ATP-dependent agonists of immune cell function as anticancer agents

Abstract

The present disclosure provides polypeptide constructs that act as agonists of immune cell function when exposed to sufficient levels of ATP to cause their assembly into dimers or higher level complexes (e.g., trimers, tetramers, etc.). The complexes of the constructs can stimulate immune cells (e.g., cytotoxic CD8+ T cells and / or NK cells) that function to promote anti-tumor immune responses. The constructs may be used as anti-cancer / therapeutic agents for the treatment of solid tumors with elevated levels of ATP.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,529, filed March 1, 2022, U.S. Provisional Patent Application No. 63 / 330,120, filed April 12, 2022, and U.S. Provisional Patent Application No. 63 / 353,896, filed June 21, 2022. (Incorporating sequence tables) The ST.26 XML format Sequence Listing entitled 2974-3_PCT_ST26.xml, having 205,746 bytes, created on March 1, 2023, prepared in accordance with 37 CFR 1.822-1.824, and filed contemporaneously with the filing of this application, is hereby incorporated by reference in its entirety.

[0002] (Introduction) Potent anti-cancer therapies remain limited due to toxic systemic effects that manifest at doses far below the ideal therapeutic dose. Immune agonists, such as cytokines and anti-T cell receptor antibodies, are particularly toxic when delivered systemically and can lead to severe adverse events such as cytokine release syndrome and death. Strategies to increase the effective dose of immunotherapeutic agents in tumors compared to systemic dose / toxicity (i.e., "therapeutic index") include intratumoral administration and targeting of immunotherapy via conjugation with antibodies against tumor-associated antigens ("TAAs"). However, intratumoral delivery is tailored and invasive, and thus limited in its ability to reach large patient populations due to its associated surgical and logistical complexities. New agents targeting TAAs attempt to impart a certain level of conditioning to the immune agonist, with the agonist accumulating to biologically effective concentrations only at sites with a sufficient density of target TAAs. TAA-targeted immune agonists suffer from serious limitations, including, but not limited to, the small number of unique and validated TAAs expressed on the cell surface accessible by immunotherapeutic agents, low efficacy due to low TAA density, cancer evasion due to loss of TAA expression, on-target / off-tumor toxicity due to TAAs that are not truly tumor-specific, and the fact that each TAA requires a different agent with a lower therapeutic reach compared to systemic therapies.

[0003] Increasing immune agonism and / or the stoichiometry of TAA binding has also been explored as a means to enhance the efficacy of TAA-targeted immunotherapy. For example, first-generation bispecific T cell engagers such as blinatumomab contain a single anti-TAA domain paired with a single anti-CD3 domain for a 1:1 stoichiometry. However, in the case of the bispecific T cell engager AFM11, a 2:2 anti-TAA:anti-CD3 stoichiometry was used instead. This format was associated with more potent T cell-mediated tumor cell killing in vitro and, importantly, reduced the number of cytotoxic effector T cells required for tumor cell killing. However, subsequent clinical trials of AFM11 revealed severe toxicity that forced the termination of the study. A conceptually similar stoichiometric increase in the Fc domain of an antibody-like molecule showed a similar increase in efficacy in vitro. Such stoichiometric modifications likely act through enhanced avidity in the TAA and immune receptor binding. In addition, cross-linking of immune receptors, such as Fc receptors or T cell receptors, is known to amplify signaling through these receptors and signaling pathways, resulting in stronger immune responses. Thus, coupling immune receptors with stoichiometrically enhanced immunotherapies may allow for more potent tumor targeting, such as reducing the requirement for effector T cells and antigen density. However, these enhanced immunotherapies have been difficult to apply clinically due to safety concerns.

[0004] Targeted immunotherapy is generalizable to most / all tumors and can circumvent the aforementioned shortcomings of current immunotherapies by taking advantage of tumor characteristics that are critical for tumor survival and highly expressed relative to normal tissues. The tumor microenvironment (TME) comprises the vasculature, immune and supportive cells, extracellular matrix, and local molecules surrounding the tumor. The TME has characteristics common to many different tumor types, such as low pH, high extracellular adenosine triphosphate (ATP) content, limited oxygen levels (hypoxia), leaky vasculature, and the presence of tumor proteases (e.g., MMPs). Among these characteristics, extracellular ATP is an excellent candidate to be exploited for the generation of novel classes of immunotherapeutics, as it is the product of an altered and essential tumor metabolic program common to most, if not all, tumors. Extracellular ATP is also observed at much higher levels in the TME (50-1000 μM) compared to normal tissues (~0.1 μM or less). Summary of the Invention

[0005] The present disclosure includes and provides nucleotide-binding domain (NBD)-containing molecular constructs that enable the use of tumor ATP as a trigger for the conditional (ATP-dependent) assembly of potent cancer therapeutics in tumors. The NBD-containing constructs form dimers or higher order complexes in the presence of ATP levels found in the TME. The NBD-containing constructs find use as therapeutics for treating various cancers, among others. Therapeutics using the constructs are designed to take advantage of the fact that ATP-driven complexation (e.g., dimerization) can result in agents that recruit immune cells to the TME and / or stimulate immune cells in the TME. Stimulation may occur, for example, by multimerizing immune-modulating protein domains presented to immune cells as a result of ATP-driven complexation of the construct, generating a more effective stimulation of immune cells than, for example, a single immune-modulating protein sequence that cannot cause receptor cross-linking. Dimers or other higher order complexes of NBD-containing constructs may also act through other mechanisms, including targeting ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity) of tumor cells. Since elevated ATP levels relative to surrounding tissues are nearly universal in the TME, the constructs provide a mechanism for treating a variety of tumor types. Furthermore, because individual constructs or higher-order constructs that have not undergone assembly into dimers have limited or no ability to provide immune stimulation, elevated ATP levels in the TME that can result in dimerization or complex formation can provide potent local stimulation with less effect in non-tumor tissues where ATP is at lower concentrations. Thus, constructs that can assemble immune-modulating amino acid sequences into a multivalent state in the presence of ATP levels found in the TME can be used to modulate immune cell action in the TME, providing selective immune-mediated anti-tumor effects.

[0006] NBD-containing constructs also offer several other advantages, including trans-targeting and cis-targeting. Cis-targeting with NBD-containing constructs can result in stimulation of tumor-infiltrating leukocytes (e.g., by providing immune-stimulating agonists such as IL-2 or CD28), resulting in amplification of anti-tumor immune responses. When trans-targeted to a TAA, the constructs result in immune-mediated cytolysis of tumor cells bearing the TAA, which adds additional tumor-specific effects beyond those resulting from local elevations in ATP levels. Regardless of the targeting scheme used (e.g., cis-targeting or trans-targeting), the constructs allow for the use of a variety of immune modulator domains, e.g., from CD40L, CD28, IFN-g, IL-12, etc., each of which acts through a receptor that requires cross-linking. Other advantages of the NBD-containing constructs described herein include their relatively small size, which allows penetration into tumor tissue, and reduced diffusion from the TME, due to the increased size at which they assemble into dimers or higher order complexes.

[0007] In addition to describing the constructs, the disclosure also provides methods for their preparation and methods of their use. [Brief description of the drawings]

[0008] [Figure 1]FIG. 1 provides in A a diagram of a construct of the present disclosure, comprising an NBD with an ATP binding site, a scaffold domain (scaffold amino acid sequence) linked by a linker sequence, and an immune cell activation domain (AD). The right side of the diagram in A also shows dimerization of the construct in the presence of a sufficient ATP concentration (e.g., a concentration in the range of the Kd of ATP binding to the NBD or higher) such that the binding site is substantially occupied. In B, FIG. 1 provides (i) a first construct comprising a tumor-specific binding agent (exemplified as a bivalent cell lineage or tumor-specific antibody, but which may also be a monovalent binding agent such as an scFv or nanobody) and a first NBD of a heterodimer-forming NBD pair linked by a linker, and (ii) a second construct comprising a second NBD of the heterodimer-forming NBD pair linked by a linker. The right side of the diagram in B shows the formation of a heterodimer of the first and second constructs by dimerization of the NBD domains in the presence of ATP.

[0009] [Diagram 2] Figure 2 provides a series of constructs (structures A-E) that include NBD and AD, with constructs C, D, and E also including scaffold sequences that may be non-dimerizing or mutually specific. The elements of each construct are linked by an optional linker that is independently selected. The figure also shows the formation of dimers of the constructs in the presence of ATP.

[0010] [Diagram 3]FIG. 3 provides a series of construct pairs (structures A-O). In A-I, the first construct of each pair includes one or more NBDs and a tumor-specific binding agent that binds to a TAA, and the second construct of the pair includes an NBD and an AD and / or a monovalent immune cell engager. In JM, the first construct of each pair includes one or more NBDs and an immune cell engager, and the second construct of the pair includes an NBD and an AD. Construct pairs E-I, L, and M each have a scaffold in at least one of the constructs. In N and O, the first construct of each pair includes a tumor-specific binding agent and two NBDs, and the second construct of the pair includes an NBD and an AD. The O construct also includes a scaffold sequence. FIG. 3 also shows the formation of a dimer of the constructs in the presence of ATP. The elements of each construct are linked by any linker that is independently selected.

[0011] [Figure 4A] Figure 4A provides a series of constructs (see A, C, and D), pairs of constructs (B, E, F, G, and I), or triplets of constructs (H) that contain two or more NBDs and can polymerize to form complexes in the presence of ATP (e.g., in the TME). In some examples, one or more of the constructs contain a scaffold sequence, and each construct can be linked by any independently selected linker. The figure also shows the formation of a complex in the presence of ATP that contains the constructs present in each case.

[0012] [Figure 4B]FIG. 4B shows an example of an NBD bound to a tumor-specific binding agent (e.g., an antibody such as anti-mesothelin) that forms homodimers in the presence of elevated ATP, as found in the tumor environment. In (A), the construct includes a bivalent antibody that may be monospecific or bispecific, thus resulting in a tetramer or tetramer-like complex in the presence of ATP. The molecule need not include an Fc domain, but may have two antigen-binding domains (e.g., a VHH or scFV tumor-specific antigen binding agent) and still remain bivalent. In (B), (C), and (D), the homodimer-forming construct includes an NBD monovalent single-chain antibody. The tumor-specific binding agent in (A)-(D) may be, for example, anti-HER2, anti-mesothelin, anti-EpCAM, anti-CLA-4, or anti-PSMA. When scaffold sequences are present, they may be immunoglobulin Fc peptides (such as IgG1 Fc) capable of binding to any one or more of the FcγRI, FcγRII, FcγRIII receptors, and / or C1q protein. Conjugates capable of binding to one or more of these proteins can provide antibody-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) directed against cells expressing a target (e.g., an antigen) to which the tumor-specific binding agent component can bind. Thus, such antibody functions can be used therapeutically to eliminate tumor cells.

[0013] [Diagram 5] FIG. 5 shows a dimer of the transporter associated with antigen processing 1 (TAP1) NBD containing two ATP molecules.

[0014] [Figure 6]FIG. 6 shows an alignment of the NBD domains of wild-type TAP1 (SEQ ID NO: 9), the ABCA1 gene product from UniProtKB-O95477 (SEQ ID NO: 1), the ABCB1 gene product from UniProtKB-P08183 (SEQ ID NO: 2), the ABCC1 gene product from UniProtKB-P33527 (SEQ ID NO: 3), the ABCD1 gene product from UniProtKB / Swiss-Prot: P33897 (SEQ ID NO: 4), the ABCE1 gene product from UniProtKB / Swiss-Prot: P61221 (SEQ ID NO: 5), the ABCF1 gene product from UniProtKB-Q8NE71 (SEQ ID NO: 6), and the ABCG2 gene product from UniProtKB / Swiss-Prot: P45844 (SEQ ID NO: 7). The Walker A region sequence from amino acids 46 to 55 of the TAP1 amino acid sequence and a portion of the LSGGQ sequence from amino acids 152 to 156 are underlined and bold. The hydrolyzed acidic amino acid (i.e., Asp at position 177) and Asp (D) or Glu (E) residues of the TAP1 NBD shown in Figure 3 are indicated.

[0015] [Figure 7] FIG. 7 shows the sequence of human TAP2 from UniProtKB-Q03519 (SEQ ID NO: 11).

[0016] [Figure 8] Figure 8 shows an SDS-polyacrylamide gel electrophoresis (PAGE) gel of TAP1 variants with N676G, S677N, Q680R, E682Q, and / or Q683R substitutions. From left to right, the lanes are molecular weight markers (M), reduced samples (R), an empty lane, and non-reduced samples (NR).

[0017] [Figure 9]FIG. 9 shows size-exclusion chromatograms of TAP1 NBD variant polypeptides having N676G, S677N, Q680R, E682Q, and / or Q683R substitutions of cysteines present in the sequence, resulting in (i) a monomeric form of the polypeptide (apparent Mw 37 kDa) in the presence of 1 mM adenosine diphosphate (ADP), and (ii) a dimeric form of the polypeptide (apparent Mw 57 kDa) in the presence of 1 mM adenosine triphosphate (ATP).

[0018] [Figure 10] Figure 10 shows the results of the molecular mass photometric evaluation of the same TAP1 NBD mutants chromatographed in Figure 9. The results obtained in the presence of 1 mM ATP indicate dimerization of the TAP1 NBD variants.

[0019] [Figure 11] FIG. 11 provides the results of dynamic light scattering (DLS) sizing of TAP1 in the presence of (A) ADP or (B) ATP.

[0020] [Figure 12] FIG. 12 provides a Coomassie blue stained gel of purified TAP1 NBD of SEQ ID NO:87 modified for homodimerization and chromatographic analysis of the purified protein in the presence of ADP and ATP.

[0021] [Figure 13] FIG. 13 shows (A) an SDS-PAGE gel of inducible expression of TAP1 NBD polypeptides provided by SEQ ID NO:87 and SEQ ID NO:88, and (B) purification of the polypeptide of SEQ ID NO:88. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] A.Definition As used herein, amino acids (singular "aa" or plural "aas") refer to naturally occurring proteinogenic amino acids incorporated into polypeptides and proteins in mammalian cell translation, unless otherwise indicated, as follows: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan). Amino acids also include hydroxyproline and selenocysteine, which are amino acids present in some proteins found in mammalian cells, but are not to be understood as included unless their presence is expressly indicated.

[0023] Substitutions of an amino acid at a particular position in a sequence are indicated by the original amino acid given in a single-letter or three-letter code, the numerical position of that amino acid, and the amino acid that is substituted into the sequence in a single-letter or three-letter code. As an example, an alanine (A) at position 12 of a sequence substituted with a proline (P) is indicated as A12P using the single-letter code, or Ala12Pro using the three-letter code. When two or more substitutions are present in the same sequence, they may be separated by a slash " / ". Thus, the substitution of V23A and Q41G and the aforementioned substitution of A12P in the same sequence may be indicated by the sequence A12P / V23A / Q41G.

[0024] The terms "polypeptide" and "protein" are used interchangeably herein and, unless otherwise specified, refer to polymeric forms of amino acids that are naturally occurring proteinogenic L-amino acids that are biosynthetically incorporated into proteins during translation in mammalian cells.

[0025] Alignments to identify corresponding residues in divergent sequences may be performed using the National Center for Biotechnology Information (NCBI) BLAST program (blast release of BLAST+ 2.9.0 available on the world wide web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) or the European Bioinformatics Institute's Clustal Omega program (version 1.2.4 available at www.ebi.ac.uk / Tools / msa / clustalo / ) using default parameters. Unless otherwise stated, Clustal Omega is used to identify corresponding residues in divergent sequences.

[0026] Percent sequence identity refers to the percentage of amino acids or nucleotides that are the same between two aligned amino acid or nucleic acid sequences. Unless otherwise specified, percentage sequence identity is determined using BLAST+ release 2.9.0 with default parameters for Blast alignment for polypeptides or Blast alignment for nucleic acids.

[0027] The term "substantially" is intended to encompass both "entirely" and "largely, but not entirely," unless otherwise indicated. For example, an Ig Fc that "does not substantially induce cytolysis via ADCC or CDC" means an Ig Fc that does not induce cytolysis at all, or that induces little, but not completely, cytolysis via ADCC or CDC.

[0028] B. Description The tumor microenvironment differs from non-malignant tissue in many ways, including differences in any one or more of its pH, ATP content, limited oxygen levels (hypoxia), and tumor protease (e.g., MMP) levels. Extracellular ATP levels in normal (non-tumor) tissues are typically in the range of 0.01-0.1 micromolar (μM) in normal tissues, while extracellular levels of ATP in solid tumors are generally in the range of 50-200 μM. The present disclosure describes and provides for the use of ATP levels found in the tumor microenvironment to effect an immune response against cells in the tumor. More specifically, some constructs described herein may include an immune cell activation domain ("AD") (i.e., an activation domain of an immunomodulatory molecule that binds to and stimulates immune cells) and at least one NBD that forms substantial amounts of dimers or other higher order complexes in the presence of ATP at levels found in the tumor microenvironment (TME). Dimerization or higher order complex formation of NBD results in a complex that presents two or more immune cell ADs. The use of immune cell ADs that can stimulate immune cells such as NK cells and CD8+ T cells when presented as dimers or higher order complexes, but do not stimulate the same immune cells to the same extent when presented alone, allows for intratumoral stimulation of an immune response within the TME.

[0029] Other constructs described herein may comprise a tumor-specific binding domain that binds to a TAA or immune cell-inducing domain, and at least one NBD that forms a substantial amount of dimers or other higher-order complexes in the presence of ATP at levels present in the tumor microenvironment. Such constructs may optionally comprise an AD. NBD dimerization or higher-order complex formation results in a dimer or higher-order complex that can recruit and / or stimulate immune cells in the TME. Such construct dimerization or other higher-order complexes allow for intratumoral stimulation of immune responses while limiting immune stimulation outside the tumor, since they do not form higher-order complexes outside the TME.

[0030] The AD, tumor-specific binding domain, and immune cell engager of the uncomplexed constructs, when presented alone, do not effectively stimulate cells such as NK cells and CD8+ T effector cells, so the constructs do not result in systemic immune activation and associated toxic effects. However, the constructs described herein provide immune stimulation that is localized to the TME where there is sufficient ATP to promote complex formation, and thus may be considered a form of ATP-dependent agonist of immune cell function whose action is restricted to the environment. In addition, the complexed forms of the constructs (e.g., dimers, trimers, etc.) have increased size and molecular weight, further reducing their diffusion from the TME and restricting their action to the TME. These dimers or higher order complexes that diffuse out of the TME dissociate into ineffective individual monomers because the ATP levels in the surrounding non-malignant tissues are too low to support the complexes. Thus, it is understood that the immune stimulation provided by the complexes of the constructs described herein is reversible and restricted to microenvironments where ATP levels are high enough to induce complexation.

[0031] C. Structures of ATP-dependent agonists of immune cell function 1. ATP-dependent agonists of immune cell function including NBD and AD In its simplest form, the NBD-containing molecular construct described herein comprises an NBD and an AD that binds and stimulates immune cells (e.g., CD8+ T cells, NK cells, etc.). The NBD comprises one or more ATP binding sites that, when occupied by ATP, two or more molecules of the construct form a duplex or higher order complex (e.g., a triplex) through interactions between the NBDs of the construct. Such molecular constructs optionally comprise one or more independently selected scaffold amino acid sequences and / or one or more independently selected linker sequences. See, for example, Structure A in FIG. 1 and Structure AD in FIG. 2. In some instances, interspecies scaffold sequences that form heterodimers may be incorporated into the construct, such that there may be more than one type of AD in the molecule, as illustrated in Structure E in FIG. 2. By providing ADs together (e.g., as homodimers or heterodimers), the ADs can be used to stimulate targeted immune cell populations, such as NK cells and / or T cells, allowing for selective stimulation of intratumoral immune responses due to high levels of ATP in the tumor microenvironment, while avoiding systemic activation (e.g., NK cells or T cells) and / or immune responses in non-tumor tissues.

