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 complex of the construct can stimulate immune cells (such as cytotoxic CD8 + T cells and / or NK cells) to play a role in promoting anti-tumor immune response. The constructs can be used as anticancer agents / therapeutic agents for the treatment of solid tumors with elevated levels of ATP.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,529 filed on March 1, 2022, U.S. Provisional Patent Application No. 63 / 330,120 filed on April 12, 2022, and U.S. Provisional Patent Application No. 63 / 353,896 filed on June 21, 2022.

[0002] I. Incorporation of Sequence Listing

[0003] The sequence listing is in ST.26 XML format, named 2974-3_PCT_ST26.xml, created on March 1, 2023, with a size of 205,746 bytes, prepared in accordance with 37 CFR 1.822 to 1.824, and submitted simultaneously with the filing of this application, and is incorporated herein by reference in its entirety.

[0004] II. Introduction

[0005] Effective anticancer therapy is still limited because systemic toxic effects are shown to be far below the ideal therapeutic dose. Immune agonists (such as cytokines and anti-T cell receptor antibodies) are particularly toxic when delivered systemically, and may cause serious adverse events, such as cytokine release syndrome and death. Strategies for increasing the effective dose (i.e., "therapeutic index") of immunotherapeutics at tumors relative to systemic dose / toxicity include intratumoral administration and targeting of immunotherapies via connections to antibodies against tumor-associated antigens ("TAA"). However, intratumoral delivery is customized and invasive, so due to the complexity of its associated surgery and logistics, the ability to reach a large number of patient populations is limited. New agents targeting TAA attempt to impose a certain degree of restriction on immune agonists, wherein the agonist accumulates to a biologically effective concentration only at sites with sufficient density of target TAA. Immune agonists targeting TAAs are severely limited, including but not limited to: a small number of unique and validated TAAs expressed on the surface of cells accessible to immunotherapies; low TAA density resulting in low efficacy; cancer escape due to loss of TAA expression; TAAs that are not truly tumor-specific resulting in on-target / off-tumor toxicity; and the fact that each TAA requires a different drug with a lower therapeutic impact (relative to systemic therapies).

[0006] Increasing the stoichiometry of immune excitation and / or TAA binding has also been explored as a means of enhancing the efficacy of immunotherapy targeting TAA. For example, the first generation of bispecific T cell engagers (such as blinatumomab) includes a single anti-TAA domain paired with a single anti-CD3 domain, with a stoichiometry of 1:1. However, in the case of the bispecific T cell engager AFM11, an anti-TAA: anti-CD3 stoichiometry of 2:2 is used instead. This form is related to more effective T cell-mediated in vitro tumor cell killing, and importantly, the number of cytotoxic effector T cells required for tumor cell killing is reduced. However, subsequent clinical trials of AFM11 showed severe toxicity and required to terminate the study. The stoichiometric increase of the Fc domain of the conceptually similar antibody-like molecule showed a similar increase in in vitro efficacy. Such stoichiometric changes may work by enhancing the affinity of TAA and immune receptor engagement. In addition, the cross-linking of known immune receptors (such as Fc receptors or T cell receptors) amplifies the signal transduction through these receptors and signal transduction pathways, producing a more effective immune response. Therefore, more effective tumor targeting, including reduced effector T cell and antigen density requirements, could potentially be achieved by coupling immune receptors with stoichiometrically enhanced immunotherapeutics. However, these enhanced immunotherapeutics have resisted clinical translation due to safety concerns.

[0007] Targeted immunotherapy can avoid the above shortcomings of current immunotherapy by exploiting tumor features that are both applicable to most / all tumors, essential for tumor survival, and highly expressed relative to normal tissues. The tumor microenvironment (TME) includes the vasculature, immune cells and supporting cells, extracellular matrix, and local molecules surrounding the tumor. The TME has common features with many different tumor types, including 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 features, extracellular ATP is an excellent candidate for the development of a new generation of immunotherapeutics because it is a product of an altered, essential tumor metabolic program that is common to most, if not all, tumors. Very high levels of extracellular ATP (50-1000 μM) are also observed in the TME relative to normal tissues (less than about 0.1 μM).

[0008] III. Summary of the Invention

[0009] The present disclosure includes and provides a molecular construct comprising a nucleotide binding domain (NBD), which enables the use of tumor ATP as a trigger for conditional (ATP-dependent) assembly of an effective cancer therapeutic agent at a tumor. The construct containing NBD forms a dimer or a higher-order complex in the presence of ATP levels found in the TME. The construct containing NBD is particularly useful as a therapeutic agent for treating various cancers. The therapy design using the construct utilizes the following fact: ATP-driven complexing (e.g., dimerization) can produce agents that recruit immune cells to the TME and / or stimulate immune cells in the TME. Stimulation can be performed by, for example, polymerizing the immunomodulatory protein domain presented to immune cells due to the complexing of the construct driven by ATP, and more effective immune cell stimulation is produced compared to a single immunomodulatory protein sequence that cannot cause, for example, cross-linking of its receptor. Dimers or other higher-order complexes of the construct containing NBD can also act by other mechanisms, including targeted ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity) of tumor cells. Because elevated ATP levels in the TME relative to surrounding tissues are virtually ubiquitous, this construct provides a mechanism for treating a variety of tumor types. In addition, because individual constructs that have not yet undergone assembly into dimers or higher-order constructs have limited or no ability to cause immune stimulation, elevated ATP levels in the TME can cause dimerization or complex formation, which can drive strong local stimulation and have less effect in non-tumor tissues with lower ATP concentrations. Therefore, constructs that can assemble immunomodulatory amino acid sequences into multivalent states in the presence of ATP levels found in the TME can be used to modulate immune cell action in the TME and cause selective immune-mediated anti-tumor effects.

[0010] Constructs containing NBD also provide several other advantages, including trans and cis targeting. The cis targeting of constructs containing NBD can affect the stimulation of tumor-infiltrating leukocytes (for example, by providing immunostimulant agonists, such as IL-2 or CD28), resulting in amplification of anti-tumor immune response. When trans targeting is carried out for TAA, the construct causes the immune-mediated cytolysis of the tumor cells carrying TAA, except for the effect produced by the increase in local ATP levels, which also increases other tumor-specific effects. Regardless of the targeting scheme adopted (for example, cis or trans targeting), constructs allow the use of a variety of immunomodulator domains derived from, for example, CD40L, CD28, IFN-g, IL-12, etc., each of which acts by requiring a cross-linked receptor. Other advantages of constructs containing NBD as described herein include its relatively small size, which allows penetration into tumor tissue, and its diffusion reduction from TME, due to the increase in size when they are assembled into dimers or higher-order complexes.

[0011] In addition to describing the constructs, this disclosure also provides methods of making and methods of using the same.

[0012] IV. Description of the Figures

[0013] Figure 1 A diagram of a construct of the present disclosure comprising an NBD having an ATP binding site, a scaffold domain (scaffold amino acid sequence), and an immune cell activation domain (AD) connected by a linker sequence is provided in A. The right side of the diagram in A also shows dimerization of the construct in the presence of an ATP concentration sufficient to substantially occupy the binding site (e.g., a concentration within the Kd range for ATP binding to the NBD or higher). Figure 1 Provided in B: (i) a first construct comprising a tumor-specific binder (exemplified as a bivalent cell lineage or tumor-specific antibody, but it can be a monovalent binder, such as an scFv or a nanobody) and a first NBD of a heterodimeric NBD pair connected by a linker; and (ii) a second construct comprising a second NBD of a heterodimeric NBD pair connected by a linker. The right side of the figure in B shows the formation of a heterodimer of the first construct and the second construct due to dimerization of the NBD domain in the presence of ATP.

[0014] Figure 2 A series of constructs (structures A to E) comprising NBD and AD are provided, with constructs of structures C, D and E further comprising a scaffold sequence that may be non-dimerizing or interspecies. The elements of each construct are connected by an independently selected optional linker. The figure also shows dimers of the constructs formed in the presence of ATP.

[0015] Figure 3 A series of construct pairs (structures A to O) are provided. In AI, the first construct of each pair comprises one or more NBDs and a tumor-specific binder that binds to a TAA, and the second construct of the pair comprises an NBD and an AD and / or a monovalent immune cell engager. In JM, the first construct of each pair comprises one or more NBDs and an immune cell engager, and the second construct of the pair comprises an NBD and an AD. Each construct pair EI, L, and M has a scaffold in at least one construct. In N and O, the first construct of each pair comprises a tumor-specific binder and two NBDs, and the second construct of the pair comprises an NBD and an AD. The construct in O also comprises a scaffold sequence. Figure 3 Also shown is the formation of dimers of the constructs in the presence of ATP. The elements of each construct are connected by independently selected optional linkers.

[0016] Figure 4AA series of constructs (see A, C and D), construct pairs (B, E, F, G and I) or construct triplets (H) are provided, which comprise two or more NBDs and can be polymerized in the presence of ATP (e.g., in the TME) to form a complex. In some cases, one or more constructs comprise a scaffold sequence, and each construct can be connected by an independently selected optional linker. The figure also shows the formation of a complex in the presence of ATP, the complex comprising the construct present in each case.

[0017] Figure 4B Examples of NBDs bound to tumor-specific binders (e.g., antibodies such as anti-mesothelin) are shown, which form homodimers in the presence of elevated ATP (such as found in the tumor environment). In (A), the construct comprises a bivalent antibody, which can be monospecific or bispecific, thus producing a tetramer or tetramer-like complex in the presence of ATP. The molecule need not contain an Fc domain, can have two antigen binding domains (e.g., VHH or scFV tumor-specific antigen binders) and still remain bivalent. In (B), (C) and (D), the homodimerization construct comprises an NBD monovalent single-chain antibody. The tumor-specific binders in (A) to (D) can be, for example, anti-HER2, anti-mesothelin, anti-EpCAM, anti-CLA-4 or anti-PSMA. When a scaffold sequence is present, the scaffold sequence can be an immunoglobulin Fc peptide (such as IgG1 Fc) that can bind any one or more of the FcγRI, FcγRII, FcγRIII receptors and / or C1q proteins. Complexes capable of binding to one or more of those proteins can induce antibody-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC) against cells expressing a target (e.g., an antigen) to which the tumor-specific binder component can bind. Thus, such antibody functions can be used therapeutically to eliminate tumor cells.

[0018] Figure 5 A dimer of the transporter associated with antigen processing 1 (TAP1) NBD containing two ATP molecules is shown.

[0019] Figure 6Shown is an alignment of the NBD domains of wild-type TAP1 (SEQ ID NO:9), the ABCA1 gene product from UniProtKB-095477 (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). A portion of the Walker A region sequence from amino acids 46 to 55 and the LSGGQ sequence from amino acids 152 to 156 of the TAP1 amino acid sequence are underlined and bolded. Figure 3 The TAP1 NBD shown in Figure 2 hydrolyzes the acidic amino acid (ie, Asp at position 177) and Asp (D) or Glu (E) residues.

[0020] Figure 7 The sequence of human TAP2 from UniProtKB-Q03519 is shown (SEQ ID NO: 11).

[0021] Figure 8 SDS-polyacrylamide gel electrophoresis (PAGE) gels of TAP1 variants carrying N676G, S677N, Q680R, E682Q and / or Q683R substitutions are shown. Lanes from left to right are: molecular weight marker (M), reduced sample (R), empty lane and non-reduced sample (NR).

[0022] Fig. 9 Size exclusion chromatograms of TAP1 NBD variant polypeptides carrying N676G, S677N, Q680R, E682Q and / or Q683R substitutions and substitution of cysteine ​​present in the sequence with serine are shown, showing that (i) a monomer form of the polypeptide is produced in the presence of 1 mM adenosine diphosphate (ADP) (apparent Mw 37 kDa) and (ii) a dimer form of the polypeptide is produced in the presence of 1 mM adenosine triphosphate (ATP) (apparent Mw 57 kDa).

[0023] Fig.10 Provides Fig. 9Results of mass photometric assessment of the same TAP1 NBD variants subjected to chromatography in Figure 2. The results obtained in the presence of 1 mM ATP indicate dimerization of the TAP1 NBD variants.

[0024] Fig.11 Provided are the results of dynamic light scattering (DLS) sizing of TAP1 in the presence of (A) ADP or (B) ATP.

[0025] Fig.12 Provided is a Coomassie blue stained gel of purified TAP1 NBD of SEQ ID NO: 87 modified to homodimerize and chromatographic analysis of the purified protein in the presence of ADP and ATP.

[0026] FIG. 13 shows an SDS-PAGE gel of: (A) induced expression of TAP1NBD polypeptides provided by SEQ ID NO: 87 and SEQ ID NO: 88; (B) purification of the polypeptide of SEQ ID NO: 88. FIG.

[0027] V. Specific Implementation Methods

[0028] A. Definition

[0029] As used herein, amino acids ("aa" singular or "aas" plural) refer to naturally occurring proteinogenic amino acids that are incorporated into polypeptides and proteins during translation in mammalian cells. Unless otherwise indicated, these amino acids are: 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 the amino acids hydroxyproline and selenocysteine, which occur in some proteins found in mammalian cells; however, they should not be construed as being included unless their presence is specifically noted.

[0030] Amino acid substitutions at specific positions in a sequence are represented by the original amino acid given in a single or three letter code, the numerical position of the amino acid, and the amino acid substituted into the sequence represented in a single or three letter code. For example, the substitution of alanine (A) at position 12 in the sequence by proline (P) is represented using a single letter code as A12P, or using a three letter code as Ala12Pro. In the case where more than one substitution occurs in the same sequence, they can be separated by a slash " / ". Thus, the above A12P substitution in the same sequence with V23A and Q41G substitutions can be represented by the sequence A12P / V23A / Q41G.

[0031] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids, which, unless otherwise specified, are the naturally occurring proteinogenic L amino acids biosynthetically incorporated into proteins during translation in mammalian cells.

[0032] Alignment can be performed using the National Center for Biotechnology Information (NCBI) BLAST program (blastp release version BLAST+2.9.0 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 to identify corresponding residues of different sequences. Unless otherwise specified, Clustal Omega was used to identify corresponding residues of different sequences.

[0033] Percent sequence identity refers to the percentage of identical amino acids or nucleotides between two aligned amino acid or nucleic acid sequences. Unless otherwise stated, percent sequence identity is determined using BLAST+ release version 2.9.0 with default parameters for blastp alignments for polypeptides or blastn alignments for nucleic acids.

[0034] Unless otherwise indicated, the term "substantially" is intended to encompass "completely" and "mostly but not completely." For example, an Ig Fc that "does not substantially induce cell lysis by ADCC or CDC" means an Ig Fc that does not induce cell lysis at all or does not mostly but not completely induce cell lysis by ADCC or CDC.

[0035] B. Description

[0036] The microenvironment within the tumor is different from non-malignant tissue in many aspects, including any one or more differences in its pH, ATP content, limited oxygen level (hypoxia) and tumor protease (e.g., MMP) level. In normal tissue, the extracellular ATP level in normal (non-tumor) tissue is generally within the range of 0.01-0.1 micromolar (μM), while the extracellular level of ATP in solid tumors is generally within the range of 50-200 μM. The present disclosure describes and provides the use of ATP levels found in the tumor microenvironment to cause an immune response to 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 engages and stimulates immune cells) and at least one NBD, which forms a large number of dimers or other higher-order complexes in the presence of ATP at the level found in the tumor microenvironment (TME). Dimerization or higher-order complex formation of NBD produces a complex presenting two or more immune cell ADs. The use of immune cell ADs, which 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 by themselves when presented alone, allows for intratumoral stimulation of immune responses within the TME.

[0037] Other constructs described herein may include a tumor-specific binding domain or an immune cell engagement domain and at least one NBD that binds to a TAA, which forms a large number of dimers or other higher-order complexes in the presence of ATP at the level found in the tumor microenvironment. Such constructs may optionally include AD. Dimerization or higher-order complex formation of NBDs produces dimers or higher-order complexes that can recruit and / or stimulate immune cells in the TME. Because the dimerization or other higher-order complexes of such constructs do not form higher-order complexes outside the TME, they allow for stimulation of immune responses within the tumor while limiting immune stimulation outside the tumor.

