ATP-dependent immune cell agonists that act as anticancer agents

Abstract

The present disclosure provides polypeptide constructs that, when exposed to sufficient levels of ATP, cause them to assemble into dimers or higher order complexes (e.g., trimers, tetramers, etc.), thereby acting as immune cell function agonists. The constructs can also be conjugated to one or more therapeutic, chemotherapeutic, or labeling agents. The complexes of the constructs are capable of stimulating immune cells (e.g., cytotoxic CD8+ T cells and / or NK cells) that function to promote an anti-tumor immune response, and delivering the conjugated agent to the tumor microenvironment. The constructs can be used as anti-cancer / therapeutic agents for the treatment of solid tumors with elevated ATP levels.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 536,921, filed on September 6, 2023.

[0002] I. Merging of sequence lists A sequence list in ST.26 XML format entitled 2974-6 PCT_05SEP2024_ST26, created on 5 September 2024, containing 521,246 bytes, prepared in accordance with 37 CFR 1.822 to 1.824, is filed concurrently with this application and is incorporated herein by reference in its entirety.

[0003] II. Introduction Potent anticancer therapies remain limited because they can produce toxic systemic stimuli even at doses far below the ideal therapeutic level. Systemic delivery of immune agonists (such as cytokines and anti-T-cell receptor antibodies) is particularly toxic, potentially leading to serious adverse events such as cytokine release syndrome and death. Strategies to increase the effective dose of tumor-site immunotherapies relative to the systemic stimuli dose / toxicity (i.e., the “therapeutic index”) include intratumoral administration and targeted immunotherapy via conjugation to anti-tumor-associated antigen (TAA) antibodies. However, intratumoral delivery is customized and invasive, thus limiting its ability to reach large patient populations due to the associated surgical and logistical complexities. Novel TAA-targeting agents attempt to impose a degree of conditionality on immune agonists, whereby the agonist accumulates to a bioeffective concentration only at sites with sufficient density of the targeted TAA. Immune agonists targeting TAAs have several serious limitations, including but not limited to: a small number of unique and validated TAAs expressed on the cell surface that can be targeted by immunotherapy; low TAA density leading to low efficacy; cancer escape due to loss of TAA expression; TAAs not being truly tumor-specific, resulting in targeted / non-tumor toxicity; and each TAA requires a different drug, which has a narrower therapeutic range compared to systemic stimulation therapy.

[0004] Increasing the stoichiometric ratio of immune agonists and / or TAA binding has also been explored as a means to enhance the potency of TAA-targeted immunotherapies. For example, first-generation bispecific T-cell conjugates such as brutinumab contain a single anti-TAA domain paired with a single anti-CD3 domain in a stoichiometric ratio of 1:1. However, in the case of the bispecific T-cell conjugate AFM11, a 2:2 anti-TAA:anti-CD3 stoichiometric ratio was used. This form was associated with more effective T-cell-mediated tumor cell killing in vitro, and more importantly, it reduced the number of cytotoxic effector T cells required for tumor cell killing. However, subsequent clinical trials of AFM11 showed severe toxicity, leading to the termination of the study. Conceptually similar increases in the stoichiometric ratio of the Fc domain of antibody-like molecules have also shown similar increases in in vitro potency. This stoichiometric modification may function by enhancing the affinity of TAAs and binding to immune receptors. Furthermore, cross-linking of known immune receptors (such as Fc receptors or T-cell receptors) can amplify signal transduction through these receptors and signaling pathways, resulting in a stronger immune response. Therefore, by binding chemodostoichiometric immunotherapies to immune receptors, it is possible to achieve more effective tumor-targeted therapy, including reducing effector T cell and antigen density requirements. However, due to safety concerns, these enhanced immunotherapies have not yet been translated into clinical applications.

[0005] Targeted immunotherapy can leverage tumor characteristics to avoid the aforementioned drawbacks of current immunotherapies. These characteristics are universal, crucial for most / all tumors, and highly expressed relative to normal tissue. The tumor microenvironment (TME) consists of blood vessels surrounding the tumor, immune and supporting cells, the extracellular matrix, and local molecules. The TME shares common features in many different tumor types, including low pH, high extracellular adenosine triphosphate (ATP) content, limited oxygen levels (hypoxia), vascular leakage, and the presence of tumor proteases (e.g., MMPs). Among these features, extracellular ATP is an excellent candidate for developing novel immunotherapies because it is a product of altered and important tumor metabolic processes common to most (if not all) tumors. Extracellular ATP levels observed in the TME are very high (50–1000 µM) compared to normal tissue (below approximately 0.1 µM, e.g., 0.01 µM to 0.1 µM).

[0006] III. Summary of the Invention This disclosure includes and provides molecular constructs comprising nucleotide-binding domains (NBDs) that enable the use of tumor ATP as a trigger for the conditional (ATP-dependent) assembly of potent cancer therapeutics at the tumor site. In the presence of ATP levels found in the tumor microenvironment (TME), NBD-containing constructs form dimers or higher-order complexes. NBD-containing constructs are particularly useful as therapeutic agents for various cancers. Therapies utilizing these constructs aim to leverage the fact that ATP-mediated complexation (e.g., dimerization) can generate agents that recruit immune cells to the TME and / or stimulate immune cells within the TME. Stimulation may occur, for example, by polymerizing and presenting immunomodulatory protein domains to immune cells through ATP-mediated construct complexation, thereby producing more effective immune cell stimulation than a single immunomodulatory protein sequence, since a single sequence cannot, for example, induce cross-linking of its receptor. Dimers or other higher-order complexes of NBD-containing constructs may also function through other mechanisms, including targeting ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity) tumor cells. Since elevated ATP levels relative to surrounding tissues are almost universally present in tumor microenvironments (TMEs), these constructs offer a mechanism for treating various tumor types. Furthermore, because individual constructs that have not yet assembled into dimers or higher-order constructs either have limited or no immunostimulatory capacity, the elevated ATP levels in the TME can promote dimerization or complex formation, driving strong local stimulation, while having a smaller effect in non-tumor tissues with lower ATP concentrations. Therefore, these constructs, capable of assembling immunomodulatory amino acid (aa) sequences into a multivalent state at the ATP levels found in the TME, can be used to modulate immune cell activity within the TME and generate selective immune-mediated antitumor effects.

[0007] NBD-containing constructs also offer several other advantages, including trans- and cis-targeting. Cis-targeting with NBD-containing constructs can stimulate tumor-infiltrating leukocytes (e.g., by providing immunostimulatory agonists such as IL-2 or CD28), thereby enhancing the anti-tumor immune response. When trans-targeting a tumor-associated antigen (TAA), these constructs can induce immune-mediated cytolysis of TAA-carrying tumor cells, adding additional tumor-specific effects beyond the effects of elevated local ATP levels. Regardless of the targeting protocol (e.g., cis- or trans-targeting), these constructs allow the use of various immunomodulatory domains derived from CD40L, CD28, IFN-γ, IL-12, etc., each of which functions through receptors requiring cross-linking. Other advantages of the NBD-containing constructs described herein include their relatively small size, which allows them to penetrate tumor tissue, and their increased size and reduced diffusion and concentration from the tumor microenvironment (TME) due to their assembly into dimers or higher-order complexes at the surface of target cells.