[0032] The NBD amino acid sequence, the scaffold amino acid sequence, and the AD can be organized in any order. In the first case, the construct comprises (e.g., from N-terminus to C-terminus) the NBD amino acid sequence, the scaffold amino acid sequence, and the AD. In the second case, the construct comprises from N-terminus to C-terminus the NBD amino acid sequence, the AD, and the scaffold amino acid sequence. In the third case, the construct comprises from N-terminus to C-terminus the AD, the scaffold amino acid sequence, and the NBD amino acid sequence. In the fourth case, the construct comprises from N-terminus to C-terminus the AD, the NBD amino acid sequence, and the scaffold amino acid sequence. In the fifth case, the construct comprises from N-terminus to C-terminus the scaffold amino acid sequence, the AD, and the NBD amino acid sequence. Finally, in the sixth case, the construct comprises from N-terminus to C-terminus the scaffold amino acid sequence, the NBD amino acid sequence, and the AD.

[0033] 2. NBD-Containing Constructs Containing Tumor-Specific Binding Agents and / or Immune Cell Engagers The NBD-containing constructs described herein may also include one or more tumor-specific binding domains and / or immune cell engaging domains in addition to the NBD. As discussed in more detail below, the tumor-specific binding domain, also referred to as the tumor-specific binding agent, is a domain of the NBD-containing construct that has affinity for a TAA expressed on the surface of a tumor cell. The immune cell engaging domain, also referred to as the immune cell engager, is a domain of the NBD-containing construct that has affinity for a cell surface molecule (e.g., an antigen) of an immune cell. Unless otherwise stated, the immune cell engagers, which may be, for example, antibody fragments, are monovalent to avoid the possibility of off-target stimulation (e.g., systemic stimulation) of the immune cells to which they bind. The NBD-containing constructs that include one or more tumor-specific binding agents and / or immune cell engagers may also include one or more ADs. See structures D, E, H, and I in FIG. 3.

[0034] The tumor-specific binding agent and immune cell engager may be incorporated into an NBD-containing construct that forms a heterodimer or other higher order complex (e.g., trimer) in the presence of ATP, for example, at concentrations found in the TME environment. Heterodimer formation may be driven by incorporating a first heterodimer-forming NBD into a first member of the pair of constructs and a second heterodimer-forming NBD into a second member of the pair of constructs. The first and second heterodimer-forming pair of NBDs act as cognate binding partners. To illustrate the types of such NBD-containing construct pairs encompassed by the present disclosure, a non-limiting set of examples of such NBD-containing construct pairs is shown in FIG. 3. In vivo, the dimerized construct directs immune cells bound by the immune cell engager to interact with target tumor cells recognized by the tumor-specific binding agent, resulting in an immune response against the target tumor cells. Such constructs require elevated ATP for complex formation, thus allowing targeting of tumor cells in the TME, thereby limiting off-target binding and stimulation of immune cells. Paired constructs comprising a tumor-specific binding agent and an immune cell engager may be enhanced by the incorporation of one or more ADs, which may be the same or different, and may require dimerization brought about by the interaction of a first and a second NBD-containing construct to stimulate target immune cells.

[0035] Entries A and B of FIG. 3 each provide a pair of constructs: a first NBD-containing construct comprising one or more first heterodimerizing NBDs linked to a tumor-specific binding agent (e.g., N- to C-terminus or C- to N-terminus) and a second NBD-containing construct comprising a cognate second heterodimerizing NBD and AD (e.g., N- to C-terminus). The elements of the constructs are linked by any linker sequence that is independently selected. Such constructs allow targeting of the AD to tumor cells in the TME, since elevated ATP is required to heterodimerize, thereby limiting off-target binding and avoiding systemic stimulation of immune cells by the AD. Additionally, the AD of the construct described in B may be the same or different and may require dimerization to function on the target immune cells. Although not shown in FIG. 3 for A or B, either or both of the heterodimerizing constructs of A or B may also include a scaffold amino acid sequence (see, e.g., entry E of FIG. 3).

[0036] An exemplary pair of constructs exhibiting the type of heterodimerization shown in entries A or B of Figure 3 may include, for example, (i) a first NBD-containing construct that includes one or more TAP1 NBDs linked to a tumor-specific binding agent having affinity for a TAA (e.g., NY ESO-1, mesothelin, CEA, PSMA, or a MAGE such as MAGE-A1, MAGE-A4, MAGE-A9, or MAGE-A11), and (ii) a second NBD-containing construct that includes a TAP2 NBD that acts as a cognate binding partner for the TAP1 NBD in the presence of ATP and IL-2 sequences. Upon exposure to ATP at levels present in the TME, such constructs can heterodimerize and act to stimulate T cells present in the TME.

[0037] Entries C and D of FIG. 3 each provide a construct of a heterodimerization pair in which a first NBD-containing construct comprises one or more first heterodimerization NBDs linked to a tumor-specific binding agent (e.g., N- to C-terminus), and a second NBD-containing construct comprises a cognate second heterodimerization NBD and an immune cell engager (e.g., N- to C-terminus). The elements of the construct are linked by an optional linker sequence, which is independently selected. Either or both of the first and second constructs of the heterodimerization pair may comprise a scaffold sequence (see, e.g., FIG. 3 in F-I). Constructs comprising a tumor-specific binding agent and / or an immune cell engager may further comprise an AD, as in entries D, E, H, and I. If the AD is present in a construct that also includes an immune cell engager, the AD may be selected from those that require presentation to T cells as a dimer or higher order complex (e.g., a TNF family member such as 4-1BBL) for immune cell stimulation to prevent off-target stimulation.

[0038] A pair of constructs, illustrated in entry C of Figure 3, includes, for example, (i) a first NBD-containing construct comprising (a) a TAP1 NBD linked to (b) a tumor-specific binding agent, and (ii) a second NBD-containing construct comprising (a) a TAP2 NBD that acts as a cognate binding partner of the TAP1 NBD in the presence of ATP, and (b) a monovalent T cell engager (e.g., an anti-CD3 antibody fragment or a single chain antibody construct such as a nanobody or scFv). The tumor-specific binding agent may be an antibody, antibody fragment, or single chain antibody construct such as a scFv or nanobody having affinity for a TAA expressed on the surface of tumor cells (e.g., NY ESO-1, mesothelin, CEA, PSMA, or a MAGE such as MAGE-A1, -A4, -A9, -A11).

[0039] Entries A-I of FIG. 3 appear as combinations of two molecular constructs. The first construct containing the tumor-specific binding agent in these combinations is separate from the second construct containing the AD and / or immune cell engager, so that a population (mixture) containing two or more types of first or second constructs can be formed. For example, a mixture of the first and second constructs shown in any one of entries A-I of FIG. 3 can be formed, the mixture containing two or more first constructs, each with a tumor-specific binding agent targeting a different TAA. A mixture of the first and second constructs shown in any one of entries A or B of FIG. 3 can be formed, the mixture containing two or more second constructs, each with one or more ADs directed to a different immune cell receptor. Similarly, a mixture of the first and second constructs shown in any one of entries C-I of FIG. 3 can be formed, the mixture containing two or more second constructs, each with a different immune cell engager directed to a different immune cell surface antigen. In addition to populations that vary in only one of the immune cell engager, tumor-specific binding agent, or AD, the mixture of first and second constructs can include two or more first and second constructs that vary in two or more elements selected from the immune cell engager, the tumor-specific binding agent, and the AD.

[0040] Entries J-M of FIG. 3 each provide a pair of constructs, where a first NBD-containing construct comprises one or more first heterodimerizing NBDs (e.g., N-terminus to C-terminus) linked to an immune cell engager, and a second NBD-containing construct comprises a cognate second heterodimerizing NBD and an AD (e.g., N-terminus to C-terminus). The elements of the constructs are linked by any linker sequence that is independently selected. Such constructs can enable activation of immune cells in the TME while avoiding substantial activation outside of the TME because they require elevated ATP to heterodimerize, thereby limiting off-target binding and avoiding systemic stimulation of immune cells by the AD. Additionally, the ADs of the constructs described in either K or M can be the same or different and require dimerization to function on the target immune cells.

[0041] An exemplary pair of constructs showing heterodimerization constructs of the type seen in Figure 3 at J and K includes, for example, (i) a first NBD-containing construct comprising one or more TAP1 NBDs linked to an immune cell engager (e.g., an antibody, antibody fragment, or single chain antibody construct such as a VHH or nanobody) with affinity for CD28 expressed on the surface of T cells, and (ii) a second NBD-containing construct comprising a TAP2 NBD that acts as a cognate binding partner for the TAP1 NBD in the presence of ATP and IL-2 sequences. When complexed into a heterodimer by ATP at levels present in the TME, such a construct can act to stimulate T cells present in that environment.

[0042] Compositions comprising mixtures of constructs find use in therapeutic applications where the use of two or more tumor-specific binding agents directed against different TAAs can avoid tumor escape from monotherapy with a single construct. Similarly, the use of two or more immune cell engagers and / or ADs can recruit two or more types of immune cells and provide a more vigorous immune response against tumor cells, thereby resulting in a more robust immune response against the tumor than occurs using monotherapy with a single construct.

[0043] 3. Formation of complexes of NBD-containing constructs containing two or more NBDs and NBD-containing molecular constructs The NBD-containing constructs described herein may contain two or more NBDs that allow the construct to effectively polymerize into a complex in the presence of ATP. The two or more NBDs may be arranged, for example, as a tandem pair. When arranged in tandem, the NBDs are adjacent to each other in the construct and are not separated by any elements of the construct other than an intervening linker amino acid sequence (e.g., no AD, scaffold, tumor-specific binding domain, or immune cell attracting domain is present between the tandem NBDs). Additional NBDs may be located in the construct such that they are not arranged in tandem. For example, NBD-containing constructs containing two or more NBDs may be formed by replacing the NBDs in the first to sixth cases described above in Section C.1 (ATP-dependent agonists of immune cell function comprising an NBD and an AD) with a tandem pair of NBDs. NBD-containing constructs containing two or more NBDs (e.g., in tandem) may also be formed from constructs containing immune cell engagers or tumor-specific binding agents.

[0044] Complexes of NBD-containing molecular constructs containing at least two NBDs can be formed by placing the constructs in an environment such as the TME where ATP levels are high enough for ATP to occupy binding sites. Such complexes are illustrated in Figure 3, N and O, and Figure 4A. Here, two or more NBD amino acid sequences are incorporated into the constructs of the present disclosure, and the constructs can, in fact, polymerize reversibly in the presence of ATP to produce complexes. The complexes formed in the presence of ATP can be dimers, trimers, tetramers, or higher order structures. See, for example, Figure 4A. Incorporating two or more NBDs into a construct can maximize potency and efficacy and can affect other properties. For example, a complex that is multivalent for an element expressed on the surface of a target cell (e.g., for a tumor-specific engager or AD) may show increased effective affinity for the target cell, and larger complexes will remain in the TME longer, thereby effectively extending the in vivo half-life of the complex, and other effects.

[0045] The complex may comprise at least two NBDs arranged in tandem, with up to a linker amino acid sequence (e.g., a rigid linker) separating the NBD amino acid sequence (Figure 4A, structures A, B, and reference E) and the AD, immune cell engager, and / or tumor-specific binding agent. Alternatively, the NBDs may be separately attached to the tumor-specific binding agent or immune cell engager (see Figure 4A, structures C and D). The NBD-containing constructs used to form the complex may contain homodimerizing or heterodimerizing NBDs. Although not shown in Figure 4A, homodimerizing NBDs in the same construct molecule may be prevented from self-associating by constructing the molecule such that a productive ATP-binding interface between the domains is not formed. This may be accomplished, for example, by restricting the distance between the NBDs or by using a rigid peptide linker between the NBDs.

[0046] NBD-containing constructs comprising at least two NBDs and an AD are illustrated by structures A and B in FIG. 4A. Structure A uses an NBD that can homodimerize in the presence of ATP to form dimers and / or larger constructs as shown in FIG. 4A by structure A. In contrast, structure B includes structures that form heterodimers or polymers in the presence of ATP. When exposed to ATP at levels present in the TME, the constructs can form large complexes (e.g., as AD homodimers or heterodimers) that present ADs. Such complexes of NBD-containing constructs can stimulate target immune cells such as NK cells and / or T cells in the TME. The ADs present in the complex can be the same (see, e.g., FIG. 4A, structure A) or different (see, e.g., FIG. 4A, structure B). For example, the sequences of the first AD and the second AD present in the complex can be identical and can result in activation of immune cell responses when presented in a dimer or higher order complex. Alternatively, the first and second ADs present in the complex may be from different molecules (e.g., different interleukins) that trigger immune cell activation when presented together. It is also possible that the sequences of the first and second ADs present in the complex are different (non-identical) and, when combined, form an active fragment of a dimeric immunomodulator, such as IL-12, IL-23, or IL-27, each of which contains two subunits (α and β polypeptide chains). NBD-containing constructs, each of which contains a single NBD, may be assembled into a complex containing two different (non-identical) ADs in the presence of ATP. Alternatively, an active AD consisting of two subunits can be assembled into an active AD using constructs each containing a single NBD in the presence of ATP (see, for example, Figure 2, structures A and B).

[0047] In addition to the NBD-containing constructs described above, the present disclosure provides NBD-containing constructs comprising at least two NBDs and a tumor-specific binding amino acid sequence (tumor-specific binder) as illustrated in FIG. 4A, Structure C, and / or an immune cell binding amino acid sequence (immune cell engager) as illustrated in FIG. 4A, Structure D. Such molecular constructs optionally comprise one or more independently selected scaffold amino acid sequences and / or one or more independently selected linker sequences. Tumor-specific binding agents include, but are not limited to, antibodies that bind to TAAs (e.g., HER-2, MAGE-2, MAGE-4, NY-ESO, etc.). Immune cell engagers include molecules including, but are not limited to, antibodies that bind to immune cell surface antigens (e.g., CD3, CD8, or CD4 on αβ T cells, TRGV9 on δγ T cells, CD16 on NK cells, etc.). Unless otherwise specified, immune cell engagers are monovalent to avoid stimulation of immune cells outside the TME and have sequences that may be modified for antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to substantially limit or prevent such responses.

[0048] When ATP levels are present in the TME that allow the NBD domains to form higher order complexes (e.g., dimers), constructs that include at least two NBDs and a tumor-specific binding agent and / or an immune cell engager form large complexes that can result in immune stimulation. Constructs that include a scaffold that includes IgFc and at least two NBDs of a tumor-specific binding agent can form complexes in the TME (see, e.g., FIG. 4A, structure C) that can result in, e.g., ADCC or CDC. Constructs that include at least two NBDs and an immune cell engager can form complexes in the TME (see, e.g., FIG. 4A, structure D) that can result in, e.g., stimulation of target immune cells by cross-linking of target antigens (e.g., CD3 on CD8+ T cells or CD16 on NK cells).

[0049] Combining an NBD-containing construct containing at least two NBDs with a tumor-specific binding agent (see, e.g., FIG. 4A, structure C), as well as an NBD-containing construct containing at least two NBDs and an immune cell engager (see, e.g., FIG. 4A, structure D), can result in a complex containing both the tumor-specific binding agent and the immune cell engager. Using a first heterodimer-forming NBD with a tumor-specific binding agent and a second heterodimer-forming NBD with an immune cell engager can ensure that the complex formed in the presence of ATP contains both the binding agent and the engager and can stimulate an immune cell response against the target tumor cells. This is exemplified with a mixture of constructs containing at least two NBDs and a tumor-specific binding agent and constructs containing at least two NBDs and an immune cell engager that form a large complex in the TME (see, e.g., FIG. 4A structures E and F where the NBDs are shown in a vertical row). The complex resulting from such a structure in the presence of sufficient ATP can bind immune cells to tumor targets via the tumor-specific binding agent and simultaneously effect immune cell activation by crosslinking receptors on the cell surface via the now-complexed monomeric immune cell engager.

[0050] The tumor-specific binding agent and / or immune cell engager amino acid sequences present in each construct used to form the complex need not be the same. Thus, the complexes of FIG. 3, Structures A-I, and FIG. 4A, Structures C-I may have two or more tumor-specific binding agents directed against different TAAs (e.g., two or more tumor-specific binding agents), and, if present, two or more immune cell engagers. The use of two or more different tumor-specific binding agents directed against different TAAs in a single complex may allow a complex formed from a construct containing two or more NBDs to be active against tumors expressing different tumor antigen targets, as well as limit tumor escape from therapy due to loss of tumor antigen expression. The use of different immune cell engagers in a single complex allows stimulation of an immune response from two or more types of immune cells (e.g., both CD8+ T cells and NK cells). It is also possible to incorporate ADs into complexes formed in the TME, including complexes containing constructs that include tumor-specific binding agents. This can be done, for example, by combining one or more constructs consisting of an AD and an NBD (e.g., two or more NBDs) in a construct such as that illustrated in FIG. 4A H, or by combining one or more constructs consisting of an AD, an NBD (e.g., two or more NBDs) and a tumor-specific binding agent in a construct such as that illustrated in FIG. 4A I.

[0051] Because the tumor-specific binding agent, immune cell engager, and AD are provided by different constructs that enter the complex formed in the TME, the composition of the complex can be controlled by mixing the different constructs (e.g., before or during administration to the patient). Thus, a single construct may be used for a combination of different therapies.

[0052] D. Elements of NBD-Containing Constructs 1. Interaction with NBD and ATP NBDs for incorporation into the constructs described herein may be selected based on several criteria. If the constructs are intended for human therapeutic use, the NBDs are preferentially derived from human ATP-binding proteins to limit their immunogenicity and preferably are capable of undergoing solution-phase dimerization or higher complex formation when exposed to sufficient ATP. If non-human proteins that are immunogenic are employed, the immunogenic portions may be humanized by modifying the immunogenic regions to more closely or completely match their human homologs. Solution-phase formation of dimers or higher complexes used in the constructs described herein occurs at ATP concentrations found in the target TME, but not in normal tissues. Thus, NBDs for use in the constructs provided herein may have a dissociation constant for ATP that is about one order of magnitude (10-fold) above the upper limit levels found in the extracellular space of normal tissues (i.e., about 1 μM or higher), to about the ATP levels found in the TME. In some cases, the affinity of the NBD is about 1 μM to about 200 μM. For example, the ATP affinity of the NBD may be in the range of about 1 μM to about 5 μM, or about 5 μM to about 25 μM. The ATP affinity of the NBD may be in the range of about 25 μM to about 50 μM. In other examples, the affinity of the NBD may be in the range of about 50 μM to about 100 μM, or about 100 μM to about 200 μM.

[0053] Some protein NBDs form homodimers in the presence of sufficient levels of ATP, while other proteins form heterodimers in the presence of sufficient levels of ATP. Both homodimer-forming and heterodimer-forming NBDs can be employed in various aspects of the polypeptide constructs described herein. If it is desired that NBDs form homodimers in solution when sufficient levels of ATP are present, amino acid residues at the interface between dimers can be modified to either remove or replace amino acid residues that prevent interface formation between two NBD molecules. Alternatively, amino acids that do not contribute to the interaction between two NBD molecules can be replaced with amino acids that contribute to the interaction that leads to homodimer formation.

