29 to I-36, wherein the cell has been modified to become an allogeneic cell.

[0303] Embodiment I-38. The cell of any one of embodiments I-29 to I-36, wherein the cell has been modified to produce an allogeneic cell.

[0304] Embodiment I-39. The cell of any one of embodiments I-29 to I-38, wherein the cell is capable of expressing or modified to express an exogenous protein.

[0305] Embodiment I-40. The cell of embodiment I-39, wherein the exogenous protein comprises an antigen receptor.

[0306] Embodiment I-41. The cell of embodiment I-39, wherein the antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR).

[0307] Embodiment I-42. The cell of any one of embodiments I-29 to I-41, wherein the cell is capable of expressing or modified to express a safety switch.

[0308] Embodiment I-43. The cell of any one of embodiments I-29 to I-41, wherein the cell is capable of expressing or modified to express a cell marker. 103 321490133Attorney Docket No. OTPC-050 / 02WO

[0309] Embodiment I-44. The cell of embodiment I-42 or I-43, wherein the cell is capable of expressing or modified to express a truncated EGFR (EGFRt).

[0310] Embodiment I-45. The cell of embodiment I-44, wherein the EGFRt comprises a sequence of SEQ ID NO: 229.

[0311] Embodiment I-46. The cell of embodiment I-42 or I-43, wherein the cell is capable of expressing or modified to express a truncated and optimized EGFR (EGFRopt).

[0312] Embodiment I-47. The cell of embodiment I-46, wherein the EGFRopt sequence comprises a sequence of one or more of SEQ ID NOs: 230-244.

[0313] Embodiment I-48. A composition comprising one or more of: (a) a polypeptide of any one of embodiments I-1 to I-14; (b) a nucleic acid sequence of any one of embodiments I-15 to I-23; (c) a vector of any one of embodiments I-24 to I-28; and (d) a cell of any one of embodiments I-29 to I-47.

[0314] Embodiment I-49. A pharmaceutical composition comprising one or more of: (a) a polypeptide of any one of embodiments I-1 to I-14, a nucleic acid sequence of any one of embodiments I-15 to I-23, a vector of any one of embodiments I-24 to I-28, a cell of any one of embodiments I-29 to I-47, and a composition of embodiment I-48; and (b) a pharmaceutically acceptable carrier.

[0315] Embodiment I-50. The pharmaceutical composition of embodiment I-49, wherein the pharmaceutical composition is formulated for systemic administration.

[0316] Embodiment I-51. The pharmaceutical composition of embodiment I-50, wherein the systemic administration comprises an intravenous injection or an intravenous infusion.

[0317] Embodiment I-52. The pharmaceutical composition of embodiment I-49, wherein the pharmaceutical composition is formulated for local administration.

[0318] Embodiment I-53. The pharmaceutical composition of embodiment I-52, wherein the local administration comprises an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

[0319] Embodiment I-54. A use of one or more of a polypeptide of any one of embodiments I-1 to I-14, a nucleic acid sequence of any one of embodiments I-15 to I-23, a vector of any one of embodiments I-24 to I-28, a cell of any one of embodiments I-29 to I-47, a composition of embodiment I-48 and a pharmaceutical composition of any one of embodiments I-49 to I-53 in the manufacture of a medicament for treating a disease or disorder. 104 321490133Attorney Docket No. OTPC-050 / 02WO

[0320] Embodiment I-55. A use of one or more of a polypeptide of any one of embodiments I-1 to I-14, a nucleic acid sequence of any one of embodiments I-15 to I-23, a vector of any one of embodiments I-24 to I-28, a cell of any one of embodiments I-29 to I-47, a composition of embodiment I-48 and a pharmaceutical composition of any one of embodiments I-49 to I-53 for treating a disease or disorder.

[0321] Embodiment I-56. The use of embodiment I-54 or I-55, wherein the disease or disorder comprises a proliferative disorder.

[0322] Embodiment I-57. The use of embodiment I-56, wherein the proliferative disorder is a metastatic disorder.

[0323] Embodiment I-58. The use of embodiment I-57, wherein the metastatic disorder comprises a solid tumor.

[0324] Embodiment I-59. A method of treating a disease or disorder comprising administering an effective amount of one or more of a polypeptide of any one of embodiments I-1 to I-14, a nucleic acid sequence of any one of embodiments I-15 to I-23, a vector of any one of embodiments I-24 to I-28, a cell of any one of embodiments I-29 to I-47, a composition of embodiment I-48 and a pharmaceutical composition of any one of embodiments I-49 to I-53 to a subject in need thereof, thereby treating the disease or disorder.

[0325] Embodiment I-60. The method of embodiment I-59, wherein the subject is at risk of developing the disease or disorder.

[0326] Embodiment I-61. The method of embodiment I-59, wherein the subject has been diagnosed with the disease or disorder.

[0327] Embodiment I-62. The method of any one of embodiments I-59 to I-61, wherein the disease or disorder comprises a proliferative disorder.

[0328] Embodiment I-63. The method of embodiment I-62, wherein the proliferative disorder is a metastatic disorder.

[0329] Embodiment I-64. The method of embodiment I-63, wherein the metastatic disorder comprises a solid tumor.

[0330] Embodiment I-65. The method of any one of embodiments I-59 to I-64, wherein treating the disease or disorder comprises one or more of: (a) decreasing the severity of a sign or symptom of the disease or disorder; (b) improving a function negatively affected by the disease or disorder; (c) improving the subject’s prognosis; (d) increasing the subject’s duration of progression-free survival (PFS); (e) reducing a quantity of cells affected by the disease or disorder; (f) inducing a remission form the disease or disorder; (g) delaying or preventing a relapse of the disease or disorder; (h) delaying or preventing a second 105 321490133Attorney Docket No. OTPC-050 / 02WO presentation of the disease or disorder; (i) increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder.

[0331] Embodiment II-1. A CD8-binding polypeptide comprising a CD8-specific single domain antibody (VHH), wherein the VHH comprises at least one modification with respect to SEQ ID NO: 314.

[0332] Embodiment II-2. The CD8-binding polypeptide of embodiment II-1, wherein the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, V79, K87, P88, A91, K124, and a combination thereof.

[0333] Embodiment II-3. The CD8-binding polypeptide of embodiment II-1, wherein the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, P88, A91, K124, and a combination thereof.

[0334] Embodiment II-4. The CD8-binding polypeptide of any one of embodiments II-1 to II-3, wherein the modification is a substitution, deletion, or insertion.

[0335] Embodiment II-5. The CD8-binding polypeptide of embodiments II-1 to II-4, wherein the at least one modification is selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, E44G, S72R, A75S, V79L, K87R, P88A, A91T, K124Q, and a combination thereof.

[0336] Embodiment II-6. The CD8-binding polypeptide of any one of embodiments II-1 to II-5, wherein the at least one modification is in the VHH complementarity-determining region 1 (CDR), CDR2, and / or CDR3.

[0337] Embodiment II-7. The CD8-binding polypeptide of any one of embodiments II-1 to II-6, wherein the at least one modification is in the VHH framework region 1 (FR), FR2, FR3, and / or FR4.

[0338] Embodiment II-8. The CD8-binding polypeptide of any one of embodiments II-1 to II-7, wherein the VHH CDR1 sequence comprises GFTFDDYAX1G (SEQ ID NO: 373), the VHH CDR2 sequence comprises X2IRVSDGSTYYADX3VKG (SEQ ID NO: 374), and the VHH CDR3 sequence comprises GSLYTX4VQSIVVVPARPYYDMDY (SEQ ID NO: 375), wherein X1 is M or I, X2 is C, S, or A, X3 is P or S, and X4 is C, S, or A.

[0339] Embodiment II-9. The CD8-binding polypeptide of embodiment II-8, wherein: (i) if X1 is I, then X2 is S or A, X3 is S, and / or X4 is S or A; (ii) if X2is C, then X1is M, X3is S, and / or X4is S or A; (iii) if X3 is P, then X1 is M, X2 is S or A, and / or X4 is S or A; and 106 321490133Attorney Docket No. OTPC-050 / 02WO (iv) if X4 is C, then X1 is M, X2 is S or A, and / or X3 is S.

[0340] Embodiment II-10. The CD8-binding polypeptide of any one of embodiments II-1 to II-9, wherein the VHH CDR1, CDR2, and CDR3 respectively comprise: (i) GFTFDDYAMG (SEQ ID NO: 14); CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); (ii) GFTFDDYAMG (SEQ ID NO: 14); CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494) (iii) GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); (iv) GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494); (v) GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and AGSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 8); (vi) GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and GSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 495); (vii) GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and AGSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 12); or (viii) GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and GSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 496).

[0341] Embodiment II-11. The CD8-binding polypeptide of any one of embodiments II-1 to II-10, wherein the at least one modification comprises: (i) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, A91T, and K124Q; (ii) Q1E, Q5V, A14P, A75S, A91T, and K124Q; (iii) Q1E, Q5V, A14P, S72R, A75S, V79L, K87R, A91T, and K124Q; (iv) Q1E, Q5V, A14P, S72R, A75S, A91T, and K124Q; (v) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, A91T, and K124Q; (vi) Q1E, Q5V, A14P, A75S, V79L, K87R, A91T, and K124Q; (vii) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; (viii) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; (ix) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, V79L, A91T, and K124Q; (x) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, A91T, and K124Q; or 107 321490133Attorney Docket No. OTPC-050 / 02WO (xi) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, K87R, P88A, A91T, and K124Q.

[0342] Embodiment II-12. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 17, 303, 1, 5, 9, 13, 21, 25, 29, 33, 37, and 315-323, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0343] Embodiment II-13. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein the VHH comprises SEQ ID NO: 17, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0344] Embodiment II-14. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein the VHH CDR1 sequence comprises DYAX5G (SEQ ID NO: 376), the VHH CDR2 sequence comprises CIRX6X7DX8X9X10YYADPX11KG (SEQ ID NO: 377), and the VHH CDR3 sequence comprises GSLX12X13CVQX14X15X16X17X18X19X20X21X22X23YDMDY (SEQ ID NO: 378), wherein X5 is I or L, X6 is A, C, F, I, L, N, S, T, V, W, or Y, X7 is F, S, W, or Y, X8 is G, R, or V, X9 is A, M, R, S, or W, X10 is D, F, S, or T, X11 is L or V, X12 is F, W, or Y, X13 is A, S, or T, X14 is G, S, W, or Y, X15 is F, H, I, L, or W, X16 is A, E, G, I, L, N, P, R, S, or V, X17 is A, G, P, S, or V, X18is D, E, or V, X19is P or S, X20is A, D, E, N, S, or T, X21is A, D, E, G, H, L, Q, R, S, or V, X22 is A, D, E, N, or P and X23 is R, V, or Y.