[0008] Compared to the monomeric counterpart (the construct formed in the absence of ATP), dimers formed in the presence of ATP exhibit significantly different properties, in addition to increased size and slower diffusion rates. For example, a dimer of a construct (molecule) consisting of NBD, a scaffold, and a tumor-specific binder (TSB) with affinity for a TAA (e.g., an antigen or neoantigen expressed by tumor cells) dissociates from a surface containing the target TAA more slowly than the same construct in monomeric form. This is reflected in the increased affinity or half-life of the dimer for dissociation from a surface containing a sufficient amount of TAA to bind the two TSBs in the dimer compared to the monomeric form. While the monomeric TSB also provides tumor homing and binding via TAA, the dimer is significantly more effective than the monomeric form in inducing responses that are absent or have very low activity and potency. In addition to the above, ATP-driven dimerization of constructs consisting of NBD, a scaffold, and TSB or immune cell binders (ICB) can lead to the clustering of molecules on cell surfaces, sometimes referred to as “capping,” “plaque formation,” or “lattice formation.” ATP-driven dimerization can unexpectedly amplify receptor signaling beyond levels associated with binding to more static monomers or gene dimers. While not wishing to be bound by any theory, this result may arise from dynamic oligomerization, as NBD-containing constructs repeatedly homodimerize with multiple adjacent NBD-containing constructs on the cell surface.

[0009] The constructs of this disclosure may also include payloads, including markers (e.g., radiolabels and / or sensitizers for use in radiotherapy and / or photodynamic therapy). The constructs of this disclosure are capable of accumulating in tumor tissue, thereby enhancing the effects of the payload and markers in vivo, particularly in cases where construct accumulation levels exceed expected levels due to kinetic and thermodynamic effects and / or ATP-mediated lattice formation on the target cell surface restricting construct release or cellular degradation. Enhanced accumulation of the construct carrying the payload means that, in addition to any effects the construct may have on shaping the antitumor response through immune and / or apoptotic actions, the payload also has the ability to enhance antitumor effects by increasing the delivery of therapeutic payloads (e.g., chemotherapeutic drugs), radionuclides, and / or sensitizers (e.g., radiosensitizers and / or photosensitizers). Furthermore, highly toxic molecules and / or molecules that produce serious side effects, used or considered as therapeutic agents (e.g., in cancer patients), can be administered as payloads attached to the NBD-containing constructs of this disclosure. The conjugation of this molecule to the NBD-containing constructs of this disclosure allows them to be administered at concentrations lower than those required to achieve therapeutic effects with the unconjugated molecule, because the NBD conjugate accumulates in the ATP-rich TME and can provide therapeutically effective levels of the molecule. Therefore, the conjugation limits negative effects on non-tumor tissues (e.g., the molecule may be systemically safe) and / or limits side effects experienced by patients.

[0010] Because the NBD-containing constructs of this disclosure form dimers or higher-order complexes (e.g., when cell surface receptor aggregation leads to capping or plaque formation), each event resulting in construct incorporation delivers at least twice the number of payload molecules to the cell compared to monomeric construct incorporation. Therefore, dimer constructs offer advantages in delivering payloads such as radioisotopes or chemotherapeutic agents compared to monomeric constructs and antibodies that specifically target epitopes appearing only once on cell surface proteins and do not promote cell surface molecular plaque formation or capping.

[0011] In addition to describing the construct, this disclosure also provides methods for its preparation and use; including using the construct to prepare a drug and / or using the construct to treat a disease or condition (e.g., cancer).

[0012] IV. Description of the attached drawings Figure 1 Figure A shows a schematic diagram of the construct disclosed herein, which includes an NBD having an ATP-binding site, a scaffold domain (scaffold aa sequence), and an immune cell activation domain (AD) linked by a linker sequence. The right side of Figure A also shows the dimerization of the construct when the binding site is adequately occupied in the presence of sufficient ATP concentration (e.g., at concentrations within the Kd range of ATP binding to NBD or higher). Figure 1 Figure B provides: (i) a first construct comprising a TSB, examples of which are bivalent cell lineage or tumor-specific antibodies, but may also be monovalent conjugates such as scFv or nanobodies, and a first NBD comprising a heterodimeric NBD pair linked by a linker; and (ii) a second construct comprising a second NBD comprising a heterodimeric NBD pair linked by a linker. The right side of Figure B shows the heterodimers of the first and second constructs formed due to the dimerization of the NBD domains in the presence of ATP. Figure 1 The construct disclosed herein is shown on the left, with the number C representing the construct built around an IgFc scaffold (KiHs-s IgFc) and comprising an NBD on one polypeptide and an ICB, AD, or TSB on another polypeptide. The construct is shown optionally with one or more payloads or tags (e.g., chemotherapeutic agents that can be bound by unstable (cleavable) linkers or radiolabeled tags). As shown in the right figure, the construct exhibits significant dimerization in the presence of ATP (e.g., levels found in the TME but not in non-tumor peripheral tissues).

[0013] Figure 2 A series of constructs (structures A through E) containing NBD and AD are provided, wherein constructs C, D, and E also contain scaffold sequences that can be non-dimerized or interspecies. The elements of each construct are linked by independently selected optional linkers. The figure also illustrates the formation of construct dimers in the presence of ATP. The sequences can be organized from the N-terminus to the C-terminus, starting from either end of the constructed shown.

[0014] Figure 3 A series of construct pairs (structures A through O) are provided. In AI, the first construct in each pair contains one or more NBDs and a TSB combined with a TAA, and the second construct in the pair contains an NBD and an AD and / or a monovalent ICB. In JM, the first construct in each pair contains one or more NBDs and an ICB, and the second construct in the pair contains one 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 in each pair contains one TSB and two NBDs, and the second construct in the pair contains one NBD and an AD. Constructs in O also contain scaffold sequences. Figure 3 The formation of construct dimers in the presence of ATP was also demonstrated. The components of each construct were connected by independently selected optional linkers.

[0015] Figure 4AA series of constructs (see A, C, and D), construct pairs (B, E, F, G, and I), or construct triplets (at H) are provided, which contain two or more NBDs and can polymerize to form a complex in the presence of ATP (e.g., in a TME). In some cases, one or more constructs contain a scaffold sequence, and each construct can be linked by an independently selected optional linker. The figure also shows the complexes formed in the presence of ATP, containing the constructs present in each case. The sequences can be organized in a direction from the N-terminus to the C-terminus, starting from either end of the constructed shown.

[0016] Figure 4B shows an example of NBD binding to a TSB (e.g., an antibody such as anti-mesothelin), which forms a homodimer in the presence of elevated ATP (as found in the TME). In (A), the construct contains a divalent antibody, which can be monospecific or bispecific, and thus produces a tetramer or tetramer-like complex in the presence of ATP. These molecules do not necessarily contain an Fc domain (e.g., VHH or scFV) and may contain two antigen-binding domains and remain divalent. In (B), (C), and (D), the homodimerized construct contains a monovalent single-chain antibody against NBD. The TSBs in (A) through (D) can be, for example, anti-HER2, anti-mesothelin, anti-EpCAM, anti-CLA-4, or anti-PSMA. When the scaffold sequence is present, they can be immunoglobulin (Ig) Fc peptides (e.g., IgG1 Fc) that can bind to any one or more of the FcγRI, FcγRII, FcγRIII receptors, and / or C1q proteins. A complex capable of binding to one or more of these proteins can stimulate Ig-mediated (Ig-dependent) effector functional responses, such as ADCC, antibody-dependent phagocytosis (ADCP), and / or CDC, targeting cells expressing a target (e.g., an antigen), to which the TSB component can bind. Therefore, this antibody function could be used therapeutically to eliminate tumor cells. Alternatively, the scaffold may contain aa sequences that do not possess the ability to stimulate one or more Ig-mediated effector responses (e.g., they are non-immunoglobulin aa sequences), or Ig molecules whose ability to stimulate Ig-mediated effector functions is significantly (e.g., completely) attenuated relative to their wild-type (wt.) Ig sequences. The sequences can be organized from the N-terminus to the C-terminus, starting from either end of the illustrated construct.