[0054] a) ATP-binding cassette as NBD Many proteins contain NBDs suitable for use as NBDs in the constructs described herein. Among these proteins, ATP-binding cassette (ABC) transporters represent suitable candidates in part because they are an abundant transporter family that is highly conserved and unlikely to be immunogenic. ABC transporters are a large superfamily of membrane proteins with diverse functions that utilize energy from ATP hydrolysis to facilitate transport of substrates into or out of the cytoplasm. See, for example, Locher, Philos Trans R Soc Lond B Biol Sci., 364(1514):239-245(2009) and references cited therein. The human genome encodes 48 or 49 distinct ABC transporters. (Ibid.) ABC transporters are divided into seven distinct family members: ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and white. Representative members of each family, including TAP1, are aligned in FIG. 6. The NBDs of the ATP-binding cassette family are conserved and contain two subdomains. (Id.) The first one is similar to the RecA protein and contains a P-loop (Walker-A motif). The second subdomain is called the "helical subdomain" and contains the LSGGQ motif. (Id.) The NBDs of the transporter assemble as dimers in a direct-tail orientation, forming two ATP-binding and hydrolysis sites sandwiched at their interface (FIG. 5). In that orientation, the LSGGQ motifs in each of the two domains flank opposing Walker-A sequences at the ATP-sandwich interface. Such an interface is described, for example, in FIG. 3A of Loo et al., (2002) J Biol Chem., 277(44): 41303-41306, and Smith et al., (2002) Mol Cell., 10(1): 139-149. When no nucleotide (ATP) is present at the binding site, there is a gap at the domain interface, but upon ATP binding, the NBDs can dimerize, hydrolyze ATP, and subsequently dissociate (see, e.g., Locher 2009, supra).Stabilized dimers can be formed by including amino acid substitutions in the NBD domain sequence that render it ATP hydrolysis deficient or substantially ATP hydrolysis deficient (see, e.g., Vakkasoglu et al., (2107) PLoS ONE, 12(5): e0178238, available at doi.org / 10.1371 / journal. pone.0178238). For example, hydrolyzable Asp or Glu residues found in NBD proteins can be replaced by Asn or Gln residues (see, e.g., FIG. 3).

[0055] If it is desired that the NBD of an ABC protein form homodimers in solution when sufficient ATP is present, residues at the interface between the dimers can be modified to either remove or replace amino acid residues that prevent interface formation, or to replace amino acids that do not contribute to the interface interaction with amino acids that may contribute to the interaction. As an example, it is known that the NBD of rat TAP1 homodimerizes in solution and can be used as a model for the formation of an ATP-sandwich interface that leads to solution-phase homodimer formation in the presence of ATP (see, for example, Vakkasoglu et al., (2107) PLoS ONE, 12(5): e0178238. https: / / doi . org / 10.1371 / journal.pone.0178238). Alignment of the NBD amino acid sequence with a sequence known to undergo homodimerization, e.g., rat TAP1 NBD, can be used to induce changes in the target sequence. Alignments can be performed using the National Center for Biotechnology Information (NCBI) BLAST program (blastp release of BLAST+ 2.9.0, available on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) or the European Bioinformatics Institute's Clustal Omega program (version 1.2.4 available at www.ebi.ac.uk / Tools / msa / clustalo / ) using default parameters. Unless otherwise stated, Clustal Omega is employed.

[0056] The NBD amino acid sequence of the ABC-containing protein can include all or a portion of the NBD domain of the phospholipid-transporting ATPase (ABC or ABCA1 transporter) encoded by the ABCA1 gene, UniProtKB-O95477 (SEQ ID NO:1). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:1.

[0057] The NBD amino acid sequence can include all or a portion of the NBD domain of the multidrug resistance protein 1 (MDR / TAP) encoded by the ABCB1 gene, UniProtKB-P08183 (SEQ ID NO:2). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:2.

[0058] The NBD amino acid sequence can include all or a portion of the NBD domain of the multidrug resistance-associated protein (MRP, multidrug resistance-associated protein 1) encoded by the ABCC1 gene, UniProtKB-P33527 (SEQ ID NO:3). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:3.

[0059] The NBD amino acid sequence can include all or a portion of the NBD domain of the ATP-binding cassette subfamily D member 1 protein (ALD, adrenoleukodystrophy protein) encoded by the ABCD1 gene, UniProtKB / Swiss-Prot:P33897 (SEQ ID NO:4). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:4.

[0060] The NBD amino acid sequence can include all or a portion of the NBD domain of the ATP-binding cassette subfamily E member 1 protein (OABP, 2'-5'-oligoadenylate binding protein) encoded by the ABCE1 gene, UniProtKB / Swiss-Prot:P61221 (SEQ ID NO:5). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:5.

[0061] The NBD amino acid sequence may comprise all or a portion of the NBD domain of the ATP-binding cassette subfamily F member 1 protein (GCN20, a TNF-alpha-inducible ATP-binding protein) encoded by the ABCF1 gene, UniProtKB-Q8NE71 (SEQ ID NO:6). The NBD may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:6.

[0062] The NBD amino acid sequence can include all or a portion of the NBD domain of the ATP-binding cassette subfamily G member 1 protein (White, Homolog Of Drosophila White) encoded by the ABCG2 gene, UniProtKB / Swiss-Prot: P45844 (SEQ ID NO:7). The NBD can include a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO:7.

[0063] (1) TAP1 and TAP2 ATP-binding cassettes as NBDs TAP1 and TAP2 proteins are cognate binding partners that act as transporters coupling ATP binding and heterodimerization with peptide translocation across the inner cell membrane. TAP1 and TAP2 are members of the ABC transporter superfamily that represent a source of NBDs for use in the constructs provided herein. The rat TAP1 NBD is known to undergo homodimerization in the presence of ATP. The NBD of human TAP1 does not undergo homodimerization in the presence of ATP, but can be induced to undergo homodimerization using previously defined substitutions. See, for example, Vakkasoglu et al. (2017) “D-helix influences dimerization of the ATP-binding cassette (ABC) transporter associated with antigen processing 1 (TAP1) nucleotide-binding domain,” PLoS ONE, 12(5): e0178238, doi.org / 10.1371 / journal. pone.0178238 on the World Wide Web. Substitutions that lead to TAP1 homodimer formation include those at one or more (e.g., each) of N676, S677, Q680, E682, and Q683, which are depicted in bold italics in SEQ ID NO:8. For example, TAP1 substitutions that lead to homodimer formation can include one or more (e.g., all) of N676G, S677N, Q680R, E682Q, and Q683R substitutions. The portions (e.g., domains) of the TAP1 and TAP2 proteins employed as NBDs can also include substitutions of one or more cysteines present in the sequence (e.g., with serine residues) to prevent undesired disulfide bond formation, resulting in non-functional folding or undesired intermolecular disulfide bond formation. The TAP1 and TAP2 NBDs can also include His tags, for example, at the C-terminus of the domains for purification of constructs containing those NBDs.

[0064] The NBD amino acid sequence from human TAP1 (SEQ ID NO: 9) is described, for example, in NCBI Reference Sequence: NP_000584.3, SEQ ID NO: 8, or in UniProtKB - Q03518-2: [ka] The NBD amino acid sequence may comprise an amino acid sequence having 90% or more, or 95% or more, sequence identity to the NBD amino acid sequence (amino acids 492-748) of.

[0065] The TAP1 NBD domain common to both the NCBI and UniProt sequences is [ka] Residues N676, S677, Q680, E682, and Q683 of SEQ ID NO:8 involved in homodimer formation appear as N185, S186, Q189, E191, and Q192 of SEQ ID NO:9 and are written in bold italics. The NBD amino acid sequence from human TAP1 consists of an amino acid sequence having 97% or more or 98% or more sequence identity to at least 230 consecutive amino acids of the NBD of the amino acid sequence set forth in, for example, NCBI Reference Sequence: NP_000584.2 (amino acids 492-748) provided as SEQ ID NO:9.

[0066] The NBD amino acid sequence from human TAP1 may, for example, contain substitutions at amino acid residues corresponding to C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO:8. The NBD amino acid sequence from human TAP1 may also contain substitutions at amino acids corresponding to one or more of N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) of SEQ ID NO:8, which are depicted in bold italics in SEQ ID NO:10 below. The NBD amino acid sequence from human TAP1 may also contain substitutions at Asp, for example, corresponding to D668 of SEQ ID NO:8, which causes an NBD ATP hydrolysis deficiency. Thus, the NBD polypeptide amino acid sequence of TAP1 may, for example, contain the amino acid sequence of SEQ ID NO:10, or a sequence thereof: [ka] The amino acid sequence may have greater than 90% or greater than 95% sequence identity to Alternatively, the NBD amino acid sequence may have greater than 97% or greater than 98% sequence identity to at least 230 or 240 contiguous amino acids of the sequence provided in SEQ ID NO:10.

[0067] An NBD amino acid sequence from human TAP1 may contain substitutions corresponding to, for example, those at C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO:8. An NBD amino acid sequence from human TAP1 may also contain substitutions corresponding to, for example, those at one or more of N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) in SEQ ID NO:8, which are depicted in bold italics in SEQ ID NO:87 below. Additionally, an NBD amino acid sequence from human TAP1 may contain a substitution with Asp corresponding to D668 in SEQ ID NO:8, which causes an NBD ATP hydrolysis deficiency. Thus, an NBD polypeptide amino acid sequence of TAP1 may contain, for example, the amino acid sequence of SEQ ID NO:87, or a sequence thereof: [ka] The amino acid sequence may have greater than 90% or greater than 95% sequence identity to Alternatively, the NBD amino acid sequence may have greater than 97% or greater than 98% sequence identity to at least 250 or 260 contiguous amino acids of the sequence provided in SEQ ID NO:87.

[0068] An NBD amino acid sequence from human TAP1 may include substitutions corresponding to those at C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO:8. An NBD amino acid sequence from human TAP1 may also include substitutions corresponding to those at one or more of N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) in SEQ ID NO:8, which are bolded and italicized in SEQ ID NO:88 below. Additionally, an NBD amino acid sequence from human TAP1 may include a substitution at an amino acid corresponding to D668 in SEQ ID NO:8 (e.g., a D668N substitution), which causes an NBD ATP hydrolysis deficiency. Thus, an NBD polypeptide amino acid sequence of TAP1 may include, for example, an amino acid sequence of SEQ ID NO:88, or a sequence thereof: [ka] The amino acid sequence may have greater than 90% or greater than 95% sequence identity to Alternatively, the NBD amino acid sequence can have greater than 97% or greater than 98% sequence identity to at least 300 or 310 contiguous amino acids of the sequence provided in SEQ ID NO: 88. The NBD provided in SEQ ID NO: 88 includes, in addition to the TAP1 NBD sequence, coupling helix 1 (CH1, amino acids 2-11) from TAP1, and coupling helix 2 (CH2, amino acids 17-25) from TAP2, as well as associated GS linkers (amino acids 12-16 and 25-30), which together constitute a "cap" on the sequence. TAP1 NBD constructs are expressed at substantially higher levels in biological expression systems, as shown in Example 3.

[0069] In some cases, NBDs from TAP proteins that do not homodimerize but rather heterodimerize with their cognate NBDs are used to form pairs of constructs (e.g., see Figure 2B and D and Figure 3) or complexes of constructs (e.g., Figure 4A) of the present disclosure. For example, a pair of constructs with human TAP1 NBD and human TAP2 NBD can be prepared. A pair of constructs containing TAP1 and TAP2 NBDs form heterodimers through the interaction of TAP1 and TAP2 in the presence of sufficient ATP. By using NBDs such as TAP1 and TAP2 that can heterodimerize, different specific combinations of activation sequences can be combined for immune cell stimulation. As an example, a construct comprising IL-12A(p35), an optional linker amino acid sequence, and TAP1 can heterodimerize with a construct comprising IL-12B(p40), an optional linker amino acid sequence, and TAP2 in the presence of sufficient ATP to form a pair of constructs that present an active IL-12AB heterodimer. Such constructs may also include a scaffold amino acid sequence, such as an Ig Fc amino acid sequence, if desired.

[0070] Figure 7 provides the sequence of human TAP2. In some cases, the NBD of the constructs of the present disclosure may comprise an amino acid sequence from the NBD from human TAP2, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to the human TAP2 NBD. For example, the NBD may comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the NBD amino acid sequence of UniProtKB-Q03519 (TAP2_HUMAN). 1 MRLPDLRPWT SLLLVDAALL WLLQGPLGTL LPQGLPGLWL EGTLRLGGLW GLLKLRGLLG 61 FVGTLLLPLC LATPLTVSLR ALVAGASRAP PARVASAPWS WLLVGYGAAG LSWSLWAVLS 121 PPGAQEKEQD QVNNKVLMWR LLKLSRPDLP LLVAAFFFLV LAVLGETLIP HYSGRVIDIL 181 GGDFDPHAFA SAIFFMCLFS FGSSLSAGCR GGCFTYTMSR INLRIREQLF SSLLRQDLGF 241 FQETKTGELN SRLSSDTTLM SNWLPLNANV LLRSLVKVVG LYGFMLSISP RLTLLSLLHM 301 PFTIAAEKVY NTRHQEVLRE IQDAVARAGQ VVREAVGGLQ TVRSFGAEEH EVCRYKEALE 361 QCRQLYWRRD LERALYLLVR RVLHLGVQML MLSCGLQQMQ DGELTQGSLL SFMIYQESVG 421 SYVQTLVYIY GDMLSNVGAA EKVFSYMDRQ PNLPSPGTLA PTTLQGVVKF QDVSFAYPNR 481 PDRPVLKGLT FTLRPGEVTA LVGPNGSGKS TVAALLQNLY QPTGGQVLLD EKPISQYEHC 541 YLHSQVVSVG QEPVLFSGSV RNNIAYGLQS CEDDKVMAAA QAAHADDFIQ EMEHGIYTDV 601 GEKGSQLAAG QKQRLAIARA LVRDPRVLIL DEATSALDVQ CEQALQDWNS RGDRTVLVIA 661 HRLQTVQRAH QILVLQEGKL QKLAQL (SEQ ID NO: 11) The NBD can include, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to the human TAP2 amino acid sequence. GTLAPTTLQG VVKFQDVSFA YPNRPDRPVL KGLTFTLRPG EVTALVGPNG SGKSTVAALL QNLYQPTGGQ VLLDEKPISQ YEHCYLHSQV VSVGQEPVLF SGSVRNNIAY GLQSCEDDKV MAAAQAAHAD DFIQEMEHGI YTDVGEKGSQ LAAGQKQRLA IARALVRDPR VLILDEATSA LDVQCEQALQ DWNSRGDRTV LVIAHRLQTV QRAHQILVLQ EGKLQK (sequence number 12). The NBD may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:12.

[0071] A mutation that removes the catalytic acidic amino acid residue corresponding to the D668N substitution in TAP1 that results in an ATP hydrolysis defect can be engineered into the TAP2 NBD amino acid sequence.

[0072] (2) CFTR ATP-binding cassette as NBD In some cases, the NBD of the constructs of the present disclosure may comprise an amino acid sequence derived from the NBD from human cystic fibrosis transmembrane conductance regulator (CFTR) or an amino acid sequence having greater than 90% or greater than 95% sequence identity to human CFTR. For example, the NBD may comprise an amino acid sequence having 90% or greater or 95% or greater sequence identity to the NBD amino acid sequence of UniProtKB - P13569 (SEQ ID NO: 13). 1 MQRSPLEKAS VVSKLFFSWT RPILRKGYRQ RLELSDIYQI PSVDSADNLS 51 EKLEREWDRE LASKKNPKLI NALRRCFFWR FMFYGIFLYL GEVTKAVQPL 101 LLGRIIASYD PDNKEERSIA IYLGIGLCLL FIVRTLLLHP AIFGLHHIGM 151 QMRIAMFSLI YKKTLKLSSR VLDKISIGQL VSLLSNNLNK FDEGLALAHF 201 VWIAPLQVAL LMGLIWELLQ ASAFCGLGFL IVLALFQAGL GRMMMKYRDQ 251 RAGKISERLV ITSEMIENIQ SVKAYCWEEA MEKMIENLRQ TELKLTRKAA 301 YVRYFNSSAF FFSGFFVVFL SVLPYALIKG IILRKIFTTI SFCIVLRMAV 351 TRQFPWAVQT WYDSLGAINK IQDFLQKQEY KTLEYNLTTT EVVMENVTAF 401 WEEGFGELFE KAKQNNNNRK TSNGDDSLFF SNFSLLGTPV LKDINFKIER 451 GQLLAVAGST GAGKTSLLMV IMGELEPSEG KIKHSGRISF CSQFSWIMPG 501 TIKENIIFGV SYDEYRYRSV IKACQLEEDI SKFAEKDNIV LGEGGITLSG 551 GQRARISLAR AVYKDADLYL LDSPFGYLDV LTEKEIFESC VCKLMANKTR 601 ILVTSKMEHL KKADKILILH EGSSYFYGTF SELQNLQPDF SSKLMGCDSF 651 DQFSAERRNS ILTETLHRFS LEGDAPVSWT ETKKQSFKQT GEFGEKRKNS 701 ILNPINSIRK FSIVQKTPLQ MNGIEEDSDE PLERRLSLVP DSEQGEAILP 751 RISVISTGPT LQARRRQSVL NLMTHSVNQG QNIHRKTTAS TRKVSLAPQA 801 NLTELDIYSR RLSQETGLEI SEEINEEDLK ECFFDDMESI PAVTTWNTYL 851 RYITVHKSLI FVLIWCLVIF LAEVAASLVV LWLLGNTPLQ DKGNSTHSRN 901 NSYAVIITST SSYYVFYIYV GVADTLLAMG FFRGLPLVHT LITVSKILHH 951 KMLHSVLQAP MSTLNTLKAG GILNRFSKDI AILDDLLPLT IFDFIQLLLI 1001 VIGAIAVVAV LQPYIFVATV PVIVAFIMLR AYFLQTSQQL KQLESEGRSP 1051 IFTHLVTSLK GLWTLRAFGR QPYFETLFHK ALNLHTANWF LYLSTLRWFQ 1101 MRIEMIFVIF FIAVTFISIL TTGEGEGRVG IILTLAMNIM STLQWAVNSS 1151 IDVDSLMRSV SRVFKFIDMP TEGKPTKSTK PYKNGQLSKV MIIENSHVKK 1201 DDIWPSGGQM TVKDLTAKYT EGGNAILENI SFSISPGQRV GLLGRTGSGK 1251 STLLSAFLRL LNTEGEIQID GVSWDSITLQ QWRKAFGVIP QKVFIFSGTF 1301 RKNLDPYEQW SDQEIWKVAD EVGLRSVIEQ FPGKLDFVLV DGGCVLSHGH 1351 KQLMCLARSV LSKAKILLLD EPSAHLDPVT YQIIRRTLKQ AFADCTVILC 1401 EHRIEAMLEC QQFLVIEENK VRQYDSIQKL LNERSLFRQA ISPSDRVKLF 1451 PHRNSSKCKS KPQIAALKEE TEEEVQDTRL

[0073] The NBD can include, for example, an amino acid sequence having greater than 80% or greater than 85% sequence identity to the human CFTR amino acid sequence. VLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGK IKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDF (SEQ ID NO: 14). Alternatively, the NBD may comprise an amino acid sequence having, for example, greater than 90% or greater than 95% sequence identity to SEQ ID NO: 14. The NBD may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 14.

[0074] The NBD can comprise, for example, an amino acid sequence having greater than 80% or greater than 85% sequence identity to the human CFTR amino acid sequence Ser-NBD1 [387-646 (D405-436)] described in Atwell et al., (2010), Protein Engineering, Design & Selection, 23(5) 375-384. SXTTTEVVMENVTAFWEEGGTPVLKDINFKIER GQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILH EGSSYFYGTFSELQNLQPDFSSKLMX (SEQ ID NO: 15), where X at position 2 can be absent or L and X at position 229 can be absent or G. Alternatively, the NBD can comprise an amino acid sequence having, for example, greater than 90% or greater than 95% sequence identity to SEQ ID NO: 15. The NBD can also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 15. An NBD may, for example, comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to amino acids 1-216 of SEQ ID NO: 16, and X at position 2 may be absent or L.

[0075] 2. Scaffolding Scaffold amino acid sequences may be incorporated into the ATP-dependent agonist constructs described herein as a means of structural structure to extend the half-life of the molecule in vivo, and may act as the basis for their effector functions if they can induce ADCC, ADCP, and / or CDC. The scaffold may be capable of forming dimers and higher order complexes (e.g., trimers) or non-dimerizable (non-dimerizing), in which case it does not form dimers or higher order complexes. The scaffolds incorporated into the constructs described herein may include, for example, immunoglobulin (Ig) amino acid sequences or non-immunoglobulin amino acid sequences.

