PD-1 single domain antibodies and therapeutic compositions thereof

Abstract

Provided herein are binding polypeptides that specifically bind PD-1. More specifically, provided herein are fusion proteins that bind PD-1, including multivalent and / or multispecific constructs and chimeric antigen receptors. Also provided are pharmaceutical compositions containing the polypeptides, nucleic acid molecules encoding the polypeptides and vectors and cells thereof, and methods of use and uses of the provided PD-1 binding polypeptides for the treatment of diseases and disorders, such as cancer.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 744,615, filed October 11, 2018, and U.S. Provisional Application No. 62 / 791,152, filed January 11, 2019, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure generally provides binding polypeptides that specifically bind PD-1. More specifically, the present disclosure relates to fusion proteins that bind at least PD-1, including multivalent and / or multispecific constructs and chimeric molecules. The present disclosure also provides nucleic acid molecules encoding the polypeptides, vectors and cells thereof, and methods of use and uses of the PD-1 binding polypeptides for treating diseases and disorders, such as cancer. BACKGROUND

[0004] PD-1 is a member of the immunoglobulin superfamily of immune cell regulatory molecules. It is expressed on the surface of activated T-cells. Expression of PD-1 on activated T-cells is targeted by tumor cells and stromal cells in the tumor microenvironment through PD-L1 to suppress the immune response, making agents that block or target PD-1 desirable therapeutic targets. There is a need for improved therapeutic molecules and agents that target PD-1. Provided herein are embodiments that meet such needs. BRIEF DESCRIPTION OF DRAWINGS

[0005] Figures 1A-1G FIG. 1 depicts the binding of 18H10 and its humanized variants hz18H10 or 1-14 to FreeStyle 293 cells expressing human PD-1 (A), cynomolgus monkey PD-1 (B), or mouse PD-1 (C). Binding to untransfected (293) cells is also assessed and shown. Figure 1A , 1D , 1E, 1F, 1G), cynomolgus monkey PD-1 (B), or mouse PD-1 (C). Binding to untransfected (293) cells is also assessed and shown. Figure 1B Figure 1C FIG. 2 depicts the binding of 18H10 and its humanized variant hz18H10v7 to activated human T-cells. Test articles were quantified by flow cytometry for binding to activated T-cells.

[0006] Figure 2 FIG. 3 depicts the ability of 18H10, its humanized variant hz18H10v7, or 1-14 to block the inhibition of T-cell receptor (TCR) signaling mediated by PD1 / PDL1 in a Jurkat reporter luciferase assay system.

[0007] FIG. 3 depicts the ability of 18H10, its humanized variant hz18H10v7, or 1-14 to block the inhibition of T-cell receptor (TCR) signaling mediated by PD1 / PDL1 in a Jurkat reporter luciferase assay system. Figure 3A FIG. 4 depicts the PD1 blockade of 18H10 and hz18H10v7, while​Figure 3B PD1 blockade of 1-14 is depicted. DETAILED DESCRIPTION

[0008] Provided herein are polypeptides that specifically bind PD-1, also referred to herein as PD-1-binding polypeptides. In certain embodiments, the provided binding polypeptides comprise at least one VHH domain that binds PD-1. In certain embodiments, the PD-1-binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that each individually bind PD-1. In certain embodiments, the PD-1-binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind PD-1. In certain embodiments, the PD-1-binding polypeptides are monospecific. In certain embodiments, the PD-1-binding polypeptides are multispecific. For example, the provided PD-1-binding polypeptides include polypeptides that bind one or more target proteins other than PD-1, which can comprise at least one VHH domain that binds PD-1 and one or more additional binding domains, such as one or more additional VHH domains.

[0009] In certain embodiments, the PD-1-binding polypeptides comprise at least one VHH domain that binds PD-1 and an Fc domain. In certain embodiments, the PD-1-binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind PD-1 and an Fc domain. In certain embodiments, the Fc domain mediates dimerization of the PD-1-binding polypeptide under physiological conditions, such that a dimer is formed that doubles the number of PD-1 binding sites. For example, a PD-1-binding polypeptide comprising three VHH domains that bind PD-1 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, such that the PD-1-binding polypeptide exists as a hexavalent dimer under such conditions.

[0010] Programmed cell death protein-1 (PD-1) is a type I membrane protein and is a member of the extended CD28 / CTLA-4 family of T cell regulators (The EMBO Journal (1992), vol. 11, no. 11, pp. 3887-3895). Expression of PD-1 has been observed on the surface of myeloid cells, including T cells or B lymphocytes activated by stimulation from an antigen receptor, or activated macrophages (International Immunology (1996), vol. 18, no. 5, pp. 765-772). It has also been demonstrated that stimulation by IFN-γ upregulates the expression of PD-1 on the cell surface. Two cell surface glycoprotein ligands for PD-1, PD-1 and PDL-2, have been identified and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Latchman et al. (2001) Nat. Immunol. 2:261-8; Carter et al. (2002) Eur. J. Immunol. 32:634-43; Ohigashi et al. (2005) Clin. Cancer Res. 11:2947-53). Both PD-1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1. PD-L1 and PD-L2 are normally expressed on the surface of T cells, B cells, and myeloid cells. PD-L1 and PD-L2 are negative regulators of immune activation and can downregulate the immune response through interaction with the PD-1 receptor. In some aspects, PD-1 is expressed on NK cells and T cells, including CD4+ and CD8+ T cells, whereby binding of PD-1 can inhibit activation cell activation, proliferation, and / or multiplication.

[0011] An exemplary sequence for human PD-1 is shown below:

[0012] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 286, signal sequence underlined)

[0013] In certain instances, the provided PD-1 binding polypeptides directly block or inhibit the interaction between PD-L1 / L2 and PD-1. In certain embodiments, the provided molecules that inhibit or reduce the interaction between PD-L1 and / or PD-L2 and PD-1 modulate an immune response. While the transmission of inhibitory signals can result in the downregulation of immune cell responses (and a corresponding downregulation of the overall immune response), blocking inhibitory signals in immune cells results in the upregulation of immune cell responses (and a corresponding upregulation of the immune response). In certain instances, the modulation achieved by enhancing the immune response can be used to treat certain diseases or conditions in which the immune response is inhibited, such as cancer. In certain embodiments, the provided PD-1 binding polypeptides can be used as a therapeutic agent to inhibit or reduce tumor cell growth or survival.

[0014] A variety of PD-1 polypeptide binding formats are provided. In certain embodiments, the PD-1 polypeptides provided herein are bivalent, such as by fusion to an Fc protein. In certain embodiments, the PD-1 binding polypeptides include a PD-1 VHH-Fc polypeptide. In certain embodiments, the Fc is an Fc that exhibits immune effector activity, such as one or more effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In other embodiments, the PD-1 polypeptides can be multispecific polypeptides containing at least one additional molecule. In certain embodiments, the additional molecule is capable of binding to another molecule in the tumor-associated microenvironment, such as a tumor-associated antigen or an immune cell, e.g., a T cell. In particular embodiments, the provided PD-1 polypeptide binding formats block the interaction between PD-1 and PD-L1 and / or PD-L1 and / or reduce, inhibit, or suppress the inhibitory signal mediated by PD-1 in a cell, such as a T cell.

[0015] In certain embodiments, the provided PD-1-binding polypeptides can be used to stimulate an immune response in a subject, which in certain aspects, treats a disease or disorder, such as a cancer, in the subject. In certain aspects, the PD-1-binding polypeptides provided herein, such as PD-1-Fc, can bind to cells expressing PD-1 and block their interaction with PD-L1 and / or PD-L2 on a neighboring cell in an immune synapse, e.g., a tumor cell, which in certain aspects, can induce an active immune response in the environment. In certain cases, the active immune response can inhibit the growth of cancer cells (e.g., block cell cycle progression).

[0016] In other aspects, VHH-binding polypeptides that exhibit multispecific binding are also provided herein. In certain cases, the binding polypeptides include polypeptides that exhibit dual affinity for PD-1 and a tumor associated antigen (TAA). Alternatively or additionally, the PD-1 binding polypeptides include polypeptides that exhibit affinity for PD-1 and a T cell antigen, such as CD3. In certain aspects, such multispecific molecules are capable of binding or activating T cells at a tumor site upon binding to the tumor or T cell, and simultaneously blocking the interaction of PD-1 and PD-L1 / PD-L2 to reduce inhibitory signals in the T cells. In particular, such molecules provided herein include molecules that exhibit restricted CD3 binding. Engineered cells, such as engineered T cells, that express a chimeric antigen receptor and are capable of secreting a PD-1 binding polypeptide are also provided herein.

[0017] All publications, including patents, scientific articles and database, mentioned in this application are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. In the event that the definition or use of a term in the present document is contrary to the definition of that term provided in a patent, application, published application, or other publication that is herein incorporated by reference, the definition or use of the term in this document controls.

[0018] Methods in Enzymology (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., 2002); the relevant chapters in the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); 60 (1983) and 154 (1987) and 101-121 (1993); PCR: THE POLYMERASE CHAIN REACTION, H. A. Erlich, ed. (1992); BIOLOGICAL SAMPLE PREPARATION: A COMPREHENSIVE TREATISE (1992); Applied Biomolecular Engineering (1993) 4: 249-307; and Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press (1986).Calos, eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993); and updated versions thereof.

[0019] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0020] I. Definition

[0021] Unless otherwise defined, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires or clearly indicates, singular terms shall include the plural, and plural terms shall include the singular. For any conflict in definitions between various sources or references, the definitions provided herein shall prevail.

[0022] It is understood that embodiments of the application described herein include "consisting" and / or "consisting essentially of embodiments. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The use of the term "or" in the context of "and / or" herein is not intended to mean an exclusive alternative, unless otherwise indicated or understood by those skilled in the art.

[0023] In this application, the use of "or" means "and / or" unless clearly indicated otherwise or understood by those skilled in the art to be exclusive in the context. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim.

[0024] The term "about" as used herein means the usual error range for values in the technical field of those skilled in the art. Reference to "about" herein includes (and describes) embodiments that are directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0025] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and mean a polymer of nucleotides. Such polymers of nucleotides can contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" means the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0026] The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, due to its origin (1) is not associated with all or a portion of the polynucleotide as it exists in nature, (2) is operably linked to a polynucleotide with which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0027] The terms "polypeptide" and "protein" are used interchangeably to mean a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural and / or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for purposes of the present disclosure, "polypeptide" means such proteins that include modifications of the native sequence, such as deletions, additions and substitutions (generally conservative in nature), as long as the protein maintains its desired activity. These modifications can be intentional, as by site-directed mutagenesis, or can be accidental, such as by mutation of the host organism producing the protein or by errors in PCR amplification.

[0028] The term "isolated protein" as used herein means that the subject protein is (1) free of at least some of the other proteins with which it is associated in nature, (2) substantially free of other proteins from the same source, e.g., from the same species, (3) expressed by a cell from a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) not associated (by covalent or noncovalent interaction) with a portion of a protein with which it is associated in nature, (6) operatively associated with a polypeptide with which it is not associated in nature (by covalent or noncovalent interaction), or (7) does not exist in nature. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can have synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially pure or substantially free of proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutically, diagnostically, prophylactically, research, or otherwise).

[0029] "Substantially pure" as used herein means that the subject material is the predominant material present (i.e., it is the most abundant, on a molar basis, in the composition) and that the substantially purified fraction is a composition in which the subject material comprises at least about 50% of all macromolecular species present (on a molar basis). Typically, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, e.g., in certain embodiments, more than about 85%, 90%, 95%, and 99%. In certain embodiments, the subject material is purified to homogeneity (contaminant materials are not detectable in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0030] The term "operably linked" as used herein means that the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0031] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods of determining such specific binding are well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds more often, more rapidly, with greater duration and / or with greater affinity to a particular cell or substance as compared to alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds other substances. For example, a sdAb or VHH-containing polypeptide that specifically or preferentially binds a PD-1 epitope is a sdAb or VHH-containing polypeptide that binds that epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds other PD-1 epitopes or non-PD-1 epitopes. It is also understood by reading this definition that, for example, a sdAb or VHH-containing polypeptide that specifically or preferentially binds a first target can or can not specifically or preferentially bind a second target. In this way, "specific binding" or "preferential binding" does not necessarily require, although it can include, exclusive binding. Often, but not necessarily, reference to binding refers to preferential binding. "Specific" denotes the ability of a binding protein to selectively bind an antigen.

[0032] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or a polypeptide containing VHH) binds. An epitope often includes chemically active surface groups, polypeptides, or sugar side chains of molecules (such as amino acids) and has specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be formed by continuous and / or adjacent non-continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed by continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. An epitope may include, but is not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In certain embodiments, the length of an epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In certain embodiments, by a certain minimum distance with the CDR residues on the antigen binding molecules, epi-position can be identified. In certain embodiments, epi-position can be identified by above-mentioned distance, and further limited to those residues involved in the bond (such as hydrogen bond) between the residue of antigen binding molecules and the antigen residue. Epi-position can also be identified by various scannings, such as alanine or arginine scanning can indicate that antigen binding molecules can interact with one or more residues. Unless clearly stated, as one group of residues of epi-position do not exclude other residues from becoming a part for the epi-position of specific antigen binding molecules. On the contrary, the existence of such a group represents the minimum series (or set of material) of epi-position. Thus, in certain embodiments, one group of residues accredited as epi-position specifies the minimum epi-position relevant to antigen, rather than the exclusive list of the residue about the epi-position on antigen.

[0033] A "non-linear epitope" or "conformational epitope" comprises a non-contiguous polypeptide, amino acid, and / or carbohydrate within an antigenic protein to which an antigen binding molecule specific for the epitope binds. In certain embodiments, at least one residue will be discontinuous with other residues of the epitope; however, one or more residues may also be continuous with other residues.

[0034] A "linear epitope" is comprised of a continuous polypeptide, amino acid, and / or sugar within an antigenic protein to which an antigen binding molecule specific for the epitope binds. It should be noted that in certain embodiments, not every residue within a linear epitope needs to be directly bound (or participate in a bond) by an antigen binding molecule. In certain embodiments, a linear epitope can be derived from immunization with a peptide effectively consisting of the sequence of the linear epitope, or from a structural portion of a protein that is relatively isolated from the rest of the protein (such that the antigen binding molecule can interact, at least primarily), such as with that sequence portion.

[0035] The terms "antibody" and "antigen binding molecule" are used interchangeably in the broadest sense and encompass various polypeptides comprising antibody-like antigen binding domains, including, but not limited to, conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, comprising only one chain, which typically resembles a heavy chain), VHH-containing polypeptides (polypeptides comprising at least one heavy chain-only antibody variable domain or VHH), and fragments of any of the foregoing, so long as they exhibit the desired antigen binding activity. In certain embodiments, an antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, heavy chain constant domains (comprising CH1, hinge, CH2, and CH3, where CH1 typically pairs with a light chain constant domain CL, and the hinge mediates dimerization) and Fc domains (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0036] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, and the like.

[0037] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy chain and light chain (V H and V L , respectively) of a native antibody generally have similar structures, each containing four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91 (2007). A single V H or V L domain can be sufficient to confer antigen-binding specificity, e.g., single domain antibodies, such as VHHs. Furthermore, antibodies that bind a particular antigen can be isolated using a V H or V L domain from an antibody that binds the antigen, to screen libraries of complementary V L or V H domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0038] "Antibody fragment" or "antigen binding fragment" means a molecule other than a conventional or intact antibody that comprises a portion of a conventional or intact antibody that contains at least the variable region that binds an antigen. Examples of antibody fragments include, but are not limited to: Fv, single chain Fv (scFv), Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-domain antibodies comprising only V H single domain antibodies of the region (VHH).

[0039] As used herein, "monovalent" with respect to a binding molecule means a binding molecule having a single antigen recognition site specific for a target antigen. Examples of monovalent binding molecules include, for example, monovalent antibody fragments, proteinaceous binding molecules having antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single chain Fv fragments (scFv).

[0040] The terms "single domain antibody," "sdAb," "VHH" are used interchangeably herein to mean an antibody having a single monomeric domain antigen binding / recognition domain. Such antibodies include camelid antibodies or shark antibodies. In certain embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In certain embodiments, a VHH can be truncated at the N- or C-terminus such that it contains only a portion of FR1 and / or FR4, or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0041] The term "VHH-containing polypeptide" denotes a polypeptide comprising at least one VHH domain. In certain embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain can be the same or different. In certain embodiments, the VHH-containing polypeptide comprises an Fc domain. In certain such embodiments, the VHH polypeptide can form a dimer. Non-limiting structures of VHH-containing polypeptides include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 can be the same or different. In some embodiments of such structures, one VHH can be connected to another VHH by a linker, or one VHH can be connected to an Fc by a linker. In certain such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In certain embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, if the structure VHH1-VHH2-Fc forms a dimer, it is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered to be hexavalent (i.e., the dimer has six VHH domains).

[0042] A PD-1-binding polypeptide, as used herein, is a polypeptide or protein that specifically binds PD-1. Typically, a PD-1-binding polypeptide herein is a VHH-containing polypeptide that contains at least one VHH domain that binds PD-1. PD-1-binding polypeptides include conjugates, including fusion proteins. PD-1-binding polypeptides include fusion proteins, including fusion proteins containing an Fc domain. In certain embodiments, a PD-1-binding polypeptide contains two, three, or four or more VHH domains that each specifically bind PD-1, where each VHH domain can be the same or different. In certain embodiments, a PD-1-binding polypeptide is multivalent. In certain embodiments, a PD-1-binding polypeptide is multispecific. In certain cases, a PD-1-binding polypeptide can contain one or more additional domains that bind one or more other or additional antigens other than PD-1.

[0043] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH-containing polypeptide), including an antibody of a population of substantially homogeneous antibody, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, each monoclonal antibody can bind the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be made by recombinant DNA methods, such as described in U.S. Patent No. 4,816,567. The monoclonal antibodies can also be isolated from a phage library using the techniques described in, for example, McCafferty et al., 1990, Nature 348:552-554.

[0044] The term "CDR" denotes a complementarity determining region as defined by at least one of the identification means of the skilled person. 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 in: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5thEd. 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 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(l):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun 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 Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme).

[0045] The boundaries of a given CDR or FR can vary with the protocol used to define them. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number based on the most common antibody region sequence length, with insertion letters, e.g., “30a,” placed at insertions, and deletions occur in some antibodies. Both schemes place certain insertions and deletions (“insertion-deletions”) in different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures, and is similar in many respects to the Chothia numbering scheme. The AbM scheme is based on a compromise between the Kabat and Chothia definitions used by Oxford Molecular’s AbM antibody modeling software.

[0046] In certain embodiments, CDRs can be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs, referred to as CDR1, CDR2, and CDR3, respectively. Table 1 below lists exemplary position boundaries for CDR-H1, CDR-H2, CDR-H3, identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FR-H1 precedes CDR-H1, FR-H2 is between CDR-H1 and CDR-H2, FR-H3 is between CDR-H2 and CDR-H3, and so on. It is noted that since the illustrated Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop varies between H32 and H34 when numbered using the illustrated Kabat numbering convention, depending on the length of the loop.

[0047]

[0048]

[0049] 1-Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD

[0050] 2-Al-Lazikani et al., (1997) JMB 273, 927-948

[0051] Thus, unless otherwise indicated, a "CDR" or "complementarity determining region" or an individual indicated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (such as a variable region thereof) is understood to encompass the (or a) complementarity determining region defined by any of the foregoing schemes. For example, when it is stated that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in a given VHH amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the VHH as defined by any of the foregoing schemes. In certain embodiments, a particular CDR sequence is specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Table 1), although it is understood that the provided antibodies can include CDRs described according to any of the other foregoing numbering schemes or other numbering schemes known to the skilled artisan.

[0052] "conjugate," "conjugation," or grammatical variants thereof as used herein means the joining or linking together of two or more compounds by any joining or linking method known in the art resulting in the formation of another compound. It can also mean a compound produced by joining or linking two or more compounds together. For example, a VHH domain directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by any other method.

[0053] An immunoglobulin Fc fusion ("Fc-fusion"), such as a VHH-Fc, is a molecule comprising one or more VHH domains operably linked to an Fc region of an immunoglobulin. The immunoglobulin Fc region can be linked to the one or more VHH domains indirectly or directly. Various linkers are known in the art and can optionally be used to link the Fc to the fusion partner to produce the Fc-fusion. In certain such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. Fc-fusions of the same species can be dimerized to form Fc-fusion homodimers, or different species are used to form Fc-fusion heterodimers. In certain embodiments, the Fc is a mammalian Fc, such as a human Fc.

[0054] The term "heavy chain constant region" as used herein means a polypeptide comprising at least three heavy chain constant domains, C H 1, hinge, C H 2, and C Hregion of SEQ ID NO: 3. Of course, deletions and alterations within the domain that do not alter functionality are encompassed within the scope of the term "heavy chain constant region" unless otherwise indicated. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a gamma 1 constant region), IgG2 (comprising a gamma 2 constant region), IgG3 (comprising a gamma 3 constant region), and IgG4 (comprising a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an alpha 1 constant region) and IgA2 (comprising an alpha 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[0055] "Fc region" as used herein means a portion of a heavy chain constant region comprising CH2 and CH3. In certain embodiments, an Fc region comprises hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. An Fc region can be of any of the antibody heavy chain constant region isotypes discussed herein. In certain embodiments, the Fc region is IgGl, IgG2, IgG3, or IgG4.

[0056] A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of B-cell receptor (BCR) expression; and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain), and can be assessed using various assays.

[0057] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0058] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region due to at least one amino acid modification. In certain embodiments, a "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In certain embodiments, a variant Fc region has at least one amino acid substitution, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions, in a native sequence Fc region or Fc region of a parent polypeptide, as compared to the native sequence Fc region or Fc region of the parent polypeptide. In certain embodiments, a variant Fc region herein will have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of the parent polypeptide, at least about 90% sequence identity therewith, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith.

[0059] Generally, the numbering of residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" means the residue numbering of the human IgGl EU antibody.

[0060] "Fc receptor" or "FcR" describes a receptor other than an antibody variable domain that binds to the Fc region of an antibody. In certain embodiments, the FcγR is a native human FcR. In certain embodiments, the FcR is an FcR that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subfamilies, including alternate forms of those receptors created by alternative splicing or alternate promoters. The FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences except in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in, e.g., Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of homeostasis of immunoglobulins in vivo. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0061] "Receptor human framework" as used herein is a heavy chain variable domain (VH) comprising a human immunoglobulin framework or a human consensus framework derived from a human immunoglobulin framework, wherein one or more hypervariable regions (HVRs) are replaced by a HVR from a non-human species. H) framework of the amino acid sequence of the framework, as discussed herein. A recipient human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can contain amino acid sequence changes. In certain embodiments, the number of amino acid changes across all human frameworks in a single antigen binding domain (such as a VHH) is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3.

[0062] A "chimeric antigen receptor" or "CAR" as used herein denotes a receptor engineered to introduce antigen specificity into the cell it is engineered into (e.g., a T cell such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) through an antigen binding domain, thereby coupling the antigen binding properties of the antigen binding domain to the T-cell activity (e.g., lytic capacity and self-renewal) of the T-cell. A CAR typically includes an extracellular antigen binding domain (extracellular domain), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain typically contains at least one IT AM signaling domain, e.g., derived from CD3 zeta, and optionally at least one costimulatory signaling domain, e.g., derived from CD28 or 4-1BB.