[0345] Embodiment II-15. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein: (i) the VHH CDR1 sequence comprises DYAX5G (SEQ ID NO: 376); (ii) the VHH CDR2 sequence comprises CIRX24X25DX26X27X28YYADPX29KG (SEQ ID NO: 379); and (iii) the VHH CDR3 sequence comprises: GSLYTCVQSX30X31X32X33PYYDMDY (SEQ ID NO: 380), GSLYTCVQX34X35X36X37X38X39PX40YDMDY (SEQ ID NO: 381), GSLYTCVQX41X42X43X44X45X46X47X48YYDMDY (SEQ ID NO: 382), GSLYTCVQSX49X50X51X52X53X54X55X56X57 X58DMDY (SEQ ID NO: 383), GSLYTCVQX59X60X61X62VX63X64X65X66X67X68YDMDY (SEQ ID NO: 384), GSLYTCVLX69X70X71X72X73X74X75X76X77X78X79X80YDMDY (SEQ ID NO: 385), or 108 321490133Attorney Docket No. OTPC-050 / 02WO GSLYTCVQX81X82X83X84X85X86 X87X88X89X90X91X92X93YDMDY (SEQ ID NO: 386); and wherein: X5 is I or L, X24 is A, C, F, I, L, N, S, T, V, W, or Y; X25 is F, S, W, or Y; X26 is A, G, I, R, or V; X27is A, M, R, S, or W; X28is D, F, S, or T; X29is V or L; X30is F, H, I, or L; X31 is A, D, G, H, I, K, L, N, Q, R, S, T, V, or Y; X32 is A, D, or G; X33 is D, E, I, K, or R; X34is F, H, L, S, W, or Y; X35is F, H, I, L, or W; X36is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W; X37 is D, P, or V; X38 is A, E, or V; X39 is P or R; X40 is E or Y; X41is F, S, or Y; X42is F, I, L, W, or Y; X43is A, G, H, K, L, M, P, R, S, V, or W; X44is P, V, or Y; X45 is D, E, H, P, or V; X46 is A, E, G, or P; X47 is A, E, G, R, or S; X48 is D, E, P, or V; X49is F, H, I, or L; X50is I, L, N, or V; X51is A, I, K, M, P, S, T, or V; X52is E, N, P, R, T, or V; X53 is G or P; X54 is A, G, H, T, or Y; X55 is E, G, L, R, or Y; X56 is D, E, or P; X57is S or Y; X58is D or Y; X59is S or Y; X60is F, I, L, or W; X61is A, E, F, G, K, L, Q, S, T, or V; X62 is A, D, E, K, M, P, S, T, or V; X63 is P or V; X64 is A, D, E, F, G, I, N, P, R, or V; X65is A, D, E, G, I, L, N, P, Q, R, S, T, V, W, or Y; X66is A, D, E, G, R, S, T, V, or Y; X67 is A, D, E, G, P, or V; X68 is L, S, or Y; X69 is H, K, L, Q, R, S, or Y; X70 is F, I, L, Q, or W; X71 is G, K, L, P, Q, R, S, or V; X72 is D, I, S, or V; X73 is D, E, N, P, or V; X74 is A, D, E, G, P, or V; X75 is A, E, R, T, or V; X76 is P, R, or V; X77 is A, D, E, K, N, P, S, T, or V; X78 is E, G, Q, R, S, V, or Y; X79 is D, E, P, S, or T; X80is R or Y; X81is H, R, S, or W; X82is F, I, or L; X83is I, N, R, V, or W; X84is P, R, S, or V; X85 is A, D, E, I, R, V, or Y; X86 is D, G, N, P, Q, or V; X87 is A, D, E, G, L, R, or V; X88is R or V; X89is D, G, or P; X90is A, D, E, G, H, L, M, P, Q, R, S, T, V, or Y; X91 is A, D, E, G, L, R, S, V, or Y; X92 is D, E, F, L, P, Q, or V; and X93 is E, I, M, R, T, V, or Y.

[0346] Embodiment II-16. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352 and 394-493, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0347] Embodiment II-17. The CD8-binding polypeptide of any one of embodiments II-1 to II-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0348] Embodiment II-18. The CD8-binding polypeptide of any one of embodiments II-1 to II-17, wherein the VHH specifically binds to CD8α. 109 321490133Attorney Docket No. OTPC-050 / 02WO

[0349] Embodiment II-19. The CD8-binding polypeptide of any one of embodiments II-1 to II-18, wherein the VHH specifically binds to human CD8α.

[0350] Embodiment II-20. The CD8-binding polypeptide of any one of embodiments II-1 to II-19, wherein the VHH comprises one or more characteristics relative to a VHH set forth in SEQ ID NO: 314, wherein the one or more characteristics is selected from the group consisting of: (i) an increased binding affinity (KD) to human CD8α; (ii) a decreased off-rate (koff) for binding to human CD8α; (iii) an increased melting temperature (Tm); (iv) an increased expression yield; and (v) an increased purity following a single-step purification.

[0351] Embodiment II-21. The CD8-binding polypeptide of any one of embodiments II-1 to II-20, wherein the polypeptide selectively targets a CD8-expressing (CD8+) cell.

[0352] Embodiment II-22. The CD8-binding polypeptide of embodiment II-21, wherein the CD8+ cell is a T cell.

[0353] Embodiment II-23. The CD8-binding polypeptide of any one of embodiments II-1 to II-22, wherein the polypeptide is operably linked to one or more of a second polypeptide, a nucleic acid, an inorganic molecule, an organic molecule, and another moiety.

[0354] Embodiment II-24. The CD8-binding polypeptide of any one of embodiments II-1 to II-22, wherein the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising a cytokine.

[0355] Embodiment II-25. The CD8-binding polypeptide of embodiment II-24, wherein the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 110 321490133Attorney Docket No. OTPC-050 / 02WO polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

[0356] Embodiment II-26. The CD8-binding polypeptide of any one of embodiments II-23 to II-24, wherein the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising an interferon.

[0357] Embodiment II-27. The CD8-binding polypeptide of embodiment II-26, wherein the interferon comprises one or more of an interferon type I, an interferon type II and an interferon type III.

[0358] Embodiment II-28. The CD8-binding polypeptide of embodiment II-26, wherein the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN- β), and interferon epsilon (IFN-ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN-γ).

[0359] Embodiment II-29. The CD8-binding polypeptide of any one of embodiments II-23 to II-24, wherein the polypeptide is operably linked to a second polypeptide comprising a designed cytokine.

[0360] Embodiment II-30. A CD8-targeted conjugate, comprising: the CD8-binding polypeptide of any one of embodiments II-1-22 and at least one second moiety, wherein the CD8-binding polypeptide is operably linked to the second moiety.

[0361] Embodiment II-31. The CD8-targeted conjugate of embodiment II-30, wherein the CD8-binding polypeptide is operably linked to the second moiety by a linker.

[0362] Embodiment II-32. The CD8-targeted conjugate of embodiment II-31, wherein the linker comprises one or more of an amino acid sequence, a nucleic acid sequence, an amino acid sequence, a small molecule, and any combination thereof.

[0363] Embodiment II-33. The CD8-targeted conjugate of any one of embodiments II-30 to II-32, wherein the at least one second moiety comprises a detection label, a cytotoxic agent, an antigen-targeting domain, a signaling domain, and / or a second polypeptide.

[0364] Embodiment II-34. The CD8-targeted conjugate of embodiment II-33, comprising the detection label.

[0365] Embodiment II-35. The CD8-targeted conjugate of embodiment II-33 or II-34, wherein the detection label comprises a radioactive isotope, an affinity tag, or a fluorescent tag. 111 321490133Attorney Docket No. OTPC-050 / 02WO

[0366] Embodiment II-36. The CD8-targeted conjugate of embodiments II-33 to II-35, comprising the cytotoxic agent, wherein the cytotoxic agent comprises a chemotherapeutic drug or a toxin.

[0367] Embodiment II-37. The CD8-targeted conjugate of any one of embodiments II-33 to II-36, comprising the antigen-targeting domain.

[0368] Embodiment II-38. The CD8-targeted conjugate of embodiment II-37, wherein the antigen-targeting domain specifically binds to an antigen expressed by an immune cell.

[0369] Embodiment II-39. The CD8-binding polypeptide of embodiment II-37, wherein the antigen-targeting domain specifically binds to a tumor-associated antigen.

[0370] Embodiment II-40. The CD8-targeted conjugate of any one of embodiments II-33 to II-39, wherein the CD8-targeted conjugate is a multi-specific antibody.

[0371] Embodiment II-41. The CD8-targeted conjugate of any one of embodiments II-33 to II-40, wherein the CD8-targeted conjugate is an antibody drug conjugate.

[0372] Embodiment II-42. The CD8-targeted conjugate of any one of embodiments II-33 to II-41, comprising the second polypeptide.

[0373] Embodiment II-43. The CD8-targeted conjugate of embodiment II-42, wherein the second polypeptide comprises a cytokine, an interferon, a chemokine, a growth factor, and / or a tumor necrosis factor.

[0374] Embodiment II-44. The CD8-targeted conjugate of embodiment II-43, wherein the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36). 112 321490133Attorney Docket No. OTPC-050 / 02WO

[0375] Embodiment II-45. The CD8-targeted conjugate of embodiment II-43, wherein the interferon comprises one or more of an interferon type I, an interferon type II and an interferon type III.

[0376] Embodiment II-46. The CD8-targeted conjugate of embodiment II-43, wherein the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN-ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN-γ).

[0377] Embodiment II-47. The CD8-targeted conjugate of embodiment II-43, wherein the second polypeptide comprises a designed cytokine.

[0378] Embodiment II-48. The CD8-targeted conjugate of embodiment II-47, wherein the designed cytokine comprises a sequence set forth in any one of SEQ ID NOs: 69-300, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0379] Embodiment II-49. A chimeric antigen receptor comprising the CD8-binding polypeptide of any one of embodiments II-1 to II-22.

[0380] Embodiment II-50. A composition comprising the CD8-binding polypeptide of any one of embodiments II-1 to II-22 and a delivery vector, wherein the CD8-binding polypeptide is operably linked to the delivery vector.

[0381] Embodiment II-51. The composition of embodiment II-50, wherein the delivery vector comprises a viral particle, a virus-like particle, a synthetic particle, a liposome, or a lipid nanoparticle.

[0382] Embodiment II-52. A nucleic acid sequence encoding the CD8-binding polypeptide of any one of embodiments II-1 to II-22.

[0383] Embodiment II-53. The nucleic acid sequence of embodiment II-52, comprising a regulatory sequence capable of driving expression of the nucleic acid sequence in a mammalian cell.

[0384] Embodiment II-54. The nucleic acid sequence of embodiment II-53, wherein the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof.

[0385] Embodiment II-55. The nucleic acid sequence of embodiment II-53 or II-54, wherein the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem. 113 321490133Attorney Docket No. OTPC-050 / 02WO

[0386] Embodiment II-56. The nucleic acid sequence of any one of embodiments II-53 to II-55, wherein the regulatory sequence comprises a constitutive promoter.

[0387] Embodiment II-57. The nucleic acid sequence of any one of embodiments II-53 to II-55, wherein the regulatory sequence comprises an inducible promoter.

[0388] Embodiment II-58. The nucleic acid sequence of any one of embodiments II-53 to II-57, wherein the regulatory sequence comprises a minimal promoter.