[0076] In some embodiments, the scaffold polypeptide sequence comprises an immunoglobulin heavy chain constant region (CH2-CH3) polypeptide sequence that functions as a dimerization or multimerization sequence. When the scaffold comprises an immunoglobulin (Ig) amino acid sequence, it can comprise an immunoglobulin heavy chain constant region (CH2-CH3) polypeptide amino acid sequence (e.g., an IgFc amino acid sequence). The scaffold amino acid sequence can comprise, for example, an Ig CH2 and / or CH3 amino acid sequence that has been modified to prevent dimerization. Any one or more cysteines involved in interchain disulfide bonds that stabilize Ig heavy chain dimers can be substituted (e.g., with alanine or serine) such that the Ig heavy chain sequence cannot homodimerize to form interchain disulfide bonds. The scaffold polypeptide can comprise a wild-type (wt) Homo sapiens IgG1 Fc polypeptide sequence of (SEQ ID NO: 56). 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK; optionally modified to prevent dimer formation. Alternatively, the scaffold may comprise, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity with SEQ ID NO: 56. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 56.

[0077] The scaffold polypeptide may comprise the Homo sapiens IgG1 Fc amino acid sequence of SEQ ID NO:57, including the LALA substitutions (L234A, L235A substitutions, appearing as amino acids 14 and 15 in bold and italics, respectively). [ka] Optionally, the scaffold is modified to prevent dimer formation. Alternatively, the scaffold may comprise, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 57. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 57.

[0078] Non-dimer-forming scaffolds may be prepared as human single-chain Fc (scFc) dimers. See, for example, Zhou et al., Biomaterials 117:24-31 (2017). Such scFc dimers may be based on IgG1 and may have the following sequence: 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGGGGG SGGGGSGGGG 241 SGGGGSGGGG SGGGGSDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV 301 DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS 361 NKALPAPIEK TISKAKGQPR EPQVYTLPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN 421 GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS 481 PG (SEQ ID NO: 102). Alternatively, the scaffold may comprise, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 102. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 102. The scaffold construct of SEQ ID NO: 102 is capable of inducing ADCC, ADCP, and CDC, although substitutions including "LALA" discussed below may be introduced to selectively alter the ability of the scFc to induce their effector functions.

[0079] The scaffold polypeptide may comprise the wt Homo sapiens IgG2 Fc polypeptide of SEQ ID NO:58 (see GenBank AAN76044 amino acids 99-325): 1 STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 61 LYSLSSVVTV PSSNFGTQTY TCNVDHKPSN TKVDKTVERK CCVECPPCPA PPVAGPSVFL 121 FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTFRV 181 VSVLTVVHQD WLNGKEYKCK VSNKGLPAPI EKTISKTKGQ PREPQVYTLP PSREEMTKNQ 241 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPMLDSDG SFFLYSKLTV DKSRWQQGNV 301 FSCSVMHEAL HNHYTQKSLS LSPGK; optionally modified to prevent dimer formation. Alternatively, the scaffold may include, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 58. The scaffold may also include an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 58.

[0080] The scaffold polypeptide can include the wt Homo sapiens IgG3 Fc polypeptide of SEQ ID NO:59 (see, e.g., GenBank AAW65947 amino acids 19-246). 1 HKPSNTKVDK RVELKTPLGD TTHTCPPCPA PELLGPSVF LFPPKPKDTL MISRTPEVTC 61 VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 121 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW 181 ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 241 SLSPGK; optionally modified to prevent dimer formation. Alternatively, the scaffold may comprise, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 59. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 59.

[0081] The scaffold polypeptide may comprise a wt Homo sapiens IgG4 Fc polypeptide of SEQ ID NO:60. 1 PPCPSCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE 61 VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP 121 REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 181 FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SPG; optionally modified to prevent dimer formation. Alternatively, the scaffold may comprise, for example, an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 60. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 60.

[0082] For example, the interactions of IgG class antibodies that result in ADCC and ADCP occur through their Fc region binding to members of the Fcγ family of receptors (FcγR). The human protein family consists of FcγRI (CD64), FcγRII (CD32, which contains the isoforms FcγRIIa, FcγRIIb, and FcγRIIc), and FcγRIII (CD16, which contains the isoforms FcγRγIIIa and FcγRIIIb). See, for example, Lazar et al. (2006) PNAS:103(11), 4005-4010.

[0083] Among the variants that can enhance one or more antibody-related effector functions (e.g., ADCC, ADCP, and / or CDC responses), there are the individual substitutions of S239D and I332E in human IgG1, as well as double and triple substitutions at S239D / I332E and S239D / I332E / A330L. The substitutions corresponding to S239D, I332E, and A330L in the human IgG1 Fc sequence (SEQ ID NO:56) are S19D, I112E, and A110L. In one embodiment, the substitution is the double mutant S239D / I332E or S239D / I332E / A330L. See Lazar et al., 2006.

[0084] Other substitutions in IgG1 antibodies that enhance one or more antibody-associated effector functions include, but are not limited to, the following: Ser298Ala / Glu333Ala / Lys334Ala; Ser239Asp / Ala330Leu / Ile332Glu; Ser239Asp / Ile332Glu; Gly236Ala / Ser239Asp / Ala330Leu / Ile332Glu; Gly236Ala; Ser239Asp / Ile332Glu / Gly236Ala; Leu234Tyr / Gly236Trp / Ser298Ala; Phe243Leu / Arg292Pro / Tyr300Leu / Val305Ile / Pro396Leu; Lys326Trp / Glu333Ser; Lys326Ala / Glu333Ser; Lys326Met / Glu333Ser; Cys221Asp / Asp222Cys; Ser267Glu / His268Phe / Ser324Thr; His268Phe / Ser324Thr; and Glu345Arg. See Saunders KO (2019) Front. Immunol. 10:1296.doi: 10.3389 / fimmu.2019.01296. The corresponding position of the IgG1 Fc sequence of SEQ ID NO: 56 can be obtained by subtracting 220 from the indicated position. In one embodiment, at least ADCC is enhanced by the substitution. In one embodiment, at least ADCP is enhanced by the substitution. In one embodiment, at least CDC is enhanced by the substitution. In one embodiment, at least complement fixation is enhanced by the substitution.

[0085] Variants that can substantially reduce one or more antibody-related effector functions (e.g., ADCC, ADCP, and / or CDC responses) of IgG1 antibodies include, but are not limited to, Leu235Glu; Leu234Ala / Leu235Ala (LALA); Ser228Pro / Leu235Glu; Leu234Ala / Leu235Ala / Pro329Gly; Pro331Ser / Leu234Glu / Leu235Phe; Asp265Ala; Gly237Ala; Glu318Ala; Glu233Pro; and Gly236Arg / Leu328Arg. As mentioned above, the corresponding positions of those substitutions in the IgG1 Fc sequence provided as SEQ ID NO: 56 can be obtained by subtracting 220 from the indicated positions. In one embodiment, the scaffold sequence comprises an IgG1 Fc sequence with a LALA substitution (see, for example, SEQ ID NO: 57). In one embodiment, at least ADCC is substantially reduced by the substitution. In one embodiment, at least ADCP is substantially reduced by the substitution. In one embodiment, at least CDC is substantially reduced by the substitution. In one embodiment, at least complement fixation is substantially reduced by the substitution. See, e.g., Saunders 2019 and citations therein. In one embodiment, the IgG1 Fc substitution may be S239D / I332E / A330L, which allows for enhanced ADCC without substantially altering CDC function (see Lazar et al., 2006).

[0086] Other variants in IgG1, IgG2, and IgG4 antibody sequences that can substantially reduce one or more antibody-associated effector functions include, but are not limited to, His268Gln / Val309Leu / Ala330Ser / Pro331Ser (IgG2m4); Val234Ala / Gly237Ala / Pro238Ser / His268Ala / Val309Leu / Ala330Ser / Pro331Ser (IgG2σ); Leu234Ala / L235Ala / Gly237Ala / P238Ser / His268Ala / Ala330Ser / Pro331Ser (IgG1σ); and S228P / Phe234Ala / Leu235Ala (IgG4PAA). See, e.g., Saunders 2019 and citations therein.

[0087] Alternative non-dimerizing scaffold domains include, but are not limited to, Xtenylated, PEGylated, and lipidated (on the World Wide Web at "www" at pubs.acs.org / doi / 10.1021 / acsmedchemlett.8b00226). See human serum albumin (HSA) fusions (www.nature.com / articles / s42003-021-01790-2) and anti-HSA binding domains, including but not limited to anti-HSA peptides (www.pubs.acs.org / doi / 10.1021 / acs.molpharmaceut.2c00106), antibodies and antibody fragments (e.g., ScFv, FAB, etc.) and VHH domains (www.semanticscholar.org / paper / Serum-albumin%E2%80%90binding-VHHs-with-variable-pH-enable-Faassen-Ryan / d34256a0d39a0ab92db9195210fa0fc7430758b6).

[0088] 3. Linker The constructs of the present disclosure optionally have a linker located between any two elements. The linker may be located between any two of the NBD amino acid sequence, the scaffold amino acid sequence, and the AD. The linkers typically consist of amino acid sequences of 4-50 amino acids in length (e.g., 4-25 amino acids or 25-50 amino acids) and may be selected for their rigidity, among other things. Each linker is independently selected.

[0089] In some examples, the linker is a flexible amino acid sequence consisting of glycine, serine, and / or alanine residues. In other examples, the linker is an amino acid sequence consisting of glycine and serine. For example, the sequence GGGS (SEQ ID NO: 16) or the sequence GGGGS (SEQ ID NO: 26) is a linker (GGGS) 1~10 (SEQ ID NO: 16-25) or (GGGGS) 1~10 (SEQ ID NOs: 26 to 35) may appear or be repeated 1 to 10 times. The linker may also contain a GGGS or GGGGS sequence, which may appear 1 to 5 or 5 to 10 times.

[0090] Rigid linkers may be used when it is desirable to maintain a substantially fixed distance or spatial separation between domains in a construct to reduce or substantially eliminate unfavorable interactions between domains (e.g., between NBDs in the same construct). For example, a pair of NBDs present in a single molecule may be kept from self-associating through the use of a rigid linker. Exemplary rigid peptide or polypeptide linkers include linkers comprising the sequence X(EAAAK)nX, where EAAAK may be present in 1-20 times (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times of SEQ ID NOs: 36-55) of the linker, and X is absent or alanine. In some cases where the linker comprises the X(EAAAK)nX sequence, n may be 1-5 or 5-10. In some cases where the linker comprises an X(EAAAK)nX sequence, n may be 11-15 or 16-20. Alternatively, the rigid peptide linker may comprise repeats of (Lys-Pro), (Glu-Pro), (Thr-Pro-Arg), and (Ala-Pro) having a length of 4-40 (e.g., 4-20 or 20-40 amino acids). Other rigid linkers include linkers that comprise 2-10 or 10-20 repeats of the dipeptide sequence KP or EP.

[0091] 4. Immune cell-inducing domain The immune cell engaging domain (also referred to in the plural as immune cell engager, ICE, or ICE) is a domain of the NBD-containing construct that can bind to immune cell surface molecules. Immune cell engagers are typically antibody-related molecules / amino acid sequences (e.g., antigen-binding fragments of antibodies, Fab, Fab', single-chain antibodies, scFv, peptide aptamers, or nanobodies). Unless otherwise stated, immune cell engagers are understood to be monovalent to prevent individual molecules from cross-linking cellular targets (e.g., immune cell surface antigens) and off-targeting stimulation of immune cells. Individual immune cell engagers generally cannot act as agonists and stimulate the immune cells to which they bind, but when multiple constructs with immune cell engagers are localized in a complex (e.g., see D or E in FIG. 4A) or immobilized on the surface of tumor cells (e.g., using constructs such as C in FIG. 3), the immune cell engagers can function as agonists of immune cell stimulation / activation.

[0092] Immune cell engagers can be directed to a number of different immune cells that appear in or can be recruited to the TME. Targets of such immune cells and their corresponding immune cell engagers include those listed in Table 1. [Table 1]

[0093] When immune cell engagers such as those listed in Table 1 are incorporated into a first NBD-containing polypeptide construct that can pair or complex with a second NBD-containing construct that includes a tumor-specific binding agent directed against a TAA, trans-targeting of tumor cells leading to their cytolysis can be observed (see, for example, Figure 3, C, D, and FH, and Figure 4A, E, F, H, and I). Incorporation of antibody-related molecules / amino acid sequences directed against immune cell surface proteins, including but not limited to CD3, TCR, and / or CD16, in such constructs and complexes can produce potent cytolytic effects. Cytolytic activity can be enhanced when immune cell engagers that interact with more than one type of immune cell are employed. Some exemplary anti-CD3 immune cell engagers are provided below.

[0094] The immune cell engager polypeptide has the following sequence: The immune cell engager may be an scFv, anti-human CD3 based on the UCHT-1 monoclonal antibody having AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVTTYADSVKGRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO:61). The immune cell engager may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:61. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:61.

[0095] The immune cell engager polypeptide has the following sequence: The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:62. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:62.

[0096] The immune cell engager polypeptide has the following sequence: The immune cell engager may be an anti-human CD3 VHH having the sequence: QVQLVESGGGLVQPGGSLRLSCAASGSIFSANTMGWYRQAPGKQRELVAGMNTSGSTVYGDSVKGRFTISRDNAKNIAYLQMNSLIPEDTAVYYCTLVQRGPNYWGQGTQVTVSS (SEQ ID NO: 63). The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 63. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 63.

[0097] The immune cell engager polypeptide has the following sequence: The immune cell engager may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGY INPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO:64). The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:64. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:64.

[0098] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence set forth in SEQ ID NO:65, and includes a disulfide bond between a cysteine ​​substitution at Q43C and G99C (G233C as shown). DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGCGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGCGTKVEIK (SEQ ID NO:65). The immune cell engager may also include an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:65. Further, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:65.

[0099] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence set forth in SEQ ID NO:66, containing a disulfide bond between a cysteine ​​substitution at Q43C and G100C (G234C shown).DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQAPGCGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGCTKVEIK (SEQ ID NO:66). The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 66. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 66.

[0100] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence set forth in SEQ ID NO:67, containing a disulfide bond between a cysteine ​​substitution (G233C shown) at G44C and:G99C:DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGCGTKVEIK (SEQ ID NO:67). The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 67. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 67.

[0101] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence set forth in SEQ ID NO:68, and containing a disulfide bond between cysteine ​​substitutions at G44C and G100C (G234C shown). 1 DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQCLEWIGY INPSRGYTNY 61 ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSG 121 GGGSGGGGSG GGGSDIVLTQ SPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW 181 IYDTSKVASG VPARFSGSGS GTDYSLTINS LEAEDAATYY CQQWSSNPLT FGGCTKVEIK (SEQ ID NO:68). The immune cell engager may also comprise a sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:68. Additionally, the immune cell engager may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:68.

[0102] GGGS (SEQ ID NO: 16) and / or GGGGS (SEQ ID NO: 26), which contain repeats of SEQ ID NOs: 61-62 and 64-68, are linker sequences between domains.

[0103] 5. Immune cell activation domain (activation domain) Activation domains ("ADs" or "AD" in the singular) that can be incorporated into the constructs of the present disclosure include various molecules or molecular fragments (e.g., fragments of cytokines) that can stimulate anti-tumor cell immune responses when presented as dimers or higher order complexes, but have limited ability to stimulate immune cells when presented singularly. Some immune cell agonists that require receptor cross-linking for maximal activity include, but are not limited to, CD40L, CD28, IFN-g, and IL-12. Anti-tumor cell immune responses can result, for example, from stimulation of NK cells or T cells (e.g., CD8+ T cells) by constructs containing ADs, and can include granule-dependent and granule-independent responses.

[0104] ADs may include, for example, the amino acid sequence of a TNF superfamily member, such as TNF, lymphotoxin alpha, lymphotoxin alpha beta, and BAFF (CD25). ADs may also include, for example, the amino acid sequence of a B7 superfamily member, such as CD80 and CD86. ADs may also include, for example, all or part of the amino acid sequence of IL-15, IL-12, IL-2, or IL-7 (e.g., all or part of the sequence of the extracellular domain). ADs may include, for example, all or part of the amino acid sequence of IL-12 (e.g., one or both subunits of human IL-12).

[0105] By way of example, constructs that homodimerize (see, e.g., Figure 2A and C) or form homomeric complexes (see, e.g., Figure 4AA) in the presence of ATP levels found in the TME include, but are not limited to, immunomodulatory amino acid sequences of IL-15, IL-12, or IL-7. Antibody-related sequences (e.g., single-chain scFV or nanobody sequences) with anti-PD1, anti-PDL1, anti-CD40, anti-CD40L, anti-CD137 / 4-1BB, anti-TGFβ, anti-IL-10, or anti-IL10R binding activity may also be used.

[0106] In addition to the above, nucleic acids containing CpG repeats or nucleic acids having one or more IMT504 sequences may be used as ADs or incorporated into NBD-containing constructs for homodimerization or heterodimerization.

[0107] The AD may, for example, comprise a CD80 polypeptide amino acid sequence. 1 VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD 61 ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT 121 SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTNHSF 181 MCLIKYGHLR VNQTFNWNTT K (SEQ ID NO: 99). Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 99. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 170 or 180 contiguous amino acids of SEQ ID NO: 99.

[0108] AD, for example, CD86 (IgV-IgC domain) 1 LKIQAYFNET ADLPCQFANS QNQSLSELVV FWQDQENLVL NEVYLGKEKF DSVHSKYMNR 61 TSFDSDSWTL RLHNLQIKDK GLYQCIIHHK KPTGMIRIHQ MNSELSVLAN FSQPEIVPIS 121 NITENVYINL TCSSIHGYPE PKKMSVLLRT KNSTIEYDGI MQKSQDNVTE LYDVSISLSV 181 SFPDVTSNMT IFCILETDKT RLLSSPFSIE LEDPQPPPDH IP (sequence number 100); or CD86 IgV domain: 1 LKIQAYFNET ADLPCQFANS QNQSLSELVV FWQDQENLVL NEVYLGKEKF DSVHSKYMGR The AD may comprise a CD86 polypeptide amino acid sequence comprising: 61 TSFDSDSWTL RLHNLQIKDK GLYQCIIHHK KPTGMIRIHQ MNSELSVLA (SEQ ID NO: 101). Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 100 or 101. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 100 or 110 contiguous amino acids of SEQ ID NO: 100 or 101.

[0109] An AD can, for example, comprise an amino acid sequence that binds IL-12 as an agonist (e.g., an IL-12 or anti-CD28 scFv or nanobody amino acid sequence). 1 IWELKKDVYV VELDWYPDAP GEMVVLTCDT PEEDGITWTL DQSSEVLGSG KTLTIQVKEF 61 GDAGQYTCHK GGEVLSHSLL LLHKKEDGIW STDILKDQKE PKNKTFLRCE AKNYSGRFTC 121 WWLTTISTDL TFSVKSSRGS SDPQGVTCGA ATLSAERVRG DNKEYEYSVE CQEDSACPAA 181 EESLPIEVMV DAVHKLKYEN YTSSFFIRDI IKPDPPKNLQ LKPLKNSRQV EVSWEYPDTW 241 STPHSYFSLT FCVQVQGKSK REKKDRVFTD KTSATVICRK NASISVRAQD RYYSSSWSEW 301 ASVPCSGGGG SGGGGSGGGG SRNLPVATPD PGMFPCLHHS QNLLRAVSNM LQKARQTLEF 361 YPCTSEEIDH EDITKDKTST VEACLPLELT KNESCLNSRE TSFITNGSCL ASRKTSFMMA 421 LCLSSIYEDL KMYQVEFKTM NAKLLMDPKR QIFLDQNMLA VIDELMQALN FNSETVPQKS The AD may comprise an IL-12 comprising 481 SLEEPDFYKT KIKLCILLHA FRIRAVTIDR VMSYLNAS (SEQ ID NO: 90), which comprises a fragment of the p40 subunit, a linker repeat of GGGGS, and a fragment of the P35 subunit. Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 90. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 480 or 500 contiguous amino acids of SEQ ID NO: 90.