[0063] "Affinity" denotes the strength of the sum total of noncovalent interactions between individual binding sites of a molecule (e.g., an antibody or VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y can be represented by the dissociation constant (K D ) or K D-表观 , respectively. Affinity can be measured by common methods known in the art, e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, including those described herein. Such methods include, but are not limited to, methods comprising or flow cytometry.

[0064] The term "K D " as used herein denotes the equilibrium dissociation constant of an antigen binding molecule / antigen interaction. When the term "K D " is used herein, it includes K D and K D-表观 .

[0065] In certain embodiments, the K D: Using cell lines expressing the antigen by flow cytometry, the mean fluorescence measured at each antibody concentration was fitted to a nonlinear single-site binding equation (Prism Software graphpad). In certain such embodiments, K D It's K D-表观 .

[0066] The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand, inducing or increasing cell proliferation (such as T cell proliferation), and inducing or increasing cytokine expression.

[0067] An "affinity matured" VHH-containing polypeptide refers to a VHH-containing polypeptide with one or more alterations in one or more CDRs, such alterations resulting in an improvement in the affinity of the VHH-containing polypeptide for antigen, compared to a parent VHH-containing polypeptide without such alterations.

[0068] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may contain residues that are not found in either the original VHH or human framework sequences, but are included to further refine and optimize the performance of VHHs or VHH-containing polypeptides. In certain embodiments, humanized VHH-containing polypeptides comprise a human Fc region. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily represent the process of generating an antibody.

[0069] As used herein, the term "substantially similar" or "substantially identical" refers to a sufficiently high degree of similarity between two or more values ​​such that, within the range of the biological characteristic measured by the values, a person skilled in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance. In certain embodiments, two or more substantially similar values ​​differ by no more than about any of 5%, 10%, 15%, 20%, 25%, or 50%.

[0070] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative changes as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted from the N- or C-terminus of the polypeptide. In certain embodiments, a variant will have at least about 80% amino acid sequence identity. In certain embodiments, a variant will have at least about 90% amino acid sequence identity. In certain embodiments, a variant will have at least about 95% amino acid sequence identity to a native sequence polypeptide.

[0071] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative changes as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0072] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0073]

[0074]

[0075] Amino acids can be grouped according to common side-chain properties:

[0076] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0077] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0078] (3) acidic: Asp, Glu;

[0079] (4) basic: His, Lys, Arg;

[0080] (5) Residues that affect chain orientation: Gly, Pro;

[0081] (6) Aromatic: Trp, Tyr, Phe.

[0082] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0083] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate expression of a polypeptide of interest (e.g., a promoter and / or enhancer), and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0084] "Host cell" means a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include Crucell and 293 and CHO cells and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include the progeny of a single host cell, and the progeny may not be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations. Host cells include cells transfected in vivo with one or more polynucleotides provided herein.

[0085] The term "isolated" as used herein means a molecule that has been separated from at least some of the components with which it is normally found or produced in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some components of the cell in which it is produced. In the case where a polypeptide is secreted by a cell following expression, physical separation of the supernatant containing the polypeptide from the cells producing the polypeptide is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide (with which it is normally found in nature) (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA), or is separated from at least some components of the cell in which it is produced (e.g., in the case of an RNA polynucleotide). Thus, a DNA polynucleotide contained within a vector in a host cell can be referred to as "isolated".

[0086] The terms "individual" and "subject" are used interchangeably herein to mean an animal; for example, a mammal. The term patient includes both human and veterinary subjects. In certain embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, swine, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. The subject can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In certain embodiments, "individual" or "subject" means an individual or subject in need of treatment for a disease or disorder. In certain embodiments, the subject to be treated can be a patient, which indicates the fact that the subject has been identified as having a disorder that is relevant to treatment, or has a sufficient risk of contracting the disorder. In particular embodiments, the subject is a human, such as a human patient.

[0087] "Disease" or "disorder" as used herein means a condition that requires and / or desires treatment.

[0088] Unless otherwise indicated, the terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein and mean a cell (or cells) that exhibits uncontrolled growth and / or abnormal increased cell survival and / or suppression of apoptosis that interferes with the normal functioning of bodily organs and systems. The definition includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases.

[0089] The terms "cancer" and "tumor" encompass solid and hematologic / lymphoid cancers, and also encompass malignant, pre-malignant, and benign neoplasms, such as dysplasias. Also included within this definition are cells with abnormal proliferation that are not impeded by the immune system (e.g., immune evasion and immune escape mechanisms), such as virus-infected cells. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung tumor, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0090] The term "non-tumor cell" as used herein refers to a normal cell or tissue. Exemplary non-tumor cells include, but are not limited to, T-cells, B-cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, interstitial kidney cells, fibroblast-like synoviocytes, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "peripheral cell or tissue" as used herein refers to a non-tumor cell that is not located in the vicinity of a tumor cell and / or within a tumor microenvironment.

[0091] The term "cell or tissue within the tumor microenvironment" as used herein refers to cells, molecules, extracellular matrix, and / or blood vessels that surround and / or supply tumor cells. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to: tumor vasculature; tumor infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T-cells (Treg cells); macrophages; neutrophils; myeloid-derived suppressor cells (MDSCs), and other immune cells located proximal to the tumor. Methods for identifying tumor cells and / or cells / tissues located within the tumor microenvironment are well known in the art, as described below.

[0092] In certain embodiments, "increasing" or "decreasing" means a statistically significant increase or decrease, respectively. As will be clear to the skilled person, "modulating" can also involve achieving an alteration (which can be an increase or decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for binding into a homo- or hetero-multimeric form, or substrates, compared to the same conditions but in the absence of the test agent; achieving a change (which can be an increase or decrease) in the sensitivity of a target or antigen to one or more conditions in the medium or environment in which the target or antigen is present, such as pH, ionic strength, presence of cofactors, etc.; and / or cell proliferation or cytokine production, compared to the same conditions but in the absence of the test agent. This can be determined in any suitable way and / or using any suitable assay known per se or described herein, depending on the target involved.

[0093] "Immune response" as used herein is intended to encompass a cellular and / or humoral immune response sufficient to inhibit or prevent the onset of a disease (e.g., cancer or cancer metastasis) or ameliorate the symptoms of the disease. "Immune response" can encompass aspects of both the innate and adaptive immune system.

[0094] The term "treatment" of a disease, disorder, or condition as used herein is an approach for obtaining beneficial or desired clinical results. "Treatment" as used herein covers any administration or use of a therapeutic agent to a mammal, including a human, for the purpose of the disease. For the purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., metastasis, such as to the lungs or lymph nodes) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, arresting its development, and remission (whether partial or total), whether irreversible or not. The methods provided herein encompass any one or more of these aspects of treatment. Consistent with the above, the term "treatment" does not require all aspects of a disorder to be removed, 100%.

[0095] The term "treatment" or "inhibition" of a cancer, "inhibition" of a cancer, as used herein in the context of cancer, means at least one of: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a decrease in the size, mass, metabolic activity, or volume of a tumor, as measured by standard criteria (e.g., but not limited to, Response Evaluation Criteria in Solid Tumors (RECIST)), or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).

[0096] "Improvement" means a lessening or amelioration of one or more symptoms as compared to not administering the therapeutic agent. "Improvement" also includes a shortening or reduction in the duration of symptoms.

[0097] "Prevention" or "preventing" of a disease or disorder means administering a pharmaceutical composition, alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of the disease or disorder, or to lessen the likelihood of onset of the disease or disorder.

[0098] The term "inhibit" means a reduction or cessation of any phenotypic characteristic, or a reduction or cessation in the incidence, extent, or likelihood of the characteristic. "Reduce" or "inhibit" is to decrease, reduce, or prevent an activity, function, and / or amount, as compared to a reference. In certain embodiments, "reduce" or "inhibit" means the ability to cause a total reduction of 10% or greater. In certain embodiments, "reduce" or "inhibit" means the ability to cause a total reduction of 50% or greater. In certain embodiments, "reduce" or "inhibit" means the ability to cause a total reduction of 75%, 85%, 90%, 95%, or greater. In certain embodiments, the inhibition or reduction is for the above amounts over a period of time, relative to a control over the same period of time.

[0099] As used herein, "delaying development of a disease" means to defer, hinder, slow, retard, postpone, stabilize, inhibit, and / or delay the development of disease (e.g., cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As will be apparent, a sufficient or significant delay can, in effect, encompass prevention, since the individual does not yet have the disease. For example, the development of late stage cancer, e.g., metastasis, can be delayed.

[0100] As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who can be predisposed to the disease but has not yet been diagnosed as having it. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not denote or require complete prevention of the stated condition at all times (but only during the measured period of time).

[0101] The term "anti-cancer agent" is used herein in its broadest sense to mean an agent used to treat one or more cancers. Illustrative classes of such agents include, but are not limited to, chemotherapeutic agents, anti-cancer biologies (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0102] The term "biological sample" refers to an amount of material obtained from a living or formerly living subject. Such material includes, but is not limited to, blood, (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0103] The term "control" or "reference" denotes a composition known to contain no analyte ("negative control") or to contain an analyte ("positive control"). A positive control can contain a known concentration of an analyte.

[0104] The terms "effective amount" or "therapeutically effective amount" mean the amount and / or concentration of a composition containing an active ingredient (e.g., a sdAb or VHH-containing polypeptide) that, when administered to a patient, either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant decrease in disease progression, e.g., by improving or eliminating symptoms and / or causes of the disease. An effective amount can be an amount that relieves, decreases, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical function in a patient. A therapeutically effective amount of a composition comprising an active agent can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual, among others. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic and / or prophylactic result.

[0105] A composition as used herein means any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0106] The terms "pharmaceutical formulation" and "pharmaceutical composition" mean a preparation that is in a form suitable for administration to a subject to be treated, and which is capable of delivering an effective amount of the active ingredient to the subject. It is a composition that is suitable for pharmaceutical use in a mammalian subject, typically a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., a sdAb or VHH-containing polypeptide) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. Such formulations can be sterile.

[0107] A "pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation, formulation auxiliary, or vehicle that is commonly used in the art for use with therapeutic agents, which together with the therapeutic agent, comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic with the dosages and concentrations employed, and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation employed.

[0108] Administration "in combination" with one or more other therapeutic agents includes simultaneous (parallel) and sequential administration in any order.

[0109] The term "concurrently" is used herein to mean administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where administration of one therapeutic agent is within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of the two agents overlap for at least a period of time.

[0110] The term "sequentially" is used herein to mean administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0111] "Concurrent with" as used herein means administration of one treatment modality in addition to another. Thus, "concurrent with" means administration of one treatment modality before, during, or after administration of the other treatment modality to an individual.

[0112] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, instructions and / or warnings concerning the use of such therapeutic products.

[0113] A "manufactured article" is any article (e.g., package or container) or kit that contains at least one reagent (e.g., a drug for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein). In certain embodiments, the manufactured article or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0114] The terms "label" and "detectable label" refer to moieties that are attached to, for example, an antibody or antigen to make the reaction (e.g., binding) between members of a specific binding pair detectable. The labeled member of a specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" denotes a protein having a label incorporated that provides for the identification of the binding protein. In certain embodiments, the label is a detectable marker that produces a signal that can be detected by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or a polypeptide attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin, which contains a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric measurement methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu,166 Ho or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridinium compounds, and fluorescence-generating moieties, such as fluorescein. In this regard, a moiety itself may not be detectably labeled, but may become detectable upon reaction with another moiety.

[0115] II. VHH Domain Binding to PD-1

[0116] Provided herein are PD-1-binding polypeptides, which are VHH-containing polypeptides containing at least one VHH domain that specifically binds to PD-1. In certain embodiments, the VHH domain binds to human PD-1. In certain embodiments, the VHH domain binds to cynomolgus monkey PD-1. In certain embodiments, the VHH domain binds to mouse PD-1. In certain embodiments, the VHH-containing polypeptides comprise multiple copies of the VHH domains provided herein. In such embodiments, the VHH-containing polypeptides may comprise multiple copies of the same VHH domain. In certain embodiments, the VHH-containing polypeptides may comprise multiple copies of different VHH domains that recognize the same epitope on PD-1. The VHH-containing polypeptides may be formed in a variety of forms, including any of those described in Section III below.

[0117] VHH domain is an antibody fragment, which is a single monomeric variable antibody domain that can selectively bind to a specific antigen. Due to its molecular weight of only 12-15kDa, VHH domain (also known as single domain antibody) is much smaller than ordinary antibodies (150-160kDa) composed of two heavy protein chains and two light chains, and is even smaller than Fab fragments (about 50kDa, one light chain and half a heavy chain) and single-chain variable fragments (about 25kDa, two variable domains, one from a light chain and one from a heavy chain).

[0118] A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, alpaca, dromedary, vicuna, shark, goat, rabbit, and / or bovine. In certain embodiments, the single domain antibodies used herein are naturally occurring single domain antibodies, which are referred to as heavy chain antibodies lacking light chains. For clarity, the variable domain derived from a heavy chain antibody naturally lacking light chains is referred to herein as a VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as in camels, llamas, dromedaries, alpacas, guanacos, and vicunas. Other species than Camelidae can produce heavy chain antibodies naturally lacking light chains; such VHHs are within the scope of the present disclosure.

[0119] Methods of screening for VHH domains (including VHH-binding polypeptides) having the desired specificity for PD-1 include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), enzyme assays, flow cytometry, and other immunologically mediated techniques known in the art.

[0120] Included among the VHH domains provided herein are PD-1 VHHs (llama or alpaca derived) and humanized sequences, such as any of those described below.

[0121] In certain embodiments, the VHH domains that bind PD-1 can be humanized. Humanized antibodies, such as VHH-containing polypeptides, are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies, which can lead to an immune response to the antibody therapeutic and reduced effectiveness of the therapeutic. Typically, a humanized antibody will comprise one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a portion of a human constant region. In certain embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues were derived), for example, to restore or improve antibody specificity or affinity.

[0122] A review of humanized antibodies and methods of making them is found, e.g., in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and further described in, e.g., Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing the "resurfacing" approach); Dall'Acqua et al., (2005) Methods 36:43-60 (describing the "FR shuffling" approach); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing the "guided selection" approach to FR shuffling).

[0123] Human framework regions that can be used include, but are not limited to, framework regions derived from a consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR regions of the VHH are replaced with human FR regions to make a humanized VHH. In certain embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. VHH domains having such replacement residues are still referred to as "humanized" herein.

[0124] Provided herein are VHH domains that bind PD-1 comprising a CDR1, CDR2, and CDR3 comprised in a VHH amino acid sequence selected from any one of SEQ ID NOS: 251-267 or 284, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid selected from any one of SEQ ID NOS: 251-267 or 284.

[0125] In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, CDR2, CDR3 comprised in a VHH domain set forth in SEQ ID NO: 284, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid set forth in SEQ ID NO: 284. In certain embodiments, the PD-1 VHH domain has the amino acid sequence set forth in SEQ ID NO: 284 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid set forth in SEQ ID NO: 284. In certain embodiments, the PD-1 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 284.

[0126] In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, CDR2, CDR3 comprised in a VHH domain set forth in SEQ ID NO: 312, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid set forth in SEQ ID NO: 312. In certain embodiments, the PD-1 VHH domain has the amino acid sequence set forth in SEQ ID NO: 312 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid set forth in SEQ ID NO: 312. In certain embodiments, the PD-1 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 312.

[0127] In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1 set forth in any one of SEQ ID NOs: 268, 272, 273, or 313, a CDR2 set forth in SEQ ID NO: 278 or 314, and a CDR3 set forth in SEQ ID NO: 283 or 315.

[0128] In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 272, 278, and 283, respectively. In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 268, 278, and 283, respectively. In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 272, 278, and 283, respectively. In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 273, 278, and 283, respectively. In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 313, 314, and 315, respectively.

[0129] In certain aspects, the VHH domain that binds PD-1 comprises a CDR1, a CDR2, and a CDR3 comprised in a VHH amino acid sequence selected from any one of SEQ ID NOs: 251-267, or a VHH amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid sequence selected from any one of SEQ ID NOs: 251-267. H The H region amino acid has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid sequence selected from any one of SEQ ID NOs: 251-267.

[0130] In certain cases, the provided PD-1 VHH domains are humanized variants that have an amino acid sequence set forth in any one of SEQ ID NOs: 251-267 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH region amino acid sequence selected from any one of SEQ ID NOs: 251-267. In certain embodiments, the PD-1 humanized VHH domains have an amino acid sequence set forth in any one of SEQ ID NOs: 251-267.

[0131] Provided herein are VHH domains that bind PD-1 comprising a CDR1, CDR2, and CDR3 comprised in the amino acid sequence selected from any one of the VHH amino acid sequences of SEQ ID NO: 287, 288, 289, or 290, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acid selected from any one of SEQ ID NO: 296, 297, 298, or 299.

[0132] In certain embodiments, the PD-1 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 296, 297, 298, or 299.

[0133] In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1 set forth in any one of SEQ ID NO: 300, 303, 306, or 309, a CDR2 set forth in SEQ ID NO: 301, 304, 307, or 310, and a CDR3 set forth in SEQ ID NO: 302, 305, 308, or 311. In certain embodiments, the PD-1 VHH domains provided herein contain a CDR1 set forth in SEQ ID NO: 300, a CDR2 set forth in SEQ ID NO: 301, and a CDR3 set forth in SEQ ID NO: 302; the PD-1 VHH domains provided herein contain a CDR1 set forth in SEQ ID NO: 303, a CDR2 set forth in SEQ ID NO: 304, and a CDR3 set forth in SEQ ID NO: 305; the PD-1 VHH domains provided herein contain a CDR1 of SEQ ID NO: 306, a CDR2 set forth in SEQ ID NO: 307, and a CDR3 set forth in SEQ ID NO: 308; or the PD-1 VHH domains provided herein contain a CDR1 set forth in SEQ ID NO: 309, a CDR2 set forth in SEQ ID NO: 310, and a CDR3 set forth in SEQ ID NO: 311.

[0134] III. Fusion proteins and conjugates containing PD-1 -binding polypeptides

[0135] Provided herein are fusion proteins and conjugates containing a PD-1-binding polypeptide containing at least one VHH domain that specifically binds PD-1, which is linked, directly or indirectly, to one or more additional domains or moieties. In certain embodiments, the fusion proteins or conjugates of the present disclosure consist of a single polypeptide. In other embodiments, the fusion proteins or conjugates of the present disclosure consist of more than one polypeptide. In certain embodiments, the PD-1-binding polypeptides of the present disclosure comprise at least one VHH domain that specifically binds PD-1. In certain aspects, the PD-1-binding polypeptides are multivalent. In certain embodiments, the PD-1-binding polypeptides include two or more copies of a VHH domain that specifically binds PD-1, e.g., three or more, four or more, five or more, or six or more copies of a VHH domain that specifically binds PD-1. In certain aspects, the PD-1-binding polypeptides are multispecific. For example, in certain instances, one or more additional domains can be one or more additional binding domains that bind one or more other antigens or proteins.

[0136] In certain embodiments, the PD-1-binding polypeptides of the present disclosure include two or more polypeptide sequences operably linked by an amino acid linker. In certain embodiments, these linkers consist primarily of the amino acids glycine and serine, denoted herein as GS-linkers. The GS-linkers of the fusion proteins of the present disclosure can have various lengths, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In certain embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 4). In certain embodiments, the linker is a flexible linker comprising glycine residues, e.g., as non-limiting examples, GG, GGG, GGGG (SEQ ID NO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In certain embodiments, the PD-1-binding polypeptides include a combination of GS-linkers and glycine linkers.

[0137] A. Fc fusions

[0138] Provided herein are PD-1-binding polypeptides that are fusion proteins containing at least one PD-1-binding VHH domain provided herein and one Fc domain. In certain embodiments, the PD-1-binding polypeptides provided herein comprise one, two, three, or four PD-1-binding VHH domains and one Fc domain.

[0139] In certain embodiments, incorporation of an immunoglobulin Fc region into a fusion protein can consist of two polypeptides that together form a dimer. In certain embodiments, the Fc domain mediates dimerization of the PD-1-binding polypeptide under physiological conditions, such as when expressed from a cell, such that a dimer is formed that multiplies the number of PD-1 binding sites. For example, a PD-1-binding polypeptide comprising three PD-1-binding VHH domains and one Fc region is trivalent as a monomer, but the Fc region can mediate dimerization such that the PD-1-binding polypeptide exists as a hexavalent dimer under such conditions. In certain embodiments, a PD-1 VHH domain is fused to an IgG Fc region and in these embodiments, the fusion protein is a bivalent that has two PD-1 VHH domains per molecule. In certain embodiments, two PD-1 binding domains (2x) are fused to an IgG Fc region and in these embodiments, the fusion protein is a tetravalent that has four PD-1 VHH domains per molecule. In certain embodiments, three PD-1 VHH domains (3x) are fused to an IgG Fc region and in these embodiments, the fusion protein is a hexavalent that has six PD-1 VHH domains per molecule.

[0140] In certain embodiments, the multivalent PD-1-binding polypeptide is a bivalent. In certain embodiments, the bivalent PD-1-binding polypeptides of the disclosure comprise two copies of a PD-1-binding polypeptide having the structure: (PD-1 VHH)-linker-Fc. In certain embodiments, the multivalent PD-1-binding polypeptide is a tetravalent. In certain embodiments, the tetravalent PD-1-binding polypeptides of the disclosure comprise two copies of a PD-1- polypeptide having the structure: (PD-1 VHH)-linker-(PD-1 VHH)-linker-Fc. In certain embodiments, the multivalent PD-1-binding polypeptide is a hexavalent. In certain embodiments, the hexavalent PD-1-binding polypeptides of the disclosure comprise two copies of a PD-1- polypeptide having the structure: (PD-1 VHH)-linker-(PD-1 VHH)-linker-(PD-1 VHH)-linker-Fc.

[0141] In certain cases, the CH3 domain of the Fc region can be used as a homodimerization domain, such that the resulting fusion protein is formed of two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein, such that the construct is an asymmetric fusion protein.

[0142] In any of the provided embodiments, the PD-1 VHH domain can be any of those described above. In certain embodiments, the PD-1 VHH domain is a humanized VHH domain that binds PD-1.

[0143] In different embodiments, the Fc domain included in the PD-1 -binding polypeptide is a human Fc domain, or is derived from a human Fc domain. In certain embodiments, the fusion protein contains an immunoglobulin Fc region. In certain embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of an IgGl isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 subclass.

[0144] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment thereof is an IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgGl isotype, which has the amino acid sequence:

[0145]

[0146] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgGl polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0147] In certain embodiments in which the fusion protein of the disclosure includes an Fc polypeptide, the Fc polypeptide is mutated or modified. In certain cases, the mutation includes one or more amino acid substitutions to reduce the effector function of the Fc polypeptide. Various examples of mutations of Fc polypeptides are known to alter (e.g., reduce) effector function, including any of those described below. In certain embodiments, unless described with reference to a particular SEQ ID NO, reference to an amino acid substitution in the Fc region is by the EU numbering of Kabat (also referred to as the Kabat numbering). The EU numbering is known, and is according to the recently updated IMGT Scientific Chart (http: / / www.imgt.org / IMGTScientificChart / IMGTScientificChart.html). In certain embodiments, the EU numbering is used to refer to the amino acid position in the Fc region of the fusion protein, unless otherwise specified. the international ImMunoGeneTics information http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: May 17, 2001, last update: January 10, 2013) and the EU index reported in Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th Ed. U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0148] In certain embodiments, for applications in which PD-1 or CD3 binding but certain effector functions (e.g., CDC and ADCC) are not necessary or are deleterious, Fc regions exhibiting reduced effector function can be desirable candidates. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the multispecific polypeptide construct and / or its cleaved components lack FcyR binding (hence can lack ADCC activity), but retain FcRn binding ability. The primary cell for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods can be employed (see, e.g., ACTI TM Non-radioactive cytotoxicity assays (Cell Technology, Inc. Mountain View, Calif; and CytoTox 96®; Promega, Madison, Wis.). TMNon-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the multispecific polypeptide construct or its cleavage component cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0149] In certain embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0150] Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In certain embodiments, the Fc region is modified at one or both of these positions using the Kabat numbering system, for example Lys326Ala and / or Glu333Ala (K326A and E333A).