[0038] Because the AD, tumor-specific binding domain and immune cell adapter of the uncompounded construct will not effectively stimulate cells, such as NK cells and CD8+T effector cells, when presented alone, the construct will not cause systemic immune activation and related toxic effects. However, the construct described herein provides an immunostimulation located in the TME, in which there is ATP sufficient to drive the formation of the complex, so it can be considered as the form of an ATP-dependent agonist of immune cell function, and its effect is limited to the environment. In addition, because the size and molecular weight of the composite form (e.g., dimer, trimer, etc.) of the construct increase, their diffusion from the TME is further reduced, and its effect is limited to the TME. Those dimers or higher-order complexes that diffuse out of the TME will dissociate into invalid single monomers because the ATP levels in the surrounding non-malignant tissues are too low to support their compounding. Therefore, the immunostimulation provided by the complex of the construct described herein is understood to be reversible and limited to a microenvironment in which the ATP level is high enough to induce the compound.

[0039] C. Structure of ATP-dependent agonists of immune cell function

[0040] 1. ATP-dependent agonists of immune cell function including NBD and AD

[0041] The molecular construct containing NBD described herein comprises NBD and AD in its simplest form, which engages and stimulates immune cells (e.g., CD8+T cells, NK cells, etc.). NBD comprises one or more ATP binding sites, and when these sites are occupied by ATP, due to the interaction between the NBD of the construct, the construct of two or more molecules forms a duplex or a higher-order complex (triplex, etc.). 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 Figure 1 The structure A and Figure 2 In some cases, interspecies scaffold sequences that form heterodimers can be incorporated into the construct so that more than one type of AD can be present in the molecule, such as in Figure 2 exemplified by structure E. Due to the high levels of ATP in the tumor microenvironment, the use of ADs allows for selective stimulation of intratumoral immune responses while avoiding systemic activation of targeted immune cells (e.g., NK cells or T cells) and / or immune responses in non-tumor tissues, which ADs can stimulate targeted immune cell (such as NK cells and / or T cells) populations when combined together (e.g., as homodimers or heterodimers).

[0042] NBD amino acid sequence, scaffold amino acid sequence and AD can be arranged in any order. In the first case, the construct comprises (for example, from N-terminus to C-terminus) NBD amino acid sequence, scaffold amino acid sequence and AD. In the second case, the construct comprises NBD amino acid sequence, AD and scaffold amino acid sequence from N-terminus to C-terminus. In the third case, the construct comprises AD, scaffold amino acid sequence and NBD amino acid sequence from N-terminus to C-terminus. In the fourth case, the construct comprises AD, NBD amino acid sequence and scaffold amino acid sequence from N-terminus to C-terminus. In the fifth case, the construct comprises scaffold amino acid sequence, AD and NBD amino acid sequence from N-terminus to C-terminus. Finally, in the sixth case, the construct comprises scaffold amino acid sequence, NBD amino acid sequence and AD from N-terminus to C-terminus.

[0043] 2. NBD-containing constructs containing tumor-specific binders and / or immune cell engagers

[0044] The NBD-containing constructs described herein may contain one or more tumor-specific binding domains and / or immune cell engaging domains in addition to the NBD. As discussed in more detail below, tumor-specific binding domains, also referred to as tumor-specific binders, are domains of NBD-containing constructs that have affinity for TAAs expressed on the surface of tumor cells. Immune cell engaging domains, also referred to as immune cell engagers, are also discussed in more detail below and are domains of NBD-containing constructs that have affinity for cell surface molecules (e.g., antigens) of immune cells. Unless otherwise specified, 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. NBD-containing constructs that contain one or more tumor-specific binders and / or immune cell engagers may also contain one or more ADs. See Figure 3 Structures D, E, H and I.

[0045] Tumor-specific binders and immune cell engagers can be incorporated into NBD-containing constructs that form heterodimers or other higher order complexes (e.g., trimers) in the presence of ATP at concentrations found in, for example, the TME environment. Heterodimer formation can be accomplished by placing a first ( 第 1) The heterodimeric NBD is incorporated into the first member of the construct pair and the second ( 第 2) The heterodimeric NBD is incorporated into the second member of the construct pair to drive. 第 1 and 第2 heterodimerizing NBD pairs act as homologous binding partners. To illustrate the types of such NBD-containing construct pairs encompassed by the present disclosure, a series of non-limiting examples of such NBD-containing construct pairs are provided in Figure 3 In the figure, it is shown. In vivo, the dimerization construct guides the immune cells bound by the immune cell engager to interact with the target tumor cells recognized by the tumor-specific binder, causing an immune response to the target tumor cells. Such constructs allow targeting tumor cells in the TME because they require elevated ATP to form complexes, thereby limiting off-target binding and stimulating immune cells. The paired constructs comprising tumor-specific binders and immune cell engagers can be enhanced by incorporating one or more ADs (which may be the same or different), and dimerization caused by the interaction of the first NBD-containing construct and the second NBD-containing construct may be required to stimulate target immune cells.

[0046] Figure 3 Items A and B of each provide a construct pair, wherein the first NBD-containing construct comprises (e.g., from the N-terminus to the C-terminus or from the C-terminus to the N-terminus) one or more 第 1 heterodimerizing NBD, and a second NBD-containing construct comprising (e.g., from N-terminus to C-terminus) homologous 第 2 Heterodimerizes NBD and AD. The elements of the construct are connected by independently selected optional linker sequences. Such constructs allow targeting of ADs to tumor cells in the TME because they require elevated ATP for heterodimerization, thereby limiting off-target binding and avoiding systemic stimulation of immune cells by ADs. In addition, the ADs of the constructs shown in B may be the same or different and may require dimerization to function on target immune cells. Although in Figure 3 , but one or both of the heterodimerization constructs in A or B may also comprise a scaffold amino acid sequence (see, e.g., Figure 3 Project E).

[0047] illustrate Figure 3An exemplary pair of heterodimerization of the type shown in item A or B of can include, for example: (i) a first NBD-containing construct comprising one or more TAP1 NBDs connected to a tumor-specific binder having affinity for TAA (e.g., NY ESO-1, mesothelin, CEA, PSMA, or MAGE (such as MAGE-A1, MAGE-A4, MAGE-A9, or MAGE-A11)); and (ii) a second NBD-containing construct comprising a TAP2 NBD and an IL-2 sequence, the TAP2 NBD acting as a cognate binding partner of the TAP1 NBD in the presence of ATP. When exposed to ATP at levels present in the TME, such constructs heterodimerize and can act to stimulate T cells present in the TME.

[0048] Figure 3 Items C and D of each provide a pair of heterodimerization constructs, wherein the first NBD-containing construct comprises (e.g., from the N-terminus to the C-terminus) one or more NBDs linked to a tumor-specific binding agent. 第 1 heterodimerizing NBD, and a second NBD-containing construct comprising (e.g., from N-terminus to C-terminus) homologous 第 2 heterodimerization NBD and immune cell adapter. The elements of the construct are connected by independently selected optional linker sequences. Either or both of the first and second constructs of the heterodimerization pair may contain a scaffold sequence (see, e.g. Figure 3 F to I). The construct comprising a tumor-specific binder and / or an immune cell engager may further comprise an AD as in items D, E, H and I. In the case where an AD is present in a construct further comprising an immune cell engager, the AD may be selected from those ADs that need to be presented to T cells as dimers or higher order complexes (e.g., TNF family members such as 4-1BBL) for immune cell stimulation to prevent off-target stimulation.

[0049] Example Figure 3The construct pair of item C of comprises, for example: (i) a first NBD-containing construct comprising (a) TAP1 NBD connected to (b) a tumor-specific binder; 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 binder can be an antibody, antibody fragment, or single-chain antibody construct (such as a scFv or nanobody) that has affinity for a TAA (e.g., NY ESO-1, mesothelin, CEA, PSMA, or MAGE (such as MAGE-A1, -A4, -A9, or -A11)) expressed on the surface of a tumor cell.

[0050] Figure 3 Items A to I of are shown as combinations of two molecular constructs. Because in those combinations the first construct comprising a tumor-specific binder is separated from the second construct comprising an AD and / or immune cell engager, a population (mixture) comprising more than one type of first construct or second construct can be formed. For example, Figure 3 A mixture of a first construct and a second construct as described in any one of items A to I, wherein the mixture comprises two or more first constructs, each first construct having a tumor-specific binding agent targeting a different TAA. Figure 3 A mixture of the first construct and the second construct as described in any one of items A or B of , wherein the mixture comprises two or more second constructs, each second construct having one or more ADs for different immune cell receptors. Similarly, Figure 3 A mixture of a first construct and a second construct as described in any one of items C to I of the present invention, wherein the mixture comprises two or more second constructs, each second construct having a different immune cell engager for a different immune cell surface antigen. In addition to the population in which only one of the immune cell engagers, tumor-specific binders or ADs is different, the mixture of the first construct and the second construct may comprise two or more first constructs and two or more second constructs, which differ in two or more elements selected from the group consisting of immune cell engagers, tumor-specific binders and ADs.

[0051] Figure 3 Items J to M of each provide a construct pair, wherein the first NBD-containing construct comprises (e.g., from the N-terminus to the C-terminus) one or more NBDs linked to an immune cell engager. 第 1 heterodimerizing NBD, and a second NBD-containing construct comprising (e.g., from N-terminus to C-terminus) homologous 第2 Heterodimerization NBD and AD. The elements of the construct are connected by independently selected optional linker sequences. Such constructs allow activation of immune cells in the TME while avoiding any large amount of activation outside the TME because they require elevated ATP for heterodimerization, thereby limiting off-target binding and avoiding systemic stimulation of immune cells by AD. In addition, the AD of the construct shown in K or M can be the same or different, and dimerization may be required to work on target immune cells.

[0052] illustrate Figure 3 The exemplary construct pairs of heterodimerization constructs of the types in J and K of the present invention include, for example: (i) a first construct containing NBD, comprising one or more TAP1 NBDs connected to an immune cell engager (e.g., an antibody, an antibody fragment or a single-chain antibody construct (such as a VHH or a nanobody)), the immune cell engager having affinity for CD28 expressed on the surface of T cells; and (ii) a second construct containing NBD, comprising a TAP2 NBD and an IL-2 sequence, the TAP2 NBD acting as a cognate binding partner of TAP1 NBD in the presence of ATP. Such constructs can act to stimulate T cells present in the environment when they are compounded into heterodimers by the ATP at the level present in the TME.

[0053] Compositions comprising mixtures of constructs can be used for therapeutic applications, where the use of more than one tumor-specific binder to different TAAs can avoid tumor escape from monotherapy with a single construct. Similarly, the use of more than one immune cell engager and / or AD can recruit more than one type of immune cell and provide a more robust immune response to tumor cells, thereby achieving a more robust immune response to the tumor than would be produced using a monotherapy with a single construct.

[0054] 3. Formation of NBD-containing constructs comprising two or more NBDs and complexes of NBD-containing molecular constructs

[0055] The construct containing NBD described herein can include two or more NBDs, which allow the construct to be effectively polymerized into a complex in the presence of ATP. Two or more NBDs can be, for example, arranged as a series pair. When placed in series, NBDs are adjacent to each other in the construct, and except for the interval joint amino acid sequence, are not separated by any element of the construct (for example, there is no AD, support, tumor-specific binding domain or immune cell engagement domain between the series NBDs). Other NBDs can be located in the construct, so they are not placed in series. For example, the construct containing NBDs comprising two or more NBDs can be formed by replacing the NBDs of the first to sixth cases mentioned above in part C.1. (ATP-dependent agonists of immune cell functions comprising NBDs and AD) with a pair of series NBDs. The construct containing NBDs comprising two or more NBDs (for example, series connection) can also be formed by the construct comprising an immune cell adapter or a tumor-specific binding agent.

[0056] Complexes of NBD-containing molecular constructs comprising at least two NBDs can be formed by placing the construct in an environment, such as the TME, where ATP levels are high enough for ATP to occupy the binding sites. Figure 3 Neutralization of N and O Figure 4A In the case where more than one NBD amino acid sequence is incorporated into a construct of the present disclosure (e.g. Figure 4A In the case of (), the construct can actually be reversibly polymerized in the presence of ATP to produce a complex. The complex formed in the presence of ATP can be a dimer, trimer, tetramer or even a higher order structure. See, e.g. Figure 4A Incorporating more than one NBD in a construct may allow for maximizing potency and efficacy, and may affect other properties. For example, complexes that become multivalent for elements expressed on the surface of target cells (e.g., for tumor-specific engagers or ADs) may show increased effective affinity for target cells, larger complexes may be retained longer in the TME, effectively extending the in vivo half-life of the complex, etc.

[0057] The complex may comprise at least two NBDs arranged in tandem, with at most one NBD having an amino acid sequence (see, Figure 4A A, B and E) and AD, immune cell engagers and / or tumor-specific binders separated by a linker amino acid sequence (e.g., a rigid linker). Alternatively, the NBD can be attached alone to a tumor-specific binder or immune cell engager (see, Figure 4A Structures C and D). The NBD-containing construct used to form the complex may contain a homodimeric or heterodimeric NBD. Figure 4ANot shown in Figure 3, self-association of homodimeric NBDs in the same construct can be prevented by constructing the molecule so that a productive ATP binding interface cannot be formed between the domains. This can be achieved, for example, by limiting the distance between the NBDs or by using a rigid peptide linker between the NBDs.

[0058] A NBD-containing construct comprising at least two NBDs and an AD is Figure 4A exemplified by structures A and B. Structure A employs an NBD that homodimerizes in the presence of ATP and can form dimers and / or larger constructs, such as Figure 4A As shown in structure A of . In contrast, structure B contains structures that form heterodimers or polymers in the presence of ATP. When exposed to ATP at levels present in the TME, the construct can form a large complex presenting the AD (e.g., as an AD homodimer or heterodimer). Such complexes containing NBD constructs can stimulate target immune cells in the TME, such as NK cells and / or T cells. The ADs present in the complex can be the same (e.g., Figure 4A structure A) or different (e.g., Figure 4A B) For example, the structure of 第 1AD and 第 The amino acid sequences of 2AD may be identical and result in activation of immune cell responses when presented in a dimer or higher order complex. Alternatively, the presence of 第 1AD and 第 The 2ADs may be derived from different molecules (e.g., different interleukins) that, when presented together, cause immune cell activation. 第 1AD and 第 The amino acid sequences of the 2ADs may also differ (not identical) and when combined form active fragments of dimeric immunomodulators (such as IL-12, 11-23 or IL-27), each of which comprises two subunits (α and β polypeptide chains). NBD-containing constructs, each comprising a single NBD, can be assembled into a complex comprising two different (not identical) ADs in the presence of ATP. Alternatively, active ADs consisting of two subunits can be assembled into active ADs in the presence of ATP using constructs, each comprising a single NBD (see, e.g. Figure 2 structures A and B).

[0059] In addition to the above-mentioned constructs containing NBD, the present disclosure also provides a construct containing NBD, which comprises at least two NBDs and a tumor-specific binding amino acid sequence (tumor-specific binder) (such as Figure 4A C) and / or immune cell binding amino acid sequence (immune cell engager) (eg Figure 4ASuch molecular constructs optionally include one or more independently selected scaffold amino acid sequences and / or one or more independently selected linker sequences. Tumor-specific binders 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 these molecules, including but not limited to antibodies that bind to immune cell surface antigens (e.g., CD3, CD8 or CD4 on αβT cells, TRGV9 on δγT cells, CD16 on NK cells, etc.). Unless otherwise specified, immune cell engagers are monovalent to avoid stimulating immune cells outside the TME, and sequences that can cause antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) are modified to substantially limit or prevent such reactions.