[0110] As mentioned above, the constructs of the present disclosure may include NBDs that heterodimerize in the presence of ATP. Thus, the complex between the first and second constructs that include heterodimer-forming NBDs can be used to assemble ADs. See, for example, Figure 2, B and D). For example, IL-12 ADs can be formed in the presence of ATP by employing the following: (i) a first construct comprising a p40 subunit (as a first immunomodulatory domain) comprising the following sequence: 1 IWELKKDVYV VELDWYPDAP GEMVVLTCDT PEEDGITWTL DQSSEVLGSG KTLTIQVKEF 61 GDAGQYTCHK GGEVLSHSLL LLHKKEDGIW STDILKDQKE PKNKTFLRCE AKNYSGRFTC 121 WWLTTISTDL TFSVKSSRGS SDPQGVTCGA ATLSAERVRG DNKEYEYSVE CQEDSACPAA 181 EESLPIEVMV DAVHKLKYEN YTSSFFIRDI IKPDPPKNLQ LKPLKNSRQV EVSWEYPDTW 241 STPHSYFSLT FCVQVQGKSK REKKDRVFTD KTSATVICRK NASISVRAQD RYYSSSWSEW 301 ASVPCS (SEQ ID NO: 91), or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 91, and (ii) a second construct comprising a p35 subunit (as a second immunomodulatory domain) comprising the following sequence: 1 RNLPVATPDP GMFPCLHHSQ NLLRAVSNML QKARQTLEFY PCTSEEIDHE DITKDKTSTV 061 EACLPLELTK NESCLNSRET SFITNGSCLA SRKTSFMMAL CLSSIYEDLK MYQVEFKTMN 121 AKLLMDPKRQ IFLDQNMLAV IDELMQALNF NSETVPQKSS LEEPDFYKTK IKLCILLHAF 181 RIRAVTIDRV MSYLNAS (SEQ ID NO:92), or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:92.

[0111] The AD can include, for example, all or a portion of an amino acid sequence that binds to PD1 (e.g., an anti-PD1 scFv or Nanobody amino acid sequence) or an amino acid sequence that binds to PDL1 (e.g., an anti-PDL1 scFv or Nanobody amino acid sequence).

[0112] An AD can include, for example, an amino acid sequence that binds to CD40 (e.g., an anti-CD40 scFv or nanobody amino acid sequence, or a CD40L amino acid sequence), or an amino acid sequence that binds to CD40L (e.g., an anti-CD40L scFv or nanobody amino acid sequence, or a CD40 amino acid sequence). A polypeptide that can be used as an AD includes the CD40L amino acid sequence: 1 GDQNPQIAAH VISEASSKTT SVLQWAEKGY YTMSNNLVTL ENGKQLTVKR QGLYYIYAQV 61 TFCSNREASS QAPFIASLCL KSPGRFERIL LRAANTHSSA KPCGQQSIHL GGVFELQPGA 121 SVFVNVTDPS QVSHGTGFTS FGLLKL (SEQ ID NO: 96), or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 96. Alternatively, a polypeptide that can be employed as an AD comprises a trimer of CD40L amino acid sequences separated by a linker sequence (e.g., a linker sequence comprising GGGGS repeats): 1 GDQNPQIAAH VISEASSKTT SVLQWAEKGY YTMSNNLVTL ENGKQLTVKR QGLYYIYAQV 61 TFCSNREASS QAPFIASLCL KSPGRFERIL LRAANTHSSA KPCGQQSIHL GGVFELQPGA 121 SVFVNVTDPS QVSHGTGFTS FGLLKLGGGGG SGGGGSGGGG SGDQNPQIAA HVISEASSKT 181 TSVLQWAEKG YYTMSNNLVT LENGKQLTVK RQGLYYIYAQ VTFCSNREAS SQAPFIASLC 241 LKSPGRFERI LLRAANTHSS AKPCGQQSIH LGGVFELQPG ASVFVNVTDP SQVSHGTGFT 301 SFGLLKLGGG GSGGGGSGGG GSGDQNPQIA AHVISEASSK TTSVLQWAEK GYYTMSNNLV 361 TLENGKQLTV KRQGLYYIYA QVTFCSNREA SSQAPFIASL CLKSPGRFER ILLRAANTHS 421 SAKPCGQQSI HLGGVFELQP GASVFVNVTD PSQVSHGTGF TSFGLLKL (SEQ ID NO:95), or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:95.

[0113] An AD can include, for example, an amino acid sequence that binds to CD137 / 4-1BB (e.g., an anti-CD137 scFv or nanobody amino acid sequence, or a CD137L / 4-1BBL amino acid sequence), or an amino acid sequence that binds to TGFβ (e.g., an anti-TGFβ scFv or nanobody amino acid sequence, or a TGFBRII amino acid sequence). A polypeptide that can be employed as an AD includes the 4-1BBL amino acid sequence: 1 ACPWAVSGAR ASPGSAASPR LREGPELSPD DPAGLLDLRQ GMFAQLVAQN VLLIDGPLSW 61 YSDPGLAGVS LTGGLSYKED TKELVVAKAG VYYVFFQLEL RRVVAGEGSG SVSLALHLQP 121 LRSAAGAAAL ALTVDLPPAS SEARNSAFGF QGRLLHLSAG QRLGVHLHTE ARARHAWQLT 181 QGATVLGLFR VTPEIPAGLP SPRSE (SEQ ID NO: 94), or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 94. Alternatively, a polypeptide that can be employed as an AD may comprise a linker sequence (e.g., a linker sequence comprising GGGGS repeats): 1 ACPWAVSGAR ASPGSAASPR LREGPELSPD DPAGLLDLRQ GMFAQLVAQN VLLIDGPLSW 61 YSDPGLAGVS LTGGLSYKED TKELVVAKAG VYYVFFQLEL RRVVAGEGSG SVSLALHLQP 121 LRSAAGAAAL ALTVDLPPAS SEARNSAFGF QGRLLHLSAG QRLGVHLHTE ARARHAWQLT 181 QGATVLGLFR VTPEIPAGLP SPRSEGGGGS GGGGSGGGGS ACPWAVSGAR ASPGSAASPR 241 LREGPELSPD DPAGLLDLRQ GMFAQLVAQN VLLIDGPLSW YSDPGLAGVS LTGGLSYKED 301 TKELVVAKAG VYYVFFQLEL RRVVAGEGSG SVSLALHLQP LRSAAGAAAL ALTVDLPPAS 361 SEARNSAFGF QGRLLHLSAG QRLGVHLHTE ARARHAWQLT QGATVLGLFR VTPEIPAGLP 421 SPRSEGGGGS GGGGSGGGGS ACPWAVSGAR ASPGSAASPR LREGPELSPD DPAGLLDLRQ 481 GMFAQLVAQN VLLIDGPLSW YSDPGLAGVS LTGGLSYKED TKELVVAKAG VYYVFFQLEL 541 RRVVAGEGSG SVSLALHLQP LRSAAGAAAL ALTVDLPPAS SEARNSAFGF QGRLLHLSAG 601 QRLGVHLHTE ARARHAWQLT QGATVLGLFR VTPEIPAGL PSPRSE (SEQ ID NO:93), or a trimer of the 4-1BBL amino acid sequence separated by an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:93.

[0114] An AD can include, for example, an amino acid sequence that binds to IL-10 (e.g., an anti-IL-10 scFv or nanobody amino acid sequence), or an amino acid sequence that binds to IL-10R (e.g., an anti-IL-10R scFv or nanobody amino acid sequence).

[0115] The AD can, for example, comprise an amino acid sequence that binds to CD3 (eg, an anti-CD3 scFv or nanobody amino acid sequence).

[0116] The AD can, for example, comprise an amino acid sequence that binds to CTLA-4 (eg, an anti-CTLA-4 scFv or nanobody amino acid sequence).

[0117] The AD may, for example, comprise an amino acid sequence that binds to CD28 (e.g., an anti-CD28 scFv or nanobody amino acid sequence). An anti-CD28 scFv that may be used as an AD may comprise the following sequence: 1 DIELTQSPAS LAVSLGQRAT ISCRASESVE YYVTSLMQWY QQKPGQPPKL LIFAASNVES 61 GVPARFSGSG SGTNFSLNIH PVDEDDVAMY FCQQSRKVPY TFGGGTKLEI KRGGGGSGG 121 GSGGGGSQVK LQQSGPGLVT PSQSLSITCT VSGFSLSDYG VHWVRQSPGQ GLEWLGVIWA 181 GGGTNYNSAL MSRKSISKDN SKSQVFLKMN SLQADDTAVY YCARDKGYSY YYSMDYWGQG 241 TTVTVSS (SEQ ID NO: 89). Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 89. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 220 or 230 contiguous amino acids of SEQ ID NO: 89.

[0118] An AD can include, for example, an interferon-gamma (IFN-gamma) polypeptide sequence, or an amino acid sequence that binds to an IFN-gamma receptor (e.g., an anti-IFN-gamma receptor scFv or nanobody amino acid sequence). A scIFN-gamma polypeptide that can be used as an IFN-gamma AD can include the following sequence: 1 MQDPYVKEAE NLKKYFNAGH SDVADNGTLF LGILKNWKEE SDRKIMQSQI VSFYFKLFKN 61 FKDDQSIQKS VETIKEDMNV KFFNSNKKKR DDFEKLTNYS VTDLNVQRKA IDELIQVMAE 121 FSTEEQQEGP YVKEAENLKK YFNAGHSDVA DNGTLFLGIL KNWKEESDRK IMQSQIVSFY 181 FKLFKNFKDD QSIQKSVETI KEDMNVKFFN SNKKKRDDFE KLTNYSVTDL NVQRKAIHEL 241 IQVMAELSPA AKTGKRKRSQ MLFRG (SEQ ID NO: 97). Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 97. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 220 or 230 consecutive amino acids of SEQ ID NO: 97.

[0119] The AD may, for example, comprise an amino acid sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody amino acid sequence). Anti-CD16 VHH polypeptides that may be used as ADs include: 1 EVQLVESGGG LVQPGESLTL SCVVAGSIFS FAMSWYRQAP GKERELVARI GSDDRVTYAD 61 SVKGRFTISR DNIKRTAGLQ MNSLKPEDTA VYYCNAQTDL RDWTVREYWG QGTQVTVSS(SEQ ID NO:98); or 1 EVQLVESGGE LVQPGGSLRL SCAASGLTFS SYNMGWFRRA PGKEREFVAS ITWSGRDTFY 61 ADSVKGRFTI SRDNAKNTVY LQMSSLKPED TAVYYCAANP WPVAAPRSGT YWGQGTQVTV 121 SS (SEQ ID NO: 103). Alternatively, the AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 98 or 103. The AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 100 or 110 contiguous amino acids of SEQ ID NO: 98 or 103.

[0120] AD does not have to be composed of an amino acid sequence, but may be composed of, for example, a nucleic acid sequence.Non-limiting examples of nucleic acid AD include nucleic acid sequences having CpG repeats (e.g., CpG oligodeoxynucleotide sequences) or IMT504 (TCATCATTTTGTCATTTTGTCATT (SEQ ID NO: 69, see, e.g., Insula et al., 2007 Stem Cells, 25:1047-1054), optionally with 1, 2 or 3 nucleotide substitutions, deletions or insertions.

[0121] 6. Tumor-specific binding domain The tumor-specific binding domain (also referred to as tumor-specific binding agent or "TSB") of the construct described herein is a polypeptide amino acid sequence that functions as a targeting sequence that directs the construct to tumor cells by binding to a target molecule expressed on the tumor cell surface. The target molecule may be a tumor-associated antigen ("TAA") (also referred to herein as a cancer-associated antigen or "CAA"), the cell surface expression of which is restricted or substantially restricted to one or more cancer cell types. In addition to molecules whose expression is restricted or substantially restricted to tumor cells, other molecules present on the surface of tumor cells may be targeted, including, but not limited to, cellular proteins expressed on normal (non-transformed) cells that may be upregulated in tumor cells. Checkpoint proteins expressed on normal and many tumor cells (e.g., transformed tumor cells) (e.g., V-domain Ig suppressor of T-cell activation or "VISTA"), T-cell immunoglobulin and mucin domain 3 or "Tim-3", and programmed death ligand 1 or PD-L1) may also function as target molecules for TSB. Both targeting checkpoint proteins, which are often upregulated by tumor cells as a means of immune evasion or escape, provide targets that can be abundantly expressed on the surface of tumor cells to provide checkpoint inhibition by TSB. Proteins that are not substantially restricted to expression on transformed tumor cells are considered targets of TSB when expressed on tumor cells. Any immune response directed by TSB is limited to the TME where ATP levels are high enough to allow pairing or complex formation of TSB to ADs or immune cell engagers, and are considered targets of TSB via NBDs bound to those elements (see, for example, Figures 3, 4A, and 4B). TSBs include, for example, polypeptides and other molecules such as antibodies, antigen-binding fragments of antibodies, Fabs, Fabs, and polypeptides containing single-chain antibodies, scFvs, peptide aptamers, or nanobody amino acid sequences.

[0122] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to, carbonic anhydrase IX (CAIX), cadherins, carcinoembryonic antigen (CEA), cell-mesenchymal epithelial transition factor (c-MET), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), epidermal growth factor receptor (EGFR) family members, epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 3 (EphA3), fibroblast activation protein alpha (FAP), folate binding protein, folate receptor alpha (FR-alpha), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), insulin-like growth factor 1 receptor (IGF-1R), integrin αVβ3, integrin α5β1, solute carrier family 39 member 6 (Liv1), melanoma-associated antigen family A members ( These include MAGEA), melanoma-associated antigen family C members (MAGEC), mesothelin, mucins (e.g., MUC1), New York esophageal squamous cell carcinoma 1 protein (NY-ESO-1, cancer / testis antigen 1A, cancer / testis antigen 1B), cancer / testis antigen 2 (NY-ESO-2, CTAG2), prostate-specific membrane antigen (PSMA), receptor activator of nuclear factor kappa B ligand (RANKL), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tenascin, TNF receptor superfamily member 10a (TRAILR1), TNF receptor superfamily member 10b (TRAILR2), and vascular endothelial growth factor receptor (VEGFR).

[0123] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to, cancer / testis antigen 1A, cancer / testis antigen 1B, or cancer / testis antigen 2 (NY-ESO-2, CTAG2);

[0124] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to, MAGEA-1, MAGEA-4, or MAGEA-9.

[0125] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to, MAGEC-1, MAGEC-2, or MAGEC-3.

[0126] Some checkpoint proteins that may act as targets for TSB include, but are not limited to, VISTA, TIM-3, PD-L1, CTLA-4 and LAG-3.

[0127] The TSB polypeptide may be an anti-human mesothelin scFv polypeptide related to the mouse-derived SS1 antibody, [ka] where X is V or G (SEQ ID NO: 70). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to that sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to that sequence.

[0128] The TSB polypeptide may comprise an anti-human mesothelin scFv (vH-vL) polypeptide related to the monoclonal antibody YP218 having the sequence: [ka] wherein X is V or G (SEQ ID NO: 71). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0129] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to the monoclonal antibody YP218 having the sequence: [ka] wherein X is V or G (SEQ ID NO: 72). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0130] The TSB polypeptide may comprise an anti-human mesothelin scFv (vH-vL) polypeptide related to monoclonal antibody 15B6; [ka] wherein X is V or G (SEQ ID NO: 73). The TSB polypeptide may also comprise a sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise a sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0131] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6; [ka] wherein X is V or G (SEQ ID NO: 74). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0132] A TSB polypeptide can include an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between positions G102C and K43C (K168C shown in the figure) of vL and vH, having the following sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0133] A TSB polypeptide can include an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between positions G102C and S44C (S169C shown in the figure) of vL and vH, having the following sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0134] A TSB polypeptide can include an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between positions G103C and K43C (K168C shown in the figure) of vL and vH, having the following sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0135] A TSB polypeptide can include an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between positions G103C and S44C (S169C shown in the figure) of vL and vH, having the following sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0136] The TSB binder polypeptide may comprise an anti-human mesothelin (VHH) polypeptide related to monoclonal antibody SD1, having the following sequence: QVQLVQSGGGLVQPGGSLRLSCAASDFDFAAYEMSWVRQA PGQGLEWVAIISHDGIDKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCLRLGAVGQGTLVTVSSS (SEQ ID NO: 79). The TSB polypeptide may also comprise a sequence having greater than 90% or greater than 95% sequence identity to that sequence. Alternatively, the TSB polypeptide may comprise a sequence having greater than 96% or greater than 98% sequence identity to that sequence.

[0137] The TSB polypeptide may comprise an anti-human EpCAM scFv (vH-vL) polypeptide related to the monoclonal antibodies MT201 and adecatumumab, and has the sequence: [ka] where X is V or G (SEQ ID NO: 80). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to that sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to that sequence.

[0138] The TSB polypeptide may comprise an anti-human EpCAM scFv (vL-vH) polypeptide related to the monoclonal antibodies MT201 and adecatumumab, and has the sequence: [ka] where X is V or G (SEQ ID NO:81). The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity to that sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity to that sequence.

[0139] The TSB polypeptide has the following sequence: [ka] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab having the following sequence: The TSB polypeptide may also comprise a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may comprise a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0140] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab, having a disulfide bond between positions Q101C and K43C (K167C as shown) in vL and vH, and having the sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0141] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab, having a disulfide bond between positions Q101C and G44C (G167C as shown) in vL and vH, and having the sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0142] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab, having a disulfide bond between positions G102C and K43C (K167C as shown) in vL and vH, and having the sequence: [ka] The tumor-specific binding agent polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the tumor-specific binding agent polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0143] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab, having a disulfide bond between positions G102C and G44C (G168C as shown) in vL and vH, and having the sequence: [ka] The TSB polypeptide may also include a sequence having greater than 90% or greater than 95% sequence identity with the sequence. Alternatively, the TSB polypeptide may include a sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0144] The linker sequences of SEQ ID NOs: 70 to 86 are written in bold italics.

[0145] E. Preparation method The present disclosure includes and provides for the preparation of the constructs disclosed herein by transcription and translation of nucleic acids encoding the constructs in mammalian cells (CHO cells), yeast cells (e.g., P. pastoris), Thoroptera cells, or bacterial cells (e.g., E. coli cells). Parts of the constructs may also be prepared separately by cellular expression, chemical synthesis, etc., as described above, and then conjugated together using one or more chemical or enzymatic processes. Indeed, enzymatic means, autocatalytic protein splicing, or chemical crosslinking (e.g., heterobifunctional crosslinkers) can be used to link (e.g., covalently link) e.g., AD to NBD, TSB to NBD, and / or NBD to immune cell engager. When the components to be linked are both composed of amino acid sequences, enzymatic means and autocatalytic protein splicing represent coupling options that allow both stoichiometry and structure of the coupling reaction, while substantially avoiding potential secondary coupling reactions. For example, sortases and split inteins may be used to add sequences containing NBDs to ADs, TSB binders, or immune cell engagers. See, e.g., Bhagawati et al., Proc Natl Acad Sci USA, 116(44):22164-22172(2019). Alternatively, the enzyme-catalyzed SpyTag / SpyCatcher system, which may use the CnaB2 domain of the FbaB protein from Streptococcus pyogenes, can be used to conjugate ADs to polypeptides containing NBDs. See, e.g., Reddington and Howarth, Curr. Opin in Chem. Biol., 29: 94-99(2015).

[0146] In addition to the purely enzymatic and chemical conjugation methods for coupling the components of the constructs described herein, affinity-induced methods that combine the specificity of protein-protein interactions with chemical conjugation may be employed. Affinity-induced chemical conjugation provides efficient, facile, and specific chemical conjugation reactions. One such method, PEptide-DIrected Photo-cross-linking (PEDIP), is described in Park et al. (2018), Bioconjug Chem., 29:3240-3244, and has been demonstrated to link polypeptides to antibody heavy chain constant regions. See also Kishomoto et al., (2019), Bioconjug Chem., 30(3):697-702.