[0151] In certain embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu 235 (L235), Asp 265 (D265), Asp 270 (D270), Ser 298 (S298), Asn 297 (N297), Asn 325 (N325) or Ala 327 (A327) or Pro 329 (P329). For example, Leu 234 Ala (L234A), Leu 235 Ala (L235A), Leu 235 Glu (L235E), Asp 265 Asn (D265N), Asp 265 Ala (D265A), Asp 270 Asn (D270N), Ser 298 Asn (S298N), Asn 297 Ala (N297A), Pro 329 Ala (P329A) or Pro 239 Gly (P329G), Asn 325 Glu (N325E) or Ala 327 Ser (A327S). In preferred embodiments, the modifications within the Fc region reduce binding to the Fc-receptor-gamma receptor while having minimal impact on binding to the neonatal Fc receptor (FcRn).

[0152] In certain embodiments, the human IgGl Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In certain embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In certain embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In certain embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In certain embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat numbering) to alter Fc receptor interactions, e.g., Asp265Ala (D265A). In certain embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A).In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In certain embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In certain embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, where Gly235 is deleted from the fusion protein. In certain embodiments, the human IgGl Fc region is modified at amino acid Gly236 to enhance interaction with CD32A, e.g., Gly236Ala (G236A). In certain embodiments, the human IgGl Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0153] In certain embodiments, the Fc region of the fusion protein lacks an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In certain embodiments, the Fc region of the fusion protein lacks an amino acid at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), and is modified at one or more of Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, the Fc region included in the PD-1-binding polypeptide is derived from a human Fc domain and comprises a deletion of three amino acids in the lower hinge corresponding to IgGl E233, L234, and L235. In certain aspects, such Fc polypeptides do not engage FcyRs and are thus referred to as“effector silenced” or“effector null.” For example, Fc deletion of these three amino acids reduces complement protein Clq binding. In certain embodiments, a polypeptide containing an Fc region with a deletion of these three amino acids retains binding to FcRn and thus has an extended half-life and transcytosis associated with FcRn-mediated recycling. Such modified Fc regions are referred to as“Fc xELL” or“Fc deletion” and have the following amino acid sequence:

[0154]

[0155] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgGl polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9.

[0156] In certain embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall'Acqua et al. 2006, J. Biol Chem vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al. 2010 Nature Biotech, vol. 28(2) 157-159), or Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively), (Kabat et al. 1991 EU index of Sequences of Proteins of Immunological Interest).

[0157] In certain embodiments, the Fc domain included in the PD-1 -binding polypeptide is derived from a human Fc domain and comprises the mutations M252Y and M428V, referred to herein as "Fc-YV". In certain embodiments, the mutated or modified Fc polypeptide comprises the following mutations: M252Y and M428L using the Kabat numbering system. In certain embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn-mediated recycling and prolonged half-life.

[0158] In certain embodiments, the Fc domain included in the PD-1-binding polypeptide is derived from a human Fc domain and comprises mutations to induce heterodimerization. In certain embodiments, such mutations include those known as "knobs" and "holes" mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain having amino acid modifications at positions Thr366, Leu368, and Tyr407 to smaller volume amino acids (e.g., Ser, Ala, and Val, respectively) ((T366S / L368A / Y407V)). In certain embodiments, the "knob" Fc domain comprises the mutation T366W. In certain embodiments, the "hole" Fc domain comprises the mutations T366S, L368A, and Y407V. Heterodimerization achieved by CH3 modifications can be further stabilized by the introduction of disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposing CH3 domains (reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15). In certain embodiments, the Fc domain for heterodimerization comprises additional mutations, such as the mutation S354C on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In certain embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K to prevent Protein A binding while maintaining FcRn binding. In certain embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is unmodified at H435. In different embodiments, the hole Fc domain comprises the modification H435R or H435K (in certain cases, referred to as "hole-R" when the modification is H435R), while the knob Fc domain does not. In certain cases, the hole-R mutation improves purification of the heterodimer relative to the homodimer hole Fc domain that can be present.

[0159] In certain embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion protein of the disclosure is monomeric. For example, modification to a charged residue at residue Thr366, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), will prevent CH3-CH3 dimerization.

[0160] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG2 isotype, which has the amino acid sequence:

[0161]

[0162] In certain embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10.

[0163] In certain embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (e.g., to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In certain embodiments, the human IgG2 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0164] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype, which has the amino acid sequence:

[0165]

[0166] In certain embodiments, the antibody or immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11.

[0167] In certain embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In certain embodiments, the human IgG3 Fc region is modified at amino acid 435 to prolong half-life, e.g., Arg435His (R435H). In certain embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0168] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype, which has the amino acid sequence:

[0169]

[0170] In certain embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0171] In certain embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype, which has the amino acid sequence:

[0172]

[0173] In certain embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13.

[0174] In certain embodiments, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In certain embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In certain embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0175] In certain embodiments, the fusion protein contains a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can contain a modified IgGl hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 14), in which Cys220, which forms a disulfide bond with the C-terminal cysteine of the light chain, is mutated to a serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 15).

[0176] In certain embodiments, the fusion protein has a modified hinge from IgG4, which is modified to prevent or reduce chain exchange, e.g., Ser228Pro (S228P), which has the sequence ESKYGPPCPPC (SEQ ID NO: 16). In certain embodiments, the fusion protein contains a linker polypeptide. In other embodiments, the fusion protein contains a linker and a hinge polypeptide.

[0177] In certain embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan chain at N297. There are a number of methods to prevent fucosylation, including, but not limited to, production in FUT8 deficient cell lines; addition of inhibitors, such as castanospermine, to the mammalian cell culture medium; and metabolic engineering of the production cell line.

[0178] In certain embodiments, the Fc region is engineered to eliminate recognition by existing antibodies found in humans. In certain embodiments, the VHH-containing polypeptides of the present disclosure are modified by mutation at position Leul l, for example Leul lGlu (Ll IE) or Leul lLys (Ll IK). In other embodiments, the single domain antibodies of the present disclosure are modified by changes in the carboxy terminal region, for example the terminal sequence has the sequence GQGTLVTVKPGG (SEQ ID NO: 17) or GQGTLVTVEPGG (SEQ ID NO: 18) or modifications thereof. In certain embodiments, the VHH-containing polypeptides of the present disclosure are modified by mutation at position 11 and changes in the carboxy terminal region.

[0179] In certain embodiments, one or more polypeptides of the fusion proteins of the present disclosure are operably linked by an amino acid linker. In certain embodiments, these linkers are composed primarily of the amino acids glycine and serine, denoted herein as GS-linkers. The GS-linkers of the fusion proteins of the present disclosure can have various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.

[0180] In certain embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of GGSGGS, i.e., (GGS)2(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 4). In certain embodiments, the linker is a flexible linker comprising glycine residues, for example, by way of non-limiting example, GG, GGG, GGGG (SEQ ID NO: 5), GGGG (SEQ ID NO: 6), and GGGGG (SEQ ID NO: 7). In certain embodiments, the fusion proteins can include a combination of GS-linkers and glycine linkers.

[0181] B. Conjugates

[0182] Provided herein are conjugates containing at least one VHH domain that specifically binds PD-1 provided herein and one or more additional moieties. The additional moieties can be therapeutic agents, such as cytotoxic agents, or can be detection agents. In certain embodiments, the moieties can be targeting moieties, small molecule drugs (non-polypeptide drugs less than 500 daltons molar mass), toxins, cytostatic agents, cytotoxic agents, immunosuppressants, radioactive agents suitable for diagnostic purposes, radioactive metal ions for therapeutic purposes, prodrug-activating enzymes, agents that increase biological half-life, or diagnostic or detectable agents.

[0183] In certain embodiments, the conjugates are antibody drug conjugates (ADCs, also known as immunoconjugates) containing one or more PD-1 VHH domains provided herein conjugated to a therapeutic agent that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In certain embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a radioactive isotope (i.e., a radioconjugate). In certain embodiments, the provided antibody drug conjugates of the present disclosure allow targeted delivery of the drug moiety to tumors. In certain cases, this can result in targeted killing of tumor cells.

[0184] In certain embodiments, a PD-1-binding conjugate is provided that comprises at least one PD-1 VHH domain provided herein conjugated to a therapeutic agent. In certain embodiments, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21 : 183-187, 1986). In certain embodiments, the therapeutic agent has intracellular activity. In certain embodiments, the PD-1-binding conjugate is internalized and the therapeutic agent is a cytotoxin that blocks protein synthesis by cells, thereby causing cell death. In certain embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity, including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, abrin, diptheria toxin, restrictocin, Pseudomonas exotoxin A, and variants thereof. In certain embodiments, where the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity, in order for the protein to be cytotoxic to cells, the PD-1-binding conjugate must be internalized after binding to the target cell.

[0185] In certain embodiments, a PD-1-binding conjugate is provided comprising at least one PD-1 VHH domain provided herein conjugated to a toxin. In certain embodiments, the toxin includes, for example, a bacterial toxin such as diphtheria toxin, a plant toxin such as ricin, a small molecule toxin such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19): 1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10: 1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13: 786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996)), and calicheamicins (Lode et al., Cancer Res. 58: 2928 (1998); Hinman et al., Cancer Res. 53: 3336-3342 (1993)). The toxin can exert its cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0186] In certain embodiments, a PD-1-binding conjugate is provided comprising at least one PD-1 VHH domain provided herein conjugated to a label that can indirectly or directly produce a detectable signal. These IgSF conjugates can be used for research or diagnostic applications, such as for in vivo detection of cancer. The label is preferably capable of directly or indirectly producing a detectable signal. For example, the label can be a radioisotope such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; an imaging agent; or a metal ion. In certain embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 123I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be complexed with various metal chelators and conjugated to antibodies, for example, for PET imaging (WO 2011 / 056983).

[0187] PD-1-binding conjugates can be prepared using any method known in the art. See, e.g., WO 2009 / 067800, WO 2011 / 133886, and U.S. Patent Application Publication No. 2014322129, incorporated by reference in their entireties.

[0188] In certain embodiments, the linkage can be covalent or non-covalent, for example, through a biotin-streptavidin non-covalent interaction. In certain embodiments, 1, 2, 3, 4, 5, or more moieties, which can be the same or different, are conjugated, linked, or fused to the PD-1 VHH domain to form a PD-1-binding conjugate. In certain embodiments, such moieties can be linked to the VHH domain using various molecular biological or chemical conjugation and linkage methods known in the art and described below. In certain embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers, or linkers that assist in conjugation reactions can be used to link or conjugate effector moieties to the variant polypeptide or immunomodulatory protein.

[0189] In certain embodiments, the PD-1 VHH domain is conjugated to one or more moieties, e.g., from about 1 to about 20 drug moieties per VHH, through a linker (L). In certain embodiments, the PD-1-binding conjugate comprises the following components: (VHH domain), (L) q and (moiety) m wherein the VHH domain is any of the VHH domains described, which is capable of binding PD-1 specifically as described; L is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or greater; and the resulting PD-1-binding conjugate binds PD-1. In particular embodiments, m is 1 to 4 and q is 0 to 8.

[0190] The linker can consist of one or more linker components. For covalent attachment of an antibody and a drug moiety, the linker typically has two reactive functional groups, i.e., bivalent in the sense of reactivity. Bivalent linker reagents useful for linking two or more functional or biologically active moieties such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods for their resulting conjugates have been described (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0191] Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p- aminobenzyloxy carbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB").

[0192] In certain embodiments, the linker can comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid residues comprising the amino acid linker components include naturally occurring residues, as well as secondary amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components can be designed and optimized with respect to their selectivity for enzymatic cleavage by particular enzymes, for example, tumor-associated proteases, cathepsins B, C, and D, and plasmin proteases.

[0193] Conjugates of VHH domains and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), azido compounds (such as

[0194] Antibody drug conjugates can be prepared by a variety of methods, such as organic chemistry reactions, conditions, and reagents known to those skilled in the art. In one embodiment, the method comprises: (1) reacting a nucleophilic group of a VHH domain with a bivalent linker reagent through a covalent bond to form VHH-L, which is then reacted with a drug moiety D; and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent through a covalent bond to form D-L, which is then reacted with a nucleophilic group of a VHH domain.

[0195] Nucleophilic groups on antibodies (including VHH domains) include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) in the case of antibodies that are glycosylated, sugar hydroxyl or amino groups. Amines, thiols, and hydroxyls are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyls, and maleimide groups. Additional nucleophilic groups can be introduced into antibodies by reaction of lysine with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine to a thiol. Reactive thiols can be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing a mutant antibody comprising one or more non-native cysteine amino acid residues).

[0196] Conjugates, such as antibody drug conjugates, can also be produced by modifying antibodies (such as VHH domains) to introduce electrophilic moieties that can react with nucleophilic substituents on linker reagents or drugs. The sugars of glycosylated antibodies can be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups that can link to amine groups of linker reagents or drug moieties. The resulting imine Schiff base groups can form stable bonds, or can be reduced, e.g., with borohydride reagents, to form stable amine bonds. In one embodiment, reaction of carbohydrate moieties of glycosylated antibodies with galactose oxidase or sodium meta-periodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on drugs (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can be reacted with sodium meta-periodate, resulting in the generation of an aldehyde at the position of the first amino acid. Such aldehydes can react with drug moieties or linker nucleophiles.

[0197] Likewise, nucleophilic groups on drug moieties include, but are not limited to, amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazides, hydrazine carboxylates, and arylhydrazide groups that can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyls, and maleimide groups.

[0198] Alternatively, fusion proteins containing a VHH domain and a cytotoxic agent can be prepared, e.g., by recombinant techniques or peptide synthesis. The DNA can comprise a length that encompasses the respective regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0199] C. Multispecific formats

[0200] Provided herein are multispecific PD-1-binding polypeptides that contain at least one VHH domain that binds PD-1 and one or more additional binding domains. Typically, the one or more additional domains bind a second antigen or protein other than PD-1. In certain aspects, the other antigen or protein can be an antigen expressed on a tumor, a molecule or receptor expressed on an immune cell (such as a T cell) (e.g., CD3), or an additional inhibitory receptor (e.g., CTLA-4, LAG3, TIM3, VISTA, TIGIT, SIRPa, NKG2A, B7H3, B7H4) or an activating receptor (e.g., OX40, GITR, 41BB, CD40, CD27, CD28, or ICOS), or confers additional specificity to a target cell (e.g., CD8 or CD4). In certain embodiments, the one or more additional domains is an antibody or antigen-binding fragment specific for the second antigen or protein. In certain embodiments, the additional domain is a VHH domain.

[0201] In certain embodiments, the multispecific PD-1-binding polypeptide comprises at least one VHH domain that binds PD-1 and at least one additional binding domain that binds a second antigen or protein. In certain embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In certain embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen is involved in enhancing or attenuating a signaling pathway in an immune cell.

[0202] In certain cases, the multispecific PD-1-binding polypeptide can further contain an Fc domain, such as any of the ones described above. In certain embodiments, the multispecific PD-1-binding polypeptides provided herein comprise at least one VHH domain that binds PD-1, at least one additional binding domain that binds a second antigen or protein, and an Fc domain. In certain embodiments, the Fc domain mediates dimerization of the multispecific PD-1-binding polypeptide under physiological conditions, such that a dimer is formed that doubles the number of binding sites for PD-1 and the additional antigen or protein.

[0203] Non-limiting exemplary multispecific PD-1-binding polypeptides are described below.

[0204] 1. Bispecific T cell engagers

[0205] In certain embodiments, the PD-1 -binding polypeptide is a bispecific construct that is or comprises at least one PD-1 VHH domain provided herein and at least one additional binding molecule that is capable of binding a surface molecule expressed on a T cell. In certain embodiments, the surface molecule is an activation component of a T cell, such as a component of a T cell receptor complex. In particular aspects, the surface molecule is an activating T cell antigen expressed on a T cell and is capable of inducing T cell activation upon interaction with an antigen binding molecule. For example, in certain aspects, interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. Suitable assays to measure T cell activation are known and include any assay that measures or assesses proliferation, differentiation, cytokine secretion, cytotoxic activity, and / or expression of one or more activation markers. In certain embodiments, simultaneous or near-simultaneous binding of such a PD-1 -binding polypeptide to its two targets (PD-1 expressed on a target cell and a T cell molecule, e.g., an activating T cell antigen, expressed on a T cell) can result in a transient interaction between the target cell and the T cell, thereby resulting in activation (e.g., cytotoxic activity) of the T cell and subsequent lysis of the target cell.

[0206] In certain embodiments, the T surface molecule, such as an activating T cell antigen, is CD3 or is CD2. Specifically, the provided bispecific PD-1 -binding polypeptides are capable of specifically binding an activating T cell antigen expressed on a human T cell, such as human CD3 or human CD3. In particular aspects, the additional binding domain specific for an activating T cell antigen (e.g., CD3 or CD2) is an antibody or antigen binding fragment. In certain embodiments, the PD-1 -binding polypeptide can be a bispecific antibody T cell-engager that contains at least one PD-1 VHH domain that specifically binds PD-1 and an additional binding molecule that is an antibody or antigen binding fragment specific for an activation component of a T cell (e.g., a T cell surface molecule, e.g., CD3 or CD2).

[0207] Bispecific antibody T cell-engagers include bispecific T cell engager (BiTE) molecules containing tandem scFv molecules fused by a flexible linker (see, e.g., Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other by, e.g., a flexible linker, and further containing an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al., Prot Eng 9, 299-305 (1996); Kipriyanov et al., J Mol Biol 293, 41-66 (1999)); dual-affinity re-targeting (DART) molecules, which can include diabody formats with C-terminal disulfide bonds; or triomabs, which include intact hybrid mouse / rat IgG molecules (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010). Using any of the PD-1 VHH domains provided herein, analogous versions of any of the above molecules can be produced.

[0208] In certain embodiments, the additional binding domain specific for an activating T cell antigen is an antigen binding fragment selected from a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In certain embodiments, the additional binding domain is monovalent for binding to an activating T cell antigen, such as CD2 or CD3.

[0209] In certain embodiments, the additional binding domain is capable of binding CD3 or the CD3 complex. The CD3 complex is a complex of at least five membrane-bound polypeptides in mature T-lymphocytes that associate with one another and with the T-cell receptor non-covalently. The CD3 complex includes gamma, delta, epsilon, zeta, and eta chains (also referred to as subunits). In certain embodiments, the additional binding molecule is an antibody or antigen-binding fragment capable of specifically binding CD3 or the CD3 complex, also referred to as a CD3-binding domain. In certain embodiments, the CD3-binding domain capable of binding CD3 or the CD3 complex includes one or more copies of an anti-CD3 Fab fragment, an anti-CD3 F(ab')2 fragment, an anti-CD3 Fv fragment, an anti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 scAb, an anti-CD3 dAb, an anti-CD3 single domain heavy chain antibody (VHH), and an anti-CD3 single domain light chain antibody. In certain embodiments, the anti-CD3 binding domain is monovalent for binding CD3.

[0210] In certain instances, the CD3-binding domain recognizes the CD3 epsilon-chain. In certain embodiments, the anti-CD3 epsilon binding domain includes one or more copies of an anti-CD3 epsilon Fab fragment, an anti-CD3 epsilon F(ab')2 fragment, an anti-CD3 epsilon Fv fragment, an anti-CD3 epsilon scFv, an anti-CD3 epsilon dsFv, an anti-CD3 epsilon scAb, an anti-CD3 epsilon dAb, an anti-CD3 epsilon single domain heavy chain antibody (VHH), and an anti-CD3 epsilon single domain light chain antibody. In certain embodiments, the anti-CD3 epsilon binding domain is monovalent for binding CD3 epsilon.

[0211] Exemplary monoclonal antibodies to CD3 or the CD3 complex include, but are not limited to, OKT3, SP34, UCHT1, or 64.1, or antigen-binding fragments thereof (see, e.g., June, et al., J. Immunol. 136:3945-3952 (1986); Yang, et al., J. Immunol. 137:1097-1100 (1986); and Hayward, et al., Immunol. 64:87-92 (1988)). In certain aspects, clustering of CD3 on T cells, e.g., by immobilized or cell-localized or tethered anti-CD3-antibodies, leads to T cell activation similar to engagement of the T cell receptor, but independent of its clonotypic specificity. In one embodiment, the CD3-binding domain monovalently and specifically binds CD3 antigen and is derived from OKT3 (ORTHOCLONE-OKT3 TM(Muromonab-CD3); humanized OKT3 (U.S. Patent No. 7,635,475 and published International Application No. WO2005040220); SP34 (Pessano et al. The EMBO Journal. 4:337-344, 1985); humanized variants of SP34 (WO2015001085); teplizumab TM (MGA031, Eli Lilly); anti-CD3 binding molecules described in US2011 / 0275787; UCHT1 (Pollard et al. 1987 J Histochem Cytochem. 35(11): 1329-38; WO2000041474); NI0401 (WO2007 / 033230); visilizumab (U.S. Patent No. 5,834,597); BC-3 (Anasetti et al., Transplantation 54:844 (1992); H2C (described in PCT Publication No. WO2008 / 119567); V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Patent No. 6,054,297)). Other anti-CD3 antibodies can also be used in the constructs provided herein, including any of those described in International Published PCT Application Nos. WO199404679, WO2018119567, WO2015095392, WO2016204966, WO2019133761, Published Patent Application Nos. US20170369563, US20180194842, US20180355038, U.S. Patent Nos. 7,728,114, 7,381,803, 7,994,289.

[0212] In certain embodiments, the CD3-binding domain contains a variable heavy (VH) chain set forth in SEQ ID NO: 19 and / or a variable light chain set forth in SEQ ID NO: 20, or a VH and / or VL sequence that is at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identical to these sequences, and specifically binds CD3. In certain embodiments, the CD3-binding domain contains CDRH1, CDRH2, and CDRH3 of the variable heavy (VH) chain set forth in SEQ ID NO: 19 and CDRL1, CDRL2, and CDRL3 of the variable light chain set forth in SEQ ID NO: 20. In certain cases, the CD3-binding region comprises a humanized version of the VH sequence set forth in SEQ ID NO: 209 and a humanized version of the VL sequence set forth in SEQ ID NO: 210. In certain embodiments, the CD3-binding region can contain a humanized OKT3-derived VH domain sequence set forth in any one of SEQ ID NOs 21, 22, 23 and / or a VL domain sequence set forth in any one of SEQ ID NOs 24, 25, 26, or a VH and / or VL sequence that is at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identical to these sequences, and specifically binds CD3. In certain embodiments, the CD3-binding domain is a Fab, scFv, Fv, or dsFv, containing any combination of the above VH and VL sequences, particularly any combination of a VH sequence set forth in any one of SEQ ID NOs: 21, 22, 23 and a VL sequence set forth in any one of SEQ ID NOs: 24, 25, 26.