[0389] Embodiment II-59. The nucleic acid sequence of any one of embodiments II-53 to II-58, wherein the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs.

[0390] Embodiment II-60. The nucleic acid sequence of any one of embodiments II-53 to II-59, wherein the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

[0391] Embodiment II-61. A vector comprising the nucleic acid sequence of any one of embodiments II-52 to II-60.

[0392] Embodiment II-62. The vector of 61, wherein the vector is an expression vector.

[0393] Embodiment II-63. The vector of 61, wherein the vector is a delivery vector.

[0394] Embodiment II-64. The vector of any one of embodiments II-61 to II-63, wherein the vector is a non-viral vector.

[0395] Embodiment II-65. The vector of any one of embodiments II-61 to II-63, wherein the vector is a viral vector.

[0396] Embodiment II-66. A cell comprising one or more of: (i) the CD8-binding polypeptide of any one of embodiments II-1 to II-29; (ii) the CD8-targeted conjugate of any one of embodiments II-30 to II-48; (iii) the chimeric antigen receptor of embodiment II-49; (iv) the composition of embodiment II-50 or II-51; (v) the nucleic acid of any one embodiments II-52 to II-60; and (vi) the vector of any one of any one of embodiments II-61 to II-65.

[0397] Embodiment II-67. The cell of embodiment II-66, wherein the cell is an immune cell.

[0398] Embodiment II-68. The cell of embodiment II-66, wherein the cell is a stem cell or a T-cell precursor cell.

[0399] Embodiment II-69. The cell of embodiment II-66, wherein the cell is an embryonic stem cell, a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell. 114 321490133Attorney Docket No. OTPC-050 / 02WO

[0400] Embodiment II-70. The cell of embodiment II-66, wherein the cell is a somatic cell or a differentiated cell.

[0401] Embodiment II-71. The cell of embodiment II-66, wherein the cell is a T cell, a Natural Killer (NK) cell, or a macrophage.

[0402] Embodiment II-72. The cell of any one of embodiments II-66 to II-71, wherein the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood.

[0403] Embodiment II-73. The cell of any one of embodiments II-66 to II-72, wherein the cell has been cryopreserved.

[0404] Embodiment II-74. The cell of any one of embodiments II-66 to II-73, wherein the cell has been modified to become an allogeneic cell.

[0405] Embodiment II-75. The cell of any one of embodiments II-66 to II-74, wherein the cell has been modified to produce an allogeneic cell.

[0406] Embodiment II-76. The cell of any one of embodiments II-66 to II-75, wherein the cell is capable of expressing or is modified to express an exogenous protein.

[0407] Embodiment II-77. The cell of embodiment II-76, wherein the exogenous protein comprises an antigen receptor.

[0408] Embodiment II-78. The cell of embodiment II-77, wherein the antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR).

[0409] Embodiment II-79. The cell of any one of embodiments II-66 to II-78, wherein the cell is capable of to II-expressing or modified to express a safety switch.

[0410] Embodiment II-80. The cell of any one of embodiments II-66 to II-78, wherein the cell is capable of expressing or modified to express a cell marker.

[0411] Embodiment II-81. The cell of any one of embodiments II-66 to II-80, wherein the cell is capable of expressing or modified to express a truncated EGFR (EGFRt).

[0412] Embodiment II-82. The cell of embodiment II-81, wherein the EGFRt comprises a sequence of SEQ ID NO: 53, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0413] Embodiment II-83. The cell of embodiment II-81 or II-82, wherein the cell is capable of expressing or modified to express a truncated and optimized EGFR (EGFRopt).

[0414] Embodiment II-84. The cell of embodiment II-83, wherein the EGFRopt sequence comprises a sequence of one or more of SEQ ID NOs: 54-68, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. 115 321490133Attorney Docket No. OTPC-050 / 02WO

[0415] Embodiment II-85. A composition, comprising: (i) the CD8-binding polypeptide of any one of embodiments II-1 to II-29; (ii) the CD8-targeted conjugate of any one of embodiments II-30 to II-48; (iii) the chimeric antigen receptor of embodiment II-49; (iv) the nucleic acid sequence of any one embodiments II-52 to II-60; (v) the vector of any one of embodiments II-61 to II-65; or (vi) the cell of any one of embodiments II-66 to II-84.

[0416] Embodiment II-86. A pharmaceutical composition comprising: (i) the CD8-binding polypeptide of any one of embodiments II-1 to II-29; the CD8-targeted conjugate of any one of embodiments II-30 to II-48; the chimeric antigen receptor of embodiment II-49; the composition of embodiment II-50 or II-51; the nucleic acid sequence of any one of embodiments II-52 to II-60; the vector of any one of embodiments II-61 to II-65; or the cell of any one of embodiments II-66 to II- 84, and (ii) a pharmaceutically acceptable carrier.

[0417] Embodiment II-87. The pharmaceutical composition of embodiment II-86, wherein the pharmaceutical composition is formulated for systemic administration.

[0418] Embodiment II-88. The pharmaceutical composition of embodiment II-87, wherein the systemic administration comprises an intravenous injection or an intravenous infusion.

[0419] Embodiment II-89. The pharmaceutical composition of embodiment II-86, wherein the pharmaceutical composition is formulated for local administration.

[0420] Embodiment II-90. The pharmaceutical composition of embodiment II-89, wherein the local administration comprises a subcutaneous injection or infusion, an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

[0421] Embodiment II-91. Use of one or more of the CD8-binding polypeptide of any one of embodiments II-1 to II-29, the CD8-targeted conjugate of any one of embodiments II-30 to II-48, the chimeric antigen receptor of embodiment II-49, the nucleic acid sequence of any one embodiments II-52 to II-60, the vector of any one of embodiments II-61 to II-65, the cell of any one of embodiments II-66 to II-84, the composition of any one of embodiments II-50 to II-51, and II-85, or the pharmaceutical composition of any one of embodiments II-86 to II- 90, in the manufacture of a medicament for treating a disease or disorder.

[0422] Embodiment II-92. Use of one or more of the CD8-binding polypeptide of any one of embodiments II-1 to II-29, the CD8-targeted conjugate of any one of embodiments II-30 to 116 321490133Attorney Docket No. OTPC-050 / 02WO II-48, the chimeric antigen receptor of embodiment II-49, the nucleic acid sequence of any one embodiments II-52 to II-60, the vector of any one of embodiments II-61 to II-65, the cell of any one of embodiments II-66 to II-84, the composition of any one of embodiments II-50 to II-51, and II-85, or the pharmaceutical composition of any one of embodiments II-86 to II- 90 for treating a disease or disorder.

[0423] Embodiment II-93. The use of embodiment II-91 or II-92, wherein the disease or disorder comprises a proliferative disorder.

[0424] Embodiment II-94. The use of embodiment II-93, wherein the proliferative disorder is a metastatic disorder.

[0425] Embodiment II-95. The use of embodiment II-94, wherein the metastatic disorder comprises a solid tumor.

[0426] Embodiment II-96. A method of treating a disease or disorder comprising administering an effective amount of one or more of the CD8-binding polypeptide of any one of embodiments II-1 to II-29, the CD8-targeted conjugate of any one of embodiments II-30 to II-48, the chimeric antigen receptor of embodiment II-49, the nucleic acid sequence of any one embodiments II-52 to II-60, the vector of any one of any one of embodiments II-61 to II- 65, the cell of any one of embodiments II-66 to II-84, the composition of embodiment II-50 to II-51, and II-85, or the pharmaceutical composition of any one of embodiments II-86 to II- 90, to a subject in need thereof, thereby treating the disease or disorder.

[0427] Embodiment II-97. The method of embodiment II-96, wherein the subject is at risk of developing the disease or disorder.

[0428] Embodiment II-98. The method of embodiment II-96, wherein the subject has been diagnosed with the disease or disorder.

[0429] Embodiment II-99. The method of any one of embodiments II-96 to II-98, wherein the disease or disorder comprises a proliferative disorder.

[0430] Embodiment II-100. The method of embodiment II-99, wherein the proliferative disorder is a metastatic disorder.

[0431] Embodiment II-101. The method of embodiment II-100, wherein the metastatic disorder comprises a solid tumor.

[0432] Embodiment II-102. The method of any one of embodiments II-96 to II-101, wherein treating the disease or disorder results in one or more of: (a) decreasing the severity of a sign or symptom of the disease or disorder; (b) improving a function negatively affected by the disease or disorder; (c) improving the subject’s prognosis; 117 321490133Attorney Docket No. OTPC-050 / 02WO (d) increasing the subject’s duration of progression-free survival (PFS); (e) reducing a quantity of cells affected by the disease or disorder; (f) inducing a remission form the disease or disorder; (g) delaying or preventing a relapse of the disease or disorder; (h) delaying or preventing a second presentation of the disease or disorder; and / or (i) increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder.

[0433] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES General Methods Humanization

[0434] Camelid anti-CD8 VHH binders were humanized by first aligning the parent VHH to germline human immunoglobulin gene segments. This search identified IGHG3-23*04 and IGHJ4*04 as closely related human germline sequences (see FIG.1). Humanizing point mutations were considered in the context of available VHH structural data, and selected combinations of mutations were introduced in framework regions and CDR boundaries. The impact of these mutations on protein yield, thermal stability, and binding to recombinant CD8 alpha (CD8a) by Octet®BLI (Satorius AG) was assessed as described below. Protein Expression and Purification

[0435] VHH and cytokine-VHH fusion proteins were expressed with either C-terminal 6xHis tag or Strep-tagII (STII, Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) in Expi293 cells (Thermo Fisher Scientific). His-tagged proteins were purified in batch format using Ni Sepharose™ excel histidine-tagged protein purification resin (Cytiva). STII-tagged proteins were purified in batch format using Strep-Tactin®XT resin (IBA Lifesciences). Untagged cytokine-VHH protein for detailed affinity measurements were expressed in Expi293 cells and purified in batch format using MonoRab™ Anti-Camelid VHH Affinity Resin (GenScript). All proteins 118 321490133Attorney Docket No. OTPC-050 / 02WO were buffer exchanged into PBS pH 7.2 and sterile-filtered prior to aliquoting and storage at - 80°C. Octet®BLI (Bio-Layer Interferometry)

[0436] Octet®BLI was used to measure binding of VHH or cytokine-VHH variants in solution to captured biotinylated human CD8a (CDA-H82E3, Acro Biosystems). For screening experiments, biotinylated human CD8a was captured to ~0.75 nm on streptavidin- coated (SA) tips (Sartorius) and tested for binding to VHH or cytokine-VHH proteins at 100 nM (first round of humanization) or 30 nM (second round of humanization) in Octet®10X Kinetics Buffer (Sartorius) using a 120 sec association time and a 480 sec dissociation time. These data were analyzed for off-rate and response at the end of association. For detailed affinity measurements, biotinylated human CD8a were captured to 0.6 nm on streptavidin- coated (SA) tips (Sartorius) and tested for binding to VHH or cytokine-VHH proteins diluted from 100 nM to 0.41 nM using a 3-fold dilution series in HBS-EP+ (Cytiva) + 0.25% BSA with 120 sec association and 480 sec dissociation. Sensograms were fitted globally using a 1:1 kinetic binding model in Octet®Analysis Studio (Sartorius). Measurements were performed in triplicate with independently prepared dilutions. GloMelt™ Thermal Shift