[0147] If the AD is not an amino acid sequence that can be translated in a biological system (e.g., it is a nucleic acid such as a CpG oligodeoxynucleotide), it may be linked to other parts of the molecule using, for example, a bifunctional crosslinker (e.g., a homobifunctional or heterobifunctional crosslinker).

[0148] The construct may be purified using various methods known in the art. For example, if the construct contains an IgFc region as a scaffold, it may be purified by protein A or protein G chromatography. The construct may be purified by nucleotide affinity chromatography or dye-ligand affinity chromatography using Cibacron Blue F3GA, Procion Blue HB, or Reactive blue 2 as affinity ligands. Size exclusion chromatography may also be used alone or in combination with affinity chromatography to purify the product.

[0149] F. Formulations and Treatment Methods The NBD-containing constructs and complexes disclosed herein (see, eg, Figures 2-4) may be formulated into compositions that include pharma- ceutically acceptable components.

[0150] The composition may be prepared for parenteral and / or other forms of administration. The construct or a composition comprising the construct may be administered by any suitable route, such as intravenously, intramuscularly, subcutaneously, intratumorally, or intralymphatically. The construct may also be administered in conjunction with one or more other agents that act synergistically or non-synergistically with the construct. For example, the construct of the present disclosure (e.g., a construct having an IL-12 activation domain) may be administered with an immune checkpoint inhibitor, such as pembrolizumab (Keytruda®, targets PD-1), avelumab (Bavencio®, targets PD-L1), or ipilimumab (Yervoy®, targets CTLA-4).

[0151] Any solid tumor that contains elevated ATP levels compared to normal tissue may be treated utilizing the constructs of the present disclosure.Treatable tumors include mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma, and germ cell tumors.Representative cancers include, but are not limited to, colorectal cancer, breast cancer, neuroendocrine tumors, lung cancer, and gastric cancer.For example, the tumor may be a lung, liver, skin, gastrointestinal, pancreatic, brain, or reproductive tissue (e.g., ovarian sarcoma) tumor. Aspects 1. A polypeptide construct comprising a nucleotide-binding domain (NBD) amino acid (aa) sequence and an activation domain (AD) amino acid sequence, optionally linked by a linker peptide sequence, wherein the NBD comprises one or more adenosine triphosphate (ATP) binding sites and is capable of homodimerizing or heterodimerizing with a cognate, non-identical NBD (e.g., an NBD of TAP1 or TAP2 that is capable of heterodimerizing) in the presence of ATP. 2. The polypeptide construct of embodiment 1, wherein the construct is organized in the N-terminal to C-terminal direction as: (i) an NBD, an optional linker, and an AD amino acid sequence; or (ii) an AD, an optional linker, and an NBD amino acid sequence. 3. A polypeptide construct according to embodiment 1 or 2, comprising an NBD amino acid sequence, a scaffold amino acid sequence, and an AD sequence, optionally linked by an independently selected linker amino acid sequence. 4. The polypeptide construct of embodiment 3, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, a scaffold amino acid sequence, and an AD amino acid sequence. 5. The polypeptide construct of embodiment 3, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, an AD amino acid sequence, and a scaffold amino acid sequence. 6. The polypeptide construct of embodiment 3, wherein the construct comprises, from N-terminal to C-terminal direction, an AD amino acid sequence, a scaffold amino acid sequence, and an NBD amino acid sequence. 7. The polypeptide construct of embodiment 3, wherein the construct comprises, from N-terminal to C-terminal direction, an AD amino acid sequence, an NBD amino acid sequence, and a scaffold amino acid sequence. 8. The polypeptide construct of embodiment 3, wherein the construct comprises, in the N-terminal to C-terminal direction, a scaffold, an AD, and an NBD amino acid sequence. 9. The polypeptide construct of embodiment 3, wherein the construct comprises, from N-terminal to C-terminal direction, a scaffold amino acid sequence, an NBD amino acid sequence, and an AD amino acid sequence. 10. The polypeptide construct of any one of aspects 1-9, comprising two or more (e.g., three or more) independently selected AD amino acid sequences, optionally arranged in tandem. 11. The polypeptide construct of any one of aspects 1-10, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally arranged in tandem (such that two or more NBDs within the same molecule are constrained not to interact to form a complex in the presence of ATP). 12. The polypeptide construct of embodiment 11, further comprising a rigid linker that substantially prevents interaction between the two or more NBD amino acid sequences in the presence of ATP. 13. A polypeptide construct according to any one of aspects 1 to 12, wherein each NBD amino acid sequence is capable of homodimerizing in the presence of ATP (e.g., comprises the same homodimer-forming amino acid sequence). 14. The polypeptide construct of embodiment 13, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence. See, e.g., Figure 2, A, C, and E. 15. A polypeptide construct according to embodiment 11 or 12, comprising two or more NBD amino acid sequences that can homodimerize to form a dimer or higher order complex of the construct in the presence of ATP. See, for example, A in Figure 4A. 16. A polypeptide construct according to any of aspects 13 to 15, wherein each NBD amino acid sequence comprises an identical or substantially identical homodimer-forming amino acid sequence. 17. A polypeptide construct according to any one of aspects 1 to 12, wherein each NBD amino acid sequence is capable of heterodimerizing with its cognate NBD in the presence of ATP. 18. The polypeptide construct of embodiment 17, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence (see, e.g., each of the constructs in Figure 2 B and D), or one or more (e.g., two or more) NBD amino acid sequences. 19. A composition according to any one of aspects 13-18, comprising a first polypeptide construct and a second polypeptide construct, wherein the first polypeptide construct comprises a first NBD amino acid sequence and the second polypeptide construct comprises a second NBD amino acid sequence, and wherein the first and second NBD amino acid sequences are cognate sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimeric or heterodimeric complex. See, e.g., Figure 2, B and D. 20. A composition comprising a first polypeptide construct and a second polypeptide construct according to embodiment 19, wherein the first polypeptide construct comprises one or more (e.g., two or more) first NBD amino acid sequences, and the second polypeptide construct comprises one or more (e.g., two or more) second NBD amino acid sequences, and the first and second NBD amino acid sequences are cognate sequences that can homodimerize or heterodimerize in the presence of ATP to form homodimers, heterodimers or higher order complexes. See, for example, Figure 2B and D, where each construct comprises an NBD (e.g., a single NBD) amino acid sequence, and Figure 4AB, where at least one of the first and second (e.g., both) constructs comprises two or more NBD amino acid sequences. 21. A polypeptide construct comprising a nucleotide-binding domain (NBD) amino acid (aa) sequence, optionally linked by a linker peptide sequence, and a tumor-specific binding agent (TSB) amino acid sequence, wherein the NBD comprises one or more adenosine triphosphate (ATP) binding sites and is capable of homodimerizing or heterodimerizing with a cognate, non-identical NBD (e.g., an NBD of TAP1 or TAP2 that can heterodimerize) in the presence of ATP, wherein the polypeptide construct optionally comprises one or more ADs. 22. The polypeptide construct of embodiment 21, wherein the construct is organized in the N-terminal to C-terminal direction as: (i) an NBD, an optional linker, and a TSB amino acid sequence; or (ii) a TSB, an optional linker, and an NBD amino acid sequence. 23. The polypeptide construct of embodiment 21 or 22, comprising an NBD amino acid sequence, a scaffold amino acid sequence, and a TSB amino acid sequence, optionally linked by an independently selected linker amino acid sequence. 24. The polypeptide construct of embodiment 23, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, a scaffold amino acid sequence, and a TSB amino acid sequence. 25. The polypeptide construct of embodiment 23, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, a TSB amino acid sequence, and a scaffold amino acid sequence. 26. The polypeptide construct of aspect 23, wherein the construct comprises, from N-terminal to C-terminal direction, a TSB amino acid sequence, a scaffold amino acid sequence, and an NBD amino acid sequence. 27. The polypeptide construct of aspect 23, wherein the construct comprises, from N-terminal to C-terminal direction, a TSB amino acid sequence, an NBD amino acid sequence, and a scaffold amino acid sequence. 28. The polypeptide construct of aspect 23, wherein the construct comprises, from N-terminal to C-terminal direction, a scaffold amino acid sequence, a TSB amino acid sequence, and an NBD amino acid sequence. 29. The polypeptide construct of embodiment 23, wherein the construct comprises, from N-terminal to C-terminal direction, a scaffold amino acid sequence, an NBD amino acid sequence, and a TSB amino acid sequence. 30. The polypeptide construct of any one of aspects 21-29, comprising two or more (e.g., three or more) independently selected TSB amino acid sequences, optionally arranged in tandem. 31. The polypeptide construct of any one of aspects 21-30, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally arranged in tandem (such that two or more NBDs within the same molecule are constrained not to interact to form a complex in the presence of ATP). 32. The polypeptide construct of embodiment 31, further comprising a rigid linker that substantially prevents interaction between the two or more NBD amino acid sequences in the presence of ATP. 33. A polypeptide construct according to any one of aspects 21 to 32, wherein each NBD amino acid sequence is capable of homodimerizing in the presence of ATP (eg, comprises an identical homodimer-forming amino acid sequence). 34. The polypeptide construct of embodiment 33, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence. See, e.g., Figure 4B, A-D. 35. A polypeptide construct according to embodiment 31 or 32, comprising two or more NBD amino acid sequences that can homodimerize to form a dimer or higher order complex of the construct in the presence of ATP. See, for example, Figure 4A. 36. The polypeptide construct of aspect 35, wherein each NBD amino acid sequence comprises identical or substantially identical homodimer-forming amino acid sequences. 37. A polypeptide construct according to any one of aspects 21 to 32, wherein each NBD amino acid sequence is capable of heterodimerizing with its cognate NBD in the presence of ATP. 38. The polypeptide construct of embodiment 37, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence, or one or more (e.g., two or more) NBD amino acid sequences. 39. A composition comprising a first polypeptide construct and a second polypeptide construct according to any of aspects 33 to 38, wherein the first polypeptide construct and the second polypeptide construct comprise a first NBD amino acid sequence, and the second polypeptide construct comprises a second NBD amino acid sequence, and wherein the first and second NBD amino acid sequences are cognate sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimeric or heterodimeric complex. 40. A composition comprising a first polypeptide construct and a second polypeptide construct according to embodiment 39, wherein the first polypeptide construct comprises one or more (e.g., two or more) first NBD amino acid sequences and the second polypeptide construct comprises one or more (e.g., two or more) second NBD amino acid sequences, the first and second NBD amino acid sequences being cognate sequences that can homodimerize or heterodimerize in the presence of ATP to form homodimers or heterodimers or higher order complexes. 41. The composition according to embodiment 39 or embodiment 40, wherein the TSB of the first polypeptide construct and the TSB of the second polypeptide construct recognize a single tumor-associated antigen (TAA) or different (non-identical) TAAs. 42. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and an immune cell engager (ICE) amino acid sequence optionally linked by a linker peptide sequence, wherein the NBD comprises one or more adenosine triphosphate (ATP) binding sites and is capable of homodimerizing or heterodimerizing with a cognate, non-identical NBD (e.g., the NBD of TAP1 or TAP2, which can heterodimerize) in the presence of ATP, and wherein the polypeptide construct optionally comprises one or more ADs. 43. The polypeptide construct of embodiment 42, wherein the construct is organized in the N-terminal to C-terminal direction as: (i) an NBD, an optional linker, and an ICE amino acid sequence, or (ii) ICE, an optional linker, and an NBD amino acid sequence. 44. The polypeptide construct of aspect 42 or 43, comprising an NBD amino acid sequence, a scaffold amino acid sequence, and an ICE amino acid sequence, optionally independently linked by a selected linker amino acid sequence. 45. The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, a scaffold amino acid sequence, and an ICE amino acid sequence. 46. ​​The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, an NBD amino acid sequence, an ICE amino acid sequence, and a scaffold amino acid sequence. 47. The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, an ICE amino acid sequence, a scaffold amino acid sequence, and an NBD amino acid sequence. 48. The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, an ICE amino acid sequence, an NBD amino acid sequence, and a scaffold amino acid sequence. 49. The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, a scaffold amino acid sequence, an ICE amino acid sequence, and an NBD amino acid sequence. 50. The polypeptide construct of aspect 43, wherein the construct comprises, from N-terminal to C-terminal direction, a scaffold amino acid sequence, an NBD amino acid sequence, and an ICE amino acid sequence. 51. The polypeptide construct of any one of aspects 42-50, comprising two or more (e.g., three or more) independently selected ICE amino acid sequences, optionally arranged in tandem. 52. The polypeptide construct of any one of aspects 42 to 51, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally arranged in tandem (such that two or more NBDs within the same molecule are constrained not to interact to form a complex in the presence of ATP). 53. The polypeptide construct according to embodiment 52, further comprising a rigid linker that substantially prevents interaction between the two or more NBD amino acid sequences in the presence of ATP. 54. A polypeptide construct according to any one of aspects 42 to 53, wherein each NBD amino acid sequence is capable of homodimerizing in the presence of ATP (e.g., comprises an identical homodimer-forming amino acid sequence). 55. The polypeptide construct of embodiment 54, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence. See, e.g., FIG. 4A. 56. A polypeptide construct according to embodiment 53 or 54, comprising two or more NBD amino acid sequences that can homodimerize to form a dimer or higher order complex of the construct in the presence of ATP. 57. A polypeptide construct according to any one of aspects 42 to 53, wherein each NBD amino acid sequence is capable of heterodimerizing with its cognate NBD in the presence of ATP. 58. A polypeptide construct according to aspect 57, wherein the construct comprises an NBD (e.g., a single NBD) amino acid sequence, or one or more (e.g., two or more) NBD amino acid sequences. 59. The composition of embodiment 58, comprising a first polypeptide construct and a second polypeptide construct, wherein the construct comprises a first NBD amino acid sequence and the second polypeptide construct comprises a second NBD amino acid sequence, and wherein the first and second NBD amino acid sequences are cognate sequences that can heterodimerize in the presence of ATP to form a heterodimeric complex. 60. The composition of embodiment 59, comprising a first polypeptide construct and a second polypeptide construct, wherein the first polypeptide construct comprises one or more (e.g., two or more) first NBD amino acid sequences, and the second polypeptide construct comprises one or more (e.g., two or more) second NBD amino acid sequences, and wherein the first and second NBD amino acid sequences are cognate sequences that can heterodimerize in the presence of ATP to form a heterodimer or higher order complex. 61. The composition according to embodiment 59 or embodiment 60, wherein the ICE of the first polypeptide construct and the ICE of the second polypeptide construct recognize a single immune cell surface molecule or different (non-identical) immune cell surface molecules. 62. A composition comprising a first polypeptide construct according to any one of aspects 1 to 18 and a second polypeptide construct according to any one of aspects 42 to 58, (i) a first polypeptide construct comprises one or more (e.g., two or more) first heterodimer-forming NBD amino acid sequences and a second polypeptide construct comprises one or more (e.g., two or more) second heterodimer-forming NBD amino acid sequences; (ii) each of the one or more first heterodimer-forming NBD amino acid sequences is a cognate binding partner of one or more second heterodimer-forming NBD amino acid sequences; (iii) a composition, wherein the first polypeptide construct and the second polypeptide construct bind to each other in the presence of ATP via an interaction between at least one of the one or more first heterodimer-forming NBD amino acid sequences and at least one of the one or more second heterodimer-forming NBD sequences to form a heterodimer or higher order complex of the first and second polypeptide constructs. 63. The composition according to embodiment 62, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second polypeptide construct comprises a single heterodimer-forming NBD. See, for example, J in Figure 3. 64. The composition of embodiment 62, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs and the second polypeptide construct comprises a single heterodimer-forming NBD. See, for example, FIG. 3, K, where the NBDs are arranged in a tandem. 65. The composition according to embodiment 62, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second polypeptide construct comprises two or more second heterodimer-forming NBDs. 66. The composition according to embodiment 62, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs, and the second polypeptide construct comprises two or more second heterodimer-forming NBDs. 67. The composition according to any one of aspects 62-66, wherein the first and / or second polypeptide construct comprises a scaffold sequence. 68. A composition comprising a first polypeptide construct according to any one of aspects 21 to 38 and a second polypeptide construct according to any one of aspects 1 to 18, (i) a first polypeptide construct comprises one or more (e.g., two or more) first heterodimer-forming NBD amino acid sequences and a second polypeptide construct comprises one or more (e.g., two or more) second heterodimer-forming NBD amino acid sequences; (ii) each of the one or more first heterodimer-forming NBD amino acid sequences is a cognate binding partner of one or more second heterodimer-forming NBD amino acid sequences; (iii) a composition, wherein the first polypeptide construct and the second polypeptide construct bind to each other in the presence of ATP via an interaction between at least one of the one or more first heterodimer-forming NBD amino acid sequences and at least one of the one or more second heterodimer-forming NBD sequences to form a heterodimer or higher order complex of the first and second polypeptide constructs. 69. The composition according to embodiment 68, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second polypeptide construct comprises a single heterodimer-forming NBD. See, for example, Figures 3, A and E. 70. The composition of embodiment 68, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs and the second polypeptide construct comprises a single heterodimer-forming NBD. See, for example, B in FIG. 3, where the NBDs are in tandem, and N and O, where the NBDs are not in tandem. 71. The composition according to embodiment 68, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second polypeptide construct comprises two or more second heterodimer-forming NBDs. 72. The composition of embodiment 68, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs and the second polypeptide construct comprises two or more second heterodimer-forming NBDs. See, for example, FIG. 4A, G. 73. The composition of any one of aspects 62-66, wherein the first and / or second polypeptide construct comprises a scaffold sequence. 74. A composition comprising a first polypeptide construct according to any one of aspects 21 to 38 and a second polypeptide construct according to any one of aspects 42 to 58, (i) a first polypeptide construct comprises one or more (e.g., two or more) first heterodimer-forming NBD amino acid sequences and a second polypeptide construct comprises one or more (e.g., two or more) second heterodimer-forming NBD amino acid sequences; (ii) each of the one or more first heterodimer-forming NBD amino acid sequences is a cognate binding partner of one or more second heterodimer-forming NBD amino acid sequences; (iii) a composition, wherein the first polypeptide construct and the second polypeptide construct bind to each other in the presence of ATP via an interaction between at least one of the one or more first heterodimer-forming NBD amino acid sequences and at least one of the one or more second heterodimer-forming NBD sequences to form a heterodimer or higher order complex of the first and second polypeptide constructs. 75. The composition according to embodiment 74, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second complex comprises a single heterodimer-forming NBD. See, for example, Figures 3, C, F, and G. 76. The composition according to embodiment 74, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs and the second complex comprises a single heterodimer-forming NBD. See, for example, in FIG. 3, M, F, G, and L, in which the NBDs are in tandem, and N and O, in which the NBDs are not in tandem. 77. The composition according to embodiment 74, wherein the first polypeptide construct comprises a single first heterodimer-forming NBD and the second complex comprises two or more second heterodimer-forming NBDs. 78. The composition according to embodiment 74, wherein the first polypeptide construct comprises two or more first heterodimer-forming NBDs and the second complex comprises two or more second heterodimer-forming NBDs. See, for example, FIG. 4A, E. 79. The composition according to any one of embodiments 62-67 and 74-78, further comprising AD. See, for example, Figure 3, D and Figure 4A, I. 80. The polypeptide construct according to any one of aspects 1-18, 21-38, and 42-58, wherein the construct comprises a scaffold. 81. The composition of any one of aspects 19-20, 39-41, and 59-61, wherein the first and / or second polypeptide construct comprises an independently selected scaffold. 82. The composition of any one of aspects 62-79, wherein the first and / or second polypeptide construct comprises an independently selected scaffold. 83. The polypeptide construct or composition of any one of aspects 80-82, wherein at least one scaffold (e.g., each scaffold present) does not comprise an immunoglobulin polypeptide amino acid sequence (e.g., does not comprise an immunoglobulin Fc, CH1, CH2, or CH3 domain). 84. The polypeptide construct or composition of any one of aspects 80-82, wherein at least one scaffold (e.g., each scaffold present) comprises an immunoglobulin polypeptide amino acid sequence (e.g., an IgFc, CH2, or CH3 amino acid sequence). 85. The polypeptide construct or composition of embodiment 84, wherein at least one (e.g., each) scaffold comprises an independently selected immunoglobulin polypeptide amino acid sequence of IgG1 IgFc of SEQ ID NO: 56 or 57, or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 56 or 57. 86. A polypeptide construct or composition according to embodiment 84, wherein at least one (e.g., each) scaffold comprises an independently selected non-dimerizing IgFc polypeptide amino acid sequence. 87. A polypeptide construct or composition according to embodiment 86, wherein at least one (e.g., each) scaffold comprises an IgFc polypeptide amino acid sequence of SEQ ID NO: 102, or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 102. 88. The polypeptide construct or composition of embodiment 84, wherein at least one (e.g., each) scaffold comprises an independently selected immunoglobulin polypeptide amino acid sequence of an IgG2, IgG3, or IgG4 IgFc of any one of SEQ ID NOs: 58-60, or a sequence having at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 58-60. 89. The polypeptide construct or composition of any one of aspects 85-88, wherein the immunoglobulin Fc (IgFc) polypeptide amino acid sequence is capable of inducing an antibody-associated effector function (e.g., ADCC, ADCP, and / or CDC) or the IgFc polypeptide sequence comprises one or more substitutions that enhance (e.g., substantially enhance) at least one (e.g., all) antibody-associated effector function selected from ADCC, ADCP, and CDC. The change in function may be assessed in comparison to a polypeptide construct lacking one or more substitutions. 90. A polypeptide construct or composition according to any one of aspects 85 to 88, wherein the immunoglobulin Fc polypeptide amino acid sequence comprises one or more substitutions (e.g., a LALA substitution) that reduce (e.g., substantially reduce) at least one (e.g., all) antibody-associated effector function selected from ADCC, ADCP, and CDC. The change in function may be assessed in comparison to a polypeptide construct lacking the one or more substitutions. 91. The polypeptide construct or composition of any of the preceding aspects, wherein, when one or more ADs are present, at least one (e.g., each) AD consists of the amino acid sequence of all or a portion of a polypeptide: CD40L, CD28, IFN-γ, a TNF superfamily member, TNF, lymphotoxin alpha, lymphotoxin alpha beta, BAFF (CD25), a B7 superfamily member, CD80, CD86, IL-15, IL-2, IL-7, IL-10, IL-12, PD1, anti-CD3, anti-CTLA4, or anti-CD28. 92. The polypeptide construct or composition according to embodiment 91, wherein at least one (e.g., each) AD comprises an amino acid sequence having greater than 90% or greater than 95% sequence identity to IL-15, IL-12, or IL-7 (e.g., greater than 97% or greater than 99% sequence identity to IL-15, IL-12, or IL-7). 93. A polypeptide construct or composition according to embodiment 91, wherein at least one (e.g. each) AD comprises (i) a single-chain IL-12 of SEQ ID NO: 90, or (ii) a single-chain IL-12 p40 subunit of SEQ ID NO: 91 and / or a p35 subunit of SEQ ID NO: 92, or an amino acid sequence having greater than 95% or greater than 98% sequence identity to any of those IL-12 sequences. 94. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to PD1 (e.g., an anti-PD1 scFv or nanobody amino acid sequence that can act as an agonist of PD1) or an amino acid sequence that binds to PD1 (e.g., an anti-PDL1 scFv or nanobody amino acid sequence). 95. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CD40 (e.g., an anti-CD40 scFv or nanobody amino acid sequence that can act as an agonist of CD40 signaling) or CD40L (e.g., an anti-CD40L scFv or nanobody amino acid sequence). 96. The polypeptide construct or composition of embodiment 95, wherein at least one (e.g., each) AD comprises a CD40L amino acid sequence of SEQ ID NO: 96, a CD40L trimer amino acid sequence of SEQ ID NO: 95, or an amino acid sequence having at least 90% or at least 95% sequence identity to either SEQ ID NO: 95 or 96 (e.g., greater than 97% or greater than 99% sequence identity to either SEQ ID NO: 95 or 96). 97. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CD137 / 4-1BB (e.g., an anti-CD137 scFv or nanobody amino acid sequence) or an amino acid sequence that binds to TGFβ (e.g., an anti-TGFβ scFv or nanobody amino acid sequence). 98. The polypeptide construct or composition of embodiment 97, wherein at least one (e.g., each) AD comprises a 4-1BBL sequence of SEQ ID NO: 94, a 4-1BBL trimer amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 90% or at least 95% sequence identity to either SEQ ID NO: 93 or 94 (e.g., greater than 97% or greater than 99% sequence identity to either SEQ ID NO: 93 or 94). 99. The polypeptide construct or composition of any of the preceding embodiments, wherein, if one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to IL-10 (e.g., an amino acid sequence that binds to an anti-IL-10 receptor ("IL-10R") (e.g., an anti-IL-10R scFv or Nanobody amino acid sequence). 100. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CTLA-4 (e.g., an anti-CTLA-4 scFv or nanobody amino acid sequence). 101. A polypeptide construct or composition according to any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CD3 (e.g., an anti-CD3 scFv or nanobody amino acid sequence). 102. A polypeptide construct or composition according to any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CD28 (e.g., an anti-CD28 scFv or nanobody amino acid sequence). 103. A polypeptide construct or composition according to embodiment 102, wherein at least one (e.g. each) AD comprises an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 89 (e.g. greater than 96% sequence identity to SEQ ID NO: 89 or greater than 99% sequence identity to SEQ ID NO: 89). 104. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to an IFN-γ receptor (e.g., an anti-IFN-γ receptor scFv or nanobody amino acid sequence). 105. A polypeptide construct or composition according to embodiment 104, wherein at least one (e.g., each) AD comprises a scIFN-γ polypeptide of SEQ ID NO: 97, or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 97 (e.g., greater than 96% or greater than 99% sequence identity to SEQ ID NO: 97). 106. A polypeptide construct or composition according to any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises an amino acid sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody amino acid sequence). 107. A polypeptide construct or composition according to embodiment 106, wherein at least one (e.g. each) AD comprises an anti-CD16 VHH polypeptide of SEQ ID NO: 98 or 103, or an amino acid sequence having at least 90% or at least 95% sequence identity to either of SEQ ID NO: 98 or 103 (e.g. greater than 96% or greater than 99% sequence identity to either of SEQ ID NO: 98 or 103). 108. The polypeptide construct or composition of any of the preceding embodiments, wherein, when one or more ADs are present, at least one (e.g., each) AD comprises a CD80 amino acid sequence of SEQ ID NO: 99, or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 99 (e.g., greater than 96% or greater than 99% sequence identity to SEQ ID NO: 99). 109. The polypeptide construct or composition of any of the preceding embodiments, wherein, if one or more ADs are present, at least one (e.g., each) AD comprises a CD86 amino acid sequence of either SEQ ID NO: 100 or 101, or an amino acid sequence having at least 90% or at least 95% sequence identity to either SEQ ID NO: 100 or 101 (e.g., greater than 96% or greater than 99% sequence identity to either SEQ ID NO: 100 or 101). 110. The polypeptide construct or composition of any of the preceding aspects, wherein, when one or more ADs are present, at least one (e.g., each) AD contains a nucleic acid having CpG repeats (e.g., a CpG oligodeoxynucleotide sequence). 111. The polypeptide construct or composition of any of the preceding embodiments, wherein, if one or more ADs are present, at least one (e.g., each) AD comprises a nucleic acid comprising the sequence TCATCATTTTGTCATTTTGTCATT (SEQ ID NO: 69), optionally with one, two, or three nucleotide substitutions, deletions, or insertions. 112. One or more (e.g., two or more) TSBs may be present in the construct or composition, and at least one (e.g., at least two, or each) TSB may be selected from the group consisting of carbonic anhydrase IX (CAIX), cadherin, carcinoembryonic antigen (CEA), cell-mesenchymal epithelial transition factor (c-MET), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), epidermal growth factor receptor (EGFR) family members, epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 3 (EphA3), fibroblast activation protein alpha (FAP), folate binding protein, folate receptor alpha (FR-alpha), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), insulin-like growth factor 1 receptor (IGF-1R), integrin αVβ3, integrin α5β1, solute carrier family 39 member 6 (Liv1), black