[0213] In certain embodiments, the anti-CD3e binding domain comprises: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 29); a VH CD2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 31); a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 32); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 33); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34). In certain embodiments, the CD3-binding domain is a Fab, scFv, Fv, or dsFv, wherein is contained a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 29); a VH CD2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 31); a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 32); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 33); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34).

[0214] In certain embodiments, the CD3-binding domain contains a variable heavy (VH) chain set forth in SEQ ID NO: 27 and / or a variable light chain set forth in SEQ ID NO: 28, or a VH and / or VL sequence that is at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identical to these sequences, and specifically binds CD3. In certain embodiments, the CD3-binding domain contains CDRH1, CDRH2, and CDRH3 of the variable heavy (VH) chain set forth in SEQ ID NO: 27 and CDRL1, CDRL2, and CDRL3 of the variable light chain set forth in SEQ ID NO: 28. In certain embodiments, the CD3-binding domain contains CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NO: 29, 30, and 31, respectively, and CDRL1, CDRL2, and CDRL3 of the variable light chain set forth in SEQ ID NO: 32, 33, and 34, respectively. In certain cases, the CD3-binding region comprises a humanized version of the VH sequence set forth in SEQ ID NO: 27 and a humanized version of the VL sequence set forth in SEQ ID NO: 28. In certain embodiments, the CD3-binding region can contain a humanized VH domain sequence set forth in any one of SEQ ID NOs 35-65 and / or a VL domain sequence set forth in any one of SEQ ID NOs: 66-84, or a VH and / or VL sequence that is at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identical to these sequences, and specifically binds CD3. In certain embodiments, the anti-CD3 binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 47 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 75.

[0215] In certain embodiments, the CD3-binding domain is a Fab, scFv, Fv, or dsFv, wherein any combination of the above VH and VL sequences are contained, particularly any combination of a VH sequence set forth in any one of SEQ ID NOs: 35-65 and a VL sequence set forth in any one of SEQ ID NOs: 66-84. In certain embodiments, the anti-CD3 binding domain is a Fab, scFv, Fv, or dsFv, wherein a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 75 are contained.

[0216] In certain embodiments, the CD3-binding domain contains a variable heavy (VH) chain as set forth in any one of SEQ ID NO: 313, 314, 317, or 318. In certain embodiments, the CD3-binding domain contains a variable light (VL) chain as set forth in any one of SEQ ID NO: 315, 316, 319, or 320.

[0217] The provided bispecific constructs can be formed in any of a number of formats containing at least one PD-1 VHH domain and at least one additional domain specific for an activating T cell antigen, such as a CD3-binding domain.

[0218] In one embodiment, the bispecific construct is a bispecific single domain antibody-linked Fab (S-Fab) containing at least one of the PD-1 VHH domains linked, directly or indirectly, to a Fab antigen binding fragment specific for a T cell activation antigen, such as an anti-CD3 Fab. The Fab against a T cell activation antigen, e.g., an anti-CD3 Fab, can contain any of the VH and VL sequences set forth. In certain embodiments, the PD-1 VHH domain is linked to the C-terminus of the VH or VL chain of the anti-CD3 Fab. In certain embodiments, the S-Fab can be further modified, such as by conjugation to polyethylene glycol (PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, a protein such as albumin, polyglutamic acid, or PASylation (Pan et al. (2018) International Journal of Nanomedicine, 2018: 3189-3201).

[0219] In another embodiment, the bispecific construct is an scFv-single domain antibody, wherein the construct contains at least one of the PD-1 VHHs linked, directly or indirectly, to an scFv containing a VH and VL antigen binding domain specific for a T cell activation antigen (e.g., CD3). The scFv against a T cell activation antigen, e.g., an anti-CD3 scFv, can contain any of the VH and VL sequences described herein. In certain embodiments, the VHH domain and the scFv are linked by a linker, such as a peptide linker. In certain embodiments, the peptide linker can be a peptide linker as described herein. In certain embodiments, the VHH domain and the scFv are each optionally linked to an Fc region, such as the N-terminus of an Fc region, by a hinge region or linker, e.g., a peptide linker. The Fc region can be any described herein, such as a human Fc region or a variant thereof, e.g., a human IgGl Fc region or a variant thereof. In particular embodiments, the Fc region is formed from a variant Fc domain (e.g., a variant human IgGl domain) that is mutated or modified to promote heterodimerization, wherein different polypeptides can dimerize to produce a heterodimer.

[0220] In another embodiment, the CD3-binding domain is a single domain antibody, such as a VHH domain that specifically binds CD3. Single domain antibodies, including VHH domains that bind CD3, are known, see, e.g., published U.S. Patent Application No. US20160280795. In certain embodiments, the CD3-binding domain is an anti-CD3 VHH set forth in SEQ ID NO: 85, or a sequence that exhibits at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 85, and specifically binds CD3. In such aspects, the bispecific constructs provided herein can include at least one PD-1 VHH domain and at least one CD3 VHH domain. To form the construct, in certain cases, each VHH domain is optionally linked to an Fc region, such as the N-terminus of an Fc region, by a hinge region or linker, e.g., a peptide linker. The Fc region can be any described herein, such as a human Fc region or a variant thereof, e.g., a human IgGl Fc region or a variant thereof. In particular embodiments, the Fc region is formed from a variant Fc domain (e.g., a variant human IgGl domain) that is mutated or modified to promote heterodimerization, wherein different polypeptides can dimerize to produce a heterodimer.

[0221] In the above embodiments, exemplary modifications of Fc regions that promote heterodimerization are known, including any of the following described, e.g., in Table 3. In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 103, 107, 115, or 117, and the other Fc polypeptide of the heterodimeric Fc contains the amino acid sequence set forth in any of SEQ ID NOs: 104, 108, 111, 113, 119, or 121. In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 105, 109, 116, or 118, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, or 122.

[0222] 2. Restricted CD3 multispecific construct

[0223] In certain embodiments, the PD-1-binding polypeptide is a multispecific polypeptide construct that is a restricted T-cell binding fusion protein. In particular aspects, the restricted multispecific constructs provided herein bind to an activating T-cell antigen, such as CD3, and PD-1. Typically, the restricted multispecific polypeptide constructs provided further contain at least one antigen binding domain that binds to a tumor associated antigen (TAA). The restricted multispecific polypeptide constructs provided herein comprise at least: a first component comprising an immunoglobulin Fc region, a second component comprising one or more copies of a binding domain that binds CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding domain, which are terms used interchangeably herein), and a linker, such as a polypeptide linker, linking the first component and the second component. In the multispecific polypeptide constructs provided, one or both of the first and second components contain at least one of the provided VHH domains that bind PD-1, and one or both of the first and second components contain at least one TAA antigen binding domain that, when engaged upon binding to an antigen, renders the restricted CD3 binding region substantially capable of binding CD3.

[0224] In certain embodiments, the restricted multispecific polypeptide constructs provided herein exist in two states with respect to their ability to bind CD3 and subsequently activate T-cells: (1) an "inactive" state occurs when there is no binding of any or all of the antigen binding domains to PD-1, such that CD3 binding is restricted and T-cell interaction is avoided or reduced, and (2) an "active" state occurs upon binding of any or all of the antigen binding domains to an antigen, such that the CD3 binding region is capable of binding CD3 and allowing T-cell interaction.

[0225] In certain embodiments, the Fc region is linked to the CD3 binding domain by one or more linkers. In certain embodiments, the Fc region is linked to the CD3 binding region by one or more linkers that are not cleavable. In certain embodiments, the Fc region is linked to the CD3 binding region by one or more linkers that are cleavable or otherwise labile. In certain embodiments, a cleavable linker is one that can be specifically cleaved in the presence of a protease. In certain aspects, enhanced CD3 binding occurs upon cleavage of the cleavable linker. In certain such aspects, the "active" state can be further amplified by several mechanisms, including by cleavage of the linker linking the CD3 binding region and the Fc region. In certain embodiments, the cleavable linker is one that contains a substrate recognition site for a protease. In certain embodiments, wherein the Fc region and the CD3 binding region are linked by a cleavable linker, enhanced CD3 binding can occur upon cleavage within the linker.

[0226] Further, in aspects wherein the Fc region and the CD3 binding region are operably linked by a cleavable linker, cleavage of the linker between the Fc region and the CD3 binding region can separate the constrained multispecific polypeptide construct into a first component and a second component. Depending on the composition of the constrained multispecific polypeptide construct, the first component and the second component can have different functionalities. In certain embodiments, the Fc region is a region that exhibits one or more effector functions, such as ADCC, CDC, or ADCP functions. In such examples, the constrained multispecific polypeptide construct of the present disclosure can be used to produce a self-amplifying system. For example, in certain aspects, the incorporation of a protease-cleavable linker between the components of the Fc and CD3 binding domains is able to amplify T-cell activation capacity by allowing full exposure of the CD3 binding domain. Depending on the specific linker included, the amplification step can be mediated by a tumor-associated protease or by granzyme released upon antigen-dependent T-cell activation. If a tumor protease-cleavable linker is included, the amplification is mediated by the tumor or tumor-microenvironment. However, if a granzyme B-cleavable linker is included, the amplification can be self-mediated by the T-cell upon antigen-dependent activation. Moreover, in cases where an effector- enabled Fc is included in the construct, the amplification can be mediated by granzyme released from NK cells, which occurs by the ADCC mechanism.

[0227] The provided constrained multispecific polypeptide constructs include configurations in which the first component containing the Fc region is N-terminal to the second component containing the CD3 binding region. In one such embodiment, the first component and the second component are linked by a linker, which is C-terminal to the end of the Fc region. In certain embodiments, the at least one PD-1 VHH domain is located on the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In certain embodiments, the at least one PD-1 VHH domain is located on the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In certain embodiments, the constrained multispecific polypeptide construct contains only one PD-1 VHH domain, which is located on the N- or C-terminal region of the multispecific polypeptide construct. In certain embodiments, the at least one TAA antigen binding domain is located on the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In certain embodiments, the at least one TAA antigen binding domain is located on the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In certain embodiments, the constrained multispecific polypeptide construct contains at least two TAA antigen binding domains, which are located on the N- and C-terminal regions of the multispecific polypeptide construct.

[0228] In certain embodiments, the constrained multispecific polypeptide construct is a dimer, in which dimerization is formed by covalent or non-covalent interactions between two polypeptide chains. In certain embodiments, the two polypeptide chains are covalently bonded to one another, for example, by interchain disulfide bonds. In certain embodiments, the Fc region mediates dimerization by interchain disulfide bonds. In particular embodiments, the constrained multispecific polypeptide construct contains a heterodimeric Fc region, in which, in certain cases, the polypeptide chains of the multispecific polypeptide construct are different (heterodimeric). In particular examples of heterodimeric multispecific polypeptide constructs, the CD3-binding region is a two-chain polypeptide containing a VH and a VL chain, such as an Fv antibody fragment containing a VH and a VL. In certain embodiments, the Fv antibody fragment includes a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0229] In certain embodiments, the constrained multispecific polypeptide construct is formed from or includes two polypeptides, including: a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a VH domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv); and a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a VL domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv). In certain embodiments, the first polypeptide contains one or two VHH domains that bind PD-1. In certain embodiments, the second polypeptide contains one or two VHH domains that bind PD-1. In certain embodiments, the constrained multispecific polypeptide construct contains only one PD-1 VHH domain. In certain embodiments, the first polypeptide contains one or two TAA antigen binding domains. In certain embodiments, the second polypeptide contains one or two TAA antigen binding domains. In certain embodiments, the constrained multispecific polypeptide construct contains at least two TAA antigen binding domains. In certain cases, at least one TAA antigen binding domain is at the N-terminus of an Fc polypeptide, and at least one TAA antigen binding domain is at the C-terminus of a chain of a CD3-binding region. In particular embodiments, the at least one VHH domain that binds PD-1 is on a separate polypeptide of the heterodimeric molecule that is different from the polypeptide containing the last at least one (e.g., two) TAA antigen binding domains.

[0230] In certain embodiments, the constrained multispecific polypeptide construct contains at least two TAA antigen binding domains and at least one VHH domain that binds PD-1. In certain embodiments, the constrained multispecific polypeptide construct contains (1) a first polypeptide comprising, in order from N- to C-terminus: a first TAA antigen binding domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a chain (e.g., a VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g., Fv or dsFv), and a second TAA antigen binding domain; and (2) a second polypeptide comprising, in order from N- to C-terminus: a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable or non-cleavable linker), the other chain (the other of VH or VL) of the anti-CD3 antibody or antigen binding fragment, and a VHH domain that binds PD-1. In certain embodiments, the constrained multispecific polypeptide construct contains (1) a first polypeptide comprising, in order from N- to C-terminus: a first TAA antigen binding domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a chain (e.g., a VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g., Fv or dsFv), and a second TAA antigen binding domain; and (2) a second polypeptide comprising, in order from N- to C-terminus: a VHH domain that binds PD-1, a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable or non-cleavable linker), and the other chain (the other of VH or VL) of the anti-CD3 antibody or antigen binding fragment.

[0231] In certain embodiments, the first polypeptide or the second polypeptide, or both the first polypeptide and the second polypeptide, further comprises a costimulatory receptor binding region (CRBR) that binds a costimulatory receptor. In certain embodiments, the CRBR of the first polypeptide and / or the second polypeptide can be located N-terminal to the Fc polypeptide and / or C-terminal to the chain of the CD3-binding region.

[0232] In certain embodiments, the constrained multispecific polypeptide construct contains at least two TAA-binding antigen binding domains, one costimulatory receptor binding region (CRBR) that binds a costimulatory receptor, and one VHH domain that binds PD-1. In certain embodiments, the constrained multispecific polypeptide construct contains (1) a first polypeptide comprising, in order from N-terminus to C-terminus: a first TAA antigen binding domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable linker), a chain (e.g., a VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g., Fv or dsFv), and a second TAA antigen binding domain; and (2) a second polypeptide comprising, in order from N-terminus to C-terminus: one of a VHH domain that binds PD-1 or a CRBR, a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (the other of VH or VL) of the anti-CD3 antibody or antigen binding fragment, and the other of the VHH domain that binds PD-1 or a CRBR.

[0233] Each component of the multispecific polypeptide constructs of the disclosure is described in more detail below.

[0234] a. Antigen binding domain

[0235] The multispecific polypeptide constructs of the disclosure include at least one antigen binding domain, such as at least a first antigen binding domain and a second antigen binding domain. In certain aspects, the antigen binding domain, or independently each antigen binding domain, is selected from an antibody or antigen binding fragment, a natural (or native) cognate binding partner, an Anticalin (engineered lipocalin), a Darpin, a Fynomer, a Centyrin (engineered fibronectin III domain), a cystine-knot domain, an Affilin, an Affibody, or an engineered CH3 domain. In certain embodiments, the natural cognate binding partner comprises an extracellular domain of a natural cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA.

[0236] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, comprises one or more copies of an antibody or antigen-binding fragment thereof. In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0237] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, is a single chain antibody. In certain embodiments, the single chain is a scFv, a scAb, a single domain heavy chain antibody, or a single domain light chain antibody.

[0238] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, comprises one or more single domain antibody (sdAb) fragments, e.g., VHH, VNAR, engineered VH or VK domains. VHHs can be generated from native camelid heavy chain only antibodies, genetically modified rodents that produce heavy chain only antibodies, or naive / synthetic camelid or humanized camelid single domain antibody libraries. VNARs can be generated from cartilaginous fish heavy chain only antibodies. Various methods have been implemented to generate monomeric sdAbs from conventional heterodimeric VH and VK domains, including interface engineering and selection of specific germline families.

[0239] In certain embodiments, the antigen binding domain or, independently, each antigen binding domain (such as the first and / or second antigen binding domain) of the multispecific polypeptide construct contains at least one sdAb or scFv that binds a TAA. In certain embodiments, the at least one scFv or sdAb that binds a TAA is located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region of the multispecific polypeptide construct. In certain embodiments, the multispecific polypeptide construct contains only one scFv or sdAb that binds a TAA, which can be located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region. In certain embodiments, the multispecific polypeptide construct contains two scFv or sdAbs that bind a TAA, located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region. In certain embodiments, the multispecific polypeptide construct contains three scFv or sdAbs, two of which are located amino-terminal relative to the Fc region or carboxy-terminal relative to the CD3 binding region, and a third located at the other end of the multispecific polypeptide construct.

[0240] In certain embodiments, the multispecific polypeptide construct is formed from or includes two polypeptides, including: a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, a VH domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv), and a scFv or sdAb that binds a tumor associated antigen; and a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, a VL domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv), and optionally the same or a different scFv or sdAb that binds a tumor associated antigen. The scFv or sdAb that binds a TAA can be located amino-terminal relative to the Fc polypeptide of the heterodimeric Fc and / or carboxy-terminal relative to the VH or VL chain of the CD3 binding region. At least one of the first and / or second polypeptide of the multispecific polypeptide construct also includes a VHH domain that binds PD-1, such as any of those described in the present disclosure. In certain aspects, at least one of the first and / or second polypeptide of the multispecific polypeptide construct can also include a CRBR or chain thereof that binds a costimulatory receptor as described.

[0241] In certain embodiments, the antigen binding domain or, independently, each antigen binding domain (such as the first and / or second antigen binding domain) of the multispecific polypeptide construct contains a binding domain that is a single domain antibody (sdAb).

[0242] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, contains more than one chain. In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and / or second antigen binding domain, of the multispecific polypeptide construct contains VH and VL sequences assembled as a FAB.

[0243] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and / or second antigen binding domain, of the multispecific polypeptide construct contains VH-CH1 (Fd) and VL-CL of a Fab antibody that binds to the TAA. In certain embodiments, the Fab antibody containing VH-CH1 (Fd) and VL-CL is located amino terminal relative to the Fc region and / or carboxy terminal relative to the CD3 binding region of the multispecific polypeptide construct. In certain embodiments, the multispecific polypeptide construct contains only one Fab antibody that contains VH-CH1 (Fd) and VL-CL that binds to the TAA, which can be located amino terminal relative to the Fc region and / or carboxy terminal relative to the CD3 binding region. In certain embodiments, the multispecific polypeptide construct contains two Fab antibody fragments, each containing VH-CH1 (Fd) and VL-CL that binds to the TAA, one located amino terminal relative to the Fc region and the other located carboxy terminal relative to the CD3 binding region.

[0244] In certain embodiments, the multispecific polypeptide construct is formed from, or comprises, three or more polypeptides, including: a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor associated antigen; a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and optionally the same VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor associated antigen; and a third polypeptide comprising the other of VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a TAA.

[0245] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, is or comprises an extracellular domain of a natural (or native) cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to the TAA.

[0246] In certain embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first and second antigen binding domains, binds the same antigen. In certain embodiments, there is more than one antigen binding domain that binds a TAA, and each antigen binding domain, such as the first and second antigen binding domains, binds a different antigen. In certain embodiments, each antigen binding domain, such as the first and second antigen binding domains, binds the same tumor associated antigen (TAA). In certain embodiments, each antigen binding domain, such as the first and second antigen binding domains, binds a different TAA. In certain embodiments, each antigen binding domain, such as the first and second antigen binding domains, binds a different epitope on the same TAA. In certain embodiments, each antigen binding domain, such as the first and second antigen binding domains, binds the same epitope on the same TAA.

[0247] In certain embodiments, the antigen binding domain, or independently each antigen binding domain that binds a TAA, results in monovalent, bivalent, trivalent, or tetravalent binding to the TAA.

[0248] In certain embodiments, the TAA is selected from: 1-92-LFA-3, 5T4, a-4 integrin, a-V integrin, a4b1 integrin, a4b7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Cryptatin-3, Cryptatin-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin-B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFN alpha, IFN beta, IFN gamma, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12R beta 1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFR alpha, PDGFR beta, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGF beta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNF alpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0249] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to a tumor associated antigen (TAA) folate receptor alpha (FRa). For example, the antigen binding domain contains a binding domain that is a sdAb that binds to FRa. An exemplary sdAb that binds to FRa is set forth in SEQ ID NO: 86, 87, or 88. The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing SEQ ID Nos and binds to FRa.

[0250] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to a tumor associated antigen (TAA) cMET. For example, the antigen binding domain contains a binding domain that is a sdAb that binds to cMET. An exemplary sdAb that binds to cMET is set forth in SEQ ID NO: 89 (U.S. Patent No. 9,346,884). The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing SEQ ID No and binds to cMET.

[0251] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to a tumor associated antigen (TAA) B7H3. For example, the antigen binding domain contains a binding domain that is a scFv that binds to B7H3. In certain embodiments, the antigen binding domain is or contains a Fab antibody fragment comprising a VH-CH1 (Fd) and a LC. An exemplary B7H3 Fd is described in PCT Publication No. WO2017 / 030926.

[0252] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to the tumor associated antigen (TAA) CD20. For example, the antigen binding domain contains a binding domain that is a scFv that binds to CD20. An exemplary scFv that binds to CD20 is set forth in SEQ ID NO: 90, or contains a VL and VH set forth in SEQ ID NOs: 91 and 92 (U.S. Pub. No. US 2005 / 0123546). The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing SEQ ID NOs and binds to CD20.

[0253] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to the tumor associated antigen (TAA) DLL3. For example, the antigen binding domain contains a binding domain that is a scFv that binds to DLL3. An exemplary scFv that binds to DLL3 is set forth in SEQ ID NOs: 93 and 94 (U.S. Pub. No. US 2017 / 0037130). In certain embodiments, the antigen binding domain is or contains a Fab antibody fragment that comprises a Fd and LC that binds to DLL3. An exemplary DLL3 Fd is set forth in SEQ ID NO: 95, and an exemplary DLL3 LC is set forth in SEQ ID NO: 96 (U.S. Pat. No. US 8,044,178). The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing SEQ ID NOs and binds to DLL3.

[0254] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to the tumor associated antigen (TAA) 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 97, and an exemplary 5T4 LC is set forth in SEQ ID NO: 98. In certain embodiments, the antibody binding domain comprises the VH and VL set forth in SEQ ID NOs: 99 and 100 (U.S. Patent No. US 8,044,178). The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing SEQ ID NOs and bind 5T4.

[0255] In certain embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to the tumor associated antigen (TAA) gpNMB. In certain embodiments, the antigen binding domain is or contains a Fab fragment, which comprises an Fd and LC chain. An exemplary gpNMB Fd is set forth in SEQ ID NO: 101, and an exemplary gpNMB LC is set forth in SEQ ID NO: 102. The antigen binding domain, or independently each antigen binding domain, in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing SEQ ID NOs and bind gpNMB.

[0256] In certain embodiments, the antigen binding domain is linked, directly or indirectly, to an Fc region and / or to a CD3 binding region. In certain embodiments, the linkage is through a linker. In certain embodiments, the linker is a linking peptide (LP), which can include any of the flexible or rigid linkers described. In certain embodiments, the linker is selected from the group consisting of: GGSGGS, i.e., (GGS)2(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 4). In certain embodiments, the linker is a flexible linker comprising glycine residues, for example, by way of non-limiting example, GG, GGG, GGGG (SEQ ID NO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In certain embodiments, the linker comprises a combination of GS-linkers and glycine linkers.

[0257] b. Fc region

[0258] The constrained multispecific polypeptide construct comprises an immunoglobulin Fc region. Typically, the constrained multispecific polypeptide construct is a dimer formed from polypeptides, each polypeptide containing an Fc. The Fc polypeptide can be any of those described above. In particular embodiments, the Fc region is formed from Fc domains that are mutated or modified to promote heterodimerization, wherein different polypeptides can dimerize to produce a heterodimer. Thus, in certain embodiments, the dimer is a heterodimer, wherein the two polypeptide chains of the multispecific polypeptide construct are different.