[0060] Once present in the TME (where ATP levels allow the NBD domains to form higher order complexes (e.g., dimers)), constructs comprising at least two NBDs and a tumor-specific binder and / or immune cell engager form large complexes that can lead to immune stimulation. Constructs comprising at least two NBDs, a scaffold containing an IgFc, and a tumor-specific binder can form complexes in the TME (see, e.g., Figure 4A Structure C), the complex can, for example, lead to ADCC or CDC. Constructs comprising at least two NBDs and an immune cell engager can form a complex in the TME (see, e.g. Figure 4A Structure D), which complex can, for example, lead to stimulation of the targeted immune cells due to cross-linking of target antigens on the surface of the targeted immune cells (e.g., CD3 on CD8+ T cells or CD16 on NK cells).

[0061] A NBD-containing construct (e.g., Figure 4A C) with a NBD-containing construct comprising at least two NBDs and an immune cell engager (e.g., Figure 4A Combining the structure D) with a tumor-specific binder can produce a complex comprising a tumor-specific binder and an immune cell engager. 第 1 Heterodimerized NBD and immune cell engagers 第 2 Heterodimerized NBDs can ensure that the complex formed in the presence of ATP contains both the binder and the adapter and can stimulate immune cell responses to target tumor cells. This is exemplified by a mixture of a construct comprising at least two NBDs and a tumor-specific binder and a construct comprising at least two NBDs and an immune cell adapter, forming a large complex in the TME (see, e.g. Figure 4AThe complexes generated by such structures in the presence of sufficient ATP can bind immune cells to tumor targets via tumor-specific binders while causing activation of immune cells by cross-linking of receptors on the cell surface via the now complexed monomeric immune cell engagers.

[0062] The tumor-specific binder and / or immune cell engager amino acid sequences present in each construct used to form the complex need not be identical. Figure 3 Structures A to I and Figure 4A The complexes of structures C to I can have more than one tumor-specific binder (e.g., two or more tumor-specific binders) for different TAAs and more than one immune cell engager when present. The use of two or more different tumor-specific binders for different TAAs in a single complex not only allows the complex formed by the construct comprising two or more NBDs to be active against tumors expressing different tumor antigen targets, but also limits tumor escape from treatment due to loss of tumor antigen expression. The use of different immune cell engagers in a single complex allows the stimulation of immune responses from more than one type of immune cells (e.g., CD8+T cells and NK cells). ADs can also be incorporated into complexes formed in the TME, including complexes comprising constructs containing tumor-specific binders. This can be accomplished by, for example, combining one or more constructs (such as ADs) comprising ADs and NBDs (e.g., two or more NBDs) in a complex. Figure 4A H), or by combining one or more constructs comprising an AD, an NBD (e.g., two or more NBDs), and a tumor-specific binder in a complex (such as Figure 4A (as exemplified in I).

[0063] Because the tumor-specific binder, 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 can be used for different therapeutic combinations.

[0064] D. Elements of NBD-containing constructs

[0065] 1. NBD and its interaction with ATP

[0066] The NBD for incorporation into the construct described herein can be selected based on several criteria. In the case where the construct is intended for human therapeutic use, the NBD is preferably derived from human ATP binding protein to limit its immunogenicity, and preferably can be formed by solution phase dimerization or higher complexes when exposed to enough ATP. When using a non-human protein with immunogenicity, the immunogenic region can be changed to be closer to or completely match its human homologue to humanize the immunogenic part. The solution phase formation of the dimer or higher complex used in the construct described herein occurs at the ATP concentration found in the target TME, but does not occur in normal tissue. Therefore, the NBD used in the construct provided herein can, for example, exceed the upper limit level found in the extracellular space of normal tissue by about one order of magnitude (10 times) (that is, about 1 μM or higher) to about ATP levels found in TME. There is a dissociation constant for ATP. In some cases, the affinity of NBD will be about 1 μM to about 200 μM. For example, the ATP affinity of the NBD can 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 can be in the range of about 25 μM to about 50 μM. In other examples, the affinity of the NBD can be in the range of about 50 μM to about 100 μM or about 100 μM to about 200 μM.

[0067] The NBD of some proteins forms homodimers in the presence of sufficient levels of ATP, while other NBDs form heterodimers in the presence of sufficient levels of ATP. Both homodimerizing NBDs and heterodimerizing NBDs can be used in various aspects of the polypeptide constructs described herein. In the case where the NBD is expected to form homodimers in solution when there are sufficient levels of ATP, the amino acid residues at the interface between the dimers can be modified to remove or replace the amino acid residues that interfere with the formation of the interface between the two NBD molecules. Alternatively, the amino acid that does not contribute to the interaction between the two NBD molecules can be replaced with an amino acid that contributes to the interaction that leads to the formation of homodimers.

[0068] a) ATP binding cassette as NBD

[0069] Many proteins contain NBDs suitable for use as NBDs in the constructs described herein. Among those proteins, ATP-binding cassette (ABC) transporters represent suitable candidates, in part because they are a large family of highly conserved transporters that are unlikely to be immunogenic themselves. ABC transporters are a large superfamily of membrane proteins with multiple functions that utilize the energy of ATP hydrolysis to facilitate substrate transport into or out of the cytoplasm. See, e.g., 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 different ABC transporters. Same as above. ABC transporters are divided into seven different family members: ABC1, MDR / TAP, MRP, ALD, OABP, GCN20 and White. Representative members of each family, including TAP1, are described in Figure 6 The NBDs of the ATP-binding cassette family are conserved and contain two subdomains. Same as above. The first is similar to the RecA protein and contains the P-loop (Walker-A motif). The second subdomain, called the "helical subdomain", contains the LSGGQ motif. Same as above. The NBDs of the transporters assemble into dimers in a head-to-tail orientation (forming two ATP binding and hydrolysis sites sandwiched at their interface) (see Figure 5 ). In this orientation, the LSGGQ motif in each of the two domains is adjacent to the opposing Walker-A sequence at the ATP sandwich interface. Such interfaces are described, for example, in Loo et al., (2002) J Biol Chem., 277(44):41303-41306 and Smith et al., (2002) Mol Cell., 10(1):139–149, Figure 3 A. When no nucleotide (ATP) is present in the binding site, there is a gap at the domain interface; however, when ATP is bound, the NBD dimerizes and hydrolyzes ATP and can then dissociate (see, e.g., Locher 2009, supra). Stable dimers can be formed by including amino acid substitutions that render the NBD domain sequence ATP-hydrolysis-deficient or substantially ATP-hydrolysis-deficient (e.g., by reducing the ATP hydrolysis rate by one or more orders of magnitude (see, e.g., Vakkasoglu et al., (2010) PLoS ONE, 12(5):e0178238, available at doi.org / 10.1371 / journal.pone.0178238). For example, the hydrolyzed Asp or Glu residues found in the NBD protein can be replaced by Asn or Gln residues (see, e.g., Figure 3 ).

[0070] In the case where the NBD of an ABC protein is expected to form a homodimer in solution when sufficient ATP is present, the residues at the interface between the dimers can be modified to remove or replace amino acid residues that interfere with interface formation, or to replace amino acids that do not contribute to interface interactions with amino acids that can contribute to interactions. For example, the NBD of rat TAP1 is known to homodimerize in solution and can be used as a model for ATP-sandwich interface formation to induce solution homodimerization in the presence of ATP (see, e.g., 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 sequences known to homodimerize (e.g., rat TAP1 NBD) can be used to guide changes in the target sequence. Alignments can be made using the National Center for Biotechnology Information (NCBI) BLAST program (blastp release version BLAST+ 2.9.0 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 specified, Clustal Omega was used.

[0071] The NBD amino acid sequence of the ABC-containing protein may comprise all or part of the NBD domain of the phospholipid transporting ATPase (ABC or ABCA1 transporter) encoded by the ABCA1 gene UniProtKB-095477 (SEQ ID NO: 1). The NBD may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 1.

[0072] The NBD amino acid sequence may comprise all or part 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 may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 2.

[0073] The NBD amino acid sequence may comprise all or part of the NBD domain of a multidrug resistance-associated protein (MRP, multidrug resistance-associated protein 1) encoded by the ABCC1 gene UniProtKB-P33527 (SEQ ID NO: 3). The NBD may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 3.

[0074] The NBD amino acid sequence may comprise all or part 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 may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 4.

[0075] The NBD amino acid sequence may comprise all or part 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 may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 5.

[0076] The NBD amino acid sequence may comprise all or part of the NBD domain of the ATP-binding cassette subfamily F member 1 protein (GCN20, TNFα-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.

[0077] The NBD amino acid sequence may comprise all or part of the NBD domain of the ATP binding cassette subfamily G member 1 protein (White, a homolog of Drosophila White) encoded by the ABCG2 gene UniProtKB / Swiss-Prot: P45844 (SEQ ID NO: 7). The NBD may comprise a sequence having at least about 90% or at least about 95% sequence identity to SEQ ID NO: 7.

[0078] (1) TAP1 and TAP2 ATP-binding cassettes as NBD

[0079] TAP1 and TAP2 proteins are homologous 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, which represent the source of the NBD for the construct provided herein. It is known that rat TAP1 NBD homodimerizes in the presence of ATP. The NBD of human TAP1 does not homodimerize in the presence of ATP, but can be induced to homodimerize 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; on the World Wide Web doi.org / 10.1371 / journal.pone.0178238. The substitutions that cause TAP1 homodimerization include those substitutions at one or more (e.g., each) of N676, S677, Q680, E682, and Q683, which are shown in bold and italics in SEQ ID NO:8. For example, the TAP1 substitutions that cause homodimerization can include one or more (e.g., all) of N676G, S677N, Q680R, E682Q, and Q683R substitutions. The portion (e.g., domain) of the TAP1 and TAP2 proteins used as NBDs can also include substitutions (e.g., with serine residues) of one or more cysteine ​​present in the sequence to prevent undesirable disulfide bond formation, resulting in non-functional folding, or undesirable intermolecular disulfide bond formation. TAP1 and TAP2 NBDs can also include His tags, such as at the C-terminus of the domain for purification of constructs containing those NBDs.

[0080] The NBD amino acid sequence from human TAP1 (SEQ ID NO: 9) may, for example, comprise the same sequence as the NCBI reference sequence: NP_000584.3 or UniProtKB-Q03518-2 of SEQ ID NO: 8:

[0081]

[0082] (reference sequence: NP_000584.2, SEQ ID NO: 8) having an amino acid sequence with at least greater than 90% or greater than 95% sequence identity to the NBD amino acid sequence (amino acids 492-748);

[0083] The TAP1 NBD domain shared by the NCBI and UniProt sequences is:

[0084]

[0085]

[0086] Residues N676, S677, Q680, E682 and Q683 of SEQ ID NO: 8 associated with homodimerization are shown as N185, S186, Q189, E191 and Q192 in SEQ ID NO: 9 and are shown in bold and italics. The NBD amino acid sequence from human TAP1 may, for example, comprise an amino acid sequence having greater than 97% or greater than 98% sequence identity to at least 230 consecutive amino acids of the NBD of the amino acid sequence shown in NCBI Reference Sequence: NP_000584.2 (amino acids 492-748) (provided as SEQ ID NO: 9).

[0087] The NBD amino acid sequence from human TAP1 may, for example, comprise 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, for example, comprise 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 shown in bold and italics in SEQ ID NO: 10 below. The NBD amino acid sequence from human TAP1 may also comprise substitutions at Asp, for example, corresponding to D668 of SEQ ID NO: 8, which renders the NBD defective in ATP hydrolysis. Thus, the NBD polypeptide amino acid sequence of TAP1 may, for example, comprise the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having greater than 90% or greater than 95% sequence identity thereto:

[0088]

[0089] 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.

[0090] The NBD amino acid sequence from human TAP1 may, for example, comprise substitutions corresponding to those at C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO: 8. The NBD amino acid sequence from human TAP1 may also, for example, comprise substitutions corresponding to those at 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 shown in bold and italics in SEQ ID NO: 87 below. In addition, the NBD amino acid sequence from human TAP1 may comprise a substitution at the Asp corresponding to D668 of SEQ ID NO: 8, which renders the NBD defective in ATP hydrolysis. Therefore, the amino acid sequence of the NBD polypeptide of TAP1 may, for example, comprise the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having greater than 90% or greater than 95% sequence identity thereto:

[0091]

[0092] 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.

[0093] The NBD amino acid sequence from human TAP1 may, for example, comprise substitutions corresponding to those at C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO: 8. The NBD amino acid sequence from human TAP1 may also, for example, comprise substitutions corresponding to those at 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 shown in bold and italics in SEQ ID NO: 88 below. In addition, the NBD amino acid sequence from human TAP1 may comprise a substitution at an amino acid corresponding to D668 of SEQ ID NO: 8 (e.g., a D668N substitution), which renders the NBD defective in ATP hydrolysis. Therefore, the amino acid sequence of the NBD polypeptide of TAP1 may, for example, comprise the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having greater than 90% or greater than 95% sequence identity thereto:

[0094]

[0095] Alternatively, the NBD amino acid sequence may have greater than 97% or greater than 98% sequence identity to at least 300 or 310 consecutive amino acids of the sequence provided in SEQ ID NO: 88. The NBD provided in SEQ ID NO: 88 comprises, 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 and the relevant GS linker (amino acids 12-16 and 25-30), which together constitute the "cap" on the sequence. The TAP1 NBD construct is expressed at significantly higher levels in biological expression systems, as shown in Example 3.

[0096] In some cases, the construct pairs of the present disclosure are prepared using an NBD from a TAP protein that does not homodimerize but heterodimerizes with a homologous NBD (see, e.g. Figure 2 B and D and Figure 3 ) or a complex of a construct (see, e.g. Figure 4A ). For example, a construct pair carrying human TAP1 NBD and human TAP2 NBD can be prepared. The construct pair comprising TAP1 and TAP2 NBD forms a heterodimer by the interaction of TAP1 and TAP2 in the presence of sufficient ATP. The use of NBDs (such as TAP1 and TAP2) that can heterodimerize allows different specific combinations of combined activation sequences to stimulate immune cells. For example, a construct comprising IL-12A (p35), an optional linker amino acid sequence and TAP1 can be heterodimerized with a construct comprising IL-12B (p40), an optional linker amino acid sequence and TAP2 in the presence of sufficient ATP to form a construct pair presenting active IL-12AB heterodimers. Such constructs can also include a scaffold amino acid sequence, such as an IgFc amino acid sequence, if desired.

[0097] Figure 7 The sequence of human TAP2 is provided. In some cases, the NBD of the construct of the present disclosure may comprise an amino acid sequence from the NBD of human TAP2, or an amino acid sequence having greater than 90% or greater than 95% sequence identity with the human TAP2 NBD. For example, the NBD may comprise the same sequence as UniProtKB-Q03519 (TAP2_HUMAN):

[0098] The NBD amino acid sequence has an amino acid sequence with greater than 90% or greater than 95% sequence identity.

[0099] The NBD may, for example, comprise the amino acid sequence of human TAP2:

[0100]

[0101] Amino acid sequences having greater than 90% or greater than 95% sequence identity.

[0102] The NBD may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:12.

[0103] A mutation that removes the catalytic acidic amino acid residue corresponding to the D668N substitution of TAP1, rendering it defective in ATP hydrolysis, could be incorporated into the TAP2 NBD amino acid sequence.

[0104] (2) CFTR ATP-binding box as NBD

[0105] In some cases, the NBD of the construct of the present disclosure may comprise an amino acid sequence of an NBD from human cystic fibrosis transmembrane conductance regulator (CFTR), or an amino acid sequence having greater than 90% or greater than 95% sequence identity with human CFTR. For example, the NBD may comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity with the NBD amino acid sequence of UniProtKB-P13569 (SEQ ID NO: 13).