[0023] The polypeptide construct or composition of any of the preceding embodiments, when independently selected to exhibit affinity for a TAA selected from the group consisting of chromoma associated antigen family A members (MAGEA), melanoma associated antigen family C members (MAGEC), mesothelin, mucins (e.g., MUC1), New York esophageal squamous cell carcinoma 1 protein (NY-ESO-1, cancer / testis antigen 1A, cancer / testis antigen 1B), cancer / testis antigen 2 (NY-ESO-2, CTAG2), prostate specific membrane antigen (PSMA), receptor activator of nuclear factor kappa B ligand (RANKL), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tenascin, TNF receptor superfamily member 10a (TRAILR1), TNF receptor superfamily member 10b (TRAILR2), and vascular endothelial growth factor receptor (VEGFR). 113. A polypeptide construct or composition according to embodiment 112, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human mesothelin scFv of SEQ ID NO: 70, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 70 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 70). 114. A polypeptide construct or composition according to embodiment 112 or 113, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human mesothelin scFv (vH-vL) of SEQ ID NO: 71, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 71 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 71). 115. A polypeptide construct or composition according to any one of aspects 112 to 114, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human mesothelin scFv (vL-vH) of SEQ ID NO: 72, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 72 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 72). 116. A polypeptide construct or composition according to any one of aspects 112 to 115, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human mesothelin scFv (vH-vL) of SEQ ID NO: 73, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 73 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 73). 117. A polypeptide construct or composition according to any one of aspects 112 to 116, wherein at least one (e.g. at least two, or each) TSB comprises an anti-human mesothelin scFv (vL-vH) of any one of SEQ ID NOs: 74 to 78, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to any one of SEQ ID NOs: 74 to 78. 118. A polypeptide construct or composition according to embodiment 117, wherein the anti-human mesothelin scFv (vL-vH) has greater than 96% or greater than 98% sequence identity to any of SEQ ID NOs: 74-78. 119. A polypeptide construct or composition according to any one of aspects 112 to 118, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human mesothelin VHH (nanobody) of SEQ ID NO: 79, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 79 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 79). 120. A polypeptide construct or composition according to any one of aspects 112 to 119, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human EpCAM scFv (vH-vL) of SEQ ID NO: 80, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 80 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 80). 121. A polypeptide construct or composition according to any one of aspects 112 to 120, wherein at least one (e.g., at least two, or each) TSB comprises an anti-human EpCAM scFv (vL-vH) of SEQ ID NO: 81, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 81 (e.g., greater than 96% or greater than 98% sequence identity to SEQ ID NO: 81). 122. A polypeptide construct or composition according to any one of aspects 112 to 121, wherein at least one (e.g. at least two, or each) TSB comprises an anti-human CTLA-4 scFv (vL-vH) of any of SEQ ID NOs: 82 to 86, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to any of SEQ ID NOs: 82 to 86. 123. A polypeptide construct or composition according to embodiment 122, wherein the anti-human CTLA-4 scFv (vL-vH) has more than 96% or more than 98% sequence identity to any of SEQ ID NOs: 82-86. 124. A polypeptide construct or composition according to any of aspects 1 to 111, wherein when one or more TSBs are present in the construct or composition, at least one (e.g. at least two, or each) TSB exhibits affinity for a checkpoint protein. 125. A polypeptide construct or composition according to embodiment 124, wherein the checkpoint protein is selected from the group consisting of V domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and mucin domain containing-3 (TIM-3), PD-L1, CTLA-4, and lymphocyte activation gene 3 (LAG-3). 126. A polypeptide construct or composition according to any of aspects 1 to 125, wherein when one or more immune cell engagers (ICEs) are present in the construct or composition, at least one (e.g., at least two, or each) independently selected ICE amino acid sequence has affinity for (e.g., binds to) a protein selected from the group consisting of CD3, CD4, CD8, αβTCR, CD2, TCR α chain, TCR β chain, δγTCR, TCR γ chain, TCR δ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34, and CD16. 127. The polypeptide construct or composition according to embodiment 126, wherein at least one ICE amino acid sequence (e.g., at least two, or each ICE sequence) is independently selected from the group consisting of an antibody, an antigen-binding fragment(s) of an antibody, a Fab, a Fab', an scFv, an aptamer, and a nanobody amino acid sequence. 128. The polypeptide construct or composition according to embodiment 126 or 127, wherein at least one (e.g., at least two, or each) ICE comprises an scFv, an anti-human CD3 comprising a sequence of SEQ ID NO: 61 or 62, or a sequence having more than 90% or more than 95% sequence identity to either of SEQ ID NO: 61 or 62 (e.g. an amino acid sequence having more than 96% or more than 98% sequence identity to either of SEQ ID NO: 61 or 62). 129. A polypeptide construct or composition according to any one of aspects 126 to 128, wherein at least one (e.g. at least two, or each) ICE comprises an anti-human CD3 VHH comprising a sequence of SEQ ID NO: 63, or a sequence having more than 90% or more than 95% sequence identity to SEQ ID NO: 63 (e.g. an amino acid sequence having more than 96% or more than 98% sequence identity to SEQ ID NO: 63). 130. A polypeptide construct or composition according to any one of aspects 126 to 129, wherein at least one (e.g. at least two, or each) ICE comprises an anti-human CD3 scFv (vH-vL) comprising the sequence of any one of SEQ ID NOs: 64-68, or a sequence having greater than 90% or greater than 95% sequence identity to any one of SEQ ID NOs: 64-68 (e.g. an amino acid sequence having greater than 96% or greater than 98% sequence identity to any one of SEQ ID NOs: 64-68). 131. The polypeptide construct or composition of any of the previous embodiments, wherein each NBD is an NBD of an ATP-binding cassette (ABC) transporter. 132. A polypeptide construct or composition according to embodiment 131, wherein the ABC transporter is a member of the ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, or White ABC transporter family. 133. A polypeptide construct or composition according to embodiment 131, wherein the ABC transporter NBD comprises a sequence selected from ABCA1 (SEQ ID NO:1), ABCB1 (SEQ ID NO:2), ABCC1 (SEQ ID NO:3), ABCD1 (SEQ ID NO:4), ABCE1 (SEQ ID NO:5), ABCF1 (SEQ ID NO:6), ABCG2 (SEQ ID NO:7), and TAP1 (SEQ ID NO:9), or an amino acid sequence having more than 90% or more than 95% sequence identity to any one of said sequences. 134. A polypeptide construct or composition according to embodiment 133, wherein the NBD comprises an amino acid sequence having more than 97% or more than 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1-7 and 9. 135. A polypeptide construct or composition according to embodiment 131, wherein the NBD comprises an aTAP2 amino acid sequence, a TAP2 amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having more than 90% or more than 95% sequence identity to the amino acid sequence of SEQ ID NO: 11. 136. A polypeptide construct or composition according to embodiment 135, wherein the NBD comprises an amino acid sequence having more than 97% or more than 98% sequence identity with the amino acid sequence of SEQ ID NO: 11. 137. A polypeptide construct or composition according to embodiment 131, wherein the NBD comprises a TAP1 NBD amino acid sequence. 138. A polypeptide construct or composition according to embodiment 137, wherein the NBD comprises a sequence of SEQ ID NO:9 or an amino acid sequence having more than 90% or more than 95% sequence identity with SEQ ID NO:9. 139. A polypeptide construct or composition according to embodiment 138, wherein the NBD comprises an amino acid sequence having more than 97% or more than 98% sequence identity to the amino acid sequence of SEQ ID NO:9. 140. A polypeptide construct or composition according to embodiment 131, wherein the NBD comprises theTAP1 amino acid sequence of any one of SEQ ID NOs: 10, 87, or 88, or an amino acid sequence having more than 90% or more than 95% sequence identity to any one of those sequences. 141. A polypeptide construct or composition according to embodiment 140, wherein the NBD comprises an amino acid sequence having more than 97% or more than 98% sequence identity to the TAP1 amino acid sequence of SEQ ID NO: 10, 87, or 88. 142. A polypeptide construct or composition according to embodiment 140, wherein the NBD comprises a TAP1 amino acid sequence of SEQ ID NO: 88, or a TAP1 amino acid sequence having more than 90% or more than 95% sequence identity to the TAP1 amino acid sequence of SEQ ID NO: 88. 143. A polypeptide construct or composition according to embodiment 142, wherein the NBD comprises a TAP1 amino acid sequence having more than 97% or more than 98% sequence identity to the TAP1 amino acid sequence of SEQ ID NO:88. 144. A polypeptide construct or composition according to any one of aspects 131-143, wherein the NBD comprises a non-homodimer-forming NBD. 145. The polypeptide construct according to embodiment 144, wherein the NBD comprises a substitution of at least one (eg at least two or at least three) of the amino acids N185, S186, Q189, E191, and Q192. 146. A polypeptide construct or composition according to any one of aspects 131-145, wherein at least one (e.g. each) NBD has a substitution that renders at least one (e.g. each) NBD substantially ATP hydrolysis defective. 147. A polypeptide construct or composition according to embodiment 146, wherein at least one (e.g., each) NBD comprises a TAP1 NBD having a D668 substitution numbered as in SEQ ID NO: 8 (e.g., D668N), or a corresponding substitution that renders at least one (e.g., each) NBD substantially ATP hydrolysis defective (e.g., any of SEQ ID NOs: 9, 10, 87, or 88). 148. The polypeptide construct or composition of any of the preceding aspects, optionally comprising one or more independently selected linkers (e.g., flexible or rigid linkers) positioned between any of the NBD amino acid sequence, the scaffold amino acid sequence, and the AD amino acid sequence. 149. The polypeptide construct or composition according to embodiment 148, wherein the one or more independently selected linkers comprise an amino acid sequence independently selected from any of SEQ ID NOs: 16-35. 150. The polypeptide construct or composition according to embodiment 148 or 149, further comprising one or more independently selected rigid linker amino acid sequences. 151. The polypeptide construct or composition according to embodiment 150, wherein the one or more independently selected rigid linker amino acid sequences are independently selected from any of SEQ ID NOs: 36-55. 152. A dimer or higher order complex of constructs comprising two or more constructs, or a first polypeptide construct and a second polypeptide construct (e.g., a composition) according to any one of embodiments 1 to 151, and at least one molecule (e.g., at least two molecules) of ATP. 153. A dimer or other higher order complex according to embodiment 152, wherein each of the constructs in the complex is identical. 154. A dimer or other higher order complex according to embodiment 153, wherein the dimer or other higher order complex is a homodimer. 155. A dimer or higher order complex according to embodiment 152, wherein each of the constructs in the complex are not identical. 156. A dimer or other higher order complex according to embodiment 155, wherein the dimer or other higher order complex is a heterodimer. 157. A population of constructs (e.g., a group or collection of constructs as a composition) comprising at least one or at least two of the dimers or higher order complexes of the constructs of any of embodiments 152-156. 158. The population according to embodiment 157, wherein at least one or at least two of the dimers or higher order complexes of the constructs comprise an immunoglobulin constant region that affects ADCC, ADCP, and / or CDC. 159. The population according to embodiment 157, wherein at least one or at least two of the dimers or higher order complexes of the construct comprise an independently selected AD. 160. The population according to embodiment 157 or 159, wherein at least one or at least two of the dimers or higher order complexes of the construct comprise an independently selected ICE. 161. The population according to any of aspects 157-160, wherein at least one or at least two of the dimers or higher order complexes of the construct comprise an independently selected TSB. 162. A polypeptide construct according to any of embodiments 1-151, having a structure as depicted in any of Figures 2, 3, 4A, or 4B. 163. A pair of constructs according to any of embodiments 1-151, having the structure shown in any of Figures 2, 3, 4A, or 4B. 164. A dimer or higher order complex according to any of embodiments 152-156, having a structure as shown in either FIG. 2 or FIG. 3. 165. A dimer or higher order complex according to any of embodiments 152-156, having a structure according to either FIG. 4 or FIG. 4B. 166. A dimer or higher order complex according to any of embodiments 152-156, having a structure as shown in any of Figures 2, 3, 4A, or 4B. 167. A dimer or higher order complex according to any of aspects 152-156, comprising a homodimer of TAP1 or TAP2 NBD amino acid sequences, or a heterodimer of TAP1 and TAP2 NBD amino acid sequences. 168. A method of stimulating CD4+ T cells, δγ T cells, NK cells, myeloid-derived suppressor cells, and / or CD8+ T cells in the presence of sufficient ATP to bind to the NBD, comprising contacting the cells with a construct or composition according to any one of aspects 1-151. 169. The method according to embodiment 168, which is carried out in vitro or outside the body of a mammal or non-mammal. 170. The method of embodiment 169, which is carried out on a cell or tissue. 171. The method according to embodiment 168, which is carried out in vivo or in a mammalian body. 172. A method for treating cancer, comprising administering to a patient or subject a construct or composition according to any one of embodiments 1 to 151. 173. The method according to any of embodiments 169 to 172, wherein the mammal is a human. 174. Use of a construct or composition according to any of embodiments 1 to 151 for the preparation of a medicament for the treatment of cancer. 175. The method or use according to any one of aspects 172 to 174, wherein the cancer comprises a solid tumor. 176. A polypeptide construct or composition according to any one of embodiments 1 to 151 for the treatment of cancer. 177. A polypeptide construct or composition according to embodiment 176, wherein the cancer comprises a solid tumor. 178. The method, use, construct or composition according to any of aspects 172 to 176, wherein the cancer is mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma, or germ cell tumor. 179. A nucleic acid comprising a sequence encoding one or more constructs or one or more constructs of a composition according to any one of embodiments 1 to 151. 180. A vector comprising the nucleic acid of embodiment 179, optionally under the control of a promoter (e.g., a constitutive promoter) or an inducible promoter. 181. A cell comprising the vector or nucleic acid of embodiment 179 or 180. 182. The cell according to aspect 181, wherein the cell is a mammalian cell, a yeast cell, an insect cell, or a bacterial cell. EXAMPLES

[0152] Example 1 To test the functionality of TAP1 NBD to form homodimers in solution, human TAP1 NBD containing (i) N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and Q683R substitutions), (ii) a cysteine ​​present in the sequence was replaced with a serine (SEQ ID NO: 10), and (iii) a C-terminal His tag for purification was expressed in E. coli and subsequently purified. Molecular weight markers and samples of purified proteins were subjected to SDS-PAGE gel analysis under reducing and non-reducing conditions as shown in Figure 8 (lanes: M-molecular weight marker, R-reduced protein sample, NR-non-reduced protein sample).