[0017] Figure 5AThe structures of a series of polypeptide constructs are shown, each containing one or two NBDs that, in the presence of ATP, can form a homodimer (H-NBD) with the NBD of a second molecule within the construct. Furthermore, each construct contains an immunoglobulin IgFc aa sequence that can stimulate one or more Ig-mediated effector functions. The constructs are oriented with the N-terminus at the top. Disulfide bonds between Ig heavy chain aa sequences in IgFc are not shown. Optional linker sequences can be chosen independently and may include rigid peptide linker sequences (e.g., repeats of Ala-Pro). Possible locations for H-NBDs are indicated by numbered circles and listed in a table below the structures. "---" in the table indicates that the location is unfilled or contains a bond or linker aa sequence. The NBDs present in the constructs may contain CAPs and disulfide bonds (not shown) for stabilization.

[0018] Figure 5B The structures of a series of constructs are shown, comprising a first (1) and a second (2) polypeptide, each containing an interspecific Ig heavy chain constant region aa sequence, which together form an IgFc structure that can stimulate one or more Ig-mediated effector functions. The first and second polypeptides together contain one or more (e.g., two) NBDs, which homodimerize with the NBD in the second molecule of the construct in the presence of ATP (H-NBD). The orientation of the constructs is N-terminus at the top. Optional linker sequences can be chosen independently and may include rigid peptide linker sequences (e.g., repeats of Ala-Pro). For structure AE, the possible locations of H-NBDs are indicated by numbered circles and listed in a table below the structure. "---" in the table indicates that the location is unfilled or is a bond or linker aa sequence. Structure FL provides a specific series of constructs. For structure AE, disulfide bonds between Ig heavy chain aa sequences in the IgFc are not shown. The disulfide bonds connecting the Ig heavy chain aa sequences of the first and second polypeptides in the lower hinge region are shown in structure FL. The NBD present in the construct may contain CAP and disulfide bonds (not shown) for stability.

[0019] Figure 5CA series of constructs of AD are shown, each comprising a first (1) and a second (2) polypeptide, each containing an interspecific Ig heavy chain constant region aa sequence, which together form an IgFc structure capable of stimulating one or more Ig-mediated effector functions. Structure E is a homodimer of the construct containing the homodimerized Ig heavy chain constant region aa sequence forming the IgFc structure. The first and second polypeptides of the construct together contain one or more (e.g., two) NBDs, which homodimerize with the NBD in the second molecule of the construct in the presence of ATP (H-NBD). The first and second polypeptides of the construct also together contain one or more (e.g., two) TSBs, which, when two TSBs are present, may target the same or different TAAs. Disulfide bonds connecting the first and second polypeptide Ig heavy chain aa sequences of the lower hinge region are shown. The orientation of the construct is N-terminus at the top. Optional linker sequences can be selected independently and may include rigid peptide linker sequences (e.g., repeats of Ala Pro). The NBDs present in the construct carry optional CAP aa sequences and contain disulfide bonds (not shown) for stabilization. Due to the interaction between NBD and ATP molecules, each construct can form a homodimer in the presence of ATP.

[0020] Figure 5D The structures of a series of constructs are shown, depicting some possible arrangements of constructs containing one or more NBDs that co-dimerize (H-NBD) in the presence of sufficient ATP. For stability, the H-NBD present in the construct, when located at the N-terminal position, may contain disulfide bonds and CAP (not shown). The constructs also contain one or more ICBs, one or more ADs, one or more TSBs, and optionally one or more scaffold sequences as elements. The elements are connected by one or more optional linker sequences, as indicated by lines between the elements. The possible locations of these elements are indicated by numbered circles and listed in a table below the structure. The possible locations of ICBs or ADs are indicated as ICB / AD. The constructs are grouped in the table such that the first number of the construct series indicates the location of the H-NBD. "---" in the table indicates that the location is either unfilled (i.e., it is a bond or linker sequence) or filled with one or more AD sequences (ADs), which may be tandem.

[0021] Figure 5EThe structure of a bispecific T cell conjugate-like (BiTE-like) construct is shown, which is built around a pair of interspecific scaffold aa sequences (displayed as an Ig KiH sequence pair with a knob and a hole) and linked by a pair of disulfide bonds. Each construct contains TSB, AD, and NBD linked to the scaffold sequences. Each construct can form a homodimer in the presence of ATP due to the interaction between NBD and ATP molecules.

[0022] Figure 5F A in this disclosure provides the protein construct. 14 The protein construct 14 contains a bispecific antibody. 10 The bispecific antibody 10 A TSB (e.g., a targeting portion) that has affinity for antigens or neoantigens expressed by tumor cells. 11 And ICBs or ADs with affinity for immune cell antigens (e.g., anti-CD3). 12 .Antibody 10 The optional connector shown in the curve is used with NBD (e.g., homodimerized NBD). 13 Conjugation. Connectors may contain elements that allow for the coupling of antibodies. 10 Connect to NBD 13 To prepare the conjugation sites for the construct. In the absence of ATP (left figure), the protein construct... 14 In its monomeric form, proteins complex into dimers in the presence of ATP. 15 Or higher-order structures, which stimulate immune cell responses more effectively than non-dimeric protein constructs. Figure 5F Figure B shows a schematic diagram of the protein construct of this disclosure, which includes TSB. 1 AD 2 and NBD 3 They are connected via independently selected optional connector sequences, which are displayed as lines between the individual components. The NBD can be constructed from a TSB. 1 and AD 2 It is part of the fusion protein, or may be present at the conjugation site. 9 Joining with them. Joining site 9 This can occur within the linker, as shown in the figure, or elsewhere on the molecule (e.g., the conjugation site may not be within the linker, but rather within the TSB or AD). The figure also shows that, in the presence of ATP, the construct forms a dimer, and the dimerized construct stimulates an immune cell response more effectively than the undimerized construct (shown on the left side of the figure) (shown on the right side of the figure).

[0023] Figure 5GA schematic diagram of a protein construct of this disclosure is provided, the construct comprising located in NBD 3 TSB on either side (N-terminus and C-terminus) 1 and AD 2 These components are linked by independently selected optional linker sequences, which are shown as lines between the elements. The figure also shows that, in the presence of ATP, the constructs form dimers, and the dimerized constructs stimulate immune cell responses more effectively than the undimerized constructs (shown on the left side of the figure) (shown on the right side of the figure).

[0024] Figure 5H A and B in the table provide TSB. 1 AD 2 and NBD 3 A diagram of the protein construct disclosed herein. TSB 1 It is in Fab form, where V H 4 CH1 5 V L 6 and C L 7 and disulfide bonds 8 All have been shown. At point A, NBD is connected to CH1. 5 AD 2 Connect to C L 7 Conversely, at point B, NBD connects to C. L 7 AD connected to CH1 5 NBD can include TSB. 1 Part of the fusion protein, or at the conjugation site. 9 The binding site can be located within the TSB or within the linker to which it is attached, as shown in the figure. The figure also shows that, in the presence of ATP, the construct forms a dimer, and the dimerized construct stimulates an immune cell response more effectively than the undimerized construct (shown on the left side of the figure) (shown on the right side of the figure).

[0025] Figure 5H C and D in the text provide TSB. 1 AD 2 and NBD 3 A diagram of the protein construct disclosed herein. AD 2 It can be in Fab form, where V H 4 CH1 5 V L 6 and C L 7and disulfide bonds 8 All have been shown. At point C, NBD is connected to CH1. 5 TSB 1 Connect to C L 7 Conversely, at point D, NBD connects to C. L 7 TSB 1 Connect to CH1 5 NBD can include AD 2 It is part of the fusion protein, or may be present at the conjugation site. 9 In combination with it, the conjugation site 9 can be in AD 2 The construct is located either within the junction to which it is attached, as shown in the figure. The figure also shows that, in the presence of ATP, the construct forms a dimer, and the dimerized construct stimulates the immune cell response more effectively than the undimerized construct (as shown on the left side of the figure) (as shown on the right side of the figure).