[0106]

[0107]

[0108] The NBD may, for example, comprise the human CFTR amino acid sequence:

[0109] VLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGK

[0110] IKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVL

[0111] GEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDF (SEQ ID NO: 14) has an amino acid sequence with greater than 80% or greater than 85% sequence identity. Alternatively, the NBD may, for example, comprise an amino acid sequence with greater than 90% or greater than 95% sequence identity to SEQ ID NO: 14. The NBD may also comprise an amino acid sequence with greater than 96% or greater than 98% sequence identity to SEQ ID NO: 14.

[0112] The NBD may, for example, comprise 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:

[0113] SXTTTEVVMENVTAFWEEGGTPVLKDINFKIER

[0114] GQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFG

[0115] VSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDAD

[0116] LYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILH

[0117] EGSSYFYGTFSELQNLQPDFSSKLMX (SEQ ID NO: 15) having greater than 80% or greater than 85% sequence identity, wherein the X at position 2 may be absent or L; and the X at position 229 may be absent or G. Alternatively, the NBD may, for example, comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO: 15. The NBD may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 15. The 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, wherein the X at position 2 may be absent or L.

[0118] 2. Bracket

[0119] The scaffold amino acid sequence can be incorporated into the ATP-dependent agonist construct described herein as a means of structural arrangement to increase the in vivo half-life of the molecule, and in the case of being able to induce ADCC, ADCP and / or CDC, serve as the basis of those effector functions. The scaffold may be able to form dimers and higher-order complexes (e.g., trimers), or may not be dimerized (non-dimerized), in which case they do not form dimers or higher-order complexes. The scaffold incorporated into the construct described herein may, for example, comprise an immunoglobulin (Ig) amino acid sequence or a non-immunoglobulin amino acid sequence.

[0120] In some embodiments, the scaffold polypeptide sequence comprises an immunoglobulin heavy chain constant region (CH2-CH3) polypeptide sequence, which acts as a dimerization or multimerization sequence. In the case where the scaffold comprises an immunoglobulin (Ig) amino acid sequence, it may 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 may, for example, comprise an Ig CH2 and / or CH3 amino acid sequence modified to prevent dimerization. Any one or more cysteines involved in stabilizing the interchain disulfide bonds of the Ig heavy chain dimers may be substituted (e.g., with alanine or serine) so that the Ig heavy chain sequence cannot homodimerize and form interchain disulfide bonds. The scaffold polypeptide may comprise a wild-type (wt) Homo sapiens IgG1 Fc polypeptide sequence (SEQ ID NO: 56):

[0121]

[0122] Optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:56. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:56.

[0123] The scaffold polypeptide may comprise the homo sapiens IgG1 Fc amino acid sequence of SEQ ID NO:57 comprising the LALA substitution (L234A, L235A substitutions, amino acids 14 and 15 shown in bold and italics, respectively):

[0124]

[0125] Optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise 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.

[0126] Non-dimerizing scaffolds can be prepared as human single-chain Fc (scFc) dimers. See, for example, Zhou et al., Biomaterials 117:24-31 (2017). Such scFc dimers can be based on IgG1 and have the following sequence:

[0127] Alternatively, the scaffold may, for example, comprise 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, but substitutions (including "LALA" discussed below) may be introduced to selectively alter the ability of the scFc to induce those effector functions.

[0128] The scaffold polypeptide may comprise the wt Homo sapiens IgG2 Fc polypeptide of SEQ ID NO:58 (see, GenBank AAN76044 amino acids 99-325):

[0129]

[0130] Optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:58. The scaffold may also comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:58.

[0131] The scaffold polypeptide may comprise the wt Homo sapiens IgG3 Fc polypeptide of SEQ ID NO:59 (see, e.g., GenBank AAW65947 amino acids 19-246):

[0132]

[0133] Optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise 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.

[0134] The scaffold polypeptide may comprise the wt Homo sapiens IgG4 Fc polypeptide of SEQ ID NO:60:

[0135]

[0136] Optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise 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.

[0137] The interaction of IgG class antibodies (which leads to, for example, ADCC and ADCP) occurs through engagement of their Fc region with members of the Fcγ receptor family (FcγR). The human protein family consists of FcγRI (CD64), FcγRII (CD32, which includes isotypes FcγRIIa, FcγRIIb, and FcγRIIc), and FcγRIII (CD16, which includes isotypes FcγRγIIIa and FcγRIIIb). See, e.g., Lazar et al. (2006) PNAS: 103(11), 4005-4010.

[0138] Variants that can enhance one or more antibody-related effector functions (e.g., ADCC, ADCP and / or CDC responses) are: single substitutions S239D and I332E in human IgG1; and 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 substitutions are double and triple mutants, S239D / I332E or S239D / I332E / A330L. See, Lazar et al., 2006.

[0139] Other substitutions in IgG1 antibodies that enhance one or more antibody-associated effector functions include, but are not limited to: Ser298Ala / Glu333Ala / Lys334Ala; Ser239Asp / Ala330Leu / Ile332Glu;

[0140] Ser239Asp / Ile332Glu; Gly236Ala / Ser239Asp / Ala330Leu / Ile332Glu; Gly236Ala;

[0141] Ser239Asp / Ile332Glu / Gly236Ala; Leu234Tyr / Gly236Trp / Ser298Ala;

[0142] Phe243Leu / Arg292Pro / Tyr300Leu / Val305Ile / Pro396Leu; Lys326Trp / Glu333Ser;

[0143] Lys326Ala / Glu333Ser; Lys326Met / Glu333Ser; Cys221Asp / Asp222Cys;

[0144] Ser267Glu / His268Phe / Ser324Thr; His268Phe / Ser324Thr; and Glu345Arg. See, Saunders KO (2019) Front.Immunol.10:1296.doi:10.3389 / fimmu.2019.01296. The corresponding position in the IgG1 Fc sequence of SEQ ID NO:56 can be obtained by subtracting 220 from the position shown. In one embodiment, at least ADCC is enhanced by substitution. In one embodiment, at least ADCP is enhanced by substitution. In one embodiment, at least CDC is enhanced by substitution. In one embodiment, at least complement fixation is enhanced by substitution.

[0145] Variants that can significantly reduce one or more antibody-related effector functions (e.g., ADCC, ADCP and / or CDC response) of an IgG1 antibody include, but are not limited to: Leu235Glu; Leu234Ala / Leu235Ala (LALA);

[0146] Ser228Pro / Leu235Glu; Leu234Ala / Leu235Ala / Pro329Gly;

[0147] Pro331Ser / Leu234Glu / Leu235Phe; Asp265Ala; Gly237Ala; Glu318Ala; Glu233Pro; and Gly236Arg / Leu328Arg. As shown 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 LALA substitutions (see, e.g., SEQ ID NO:57). In one embodiment, at least ADCC is significantly reduced by the substitutions. In one embodiment, at least ADCP is significantly reduced by the substitutions. In one embodiment, at least CDC is significantly reduced by the substitutions. In one embodiment, at least complement binding is significantly reduced by the substitutions. See, e.g., Saunders 2019 and the citations therein. In one embodiment, the IgG1 Fc substitution can be S239D / I332E / A330L, which allows enhanced ADCC with substantially no change in CDC function (see, Lazar et al., 2006).

[0148] Other variants of IgG1, IgG2, and IgG4 antibody sequences can significantly reduce one or more antibody-related effector functions, including but not limited to: His268Gln / Val309Leu / Ala330Ser / Pro331Ser (IgG2m4); Val234Ala / Gly237Ala / Pro238Ser / His268Ala / Val309Leu / Ala330Ser / Pro331Ser (IgG2σ);

[0149] Leu234Ala / L235Ala / Gly237Ala / P238Ser / His268Ala / Ala330Ser / Pro331Ser (IgG1σ); and S228P / Phe234Ala / Leu235Ala (IgG4PAA). See, e.g., Saunders 2019 and the citations therein.

[0150] Alternative non-dimerizing scaffold domains include, but are not limited to, XTENylation, PEGylation, esterification (see, on the World Wide Web at "www" pubs.acs.org / doi / 10.1021 / acsmedchemlett.8b00226), human serum albumin (HSA) fusion proteins (see, on www nature.com / articles / s42003-021-01790-2), and anti-HSA binding domains (including, but not limited to, anti-HSA peptides (see, on www pubs.acs.org / doi / 10.1021 / acs.molpharmaceut.2c00106), antibodies and antibody fragments (e.g., ScFvs, FABs, etc.), and VHH domains (see, at www.semanticscholar.org / paper / Serum-albumin%E2%80%90binding-VHHs-with-variable-pH-enable-Faassen-Ryan / d34256a0d39a0ab92db9195210fa0fc7430758b6)).

[0151] 3. Connector

[0152] The constructs disclosed herein optionally have a joint between any two elements. The joint may be between any two of the NBD amino acid sequence, the scaffold amino acid sequence and the AD. The joint is generally composed of an amino acid sequence of 4 to 50 amino acids (e.g., 4 to 25 amino acids or 25 to 50 amino acids) in length and may be selected based on, among other things, its rigidity. The joints are each independently selected.

[0153] In some cases, the linker is a flexible amino acid sequence consisting of glycine, serine and / or alanine residues. In other cases, 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) can be in the linker (GGGS) 1-10 (SEQ ID NO: 16-25) or (GGGGS) 1-10 (SEQ ID NO: 26-35) appears or is repeated 1-10 times. In some cases where the linker comprises a GGGS or GGGGS sequence, these sequences may appear 1-5 times or 5-10 times.

[0154] In cases where it is desired to maintain a substantially fixed distance or spatial spacing between domains to reduce or substantially eliminate adverse interactions between domains of a construct (e.g., between NBDs in the same construct), a rigid linker may be employed. For example, NBD pairs present in a single molecule may be prevented from self-association by the use of a rigid linker. Exemplary rigid peptide or polypeptide linkers include linkers comprising the sequence X(EAAAK)nX, wherein EAAAK may appear 1-20 times in the linker (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, SEQ ID NOs: 36-55), and wherein X is absent or is alanine. In some cases where the linker comprises the sequence X(EAAAK)nX, n may be 1-5 or 5-10. In some cases where the linker comprises the sequence X(EAAAK)nX, n may be 11-15 or 16-20. Alternatively, the rigid peptide linker can comprise a length of 4 to 40 (e.g., 4-20 or 20-40 amino acids) of (Lys-Pro), (Glu-Pro), (Thr-Pro-Arg) and (Ala-Pro) repeats. Other rigid linkers include linkers comprising 2 to 10 or 10 to 20 repeats of the dipeptide sequence KP or EP.

[0155] 4. Immune cell engagement domain

[0156] An immune cell engaging domain (also referred to as an immune cell engager, "ICE" or "ICEs" when plural) is a domain of a construct containing an NBD that can bind to an immune cell surface molecule. An immune cell engager is typically an antibody-related molecule / amino acid sequence (e.g., an antigen-binding fragment of an antibody, Fab, Fab', single-chain antibody, scFv, peptide aptamer, or nanobody). Unless otherwise specified, an immune cell engager is understood to be monovalent to prevent single molecule cross-linking of cellular targets (e.g., immune cell surface antigens) and off-target stimulation of immune cells. Single immune cell engagers are generally unable to act as agonists and stimulate the immune cells to which they bind; however, when multiple constructs carrying immune cell engagers are positioned in a complex (see, e.g. Figure 4A D or E) or immobilized on the surface of tumor cells (e.g., using constructs such as Figure 3 C), the immune cell engager can act as an agonist for immune cell stimulation / activation.

[0157] Immune cell engagers can target many different immune cells that appear or may be recruited into the TME. Targets of such immune cells and their corresponding immune cell engagers include those shown in Table 1.

[0158] Table 1: Some immune cells and their corresponding immune cell engagers

[0159]

[0160] Antibody-related molecules include, but are not limited to, antigen-binding fragments of antibodies, Fab, Fab', and polypeptides comprising single-chain antibody, scFv, peptide aptamer, or nanobody amino acid sequences.

[0161] When an immune cell engager, such as those listed in Table 1, is incorporated into a first NBD-containing polypeptide construct, which may be paired or complexed with a second NBD-containing construct comprising a tumor-specific binder to a TAA, then trans-targeting of tumor cells leading to their cytolysis may be observed (see, e.g. Figure 3 C, D and FH, and Figure 4A Incorporation of antibody-related molecules / amino acid sequences against immune cell surface proteins (including but not limited to CD3, TCR and / or CD16) into such constructs and complexes can produce effective cytolysis. When an immune cell engager that interacts with more than one type of immune cell is employed, cytolytic activity can be enhanced. Some exemplary anti-CD3 immune cell engagers are provided below.

[0162] The immune cell engager polypeptide may be an anti-human CD3 scFv based on the UCHT-1 monoclonal antibody having the following sequence: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKA PKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVTTYADSVKGRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (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 scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:61.

[0163] The immune cell engager polypeptide may be an anti-human CD3 scFv based on the UCHT-1 monoclonal antibody having the following sequence: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKA PKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 62). 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: 62. Additionally, the immune cell engager scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:62.

[0164] The immune cell engager polypeptide may be an anti-human CD3 VHH having the following sequence:

[0165] QVQLVESGGGLVQPGGSLRLSCAASGSIFSANTMGWYRQAPGKQRELVAGMN TSGSTVYGDSVKGRFTISRDNAKNIAYLQMNSLIPEDTAVYYCTLVQRGPNYWGQG TQVTVSS (SEQ ID NO: 63). 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: 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.

[0166] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the following sequence: DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO: 64). 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: 64. Additionally, the immune cell engager scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:64.

[0167] The immune cell engager polypeptide can be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence shown in SEQ ID NO:65, comprising a disulfide bond between cysteine ​​substitutions at Q43C and G99C (G233C as shown): DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGCGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGCGTKVEIK (SEQ ID NO:65). 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: 65. Additionally, the immune cell engager scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 65.

[0168] The immune cell engager polypeptide can be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence shown in SEQ ID NO:66, comprising a disulfide bond between the cysteine ​​substitutions at Q43C and G100C (G234C as shown): DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQAPGCGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGCTKVEIK (SEQ ID NO:66). 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: 66. Additionally, the immune cell engager scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO: 66.

[0169] The immune cell engager polypeptide can be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence shown in SEQ ID NO:67, comprising a disulfide bond between the cysteine ​​substitutions at G44C and G99C (G233C as shown): DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWRVQAPGQCLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGCGTKVEIK (SEQ ID NO:67). 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: 67. Additionally, the immune cell engager scaffold may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:67.

[0170] The immune cell engager polypeptide may be an anti-human CD3 scFv (vH-vL) based on the L2K antibody having the sequence shown in SEQ ID NO:68, comprising a disulfide bond between the cysteine ​​substitutions at G44C and G100C (G234C as shown):

[0171] 1DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQCLEWIGY INPSRGYTNY

[0172] 61ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSG121GGGSGGGGSG GGGSDIVLTQ SPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW181IYDTSKVASG VPARFSGSGS GTDYSLTINS LEAEDAATYY CQQWSSNPLT FGGCTKVEIK (SEQ ID NO: 68). 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: 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.

[0173] The repeating sequences containing GGGS (SEQ ID NO: 16) and / or GGGGS (SEQ ID NO: 26) in SEQ ID NOs: 61-62 and 64-68 are linker sequences between the domains.

[0174] 5. Immune cell activation domain (activation domain)

[0175] Activation domains ("ADs" or "AD" when singular) that can be incorporated into the constructs of the present disclosure include a variety of molecules or fragments of molecules (e.g., fragments of cytokines) that are capable of stimulating anti-tumor cell immune responses when presented as dimers or higher order complexes, but have limited ability to stimulate immune cells when presented alone. Some immune cell agonists that require receptor cross-linking for maximum activity include, but are not limited to, CD40L, CD28, IFN-g, and IL-12. Anti-tumor cell immune responses can be generated by, for example, stimulating NK cells or T cells (e.g., CD8+ T cells) with constructs comprising ADs, and can include both particle-dependent and particle-independent responses.