[0259] A variety of methods for promoting heterodimerization of complementary Fc polypeptides are known, see, e.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al., Nat. Biotechnol. 16(7):677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285: 19637-46; Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sel, 23:195-202; published international PCT applications WO 1998 / 050431, WO 2009 / 089004, WO 2011143545 WO 2014 / 067011, WO 2012 / 058768, WO 2018027025; published U.S. patent applications US 20140363426, US 20150307628, US 20180016354, US 20150239991; and U.S. patents US 5731168, US 7183076, US 9701759, US 9605084, and US 9650446. Methods for promoting heterodimerization of Fc chains include mutagenesis of the Fc region, such as by including a set of “knob-into-hole” mutations, or by including mutations to enable electrostatic steering of the Fc to favor attractive interaction between different polypeptide chains. For example, in certain embodiments, the Fc polypeptides of the heterodimer include mutations to alter the charge polarity between the Fc dimer interface, such that co-expression of electrostatically matched Fc chains supports a favorable attractive interaction, thereby promoting the formation of the desired Fc heterodimer, while unfavorable repulsive charge interactions discourage the formation of the unwanted Fc homodimer (Guneskaran et al. (2010) JBC, 285: 19637-19646). When co-expressed in a cell, binding between the chains is possible, but the chains do not substantially self-associate due to charge repulsion. Other strategies for producing heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to produce complementary CH3 heterodimers, which are known as SEED Fc.

[0260] In certain embodiments, to promote heterodimerization, the two polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary paired amino acid modifications of the polypeptides of the Fc fusion are shown in Table 3.

[0261]

[0262]

[0263] In certain embodiments, the modifications include the introduction of a protuberance (bump) into the first Fc polypeptide and the introduction of a cavity (hole) into the second Fc polypeptide, such that the protuberance can be positioned in the cavity to facilitate complexing of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create the protuberance or cavity in the polypeptide are typically interface amino acids that interact or contact one or more amino acids in the interface of the second polypeptide.

[0264] In certain embodiments, the first Fc polypeptide modified to contain a protuberance (bump) amino acid includes the replacement of a native or original amino acid with a particular amino acid having at least one side chain that protrudes from the interface of the first Fc polypeptide and thus can be positioned in a complementary cavity (hole) in the adjacent interface of the second polypeptide. Most often, the replacement amino acid is one having a larger side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or assess the properties of amino acid residues to identify those that are ideal replacement amino acids to create a protuberance. In certain embodiments, the replacement residue used to form the protuberance is a naturally occurring amino acid residue and includes, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In certain embodiments, the original residue identified for replacement is one having a small side chain, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0265] In certain embodiments, the second Fc polypeptide modified to contain a cavity (hole) is one that includes the replacement of a native or original amino acid with a particular amino acid having at least one side chain that is recessed from the interface of the second polypeptide and thus is capable of accommodating a corresponding protuberance from the interface of the first polypeptide. Most often, the replacement amino acid is one having a smaller side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or assess the properties of amino acid residues to identify those that are ideal replacement residues to create a cavity. Typically, the replacement residue used to form the cavity is a naturally occurring amino acid residue and includes, for example, alanine (A), serine (S), threonine (T), and valine (V). In certain embodiments, the original amino acid identified for replacement is one having a large side chain, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0266] For example, the CH3 interface of human IgGl comprises sixteen residues on each domain that lie on four anti-parallel beta-strands that are buried from each surface by 10902 (see, e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modifications to the CH3 domain to create knobs or cavities are described in, e.g., U.S. Patent No. 5,731,168; International Patent Application WO 98 / 50431 and WO 2005 / 063816; and Ridgway et al., (1996) Prot. Engin., 9:617-621. In certain embodiments, modifications to the CH3 domain to create knobs or cavities typically target residues that lie on the two central anti-parallel beta-strands. The goal is to minimize the risk that created knobs can accommodate by protruding into the surrounding solvent, rather than by fitting into a complementary cavity in a partner CH3 domain.

[0267] For example, in certain embodiments, the heterodimeric Fc comprises a polypeptide with an amino acid modification within the CH3 domain at Thr366 that, when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain that has amino acid modifications at positions Thr366, Leu368, and Tyr407 to smaller volume amino acids (e.g., Ser, Ala, and Val, respectively) (T366S / L368A / Y407V). The heterodimerization via CH3 modifications can be further stabilized by introducing disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on the opposing CH3 domain (reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0268] The resulting constrained multispecific polypeptide construct can be purified by any suitable method, e.g., by affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, both homodimer and heterodimer formation can occur. Expression conditions can be adjusted so that heterodimer formation is favored over homodimer formation.

[0269] Techniques for recovering heterodimers from homodimers based on differential affinity of the heterodimer pair for an affinity reagent are known. In certain aspects, such techniques include designing the heterodimer such that one of the Fc polypeptide chains does not bind the affinity reagent protein A. In certain cases, one of the polypeptide chains can contain one or more amino acid substitutions to eliminate or reduce affinity for the protein A reagent in the one of the polypeptide chains of the Fc heterodimer, see, e.g., WO2017134440, WO2010151792, Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some of these embodiments, the Fc region can be modified at the protein-A binding site on one member of the heterodimer, thereby preventing protein-A binding and thereby enabling more efficient purification of the heterodimer fusion protein. An exemplary modification within this binding site is Ile253, e.g., Ile253Arg (I253R). In certain embodiments, the modification can be H435R or H435R / Y436F. In certain embodiments, the Fc polypeptide of the Fc heterodimer can contain modifications such that it is able to bind protein A but not protein G (pA+ / pG-). Exemplary pA+ / pG- amino acid modifications include: an Fc containing a serine at position 428, a serine at position 434, optionally a histidine at position 436, with reference to human IgGl, or an Fc containing these residues at the corresponding positions in human IgG 2, 3, or 4. In certain aspects, such amino acid modifications in the IgG Fc polypeptide at positions 428, 434, and optionally 436 reduce or prevent binding of protein G, thereby enhancing purification of the protein.

[0270] In certain embodiments, any such modification that confers differential affinity for an affinity reagent can be combined with any one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with the T366S / L368A / Y407V modification, or with the T366W modification. The T366S / L368A / Y407V modified Fc is able to form a homodimer because there is no steric obstruction of the dimerization interface, as is the case with the T336W modified Fc. Thus, in certain embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc to prohibit any homodimer Fc that can have formed from being purified. Similar modifications can be employed by combining the T366S / L368A / Y407V and H453R.

[0271] In certain embodiments, the Fc region of the heterodimeric molecule can additionally contain one or more other Fc mutations, such as any of those described above. In certain embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function.

[0272] In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 103, 107, 115, or 117, and the other Fc polypeptide of the heterodimeric Fc contains the amino acid sequence set forth in any of SEQ ID NOs: 104, 108, 111, 113, 119, or 121. In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 105, 109, 116, or 118, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, or 122.

[0273] In certain embodiments, the Fc region of the provided multispecific polypeptide construct exhibits one or more effector functions. In certain cases, the Fc region is capable of providing Fc-mediated effector functions, e.g., ADCC (e.g., NK cell release of granzyme B), ADCP, and / or CDC. In general, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), as well as antigen binding ability, the latter being the primary function of immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating IgG levels in serum by conjugation to the FcRn receptor in vivo, increasing half-life in vivo. In certain embodiments in which the multispecific polypeptide construct contains a cleavable linker, cleavage of the linker can result in two components each having biological activity: a CD3-binding region, which is capable of binding and engaging CD3 on a T cell, which in certain aspects can also contain a CRBR (for inducing a costimulatory signal on a T cell) and / or a VHH domain that binds PD-1 (for blocking an inhibitory signal on a T cell), and an Fc region linked to a TAA antigen binding domain, which can exhibit target-specific effector functions.

[0274] In certain embodiments, the Fc region comprises an Fc polypeptide that is mutated or modified to alter one or more effector functions. Thus, in certain cases, in the Fc used with the provided constrained multispecific polypeptide construct, effector functions such as one or more of ADCC, ADCP, and / or CDC can be altered (e.g., diminished or enhanced). Exemplary mutations that diminish effector function include any of those described above.

[0275] c.CD3 binding domain

[0276] Restricted multispecific polypeptide constructs include a CD3 binding domain. Anti-CD3 binding domains of the present disclosure activate T cells by engaging CD3 or a member of the CD3 complex on a T cell. In preferred embodiments, anti-CD3 binding domains of the present disclosure specifically bind the epsilon chain of CD3, also referred to as CD3 epsilon. Anti-CD3 epsilon binding domains of the present disclosure activate T cells by engaging CD3 epsilon on a T cell. Anti-CD3 binding domains of the present disclosure agonize, stimulate, activate, and / or otherwise enhance CD3-mediated T cell activation. Biological activities of CD3 include, for example, T cell activation and other signaling effected by the interaction between CD3 and the antigen binding subunit of the T-cell receptor (TCR). For example, by partially or completely modulating (e.g., agonizing, stimulating, activating, or otherwise enhancing) CD3-mediated T cell activation, anti-CD3 binding domains of the present disclosure completely or partially activate T cells by engaging CD3 epsilon on a T cell.

[0277] The CD3 binding domain can be any of those described above. In particular embodiments, the CD3 binding domain is an Fv antibody fragment that binds CD3 epsilon (referred to herein as an anti-CD3 epsilon Fv fragment). In certain embodiments, the anti-CD3 epsilon Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In certain embodiments, the anti-CD3 binding domain is monovalent for binding CD3.

[0278] In certain embodiments, the CD3 binding region is an Fv antibody fragment containing a variable heavy chain (Hv, also referred to as VH) and a variable light chain (Lv, also referred to as VL), such as any of those described above. In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region containing two different Fc polypeptides that are capable of effecting heterodimeric binding between the two polypeptides of the Fc heterodimer, such as any of those described above. In such embodiments, the variable heavy chain (VH) and the variable light chain (VL) of the CD3 binding region are linked on opposite chains of the heterodimeric Fc.

[0279] In certain embodiments, the CD3 binding region is an Fv or dsFv of SP34 (Pessano et al. The EMBO Journal. 4:337-344, 1985) or an Fv or dsFv of a humanized variant of SP34 (WO2015001085).

[0280] In certain embodiments, the anti-CD3 binding domain thereof is a Fv or dsFv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In certain embodiments, the CD3-binding domain is a Fv or dsFv fragment, wherein contained are: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 29); a VH CD2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 31); a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 32); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 33); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34). In certain embodiments, the anti-CD3 binding domain thereof is a Fv or dsFv fragment comprising a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 35-65 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84, 285. In certain embodiments, the anti-CD3 binding domain thereof is a Fv or dsFv fragment comprising a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-65 and a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84, 285. In certain embodiments, the anti-CD3 binding domain is a Fv or dsFv, wherein contained are a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 285.

[0281] d. linker

[0282] The constrained multispecific polypeptide construct contains a linker that connects or couples a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region. In certain embodiments, the linker is at the end of the C-terminal region of the Fc region, such that the Fc region is N-terminal to the CD3 binding region. It will be appreciated that because the provided constrained multispecific polypeptide construct is multimeric, such as a dimer containing a first polypeptide and a second polypeptide that together form the first component and the second component, the provided construct includes a linker that connects the Fc portion of the first polypeptide and the CD3 binding region and a linker that connects the Fc portion of the second polypeptide and the CD3 binding region. In certain embodiments, the first polypeptide includes a first Fc polypeptide of a heterodimeric Fc region, a linker, and a first domain of a CD3 binding region (e.g., a VH), and the second polypeptide includes a second Fc polypeptide of a heterodimeric Fc region, a linker, and a second domain of a CD3 binding region (e.g., a VL). Typically, the linker present in the first polypeptide and the second polypeptide of the constrained multispecific polypeptide construct is the same. Thus, in certain embodiments, each domain of the CD3 binding domain is connected to the opposite polypeptide of the Fc (such as a heterodimeric Fc) by a linker (such as the same linker).

[0283] A variety of polypeptide linkers for use in fusion proteins are known (see, e.g., Chen et al. (2013) Adv. Drug. Deliv. 65: 1357-1369; and International PCT Publication Nos. WO 2014 / 099997, WO 2000 / 24884; U.S. Patent No. 5,258,498; U.S. Patent No. 5,525,491; U.S. Patent No. 5,525,491, U.S. Patent No. 6,132,992).

[0284] In certain embodiments, the linker is chosen such that when the CD3 binding region is linked to the Fc region of a multispecific polypeptide conjugate, the CD3 binding region is constrained and cannot or substantially cannot bind or engage CD3 on the surface of a cell (e.g., a T cell) after the multispecific polypeptide construct is contacted with the cell. A variety of assays can be used to assess the binding or engagement of CD3 by a multispecific polypeptide construct, including assays that assess T cell binding, NFAT activation using a reporter system, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers. Exemplary assays are shown in the Examples provided. Generally, the linker is also one that ensures proper folding of the polypeptide construct, does not display a charge that is inconsistent with the activity or function of the linked polypeptide, and does not form bonds or other interactions with amino acid residues in one or more of the domains that would hinder or alter the activity of the linked polypeptide. In certain embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker or a rigid linker or a combination of the two. In certain aspects, the linker is a short, medium, or long linker. In certain embodiments, the linker is at most 40 amino acids in length. In certain embodiments, the linker is at most 25 amino acids in length. In certain embodiments, the linker is at least or is at least about 2 amino acids in length. In certain aspects, a suitable length is, for example, at least one and generally less than about 40 amino acid residues in length, such as 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acids. In certain embodiments, the linker is from or from about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids in length. In certain embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0285] In certain aspects, the longer the linker length, the greater the CD3 binding when the multispecific polypeptide conjugate is bound to its antigen (e.g., TAA). Thus, in certain aspects, the linker is greater than 12 amino acids in length, such as greater than 13, 14, 15, 16, 17, or 18 amino acids in length. In certain embodiments, the linker is 12 to 40 amino acids in length, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30 amino acids, or 30 to 40 amino acids.

[0286] The linker can be naturally occurring, synthetic, or a combination of both. Particularly suitable linker polypeptides include primarily amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker can contain at least 75% (calculated based on the total number of residues present in the peptide linker) such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker can also consist of only Gly, Ser, Ala, and / or Thr residues. In certain embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In certain aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In certain embodiments, the peptide linker comprises only glycine residues. In certain embodiments, the peptide linker comprises only glycine and serine residues.

[0287] In certain embodiments, these linkers consist primarily of the amino acids glycine and serine, denoted herein as GS-linkers. In certain embodiments, the linker contains (GGS)n, where n is 1 to 10, such as 1 to 5, for example 1 to 3, such as GGS(GGS)n(SEQ ID NO: 123), where n is 0 to 10. In particular embodiments, the linker contains the sequence (GGGGS)n(SEQ ID NO: 123), where n is 1 to 10 or n is 1 to 5, such as 1 to 3. In other embodiments, the linker contains (GGGGGS)n(SEQ ID NO: 124), where n is 1 to 4, such as 1 to 3. The linker can include any combination of the above, such as can combine 2, 3, 4, or 5 repeats of the GS, GGS, GGGGS, and / or GGGGGS linkers. In certain embodiments, such linkers are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0288] In certain embodiments, the linker is (in single letter amino acid code): GGS, GGGGS (SEQ ID NO: 125), or GGGGGS (SEQ ID NO: 126). In certain embodiments, the GS-linker comprises the following amino acid sequences: GGSGGS, i.e., (GGS)2(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 3); GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 4); GGGGGSGGGGGSGGGGGS, i.e., (G5S)3(SEQ ID NO: 127), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 129), and GGGGSGGGGSGGGGS (SEQ ID NO: 128). In certain embodiments, the linker is GGGG (SEQ ID NO: 5). In any of the above examples, the serine can be replaced with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0289] In certain embodiments, the linker includes the amino acid sequence Gly x Xaa-Gly y -Xaa-Gly z(SEQ ID NO: 130), wherein each Xaa is independently selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg), histidine (His), aspartate (Asp) and glutamate (Glu), and wherein x, y and z are each an integer in the range of 1 to 5. In certain embodiments, each Xaa is independently selected from Ser, Ala and Thr. In a specific variation, each of x, y, and z is equal to 3 (thereby generating a peptide linker having the amino acid sequence Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO: 131), wherein each Xaa is selected as above.

[0290] In certain embodiments, the linker is a serine-rich linker based on repetitions of the (SSSSG)y (SEQ ID NO: 132) motif, wherein y is at least 1, although y can be 2, 3, 4, 5, 6, 7, 8, and 9.

[0291] In some cases, it may be desirable to provide a certain rigidity in the peptide linker. This can be achieved by including proline residues in the amino acid sequence of the peptide linker. Therefore, in certain embodiments, the linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, the peptide linker can have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In a specific embodiment, the peptide linker comprises only proline residues.

[0292] In some aspects, the peptide linker comprises at least one cysteine ​​residue, such as a cysteine ​​residue. For example, in certain embodiments, the linker comprises at least one cysteine ​​residue and an amino acid residue selected from Gly, Ser, Ala and Thr. In some such embodiments, the linker comprises a glycine residue and a cysteine ​​residue, such as only a glycine residue and a cysteine ​​residue. Typically, each peptide linker includes only one cysteine ​​residue. An example of a specific linker comprising a cysteine ​​residue includes a linker having the amino acid sequence Gly m -Cys-Gly nwherein n and m are each an integer from 1 to 12, for example, 3 to 9, 4 to 8, or 4 to 7. In a specific variant, such a peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO: 133).

[0293] In certain embodiments, the linker of the fusion protein is a structured or constrained linker. In a specific embodiment, the structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID NO: 134), wherein n is 2 to 20, preferably 4 to 10, including, but not limited to, AS-(AP)n-GT (SEQ ID NO: 135) or AS-(EAAAK)n-GT (SEQ ID NO: 136), wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments, the linker comprises the sequence (GGGGA)n (SEQ ID NO: 137), (PGGGS)n (SEQ ID NO: 138), (AGGGS)n (SEQ ID NO: 139), or GGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 140, wherein n is 2 to 20. In certain embodiments, the linker is SSSASASSA (SEQ ID NO: 141), GSPGSPG (SEQ ID NO: 142), or ATTTGSSPGPT (SEQ ID NO: 143). In certain embodiments, due to their structure, such linkers may be more resistant to proteolytic degradation, thereby providing an advantage when injected in vivo.

[0294] In certain embodiments, the joint is not a cleavable joint, also referred to as a non-cleavable joint. In certain embodiments, the joint can not be cleaved by protease. In certain embodiments, the joint is not a cleavable joint or a joint that can not be cleaved by protease is a stable joint usually for in vivo delivery or recombinant production. In some aspects, the joint that can not be cleaved by protease comprises a joint that does not contain at least one peptide bond, and the peptide bond is preferably located in a cleavable peptide sequence or the recognition site of protease. In specific embodiments, the non-cleavable joint is not the target substrate of protease, so that compared with the joint that contains the substrate recognition site of the same protease, it is not preferentially or specifically cut by protease.

[0295] In certain embodiments, the joint does not contain a substrate recognition site or a cleavage site for a specific protease, and the site is a sequence that is cleaved by a protease, that is recognized by the active site of the protease. Typically, for example, for serine proteases, the cleavage sequence is composed of the P1-P4 and P1'-P4' amino acids in the substrate, and wherein the cleavage occurs after the P1 position. Typically, the cleavage sequence for serine proteases is a length of six residues to match the extended substrate specificity of many proteases, but can be longer or shorter depending on the protease. Typically, the joint does not include the P1-P1' key sequence that is easily cut off by the protease recognition. In some aspects, a non-cleavable joint or a joint that does not include a substrate recognition site that is specifically recognized by the protease to be cut is a joint that is significantly less than the protease to the cutting of the target substrate.

[0296] In certain embodiments, the joint is a cleavable joint. In some aspects, a cleavable joint is a joint such as any one of the above, which further comprises a sequence that is a substrate for proteolytic enzymes because there is at least one bond that can break under physiological conditions. In some cases, a cleavable joint is easy to cut or is sensitive to cutting under the specific conditions present in vivo, for example, after being exposed to extracellular proteases, is included in those conditions that exist in the cell environment in vivo. In some cases, proteolytic enzymes can be present in a specific physiological microenvironment, for example, in a tumor microenvironment, thereby limiting the site where cutting may occur.

[0297] Compared with another non-target substrate, protease usually shows specificity or preference to the cutting of specific target substrate. Such specificity degree can be determined based on the rate constant of the cutting of sequence (for example joint), which is the measurement of the preference of protease to its substrate and the efficiency of enzyme. Any method that determines the rate that cutting increases over time under the existence of different concentration substrates can be used to calculate the specificity constant. For example, substrate is connected to fluorescent moiety, and this fluorescent moiety is released after being cut by protease. By determining the cutting rate under different protease concentrations, the specificity constant (k) about the cutting of specific protease to specific joint can be determined. cat / K m In certain embodiments, a cleavable linker is one that is capable of cleaving at a rate of about at least 1×10 4 M -1 S -1 or at least 5×10 4 M -1 S, at least 10×10 4 M -1 S, at least 10×10 5 M -1 A linker that is specifically cleaved by a protease at a rate of 5s or greater.

[0298] In certain embodiments, the constrained multispecific polypeptide constructs of the present disclosure include a cleavable linker connecting the first component and the second component. In certain embodiments, the cleavable linker includes an amino acid sequence that can serve as a substrate for a protease, often an extracellular protease. For example, the cleavable linker can include at least one cleavage sequence of a peptide bond, which is preferably located within a cleavable peptide sequence of a protease. Suitable proteases include, for example, matrix metalloproteinases (MMPs), cysteine proteases, serine proteases, and plasmin activators, which are formed or activated in an exaggerated manner in diseases such as rheumatoid arthritis or cancer, resulting in excessive tissue degradation, inflammation, and metastasis. In particular embodiments, the protease is one produced by a tumor, an activated immune effector cell (e.g., a T cell or NK cell), or a cell in the tumor microenvironment. In certain embodiments, the protease is granzyme B, matriptase, or an MMP, such as MMP-2.

[0299] The cleavable linker can be selected based on a protease produced by a tumor that is proximal to a cell expressing a target, and / or produced by a tumor that is co-localized with a desired target of the multispecific polypeptide construct in a tissue. Increased levels of proteases with known substrates have been reported in the literature in many cancers, e.g., solid tumors. See, e.g., La Rocca et al. (2004) British J. of Cancer 90(7): 1414-1421.

[0300] In certain embodiments, the cleavable linker connecting the first component and the second component of the limited multispecific polypeptide construct is cleaved by a protease produced by an immune effector cell that is activated by one of the components. For example, a multispecific polypeptide construct comprising an effector primed or enhanced IgG Fc region is capable of eliciting ADCC upon engagement with a target antigen. Central to ADCC is the release of granzyme B and perforin from effector cells (i.e., NK cells and cytotoxic T-cells). Upon release, granzyme B enters the target cell in a perforin-dependent manner, where it mediates apoptosis. Importantly, granzyme B is active in the extracellular synapse between the effector cell and the target cell. In certain embodiments, the cleavable linker connecting the first component and the second component of the multispecific polypeptide construct is cleaved by granzyme B. During the process of effector cell activation mediated by one of the components of the multispecific polypeptide construct, granzyme B is released. In certain embodiments, granzyme B and other proteases can be produced by immune effector cells, including activated T-cells or NK cells. In certain embodiments, activation of T-cells by CD3 engagement upon binding of the multispecific polypeptide construct to a TAA can release such proteases, which can then cleave specific cleavable linkers, thereby enhancing or increasing the activity of the CD3-binding molecule in engaging CD3. In certain embodiments, the cleavage can amplify or increase the activity achieved by the multispecific construct when bound to the TAA in an uncleaved state.