[0026] Figure 1-5H The structure shown can be understood as an arrangement of elements from the N-end at the top of the page to the C-end at the bottom of the page; however, as long as the order of the elements remains unchanged, it can also be understood as a structure in which the supporting elements are arranged with the C-end at the top of the page and the N-end at the bottom of the page.

[0027] Figure 6 The sequence alignments containing the following NBD domain elements are shown: ABCA1 gene product from UniProtKB-O95477 (SEQ ID NO: 1), ABCB1 gene product from UniProtKB-P08183 (SEQ ID NO: 2), ABCC1 gene product from UniProtKB-P33527 (SEQ ID NO: 3), ABCD1 gene product from UniProtKB / Swiss-Prot:P33897 (SEQ ID NO: 4), ABCE1 gene product from UniProtKB / Swiss-Prot:P61221 (SEQ ID NO: 5), ABCF1 gene product from UniProtKB-Q8NE71 (SEQ ID NO: 6), and ABCG2 gene product from UniProtKB / Swiss-Prot:P45844 (SEQ ID NO: 5). 7), and wt. TAP1 (SEQ ID NO:9) from UniProtKB-O95477. Partial Walker A region sequences of aa 46 to 55 and LSGGQ motif sequences of aa 152 to 156 of the TAP1 aa sequence are underlined and bolded. Figure 6The hydrolyzed acidic amino acid (i.e., Asp at position 177) and Asp (D) or Glu (E) residues of TAP1 NBD shown are underlined.

[0028] Figure 7 The sequence of human TAP2 from UniProtKB-Q03519 is shown (SEQ ID NO: 21).

[0029] Figure 8 SDS-polyacrylamide gel electrophoresis (PAGE) gels of TAP1 variants substituted with N676G, S677N, Q680R, E682Q and / or Q683R are shown. From left to right, the lanes are molecular weight marker (M), reduced sample (R), blank lane and non-reduced sample (NR).

[0030] Figure 9 Size exclusion chromatograms of the TAP1 NBD variant peptide with N676G, S677N, Q680R, E682Q and / or Q683R substitutions, and the substitution of serine for cysteine ​​residues present in the sequence, (i) in the presence of 1 mM adenosine diphosphate (ADP), resulting in the monomeric form of the peptide (apparent Mw 37 kDa), and (ii) in the presence of 1 mM ATP, resulting in the dimer form of the peptide (apparent Mw 57 kDa).

[0031] Figure 10 Gives a comparison with Figure 9 The results were evaluated by mass spectrometry analysis of the same TAP1 NBD variant using chromatographic analysis. The results obtained in the presence of 1 mM ATP indicated that the TAP1 NBD variant underwent dimerization.

[0032] Figure 11 The results of dynamic light scattering (DLS) size determination of TAP1 in the presence of (A) ADP or (B) ATP are presented.

[0033] Figure 12 A purified TAP1 NBD (SEQ ID NO: 118) gel stained with Coomassie Brilliant Blue is provided. This TAP1 NBD is modified to form homodimers, and the purified protein is subjected to chromatographic analysis in the presence of ADP and ATP.

[0034] Figure 13 SDS-PAGE gels from the following samples are shown: (A) induced expression of TAP1 NBD peptides provided by SEQ ID NO: 118 and SEQ ID NO: 119; and (B) purification of the peptide of SEQ ID NO: 119.

[0035] Figure 14A A shows a band diagram of a portion of the TAP1 NBD. At B, the TAP1 construct is represented in linear form with the N-terminus at the top. Small horizontal rectangles indicate approximate locations where substitutions can occur, including cysteine ​​substitutions. The diagram shows the catalytic aspartic residue (D668 in human TAP1) and the D-helix. The dashed lines on the left indicate disulfide bonds that may exist between cysteine ​​residues present or substituted in coupling helix 1 and those present or substituted in the NBD (see, for example, construct 308, where disulfide bonds between cysteine ​​residues are formed due to substitutions of E274C and P513C). The dashed lines on the right indicate disulfide bonds that may exist between cysteine ​​residues within the NBD, such as the disulfide bonds between substituted cysteine ​​residues in construct 182 (L493C:E573C). The numbers provided in A and B are construct numbers, indicating approximate locations within the protein affected by changes occurring in the specified construct. Constructs 175-179 and 211 involve C-terminal deletions in the NBD. Construct 179 contains a deletion approximately up to the N-terminus of α-helix 9 (a9), and construct 211 contains a deletion approximately up to the N-terminus of α-helix 8 (a8). Therefore, construct 179 substantially lacks the a9 helix to the NBD C-terminus, while construct 211 substantially lacks the a8 helix (including the a9 helix) to the NBD C-terminus. The human TAP1 a8 helix is ​​composed of the aa sequence THQQLME (SEQ ID NO: 30), and the a9 helix is ​​composed of the aa sequence CYWAMVQ (SEQ ID NO: 31). This figure and... Figure 12 and Figure 15 The TEV site described herein is a protease cleavage site specific to tobacco etch virus. The His tag may consist of a series of eight histidine residues (HHHHHHHH, SEQ ID NO: 151).

[0036] Figure 14B A series of TAP construct sequences are provided. The coupled helix from TAP1 (coupled helix 1) contains the aa sequence eteffqqnqt (SEQ ID NO: 17), and the coupled helix from TAP2 (coupled helix 2) contains the aa sequence GLQTVRSFG (SEQ ID NO: 18). Sequences annotated with “modified D-helix” include the rat TAP1 D-helix sequence GNQLRVQRLL (SEQ ID NO: 26). No adapter sequence is present, i.e., “no adapter”, denoted by the abbreviation “(NL)”.

[0037] Figure 15Figure A shows an SDS-PAGE analysis of the exemplary protein construct (construct 303) expressed in *E. coli*, before (lane 4) and after (lane 5) induction with isopropyl β-D-1-thiogalactoside (IPTG). The figure also shows reduced (lane 7) and non-reduced (“NR”, lane 9) protein construct samples purified on Talon® resin. Figure 15 B in the figure shows size exclusion chromatography, which demonstrates the dimerization behavior of construct 303 in the presence of ADP and ATP. At C, Figure 15 The melting point of component 303, as determined by differential scanning calorimetry (DSC), is shown.

[0038] Figure 16 A graph showing the effect of C-terminal amino acid deletion on the melting points of various TAP constructs as determined by DSC is presented. The graph shows the melting points of several constructs containing native TAP1 (solid circles) and those with added disulfide bonds (solid cubes). The melting point of construct 451, which lacks an N-terminal CAP (i.e., it lacks the N-terminal sequence consisting of coupling helix 1 and coupling helix 2), is also marked with an asterisk.

[0039] Figure 17 A table is provided that summarizes the structural characteristics, expression test results, dimerization in the presence of ATP, and melting point of various TAP1 constructs.

[0040] Figure 18 Constructs 156-159 are shown, whose elements include NBD, anti-B cell maturation antigen (anti-BCMA) VHH, and anti-CD3 aa sequence, the order of which is further described in Example 6.

[0041] Figure 19 SDS-PAGE gels of constructs 156, 157, and 159 purified under reducing and non-reducing (oxidizing) conditions are shown. Each construct produced a single band in both the reduced and non-reduced states.

[0042] Figure 20 Size exclusion chromatograms of constructs 156, 157, and 159 in the presence of ADP or ATP are shown.

[0043] Figure 21 Figure A shows a schematic diagram of three different constructs, illustrating the ability to select the ATP concentration (ATP set point) that leads to construct dimerization. At point B, the figure shows the residence time (1 / kJ) of each of the three constructs from its target (immobilized mesothelin peptide) at different ATP concentrations. dis The curve graph of ).