[0437] Stability of VHH and cytokine-VHH variants during thermal denaturation was measured using GloMelt™ dye (Biotium) on a Bio-Rad CFX384 Real-Time PCR Instrument using the SYBR channel. Proteins were diluted to a final concentration of 0.5 mg / mL in PBS pH 7.2 + / - 1 mM TCEP with 1X GloMelt™ dye in a final volume of 20 μL in thin-wall, hard shell 384-well PCR plates with microseal ‘B’ seals (Bio-Rad). Proteins were tested both in non-reducing conditions to capture native stability as well as in the presence of 1 mM TCEP (Tris(2-carboxyethyl)phosphine) to amplify the fluorescent signal upon denaturation. The temperature ramp was performed by first holding at 25°C for 30 sec then increasing temperature in 0.5°C steps with a 20 sec hold at each step in addition to the measurement time at each step up to 95°C. Melting temperatures (Tms) under reducing and non-reducing conditions were calculated as the temperature at which the first derivative of the fluorescence-temperature curve reaches a minimum. Measurements were performed in duplicate. 119 321490133Attorney Docket No. OTPC-050 / 02WO pSTAT5 (phosphorylated signal transducer and activator of transcription 5) Assay

[0438] Leukopak-isolated T cells were thawed and rested overnight in the absence of exogenous cytokines, then mixed with media containing the indicated concentration of recombinant cytokines and incubated for 15 minutes at 37°C. Cells were transferred onto ice for 5 min, washed 1x with cold Cell Staining Buffer (BioLegend®), and stained for surface markers for 20 min at 4°C. Cells were then fixed with Cytofix™ Fixation Buffer (BD Biosciences) at 37°C for 10 min, washed, permeabilized with Phosflow™ Perm Buffer III (BD Biosciences) for 30 min at 4°C, and stained for intracellular markers including pStat5 (pY694) (BD Biosciences). Spheroid Coculture Plasmid

[0439] Lentiviral constructs were constructed using a third generation backbone containing an inducible single promoter driving a MSLN-targeted CAR, the EGFRopt transduction marker (Shabaneh et al., Front. Mol. Med., 11 October 2022, Vol.2, pp 1-11), and the engineered cytokine linked to the indicated anti-hCD8 VHH. Lentivirus

[0440] VSV-pseudotyped third generation lentiviral vector particles were generated using polyethylenimine (PEI)-mediated transient transfection of in-house suspension-adapted HEK293Ts (ATCC) grown in shake plates in FreeStyle™ medium (Thermo Fisher Scientific). Lentiviral supernatant was harvested, pooled, clarified, and filter-sterilized using 0.2 µm filtration. Lentiviral supernatant was then concentrated in 50 mL conicals for 4h at 4°C at 10,000 x g in 10% sucrose buffer (Jiang et al., Nature Scientific Reports, 8 September 2015, pp.1-9). The volume was then decanted and pellets were resuspended to equal ~60- 100x concentration before vortexing vigorously. Concentrated lentivirus was aliquoted and stored at -80°C. Lentiviral batch titers were evaluated by limited dilution on primary T cells and measured by flow cytometry against surface expression of the transduction marker EGFRopt. 120 321490133Attorney Docket No. OTPC-050 / 02WO Cells

[0441] To generate CAR-T cells expressing the indicated constructs, leukopak-isolated T cells were cultured in T Cell Media (TCM) with added cytokines: OpTmizer™ medium (Thermo Fisher Scientific) supplemented with CTS™ Immune Cell Serum Replacement (Thermo Fisher Scientific), 2 mM Gibco L-glutamine (Thermo Fisher Scientific), 2 mM Gibco GlutaMAX™ (Thermo Fisher Scientific), 200 IU / ml IL-2 (R&D Systems), 1200 IU / mL IL-7 (R&D Systems), and 200 IU / mL IL-15 (R&D Systems). T cells were stimulated with 1:100 dilution of T cell TransAct™ (Miltenyi Biotec) for 24h, followed by incubation with lentivirus. Stimulation and viral infection were terminated after 24h by addition of 7 volumes of fresh TCM with cytokines, without TransAct™, and cells were expanded for 5 additional days before cryopreservation. Chronic Stimulation

[0442] Tissue culture plates were coated with approximately 155 μL / cm2of 5 μg / mL of recombinant human mesothelin (MSLN, manufactured in-house) and 2 μg / mL each of recombinant human CD58-Fc and ICAM-1-Fc (R&D Systems). Plates were incubated at either 37°C for 2 hours or overnight at 4°C. Residual coating solution was removed and 1- 2E6 CAR T cells / mL in TCM with cytokines were added to the wells. CAR T cells were continuously cultured on MSLN / CD58 / ICAM-1 coated wells for 10 days. To ensure continual access to stimulation proteins, CAR T cells were enumerated and replated on freshly MSLN / CD58 / ICAM-1 coated wells every 2-3 days (at 1-2E6 CAR T cells / mL) for a total of 4 rounds of stimulation. Coculture

[0443] Tumor spheroids were generated by adding mKate2-expressing H226 cells to a 96- well round-bottom ultra-low attachment plate (S-bio) in 100 μL R10 media: RPMI 1640 + Glutamax-I (Gibco) with 10% FBS (Gibco). PBS (Gibco) was added to edge wells. The plate was centrifuged at 1000xg for 10 min before transferring to a 37°C incubator for 72h. Subsequently, chronically-stimulated T cells were added to tumor spheroids (250 CAR T cells / well for a 1:20 E:T ratio) in 100 μL R10. Plates were imaged in an Incucyte Sx5 (Sartorius) every 6 hours for 7 days. Mean total red fluorescence integrated intensity (RCU x μm2) was calculated for 3 technical replicates per sample and normalized to baseline for visualization. 121 321490133Attorney Docket No. OTPC-050 / 02WO Affinity Maturation Library Cloning

[0444] In certain embodiments, affinity-maturation libraries were generated by a saturation-mutagenesis strategy. For each position pair within CDR-H1 (Chothia / Kabat), CDR-H2 (Kabat), or CDR-H3 (Kabat), 35- to 45-mer single-stranded oligonucleotides were synthesized by Integrated DNA Technologies, each oligonucleotide containing dual NNK degeneracies and / or up to 3 deletions of an amino acid position, and outward-facing Esp3 I recognition sites flanking the variable region. Oligonucleotides were converted to double- stranded DNA by high-fidelity amplification and purified by silica-membrane chromatography. Amplicons were Golden-Gate assembled into an Aga2p surface-display vector bearing an inducible GAL1 promoter and a tryptophan (TRP1) auxotrophic selection cassette. The ligation mixtures were transformed into electrocompetent Escherichia coli DH10-beta, recovered in SOC for 1hr, and expanded overnight in luria broth plates supplemented with 100 µg mL⁻¹ ampicillin at 37°C with agitation. The following day plates were scraped, and the plasmid libraries were isolated using the Thermo Fisher GeneJET Midiprep kit, quantified spectrophotometrically, and linearized by restriction at the TRP1 locus. Approximately 5–10 µg of the digested plasmid pool was co-electroporated into Saccharomyces cerevisiae strain EBY100 together with a 300bp PCR-generated homology- directed repair fragment encoding functional TRP1, thereby reconstituting the selection marker in vivo and promoting high-efficiency gap repair. Transformants were recovered in SDCAA medium lacking tryptophan and cultured at 30°C for 24 h, yielding libraries that retained ≥10-fold coverage of the theoretical diversity for each two-site NNK sub-library. Fluorescence-activated cell sorting of CDR1, 2, & 3 Libraries (FACS)

[0445] Yeast libraries displaying Aga2-fused VHH variants were induced for 18 h in galactose-containing medium, harvested, and washed in phosphate-buffered saline supplemented with 0.5% (w / v) bovine serum albumin. Cells were dual-stained for 30 min at 4°C with (i) a monoclonal antibody specific for the c-Myc epitope fused to fluorescein and (ii) biotinylated target antigen followed by streptavidin-phycoerythrin. After two washes, approximately 2×107events were analyzed and sorted on a Sony SH800 cell sorter equipped with 70 μm chip. Dual-positive events corresponding to the PE^high / FITC^high population 122 321490133Attorney Docket No. OTPC-050 / 02WO were collected into selective SDCAA medium, expanded, and subjected to additional rounds of affinity-based enrichment with progressively decreasing antigen concentrations. CDR Assembly

[0446] Plasmid was isolated from sorted yeast by Zymoprep™ mini-prep. CDR-H1, H2 and H3 segments were individually PCR-amplified with primers flanked by overlap segments. Equimolar pools of the three matured CDR fragments and the constant frameworks were stitched by three-piece overlap-extension PCR to create a secondary “combinatorial- matured” library. The product was cloned back into pCT-con2 and transformed into EBY100 as above, yielding ~1 × 107independent clones. Fluorescence-activated cell sorting of fixed and variable length CDR Combinatorial Libraries (FACS)

[0447] The combinatorial-matured library underwent additional FACS selection as described above. The dual-positive events (PE^high / FITC^high) were collected into SD-CAA medium lacking tryptophan, expanded overnight, and subjected to an additional two-round affinity-based enrichment of the combinatorial library with stepwise antigen reduction. Specifically, the recombined pool was first sorted at 1 nM antigen, and the upper PE^high / FITC^high population was recovered. After expansion and re-induction, a second sort was performed at 0.5 nM antigen; during this round an edge-and-diversity gate encompassing the diagonal PE intensity shoulder was included alongside the principal PE^high cluster to retain sequence breadth while enriching for higher-affinity clones. The resulting populations were propagated under selection and taken forward for plasmid extraction and next-generation sequencing. Next-generation sequencing (NGS)

[0448] Plasmid DNA was isolated from the enriched S. cerevisiae pools with a silica-spin yeast miniprep kit (Zymo Research, Zymoprep™). VHH inserts were first amplified in 50 µL reactions (KAPA HiFi polymerase) using locus-specific primers that appended the Nextera adapter sequences. A second, minimum-cycle indexing PCR was performed directly on the purified amplicons with i5 / i7 indexing primers complementary to the added Nextera overhangs, thereby introducing full P5 / P7 flow-cell adapters and dual combinatorial indices. Libraries were quantified fluorometrically, pooled at equimolar concentrations, and sequenced on an Illumina MiSeq instrument with a 600-cycle v3 kit (2 × 300 bp). 123 321490133Attorney Docket No. OTPC-050 / 02WO Demultiplexed FASTQ reads (bcl2fastq, default parameters) were merged, translated in- frame, tabulated to determine variant frequencies and enrichment factors, and clustered on 90% VDJ identity for downstream lead selection. Example 1: Generation and Biophysical Characterization of Humanized Anti-CD8 VHH

[0449] Camelid anti-CD8 VHH binders were humanized according to the methods described herein. DNA fragments were then synthesized and inserted into mammalian expression vectors downstream of a CMV promoter and hIgK signal peptide and expression plasmids were confirmed by sequencing. VHH and cytokine-VHH fusion proteins were then expressed according to the methods described herein prior to measuring binding affinities (Octet®BLI) and thermal stability (GloMelt™ Thermal Shift).