[0176] AD may comprise, for example, the amino acid sequence of a TNF superfamily member, such as TNF, lymphotoxin α, lymphotoxin αβ, and BAFF (CD25). AD may also comprise, for example, the amino acid sequence of a B7 superfamily member, such as CD80 and CD86. AD may also comprise, 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 extracellular domain). AD may comprise, for example, all or part of the amino acid sequence of IL-12 (e.g., one or two subunits of human IL-12).

[0177] For example, constructs that homodimerize in the presence of ATP levels found in the TME (see, e.g. Figure 2 A and C) or form homopolymeric complexes (see, e.g. Figure 4A Some ADs of A)) include but are not limited to the immunomodulatory amino acid sequences of the following: 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 can also be used.

[0178] In addition to the foregoing, nucleic acids (including nucleic acids having CpG repeat sequences or one or more IMT504 sequences) can be used as ADs and can be incorporated into homodimeric or heterodimeric NBD-containing constructs.

[0179] AD may comprise, for example, the CD80 polypeptide amino acid sequence:

[0180]

[0181] Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 99. AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 170 or 180 consecutive amino acids of SEQ ID NO:99.

[0182] AD may comprise, for example, a CD86 polypeptide amino acid sequence comprising CD86 (IgV-IgC domain):

[0183]

[0184] or CD86 IgV domain:

[0185] 1LKIQAYFNET ADLPCQFANS QNQSLSELVV FWQDQENLVL NEVYLGKEKF DSVHSKYMGR

[0186] 61TSFDSDSWTL RLHNLQIKDK GLYQCIIHHK KPTGMIRIHQ MNSELSVLA, (SEQ ID NO: 101). Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 100 or 101. AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 100 or 110 consecutive amino acids of SEQ ID NO: 100 or 101.

[0187] AD may comprise, for example, an amino acid sequence that binds to IL-12 as an agonist (e.g., IL-12 or anti-CD28 scFv or nanobody amino acid sequence). AD may comprise, for example, a single-chain IL-12 comprising the following sequence:

[0188] It comprises a fragment of the p40 subunit, a linker repeat sequence of GGGGS and a fragment of the p35 subunit. Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 90. AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 480 or 500 consecutive amino acids of SEQ ID NO: 90.

[0189] As discussed above, the constructs of the present disclosure may comprise an NBD that heterodimerizes in the presence of ATP. Thus, the complex between the first construct comprising the heterodimerizing NBD and the second construct may be used to assemble an AD, see, e.g. Figure 2 B and D). For example, IL-12AD can be formed in the presence of ATP by using the following construct:

[0190] (i) contains p40 subunit (as 第 1 immunomodulatory domain), the p40 subunit comprising the following sequence:

[0191]

[0192] or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:91; and

[0193] (ii) contains the p35 subunit (as 第2 immunomodulatory domain), the p35 subunit comprising the following sequence:

[0194]

[0195] Or a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:92.

[0196] AD may comprise, for example, all or part 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).

[0197] AD may comprise, 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 useful as AD may comprise a CD40L amino acid sequence:

[0198]

[0199] or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 96. Alternatively, a polypeptide useful as AD comprises a trimer of CD40L amino acid sequences separated by a linker sequence (e.g., a linker sequence comprising a GGGGS repeat sequence):

[0200]

[0201] Or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:95.

[0202] 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 used as AD can include a 4-1BBL amino acid sequence:

[0203]

[0204] Or an amino acid sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 94. Alternatively, a polypeptide useful as AD comprises a trimer of 4-1BBL amino acid sequences separated by a linker sequence (e.g., a linker sequence comprising a GGGGS repeat sequence):

[0205]

[0206] Or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:93.

[0207] The AD may comprise, for example, an amino acid sequence that binds to IL-10 (eg, an anti-IL-10 scFv or Nanobody amino acid sequence) or an amino acid sequence that binds to IL-10R (eg, an anti-IL-10R scFv or Nanobody amino acid sequence).

[0208] The AD may comprise, for example, an amino acid sequence that binds to CD3 (eg, an anti-CD3 scFv or nanobody amino acid sequence).

[0209] The AD may comprise, for example, an amino acid sequence that binds to CTLA-4 (eg, an anti-CTLA-4 scFv or nanobody amino acid sequence).

[0210] AD can include, for example, an amino acid sequence that binds to CD28 (e.g., an anti-CD28 scFv or nanobody amino acid sequence). Anti-CD28 scFv that can be used as AD can include the following sequence:

[0211]

[0212] 241TTVTVSS (SEQ ID NO: 89). Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 89. 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: 89.

[0213] AD can comprise, for example, an interferon-γ (IFN-γ) polypeptide sequence or an amino acid sequence that binds to an interferon γ receptor (e.g., an anti-IFN-γ receptor scFv or nanobody amino acid sequence). ScIFN-γ polypeptides that can be used as IFN-γ AD can comprise the following sequence:

[0214]

[0215] Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 97. 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.

[0216] AD may comprise, for example, an amino acid sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody amino acid sequence). Anti-CD16 VHH polypeptides useful as AD may comprise one of the following:

[0217]

[0218] Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:98 or 103. AD may also comprise a sequence having at least 96% or at least 98% sequence identity to at least 100 or 110 consecutive amino acids of SEQ ID NO:98 or 103.

[0219] AD does not have to be composed of an amino acid sequence, and may be composed of, for example, a nucleic acid sequence. Non-limiting examples of nucleic acid AD include a nucleic acid sequence having a CpG repeat sequence (e.g., a CpG oligodeoxynucleotide sequence) 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.

[0220] 6. Tumor-specific binding domain

[0221] The tumor-specific binding domain (also referred to as a tumor-specific binder or "TSB") of the construct described herein is a polypeptide amino acid sequence that acts as a targeting sequence to guide the construct to the tumor cell by binding to a target molecule expressed on the surface of the tumor cell. The target molecule can be a tumor-associated antigen ("TAA") (also referred to herein as a cancer-associated antigen or "CAA"), whose cell surface expression is limited to or substantially limited to one or more cancer cell types. In addition to molecules whose expression is limited to or substantially limited to tumor cells, other molecules present on the surface of tumor cells can also be targeted, including but not limited to cellular proteins expressed on normal (non-transformed) cells, which may be upregulated in tumor cells. Checkpoint proteins expressed on normal cells and many tumor cells (e.g., transformed tumor cells) (e.g., V domain Ig inhibitor of T cell activation or "VISTA", T cell immunoglobulin and mucin domain 3 or "Tim-3" and programmed death ligand 1 or "PD-L1") can also be used as target molecules for TSB. Targeted checkpoint proteins are often upregulated by tumor cells as a means of immune evasion or escape, providing both a target that can be expressed in large quantities on the surface of tumor cells and a checkpoint inhibitory effect of TSB. When expressed on tumor cells, proteins that are not essentially limited to expression on transformed tumor cells are considered targets of TSB. They are considered targets of TSB because any immune response directed by TSB is limited to the TME, where ATP levels are high enough to allow TSB to pair or complex with AD or immune cell engagers and to be activated by NBDs coupled to those elements (see, e.g. Figure 3 , 4A and 4B). TSB include, for example, polypeptides and other molecules such as antibodies, antigen-binding fragments of antibodies, Fab, Fab', and polypeptides comprising the amino acid sequence of a single-chain antibody, scFv, peptide aptamer or nanobody.

[0222] 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-α), 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 3 9 member 6 (Liv1), melanoma-associated antigen family A member (MAGEA), melanoma-associated antigen family C member (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 κB 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).

[0223] 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).

[0224] 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.

[0225] 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.

[0226] Some checkpoint proteins that can be used as targets for TSB include, but are not limited to, VISTA, TIM-3, PD-L1, CTLA-4, and LAG-3.

[0227] The TSB polypeptide may be an anti-human mesothelin scFv polypeptide related to the murine SS1 antibody, also used in Amatuximab, having the following sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGY TMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSGXGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK, wherein X is V or G (SEQ ID NO: 70). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0228] The TSB polypeptide may comprise an anti-human mesothelin scFv (vH-vL) polypeptide related to monoclonal antibody YP218 having the following sequence: QEQLVESGGGLVQPGASLTLTCTASGIDFSRYYMCWVRQ APGKGLEGIACIYIGGSGSTYYASWAKGRFTISKASSTTVTLQMTSLTAADTATYFCARGTNLNYIFRLWGPGTLVTVSSGXGGSGGGGSGGGGSDVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVV, wherein X is V or G (SEQ ID NO: 71). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0229] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody YP218 having the following sequence: DVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQK PGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVVGXGGSGGGGSGGGGSQEQLVESGGGLVQPGASLTLTCTASGIDFSRYYMCWVRQAPGKGLEGIACIYIGGSGSTYYASWAKGRFTISKASSTTVTLQMTSLTAADTATYFCARGTNLNYIFRLWGPGTLVTVSS, wherein X is V or G (SEQ ID NO: 72). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0230] The TSB polypeptide may comprise an anti-human mesothelin scFv (vH-vL) polypeptide related to monoclonal antibody 15B6 having the following sequence:

[0231] wherein X is V or G (SEQ ID NO: 73). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0232] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having the following sequence:

[0233] wherein X is V or G (SEQ ID NO: 74). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0234] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between vL and vH positions G102C and K43C (K168C as shown) having the following sequence:

[0235] The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0236] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6, having a disulfide bond between vL and vH positions G102C and S44C (S169C as shown), having the following sequence: QAVVTQESALTTSPGETVTLTCRSSTGAVTTGNYPNWVQEKPDHLFTGLIAGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWFSSHWVFGCGTKLTVLGGGGGSGGGGSGGGGSEVQLQQSGPVLVKPGASVKISCKASGYSFTGYYMHWVRQSNGKCLEWIGRINPYTGVPSYKHNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARELGGYWGQGTTLTVSS (SEQ ID NO: 76). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0237] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between vL and vH positions G103C and K43C (K168C as shown) having the following sequence:

[0238] The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0239] The TSB polypeptide may comprise an anti-human mesothelin scFv (vL-vH) polypeptide related to monoclonal antibody 15B6 having a disulfide bond between vL and vH positions G103C and S44C (S169C as shown) having the following sequence:

[0240] The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0241] The TSB binder polypeptide may comprise an anti-human mesothelin (VHH) polypeptide related to the monoclonal antibody SD1 having the following sequence: QVQLVQSGGGLVQPGGSLRLSCAASDFDFAAYEMSWVRQA PGQGLEWVAIISHDGIDKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCL RLGAVGQGTLVTVSSS (SEQ ID NO: 79). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0242] The TSB polypeptide may comprise an anti-human EpCAM scFv (vH-vL) polypeptide related to monoclonal antibody MT201 and Adecatumumab, having the following sequence:

[0243] wherein X is V or G (SEQ ID NO: 80). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0244] The TSB polypeptide may comprise an anti-human EpCAM scFv (vL-vH) polypeptide related to monoclonal antibody MT201 and Adecatumumab, having the following sequence:

[0245] wherein X is V or G (SEQ ID NO: 81). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0246] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to Ipilimumab having the following sequence:

[0247]

[0248] The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0249] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to Ipilimumab having a disulfide bond between vL and vH positions Q101C and K43C (K167C as shown) having the following sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGCGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGCGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 83). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0250] The TSB polypeptide may comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab having a disulfide bond between vL and vH positions Q101C and G44C (G167C as shown) having the following sequence: IVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIEPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGCGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKCLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 84). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to this sequence.

[0251] The TSB polypeptide can comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab having a disulfide bond between vL and vH positions G102C and K43C (K167C as shown) having the following sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQCTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGCGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO:85). The tumor-specific binding 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 polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity with the sequence.

[0252] The TSB polypeptide can comprise an anti-human CTLA-4 scFv (vL-vH) polypeptide related to ipilimumab having a disulfide bond between vL and vH positions G102C and G44C (G168C as shown) having the following sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQCTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKCLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO:86). The TSB polypeptide may also comprise an amino acid sequence having greater than 90% or greater than 95% sequence identity to the sequence. Alternatively, the TSB polypeptide may comprise an amino acid sequence having greater than 96% or greater than 98% sequence identity to the sequence.

[0253] The linker sequences in SEQ ID NOs:70-86 are shown in bold and italics.

[0254] E. Preparation method

[0255] The present disclosure includes and provides for the preparation of the constructs disclosed herein by transcribing and translating nucleic acids encoding the constructs in mammalian cells (CHO cells), yeast cells (e.g., Pichia pastoris), sodoptera cells, or bacterial cells (e.g., E. coli cells). Portions of the constructs may also be prepared separately, such as by cell expression, chemical synthesis, etc., as discussed above, and then conjugated together using one or more chemical or enzymatic processes. In fact, enzymatic means, autocatalytic protein splicing, or chemical cross-linking (e.g., with heterobifunctional cross-linking agents) may be used to couple (e.g., covalently attach) AD to NBD, TSB to NBD, and / or NBD to immune cell adapters. In the case where the components to be coupled are all composed of amino acid sequences, enzymatic means and autocatalytic protein splicing represent coupling options that allow for the stoichiometry and structure of the coupling reaction while substantially avoiding potential secondary coupling reactions. For example, a sequence comprising an NBD may be attached to an AD, TSB conjugate, or immune cell adapter using a localized enzyme and a split intein. See, e.g., Bhagawati et al., Proc Natl Acad Sci USA, 116(44):22164-22172 (2019). Alternatively, an enzyme-catalyzed SpyTag / SpyCatcher system (which can employ the CnaB2 domain of the FbaB protein from Streptococcus pyogenes) can be used to conjugate AD to a polypeptide comprising an NBD. See, e.g., Reddington and Howarth, Curr. Opin in Chem. Biol., 29:94–99 (2015).

[0256] In addition to purely enzymatic and chemical conjugation methods for coupling the components of the constructs described herein, affinity-guided methods that combine the specificity of protein-protein interactions with chemical conjugation can also be used. Affinity-guided chemical conjugation provides efficient, simple and specific chemical conjugation reactions. Park et al. (2018), Bioconjug Chem., 29: 3240-3244 have described one such method, peptide-directed photocrosslinking (PEDIP), and have been demonstrated for coupling polypeptides to antibody heavy chain constant regions. See also Kishomoto et al., (2019), Bioconjug Chem., 30 (3): 697-702.

[0257] In cases where 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), the AD can be coupled to the rest of the molecule using, for example, a bifunctional cross-linker (e.g., a homobifunctional or heterobifunctional cross-linker).

[0258] Constructs can be purified using a variety of methods known in the art. For example, in the case where the construct comprises an IgFc region as a scaffold, the construct can be purified by protein A or protein G chromatography. Constructs can 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 can also be used alone or in combination with affinity chromatography to purify the product.

[0259] F. Preparations and Methods of Treatment

[0260] The NBD-containing constructs and complexes disclosed herein (see, e.g. Figure 2 - Figure 4) can be formulated into a composition comprising pharmaceutically acceptable components.

[0261] The composition can be prepared for parenteral and / or other forms of administration. The construct or composition comprising the construct can be administered by any suitable route, such as intravenous, intramuscular, subcutaneous, intratumoral or intralymphatic. The construct can also be co-administered with one or more other agents that act synergistically or non-synergistically with the construct. For example, the constructs of the present disclosure (e.g., those with IL-12 activation domains) can be co-administered with immune checkpoint inhibitors such as pembrolizumab ( Targeting PD-1), Avelumab ( Targeting PD-L1) or ipilimumab ( targeting CTLA-4).

[0262] Any solid tumor containing elevated levels of ATP relative to normal tissue can be treated using the constructs disclosed herein. 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 can be a tumor of the lung, liver, skin, gastrointestinal, pancreatic, brain, or reproductive tissue (e.g., ovarian sarcoma).