[0044] Figure 22The diagram shows the TAP1 NBD-scFc fusion protein construct (436) at point A, and the construct at point B, labeled "571:476," consisting of a TAP1NBD-IgG knob fusion peptide (571) paired with an IgFc pore peptide (476). Point C shows three replicates of the dynamic light scattering (DLS) data of the constructs in the presence of ADP only (top panel) and in the presence of both ADP and ATP (bottom panel). Figure 22 The D in the figure provides the time course of serum concentrations for construct 571:476.

[0045] Figure 23 Three different constructs are shown, each containing a pair of peptides. The peptide numbers within each construct are shown above each peptide, with pairs of peptides separated by colons. A represents construct 590:587, B represents construct 473:587, C represents a genetic dimer of the single-stranded (sc)4-1BBL trimer repeat sequence, and D represents… Figure 23 The diagram shows the luminescence of luciferase expression in Jurkat reporter cells stimulated with 4-1BB, with the left side showing the specified concentrations of construct 590:587 and the negative control 473:587, and the right side showing anti-41BB (Urelmab) and the 4-1BBL genetic dimer (“dimer-41BBL”, see C). Section E shows triplicate luminescence measurements of the same Jurkat cell line stimulated with construct 590:587 containing and without adenosine triphosphate diphosphatase, and with the negative control construct 473:587; Section F shows triplicate luminescence measurements of the same cells in the presence of construct 590:587, the negative control construct 473:587, and a modified construct 590:587* with reduced activity.

[0046] V. Detailed Implementation A. Definition The amino acids used in this article (“aa” singular or “aas” plural) refer to naturally occurring protein amino acids incorporated into polypeptides and proteins during mammalian cell translation. Unless otherwise stated, 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). Aa also includes aa-hydroxyproline and selenocysteine, which are found in some proteins found in mammalian cells; however, they are not considered to be included unless their presence is explicitly stated.

[0047] A substitution of aa at a specific position in a sequence is represented by a single- or three-letter code, which indicates the original aa, the numerical position of that aa, and the aa that has been substituted into the sequence. For example, if alanine (A) at position 12 of a sequence is replaced by proline (P), it is represented by the single-letter code A12P or the three-letter code Ala12Pro. If more than one substitution occurs in the same sequence, they can be separated by a forward slash " / ". Therefore, the above A12P substitution, along with V23A and Q41G substitutions, is in the same sequence and can be represented by the sequence A12P / V23A / Q41G.

[0048] Amino acid residues appearing in the wt. protein or reference sequence are indicated by their single-letter codes and positions within the sequence. For example, cysteine ​​at position 372 is represented as C372. Substitutions of amino acids in the sequence are indicated by abbreviations beginning with the single-letter code of the original amino acid, followed by its numerical position, and then the single-letter code of the substituted amino acid (e.g., replacing cysteine ​​at position 372 with serine is represented as C372S). When the sequence of the wt. polypeptide is altered, whether by adding or deleting one or more amino acids, a specific residue or residue number still refers to the same specific amino acid in the altered polypeptide (e.g., adding an amino acid at the N-terminus of the peptide chain, where position 2 is glycine (G2), then a reference to position G2 still refers to the glycine now located at position 3, unless otherwise stated).

[0049] As used herein, “conservative amino acid substitution” refers to the substitution of an amino acid in a protein with an amino acid having a similar side chain in the subsequent group. The group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine. The group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine. The group of amino acids with amide-containing side chains consists of asparagine and glutamine. The group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan. The group of amino acids with basic side chains includes lysine, arginine, and histidine. The group of amino acids with acidic side chains consists of glutamic acid and aspartic acid. The group of amino acids with sulfur side chains consists of cysteine ​​and methionine. Therefore, conservatively substituted amino acids refer to substitution with an amino acid within the groups defined above. Conversely, “non-conservative amino acid substitution” as used herein refers to substitution using amino acids from different groups.

[0050] In this article, the terms “peptide” and “protein” are used interchangeably and refer to the polymeric form of protein aa, unless otherwise stated. They refer to naturally occurring protein L-aa that are incorporated into proteins through biosynthesis during mammalian cell translation.

[0051] In the aa sequences used in this article, the colons between cysteine ​​positions indicate the presence of a disulfide bond (in the non-reduced state) between these positions. For example, C735:R721C represents a disulfide bond formed between a cysteine ​​residue at position 735 and a cysteine ​​residue that replaces arginine at position 721. The order can be reversed (e.g., R721C:C735), but the meaning remains the same.

[0052] As used herein, the term "antibody" includes, but is not limited to, antibodies derived from, for example, mammals, sharks, and camels, such as human IgG (hIgG) and heavy-chain-only antibodies. Antibodies also include their antigen-binding fragments, including but not limited to Fab' and Fv fragments. Monovalent antibody fragments include, but are not limited to, Fab and Fv fragments. Antibodies also include monovalent single-chain (sc) antibodies and antibody constructs containing the antigen-binding portion of the antibody, including but not limited to scFv (scFv(vH-vL) or scFv(vL-vH)), nanobodies (VHH or VHH), and antibody peptides containing only the VH heavy chain. Throughout this disclosure, the term "antibody" may be cited in conjunction with a variety of antibody fragments or constructs. These citations are not intended to change the definition of an antibody, but rather to illustrate the possible types of antibody sequences considered in various aspects of this disclosure and / or as prerequisites.

[0053] You can use the NCBI BLAST program (BLAST+ 2.9.0, blastp version, available at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) or the CERI Clustal Omega program (version 1.2.4, available at www.ebi.ac.uk / Tools / msa / clustalo / ) with the default parameters at the time of submission to perform an alignment to identify corresponding residues in different sequences. Unless otherwise stated, Clustal Omega is used to identify corresponding residues in different sequences.

[0054] Sequence identity percentage refers to the percentage of identical amino acids or nucleotides in two aligned aa or nucleic acid sequences. Unless otherwise specified, sequence identity percentage is determined using BLAST+ version 2.9.0, where peptides are aligned using blastp and nucleic acids using blastn, with parameters set to the default values ​​at the time of submission.

[0055] Unless otherwise stated, the term "significant" is intended to include both "all" and "most but not all". For example, an Ig Fc that "does not induce cell lysis by ADCC or CDC" means an Ig Fc that does not induce cell lysis at all, or that does not induce cell lysis by ADCC or CDC most but not entirely.

[0056] As used herein, the term “about” in conjunction with a quantity indicates that the quantity can vary by 10%. For example, “about 100” means a quantity between 90 and 110. When “about” is used in the context of a range, “about” for a lower quantity in the range means that the lower quantity includes a quantity 10% lower than the lower quantity in the range, and “about” for a higher quantity in the range means that the higher quantity includes a quantity 10% higher than the higher quantity in the range. For example, “from about 100 to about 1000” means a range from 90 to 1100.

[0057] The terms "patient" and "subject" are used interchangeably.

[0058] The term "payload" includes molecules or atoms that can be covalently or non-covalently (e.g., by chelation) attached to the constructs of this disclosure. The notation represents a subclass of payload that represents a molecule or a detectable position of a molecule.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0060] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, reference to “a T cell” includes a plurality of such T cells. This statement is intended to serve as a presupposition for the use of exclusive terms such as “a,” “solely,” and “only” in relation to the description of elements of the claim, and for the use of “negative” limitations that exclude the limitation of the claim.

[0061] It should be understood that, for clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of this disclosure described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to this disclosure are specifically included in this disclosure and disclosed herein as if each combination were separately and explicitly disclosed. Furthermore, all sub-combinations of various embodiments and their elements are also specifically included in this disclosure and disclosed herein as if each such sub-combination were separately and explicitly disclosed herein.