[0450] Sequence alignment of the parental VHH sequence against human germline sequences identified IGHGV3-23*04 and IGHJ4*01 as the most closely related human germline gene segments. Alignment of the parental sequence with these gene segments identified possible point mutations to increase the degree of similarity to human sequences and reduce the potential for immunogenicity (FIG.1).

[0451] Consideration of possible point mutants in the context of available structural data for VHHs allowed for the identification of four VHH variants containing different combinations of humanizing mutations. These VHH variants were initially selected for expression and biophysical characterization (FIG.2). While some of the tested mutations had a negative impact on yield and binding (e.g., V79L+K87R), the initial screen of humanizing mutations identified combinations of mutations that were well-tolerated. For example, an intermediate variant, VHH 1.4.0, displayed ~10% higher yield and increased thermostability in the presence of the reducing agent TCEP (tris(2-carboxyethyl)phosphine) compared to that of the parent, VHH 1, but showed a slightly weaker binding to CD8a by Octet®BLI that was based on a reduced response at the end of association and a ~1.5-fold faster apparent off-rate compared to the parent.

[0452] The next steps involved identifying a core set of humanizing mutations that were well-tolerated (e.g., VHH 1.4.0) in the context of the VHH and testing additional rational humanizing mutations for their impact on binding and stability in the context of the VHH alone (FIG.3A) and in the context of a cytokine-VHH fusion (FIG.3B). This round of expression and testing identified a combination of mutations (I34M, E44G, and P63S) that maintained the increased thermostability of VHH 1.4.0 relative to the parental VHH, while 124 321490133Attorney Docket No. OTPC-050 / 02WO restoring some of the favorable binding properties of the parental VHH to CD8a (e.g., a slower off-rate and higher response at the end of the association phase compared to VHH1.4.0). The V79L and K87R mutations were tested individually in this round, and they again negatively impacted binding and stability, respectively. The relative effect of incrementally adding humanizing mutations was similar in the context of VHH alone or the cytokine-VHH fusion. Based on its favorable stability and binding properties, the humanized variant VHH 1.4.0+ was selected for further affinity characterization and functional testing.

[0453] The affinity of VHH 1.4.0+ for human CD8a was measured by Octet®BLI in the context of a VHH alone or a cytokine-VHH fusion (FIGS.4A-4D). While the humanized VHH alone showed a ~7-fold weaker affinity for CD8a compared to the parent, VHH 1.4.0+ maintained a high affinity of 2.2 ± 0.2 nM. The humanized cytokine-VHH fusion similarly showed a ~5-fold weaker affinity for CD8a compared to the parent cytokine-VHH fusion but maintained a high affinity of 6.5 ± 0.1 nM.

[0454] These results indicate that incorporation of selected humanizing mutations into a parental camelid anti-CD8a VHH maintained high affinity binding to CD8a and improved thermostability, while also reducing the number of non-human residues that may pose some risk for immunogenicity. Example 2: Affinity Maturation of the Humanized Anti-CD8 VHH and Biophysical Characterization

[0455] Affinity matured anti-hCD8a VHHs were developed using yeast display. For example, a DNA library was constructed using NNKNNK degeneracies, along with up to three insertions or deletions, and were systematically tiled across CDR-H1 (Chothia / Kabat), CDR-H2 (Kabat), and CDR-H3 (Kabat). Double stranded DNA was Golden Gate cloned into a yeast display shuttle vector, linearized, and then electroporated into EBY100 using a repair template. Yeast libraries displaying Aga2-fused VHH were dual-stained (i) a monoclonal antibody specific for the c-Myc epitope fused to fluorescein and (ii) biotinylated target antigen followed by streptavidin-phycoerythrin. Dual-positive events corresponding to the PE^high / FITC^high population were collected into selective SDCAA medium, expanded, and subjected to additional rounds of affinity-based enrichment with progressively decreasing antigen concentrations. Following two rounds of sorting the plasmid DNA was isolated and CDR-H1, H2 and H3 segments were individually PCR-amplified with primers flanked by overlap segments and stitched together by three-piece overlap-extension PCR to create two 125 321490133Attorney Docket No. OTPC-050 / 02WO secondary “combinatorial-matured” libraries, combinatorial library 1 had a fixed 23 amino acid long CDR-H3 while combinatorial library 2 had CDR-H3s with lengths that spanned 20- 26 amino acids. The combinatorial libraries underwent two rounds of FACS selection. Specifically, the recombined pools were first sorted at 1 nM antigen, and the upper PE^high / FITC^high population was recovered. After expansion and re-induction, a second sort was performed at 0.5 nM antigen. The resulting populations were propagated under selection and selected for plasmid extraction and next-generation sequencing (NGS) as described herein.

[0456] The frequency of CDR sequence that arose in >1% of the enriched combinatorial library represents CDR residues that may positively contribute to binding. Based on NGS analysis, CDR1 may be any combination of any of the following residues at the specified positions as identified from the CDR3 fixed and variable length combinatorial libraries (FIGS.5A and 5D): residue 1 may be D; residue 2 may be Y; residue 3 may be A; residue 4 may be selected from the group consisting of I or L; residue 5 may be G;

[0457] CDR2 may be any combination of any of the following residues at the specified positions as identified from the CDR3 fixed and variable length combinatorial libraries (FIGS.5B and 5E) : residue 1 may be C; residue 2 may be I; residue 3 may be R; residue 4 may be selected from the group consisting of A, C, F, I, L, N, S, T, V, W, or Y; residue 5 may be selected from the group consisting of F, S, W, or Y; residue 6 may be D; residue 7 may be selected from the group consisting of A, G, I, R, or V; residue 8 may be selected from the group consisting of A, M, R, S, or W; residue 9 may be selected from the group consisting of D, F, S, or T; residue 10 may be Y; residue 11 may be Y; residue 12 may be A; residue 13 may be D; residue 14 may be s P; residue 15 may be selected from the group consisting of V or L; residue 16 may be K; residue 17 may be G;

[0458] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 fixed length combinatorial library (FIG.5C): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be selected from the group consisting of F, W, or Y; residue 5 may be selected from the group consisting of A, S, or T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be selected from the group consisting of G, S, W, or Y; residue 10 may be selected from the group consisting of F, H, I, L, or W; residue 11 may be selected from the group consisting of A, E, G, I, L, N, P, R, S, or V; residue 12 may be selected from the group consisting of A, G, P, S, or V; residue 13 may be selected from the group consisting of D, E, or V; residue 14 may be selected from the group consisting of P or S; residue 15 may be selected from the group consisting of A, D, 126 321490133Attorney Docket No. OTPC-050 / 02WO E, N, S, or T; residue 16 may be selected from the group consisting of A, D, E, G, H, L, Q, R, S, or V; residue 17 may be selected from the group consisting of A, D, E, N, or P; residue 18 may be selected from the group consisting of R, V, or Y; residue 19 may be Y; residue 20 may be D; residue 21 may be M; residue 22 may be D; residue 23 may be Y;

[0459] CDR3 may be any combination of any of the following residues at the specified positions as identified from the CDR3 length 20 combinatorial library (FIG.5F): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be S; residue 10 may be selected from the group consisting of F, H, I, or L; residue 11 may be selected from the group consisting of A, D, G, H, I, K, L, N, Q, R, S, T, V, or Y; residue 12 may be selected from the group consisting of A, D, or G; residue 13 may be selected from the group consisting of D, E, I, K, or R; residue 14 may be P; residue 15 may be Y; residue 16 may be Y; residue 17 may be D; residue 18 may be M; residue 19 may be D; residue 20 may be Y;

[0460] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 21 combinatorial library (FIG.5G): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be selected from the group consisting of F, H, L, S, W, or Y; residue 10 may be selected from the group consisting of F, H, I, L, or W; residue 11 may be selected from the group consisting of A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W; residue 12 may be selected from the group consisting of D, P, or V; residue 13 may be selected from the group consisting of A, E, or V; residue 14 may be selected from the group consisting of P or R; residue 15 may be P; residue 16 may be selected from the group consisting of E or Y; residue 17 may be Y; residue 18 may be D; residue 19 may be M; residue 20 may be D; residue 21 may be Y;

[0461] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 22 combinatorial library (FIG.5H): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be f Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be selected from the group consisting of F, S, or Y; residue 10 may be selected from the group consisting of F, I, L, W, or Y; residue 11 may be selected from the group consisting of A, G, H, K, L, M, P, R, S, V, or W; residue 12 may be selected from the group consisting of P, V, or Y; residue 13 may be selected from the group consisting of D, E, H, P, or V; residue 14 may be selected from the group consisting of A, E, G, or P; residue 15 may be selected from the group consisting of A, E, G, R, or S; residue 16 may be selected from the group consisting of D, E, 127 321490133Attorney Docket No. OTPC-050 / 02WO P, or V; residue 17 may be Y; residue 18 may be Y; residue 19 may be D; residue 20 may be M; residue 21 may be D; residue 22 may be Y;

[0462] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 23 combinatorial library (FIG.5I): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be S; residue 10 may be selected from the group consisting of F, H, I, or L; residue 11 may be selected from the group consisting of I, L, N, or V; residue 12 may be selected from the group consisting of A, I, K, M, P, S, T, or V; residue 13 may be selected from the group consisting of E, N, P, R, T, or V; residue 14 may be selected from the group consisting of G or P; residue 15 may be selected from the group consisting of A, G, H, T, or Y; residue 16 may be selected from the group consisting of E, G, L, R, or Y; residue 17 may be selected from the group consisting of D, E, or P; residue 18 may be selected from the group consisting of S or Y; residue 19 may be selected from the group consisting of D or Y; residue 20 may be D; residue 21 may be M; residue 22 may be D; residue 23 may be Y;

[0463] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 24 combinatorial library (FIG.5J): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be S, Y; residue 10 may be selected from the group consisting of F, I, L, or W; residue 11 may be selected from the group consisting of A, E, F, G, K, L, Q, S, T, or V; residue 12 may be selected from the group consisting of A, D, E, K, M, P, S, T, or V; residue 13 may be V; residue 14 may be selected from the group consisting of P or V; residue 15 may be selected from the group consisting of A, D, E, F, G, I, N, P, R, or V; residue 16 may be selected from the group consisting of A, D, E, G, I, L, N, P, Q, R, S, T, V, W, or Y; residue 17 may be selected from the group consisting of A, D, E, G, R, S, T, V, or Y; residue 18 may be selected from the group consisting of A, D, E, G, P, or V; residue 19 may be selected from the group consisting of L, S, or Y; residue 20 may be Y; residue 21 may be D; residue 22 may be M; residue 23 may be D; residue 24 may be Y;