[0301] Exemplary substrates include, but are not limited to, substrates that can be cleaved by one or more of the following enzymes or proteases: ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine cathepsins, e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, e.g., Cruzipain; Legumain; Otubain-2; KLKs, e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteases, e.g., Meprin; Kidney androgens-inducible lysosomal protease; PSMA; BMP-1; MMPs, e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, e.g., activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, Guanidinobenzoatase, HtrAl, human neutrophil elastase, lactotransferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, trypsin-like proteases, uPA; Type II transmembrane serine proteases (TTSPs), e.g., DESC1, DPP-4, FAP, Hepsin, Matriptase-2, Matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof.

[0302] In certain embodiments, the cleavable linker is cleaved by multiple proteases, e.g., 2 or more proteases, 3 or more proteases, 4 or more proteases, and the like.

[0303] In certain embodiments, the cleavable linker is selected for use with a particular protease, e.g., a protease known to be produced by a tumor adjacent to cells expressing a target and / or a protease produced by a tumor co-localized with a target of the multi-specific polypeptide construct.

[0304] In certain embodiments, the cleavable linker contains a substrate recognition site or cleavage site for a specific protease, which is a sequence that is cleaved by a protease, recognized by the active site of the protease. Typically, e.g., for serine proteases, the cleavage sequence consists of P1-P4 and P1'-P4' amino acids in the substrate, with cleavage occurring after the P1 position. Typically, the cleavage sequence for serine proteases is six residues in length to match the extended substrate specificity of many proteases, but can be longer or shorter depending on the protease. Typically, the cleavable linker includes a P1-P1' scissile bond sequence that is recognized by the protease. In certain aspects, the cleavable linker is engineered to introduce a peptide bond that is capable of being cleaved by a specific protease, e.g., by introducing a substrate recognition site sequence or cleavage sequence for the protease.

[0305] In certain embodiments, the cleavable linker includes a combination of two or more substrate sequences. In certain embodiments, each substrate sequence is cleaved by the same protease. In certain embodiments, at least two substrate sequences are cleaved by different proteases. In certain embodiments, the cleavable linker comprises amino acids that are a substrate for granzyme B. In certain embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 144), wherein P4 is an amino acid I, L, Y, M, F, V, or A; P3 is an amino acid A, G, S, V, E, D, Q, N, or Y; P2 is an amino acid H, P, A, V, G, S, or T; P1 is an amino acid D or E; and P1' is an amino acid I, L, Y, M, F, V, T, S, G, or A. In certain embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 145), wherein P4 is an amino acid I or L; P3 is an amino acid E; P2 is an amino acid P or A; P1 is an amino acid D; and P1' is an amino acid I, V, T, S, or G.

[0306] In certain embodiments, the substrate for granzyme B comprises the amino acid sequence LEAD (SEQ ID NO: 146), LEPG (SEQ ID NO: 147), or LEAE (SEQ ID NO: 148). In certain embodiments, the cleavable linker contains the amino acid sequence IEPDI (SEQ ID NO: 149), LEPDG (SEQ ID NO: 150), LEADT (SEQ ID NO: 151), IEPDG (SEQ ID NO: 152), IEPDV (SEQ ID NO: 153), IEPDS (SEQ ID NO: 154), IEPDT (SEQ ID NO: 155), IEPDP (SEQ ID NO: 144), LEPDG (SEQ ID NO: 152), or LEADG (SEQ ID NO: 153).

[0307] In certain embodiments, the cleavable linker comprises an amino acid that is a substrate for matriptase. In certain embodiments, the cleavable linker comprises the sequence P4QAR↓(A / V) (SEQ ID NO: 156), where P4 is any amino acid. In certain embodiments, the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 157). In certain embodiments, the substrate for matriptase comprises the amino acid sequence RQAR (SEQ ID NO: 158). In certain embodiments, the cleavable linker comprises the amino acid sequence RQARV (SEQ ID NO: 159).

[0308] In certain embodiments, the cleavable linker comprises an amino acid that is a substrate for one or more matrix metalloproteinases (MMPs). In certain embodiments, the MMP is MMP-2. In certain embodiments, the cleavable linker contains the general formula P3 P2 P1↓P1’ (SEQ ID NO: 160), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1’ is L, I, or M. In certain embodiments, the cleavable linker contains the general formula P3 P2 P1↓P1’ (SEQ ID NO: 161), where P3 is P; P2 is Q or D; P1 is A or N; and P1’ is L or I. In certain embodiments, the substrate for MMP comprises the amino acid sequence PAGL (SEQ ID NO: 162).

[0309] In certain embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B and an amino acid sequence that is a substrate for matriptase. In certain embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 146) and the amino acid sequence RQAR (SEQ ID NO: 158).

[0310] In certain embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B and an amino acid sequence that is a substrate for MMP. In certain embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 146) and the amino acid sequence PAGL (SEQ ID NO: 162).

[0311] In certain embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for matriptase and an amino acid sequence that is a substrate for MMP. In certain embodiments, the cleavable linker comprises a combination of the amino acid sequence RQAR (SEQ ID NO: 158) and the amino acid sequence PAGL (SEQ ID NO: 162).

[0312] In certain embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B, an amino acid sequence that is a substrate for matriptase, and an amino acid sequence that is a substrate for MMP. In certain embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B and an amino acid sequence that is a substrate for MMP. In certain embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 146), the amino acid sequence RQAR (SEQ ID NO: 158), and the amino acid sequence PAGL (SEQ ID NO: 162).

[0313] The cleavable linker can comprise any known linker. Examples of cleavable linkers are described in Be’liveau et al. (2009) FEBS Journal, 276, US published application numbers US20160194399, US20150079088, US20170204139, US20160289324, US20160122425, US20150087810, US20170081397, US patent number US9644016.

[0314] In certain embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of: TGLEADGSPAGLGRQARVG (SEQ ID NO: 163); TGLEADGSRQARVGPAGLG (SEQ ID NO: 164); TGSPAGLEADGSRQARVGS (SEQ ID NO: 162); TGPAGLGLEADGSRQARVG (SEQ ID NO: 166); TGRQARVGLEADGSPAGLG (SEQ ID NO: 167); TGSRQARVGPAGLEADGS (SEQ ID NO: 168); and TGPAGLGSRQARVGLEADGS (SEQ ID NO: 169); GPAGLGLEPDGSRQARVG (SEQ ID NO: 170); GGSGGGGIEPDIGGSGGS (SEQ ID NO: 171); GGSGGGGLEADTGGSGGS (SEQ ID NO: 172); GSIEPDIGS (SEQ ID NO: 173); GSLEADTGS (SEQ ID NO: 174); GGSGGGGIEPDGGGSGGS (SEQ ID NO: 175); GGSGGGGIEPDVGGSGGS (SEQ ID NO: 176); GGSGGGGIEPDSGGSGGS (SEQ ID NO: 177); GGSGGGGIEPDTGGSGGS (SEQ ID NO: 178); GGGSLEPDGSGS (SEQ ID NO: 179); and GPAGLGLEADGSRQARVG (SEQ ID NO: 180), GGEGGGGSGGSGGGS (SEQ ID NO: 181); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 182); GGSGGGGLEAEGSGGGGS (SEQ ID NO: 183); GGSGGGGIEPDPGGSGGS (SEQ ID NO: 184); TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 185).

[0315] e. Anti-PD-1 VHH Domains

[0316] The constrained multispecific polypeptide constructs of the present disclosure include at least one PD-1 VHH domain from any one provided herein. In certain embodiments, the PD-1 VHH domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 245-287, 294-299, and 312-315.

[0317] In particular embodiments, the constrained multispecific polypeptide construct contains at least two PD-1 VHH domains, such as any of those described. In certain cases, at least one PD-1 VHH domain is located amino-terminal to the Fc polypeptide of a heterodimeric Fc, and at least one PD-1 VHH domain is located carboxy-terminal to the VH or VL chain of a CD3 binding region. In certain aspects, each of the two PD-1 VHH domains is identical.

[0318] In particular embodiments, the constrained multispecific polypeptide construct contains only a PD-1 domain. In certain cases, the PD-1 VHH domain is located amino-terminal to the Fc polypeptide of a heterodimeric Fc. In certain embodiments, the PD-1 VHH domain is located carboxy-terminal to the VH or VL chain of a CD3 binding region.

[0319] In certain embodiments, the anti-PD-1 VHH domain is connected to the Fc region and / or to the CD3 binding region, either directly or indirectly through a linker. In certain embodiments, the connection is through a linker. In certain embodiments, the linker is a connecting peptide (LP), which can include any of the flexible or rigid linkers described. In certain embodiments, the linker is selected from the group consisting of: GGSGGS, i.e., (GGS)2(SEQ ID NO: 244); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 245); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 246); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 247). In certain embodiments, the linker is a flexible linker comprising glycine residues, for example, as non-limiting examples, GG, GGG, GGGG (SEQ ID NO: 248), GGGG (SEQ ID NO: 249), and GGGGG (SEQ ID NO: 7). In certain embodiments, the linker comprises a combination of GS-linkers and glycine linkers.

[0320] f. Co-stimulatory binding domains

[0321] The multispecific polypeptide constructs of the present disclosure include one or more costimulatory receptor binding regions (CRBRs) that bind to costimulatory receptors. In certain embodiments, one or more CRBRs of the multispecific polypeptide constructs provided bind to costimulatory receptors expressed on T cells. In certain embodiments, the costimulatory receptors are upregulated, induced, or expressed on the surface of activated T cells. In certain aspects, the CRBRs bind to costimulatory receptors and stimulate costimulatory receptors. In certain embodiments, the agonistic binding of costimulatory receptors to the CRBRs of the multispecific polypeptides induces downstream signaling in T cells to enhance or increase T cell activation or functionality after engaging CD3. In certain embodiments, the CRBRs or each CRBR independently is an antibody or antigen-binding fragment, a natural cognate binding partner of a costimulatory receptor, an anticalin (engineered lipocalin), a darpin, a fynomer, a centyrin (engineered fibroneticin III domain), a cystine-knot domain, an affilin, an affibody, or an engineered CH3 domain.

[0322] In certain embodiments, the CRBR or each CRBR independently, such as the first CRBR and the second CRBR, comprises one or more copies of an antibody or antigen-binding fragment thereof. In certain embodiments, the CRBR or each CRBR independently, such as the first antigen-binding domain and the second CRBR, comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0323] In certain embodiments, the CRBR or independently each CRBR, such as the first CRBR and the second CRBR, is a single chain antibody. In certain embodiments, the single chain is a scFv, scAb, a single domain heavy chain antibody, or a single domain light chain antibody.

[0324] In certain embodiments, the CRBR or independently each CRBR, such as the first CRBR and the second CRBR, comprises one or more single domain antibody (sdAb) fragments, such as V H H, V NAR , engineered V H or V K Domain. V H H can be generated from natural camelid heavy chain-only antibodies, genetically modified rodents producing heavy chain-only antibodies, or naive / synthetic camelid or humanized camelid single domain antibody libraries. NARCan be generated from cartilaginous fish only heavy chain antibodies. Various methods have been implemented to generate monomeric sdAbs from conventional hetero-dimeric V H and V K domains, including interface engineering and selection of specific germline families.

[0325] In certain embodiments, a CRBR or, independently, each CRBR (such as a first CRBR and / or a second CRBR) of a multispecific polypeptide construct contains at least one sdAb or scFv that binds a costimulatory receptor. In certain embodiments, the at least one scFv or sdAb that binds a costimulatory receptor is located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region of the multispecific polypeptide construct. In certain embodiments, the multispecific polypeptide construct contains only one scFv or sdAb that binds a costimulatory receptor, which can be located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region. In certain embodiments, the multispecific polypeptide construct contains two scFv or sdAbs that bind a costimulatory receptor, which are located amino-terminal relative to the Fc region and / or carboxy-terminal relative to the CD3 binding region.

[0326] In certain embodiments, the multispecific polypeptide construct is formed from or includes two polypeptides, including: a first polypeptide comprising a first Fc polypeptide of a hetero-dimeric Fc region, a linker, a VH domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv), and a scFv or sdAb that binds a costimulatory receptor; and a second polypeptide comprising a second Fc polypeptide of a hetero-dimeric Fc region, a linker, a VL domain of an anti-CD3 antibody or antigen binding fragment (e.g., Fv), and optionally another identical or different scFv or sdAb that binds a costimulatory receptor. The scFv or sdAb that binds a costimulatory receptor can be located amino-terminal relative to the Fc polypeptides of the hetero-dimeric Fc and / or carboxy-terminal relative to the VH or VL chain of the CD3 binding region. At least one of the first polypeptide and / or the second polypeptide of the multispecific polypeptide construct also includes a TAA binding antigen binding domain or chain thereof described in Section II.4. In certain embodiments, the TAA binding antigen binding domain is a scFv or sdAb and is included as part of the first polypeptide and / or the second polypeptide of the multispecific polypeptide construct. In certain embodiments, the TAA binding antigen binding domain is a Fab and the multispecific polypeptide construct is additionally formed from a third polypeptide, wherein at least the first polypeptide and the second polypeptide include a chain of the Fab that binds the TAA (e.g., VH-CH1 or VL-CL of the Fab), and the third polypeptide contains the other chain of the Fab that binds the TAA (e.g., the other of VH-CH1 or VL-CL of the Fab).

[0327] In certain embodiments, the CRBR or independently each CRBR, such as the first CRBR and / or the second CRBR, contains more than one chain. In certain embodiments, the CRBR or independently each CRBR of the multispecific polypeptide construct, such as the first CRBR and / or the second CRBR, contains a VH and a VL sequence assembled as a FAB.

[0328] In certain embodiments, the CRBR antigen binding domain or independently each CRBR antigen binding domain of the multispecific polypeptide construct, such as the first antigen binding domain and / or the second antigen binding domain, contains a VH-CH1 (Fd) and a VL-CL of a Fab antibody that binds a costimulatory receptor. In certain embodiments, the Fab antibody containing a VH-CH1 (Fd) and a VL-CL is located amino terminal relative to the Fc region and / or carboxy terminal relative to the CD3 binding region of the multispecific polypeptide construct. In certain embodiments, the multispecific polypeptide construct contains only one Fab antibody (which contains a VH-CH1 (Fd) or a VL-CL) that binds a costimulatory receptor, which can be located amino terminal relative to the Fc region and / or carboxy terminal relative to the CD3 binding region. In certain embodiments, the multispecific polypeptide construct contains two Fab antibody fragments that bind a costimulatory receptor, each containing a VH-CH1 (Fd) and a VL-CL, one of which is located amino terminal relative to the Fc region and the other of which is located carboxy terminal relative to the CD3 binding region.

[0329] In certain embodiments, the multispecific polypeptide construct is formed from or includes three or more polypeptides, including: a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH-CH1 (Fd) or a VL-CL of a Fab antibody fragment that binds a costimulatory receptor; a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and optionally the same VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds a costimulatory receptor; and a third polypeptide comprising the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds a costimulatory receptor. The first, second, and / or third polypeptide of the multispecific polypeptide construct can also include a PD-1 VHH domain, such as any of those described.

[0330] In certain embodiments, the CRBR or independently each CRBR is or includes a natural (native) cognate binding partner (e.g., natural ligand) of the costimulatory receptor or a variant thereof that exhibits binding activity to the costimulatory receptor.

[0331] In certain embodiments, one or more of the CRBRs of the provided multispecific polypeptide construct binds a costimulatory receptor expressed on a T cell. In certain embodiments, there is more than one CRBR that binds a costimulatory receptor, and each CRBR (such as a first CRBR and a second CRBR) binds the same costimulatory receptor. In certain embodiments, each CRBR (such as a first CRBR and a second CRBR) binds a different costimulatory receptor. In certain embodiments, each CRBR (such as a first CRBR and a second CRBR) binds a different epitope on the same costimulatory receptor. In certain embodiments, each CRBR (such as a first antigen-CRBR and a CRBR) binds the same epitope on the same costimulatory receptor.

[0332] In certain embodiments, the CRBR or independently each CRBR that binds a costimulatory receptor results in monovalent, bivalent, trivalent, or tetravalent binding to the costimulatory receptor.

[0333] In certain embodiments, the costimulatory receptor is expressed on a T cell, such as a primary T cell from a subject. In certain embodiments, the costimulatory receptor is expressed on a human T cell, such as a primary human T cell from a subject.

[0334] In certain embodiments, the costimulatory receptor is a member of the tumor necrosis factor (TNF) receptor family. In certain embodiments, the costimulatory receptor is a member of the immunoglobulin superfamily (IgSF). In certain embodiments, the costimulatory receptor is a member of the B7 family of receptors.

[0335] In certain embodiments, the costimulatory receptor is selected from 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR), CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and NKG2D. In certain embodiments, the costimulatory receptor is selected from 41BB, OX40, GITR, ICOS, or CD28. In certain embodiments, the costimulatory receptor is selected from 41BB, OX40, or GITR.

[0336] In certain embodiments, the costimulatory receptor is 41BB. In certain embodiments, the costimulatory receptor is OX40. In certain embodiments, the costimulatory receptor is GITR. In certain embodiments, the costimulatory receptor is ICOS. In certain embodiments, the costimulatory receptor is CD28.

[0337] In certain embodiments, a CRBR of a multispecific polypeptide is or comprises an agonistic binding molecule to a costimulatory receptor. A CRBR can bind to a costimulatory receptor and elicit, induce, or stimulate a response or activity that is similar or identical to a response or activity elicited, induced, or stimulated by a natural ligand of the receptor. In certain aspects, binding of a CRBR to a costimulatory receptor induces or stimulates a downstream signal that is more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100% of a signal elicited, induced, or stimulated by a natural ligand of the receptor.

[0338] In certain embodiments, the one or more CRBRs are antibodies or fragments thereof that bind to a costimulatory receptor 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-induced TNFR- related protein (GITR), CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cell maturation antigen (BCMA). In certain embodiments, the one or more CRBRs are antibodies or fragments thereof that bind to a costimulatory receptor 41BB, OX40, GITR, ICOS, or CD28. In certain embodiments, the one or more CRBRs are antibodies or fragments thereof that bind to a costimulatory receptor 41BB, OX40, or GITR. Exemplary polypeptides that bind to 41BB, OX40, and GITR are described in PCT Publication Nos. WO2017123650, WO2017123673, and WO2017015623, respectively. In certain embodiments, the one or more CRBRs are single domain antibodies (sdAbs) that bind to a costimulatory receptor, such as those described in PCT Publication Nos. WO2017123650, WO2017123673, and WO2017015623.

[0339] In certain embodiments, the co-stimulatory receptor binding region (CRBR) binds or comprises 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-induced TNFR- related protein (GITR), CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cell maturation antigen (BCMA), Transmembrane activator and CAML interactor (TACI), a natural cognate binding partner of NKG2D. In certain embodiments, the natural cognate binding partner is selected from 41BB ligand (41BBL), OX40L (CD252), CD70, GITR ligand / TNFSF18, CD80 (B7-1), CD86 (B7-2), ICOS ligand (ICOSL), CD154 (CD40L), B-cell activating factor (BAFF), A proliferation-inducing ligand (APRIL), NKG2D ligand, or a functional fragment thereof.

[0340] Exemplary sequences of CRBRs are shown in Table 4.

[0341] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor 41BB. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds 41BB, such as an sdAb or fragment (e.g., scFv) containing a VH and VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand of 41BB or is a functional binding fragment thereof. In certain embodiments, at least one CRBR or independently each CRBR is an Anticalin. Exemplary examples of such CRBRs that bind 41BB are shown in any one of SEQ ID NOs: 186-210. In certain embodiments, the CRBR that binds 41BB contains a VH shown in any one of SEQ ID NOs: 187, 189, and 191 and a VL shown in any one of SEQ ID NOs: 188, 190, or 192. The CRBR or independently each CRBR in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the foregoing SEQ ID NOs and bind 41BB.

[0342] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor OX40. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds OX40, such as a sdAb or fragment (e.g., scFv) containing a VH and a VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand for OX40 or is a functional binding fragment thereof. Illustrative examples of CRBRs that bind OX40 are shown in any one of SEQ ID NOs: 211-220. In certain embodiments, the CRBR that binds OX40 contains a VH shown in any one of SEQ ID NOs: 216 and 218 and a VL shown in any one of SEQ ID NOs: 217 and 219. The CRBR or independently each CRBR in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the foregoing SEQ ID NOs and bind OX40.

[0343] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor GITR. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds GITR, such as a sdAb or fragment (e.g., scFv) containing a VH and a VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand for GITR or is a functional binding fragment thereof. Illustrative examples of CRBRs that bind GITR are shown in any one of SEQ ID NOs: 221-230. In certain embodiments, the CRBR that binds GITR contains a VH shown in any one of SEQ ID NOs: 222, 224, 226, and 228 and a VL shown in any one of SEQ ID NOs: 223, 225, 227, and 229. The CRBR or independently each CRBR in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the foregoing SEQ ID NOs and bind GITR.

[0344] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor CD27. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds CD27, such as a sdAb or fragment (e.g., scFv) containing a VH and a VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand for CD27 or is a functional binding fragment thereof. Exemplary examples of such CRBRs that bind CD27 are shown in any one of SEQ ID NOs: 231. In certain embodiments, the CRBR that binds CD27 contains a VH shown in SEQ ID NO: 232 and a VL shown in SEQ ID NO: 233. The CRBR or independently each CRBR in the provided multispecific polypeptide constructs can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the foregoing SEQ ID NOs and bind CD27.

[0345] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor ICOS. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds ICOS, such as a sdAb or fragment (e.g., scFv) containing a VH and a VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand for ICOS or is a functional binding fragment thereof. Exemplary CRBR sequences that bind ICOS are shown in SEQ ID NO: 234.

[0346] In certain embodiments, at least one CRBR or independently each CRBR binds the co-stimulatory receptor CD28. In certain embodiments, the CRBR is or contains an antibody or antigen binding fragment that is specific for or binds CD28, such as a sdAb or fragment (e.g., scFv) containing a VH and a VL. In certain embodiments, at least one CRBR or independently each CRBR is a natural ligand for CD28 or is a functional binding fragment thereof. Exemplary CRBR sequences that bind CD28 are shown in SEQ ID NO: 235.

[0347]

[0348]

[0349]

[0350] In certain embodiments, one or more CRBRs are linked to the Fc region, directly or indirectly through a linker, and / or to the CD3 binding region. In certain embodiments, the linkage is through a linker. In certain embodiments, the linker is a connecting peptide (LP), which can include any flexible or rigid linker as described herein, although typically the peptide connecting the CRBR or region is not a cleavable linker.

[0351] In certain embodiments, the multispecific polypeptide construct comprises a connecting peptide (LP) between the CRBR and the Fc region. In certain embodiments, the multispecific polypeptide construct comprises a connecting peptide (LP) between the CD3 binding region and the CRBR.

[0352] 3. NK recruitment

[0353] In certain embodiments, the PD-1 -binding polypeptide is a bispecific construct that is or comprises at least one PD-1 VHH domain provided herein and at least one additional binding molecule capable of binding a surface molecule expressed on natural killer (NK) cells and / or recruiting NK cells. In particular aspects, the multispecific construct is bispecific for PD-1 and a NK cell surface molecule. In certain embodiments, the surface molecule is CD16 (FcyRIII). Specifically, the provided bispecific PD-1 -binding polypeptides are capable of specifically binding to an NK activating receptor expressed on human NK cells, such as human CD16a.