[0062] B. Description The tumor microenvironment differs from that of non-malignant tissues in many ways, including differences in one or more aspects such as pH, ATP content, oxygen levels (hypoxia), and tumor protease (e.g., MMP) levels. Extracellular ATP levels in normal (non-tumor) tissues are typically in the range of 0.01–0.1 μM, while those in solid tumors are typically in the range of 50–200 μM. This disclosure describes and provides methods for inducing an immune response against tumor cells using the ATP levels found in the tumor microenvironment (TME). More specifically, certain constructs described herein may include an immune cell activation domain (AD) (i.e., an AD that binds to and stimulates immune cell immunomodulatory molecules) and at least one non-tumor brainstem (NBD) that forms a large number of dimers or other higher-order complexes in the presence of ATP levels found in the TME. The dimerization of the NBD or the formation of higher-order complexes results in the formation of a complex containing two or more immune cell ADs. When immune cells present as dimers or higher-order complexes, they can stimulate immune cells such as NK cells and CD8+ T cells. However, when they are presented individually, they cannot stimulate the same immune cells to the same extent. This makes it possible to stimulate intratumoral immune responses within the tumor microenvironment (TME).

[0063] Other constructs described herein may include a TSB that binds to TAA and ICB, and at least one NBD that forms a dimer or other higher-order complex in the presence of ATP levels found in the TME. Such constructs may optionally include AD. The dimerization of the NBD or the formation of higher-order complexes leads to the production of dimers or higher-order complexes that can recruit and / or stimulate immune cells in the TME. Because the dimerization of such constructs or the formation of other higher-order complexes outside the TME does not result in the formation of higher-order complexes outside the TME, they allow for intratumoral stimulation of the immune response while limiting extratumor immune stimulation.

[0064] Because the AD, tumor-specific binding domain, and ICB of the construct presented alone (as monomers) do not stimulate cells such as NK cells and CD8+ T effector cells as much as those presented as ATP-induced dimers (see, for example) Figure 1-3 and 4B) or higher-order complexes (see, for example) Figure 4A While effective when presented in pairs or multiples, the application of monomeric constructs limits or significantly restricts systemic stimulation-induced immune activation and associated toxic effects (e.g., these molecules may be safe for systemic stimulation). However, the constructs described herein do provide immune stimulation confined to the TME because there is sufficient ATP in the TME to drive complex formation, thus they can be considered ATP-dependent immune cell function agonists whose effects are limited to this environment. Dimers and higher-order complexes are known to have higher affinity for cells in the TME because the binding energy of two or more binding interactions is greater than the binding energy of a single (isolated) binding interaction of a single construct. The difference in binding energy between a single construct and a dimer or higher-order complex is reflected in the higher binding affinity (Kd value) of dimers and higher-order complexes compared to any single construct constituting a dimer or complex. Furthermore, the diffusion of complex forms of constructs (e.g., dimers, trimers, etc.) from the TME is reduced due to their increased size and molecular weight, thus limiting their role in the TME. Those dimers or higher-order complexes that diffuse out of the TME will dissociate into individual monomers, which are ineffective or less effective in stimulating cellular responses because the ATP levels in the surrounding non-malignant tissue are too low to support their recombination. Therefore, the immunostimulation provided by the complexes of the constructs described herein is considered reversible and limited to microenvironments with sufficiently high ATP levels to induce complex formation.

[0065] The binding interactions between the ATP-mediated complexes of the constructs described herein can also be described in kinetic terms, rather than the thermodynamic terms described above. Kinetically, the binding rate of a single construct to tumor cells is not understood to be affected by the presence or absence of bound ATP, because there is little difference in size or molecular weight between a single construct and a single construct with bound ATP, and therefore little difference in their diffusion rate and accessibility (entry) into the TME. Conversely, once a construct binds to cells in the TME and forms a dimer or higher-order complex due to high ATP levels, the effective affinity for the cell increases, and the effective dissociation rate of the construct from the cell decreases relative to the dissociation rate of a single (monomer) construct bound to the same cell surface antigen. An unexpectedly large increase in binding affinity to surface-carrying target antigens (e.g., TAAs) was observed.

[0066] While not wishing to be bound by any particular theory, the binding of multiple constructs to the cell surface, even with only a single NBD in homodimers or heterodimers, can unexpectedly lead to cell capping or plaque formation, where the cell surface target molecules bound to the constructs accumulate or cluster on a portion of the cell surface. For many divalent binding molecules like the dimers described herein, their failure to bind to repetitive epitopes on the surface of a single cell surface antigen is unexpected and could be due to the reversible entry of NBDs with more than one other molecule within the binding distance into the alternative dimer complex. The consequence of such clustering can be a localized increase in the concentration of the construct and the cell surface molecules bound to it, and potentially a localized increase in ATP levels relative to the TME. This increased concentration, both kinetically and thermodynamically, leads to an unexpected increase in the effective binding affinity (affinity) of the construct to the cell surface, potentially allowing it to continue to influence the cell through interactions with cell surface proteins. For example, the formation of cell surface clusters may significantly alter (e.g., reduce, increase, or even block) the internalization of cell surface molecules that form complexes with the construct, thereby enabling the construct to be present on the target cell surface and induce a response in cells within the TME (e.g., tumor cells, T cells, NK cells, macrophages, etc.). Constructs with more than one NBD can also be used to induce or enhance capping or plaque formation on cells (see, for example...). Figure 4A ).

[0067] Regardless of the mechanism, and any unintended increase in the effective binding constant, the high levels of ATP present in the TME will lead to a biodistribution favorable for the accumulation of the construct within the TME. When measured after a single dose (e.g., intravenous injection), the maximum concentration of the construct found in the TME can be at least about 1.5 to 2 times the circulating level of the construct in the patient's plasma. The maximum level at which the construct of this disclosure can accumulate in the TME is at least 3 to 4 times the circulating level of the construct in the patient's plasma after a single dose. The maximum level at which the construct of this disclosure can accumulate in the TME is at least 5 to 8 times the circulating level of the construct in the patient's plasma after a single dose. The maximum level at which the construct of this disclosure can accumulate in the TME is at least 12 to 20 times the circulating level of the construct in the patient's plasma after a single dose. The maximum level at which the construct of this disclosure can accumulate in the TME is in the range of about 1.5 to about 2 times or about 2 to about 4 times the circulating level of the construct in the patient's plasma after a single dose. The maximum level at which the construct of this disclosure can accumulate in the TME is approximately 4 to 8 times, or approximately 8 to 20 times, the circulating level of the construct in plasma after a single dose of the construct in a patient. The maximum level at which the construct of this disclosure can accumulate in the TME is approximately 20 times or more the circulating level in plasma after a single dose of the construct in a patient. The amount of construct accumulation in tumor tissue can also be calculated relative to whole blood or serum, as previously described. Similarly, the accumulation of the construct in tumor tissue can also be relative to peritumoral tissue or normal (untransformed) tissue of the tumor-bearing organ. The aforementioned accumulation levels in tumor tissue can be observed regardless of whether the construct is administered intravenously or via a non-intravenous route other than intratumoral injection. Regardless of the comparative tissue / fluid used, the construct of this disclosure can continue to accumulate after subsequent injections (administrations) and may persist in tumor tissue for a longer period after administration than equivalent constructs without the ability to dimerize (or form higher-order complexes) of NBD. Compared to equivalent constructs that do not have NBD capable of dimerizing (or forming higher-order complexes), constructs containing NBD administered via intratumoral injection may persist in tumor tissue for a longer period of time.

[0068] The constructs described herein may also contain NBD and Ig heavy chain aa sequences (e.g., as a scaffold) capable of stimulating Ig-mediated Fc responses, including but not limited to ADCC, ADCP, and / or CDC. When compounded in the presence of ATP, the constructs will more effectively stimulate at least one Ig-mediated effector function, thereby effectively making the scaffold an AD-like domain. Constructs with aa sequences capable of stimulating effector functions do not need to target specific cells in the TME to have antitumor activity; however, they may contain one or more TSBs that target the construct to one or more TAAs (e.g., two independently selected TAAs) that may be present on tumor cells in the TME, thereby concentrating the response on tumor cells.