[0263] VI. Aspects

[0264] 1. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and an activation domain (AD) amino acid sequence, optionally connected by a linker peptide sequence; wherein the NBD comprises one or more adenosine triphosphate (ATP) binding sites and can homodimerize or heterodimerize with a homologous non-identical NBD (e.g., the NBD of TAP1 or TAP2 that can heterodimerize) in the presence of ATP.

[0265] 2. A polypeptide construct as described in aspect 1, wherein the construct is arranged in the N-terminal to C-terminal direction as (i) NBD, an optional linker and AD amino acid sequence, or (ii) AD, an optional linker and NBD amino acid sequence.

[0266] 3. The polypeptide construct of aspect 1 or 2, comprising an NBD amino acid sequence, a scaffold amino acid sequence and an AD sequence, optionally connected by independently selected linker amino acid sequences.

[0267] 4. The polypeptide construct of aspect 3, wherein the construct comprises NBD, scaffold and AD amino acid sequences in the N-terminal to C-terminal direction.

[0268] 5. The polypeptide construct of aspect 3, wherein the construct comprises NBD, AD and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0269] 6. The polypeptide construct of aspect 3, wherein the construct comprises AD, scaffold and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0270] 7. The polypeptide construct of aspect 3, wherein the construct comprises AD, NBD and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0271] 8. The polypeptide construct of aspect 3, wherein the construct comprises a scaffold, AD and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0272] 9. The polypeptide construct of aspect 3, wherein the construct comprises a scaffold, NBD and AD amino acid sequences in the N-terminal to C-terminal direction.

[0273] 10. The polypeptide construct of any one of aspects 1-9, comprising two or more (eg, three or more) independently selected AD amino acid sequences, wherein the AD amino acid sequences are optionally placed in tandem.

[0274] 11. A polypeptide construct as described in any of aspects 1-10, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally placed in tandem (constrained so that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP).

[0275] 12. The polypeptide construct of aspect 11, further comprising a rigid linker that substantially prevents interaction between two or more NBD amino acid sequences in the presence of ATP.

[0276] 13. The polypeptide construct of any one of aspects 1-12, wherein each NBD amino acid sequence can homodimerize in the presence of ATP (eg, comprises the same homodimerization amino acid sequence).

[0277] 14. The polypeptide construct of aspect 13, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence. See, e.g. Figure 2 A, C and E.

[0278] 15. A polypeptide construct according to aspect 11 or 12, comprising two or more NBD amino acid sequences, which can homodimerize in the presence of ATP to form a dimer or higher order complex of the construct. See, e.g. Figure 4A A.

[0279] 16. The polypeptide construct of any one of aspects 13-15, wherein each NBD amino acid sequence comprises the same or substantially the same homodimerization amino acid sequence.

[0280] 17. The polypeptide construct of any one of aspects 1-12, wherein each NBD amino acid sequence can heterodimerize with a homologous NBD in the presence of ATP.

[0281] 18. The polypeptide construct of aspect 17, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence (see, e.g. Figure 2 Each of constructs B and D) or one or more (e.g., two or more) NBD amino acid sequences.

[0282] 19. A composition comprising a first polypeptide construct and a second polypeptide construct as described in any one of aspects 13-18, 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 the first NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimer or heterodimer complex. See, e.g. Figure 2 B and D.

[0283] 20. A composition comprising a first polypeptide construct and a second polypeptide construct as described in aspect 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 NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can homodimerize or heterodimerize in the presence of ATP to form homodimers, heterodimers or higher order complexes. See, e.g. Figure 2 B and D, wherein each construct comprises an NBD (e.g., a single NBD) amino acid sequence, and Figure 4A B, wherein at least one (eg, both) of the first construct and the second construct comprises two or more NBD amino acid sequences.

[0284] 21. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and a tumor-specific binder (TSB) amino acid sequence, optionally connected by a linker peptide sequence, wherein: the NBD comprises one or more adenosine triphosphate (ATP) binding sites and can homodimerize or heterodimerize with a homologous non-identical NBD (e.g., the NBD of TAP1 or TAP2 that can heterodimerize) in the presence of ATP; and wherein the polypeptide construct optionally comprises one or more ADs.

[0285] 22. The polypeptide construct of aspect 21, wherein the construct is arranged in the N-terminal to C-terminal direction as (i) NBD, an optional linker and TSB amino acid sequence, or (ii) TSB, an optional linker and NBD amino acid sequence.

[0286] 23. The polypeptide construct of aspect 21 or 22, comprising a NBD amino acid sequence, a scaffold amino acid sequence and a TSB amino acid sequence, optionally connected by independently selected linker amino acid sequences.

[0287] 24. The polypeptide construct of aspect 23, wherein the construct comprises NBD, scaffold and TSB amino acid sequences in the N-terminal to C-terminal direction.

[0288] 25. The polypeptide construct of aspect 23, wherein the construct comprises NBD, TSB and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0289] 26. The polypeptide construct of aspect 23, wherein the construct comprises TSB, scaffold and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0290] 27. The polypeptide construct of aspect 23, wherein the construct comprises TSB, NBD and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0291] 28. The polypeptide construct of aspect 23, wherein the construct comprises the Scaffold, TSB and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0292] 29. The polypeptide construct of aspect 23, wherein the construct comprises Scaffold, NBD and TSB amino acid sequences in the N-terminal to C-terminal direction.

[0293] 30. The polypeptide construct of any one of aspects 21-29, comprising two or more (eg, three or more) independently selected TSB amino acid sequences, wherein the TSB amino acid sequences are optionally placed in tandem.

[0294] 31. A polypeptide construct as described in any of aspects 21-30, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally placed in tandem (constrained so that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP).

[0295] 32. The polypeptide construct of aspect 31, further comprising a rigid linker that substantially prevents interaction between two or more NBD amino acid sequences in the presence of ATP.

[0296] 33. The polypeptide construct of any one of aspects 21-32, wherein each NBD amino acid sequence can homodimerize (eg, comprises the same homodimerization amino acid sequence) in the presence of ATP.

[0297] 34. The polypeptide construct of aspect 33, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence. See, e.g. Figure 4B AD.

[0298] 35. A polypeptide construct according to aspect 31 or 32, comprising two or more NBD amino acid sequences that can homodimerize in the presence of ATP to form a dimer or higher order complex of the construct. See, e.g. Figure 4A C.

[0299] 36. The polypeptide construct of aspect 35, wherein each NBD amino acid sequence comprises the same or substantially the same homodimerization amino acid sequence.

[0300] 37. The polypeptide construct of any one of aspects 21-32, wherein each NBD amino acid sequence can heterodimerize with a homologous NBD in the presence of ATP.

[0301] 38. The polypeptide construct of aspect 37, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence or one or more (eg, two or more) NBD amino acid sequences.

[0302] 39. A composition comprising a first polypeptide construct and a second polypeptide construct as described in any one of aspects 33-38, 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 the first NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimer or heterodimer complex.

[0303] 40. A composition comprising a first polypeptide construct and a second polypeptide construct as described in aspect 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, and the first NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can homodimerize or heterodimerize in the presence of ATP to form a homodimer or heterodimer or a higher order complex.

[0304] 41. The composition of aspect 39 or aspect 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.

[0305] 42. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and an immune cell engager (ICE) amino acid sequence, optionally connected by a linker peptide sequence, wherein: the NBD comprises one or more adenosine triphosphate (ATP) binding sites and can homodimerize or heterodimerize with a homologous non-identical NBD (e.g., the NBD of TAP1 or TAP2 that can heterodimerize) in the presence of ATP; and wherein the polypeptide construct optionally comprises one or more ADs.

[0306] 43. The polypeptide construct of aspect 42, wherein the construct is arranged in the N-terminal to C-terminal direction as (i) NBD, an optional linker and ICE amino acid sequence, or (ii) ICE, an optional linker and NBD amino acid sequence.

[0307] 44. The polypeptide construct of aspect 42 or 43, comprising a NBD amino acid sequence, a scaffold amino acid sequence and an ICE amino acid sequence, optionally connected by independently selected linker amino acid sequences.

[0308] 45. The polypeptide construct of aspect 43, wherein the construct comprises NBD, scaffold and ICE amino acid sequences in the N-terminal to C-terminal direction.

[0309] 46. ​​The polypeptide construct of aspect 43, wherein the construct comprises NBD, ICE and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0310] 47. The polypeptide construct of aspect 43, wherein the construct comprises ICE, scaffold and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0311] 48. The polypeptide construct of aspect 43, wherein the construct comprises ICE, NBD and a scaffold amino acid sequence in the N-terminal to C-terminal direction.

[0312] 49. The polypeptide construct of aspect 43, wherein the construct comprises the Scaffold, ICE and NBD amino acid sequences in the N-terminal to C-terminal direction.

[0313] 50. The polypeptide construct of aspect 43, wherein the construct comprises, in the N-terminal to C-terminal direction, a scaffold, NBD and ICE amino acid sequences.

[0314] 51. The polypeptide construct of any one of aspects 42-50, comprising two or more (eg, three or more) independently selected ICE amino acid sequences, wherein the ICE amino acid sequences are optionally placed in tandem.

[0315] 52. A polypeptide construct as described in any of aspects 42-51, comprising two or more (e.g., three or more) independently selected NBD amino acid sequences, optionally placed in tandem (constrained so that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP).

[0316] 53. The polypeptide construct of aspect 52, further comprising a rigid linker that substantially prevents interaction between two or more NBD amino acid sequences in the presence of ATP.

[0317] 54. A polypeptide construct as described in any of aspects 42-53, wherein each of the NBD amino acid sequences can homodimerize in the presence of ATP (e.g., comprises the same homodimerization amino acid sequence).

[0318] 55. The polypeptide construct of aspect 54, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence. See, e.g. Figure 4A D.

[0319] 56. The polypeptide construct of aspect 53 or 54, comprising two or more NBD amino acid sequences that can homodimerize in the presence of ATP to form a dimer or a higher order complex of the construct.

[0320] 57. A polypeptide construct as described in any of aspects 42-53, wherein each NBD amino acid sequence can heterodimerize with a homologous NBD in the presence of ATP.

[0321] 58. The polypeptide construct of aspect 57, wherein the construct comprises a NBD (eg, a single NBD) amino acid sequence or one or more (eg, two or more) NBD amino acid sequences.

[0322] 59. A composition comprising a first polypeptide construct and a second polypeptide construct as described in aspect 58, wherein the construct comprises a first NBD amino acid sequence, the second polypeptide construct comprises a second NBD amino acid sequence, and the first NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can heterodimerize in the presence of ATP to form a heterodimer complex.

[0323] 60. A composition comprising a first polypeptide construct and a second polypeptide construct as described in aspect 59, 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 NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences that can heterodimerize in the presence of ATP to form a heterodimer or a higher order complex.

[0324] 61. The composition of aspect 59 or aspect 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.

[0325] 62. A composition comprising a first polypeptide construct as described in any one of aspects 1-18 and a second polypeptide construct as described in any one of aspects 42-58, wherein:

[0326] (i) the first polypeptide construct comprises one or more (e.g., two or more) first heterodimeric NBD amino acid sequences, and the second polypeptide construct comprises one or more (e.g., two or more) second heterodimeric NBD amino acid sequences;

[0327] (ii) each of the one or more first heterodimeric NBD amino acid sequences is a cognate binding partner of the one or more second heterodimeric NBD amino acid sequences; and

[0328] (iii) the first polypeptide construct and the second polypeptide construct bind to each other through the interaction between at least one of the one or more first heterodimeric NBD amino acid sequences and at least one of the one or more second heterodimeric NBD sequences in the presence of ATP to form a heterodimer or a higher order complex of the first polypeptide construct and the second polypeptide construct.

[0329] 63. The composition of aspect 62, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second polypeptide construct comprises a single heterodimeric NBD. See, e.g. Figure 3 J.

[0330] 64. The composition of aspect 62, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second polypeptide construct comprises a single heterodimeric NBD. See, e.g. Figure 3 K, where NBD is connected in series.

[0331] 65. The composition of aspect 62, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second polypeptide construct comprises two or more second heterodimeric NBDs.

[0332] 66. The composition of aspect 62, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second polypeptide construct comprises two or more second heterodimeric NBDs.

[0333] 67. The composition of any one of aspects 62-66, wherein the first polypeptide construct and / or the second polypeptide construct comprises a scaffold sequence.

[0334] 68. A composition comprising a first polypeptide construct as described in any one of aspects 21-38 and a second polypeptide construct as described in any one of aspects 1-18, wherein:

[0335] (i) the first polypeptide construct comprises one or more (e.g., two or more) first heterodimeric NBD amino acid sequences, and the second polypeptide construct comprises one or more (e.g., two or more) second heterodimeric NBD amino acid sequences;

[0336] (ii) each of the one or more first heterodimeric NBD amino acid sequences is a cognate binding partner of the one or more second heterodimeric NBD amino acid sequences; and

[0337] (iii) the first polypeptide construct and the second polypeptide construct bind to each other through the interaction between at least one of the one or more first heterodimeric NBD amino acid sequences and at least one of the one or more second heterodimeric NBD sequences in the presence of ATP to form a heterodimer or a higher order complex of the first polypeptide construct and the second polypeptide construct.

[0338] 69. The composition of aspect 68, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second polypeptide construct comprises a single heterodimeric NBD. See, e.g. Figure 3 A and E.

[0339] 70. The composition of aspect 68, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second polypeptide construct comprises a single heterodimeric NBD. See, e.g. Figure 3 B, where NBD is connected in series, and N and O, where NBD is not connected in series.

[0340] 71. The composition of aspect 68, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second polypeptide construct comprises two or more second heterodimeric NBDs.

[0341] 72. The composition of aspect 68, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second polypeptide construct comprises two or more second heterodimeric NBDs. See, e.g. Figure 4A G.

[0342] 73. The composition of any one of aspects 62-66, wherein the first polypeptide construct and / or the second polypeptide construct comprises a scaffold sequence.

[0343] 74. A composition comprising a first polypeptide construct as described in any one of aspects 21-38 and a second polypeptide construct as described in any one of aspects 42-58, wherein:

[0344] (i) the first polypeptide construct comprises one or more (e.g., two or more) first heterodimeric NBD amino acid sequences, and the second polypeptide construct comprises one or more (e.g., two or more) second heterodimeric NBD amino acid sequences;

[0345] (ii) each of the one or more first heterodimeric NBD amino acid sequences is a cognate binding partner of the one or more second heterodimeric NBD amino acid sequences; and

[0346] (iii) the first polypeptide construct and the second polypeptide construct bind to each other through the interaction between at least one of the one or more first heterodimeric NBD amino acid sequences and at least one of the one or more second heterodimeric NBD sequences in the presence of ATP to form a heterodimer or a higher order complex of the first polypeptide construct and the second polypeptide construct.

[0347] 75. The composition of aspect 74, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second complex comprises a single heterodimeric NBD. See, e.g. Figure 3 C, F and G.

[0348] 76. The composition of aspect 74, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second complex comprises a single heterodimeric NBD. See, e.g. Figure 3 M, F, G and L, where NBD are connected in series, and N and O, where NBD are not connected in series.

[0349] 77. The composition of aspect 74, wherein the first polypeptide construct comprises a single first heterodimeric NBD and the second complex comprises two or more second heterodimeric NBDs.

[0350] 78. The composition of aspect 74, wherein the first polypeptide construct comprises two or more first heterodimeric NBDs and the second complex comprises two or more second heterodimeric NBDs. See, e.g. Figure 4A E.

[0351] 79. The composition of any one of aspects 62-67 and 74-78, further comprising AD. See, e.g., D of 3 and Figure 4A I.

[0352] 80. The polypeptide construct of any one of aspects 1-18, 21-38 and 42-58, wherein the construct comprises a scaffold.

[0353] 81. The composition of any one of aspects 19-20, 39-41 and 59-61, wherein the first polypeptide construct and / or the second polypeptide construct comprises an independently selected scaffold.

[0354] 82. The composition of any one of aspects 62-79, wherein the first polypeptide construct and / or the second polypeptide construct comprise independently selected scaffolds.