[0153] Samples of purified TAP1 NBD were exposed to 1 mM ADP or 1 mM ATP and subjected to size exclusion chromatography. The results shown in Figure 9 show a protein chromatograph with an effective molecular weight of 37 kDa in the presence of ADP and 57 kDa in the presence of ATP. The results show that in the presence of ATP, TAP1 protein dimerizes. Mass spectrophotometry and dynamic light scattering (DLS) were used to confirm dimer formation and determine the molecular weight. Mass measurements yielded an effective molecular weight of 62 kDa in the presence of 1 mM ATP, consistent with the chromatographic results (see Figure 10). DLS in 20 mM Tris (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 10% glycerol in the presence of 1 mM ADP (Figure 11 in A) or 1 mM ATP (Figure 11 in B) gave a size of 5.517 in the presence of ADP and 8.057 in the presence of ATP, consistent with dimerization of the protein in the presence of 1 mM ATP. The DLS results are shown in the table below. [Table 2]

[0154] Example 2 TAP1 NBD of SEQ ID NO:87 with SpyTag3, TEV protease, and a peptide containing a His-tag added to the C-terminus was expressed in E. coli, and cells were pelleted and frozen until analysis. TAP1 NBD fragments were purified by cell lysis followed by chromatography on a HisTrap column. For analysis, cell pellets were thawed and resuspended in 200 ml of lysis buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 5 mM MgCl2, 10% glycerol, 2 mM ATP) supplemented with 100 μg / ml lysozyme, two protease inhibitor tablets (Thermo Pierce A32965), and Universal Nuclease (Thermo Pierce 88702) added to 12.5 U / ml. The cells were incubated at 4° C. for 30 min with agitation and then lysed by sonication at 80% intensity for a total of 4 min active time (5 s on, 15 s rest on ice per cycle). The lysate was then clarified by centrifugation at 12,000×g for 1 h at 4° C., then 0.22-μm filtered and loaded onto a 0.5 ml / min HisTrap HP column (Cytiva) at 0.5 ml / min and recirculated overnight with a peristaltic pump. The next day, the column was switched to flow-through and the lysate was loaded completely at 1 ml / min, after which the column was washed with 10 column volumes (CV) of lysis buffer supplemented with 7 mM β-mercaptoethanol and 20 mM imidazole. The column was then transferred to an AKTAxpress FPLC and bound protein was eluted with a 0-500 mM imidazole gradient over 40 CV in binding buffer (20 mM Tris-Cl pH 8.0, 200 mM NaCl, 5 mM MgCl2, 10% glycerol, 2 mM ATP, 0.5 mM PMSF). Column fractions were assessed by SDS-PAGE and fractions containing the target protein were pooled, concentrated to 10 ml (10 kDa MWCO) and then loaded as three injections (approximately 3.3 ml per injection) onto a Superdex 200 16 / 600 column equilibrated in target buffer (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol, 1 mM ATP).Elution peak fractions were assessed by SDS-PAGE and target protein-containing fractions were pooled and concentrated to 10 mg / ml (10 kDa molecular weight cutoff) before being aliquoted (500 μl / tube) and stored at 4°C.

[0155] The purified proteins were subjected to SDS-PAGE along with molecular weight samples. A Coomassie blue stained gel is provided in Figure 12, with the leftmost lane being the molecular weight sample and the rightmost lane being the purified protein.

[0156] Samples of purified proteins (1 mg each) were buffer exchanged on PD-10 columns (Cytiva 17085101) according to the manufacturer's instructions. For buffer exchange, PD-10 desalting columns were equilibrated with target buffer (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol) containing either 1 mM ATP or 1 mM ADP. One milligram (100 μl of 10 mg / ml stock) of protein was added to each column along with 2.4 ml of target buffer, the columns were centrifuged at 1000×g for 2 min in 50 ml conical tubes, and the eluate was collected. The buffer exchanged protein was then concentrated to 0.5 ml (approximately 60 μM) and loaded onto a Superdex S200 Increase column (Cytiva) equilibrated in the appropriate buffer containing ATP or ADP and eluted at 0.5 ml / min. The ATP-containing sample eluted at a volume of 15.75 ml, corresponding to a molecular weight of approximately 65 kDa, while the ADP sample eluted at 16.81 ml, corresponding to a molecular weight of approximately 38 kDa. The chromatographic analysis traces are shown in FIG.

[0157] For molecular weight determination by size-exclusion chromatography in the presence of ADP and ATP, purified protein samples (1 mg each) were buffer exchanged onto PD-10 columns (Cytiva 17085101) according to the manufacturer's instructions. Briefly, PD-10 desalting columns were equilibrated with target buffer (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol) containing either 1 mM ATP or ADP. One milligram (100 μl of 10 mg / ml stock) of protein was added to each column along with 2.4 ml of target buffer, the columns were centrifuged at 1000×g for 2 min in 50 ml conical tubes, and the eluate was collected. The buffer-exchanged protein was then concentrated to 0.5 ml (approximately 60 uM) and loaded onto a Superdex S200 Increase column (Cytiva) and equilibrated at 0.5 ml / min in the appropriate buffer containing ATP or ADP. The ATP-containing sample eluted at a volume of 15.75 ml, corresponding to a molecular weight of approximately 65 kDa, while the ADP sample eluted at 16.81 ml, corresponding to a molecular weight of approximately 38 kDa.

[0158] Example 3 For evaluation of uninduced and inducible expression of NBD polypeptides, samples of E. coli BL21(DE3) were transformed with a vector allowing isopropyl-beta-D-thiogalactoside (IPTG)-inducible expression of the NBD of SEQ ID NO:87 or SEQ ID NO:88 using a peptide containing SpyTag3, a TEV protease site, and His tagged at the C-terminus. For analysis of uninduced and induced fractions, the optical density (OD) at 600 nm was monitored for each culture until induction at 0.4 OD, when an aliquot corresponding to 1 OD-ml (e.g., 0.4 OD of 2.5 ml) was taken. The cells in the aliquots were pelleted at 6800×g for 3 min, the pellet resuspended in 300 μl of 1×PBS, and the resuspended material was then frozen at −80° C. After induction with 80 μM IPTG, cells were grown and samples of 1 OD-ml (e.g., 0.25 ml of 4OD) equivalent were collected during the final harvest after induction. For analysis of induction, frozen samples were thawed and cells in all samples were lysed by mixing with an equal volume of 2× lysis buffer (1× PBS containing 0.2% Triton X-100, 100 μg / ml lysozyme, and 2× concentration of universal nuclease from Pierce). Samples were incubated on a rocker at 4° C. for 30 minutes, after which the lysates were spun at 21,000×g for 10 minutes to pellet debris. A sample of the supernatant (30 μl) was mixed with 10 μl of 4× SDS-PAGE loading buffer, heated at 70° C. for approximately 5 minutes, and then loaded onto an SDS-PAGE gel. The results are shown in SDS-PAGE gel A in FIG. 13.

[0159] To determine expression levels and molecular weight by size exclusion chromatography, samples of the NBD polypeptides of SEQ ID NOs:87 and 88 were expressed in E. coli and purified for SDS-PAGE and chromatographic analysis as in Example 2. The results of chromatographic molecular weight determination and protein yield, approximately two orders of magnitude (90-fold) higher for the "capped" NBD of SEQ ID NO:88 compared to that of SEQ ID NO:87, are provided in the table below. [Table 3]

[0160] Figure 13 provides an SDS PAGE analysis of induced and uninduced expression in A, and an analysis of purified protein in B. The lanes in the reduced SDS-PAGE gel image provided in Figure 13 are as follows: [Table 4]

Claims

1. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid sequence and a tumor-specific binding agent (TSB) amino acid sequence, the NBD amino acid sequence has at least 90% sequence identity to an amino acid sequence selected from any of SEQ ID NOs: 1-7, 9, 10, 12, 15, 87, or 88, and comprises two adenosine triphosphate (ATP) binding sites of an ATP-binding cassette transporter protein, and is capable of homodimerizing or heterodimerizing with a cognate, non-identical NBD sequence in the presence of ATP; and The polypeptide construct, wherein the TSB amino acid sequence comprises the sequence of an antibody, an antigen-binding fragment of an antibody, Fab, Fab', scFv, nanobody, or aptamer having affinity for a tumor-associated antigen (TAA) expressed on the surface of tumor cells.

2. The polypeptide construct of claim 1, further comprising a scaffold amino acid sequence.

3. A composition comprising the first polypeptide construct and second polypeptide construct of claim 2, wherein the first polypeptide construct comprises a first NBD amino acid sequence, the second polypeptide construct comprises a second NBD amino acid sequence, and the first and second NBD amino acid sequences are homologous sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimer or heterodimer complex.

4. The composition of claim 3 , wherein the first and second polypeptide constructs form a homodimer.

5. The TSB of the first polypeptide construct and the TSB of the second polypeptide construct are selected from the group consisting of PD-L1, mesothelin, epithelial cell adhesion molecule (EpCAM), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), carbonic anhydrase IX (CAIX), cadherin, carcinoembryonic antigen (CEA), cell-mesenchymal-epithelial transition factor (c-MET), epidermal growth factor receptor (EGFR) family members, ephrin type A receptor 3 (EphA3), fibroblast activation protein alpha (FAP), folate binding protein, folate receptor alpha (FR-alpha), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), insulin-like growth factor 1 receptor (IGF-1R), integrin αVβ3, integrin α5β1, solute carrier family members, and / or integrin αVβ1.

5. The composition of claim 4, which binds to a tumor-associated antigen (TAA) selected from the group consisting of Li-39 member 6 (Liv1), melanoma-associated antigen family A member (MAGEA), melanoma-associated antigen family C member (MAGEC), mucin (e.g., MUC1), New York esophageal squamous cell carcinoma 1 protein (NY-ESO-1, cancer / testis antigen 1A, cancer / testis antigen 1B), cancer / testis antigen 2 (NY-ESO-2, CTAG2), prostate-specific membrane antigen (PSMA), receptor activator of nuclear factor kappa B ligand (RANKL), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tenascin, TNF receptor superfamily member 10a (TRAILR1), TNF receptor superfamily member 10b (TRAILR2), and vascular endothelial growth factor receptor (VEGFR).

6. 6. The composition of claim 5, wherein each scaffold comprises an immunoglobulin polypeptide sequence.

7. 7. The composition of claim 6, wherein each scaffold comprises an immunoglobulin Fc (IgFc) amino acid sequence.

8. 8. The composition of claim 7, wherein each scaffold comprises an independently selected non-dimerized IgFc.

9. The composition of claim 8, wherein each polypeptide amino acid sequence is capable of inducing an antibody-associated effector function, or each IgFc polypeptide amino acid sequence contains one or more substitutions that enhance at least one antibody-associated effector function selected from antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity.

10. 10. The composition of claim 9, wherein each NBD comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from any of SEQ ID NOs: 1-7, 9, 10, 12, 15, 87, or 88.

11. The composition of claim 9 , wherein the NBD comprises a TAP1 NBD.

12. The composition of claim 11, wherein the TAP1 NBD comprises the amino acid sequence of any one of SEQ ID NOs: 10, 87, or 88, or a sequence having greater than 95% sequence identity to any one of those sequences.

13. The composition of claim 3, wherein the TSB of the first polypeptide construct and the TSB of the second polypeptide construct bind to a tumor-associated antigen (TAA) selected from the group consisting of: mesothelin and EpCAM.

14. 2. The polypeptide construct of claim 1, wherein when the polypeptide construct is present as a monomer, homodimer, or in a heterodimer, at least one of the polypeptide constructs further comprises an immune cell engager (ICE) amino acid sequence having affinity for a protein selected from the group consisting of CD3, CD4, CD8, αβ TCR, CD2, TCR α chain, TCR β chain, δγ TCR, TCR γ chain, TCR δ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34, and CD16.

15. 2. The polypeptide construct of claim 1, wherein when the polypeptide construct is present as a monomer, homodimer, or in a heterodimer, at least one of the polypeptide constructs further comprises an activation domain (AD) amino acid sequence selected from the group consisting of 4-1BBL, anti-4-1BB, CD40L, CD28, IFN-γ, a TNF superfamily member, TNF, lymphotoxin α, lymphotoxin αβ, BAFF (CD25), a B7 superfamily member, CD80, CD86, IL-15, IL-2, IL-7, IL-10, IL-12, PD1, anti-CD3, anti-CTLA4, and anti-CD28 amino acid sequences.

16. A pharmaceutical composition for treating cancer in a patient or subject, comprising the polypeptide construct of claim 1.

17. A pharmaceutical composition for the treatment of cancer in a patient or subject, comprising the polypeptide construct of claim 2.

18. 10. Use of the composition of claim 7 for the preparation of a medicament for the treatment of cancer.

19. 19. The use of claim 18, wherein the cancer is mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma, or germ cell tumor.

20. A nucleic acid comprising a nucleotide sequence encoding one or more of the constructs of claim 1, or a vector comprising said nucleic acid.

21. A cell comprising the vector or nucleic acid of claim 20.

22. 1. A composition comprising: (i) a first polypeptide construct according to claim 1; and (ii) a second polypeptide construct comprising an NBD amino acid sequence that comprises two ATP-binding sites of an ATP-binding cassette transporter protein and that is capable of heterodimerizing with a cognate, non-identical NBD of the first polypeptide construct in the presence of ATP; and an immune cell engager (ICE) amino acid sequence, (i) the NBD amino acid sequence of the first polypeptide construct constitutes a first heterodimerization NBD amino acid sequence, and the NBD amino acid sequence of the second polypeptide construct constitutes a second heterodimerization NBD amino acid sequence; (ii) the first heterodimerization NBD amino acid sequence is the cognate binding partner of the second heterodimerization NBD amino acid sequence; and (iii) The composition, wherein the first polypeptide construct and the second polypeptide construct bind to each other in the presence of ATP through an interaction between the first heterodimerization NBD amino acid sequence and the second heterodimerization NBD amino acid sequence to form a heterodimer or higher order complex of the first and second polypeptide constructs.

23. The TSB of the first polypeptide construct may be selected from the group consisting of mesothelin, epithelial cell adhesion molecule (EpCAM), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), carbonic anhydrase IX (CAIX), cadherin, carcinoembryonic antigen (CEA), cell-mesenchymal-epithelial transition factor (c-MET), epidermal growth factor receptor (EGFR) family members, ephrin type A receptor 3 (EphA3), fibroblast activation protein alpha (FAP), folate binding protein, folate receptor alpha (FR-alpha), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), insulin-like growth factor 1 receptor (IGF-1R), integrin αVβ3, integrin α5β1, solute carrier family 39 member 6 (L iv1), melanoma-associated antigen family A member (MAGEA), melanoma-associated antigen family C member (MAGEC), mucin (e.g., MUC1), New York esophageal squamous cell carcinoma 1 protein (NY-ESO-1, cancer / testis antigen 1A, cancer / testis antigen 1B), cancer / testis antigen 2 (NY-ESO-2, CTAG2), prostate-specific membrane antigen (PSMA), receptor activator of nuclear factor kappa B ligand (RANKL), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tenascin, TNF receptor superfamily member 10a (TRAILR1), TNF receptor superfamily member 10b (TRAILR2), and vascular endothelial growth factor receptor (VEGFR); and 23. The composition of claim 22, wherein the ICE of the second polypeptide construct comprises a Fab, Fab', scFv, aptamer, or nanobody having affinity for a protein selected from the group consisting of CD3, CD4, CD8, αβ TCR, CD2, TCR α chain, TCR β chain, δγ TCR, TCR γ chain, TCR δ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34, and CD16.

24. 23. The composition of claim 22, wherein the TSB of the first polypeptide construct binds to a TAA from the group consisting of mesothelin and EpCAM.

25. 23. The composition of claim 22, wherein the ICE has affinity for a protein selected from the group consisting of CD3, CD4, CD8, αβ TCR, CD2, TCR α chain, TCR β chain, δγ TCR, TCR γ chain, TCR δ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34, and CD16.

26. 23. The composition of claim 22, wherein the first and / or second polypeptide further comprises an activation domain (AD) amino acid sequence independently selected from the group consisting of 4-1BBL, anti-4-1BB, CD40L, CD28, IFN-γ, a TNF superfamily member, TNF, lymphotoxin α, lymphotoxin αβ, BAFF (CD25), a B7 superfamily member, CD80, CD86, IL-15, IL-2, IL-7, IL-10, IL-12, PD1, anti-CD3, anti-CTLA4, and anti-CD28 amino acid sequences.

27. 23. The composition of claim 22 for treating cancer in a patient or subject.

28. 23. Use of the composition of claim 22 for the preparation of a medicament for the treatment of cancer.

29. 29. The use of claim 28, wherein the cancer is mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma, or germ cell tumor.

30. A nucleic acid comprising a sequence encoding one or more of the first and second polypeptide constructs of the composition of claim 22, or a vector comprising said nucleic acid.

31. A cell comprising the vector or nucleic acid of claim 30.

32. 23. The composition of claim 22, wherein the first and second polypeptide constructs each comprise an independently selected scaffold amino acid sequence.

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