[0464] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 25 combinatorial library (FIG.5K): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be L, Q; residue 9 may be selected from the group consisting of H, K, L, Q, R, S, or Y; residue 10 may be selected from the 128 321490133Attorney Docket No. OTPC-050 / 02WO group consisting of F, I, L, Q, or W; residue 11 may be selected from the group consisting of G, K, L, P, Q, R, S, or V; residue 12 may be selected from the group consisting of D, I, S, or V; residue 13 may be selected from the group consisting of D, E, N, P, or V; residue 14 may be selected from the group consisting of A, D, E, G, P, or V; residue 15 may be selected from the group consisting of A, E, R, T, or V; residue 16 may be selected from the group consisting of P, R, or V; residue 17 may be selected from the group consisting of A, D, E, K, N, P, S, T, or V; residue 18 may be selected from the group consisting of E, G, Q, R, S, V, or Y; residue 19 may be selected from the group consisting of D, E, P, S, or T; residue 20 may be selected from the group consisting of R or Y; residue 21 may be Y; residue 22 may be D; residue 23 may be M; residue 24 may be D; residue 25 may be Y; and

[0465] CDR3 may be any combination of any of the following residues at the specified positions identified from the CDR3 length 26 combinatorial library (FIG.5L): residue 1 may be G; residue 2 may be S; residue 3 may be L; residue 4 may be Y; residue 5 may be T; residue 6 may be C; residue 7 may be V; residue 8 may be Q; residue 9 may be selected from the group consisting of H, R, S, or W; residue 10 may be selected from the group consisting of F, I, or L; residue 11 may be selected from the group consisting of I, N, R, V, or W; residue 12 may be selected from the group consisting of P, R, S, or V; residue 13 may be selected from the group consisting of A, D, E, I, R, V, or Y; residue 14 may be selected from the group consisting of D, G, N, P, Q, or V; residue 15 may be selected from the group consisting of A, D, E, G, L, R, or V; residue 16 may be selected from the group consisting of R or V; residue 17 may be selected from the group consisting of D, G, or P; residue 18 may be selected from the group consisting of A, D, E, G, H, L, M, P, Q, R, S, T, V, or Y; residue 19 may be selected from the group consisting of A, D, E, G, L, R, S, V, or Y; residue 20 may be selected from the group consisting of D, E, F, L, P, Q, or V; residue 21 may be selected from the group consisting of E, I, M, R, T, V, or Y; residue 22 may be Y; residue 23 may be D; residue 24 may be M; residue 25 may be D: residue 26 may be Y.

[0466] Based on NGS of the variable and fixed-length CDR combinatorial libraries, 20 representative VHH sequences were selected or designed for biophysical characterization.16 VHHs used the parental FW residues (FIG.6A). The VDJs in VHH 1.4.0_aff.16 (SEQ ID NO: 348) and VHH 1.4.0_aff.1 (SEQ ID NO: 333) were the most frequent clones in the sorted variable length combinatorial library. To assess the contribution of the CDR2 shared by VHH 1.4.0_aff.16 (SEQ ID NO: 348) and VHH 1.4.0_aff.1 (SEQ ID NO: 333), VHH 1.4.0_aff.2 (SEQ ID NO: 334) was engineered to incorporate this CDR2 while retaining the parental CDR3. VDJs from the fixed-length library often enriched on clones that introduced 129 321490133Attorney Docket No. OTPC-050 / 02WO tryptophan(s) in CDR2 or featured CDR3s with unusually high hydrophobicity. VHH 1.4.0_aff.3 (SEQ ID NO: 335) was selected despite its relatively low frequency in the fixed length library because it lacked both of these liabilities, whereas VHH 1.4.0_aff.4 (SEQ ID NO: 336) and VHH 1.4.0_aff.5 (SEQ ID NO: 337) were prioritized due to their high clonal frequencies. Because of the percent similarity between the most represented VHH clones, additional clones from distinct VDJ clusters with lower frequency were also nominated to sample alternative sequence space: VDJs in VHH 1.4.0_aff.7 to VHH 1.4.0_aff.15 (SEQ ID NOs: 339-347). Finally, CDRs from four clones nominated in FIG.6A from the variable length library with the most divergent sequence identities were grafted onto a more humanized VHH scaffold than the original library parent (FIG.6B).

[0467] As a result, twenty VDJs, either directly selected from or informed by combinatorial affinity maturation libraries with diverse CDR identities, were chosen for further downstream characterization. Example 3: Functional characterization of humanized and affinity matured CD8 Targeted Engineered Cytokines

[0468] Selected VHHs were fused to an engineered not-alpha human IL2 as a potential way to increase the efficacy of IL-2R signaling. These cytokine-fusion molecules were assessed based on their biophysical stability, their ability to stimulate the IL-2R as recombinant proteins, and to armor anti-MSLN CAR-T cells.

[0469] DNA fragments were synthesized and inserted into mammalian expression vectors downstream of a CMV promoter and hIgK signal peptide. Expression plasmids were confirmed by sequencing. Cytokine-VHH fusion proteins were then expressed with C- terminal STII tags in Expi293 cells. STII-tagged proteins were purified in batch format using Streptactin-XT resin (IBA). All proteins were buffer exchanged into PBS pH 7.2 and sterile- filtered prior to aliquoting and storage at -80°C.

[0470] Octet®BLI was then used to measure binding of cytokine-VHH variants in solution to captured biotinylated human CD8a (Acro CDA-H82E3) as described herein. Additionally, the stability of cytokine-VHH variants during thermal denaturation was measured using GloMelt™ dye (Biotium) on a Bio-Rad CFX384 Real-Time PCR Instrument using the SYBR channel as described herein. 130 321490133Attorney Docket No. OTPC-050 / 02WO

[0471] The pSTAT assay, as well as the steps of plasmid generation, lentiviral construction, cell generation, chronic stimulation and spheroid coculture preparation, were performed according to the methods described herein.

[0472] Based on CDR3 clustering and frequency in the sequenced sorted combinatorial affinity maturation library, 15 VHHs (FIG.6A) and 4 VHH 1.4.0+ scaffolds (FIG.6B) with affinity matured CDR grafts were selected for further characterization as cytokine-VHH-STII fusions, including their binding to hCD8a (Octet response, koff), thermal stability (Tm), purity from single step purification (% monomer on HPLC-SEC), and yield from mammalian expression. All affinity variants showed slower koff and maintained reasonable biophysical characteristics.

[0473] To assess how the affinity variants of anti-CD8a VHH molecules affect the potency of cytokine-VHH fusions, CD8+ T cells were incubated with a 7-point dilution series of cytokine or cytokine-VHH fusion proteins, and activation of IL-2R signaling was measured by STAT5 phosphorylation as determined by flow cytometry (FIG.7). When fused with designed cytokine, anti-CD8a VHH affinity variants increased activation of CD8+ T cells, as indicated by percentage of pSTAT5+ CD8+ T cells, compared to the untargeted wt IL-2 cytokine (SEQ ID NO: 389). In addition, when fused with designed cytokine, several anti- CD8 VHH affinity variants showed increased potency in activation of IL-2R signaling in CD8+ T cells, when compared to VHH 1, including Cytokine-VHH 1.4.0+ (SEQ ID NO: 327), Cytokine-VHH 1.4.0_aff.1 (SEQ ID NO: 353), Cytokine-VHH 1.4.0_aff.9 (SEQ ID NO: 361), Cytokine-VHH 1.4.0_aff.11 (SEQ ID NO: 363), Cytokine-VHH 1.4.0_aff.13 (SEQ ID NO: 365), Cytokine-VHH 1.4+_aff.3 (SEQ ID NO: 371), Cytokine-VHH1.4+_aff.4 (SEQ ID NO: 372). Based on phosphorylation of STAT5 and biophysical characteristics, Cytokine- VHH1.4.0+ (SEQ ID NO: 17 and 303), Cytokine-VHH 1.4.0_aff.1 (SEQ ID NO: 353) and Cytokine-VHH 1.4.0_aff.11 (SEQ ID NO: 363) were nominated as armoring strategies for anti-MSLN CAR-T cells and enrolled in a spheroid cytotoxicity assay to assess efficacy.

[0100] To that end, 250 chronically stimulated anti-MSLN CAR-T cells armored with cytokine-VHH were cocultured with MSLN- and mKate2-positive H226 tumor spheroids at an effector target (E:T) ratio of 1:20. When armored with cytokine-VHH molecule, CAR-T cells showed superior cytotoxic activities in clearing spheroid tumors as compared to CAR-T cells alone. Furthermore, MSLN CAR-P2A-EGFRopt-T2A-cytokine-VHH 1.4.0_aff.1 (SEQ ID NO:392 that includes Cytokine-VHH 1.4.0_aff.1 or SEQ ID NO: 353) and MSLN CAR- P2A-EGFRopt-T2A-cytokine-VHH 1.4.0_aff.11 (SEQ ID NO:393 that includes Cytokine- VHH 1.4.0_aff.11 or SEQ ID NO: 363) showed at least comparable spheroid clearance to 131 321490133Attorney Docket No. OTPC-050 / 02WO MSLN CAR-P2A-EGFRopt-T2A-cytokine-VHH-armored CAR-T cells (SEQ ID NO: 390) (FIG.8). As a result, STAT5 phosphorylation and spheroid cytotoxicity experiments suggest that affinity matured anti-human CD8a targeting may further improve the potency of cytokine-VHH fusion molecules. ADDITIONAL SEQUENCES OF THE DISCLOSURE Sequence SEQ Name / identifier ID NO EVQLVESGGGLVQPGGSLRLSCAASGFTSSYAMSWVRQAPGKGLEWVSAIS321490133Attorney Docket No. OTPC-050 / 02WO Sequence SEQ Name / identifier ID NO ENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK133 321490133

Claims

Attorney Docket No. OTPC-050 / 02WO CLAIMS 1. A CD8-binding polypeptide comprising a CD8-specific single domain antibody (VHH), wherein the VHH comprises at least one modification with respect to SEQ ID NO:

314.

2. The CD8-binding polypeptide of claim 1, wherein the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, V79, K87, P88, A91, K124, and a combination thereof.

3. The CD8-binding polypeptide of claim 1, wherein the at least one modification occurs at a position of SEQ ID NO: 314 selected from the group consisting of I34, P63, Q1, Q5, A14, E44, S72, A75, P88, A91, K124, and a combination thereof.

4. The CD8-binding polypeptide of any one of claims 1-3, wherein the modification is a substitution, deletion, or insertion.

5. The CD8-binding polypeptide of claim1-4, wherein the at least one modification is selected from the group consisting of: I34M, P63S, Q1E, Q5V, A14P, E44G, S72R, A75S, V79L, K87R, P88A, A91T, K124Q, and a combination thereof.

6. The CD8-binding polypeptide of any one of claims 1-5, wherein the at least one modification is in the VHH complementarity-determining region 1 (CDR), CDR2, and / or CDR3.

7. The CD8-binding polypeptide of any one of claim 1-6, wherein the at least one modification is in the VHH framework region 1 (FR), FR2, FR3, and / or FR4.