[0355] 83. The polypeptide construct or composition of any 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., it does not comprise an immunoglobulin Fc, CH1, CH2 or CH3 domain).

[0356] 84. The polypeptide construct or composition of any of aspects 80-82, wherein at least one scaffold (eg, each scaffold present) comprises an immunoglobulin polypeptide amino acid sequence (eg, an IgFc, CH2 or CH3 amino acid sequence).

[0357] 85. The polypeptide construct or composition of aspect 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.

[0358] 86. The polypeptide construct or composition of aspect 84, wherein at least one (eg, each) scaffold comprises an independently selected non-dimerizing IgFc polypeptide amino acid sequence.

[0359] 87. The polypeptide construct or composition of aspect 86, wherein the at least one (eg, each) scaffold comprises the 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.

[0360] 88. The polypeptide construct or composition of aspect 84, wherein at least one (e.g., each) scaffold comprises an independently selected immunoglobulin polypeptide amino acid sequence of 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 SEQ ID NOs: 58-60.

[0361] 89. A polypeptide construct or composition as described in any one of aspects 85-88, wherein the immunoglobulin Fc (IgFc) polypeptide amino acid sequence can induce antibody-related effector functions (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-related effector functions selected from ADCC, ADCP and CDC. The functional change can be assessed relative to a polypeptide construct lacking the one or more substitutions.

[0362] 90. A polypeptide construct or composition as described in any of aspects 85-88, wherein the immunoglobulin Fc polypeptide amino acid sequence comprises one or more substitutions (e.g., LALA substitutions) that reduce (e.g., substantially reduce) at least one (e.g., all) antibody-associated effector functions selected from ADCC, ADCP, and CDC. The functional change can be assessed relative to a polypeptide construct lacking the one or more substitutions.

[0363] 91. A polypeptide construct or composition as described in any of the preceding aspects, wherein when one or more ADs are present, at least one (e.g., each) AD comprises all or part of the amino acid sequence of the following polypeptides: CD40L, CD28, IFN-γ, TNF superfamily members, TNF, lymphotoxin α, lymphotoxin αβ, BAFF (CD25), B7 superfamily members, CD80, CD86, IL-15, IL-2, IL-7, IL-10, IL-12, PD1, anti-CD3, anti-CTLA4, anti-CD28.

[0364] 92. A polypeptide construct or composition as described in aspect 91, wherein at least one (e.g., each) AD comprises an amino acid sequence having greater than 90% or greater than 95% sequence identity with IL-15, IL-12 or IL-7 (e.g., greater than 97% or greater than 99% sequence identity with IL-15, IL-12 or IL-7).

[0365] 93. A polypeptide construct or composition as described in aspect 91, wherein at least one (e.g., each) AD comprises (i) a single-chain IL-12 of SEQ ID NO: 90 or (ii) an 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 with any of those IL-12 sequences.

[0366] 94. A polypeptide construct or composition as described in any preceding aspect, 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 PDL1 (e.g., an anti-PDL1 scFv or nanobody amino acid sequence).

[0367] 95. A polypeptide construct or composition as described in any preceding aspect, 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 an amino acid sequence that binds to CD40L (e.g., an anti-CD40L scFv or nanobody amino acid sequence).

[0368] 96. A polypeptide construct or composition as described in aspect 95, wherein at least one (e.g., each) AD comprises the CD40L amino acid sequence of SEQ ID NO: 96, the 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 with SEQ ID NO: 95 or 96 (e.g., having greater than 97% or greater than 99% sequence identity with SEQ ID NO: 95 or 96).

[0369] 97. A polypeptide construct or composition as described in any of the preceding aspects, 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).

[0370] 98. A polypeptide construct or composition as described in aspect 97, wherein at least one (e.g., each) AD comprises the 4-1BBL amino acid sequence of SEQ ID NO: 94, the 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 with SEQ ID NO: 93 or 94 (e.g., having greater than 97% or greater than 99% sequence identity with SEQ ID NO: 93 or 94).

[0371] 99. A polypeptide construct or composition as described in any preceding aspect, wherein when 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 anti-IL-10 scFv or nanobody amino acid sequence) or an amino acid sequence that binds to an IL-10 receptor ("IL-10R") (e.g., an anti-IL-10R scFv or nanobody amino acid sequence).

[0372] 100. A polypeptide construct or composition as described in any preceding aspect, 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).

[0373] 101. A polypeptide construct or composition as described in any preceding aspect, 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).

[0374] 102. A polypeptide construct or composition as described in any preceding aspect, 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).

[0375] 103. The polypeptide construct or composition of aspect 102, wherein at least one (e.g., each) AD comprises the anti-CD28 scFv sequence of SEQ ID NO: 89, or an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 89 (e.g., having greater than 96% or greater than 99% sequence identity to SEQ ID NO: 89).

[0376] 104. A polypeptide construct or composition as described in any preceding aspect, 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).

[0377] 105. The polypeptide construct or composition of aspect 104, wherein the 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., having greater than 96% or greater than 99% sequence identity to SEQ ID NO: 97).

[0378] 106. A polypeptide construct or composition as described in any preceding aspect, 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).

[0379] 107. The polypeptide construct or composition of aspect 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 SEQ ID NO: 98 or 103 (e.g., having greater than 96% or greater than 99% sequence identity to SEQ ID NO: 98 or 103).

[0380] 108. A polypeptide construct or composition as described in any preceding aspect, wherein when one or more ADs are present, at least one (e.g., each) AD comprises the CD80 amino acid sequence of SEQ ID NO: 99, or an amino acid sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 99 (e.g., having greater than 96% or greater than 99% sequence identity with SEQ ID NO: 99).

[0381] 109. A polypeptide construct or composition as described in any of the preceding aspects, wherein when one or more ADs are present, at least one (e.g., each) AD comprises the CD86 amino acid sequence of SEQ ID NO: 100 or 101, or an amino acid sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 100 or 101 (e.g., having greater than 96% or greater than 99% sequence identity with SEQ ID NO: 100 or 101).

[0382] 110. The polypeptide construct or composition of any preceding aspect, wherein when one or more ADs are present, at least one (eg, each) AD comprises a nucleic acid having a CpG repeat sequence (eg, a CpG oligodeoxynucleotide sequence).

[0383] 111. A polypeptide construct or composition as described in any preceding aspect, wherein when 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 having 1, 2 or 3 nucleotide substitutions, deletions or insertions.

[0384] 112. A polypeptide construct or composition as described in any of the preceding aspects, wherein when one or more (e.g., two or more) TSBs are present in the construct or composition, at least one (e.g., at least two or each) TSB is independently selected to exhibit affinity for a TAA selected from the group consisting of carbonic anhydrase IX (CAIX), cadherin, carcinoembryonic antigen (CEA), 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-α), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), 3), insulin-like growth factor 1 receptor (IGF-1R), integrin αVβ3, integrin α5β1, solute carrier family 39 member 6 (Liv1), melanoma-associated antigen family A member (MAGEA), melanoma-associated antigen family C member (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 κB 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).

[0385] 113. A polypeptide construct or composition as described in aspect 112, wherein at least one (e.g., at least two or each) TSB comprises the anti-human mesothelin scFv of SEQ ID NO:70, or an amino acid sequence having greater than 90% or greater than 95% sequence identity with SEQ ID NO:70 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:70).

[0386] 114. A polypeptide construct or composition as described in aspect 112 or 113, wherein at least one (e.g., at least two or each) TSB comprises the 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 with SEQ ID NO:71 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:71).

[0387] 115. A polypeptide construct or composition as described in any of aspects 112-114, wherein at least one (e.g., at least two or each) TSB comprises the 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 with SEQ ID NO:72 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:72).

[0388] 116. A polypeptide construct or composition as described in any of aspects 112-115, wherein at least one (e.g., at least two or each) TSB comprises the 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 with SEQ ID NO:73 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:73).

[0389] 117. A polypeptide construct or composition as described in any of aspects 112-116, wherein at least one (e.g., at least two or each) TSB comprises an anti-human mesothelin scFv (vL-vH) of SEQ ID NO: 74-78, or an amino acid sequence having greater than 90% or greater than 95% sequence identity with any one of SEQ ID NO: 74-78.

[0390] 118. The polypeptide construct or composition of aspect 117, wherein the anti-human mesothelin scFv (vL-vH) has greater than 96% or greater than 98% sequence identity to any one of SEQ ID NOs: 74-78.

[0391] 119. A polypeptide construct or composition as described in any 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 with SEQ ID NO: 79 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO: 79).

[0392] 120. A polypeptide construct or composition as described in any of aspects 112-119, 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 with SEQ ID NO:80 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:80).

[0393] 121. A polypeptide construct or composition as described in any of aspects 112-120, 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 with SEQ ID NO:81 (e.g., greater than 96% or greater than 98% sequence identity with SEQ ID NO:81).

[0394] 122. A polypeptide construct or composition as described in any of aspects 112-121, wherein at least one (e.g., at least two or each) TSB comprises an anti-human CTLA-4 scFv (vL-vH) of SEQ ID NO:82-86, or an amino acid sequence having greater than 90% or greater than 95% sequence identity with any one of SEQ ID NO:82-86.

[0395] 123. The polypeptide construct or composition of aspect 122, wherein the anti-human CTLA-4 scFv (vL-vH) has greater than 96% or greater than 98% sequence identity to any one of SEQ ID NOs: 82-86.

[0396] 124. A polypeptide construct or composition as described in any of aspects 1-111, 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.

[0397] 125. A polypeptide construct or composition as described in aspect 124, wherein the checkpoint protein is selected from the group consisting of: V-domain Ig inhibitor 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).

[0398] 126. A polypeptide construct or composition as described in any of Aspects 1-125, 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, TCRα chain, TCRβ chain, δγTCR, TCRγ chain, TCRδ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34 and CD16.

[0399] 127. A polypeptide construct or composition as described in aspect 126, wherein the 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 of an antibody, a Fab, a Fab', a scFv, an aptamer and a nanobody amino acid sequence.

[0400] 128. The polypeptide construct or complex of aspect 126 or 127, wherein at least one (e.g., at least two or each) ICE comprises an anti-human CD3 scFv comprising the sequence of SEQ ID NO: 61 or 62, or a sequence having greater than 90% or greater than 95% sequence identity with SEQ ID NO: 61 or 62 (e.g., an amino acid sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 61 or 62).

[0401] 129. A polypeptide construct or composition as described in any of aspects 126 to 128, wherein at least one (e.g., at least two or each) ICE comprises an anti-human CD3 VHH comprising the sequence of SEQ ID NO: 63, or a sequence having greater than 90% or greater than 95% sequence identity with SEQ ID NO: 63 (e.g., an amino acid sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 63).

[0402] 130. The polypeptide construct or composition of any 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 with any one of SEQ ID NOs: 64-68 (e.g., an amino acid sequence having greater than 96% or greater than 98% sequence identity with any one of SEQ ID NOs: 64-68).

[0403] 131. The polypeptide construct or composition of any preceding aspect, wherein each NBD is an NBD of an ATP-binding cassette (ABC) transporter.

[0404] 132. The polypeptide construct or composition of aspect 131, wherein the ABC transporter is a member of the ABC1, MDR / TAP, MRP, ALD, OABP, GCN20 or White ABC transporter family.

[0405] 133. The polypeptide construct or composition of aspect 131, wherein the ABC transporter NBD comprises an amino acid 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 a sequence having greater than 90% or greater than 95% sequence identity to any of these sequences.

[0406] 134. The polypeptide construct or composition of aspect 133, wherein the NBD comprises an amino acid sequence having greater than 97% or greater than 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1-7 and 9.

[0407] 135. The polypeptide construct or composition of aspect 131, wherein the NBD comprises a TAP2 amino acid sequence, the TAP2 amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having greater than 90% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0408] 136. The polypeptide construct or composition of aspect 135, wherein the NBD comprises an amino acid sequence having greater than 97% or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0409] 137. The polypeptide construct or composition of aspect 131, wherein the NBD comprises a TAP1 NBD amino acid sequence.

[0410] 138. The polypeptide construct or composition of aspect 137, wherein the NBD comprises the amino acid sequence of SEQ ID NO: 9, or a sequence having greater than 90% or greater than 95% sequence identity to the sequence of SEQ ID NO: 9.

[0411] 139. The polypeptide construct or composition of aspect 138, wherein the NBD comprises an amino acid sequence having greater than 97% or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0412] 140. The polypeptide construct or composition of aspect 131, wherein the NBD comprises the TAP1 amino acid sequence of any one of SEQ ID NO: 10, 87 or 88, or a sequence having greater than 90% or greater than 95% sequence identity to any of these sequences.

[0413] 141. The polypeptide construct or composition of aspect 140, wherein the NBD comprises an amino acid sequence having greater than 97% or greater than 98% sequence identity to the TAP1 amino acid sequence of SEQ ID NO: 10, 87 or 88.

[0414] 142. The polypeptide construct or composition of aspect 140, wherein the NBD comprises the TAP1 amino acid sequence of SEQ ID NO: 88, or a TAP1 amino acid sequence having greater than 90% or greater than 95% sequence identity to the TAP1 amino acid sequence of SEQ ID NO: 88.

[0415] 143. The polypeptide construct or composition of aspect 142, wherein the NBD comprises a TAP1 amino acid sequence having greater than 97% or greater than 98% sequence identity to the TAP1 amino acid sequence of SEQ ID NO: 88.

[0416] 144. The polypeptide construct or composition of any one of aspects 131-143, wherein the NBD comprises a non-homodimeric NBD.

[0417] 145. The polypeptide construct of aspect 144, wherein the NBD comprises a substitution of at least one (eg, at least two or at least three) of amino acids N185, S186, Q189, E191 and Q192.

[0418] 146. The polypeptide construct or composition of any of aspects 131-145, wherein at least one (e.g., each) NBD has a substitution that renders the at least one (e.g., each) NBD substantially ATP hydrolysis deficient.

[0419] 147. A polypeptide construct or composition as described in aspect 146, wherein the at least one (e.g., each) NBD comprises a TAP1 NBD having a D668 substitution (e.g., D668N) as numbered in SEQ ID NO:8, or a corresponding substitution (in, e.g., any one of SEQ ID NO:9, 10, 87 or 88), wherein the substitution renders the at least one (e.g., each) NBD substantially ATP hydrolysis deficient.

[0420] 148. A polypeptide construct or composition as described in any preceding aspect, comprising one or more independently selected linkers (e.g., flexible or rigid linkers), optionally located between any of the NBD amino acid sequence, the scaffold amino acid sequence and the AD amino acid sequence.

[0421] 149. The polypeptide construct or composition of aspect 148, wherein the one or more independently selected linkers comprise an amino acid sequence independently selected from any one of SEQ ID NOs: 16-35.

[0422] 150. The polypeptide construct or composition of aspect 148 or 149, further comprising one or more independently selected rigid linker amino acid sequences.

[0423] 151. The polypeptide construct or composition of aspect 150, wherein the one or more independently selected rigid linker amino acid sequences are independently selected from any one of SEQ ID NOs: 36-55.

[0424] 152. A dimer or higher order complex of a construct comprising two or more constructs or a first polypeptide construct and a second polypeptide construct (e.g., a composition) as described in any one of aspects 1-151 and at least one molecule (e.g., at least two molecules) of ATP.

[0425] 153. A dimer or other higher order complex as described in aspect 152, wherein each construct in the complex is identical.

[0426] 154. The dimer or other higher order complex of aspect 153, wherein the dimer or other higher order complex is a homodimer.

[0427] 155. The dimer or higher order complex of aspect 152, wherein each construct in the complex is different.

[0428] 156. The dimer or other higher order complex of aspect 155, wherein the dimer or other higher order complex is a heterodimer.

[0429] 157. A population of constructs (eg, 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 aspects 152-156.