[0069] C. Structure of ATP-dependent immune cell agonists ATP-dependent immune cell agonists can exist in multiple forms, allowing them to interact with immune system cells through one or more different mechanisms. These mechanisms include, but are not limited to, stimulating ADCC, ADCP, and / or CDC-mediated responses in the tumor cell environment.

[0070] The constructs described herein include one or more NBDs and one or more Ig sequences, which can also serve as scaffold sequences capable of stimulating Ig-mediated effector functions. When these constructs are present in a TME containing a sufficient concentration of ATP, the formation of dimers or multimers of these constructs enhances their ability to stimulate Ig-mediated effector functions. By integrating TSBs that target antigens on tumor cells, responses generated by cells such as T effector cells and / or NK cells can be directly focused on tumor cells.

[0071] The second set of constructs described herein includes one or more NBDs and one or more ICBs and / or one or more ADs, and optionally one or more TSBs. These constructs may also contain scaffold sequences (e.g., Ig scaffolds). Such constructs may contain (i) one or more ICBs or ADs, and (ii) one or more TSBs. When the TSBs and ICBs or ADs are located in a single construct (e.g., they are part of a single polypeptide or bound to a pair of interspecific scaffold aa sequences), the molecule can be considered a BiTE-like molecule. When the ICB is part of a first polypeptide having a first NBD, and the TSB is part of a second peptide having a second NBD, the first and second polypeptides undergo heterodimerization via their NBDs in the presence of ATP, thereby forming a BiTE-like construct (see, for example...). Figure 3 (C, D, and FI in the original text). Constructs containing one or more ICBs and / or one or more TSBs may also contain one or more ADs.

[0072] The third type of construct described in this paper contains one or more NBDs and one or more TSBs. This construct may contain one or more scaffold sequences.

[0073] 1. An NBD-containing construct containing an Ig sequence capable of stimulating Ig-mediated effector functions. The constructs disclosed herein act as ATP-dependent immune cell agonists by stimulating one or more antibody-associated effector functions (e.g., ADCC, ADCP, and / or CDC), and in their simplest form comprise at least one homodimeric NBD aa sequence linked to an IgFc aa sequence via an optional linker; see, for example Figure 5A A and B in the example. These constructs can also include a second copy of NBD (see example). Figure 5A (CE in the text). When using two peptides, each containing one of a pair of heterodimerized NBDs, this construct can be made asymmetric ( Figure 5A (Not shown in the image).

[0074] Constructs capable of stimulating one or more Ig-mediated effector functions may also comprise a pair of polypeptides, wherein: (i) a first polypeptide consisting of a first Ig heavy chain constant region aa sequence (e.g., comprising an Ig CH2-CH3 aa sequence) and an optional NBD aa sequence, the NBD aa sequence being directly or indirectly linked to the first Ig heavy chain constant region aa sequence via an optional adaptor peptide aa sequence; and (ii) a second polypeptide consisting of a second Ig heavy chain constant region aa sequence (e.g., comprising an Ig CH2-CH3 aa sequence) and an optional NBD aa sequence, the NBD aa sequence being directly or indirectly linked to the second Ig heavy chain constant region aa sequence via an optional adaptor peptide aa sequence; wherein at least one (e.g., both) of the first and second polypeptides comprises NBD (see example...) Figure 5B In this construct, the Ig heavy chain aa sequences of the first and second polypeptide chains form IgFc (e.g., heterodimeric IgFc, where they are interspecies sequence pairs) capable of stimulating immune cell effector functions. The first and second IgFc sequences can be interspecies sequence pairs, such as knob-in-hole structures (KiH) or KiHs with disulfide bonds (KiHs-s). Other interspecies heterodimeric immunoglobulin heavy chain scaffold aa sequences will be described below. The first and second polypeptides of the construct may contain one or more NBDs, or two or more NBDs. Figure 5BThe structure FL in this disclosure represents a construct with specific interspecific Ig heavy chain aa sequence pairs that together constitute two homodimeric NBDs. Stable disulfide bonds may exist between Ig heavy chain sequences in the constructs disclosed herein, such as disulfide bonds in the lower hinge region and / or disulfide bonds between interspecific Ig heavy chain aa sequences (e.g., KiHs-s or EW-RVTs-s).

[0075] When two or more NBD aa sequences are present as part of the same polypeptide (e.g., Figure 5B When F is present in the construct, or when it is part of two different polypeptides (e.g., F in the construct), Figure 5C The NBDs (G) in the construct are positioned such that they cannot form intramolecular dimers in the presence of ATP, but instead form intermolecular associations with the NBDs on the second molecule in the construct. This restriction on NBDs can be achieved by, for example, positioning them on the molecule at sufficient distances from each other, or by using rigid linkers to position them in orientations that prevent dimerization. It should be recognized that when two or more NBDs are present in the construct, the molecules can form intermolecular dimers and / or higher-order complexes, such as trimers, tetramers, etc.

[0076] When constructs of IgFc capable of stimulating one or more effector functions dimerize in the presence of ATP (e.g., in the TME), their size / molecular weight increases, reducing their diffusion out of the tissue (e.g., out of the TME) and thus allowing accumulation. The formation of dimers or higher-order constructs can also enhance the stimulation of any one or more effector functions.

[0077] In addition to accumulating constructs in environments with sufficiently high ATP concentrations to drive the formation of dimers or higher-order complexes, constructs and their ATP-mediated complexes may also contain TSBs as target sequences (see example...). Figure 4A (Structure C). Alternatively, these constructs may contain one or more (e.g., two or more) independently selected TSBs that accumulate not only in the TME but also on the cell surface of a specific target (TAA) containing TSB binding. Multiple TAAs can be targeted, including but not limited to those listed below. One or more (e.g., two or more) independently selected TSBs can be added to... Figure 5A and 5BIn any of the constructs listed (e.g., the N-terminus and / or C-terminus of one or more peptides in these constructs). Two or more TSBs can be incorporated into constructs that stimulate one or more Ig-mediated effector functions to prevent tumor cells from escaping targeting Ig-mediated effector cell functions, for example, by mutating a single targeting TAA or inhibiting the expression of a targeting TAA. Incorporation of two or more TSBs with affinity for different TAAs also allows for targeting a wider range of tumor cells. Figure 5C Some specific instances of constructs are listed that contain one or two NBDs and also one or more independently selected TSB aa sequences.

[0078] The constructs described herein that stimulate effector function may include IgFc scaffold aa sequences of any suitable antibody isotype (e.g., IgG or IgM) or its subtype (e.g., IgG1, IgG2, etc.), provided they can stimulate the desired effector function. The IgFc portion of the construct may contain a single-chain IgFc (scFc) aa sequence having two CH2-CH3 sequences linked by a sufficiently long linker that can pair to form a structure consistent with that of a natural heavy-chain antibody (see, for example, SEQ ID NO: 63). The IgFc portion of the construct may also be formed from two polypeptides that can homodimerize or heterodimerize as an interspecies pair.

[0079] Whether the IgFc sequence is formed in the form of scFc or from a pair of homodimerized or heterodimerized Ig heavy chain sequences, the resulting IgFc may have a wt. effector function or may contain one or more substitutions that enhance its effector function. Therefore, the construct can use a wt. IgFc sequence (e.g., a sequence containing the wt. IgG sequences of SEQ ID NO: 59 and 64-66). Alternatively, the IgFc sequence may contain one or more substitutions relative to the wt. sequence, thereby enhancing its ability to stimulate one or more effector functions (e.g., increasing the potency and / or efficacy of IgFc effector stimulation). The IgFc of the construct may contain one or more substitutions that enhance one or more of ADCC, ADCP, and / or CDC compared to the same construct lacking substitutions. Homodimerized or heterodimerized Ig heavy chain constant region aa sequences present in scFc as interspecies pairs may contain substitutions, alone or in pairs, that enhance one or more effector functions.