8. The CD8-binding polypeptide of any one of claims 1-7, wherein the VHH CDR1 sequence comprises GFTFDDYAX1G (SEQ ID NO: 373), the VHH CDR2 sequence comprises X2IRVSDGSTYYADX3VKG (SEQ ID NO: 374), and the VHH CDR3 sequence comprises GSLYTX4VQSIVVVPARPYYDMDY (SEQ ID NO: 375), wherein X1is M or I, X2 is C, S, or A, X3 is P or S, and X4 is C, S, or A. 134 321490133Attorney Docket No. OTPC-050 / 02WO 9. The CD8-binding polypeptide of claim 8, wherein: (i) if X1is I, then X2is S or A, X3is S, and / or X4is S or A; (ii) if X2 is C, then X1 is M, X3 is S, and / or X4 is S or A; (iii) if X3is P, then X1is M, X2is S or A, and / or X4is S or A; and (iv) if X4 is C, then X1 is M, X2 is S or A, and / or X3 is S.

10. The CD8-binding polypeptide of any one of claims 1-9, wherein the VHH CDR1, CDR2, and CDR3 respectively comprise: (i) GFTFDDYAMG (SEQ ID NO: 14); CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); (ii) GFTFDDYAMG (SEQ ID NO: 14); CIRVSDGSTYYADSVKG (SEQ ID NO: 15), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494) (iii) GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and AGSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 4); (iv) GFTFDDYAIG (SEQ ID NO: 2), CIRVSDGSTYYADPVKG (SEQ ID NO: 3), and GSLYTCVQSIVVVPARPYYDMDY (SEQ ID NO: 494); (v) GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and AGSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 8); (vi) GFTFDDYAIG (SEQ ID NO: 2), SIRVSDGSTYYADPVKG (SEQ ID NO: 7), and GSLYTSVQSIVVVPARPYYDMDY (SEQ ID NO: 495); (vii) GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and AGSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 12); or (viii) GFTFDDYAIG (SEQ ID NO: 2), AIRVSDGSTYYADPVKG (SEQ ID NO: 11), and GSLYTAVQSIVVVPARPYYDMDY (SEQ ID NO: 496).

11. The CD8-binding polypeptide of any one of claims 1-10, wherein the at least one modification comprises: (i) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, A91T, and K124Q; (ii) Q1E, Q5V, A14P, A75S, A91T, and K124Q; (iii) Q1E, Q5V, A14P, S72R, A75S, V79L, K87R, A91T, and K124Q; (iv) Q1E, Q5V, A14P, S72R, A75S, A91T, and K124Q; (v) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, A91T, and K124Q; (vi) Q1E, Q5V, A14P, A75S, V79L, K87R, A91T, and K124Q; (vii) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; 135 321490133Attorney Docket No. OTPC-050 / 02WO (viii) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, K87R, P88A, A91T, and K124Q; (ix) Q1E, Q5V, A14P, I34M, P63S, S72R, A75S, V79L, A91T, and K124Q; (x) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, A91T, and K124Q; or (xi) Q1E, Q5V, A14P, I34M, E44G, P63S, S72R, A75S, V79L, K87R, P88A, A91T, and K124Q.

12. The CD8-binding polypeptide of any one of claims 1-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 17, 303, 1, 5, 9, 13, 21, 25, 29, 33, 37, and 315-323, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

13. The CD8-binding polypeptide of any one of claims 1-11, wherein the VHH comprises SEQ ID NO: 17, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

14. The CD8-binding polypeptide of any one of claims 1-11, wherein the VHH CDR1 sequence comprises DYAX5G (SEQ ID NO: 376), the VHH CDR2 sequence comprises CIRX6X7DX8X9X10YYADPX11KG (SEQ ID NO: 377), and the VHH CDR3 sequence comprises GSLX12X13CVQX14X15X16X17X18X19X20X21X22X23YDMDY (SEQ ID NO: 378), wherein X5 is I or L, X6 is A, C, F, I, L, N, S, T, V, W, or Y, X7 is F, S, W, or Y, X8 is G, R, or V, X9is A, M, R, S, or W, X10is D, F, S, or T, X11is L or V, X12is F, W, or Y, X13is A, S, or T, X14 is G, S, W, or Y, X15 is F, H, I, L, or W, X16 is A, E, G, I, L, N, P, R, S, or V, X17 is A, G, P, S, or V, X18is D, E, or V, X19is P or S, X20is A, D, E, N, S, or T, X21is A, D, E, G, H, L, Q, R, S, or V, X22 is A, D, E, N, or P and X23 is R, V, or Y.

15. The CD8-binding polypeptide of any one of claims 1-11, wherein: (i) the VHH CDR1 sequence comprises DYAX5G (SEQ ID NO: 376); (ii) the VHH CDR2 sequence comprises CIRX24X25DX26X27X28YYADPX29KG (SEQ ID NO: 379); and (iii) the VHH CDR3 sequence comprises: GSLYTCVQSX30X31X32X33PYYDMDY (SEQ ID NO: 380), GSLYTCVQX34X35X36X37X38X39PX40YDMDY (SEQ ID NO: 381), 136 321490133Attorney Docket No. OTPC-050 / 02WO GSLYTCVQX41X42X43X44X45X46 X47X48YYDMDY (SEQ ID NO: 382), GSLYTCVQSX49X50X51X52X53X54X55X56X57X58DMDY (SEQ ID NO: 383), GSLYTCVQX59X60X61X62VX63X64X65X66X67X68YDMDY (SEQ ID NO: 384), GSLYTCVLX69X70X71X72X73X74X75X76X77X78X79X80YDMDY (SEQ ID NO: 385), or GSLYTCVQX81X82X83X84X85X86X87X88X89X90X91X92X93YDMDY (SEQ ID NO: 386); and wherein: X5is I or L, X24is A, C, F, I, L, N, S, T, V, W, or Y; X25is F, S, W, or Y; X26is A, G, I, R, or V; X27 is A, M, R, S, or W; X28 is D, F, S, or T; X29 is V or L; X30 is F, H, I, or L; X31 is A, D, G, H, I, K, L, N, Q, R, S, T, V, or Y; X32is A, D, or G; X33is D, E, I, K, or R; X34is F, H, L, S, W, or Y; X35 is F, H, I, L, or W; X36 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W; X37 is D, P, or V; X38is A, E, or V; X39is P or R; X40is E or Y; X41is F, S, or Y; X42is F, I, L, W, or Y; X43 is A, G, H, K, L, M, P, R, S, V, or W; X44 is P, V, or Y; X45 is D, E, H, P, or V; X46 is A, E, G, or P; X47is A, E, G, R, or S; X48is D, E, P, or V; X49is F, H, I, or L; X50is I, L, N, or V; X51 is A, I, K, M, P, S, T, or V; X52 is E, N, P, R, T, or V; X53 is G or P; X54 is A, G, H, T, or Y; X55 is E, G, L, R, or Y; X56 is D, E, or P; X57 is S or Y; X58 is D or Y; X59 is S or Y; X60 is F, I, L, or W; X61 is A, E, F, G, K, L, Q, S, T, or V; X62 is A, D, E, K, M, P, S, T, or V; X63 is P or V; X64 is A, D, E, F, G, I, N, P, R, or V; X65 is A, D, E, G, I, L, N, P, Q, R, S, T, V, W, or Y; X66 is A, D, E, G, R, S, T, V, or Y; X67is A, D, E, G, P, or V; X68is L, S, or Y; X69is H, K, L, Q, R, S, or Y; X70 is F, I, L, Q, or W; X71 is G, K, L, P, Q, R, S, or V; X72 is D, I, S, or V; X73 is D, E, N, P, or V; X74is A, D, E, G, P, or V; X75is A, E, R, T, or V; X76is P, R, or V; X77is A, D, E, K, N, P, S, T, or V; X78 is E, G, Q, R, S, V, or Y; X79 is D, E, P, S, or T; X80 is R or Y; X81 is H, R, S, or W; X82is F, I, or L; X83is I, N, R, V, or W; X84is P, R, S, or V; X85is A, D, E, I, R, V, or Y; X86 is D, G, N, P, Q, or V; X87 is A, D, E, G, L, R, or V; X88 is R or V; X89 is D, G, or P; X90is A, D, E, G, H, L, M, P, Q, R, S, T, V, or Y; X91is A, D, E, G, L, R, S, V, or Y; X92is D, E, F, L, P, Q, or V; and X93 is E, I, M, R, T, V, or Y.

16. The CD8-binding polypeptide of any one of claims 1-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352 and 394-493, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

17. The CD8-binding polypeptide of any one of claims 1-11, wherein the VHH comprises a sequence selected from the group consisting of SEQ ID NOs: 333-352, or a sequence 137 321490133Attorney Docket No. OTPC-050 / 02WO having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

18. The CD8-binding polypeptide of any one of claims 1-17, wherein the VHH specifically binds to CD8α.

19. The CD8-binding polypeptide of any one of claims 1-18, wherein the VHH specifically binds to human CD8α.

20. The CD8-binding polypeptide of any one of claims 1-19, wherein the VHH comprises one or more characteristics relative to a VHH set forth in SEQ ID NO: 314, wherein the one or more characteristics is selected from the group consisting of: (i) an increased binding affinity (KD) to human CD8α; (ii) a decreased off-rate (koff) for binding to human CD8α; (iii) an increased melting temperature (Tm); (iv) an increased expression yield; and (v) an increased purity following a single-step purification.

21. The CD8-binding polypeptide of any one of claims 1-20, wherein the polypeptide selectively targets a CD8-expressing (CD8+) cell.

22. The CD8-binding polypeptide of claim 21, wherein the CD8+ cell is a T cell.

23. The CD8-binding polypeptide of any one of claims 1-22, wherein the polypeptide is operably linked to one or more of a second polypeptide, a nucleic acid, an inorganic molecule, an organic molecule, and another moiety.

24. The CD8-binding polypeptide of any one of claims 1-22, wherein the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising a cytokine.

25. The CD8-binding polypeptide of claim 24, wherein the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), 138 321490133Attorney Docket No. OTPC-050 / 02WO interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

26. The CD8-binding polypeptide of any one of claims 23-24, wherein the polypeptide is operably linked to a second polypeptide comprising a sequence isolated or derived from a sequence encoding or comprising an interferon.

27. The CD8-binding polypeptide of claim 26, wherein the interferon comprises one or more of an interferon type I, an interferon type II and an interferon type III.

28. The CD8-binding polypeptide of claim 26, wherein the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN- ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN- γ).

29. The CD8-binding polypeptide of any one of claims 23-24, wherein the polypeptide is operably linked to a second polypeptide comprising a designed cytokine.

30. A CD8-targeted conjugate, comprising: the CD8-binding polypeptide of any one of claims 1-22 and at least one second moiety, wherein the CD8-binding polypeptide is operably linked to the second moiety. 139 321490133Attorney Docket No. OTPC-050 / 02WO 31. The CD8-targeted conjugate of claim 30, wherein the CD8-binding polypeptide is operably linked to the second moiety by a linker.

32. The CD8-targeted conjugate of claim 31, wherein the linker comprises one or more of an amino acid sequence, a nucleic acid sequence, an amino acid sequence, a small molecule, and any combination thereof.