[0354] CD16, a low affinity receptor for the Fc portion of certain IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), is the best characterized membrane receptor responsible for triggering lysis of target cells by NK cells (Mandelboim et al., 1999, PNAS 96:5640-5644). Typically, the majority (about 90%) of human NK cells express CD56 at low density (CD56dim) and high levels of FcyRIII (CD16) (Cooper et al., 2001, Trends Immunol. 22:633-640). Human FcyRIII exists as two isoforms, CD16a (FcyRIIIA) and CD16b (FcyRIIIB), which have 96% sequence identity in their extracellular immunoglobulin binding regions (van de Winkel and Capel, 1993, Immunol. Today 14(5):215-221). In particular embodiments, the additional binding molecule is capable of specifically binding CD16a.

[0355] CD16a is expressed as a transmembrane receptor on macrophages, mast cells, and NK cells. On NK cells, the alpha chain of CD16a associates with immunoreceptor tyrosine-based activation motifs (ITAMs) containing FcεRI γ-chain and / or T-cell receptor (TCR) / CD3 ζ-chain to mediate signal transduction (Wirthmueller et al., 1992, J. Exp. Med. 175: 1381-1390). Interaction of CD16a with different combinations of homodimers and heterodimers of γ and ζ chains has been observed in NK cells, indicating the ability to mediate signal transduction via different signaling pathways through variations in the CD16a complex in NK cells (Anderson et al., 1990, PNAS 87(6): 2274-2278; Ackerly et al., 1992, Int. J. Cancer Suppl. 7: 11-14). Effector cells expressing FcγRs have been shown to participate in the destruction of tumor cells by ADCC. For example, engagement of CD16a with an agonist binding molecule, such as one that is capable of specifically binding CD16a, can result in the activation of NK cells expressing CD16a, thereby eliciting a biological response, particularly a signal transduction response. In certain instances, the binding molecule is capable of triggering cell killing by its binding to such cells in a manner similar to antibody-dependent cellular cytotoxicity (ADCC).

[0356] In particular embodiments, the PD-1-binding polypeptide comprises a bispecific molecule that can specifically bind PD-1 and CD16a, which can target NK cells to cells bearing such antigens, such that the cells bearing the antigens can be eradicated by NK cell-mediated cell killing. For example, a binding molecule that specifically binds PD-1 expressed on tumor cells can target NK-cells to the tumor cells. In certain instances, activation of NK cells by the binding molecule that binds CD16a can result in killing of the tumor cells.

[0357] In certain embodiments, the additional binding domain specific for an activating NK cell receptor, such as CD16a, is an antigen binding fragment selected from a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In certain embodiments, the additional binding domain is monovalent for binding to an activating NK cell receptor, such as CD16a.

[0358] In certain instances, the additional binding domain recognizes CD16a. In certain embodiments, the anti-CD16a binding domain comprises one or more copies of an anti-CD16 Fab fragment, an anti-CD16a F(ab')2 fragment, an anti-CD16a Fv fragment, an anti-CD16a scFv, an anti-CD16a dsFv, an anti-CD16a scAb, an anti-CD16a dAb, an anti-CD16a single domain heavy chain antibody (VHH), and an anti-CD16a single domain light chain antibody. In certain embodiments, the anti-CD16a binding domain is monovalent for binding CD16a. In certain embodiments, the BH73-binding polypeptide is a bispecific construct that binds BH73 and activates the activity of CD16a.

[0359] Antibodies and antigen-binding fragments thereof specific for CD16a are known and include, for example, NM3E2 (McCall et al. (1999) Mol. Immunol., 36:433-045. Other anti-CD16a antibodies can also be used in the constructs provided herein, including any of those described in published U.S. Patent Application No. US10160280795; U.S. Patent No. 9,701,750; Behar et al. (2008) Protein Eng Des Sel. 21 : 1-10; Arndt et al., (1999) Blood 94:2562-2568. In particular embodiments, the anti-CD16a is an anti-CD16a scFv. In certain embodiments, the anti-CD16a is an anti-CD16a antibody included in a TandAb molecule (see, e.g., Reush et al. (2014) Mabs, 6:727-738). In certain aspects, the anti-CD16a is an anti-CD16a or antigen-binding fragment, such as a scFv, described in U.S. Patent No. 9,035,026.

[0360] The bispecific constructs provided can be formed in any of a number of formats containing at least one PD-1 VHH domain and at least one additional domain specific for an activating NK cell receptor, such as a CD16a-binding domain.

[0361] In one embodiment, the bispecific construct is a bispecific single domain antibody- linked Fab (S-Fab) containing at least one of the PD-1 VHH domains directly or indirectly linked to a Fab antigen binding fragment specific for an NK cell activating receptor (e.g., CD16a), such as an anti-CD16a Fab. In certain embodiments, the PD-1 VHH domain is linked to the C-terminus of the VH or VL chain of the anti-C16a Fab. In certain embodiments, the S-Fab can be further modified, such as by conjugation to polyethylene glycol (PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, a protein such as albumin, polyglutamic acid, or PASylation (Pan et al. (2018) International Journal of Nanomedicine, 2018: 3189-3201).

[0362] In another embodiment, the bispecific construct is an scFv-single domain antibody, wherein the construct contains at least one of the PD-1 VHHs directly or indirectly linked to an scFv containing the VH and VL of an antigen binding domain specific for an NK cell activating receptor (e.g., CD16a). The scFv against an NK cell activating receptor, e.g., an anti-CD16a scFv, can contain any of the VH and VL sequences described. In certain embodiments, the VHH domain and the scFv are linked by a linker, such as a peptide linker. In certain embodiments, the peptide linker can be a peptide linker as described herein. In certain embodiments, the VHH domain and the scFv are each optionally linked to an Fc region, such as the N-terminus of an Fc region, by a hinge region or linker, e.g., a peptide linker. The Fc region can be any described herein, such as a human Fc region or a variant thereof, e.g., a human IgGl Fc region or a variant thereof. In particular embodiments, the Fc region is formed from a variant Fc domain (e.g., a variant human IgGl domain) that is mutated or modified to promote heterodimerization, wherein different polypeptides can dimerize to produce a heterodimer.

[0363] In another embodiment, the antigen binding domain specific for an NK cell activating receptor (e.g., CD16a) is a single domain antibody, such as a VHH domain that specifically binds CD16a. Single domain antibodies, including VHH domains that bind CD16a, are known, see, e.g., published U.S. Patent Application No. US20160280795. In such aspects, the bispecific constructs provided herein can include at least one PD-1 VHH domain and at least one CD16a VHH domain. To form the construct, in certain cases, each VHH domain is optionally linked to an Fc region, such as the N-terminus of an Fc region, by a hinge region or linker, e.g., a peptide linker. The Fc region can be any described herein, such as a human Fc region or a variant thereof, e.g., a human IgGl Fc region or a variant thereof. In particular embodiments, the Fc region is formed from a variant Fc domain, e.g., a variant human IgGl domain, that is mutated or modified to promote heterodimerization, where different polypeptides can dimerize to produce a heterodimer.

[0364] In the above embodiments, exemplary modifications of the Fc region that promote heterodimerization are known, including any described below, e.g., in Table 1. In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 103, 107, 115, or 117, and the other Fc polypeptide of the heterodimeric Fc contains the amino acid sequence set forth in any of SEQ ID NO: 104, 108, 111, 113, 119, or 121. In certain embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 105, 109, 116, or 118, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 106, 110, 112, 114, 120, or 122.

[0365] 4. Cytokine fusions and / or cytokine receptor targeting

[0366] In certain embodiments, the PD-1-binding polypeptide is a multispecific polypeptide construct as a cytokine-antibody fusion protein (also referred to as a PD-1 VHH-cytokine fusion). In certain aspects, at least one PD-1 VHH domain provided herein is linked, directly or indirectly, to at least one cytokine, such as an interferon. In particular embodiments, the cytokine is an interferon capable of exhibiting anti-proliferative activity, apoptotic activity, and / or anti-viral activity. In certain embodiments, the interferon of a PD-1 VHH-cytokine fusion provided herein is capable of binding to a receptor composed of IFNAR1 and / or 2. Any of a variety of assays can be used to assess the following effects of such fusion proteins: binding to IFNAR1 and / or 2, reducing or decreasing the growth rate and / or proliferation rate of cancer cells, reducing tumor size, eliminating a tumor, or inducing death of cancer cells (e.g., by apoptosis). Such assays include in vitro assays using various cancer cell lines known to express PD-1 or in vivo assays employing animal tumor models.

[0367] In certain embodiments, the interferon is a Type I interferon, such as a human Type I interferon or a variant thereof. In certain aspects, the human Type I interferon is a variant as a truncated human Type I interferon or a human mutant Type I interferon. In certain embodiments, the Type I interferon or variant thereof is wild-type human IFN-a (IFN-a; a2 and natural higher affinity variants such as a14), interferon beta (IFN-b), and mutants and / or truncated forms thereof. In certain embodiments, the interferon is a Type II interferon, such as a human Type II interferon or a variant thereof. In certain aspects, the human Type II interferon is a variant as a truncated human Type II interferon or a human mutant Type II interferon. In certain embodiments, the Type II interferon or variant thereof is wild-type human interferon gamma (IFN-g) and mutants and / or truncated forms thereof. In certain embodiments, the cytokine-antibody fusion proteins provided can be used to inhibit the growth and / or proliferation of target cells (e.g., cancer cells) expressing or overexpressing PD-1.

[0368] In certain embodiments, the PD-1 VHH-cytokine fusion protein is similar in form to any of those described in International PCT Published Application No. WO2014194100; U.S. Patent No. 9,803,021; Valedkarimi et al. (2017) Biomed Pharmacother., 95:731-742; or Young et al. (2014) Semin Oncol., 41 :623-636.

[0369] In particular embodiments, the interferon, e.g., a Type I interferon, such as a human Type I interferon (e.g., IFN-a, IFN-b or IFN-g), is an interferon having the endogenous binding affinity and / or activity of a natural or wild-type interferon, preferably at least 60% or at least or at least about 80% of the level of a natural wild-type interferon (in its isolated form), such as at least 90%, 95%, 98%, 99%, 100%, or greater.

[0370] Interferons and interferon mutants are a well-known and well-characterized collection of cytokines (see, e.g., WO 2002 / 095067; WO 2002 / 079249; WO 2002 / 101048; WO 2002 / 095067; WO 2002 / 083733; WO 2002 / 086156; WO 2002 / 083733; WO 2003 / 000896; WO 2002 / 101048; WO 2002 / 079249; WO 2003 / 000896; WO 2004 / 022593; WO 2004 / 022747; WO 2003 / 023032; WO 2004 / 022593, and Kim et al. (2003) Cancer Lett. 189(2): 183-188; Hussain et al. (2000) J. Interferon Cytokine Res. 20(9): 763-768; Hussain et al. (1998) J. Interferon Cytokine Res. 18(7): 469-477; Nyman et al. (1988) Biochem. J. 329(Pt 2): 295-302; Golovleva et al. (1997) J. Interferon Cytokine Res. 17(10): 637-645; Hussain et al. (1997) J. Interferon Cytokine Res. 17(9): 559-566; Golovleva et al. (1997) Hum. Hered. 47(4): 185-188; Kita et al. (1991) J. Interferon Cytokine Res. 17(3): 135-140; Golovleva et al. (1996) Am. J. Hum. Genet. 59(3): 570-578; Hussain et al. (1996) J. Interferon Cytokine Res. 16(7): 523-529; Linge et al. (1995) Biochim Biophys Acta. Any of these can be used in the provided cytokine-antibody fusion proteins.

[0371] In certain embodiments, the interferon is a human type I interferon. Alleles of the human interferon family of genes / proteins are known, see, e.g., Pestka (1983) Arch Biochem Biophys., 221 : 1-37; Diaz et al. (1994) Genomics, 22:540-52; Pestka (1986) Meth. Enzymol, 199:3-4; and Krause et al. (2000) J. Biol. Chem., 275:22995-3004.

[0372] In certain embodiments, the interferon is a full-length IFN-a (e.g., human IFN-a), a full-length IFN-β (e.g., human IFN-β), or a full-length IFN-γ (e.g., human IFN-γ). In certain embodiments, the interferon is a biologically active truncated IFN-a (e.g., human IFN-a), a biologically active truncated IFN-β (e.g., human IFN-β), or a biologically active truncated IFN-γ (e.g., human IFN-γ). In certain embodiments, a biologically active truncated interferon contains a contiguous sequence of amino acids of a wild-type or natural interferon that is truncated at the N- and / or C-terminus and comprises at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the length of the natural or wild-type interferon. Any of a variety of standard assays for assessing the biological activity of an interferon can be used. For example, IFN-a activity can be determined by measuring antiviral activity against a particular test virus. Kits for determining IFN-a activity are commercially available (see, e.g., ILITE® kits from Neutek bio, Ireland). In certain embodiments, the interferon is a biologically active truncated IFN-a (e.g., human IFN-a), a biologically active truncated IFN-β (e.g., human IFN-β), or a biologically active truncated IFN-γ (e.g., human IFN-γ). In certain embodiments, a biologically active truncated interferon contains a contiguous sequence of amino acids of a wild-type or natural interferon that is truncated at the N- and / or C-terminus and comprises at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the length of the natural or wild-type interferon. Any of a variety of standard assays for assessing the biological activity of an interferon can be used. For example, IFN-a activity can be determined by measuring antiviral activity against a particular test virus. Kits for determining IFN-a activity are commercially available (see, e.g., ILITE TMIFN-a2a (e.g., Acc. No. CAA23805), IFN-a-c (Acc. No. P01566), IFN-a-d (Acc. No. AAB59403); IFNa-5 (Acc. No. CAA26702); IFNa-6 (Acc. No. AA26704); IFNa-4 (Acc. No. NP_066546); IFNa-4b (Acc. No. CAA26701); IFNa-l (Acc. No. AAA52725); IFNa-J (Acc. No. CAA23792); IFNa-H (Acc. No. CAA23794); IFNa-F (Acc. No. AAA52718); IFNa-7 (Acc. No. CAA26903), or a biologically active fragment thereof. In certain aspects, the IFN-β is IFN-β as set forth in Acc. No. AAC41702 or a biologically active fragment thereof. In certain aspects, the IFN-γ is IFN-γ as set forth in Acc. No. P01579 or a biologically active fragment thereof.

[0373] In certain embodiments, the provided PD-1 VHH-cytokine fusions contain a variant or mutant interferon alpha 2 (IFNa2). Certain mutants include mutations of His at position 57 and / or E at position 58 and / or Q at position 61. In certain embodiments, the mutants include mutations H57Y and / or E58N and / or Q61S. In certain embodiments, the mutants include a mutated IFNa2 with mutations H57Y, E58N and Q61S (YNS) (see, e.g., Kalie et al. (2007) J. Biol. Chem., 282: 11602-11611). In other embodiments, the mutants include mutations of His at position 57 and / or E at position 58 and / or Q to A (alanine) at position 61. In certain embodiments, the mutants include a mutated IFNa2 with mutations H57A, E58A and Q61A (HEQ) (see, e.g., Jaitin et al. (2006) MoI. Cellular Biol, 26(5): 1888-1897). In certain embodiments, the mutant interferons include mutations of His at position 57 to A, Y or M, and / or E at position 58 to A or N or D or L, and / or Q at position 61 to A or S or L or D. In certain embodiments, the mutants include mutants of interferon alpha 8 (IFN-a8), such as variants with amino acid substitutions corresponding to R145 to V, I or L, and / or A146 to N or S, and / or M149 to Y, e.g., R145V / A146N / M149Y), R145I, / A146S / M149Y or R145L / A146S / M149Y (see, e.g., Yamamoto et al. (2009) J. Interferon & Cytokine Res, 29: 161-170.

[0374] In certain embodiments, the provided PD-1 VHH-cytokine fusions include a mutant or variant IFN-β containing a serine substitution for the naturally occurring cysteine at amino acid 17 (see, e.g., Hawkins et al. (1985) Cancer Res., 45, 5914-5920).

[0375] In certain embodiments, the provided PD-1 VHH-cytokine fusions contain a truncated interferon. In one embodiment, the truncated interferon includes human IFN-a with a deletion of up to the first 15 amino-terminal amino acid residues and / or up to the last 10-13 carboxy-terminal amino acid residues, which have been shown to retain the activity of native or wild-type human IFN-a (see, e.g., Ackerman (1984) Proc. Natl. Acad. Sci, USA, 81 : 1045-1047). In certain embodiments, the truncated human IFN-a has a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carboxy-terminal amino acid residues and / or a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino-terminal amino acid residues.

[0376] In certain embodiments, the provided PD-1 VHH-cytokine fusions contain a truncated interferon, such as described in published U.S. Patent Application No. US2009 / 0025106. In certain embodiments, the provided PD-1 VHH-cytokine fusions contain a truncated IFN-gamma containing N- and / or C-terminal deletions, such as described in Lundell et al. (1991) Protein Neg., 4:335-341; Pan et al. (1987) Eur. J. Biochem., 166: 145-149.

[0377] In certain embodiments, the interferon (e.g., human interferon) is a mutant interferon that is resistant to proteolysis compared to the unmodified, typically wild-type protein, see, e.g., U.S. Patent No. 7,998,469; U.S. Patent No. 8,052,964; U.S. Patent No. 4,832,959 U.S. Patent No. 6,120,762; WO 1992 / 008737; and EP 219781.

[0378] In aspects of the provided PD-1 VHH-cytokine fusion proteins, the antibody and cytokine (e.g., interferon) are linked directly or indirectly through a linker, such as a peptide linker. The linkage can be to the N- or C-terminus of the VHH domain, so long as the linkage does not interfere with binding of the antibody to PD-1. Any linker described herein can be used, e.g., a peptide linker. In certain embodiments, the linker is a GS-linker comprising an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 4). In certain embodiments, the linker is a flexible linker comprising glycine residues, e.g., as non-limiting examples, GG, GGG, GGGG (SEQ ID NO: 5), GGGG (SEQ ID NO: 6), and GGGGG (SEQ ID NO: 7). In certain embodiments, the fusion protein can include a combination of a GS-linker and a glycine linker.

[0379] D. Engineered Cells

[0380] Provided herein are engineered cells that express any of the provided PD-1 binding molecules described herein. In particular embodiments, the provided PD-1 binding molecules are secreted by the cells. In certain embodiments, the PD-1 binding molecules (e.g., containing an anti-PD-1 VHH domain provided herein) comprise a signal peptide (e.g., an antibody signal peptide) or other effective signal sequence to get the domain to the extracellular. When the PD-1 binding molecule comprises a signal peptide and is expressed by the engineered cell, the signal peptide causes the immunomodulatory protein to be secreted by the engineered cell. Typically, the signal peptide or portion of the signal peptide is cleaved from the binding molecule upon secretion. The PD-1 binding molecule can be encoded by a nucleic acid, which can be part of an expression vector. In certain embodiments, the PD-1 binding molecule is expressed and secreted by a cell, such as an immune cell, e.g., a primary immune cell.

[0381] In certain embodiments, the provided engineered cells further contain a chimeric antigen receptor (CAR). CARs are synthetic receptors that typically contain an extracellular targeting / binding moiety that is combined with one or more signaling domains in a single fusion molecule and expressed on the surface of a cell, such as a T cell. As such, CARs combine antigen specificity and T cell activation properties in a single fusion molecule. First generation CARs typically include the cytoplasmic region of CD3 zeta or FcI receptor gamma chain as their signaling domain. First generation CARs have been tested in phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, where they have induced modest responses (reviewed in Sadelain et al., Curr Opin Immunol, 21(2):215-223, 2009). Second generation CARs, which contain the signaling domains of costimulatory molecules such as CD28 and CD3 zeta, provide dual signaling to direct combined activation and costimulatory signals. Third generation CARs are more complex, with three or more signaling domains (reviewed in Sadelain et al., Cancer Discovery (3), 388-398, 2013 and Dotti et al., Immuno. Rev, 257(1), 1-36, 2014).

[0382] In certain embodiments, the CAR contains an extracellular domain comprising one or more antigen binding domains specific for a tumor antigen. In certain aspects, the tumor antigen and / or antigen binding domain specific for a TAA can be any as described herein. CAR constructs include an extracellular domain containing one or more extracellular antigen binding domains, a transmembrane domain, and an intracellular signaling region. In certain cases, the extracellular antigen binding domain is a scFv or a single domain antibody (VHH). Generally, the extracellular antigen binding domains forming the antigen binding unit of the CAR "bind" or are "capable of binding" (i.e., target) a target antigen with sufficient affinity so that the CAR is useful in therapies that target cells or tissues expressing the target antigen.

[0383] The transmembrane domain of a CAR is a domain that normally traverses, or is capable of traversing or spanning, the plasma membrane and is linked, directly or indirectly (e.g., via a spacer such as an immunoglobulin hinge sequence), to the extracellular antigen binding domain and the endoplasmic portion containing intracellular signaling domains. In one embodiment, the transmembrane domain of a CAR is a transmembrane region of a transmembrane protein (e.g., a Type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. In one embodiment, the transmembrane domain comprises a CD3 zeta domain or a CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art and can be used in conjunction with the embodiments of CARs provided herein.

[0384] The intracellular signaling region of a CAR provided herein contains one or more intracellular signaling domains that, upon engagement of the antigen binding domain of the CAR, e.g., upon binding of an antigen, transmits a signal into the T cell. In certain embodiments, the intracellular region contains an intracellular signaling domain that is or contains an IT AM signaling domain. Exemplary intracellular signaling domains include, e.g., signaling domains derived from any of the zeta chain of the T-cell receptor complex or its homologs (e.g., eta chain, FcsRIy and beta chain, MB 1 (Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3 polypeptides (delta, delta, and epsilon), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T-cell transduction (such as CD2, CD5, OX40, and CD28). In particular embodiments, the intracellular signaling region contains an intracellular signaling domain derived from the human CD3 zeta chain.

[0385] In certain embodiments, the intracellular domain comprises a CD3-zeta signaling domain. In certain embodiments, the CD3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 236 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 236 and retains activity for T cell signaling.

[0386] In certain embodiments, the intracellular signaling region of CAR may further contain an intracellular signaling domain derived from a costimulatory molecule. In such embodiments, after antigen-specific engagement, such a signaling domain can enhance CAR-T cell activity, such as by enhancing the proliferation, survival and / or development of memory cells, for example, compared to a CAR (e.g., CD3ζ) containing only an ITAM containing a signaling domain. In certain embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of CD28, CD137 (4-IBB), CD134 (OX40), DapIO, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1 1a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof. In a specific embodiment, the costimulatory signaling domain is derived from or is derived from a human protein. In some aspects, the costimulatory signaling domain is derived from or is derived from human CD28 or human CD137 (4-IBB).

[0387] In certain embodiments, the costimulatory signaling domain is derived from CD28 or 4-1BB and comprises an amino acid sequence shown in any one of SEQ ID NOs: 237-240, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NOs: 237-240, and retains T cell costimulatory signaling activity.

[0388] In certain embodiments, the CAR further comprises a hinge or spacer region connecting the extracellular antigen binding domain and the transmembrane domain. The hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of hinge or spacer regions that can be used include, but are not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a C fragment of an antibody, or a fragment or derivative thereof. H 2 regions, antibody C H 3 regions, artificial spacer sequences (e.g., peptide sequences), or combinations thereof. Other hinge or spacer regions will be apparent to those skilled in the art and may be used. In one embodiment, the hinge is an IgG4 hinge or a CD8A hinge.