[0430] 158. A population according to aspect 157, wherein at least one or at least two of the dimers or higher order complexes of the constructs comprises an immunoglobulin constant region that affects ADCC, ADCP and / or CDC.

[0431] 159. The population of aspect 157, wherein at least one or at least two of the dimers or higher order complexes of the constructs comprise independently selected ADs.

[0432] 160. The population of aspect 157 or 159, wherein at least one, or at least two, of the dimers or higher order complexes of the constructs comprise independently selected ICEs.

[0433] 161. A population as described in any of aspects 157-160, wherein at least one or at least two of the dimers or higher order complexes of the constructs comprise independently selected TSBs.

[0434] 162. A polypeptide construct as described in any one of aspects 1-151, which has Figure 2 , Figure 3 , Figure 4A or Figure 4B The structure shown in any one of them.

[0435] 163. A construct pair as described in any one of aspects 1-151, having Figure 2 , Figure 3 , Figure 4A or Figure 4B The structure shown in any one of them.

[0436] 164. A dimer or higher order complex as described in any one of aspects 152-156, having Figure 2 or Figure 3 The structure shown in any one of them.

[0437] 165. A dimer or higher order complex as described in any one of aspects 152-156, having Figure 4A or Figure 4B The structure shown in any one of them.

[0438] 166. A dimer or higher order complex as described in any one of aspects 152-156, having Figure 2 , Figure 3 , Figure 4A or Figure 4B The structure shown in any one of them.

[0439] 167. The dimer or higher order complex of any one of aspects 152 to 156, comprising a homodimer of TAP1 or TAP2 NBD amino acid sequence or a heterodimer of TAP1 and TAP2 NBD amino acid sequences.

[0440] 168. A method of stimulating CD4+T cells, δγT cells, NK cells, myeloid-derived suppressor cells and / or CD8+T cells, the method comprising contacting the cells with a construct or composition as described in any of aspects 1-151 in the presence of sufficient ATP to bind to the NBD.

[0441] 169. The method of aspect 168, which is performed in vitro or outside the body of a mammal or non-mammal.

[0442] 170. The method of aspect 169, which is performed in a cell or tissue.

[0443] 171. The method of aspect 168, which is performed in vivo or in the body of a mammal.

[0444] 172. A method of treating cancer, the method comprising administering to a patient or subject a construct or composition as described in any of aspects 1-151.

[0445] 173. The method of any one of aspects 169-172, wherein the mammal is a human.

[0446] 174. Use of the construct or composition of any one of aspects 1-151 for the preparation of a medicament for treating cancer.

[0447] 175. The method or use of any one of aspects 172-174, wherein the cancer comprises a solid tumor.

[0448] 176. The polypeptide construct or composition of any one of aspects 1-151 for use in treating cancer.

[0449] 177. The polypeptide construct or composition of aspect 176, wherein the cancer comprises a solid tumor.

[0450] 178. The method, use, construct or composition of any one of aspects 172 to 176, wherein the cancer is mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma or germ cell tumor.

[0451] 179. A nucleic acid comprising a sequence encoding one or more constructs of one or more constructs or compositions of any of aspects 1-151.

[0452] 180. A vector comprising the nucleic acid of aspect 179, optionally under the control of a promoter (eg, a constitutive promoter) or an inducible promoter.

[0453] 181. A cell comprising the vector or nucleic acid of aspect 179 or 180.

[0454] 182. The cell of aspect 181, wherein the cell is a mammalian cell, a yeast cell, an insect cell, or a bacterial cell.

[0455] VII. Examples

[0456] Example 1.

[0457] To test the functionality of TAP1 NBD homodimerization in solution, human TAP1 NBD comprising: (i) N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and Q683R substitutions, (ii) cysteine ​​present in the sequence was replaced with serine (SEQ ID NO: 10), and (iii) a C-terminal His tag for purification was expressed in E. coli and subsequently purified as described above. Figure 8 Samples of molecular weight markers and purified proteins were subjected to SDS-PAGE gel analysis under reducing and non-reducing conditions as indicated (lanes: M - molecular weight marker, R - reduced protein sample, NR non-reduced protein sample).

[0458] Purified TAP1 NBD samples were exposed to 1 mM ADP or 1 mM ATP and subjected to size exclusion chromatography. Fig. 9 As shown, the results show that the effective molecular weight of the protein chromatogram in the presence of ADP is 37 kDa, while the effective molecular weight of the protein chromatogram in the presence of ATP is 57 kDa. The results show that the TAP1 protein dimerizes in the presence of ATP. The dimerization was confirmed using mass photometry and dynamic light scattering (DLS) to determine the molecular weight. In the presence of 1 mM ATP, mass photometry gave an effective molecular weight of 62 kDa (see Fig.10 ), which is consistent with the chromatographic results. Fig.11 A) or 1 mM ATP ( Fig.11 In the presence of B), DLS in 20 mM Tris (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 10% glycerol gave a size of 5.517 in the presence of ADP and 8.057 in the presence of ATP, again consistent with dimerization of the protein in the presence of 1 mM ATP. The DLS results are provided in the table below.

[0459] Sample name Polydispersity Index (PI) Volume size distribution (nm) NBD1 in ADP buffer 0.3356 5.157 NBD1 in ATP buffer 0.6278 8.057

[0460] Example 2.

[0461] The TAP1 NBD of SEQ ID NO:87 of the peptide of the His tag of the C-terminus is expressed in Escherichia coli, and the cells are precipitated and frozen until analysis. The TAP1 NBD fragment is passed through cell lysis, and then purified by chromatography on HisTrap columns. In order to analyze, the cell pellet is thawed, and 200ml lysis buffer (20mM Tris-Cl pH 8.0, 500mM NaCl, 5mM MgCl2, 10% glycerol, 2mM ATP) is resuspended with 100 μg / ml lysozyme, 2 kinds of protease inhibitor tablets (Thermo Pierce A32965) added to 12.5U / ml and universal nuclease (ThermoPierce 88702) are added. Cells are stirred and incubated for 30 minutes at 4°C, and then the ultrasonic treatment lysis by 80% intensity continues a total of 4 minutes of active time (5 seconds per cycle, standing on ice for 15 seconds). The lysate was then clarified by centrifugation at 12,000 x g for 1 hour at 4°C, then 0.22 μm filtered and loaded onto a 1 ml HisTrap HP column (Cytiva) at 0.5 ml / min, recirculated overnight with a peristaltic pump. The next day, the column was switched to flow-through, and after complete lysate loading at 1 ml / min, 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 FPLC, and eluted the bound protein with a gradient of 0 to 500 mM imidazole in a binding buffer (20 mM Tris-Cl pH 8.0, 200 mM NaCl, 5 mM MgCl2, 10% glycerol, 2 mM ATP, 0.5 mM PMSF) over 40 CV. Column fractions were evaluated by SDS-PAGE, and fractions containing the target protein were combined and concentrated (10 kDa MWCO) to 10 ml, and then injected 3 times (about 3.3 ml per injection) onto a Superdex 20016 / 600 column (20 mMTris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol, 1 mM ATP) equilibrated in the target buffer. The elution peak fractions were evaluated by SDS-PAGE, and the fractions containing the target protein were pooled and concentrated (molecular weight cutoff 10 kDa) to 10 mg / ml, then aliquoted (500 μl / tube) and stored at 4°C.

[0462] The purified protein was subjected to SDS-PAGE along with molecular weight samples. The Coomassie blue stained gel was Fig.12 The leftmost lane is the molecular weight sample and the rightmost lane is the purified protein.

[0463] Purified protein samples (1 mg each) were buffer exchanged on a PD-10 column (Cytiva 17085101) according to the manufacturer's instructions. For buffer exchange, a PD-10 desalting column was equilibrated with a target buffer (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol) containing 1 mM ATP or 1 mM ADP. One milligram (100 μl of 10 mg / ml stock solution) of protein was added to each column along with 2.4 ml of target buffer, the column was centrifuged at 1000 x g for 2 minutes in a 50 ml conical tube, 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 sample containing ATP 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 approximately 38 kDa. The chromatographic trace is Fig.12 Available in.

[0464] To determine the molecular weight in the presence of ADP and ATP by size exclusion chromatography, the purified protein samples (1 mg each) were buffer exchanged on a PD-10 column (Cytiva 17085101) according to the manufacturer's instructions. Briefly, a PD-10 desalting column was equilibrated with a target buffer (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol) containing 1 mM ATP or ADP. One milligram (100 μl of 10 mg / ml stock solution) of protein was added to each column along with 2.4 ml of target buffer, the column was centrifuged at 1000 x g for 2 minutes in a 50 ml conical tube, and the eluate was collected. The buffer-exchanged protein was then concentrated to 0.5 ml (about 60 uM), loaded onto a Superdex S200 Increase column (Cytiva), and equilibrated at 0.5 ml / min in the appropriate buffer containing ATP or ADP. The sample containing ATP 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 approximately 38 kDa.

[0465] Example 3.

[0466] In order to evaluate the non-induced and induced expression of NBD polypeptides, the E. coli BL21 (DE3) samples were transformed with a vector that allows the isopropyl-β-D-thiogalactoside (IPTG)-induced expression of NBD of SEQ ID NO:87 or SEQ ID NO:88, and the NBD had a peptide containing SpyTag3, a TEV protease site and a His tag at the C-terminus. In order to analyze the non-induced and induced fractions, the optical density (OD) of each culture at 600nm was monitored until induced at 0.4OD, at which time an aliquot of 1OD-ml (e.g., 2.5ml of 0.4OD) was taken out. The cells in the aliquots were precipitated at 6800xg for 3 minutes, and the precipitate was resuspended in 300μl of 1x PBS, after which the resuspended material was frozen at -80°C. After induction with 80μM IPTG, the cells were grown and comparable 1OD-ml samples (e.g., 0.25ml of 4OD) were collected during the final harvest after induction. In order to analyze induction, the frozen samples were thawed and the cells in all samples were lysed by mixing with an equal volume of 2x lysis buffer (1x PBS with 0.2% TritonX-100, 100 μg / ml lysozyme and 2x concentration of universal nuclease from Pierce). The samples were incubated on a shaker at 4°C for 30 minutes, after which the lysate was rotated at 21,000xg for 10 minutes to precipitate debris. The sample (30 μl) of the supernatant was mixed with 10 μl of 4x SDS-PAGE loading buffer, heated at 70°C for about 5 minutes, and then loaded onto the SDS-PAGE gel. The results are shown in the SDS-PAGE gel A of Figure 13.

[0467] To determine expression levels and molecular weights 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 chromatography analysis as in Example 2. The results of the chromatographic molecular weight determination and protein yield are provided in the table below, with protein yields of the "capped" NBD of SEQ ID NO: 88 being approximately two orders of magnitude (90-fold) higher relative to SEQ ID NO: 87.

[0468]

[0469] Figure 13 provides SDS PAGE analysis of induced and non-induced expression in A and analysis of purified protein in B. The lanes of the reducing SDS-PAGE gel image provided in Figure 13 are as follows.

[0470]

Claims

1. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and a tumor-specific binder (TSB) amino acid sequence, optionally connected 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 homologous non-identical NBD (e.g., an NBD of TAP1 or TAP2 capable of heterodimerization) in the presence of ATP.

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

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

4. The composition of claim 3, wherein the first polypeptide construct and the second polypeptide construct homodimerize.

5. The composition of claim 4, wherein the TSB of the first polypeptide construct and the TSB of the second polypeptide construct recognize (bind to) a single tumor-associated antigen (TAA), the single tumor-associated antigen (TAA) being 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), 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-α), 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 member (MAGEA), melanoma-associated antigen family C member (MAGEC), 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 κB 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. The composition of claim 5, wherein each scaffold comprises an immunoglobulin polypeptide sequence.

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

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

9. The composition of claim 8, wherein the immunoglobulin Fc (IgFc) polypeptide amino acid sequence is capable of inducing antibody-associated effector functions, or the IgFc polypeptide amino acid sequence comprises one or more substitutions that enhance at least one antibody-associated effector function selected from ADCC, ADCP and CDC.

10. The composition of claim 9, wherein each NBD is an NBD of an ATP-binding cassette (ABC) transporter.

11. The composition of claim 10, 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 90% or greater than 95% sequence identity to any of these sequences.

13. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and an activation domain (AD) amino acid sequence, optionally connected 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 homologous non-identical NBDs in the presence of ATP.

14. The polypeptide construct of claim 13, further comprising a scaffold amino acid sequence.

15. The polypeptide construct of claim 13, wherein the AD is selected from: (i) IL-7, IL-15, IL-12 or their subunits; (ii) an amino acid sequence that binds to one of PD1, CD40, 4-1BB, IL-10R, CTLA-4, or CD28.

16. A composition comprising the first polypeptide construct and the second polypeptide construct as described in any one of claims 13 to 15, 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 the first NBD amino acid sequence and the second NBD amino acid sequence are homologous sequences capable of heterodimerization; and The first polypeptide construct comprises the p40 subunit of IL-12, and the second polypeptide construct comprises the p35 subunit of IL-12.

17. The composition of claim 16, wherein the first NBD comprises a TAP1 NBD amino acid sequence and the second NBD comprises a TAP2 NBD amino acid sequence.

18. A polypeptide construct comprising a nucleotide binding domain (NBD) amino acid (aa) sequence and an immune cell engager (ICE) amino acid sequence, optionally connected by a linker peptide sequence; wherein the NBD comprises one or more adenosine triphosphate (ATP) binding sites and is capable of heterodimerizing with a homologous non-identical NBD (e.g., an NBD of TAP1 or TAP2 capable of heterodimerization) in the presence of ATP.

19. The polypeptide construct of claim 18, further comprising a scaffold amino acid sequence.

20. A composition comprising the first polypeptide construct of claim 1 or 2 and the second polypeptide construct of claim 18 or 19, wherein: (i) the first polypeptide construct comprises one or more first heterodimeric NBD amino acid sequences, and the second polypeptide construct comprises one or more second heterodimeric NBD amino acid sequences; (ii) each of the one or more first heterodimeric NBD amino acid sequences is a cognate binding partner of the one or more second heterodimeric NBD amino acid sequences; (iii) the first polypeptide construct and the second polypeptide construct bind to each other through the interaction between at least one of the one or more first heterodimeric NBD amino acid sequences and at least one of the one or more second heterodimeric NBD sequences in the presence of ATP to form a heterodimer or a higher order complex of the first polypeptide construct and the second polypeptide construct; (iv) the TSB of the first polypeptide construct binds to a tumor-associated antigen (TAA) 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), 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-α), Erb-B2 receptor tyrosine kinase 2 (HER2), Erb-B2 receptor tyrosine kinase 3 (HER3), insulin-like growth factor 1 receptor (IGF-1R), integrin, protein αVβ3, integrin α5β1, solute carrier family 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); and (v) 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, αβTCR, CD2, TCRα chain, TCRβ chain, δγTCR, TCRγ chain, TCRδ chain, TRGV9, CD84, CXCR1, CD13, CD33, CD34 and CD16.

21. A method of treating cancer, the method comprising administering to a patient or subject a construct or composition as described in any one of aspects 1-20.

22. Use of the construct or composition of any one of aspects 1 to 20 for the preparation of a medicament for treating cancer.

23. A polypeptide construct or composition according to any one of aspects 1 to 20, for use in treating cancer.

24. The method, use, construct or composition of claims 21-23, wherein the cancer comprises a solid tumor.

25. The method, use, construct or composition of claim 24, wherein the cancer is mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma or germ cell tumor.

26. A nucleic acid comprising a sequence encoding one or more constructs of one or more constructs or compositions as described in any one of aspects 1-20.

27. A vector comprising the nucleic acid of claim 26, optionally under the control of a promoter (eg, a constitutive promoter) or an inducible promoter.

28. A cell comprising the vector or nucleic acid of claim 26 or 27.

29. The cell of claim 28, wherein the cell is a mammalian cell, a yeast cell, an insect cell, or a bacterial cell.

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