33. The CD8-targeted conjugate of any one of claims 30-32, wherein the at least one second moiety comprises a detection label, a cytotoxic agent, an antigen-targeting domain, a signaling domain, and / or a second polypeptide.

34. The CD8-targeted conjugate of claim 33, comprising the detection label.

35. The CD8-targeted conjugate of claim 33 or 34, wherein the detection label comprises a radioactive isotope, an affinity tag, or a fluorescent tag.

36. The CD8-targeted conjugate of claims 33-35, comprising the cytotoxic agent, wherein the cytotoxic agent comprises a chemotherapeutic drug or a toxin.

37. The CD8-targeted conjugate of any one of claims 33-36, comprising the antigen- targeting domain.

38. The CD8-targeted conjugate of claim 37, wherein the antigen-targeting domain specifically binds to an antigen expressed by an immune cell.

39. The CD8-binding polypeptide of claim 37, wherein the antigen-targeting domain specifically binds to a tumor-associated antigen.

40. The CD8-targeted conjugate of any one of claims 33-39, wherein the CD8-targeted conjugate is a multi-specific antibody.

41. The CD8-targeted conjugate of any one of claims 33-40, wherein the CD8-targeted conjugate is an antibody drug conjugate. 140 321490133Attorney Docket No. OTPC-050 / 02WO 42. The CD8-targeted conjugate of any one of claims 33-41, comprising the second polypeptide.

43. The CD8-targeted conjugate of claim 42, wherein the second polypeptide comprises a cytokine, an interferon, a chemokine, a growth factor, and / or a tumor necrosis factor.

44. The CD8-targeted conjugate of claim 43, wherein the cytokine comprises one or more of an interleukin 1 polypeptide (IL-1), interleukin 2 polypeptide (IL-2), interleukin 3 polypeptide (IL-3), interleukin 4 polypeptide (IL-4), interleukin 5 polypeptide (IL-5), interleukin 6 polypeptide (IL-6), interleukin 7 polypeptide (IL-7), interleukin 8 polypeptide (IL-8), interleukin 9 polypeptide (IL-9), interleukin 10 polypeptide (IL-10), interleukin 11 polypeptide (IL-11), interleukin 12 polypeptide (IL-12), interleukin 13 polypeptide (IL-13), interleukin 14 polypeptide (IL-14), interleukin 15 polypeptide (IL-15), interleukin 16 polypeptide (IL-16), interleukin 17 polypeptide (IL-17), interleukin 18 polypeptide (IL-18), interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

45. The CD8-targeted conjugate of claim 43, wherein the interferon comprises one or more of an interferon type I, an interferon type II and an interferon type III.

46. The CD8-targeted conjugate of claim 43, wherein the interferon comprises one or more of an interferon alpha (IFN-α), an interferon beta (IFN-β), and interferon epsilon (IFN- ε), an interferon kappa (IFN-κ), an interferon omega (IFN-ω), and an interferon gamma (IFN- γ).

47. The CD8-targeted conjugate of claim 43, wherein the second polypeptide comprises a designed cytokine. 141 321490133Attorney Docket No. OTPC-050 / 02WO 48. The CD8-targeted conjugate of claim 47, wherein the designed cytokine comprises a sequence set forth in any one of SEQ ID NOs: 69-300, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

49. A chimeric antigen receptor comprising the CD8-binding polypeptide of any one of claims 1-22.

50. A composition comprising the CD8-binding polypeptide of any one of claims 1-22 and a delivery vector, wherein the CD8-binding polypeptide is operably linked to the delivery vector.

51. The composition of claim 50, wherein the delivery vector comprises a viral particle, a virus-like particle, a synthetic particle, a liposome, or a lipid nanoparticle.

52. A nucleic acid sequence encoding the CD8-binding polypeptide of any one of claims 1-22.

53. The nucleic acid sequence of claim 52, comprising a regulatory sequence capable of driving expression of the nucleic acid sequence in a mammalian cell.

54. The nucleic acid sequence of claim 53, wherein the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof.

55. The nucleic acid sequence of claim 53 or 54, wherein the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem.

56. The nucleic acid sequence of any one of claims 53-55, wherein the regulatory sequence comprises a constitutive promoter.

57. The nucleic acid sequence of any one of claims 53-55, wherein the regulatory sequence comprises an inducible promoter. 142 321490133Attorney Docket No. OTPC-050 / 02WO 58. The nucleic acid sequence of any one of claims 53-57, wherein the regulatory sequence comprises a minimal promoter.

59. The nucleic acid sequence of any one of claims 53-58, wherein the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs.

60. The nucleic acid sequence of any one of claims 53-59, wherein the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

61. A vector comprising the nucleic acid sequence of any one of claims 52-60.

62. The vector of 61, wherein the vector is an expression vector.

63. The vector of 61, wherein the vector is a delivery vector.

64. The vector of any one of claims 61-63, wherein the vector is a non-viral vector.

65. The vector of any one of claims 61-63, wherein the vector is a viral vector.

66. A cell comprising one or more of: (i) the CD8-binding polypeptide of any one of claims 1-29; (ii) the CD8-targeted conjugate of any one of claims 30-48; (iii) the chimeric antigen receptor of claim 49; (iv) the composition of claim 50 or 51; (v) the nucleic acid of any one claims 52-60; and (vi) the vector of any one of any one of claims 61-65.

67. The cell of claim 66, wherein the cell is an immune cell.

68. The cell of claim 66, wherein the cell is a stem cell or a T-cell precursor cell.

69. The cell of claim 66, wherein the cell is an embryonic stem cell, a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell. 143 321490133Attorney Docket No. OTPC-050 / 02WO 70. The cell of claim 66, wherein the cell is a somatic cell or a differentiated cell.

71. The cell of claim 66, wherein the cell is a T cell, a Natural Killer (NK) cell, or a macrophage.

72. The cell of any one of claims 66-71, wherein the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood.

73. The cell of any one of claims 66-72, wherein the cell has been cryopreserved.

74. The cell of any one of claims 66-73, wherein the cell has been modified to become an allogeneic cell.

75. The cell of any one of claims 66-74, wherein the cell has been modified to produce an allogeneic cell.

76. The cell of any one of claims 66-75, wherein the cell is capable of expressing or is modified to express an exogenous protein.

77. The cell of claim 76, wherein the exogenous protein comprises an antigen receptor.

78. The cell of claim 77, wherein the antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR).

79. The cell of any one of claims 66-78, wherein the cell is capable of expressing or modified to express a safety switch.

80. The cell of any one of claims 66-78, wherein the cell is capable of expressing or modified to express a cell marker.

81. The cell of any one of claims 66-80, wherein the cell is capable of expressing or modified to express a truncated EGFR (EGFRt). 144 321490133Attorney Docket No. OTPC-050 / 02WO 82. The cell of claim 81, wherein the EGFRt comprises a sequence of SEQ ID NO: 53, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

83. The cell of claim 81 or 82, wherein the cell is capable of expressing or modified to express a truncated and optimized EGFR (EGFRopt).

84. The cell of claim 83, wherein the EGFRopt sequence comprises a sequence of one or more of SEQ ID NOs: 54-68, or a sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

85. A composition, comprising: (i) the CD8-binding polypeptide of any one of claims 1-29; (ii) the CD8-targeted conjugate of any one of claims 30-48; (iii) the chimeric antigen receptor of claim 49; (iv) the nucleic acid sequence of any one claims 52-60; (v) the vector of any one of claims 61-65; or (vi) the cell of any one of claims 66-84.

86. A pharmaceutical composition comprising: (i) the CD8-binding polypeptide of any one of claims 1-29; the CD8-targeted conjugate of any one of claims 30-48; the chimeric antigen receptor of claim 49; the composition of claim 50 or 51; the nucleic acid sequence of any one of claims 52-60; the vector of any one of claims 61-65; or the cell of any one of claims 66-84, and (ii) a pharmaceutically acceptable carrier.

87. The pharmaceutical composition of claim 86, wherein the pharmaceutical composition is formulated for systemic administration.

88. The pharmaceutical composition of claim 87, wherein the systemic administration comprises an intravenous injection or an intravenous infusion. 145 321490133Attorney Docket No. OTPC-050 / 02WO 89. The pharmaceutical composition of claim 86, wherein the pharmaceutical composition is formulated for local administration.

90. The pharmaceutical composition of claim 89, wherein the local administration comprises a subcutaneous injection or infusion, an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

91. Use of one or more of the CD8-binding polypeptide of any one of claims 1-29, the CD8-targeted conjugate of any one of claims 30-48, the chimeric antigen receptor of claim 49, the nucleic acid sequence of any one claims 52-60, the vector of any one of claims 61-65, the cell of any one of claims 66-84, the composition of any one of claims 50-51, and 85, or the pharmaceutical composition of any one of claims 86-90, in the manufacture of a medicament for treating a disease or disorder.

92. Use of one or more of the CD8-binding polypeptide of any one of claims 1-29, the CD8-targeted conjugate of any one of claims 30-48, the chimeric antigen receptor of claim 49, the nucleic acid sequence of any one claims 52-60, the vector of any one of claims 61-65, the cell of any one of claims 66-84, the composition of any one of claims 50-51, and 85, or the pharmaceutical composition of any one of claims 86-90 for treating a disease or disorder.

93. The use of claim 91 or 92, wherein the disease or disorder comprises a proliferative disorder.

94. The use of claim 93, wherein the proliferative disorder is a metastatic disorder.

95. The use of claim 94, wherein the metastatic disorder comprises a solid tumor.

96. A method of treating a disease or disorder comprising administering an effective amount of one or more of the CD8-binding polypeptide of any one of claims 1-29, the CD8- targeted conjugate of any one of claims 30-48, the chimeric antigen receptor of claim 49, the nucleic acid sequence of any one claims 52-60, the vector of any one of any one of claims 61- 65, the cell of any one of claims 66-84, the composition of claim 50-51, and 85, or the 146 321490133Attorney Docket No. OTPC-050 / 02WO pharmaceutical composition of any one of claims 86-90, to a subject in need thereof, thereby treating the disease or disorder.

97. The method of claim 96, wherein the subject is at risk of developing the disease or disorder.

98. The method of claim 96, wherein the subject has been diagnosed with the disease or disorder.

99. The method of any one of claims 96-98, wherein the disease or disorder comprises a proliferative disorder.

100. The method of claim 99, wherein the proliferative disorder is a metastatic disorder.

101. The method of claim 100, wherein the metastatic disorder comprises a solid tumor.

102. The method of any one of claims 96-101, wherein treating the disease or disorder results in one or more of: (a) decreasing the severity of a sign or symptom of the disease or disorder; (b) improving a function negatively affected by the disease or disorder; (c) improving the subject’s prognosis; (d) increasing the subject’s duration of progression-free survival (PFS); (e) reducing a quantity of cells affected by the disease or disorder; (f) inducing a remission form the disease or disorder; (g) delaying or preventing a relapse of the disease or disorder; (h) delaying or preventing a second presentation of the disease or disorder; and / or (i) increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder. 147 321490133

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