[0389] In certain embodiments, the spacer and transmembrane domain are a hinge and transmembrane domain derived from CD8, such as having an exemplary sequence set forth in SEQ ID NOs: 241-243 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOs: 241-243.

[0390] Also provided herein are isolated nucleic acid constructs comprising a polynucleotide encoding a PD-1 binding molecule provided herein. Also provided herein are isolated nucleic acid constructs comprising a polynucleotide encoding a CAR and a polynucleotide encoding a PD-1 binding molecule provided herein. In certain aspects, the first nucleic acid encoding the CAR is separated from the second nucleic acid encoding the PD-1 binding molecule by a bi-cistronic element such as an IRES or a ribosomal skip sequence (e.g., T2A or P2A). In certain aspects, the construct is an expression vector for expressing the PD-1 binding molecule and / or the CAR in a cell. The expression vector can be a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral-based systems have been developed for the transfer of genes into mammalian cells. For example, retroviruses, such as adenovirus vectors are used. In one embodiment, a lentivirus vector is used.

[0391] In another aspect, also provided are isolated cells or populations of cells comprising one or more nucleic acid constructs as described above. Also provided are isolated cells or populations of cells that have been genetically modified to express a PD-1 binding molecule and / or a CAR provided herein. Thus, provided herein are genetically engineered cells comprising (e.g., stably expressing) a CAR provided herein. In one embodiment, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a hematopoietic stem cell, and / or a pluripotent embryonic / induced stem cell. In certain cases, the cell is a T cell, such as a CD4 and / or CD8 T cell. In certain embodiments, the cell is autologous to the subject. For example, in certain embodiments, a T cell (also referred to as a primary T cell) can be isolated from a patient for engineering, e.g., transfection or transduction, with a CAR nucleic acid construct.

[0392] In one exemplary instance, primary T-cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with a TCR / CD28 agonist such as anti-CD3 / anti- CD28 coated beads. Following a 2 or 3 day activation process, a recombinant expression vector encoding a CAR can be stably introduced into the primary T-cells by standard lentivirus or retrovirus transduction protocols or plasmid electroporation strategies. Cells can be monitored for secretion of PD-1 binding molecules and / or CAR expression, for example, by flow cytometry using anti-epitope tag or antibodies cross-reactive with the native parent molecule. CAR-expressing T-cells can be enriched by sorting with anti-epitope tag antibodies, or enriched for high or low expression, as appropriate.

[0393] PD-1 binding molecules and / or CAR engineered T-cells can be assayed for appropriate function by a variety of means. In some cases, in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-γ expression) can be used to assess the function of engineered T-cells. Exemplary standard endpoints are % lysis of tumor lines, proliferation of engineered T-cells, or IFN-γ protein expression in culture supernatants. In some cases, the ability of stimulated T-cells to activate upon stimulation of the CAR (e.g., by antigen) can be assessed, for example, by monitoring expression of activation markers such as CD69, CD44, or CD62L, proliferation, and / or cytokine production.

[0394] Also provided herein are methods for preventing and / or treating a disease or disorder, such as a cancer, in a subject comprising administering to the subject an engineered cell provided herein. Typically, the subject is in need of treatment of the disease or disorder with a pharmaceutically active amount of the cell and / or pharmaceutical composition of the application.

[0395] IV. Polypeptide Expression and Production

[0396] Provided are nucleic acid molecules comprising a polynucleotide encoding any of the provided sdAbs and PD-1 -binding polypeptides. In certain embodiments, the provided nucleic acid sequences and particularly DNA sequences encode the fusion proteins provided herein. In any of the foregoing embodiments, the nucleic acid molecule can also encode a leader sequence that directs secretion of the PD-1 -binding polypeptide, which leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be a native heavy chain (or VHH) leader sequence, or can be another heterologous leader sequence.

[0397] The nucleic acid molecules can be constructed using recombinant DNA technology as is routine in the art. In certain embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0398] Vectors are provided that comprise nucleic acids encoding the PD-1 -binding polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, bacteriophage vectors, viral vectors, retroviral vectors, and the like. In certain embodiments, vectors are selected that are optimized for expression of the polypeptides in desired cell types, such as CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described in, e.g., Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0399] In particular, DNA vectors encoding the desired PD-1 -binding polypeptides, such as fusion proteins, can be used to facilitate methods of making and obtaining large quantities of the PD-1 -binding polypeptides described herein. The DNA sequence can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. A variety of host-vector systems can be utilized to express the protein coding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. Depending on the host-vector system utilized, any of a number of suitable transcription and translation elements can be used.

[0400] The present disclosure also provides methods of producing a PD-1 -binding polypeptide by culturing a cell under conditions that result in expression of the polypeptide, wherein the cell comprises an isolated nucleic acid molecule encoding a PD-1 -binding polypeptide described herein, and / or a vector comprising these isolated nucleic acid sequences.

[0401] In certain embodiments, the PD-1 -binding polypeptides can be expressed in prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be performed, e.g., according to protocols known in the art. Exemplary eukaryotic cells that can be used to express the polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec 13 CHO cells, and FUT8 CHO cells; CHO cells; and NSO cells. In certain embodiments, a PD-1-binding polypeptide can be expressed in yeast. See, e.g., U.S. Pub. No. US 2006 / 0270045 Al. In certain embodiments, a particular eukaryotic host cell is chosen for its ability to perform desired post-translational modifications on a polypeptide. For example, in certain embodiments, CHO cells produce a PD-1-binding polypeptide with a higher level of sialylation than the same polypeptide produced in 293 cells.

[0402] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, and the like. Non-limiting exemplary methods are described in, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rded. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in a desired host cell according to any suitable method.

[0403] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In certain embodiments, host cells expressing a PD-1-binding polypeptide described herein are provided. A PD-1-binding polypeptide expressed in a host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind Fc regions. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind Fc regions and purify PD-1-binding polypeptides comprising Fc regions. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying certain polypeptides such as antibodies. Ion exchange chromatography, e.g., anion exchange chromatography and / or cation exchange chromatography, can also be suitable for purifying certain polypeptides such as antibodies. Mixed mode chromatography, e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, and the like, can also be suitable for purifying certain polypeptides such as antibodies. Many methods of purifying polypeptides are known in the art.

[0404] In certain embodiments, the PD-1-binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21 :695-713 (2003).

[0405] In certain embodiments, a PD-1-binding polypeptide produced by the above methods is provided. In certain embodiments, the PD-1-binding polypeptide is produced in a host cell. In certain embodiments, the PD-1-binding polypeptide is produced in a cell-free system. In certain embodiments, the PD-1-binding polypeptide is purified. In certain embodiments, a cell culture medium comprising a PD-1-binding polypeptide is provided.

[0406] In certain embodiments, a composition comprising an antibody produced by the above methods is provided. In certain embodiments, the composition comprises a PD-1-binding polypeptide produced in a host cell. In certain embodiments, the composition comprises a PD-1-binding polypeptide produced in a cell-free system. In certain embodiments, the composition comprises a purified PD-1-binding polypeptide.

[0407] V. Pharmaceutical Compositions and Formulations

[0408] Provided herein are pharmaceutical compositions containing any of the PD-1-binding polypeptides provided herein or engineered cells expressing them. In certain embodiments, the PD-1-binding polypeptides, such as the fusion proteins of the present disclosure (also referred to herein as "active compounds"), and derivatives, fragments, analogs, and homologs thereof, can be incorporated into a pharmaceutical composition suitable for administration. In certain embodiments, engineered cells expressing chimeric receptors, such as chimeric antigen receptors, containing the PD-1-binding polypeptides provided herein can be incorporated into a pharmaceutical composition suitable for administration.

[0409] Such compositions typically contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0410] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intratumoral, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0411] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0412] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or more of the ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0413] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0414] For inhalation administration, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser (which contains a suitable propellant, e.g., a gas such as carbon dioxide) or a nebulizer.

[0415] Systemic administration can also be by way of transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into

[0416] The compounds can also be formulated in suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or in rectal gels for rectal delivery.

[0417] In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0418] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms according to the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0419] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in a diagnostic kit together with instructions for use.

[0420] The pharmaceutical composition is administered in an amount effective to treat or prevent the particular indication. The therapeutically effective amount will generally depend on the subject being treated, his or her physical or health condition, the severity of the disorder to be treated, and the age of the subject being treated. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 50 pg / kg body weight to about 50 mg / kg body weight per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 100 pg / kg body weight to about 50 mg / kg body weight per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 100 pg / kg body weight to about 20 mg / kg body weight per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

[0421] In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 10 mg to about 1,000 mg per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 20 mg to about 500 mg per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 20 mg to about 300 mg per dose. In certain embodiments, the pharmaceutical composition can be administered in an amount ranging from about 20 mg to about 200 mg per dose.

[0422] The pharmaceutical composition can be administered to the subject as needed. In certain embodiments, an effective dose of the pharmaceutical composition is administered to the subject one or more times. In different embodiments, an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, e.g., once every two months, once every three months, or once every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, e.g., once every two weeks, once a week, twice a week, three times a week, once a day, or multiple times a day. An effective dose of the pharmaceutical composition is administered to the subject at least once. In certain embodiments, an effective dose of the pharmaceutical composition can be administered multiple times, including for a period of at least one month, at least six months, or at least one year. In certain embodiments, the pharmaceutical composition is administered to the subject as needed to alleviate one or more symptoms of the disorder.

[0423] VI. Methods of Treatment and Uses

[0424] The PD-1-binding polypeptides or engineered cells expressing them described herein can be used for a variety of therapeutic, diagnostic, and prophylactic indications. For example, the PD-1-binding polypeptides or engineered cells can be used to treat a variety of diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses, e.g., involving administration of the molecules or engineered cells or compositions containing them to a subject having a disease, condition, or disorder, such as a tumor or cancer. In certain embodiments, the molecules or engineered cells are administered in an effective amount to effect treatment of a disease or disorder. Uses include the use of molecules or engineered cells containing PD-1-binding polypeptides in such methods and treatments, and the use in the manufacture of a medicament for effecting such therapeutic methods. In certain embodiments, the methods are effected by administering a PD-1-binding polypeptide or engineered cell or composition containing them to a subject having or suspected of having a disease or condition. In certain embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0425] In one embodiment, the PD-1-binding polypeptides or engineered cells of the present disclosure can be used as a therapeutic agent. Such agents will generally be used to diagnose, prognose, monitor, treat, alleviate, and / or prevent a disease or pathology in a subject. A treatment regimen is implemented by identifying a subject, e.g., a human patient or other mammal, who has a disorder (or is at risk of developing a disorder) using standard methods. In certain cases, a subject known, suspected, or identified to have a tumor expressing PD-1 is selected. The PD-1-binding polypeptide or engineered cell is administered to the subject. The PD-1-binding polypeptide or engineered cell is administered to the subject and, due to its binding to the target, generally has an effect.

[0426] In certain embodiments, the provided PD-1 polypeptide multispecific polypeptide constructs or engineered cells are capable of modulating (e.g., increasing) an immune response when administered to a subject, e.g., by engagement of CD3 and / or CD3 signaling in a cell and / or by blocking the interaction of PD-1 and PD-L1 / PD-L2. In certain embodiments, provided herein are methods of modulating an immune response in a subject by administering a therapeutically effective amount of a provided multispecific construct or engineered cell or a pharmaceutical composition thereof. In certain embodiments, the methods of modulating an immune response increase or enhance an immune response in a subject. For example, the increased or enhanced response can be an increase in cell-mediated immunity. In certain embodiments, the methods increase T-cell activity, such as cytolytic T-cell (CTL) activity. In certain embodiments, the modulated (e.g., increased) immune response is against a tumor or cancer.

[0427] In certain embodiments, administration of a PD-1-binding polypeptide, such as a PD-1-Fc fusion protein or a multi-specific construct containing an Fc region, can activate innate immune cells via engagement of FcyRs by the Fc-region of the multi-specific polypeptide construct. Administration of such multi-specific polypeptide constructs can agonize, stimulate, activate, and / or increase innate immune cell effector functions, including ADCC, cytokine release, degranulation, and / or ADCP. In the case of a constrained multi-specific polypeptide construct, administration of such a multi-specific polypeptide construct can activate T-cells once the linker connecting the first and second components is cleaved by a protease and / or upon binding of the tumor antigen on a target cell (e.g., a tumor cell), thereby allowing the anti-CD3 binding moiety to bind to CD3 epsilon on the T cell. In certain cases, administration of a multi-specific polypeptide construct can agonize, stimulate, activate, and / or increase CD3-mediated T cell activation, cytotoxicity, cytokine release, and / or proliferation.

[0428] In certain embodiments, the provided methods are used to treat a disease or disorder in a subject by administering a therapeutically effective amount of any of the provided PD-1-binding polypeptides or engineered cells or pharmaceutical compositions thereof. In certain embodiments, the disease or disorder is a tumor or cancer. Generally, reduction or treatment of a disease or disorder involves reduction of one or more symptoms or medical issues associated with the disease or disorder. For example, in the case of a cancer, a therapeutically effective amount of an agent can achieve one or more of the following: reduce the number of cancer cells; reduce tumor size; inhibit (i.e., reduce to some extent and / or stop) cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more symptoms associated with the cancer. In certain embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in a subject, e.g., a human or other mammal, such as a non-human primate, a companion animal (e.g., cat, dog, horse), a farm animal, a work animal, or a zoo animal. The terms subject and patient are used interchangeably herein.

[0429] In certain embodiments, the PD-1-binding polypeptides or engineered cells or pharmaceutical compositions thereof can be used to inhibit the growth of mammalian cancer cells, such as human cancer cells. A method of treating a cancer can include administering to a subject having a cancer an effective amount of any of the pharmaceutical compositions described herein. An effective amount of a pharmaceutical composition can be administered to inhibit, stop, or reverse the progression of a cancer. Human cancer cells can be treated in vivo or ex vivo. In ex vivo treatment of a human patient, tissue or fluid containing cancer cells is treated outside the body and the tissue or fluid is then reintroduced into the patient. In certain embodiments, a cancer is treated in a human patient by administering a therapeutic composition into the patient.

[0430] Non-limiting examples of diseases include: all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, ischemia, etc. For example, indications will include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic disorders, including multiple myeloma, and solid tumors, including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple negative breast cancer. For example, indications include bone disease or cancer metastasis, regardless of the origin of the primary tumor; breast cancer, including, as non-limiting examples, ER / PR+ breast cancer, Her2+ breast cancer, triple negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as, as non-limiting examples, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, such as, as non-limiting examples, renal cell carcinoma; and / or skin cancer, such as, as non-limiting examples, squamous cell carcinoma, basal cell carcinoma, or melanoma. In certain embodiments, the cancer is squamous cell carcinoma. In certain embodiments, the cancer is cutaneous squamous cell carcinoma. In certain embodiments, the cancer is esophageal squamous cell carcinoma. In certain embodiments, the cancer is head and neck squamous cell carcinoma. In certain embodiments, the cancer is lung squamous cell carcinoma.

[0431] In certain embodiments, the PD-1 -binding polypeptide or engineered cell or pharmaceutical composition thereof can be used to treat, ameliorate, alleviate symptoms of, and / or delay progression of a cancer or other neoplastic disorder. In certain embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, blastoma cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer. In certain embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0432] In certain embodiments, a therapeutically effective amount of a PD-1 -binding polypeptide of the present disclosure, such as a fusion protein or a multi-specific polypeptide construct, relates generally to the amount necessary to achieve the intended treatment goal. Generally, a physician will determine the precise amount of a composition of the present disclosure to be administered, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0433] In certain embodiments, a therapeutically effective dose can be, by way of non-limiting example, from about 0.01 pg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the therapeutically effective dose can be, by way of non-limiting example, from about 0.01 mg / kg body weight to about 5-10 mg / kg body weight. Common administration frequencies can range, for example, from twice a day to once a week.

[0434] In certain embodiments, a therapeutically amount of the engineered cell composition of the present disclosure is administered. It can be generally stated that a dose of 10 4 -10 9 cells / kg body weight (such as 10 5 -10 6 cells / kg body weight) of a pharmaceutical composition comprising engineered cells (e.g., T cells) as described herein can be administered, including all integer values within those ranges. The engineered cell composition (such as a T cell composition) can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by monitoring the patient's disease signs and adjusting the treatment accordingly, by those skilled in the medical arts.

[0435] The effectiveness of the treatment is determined in conjunction with any known method for diagnosing or treating the particular disorder. Methods for screening PD-1 -binding polypeptides or engineered cells containing them for the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known in the art. Various methods are known to determine whether administration of a provided PD-1 -binding polypeptide or engineered cell adequately modulates immune activity by eliminating, sequestering, or inactivating immune cells that mediate or are capable of mediating an unwanted immune response; inducing, producing, or turning on immune cells that mediate or are capable of mediating a protective immune response; altering the physical or functional properties of immune cells; or a combination of these effects. Examples of measurements of immune activity modulation include, but are not limited to: checking for the presence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measuring the functional capacity of immune cells, including the ability or resistance to proliferate or divide in response to a signal (such as using T-cell proliferation assays and peptidescan analysis, which is based on 3H-thymidine incorporation following stimulation with anti-CD3 antibodies, anti-T-cell receptor antibodies, anti-CD28 antibodies, calcium ionophore, PMA (phorbol 12-myristate 13-acetate) antigen presenting cells loaded with peptide or protein antigens; B-cell proliferation assays); measuring the ability to kill or lyse other cells (such as cytotoxic T-cell assays); measuring cytokines, chemokines, cell surface molecules, antibodies, and other cellular products (e.g., by flow cytometry, enzyme-linked immunosorbent assays, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measuring biochemical markers of immune cell activation or signaling pathways within immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine, or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array analysis; DNA transcription, mapping or subtractive hybridization using DNA arrays); measuring cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assays, gel electrophoresis to measure DNA laddering, histology; fluorescent caspase assays, Western blot analysis of caspase substrates); measuring genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assays, flow cytometry); and measuring clinical symptoms or outcomes, such as improvement in autoimmunity, neurodegeneration, and other diseases involving self-proteins or self-polypeptides (clinical scores, requirement for other therapies, functional status, imaging studies), for example, by measuring relapse rate or disease severity.

[0436] The provided PD-1-binding polypeptides can also be used in a variety of diagnostic and prophylactic formulations. In one embodiment, the PD-1-binding polypeptides are administered to a patient at risk for developing one or more of the aforementioned disorders. The patient's or an organ's predisposition to one or more disorders can be determined using genotypic, serological, or biochemical markers.

[0437] In another embodiment of the present disclosure, a PD-1-binding polypeptide or engineered cell is administered to a human subject diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Following diagnosis, such a therapeutic agent is administered to alleviate or reverse the effects of the clinical indication.

[0438] Combination therapy

[0439] The PD-1-binding polypeptides or engineered cells of the present disclosure can be administered alone or in combination with other treatment modalities (e.g., other anticancer agents). They can be provided before, substantially simultaneously, or after (i.e., in parallel or sequentially) other treatment modalities. In certain embodiments, the methods of treatment described herein may further include administration of: radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, antisense agents for tumor targets, siRNA agents for tumor targets, microRNA agents for tumor targets, or anticancer agents / tumor agents or biological agents, such as antibodies, cytokines, or receptor extracellular domain-Fc fusions.

[0440] In certain embodiments, the PD-1 -binding polypeptides provided herein are administered concurrently with one or more chemotherapeutic agents, CAR-T (chimeric antigen receptor T-cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules (such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.).

[0441] In certain embodiments, the PD-1-binding polypeptides or engineered cells of the present disclosure are used in combination with other anti-tumor agents, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib),...

Claims

1. A PD-1-binding polypeptide construct comprising at least one heavy chain variable domain only (PD-1 VHH domain) that specifically binds to PD-1, wherein the PD-1 VHH domain comprises: a) the complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 268, the complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 278, and the complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 283; b) CDR1 set forth in SEQ ID NO: 272, CDR2 set forth in SEQ ID NO: 278, and CDR3 set forth in SEQ ID NO: 283; or c) CDR1 shown in SEQ ID NO: 273, CDR2 shown in SEQ ID NO: 278, and CDR3 shown in SEQ ID NO:

283.

2. The PD-1-binding polypeptide construct of claim 1 , wherein the at least one PD-1 VHH domain comprises the sequence shown in any one of SEQ ID NOs: 251-267 or 284, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 251-267 or 284, and binds to PD-1.

3. The PD-1-binding polypeptide construct according to claim 1 or 2, comprising one or more additional binding domains that bind to targets other than PD-1. 4 . The PD-1-binding polypeptide construct according to claim 1 , wherein the PD-1 VHH domain that specifically binds to PD-1 blocks the interaction between PD-1 and PD-L1.

5. The PD-1 -binding polypeptide construct of claim 3, wherein the one or more additional binding domains bind to a co-stimulatory molecule.

6. The PD-1 -binding polypeptide construct of claim 3, wherein the one or more additional binding domains bind to a T-cell surface marker. The PD-1-binding polypeptide construct according to claim 6 , wherein the T-cell surface marker is CD8. The PD-1-binding polypeptide construct according to claim 6 , wherein the T-cell surface marker is CD4.

9. The PD-1-binding polypeptide construct of claim 3, wherein the one or more additional binding domains bind to an immune checkpoint other than PD1.

10. The PD-1 -binding polypeptide construct of claim 3, wherein the one or more additional binding domains bind to an activating receptor on an immune cell.

11. The PD-1 -binding polypeptide construct of claim 3, wherein the one or more additional domains bind to a tumor-associated antigen (TAA).

12. The PD-1 -binding polypeptide construct of claim 3, wherein the one or more additional binding domains bind to a cytokine receptor.

13. The PD-1-binding polypeptide construct according to any one of claims 1 to 3, wherein the polypeptide comprises an immunoglobulin Fc region.

14. An isolated single-domain antibody that binds PD-1, wherein the single-domain antibody comprises: a) the complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 268, the complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 278, and the complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 283; b) CDR1 set forth in SEQ ID NO: 272, CDR2 set forth in SEQ ID NO: 278, and CDR3 set forth in SEQ ID NO: 283; or c) CDR1 shown in SEQ ID NO: 273, CDR2 shown in SEQ ID NO: 278, and CDR3 shown in SEQ ID NO:

283.

15. The isolated single domain antibody according to claim 14, comprising the amino acid sequence shown in any one of SEQ ID NOs: 251-267 or 284, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 251-267 or 284, and binds to PD-1.

16. The isolated single domain antibody of claim 15, wherein the sdAb comprises the amino acid sequence shown in SEQ ID NO: 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 284. A polynucleotide encoding the PD-1-binding polypeptide according to any one of claims 1 or 2.

18. A polynucleotide encoding the single domain antibody according to any one of claims 14-16.

19. A vector comprising the polynucleotide according to any one of claims 17 or 18.

20. A cell comprising one or more polynucleotides according to any one of claims 17 or 18 or a vector according to claim 19.

21. A method for producing a polypeptide, the method comprising: One or more polynucleotides according to any one of claims 17 or 18 or a vector according to claim 19 are introduced into a cell, and the cell is cultured under conditions in which the polypeptide construct is produced.

22. An engineered immune cell comprising a binding molecule comprising the PD-1 -binding polypeptide of any one of claims 1 or 2 or the single domain antibody of any one of claims 14-16, optionally wherein the binding molecule is secretable from the cell.

23. A pharmaceutical composition comprising the PD-1-binding polypeptide according to any one of claims 1 or 2, the single domain antibody according to any one of claims 14-16, or the engineered immune cell according to claim 22.

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