[0080] Any construct that stimulates one or more effector functions may contain an N-terminal CAP and / or a stable disulfide bond connected to the NBD. The TAP1 and / or TAP2 NBDs present in the construct may contain an N-terminal CAP formed by the coupling helix of TAP1 and the coupling helix of TAP2 linked by an internal linker and / or a stable disulfide bond. Stable disulfide bonds may form between cysteine ​​residues present or substituted at L493C:E573C, C735:R721C, or C662:Q580C (or the corresponding position in TAP2) in the TAP1 aa sequence. The TAP1 NBD aa sequence may terminate at approximately Q741 of SEQ ID NO:8, thereby deleting the C-terminal sequence PADAPE (SEQ ID NO: 11).

[0081] 2. NBD-containing constructs including ICB, AD, and optional TSB. In addition to containing one or more NBDs, the NBD-containing constructs described herein may also contain one or more ICBs, one or more ADs, and / or one or more TSBs, as well as optional one or more scaffold sequences. As will be discussed in more detail below, ICBs and ADs are part of the NBD-containing construct (e.g., aa sequences) and have affinity for cell surface molecules (e.g., antigens) of immune cells, where ADs can induce a response in immune cells. Similarly, TSBs are part of the NBD-containing construct (e.g., aa sequences) and have affinity for cell surface molecules of tumor cells. ICBs and ADs (e.g., antibody fragments) are monovalent to avoid off-target stimulation (e.g., systemic stimulation) on the immune cells to which they bind. NBD-containing constructs containing one or more NBDs and / or one or more ICBs and optional one or more TSBs can appear in various forms, such as... Figure 2 -4B and Figure 5D As shown.

[0082] A construct containing one or more ICBs, one or more ADs and / or one or more TSBs, and optionally one or more scaffold sequences as elements, may have those elements arranged in any order. Figure 5DThe diagram shows the structures of a series of constructs, depicting some possible arrangements of constructs containing one or more NBDs that co-dimerize (H-NBD) in the presence of sufficient ATP. For stability, the H-NBD present in the construct, when located at the N-terminal position, may contain disulfide bonds and CAP (not shown). The construct also contains one or more ICBs, one or more ADs, one or more TSBs, and optionally one or more scaffold sequences. Elements of the construct can be linked by one or more independently selected optional linker sequences, as indicated by lines between elements. For the constructs in this figure, the ICBs and TSBs can be monovalent (e.g., scFv) or divalent (e.g., IgG). Each of the ICB, TSB, and AD sequences is independently selected. The possible locations of the construct elements are indicated by numbered circles and listed in a table below the structure. The constructs are grouped in the table such that the first number in the construct series indicates the location of the H-NBD. "---" in the table indicates that the location is either unfilled (i.e., it is a bond or linker sequence) or filled with one or more AD sequences (ADs), which may be tandem. Except where AD sequences are directly linked in tandem or separated by amino acid linker sequences, other elements can be added to the N-terminus, C-terminus, or between any two elements. The scaffold aa sequence is optional and may be absent (structure G); if present, it may be homodimerized or nondimerized (structure A), or heterodimerized (as shown in structures C–F, H, and I). The orientation of the construct is with the N-terminus at the top. Constructs containing tumor-specific antibody sequences but not ICB may contain scaffolds with wild-type or enhanced ADCC, CDC, and / or ADCP functions. Constructs containing ICB or AD should not contain scaffolds with the ability to stimulate Ig-mediated effector functions (e.g., ADCC, CDC, or ADCP) in a wild-type or enhanced manner, and may have the ability to significantly attenuate and / or completely eliminate the stimulation of these effector functions.

[0083] a) ATP-dependent immune cell agonists, including NBD and ICB and / or AD. The NBD-containing molecular constructs described herein may contain one or more NBDs and one or more ICBs and / or one or more ADs, which bind to or stimulate immune cells (e.g., CD8+ T cells, NK cells, etc.). The NBDs contain one or more ATP-binding sites, which, when occupied by ATP, result in the formation of a double-stranded or higher-order complex (triplexed, etc.) between the NBDs of the constructs due to interactions between them. Such molecular constructs optionally contain one or more independently selected scaffold aa sequences and / or one or more independently selected linker sequences. See, for example... Figure 1 Structure A and Figure 2The structure of AD. In some cases, interspecific scaffold sequences that form heterodimers can be incorporated into the construct, allowing more than one type of ICB or AD to exist in the molecule, such as Figure 2 The structure E is shown in the diagram. The use of ICB or AD (optionally denoted as "ICB / AD" in the singular and "ICB / AD" in both singular and plural forms) when they aggregate together (e.g., as homodimers or heterodimers) can stimulate targeted immune cell populations, such as NK cells and / or T cells, thereby selectively stimulating intratumoral immune responses due to the high levels of ATP in the TME, while avoiding systemic stimulatory activation of targeted immune cells (e.g., NK cells or T cells) and / or immune responses in non-tumor tissues.

[0084] Constructs containing NBD and ICB / AD can be arranged in the N-to-C direction as follows: NBD followed by ICB / AD; or ICB / AD followed by NBD (see example). Figure 2 A and Figure 5D Structure G, as in serial numbers 1-1a to 1-1d, is replaced.

[0085] Constructs containing NBD and ICB / AD may also contain one or more scaffold sequences that do not dimerize with other scaffold sequences (see, for example) Figure 2 The C in the construct has a structure containing homodimeric NBD, and the D has a structure containing heterodimeric NBD. These constructs can contain those elements arranged in six different orders. First, the construct can contain the NBD aa sequence, the scaffold aa sequence, and the ICB / AD from the N-terminus to the C-terminus. Second, the construct can contain the NBD aa sequence, the ICB / AD, and the scaffold aa sequence from the N-terminus to the C-terminus. Third, the construct can contain the ICB / AD, the scaffold aa sequence, and the NBD aa sequence from the N-terminus to the C-terminus. Fourth, the construct can contain the ICB / AD, the NBD aa sequence, and the scaffold aa sequence from the N-terminus to the C-terminus. Fifth, the construct can contain the scaffold aa sequence, the ICB / AD, and the NBD aa sequence from the N-terminus to the C-terminus. Finally, the construct can contain the scaffold aa sequence, the NBD aa sequence, and the ICB / AD from the N-terminus to the C-terminus. When two such constructs are combined, capable of heterodimerizing NBD, they can form a complex in the presence of ATP (see, for example...). Figure 2 (D in the middle).

[0086] Constructs containing NBD, ICB / AD, and one or more scaffold aa sequences may include: (i) A first polypeptide comprising a first scaffold aa sequence (e.g., containing an Ig heavy chain constant CH2-CH3 aa sequence) and a first NBD aa sequence optionally linked directly or via a linker aa sequence to the first scaffold aa sequence, and (ii) A second polypeptide comprising a second scaffold aa sequence (e.g., containing an Ig heavy chain constant CH2-CH3 aa sequence) and an optional second NBD aa sequence connected directly or via a linker aa sequence to the second scaffold aa sequence; in (i) At least one of the first and second polypeptides contains an NBD aa sequence. (ii) At least one of the first and second polypeptides contains an ICB or AD aa sequence. (iii) Each NBD aa sequence contains one or more ATP-binding sites and, in the presence of ATP, can homodimerize or heterodimerize with a homologous but not identical second NBD. (iv) The first and second scaffold sequences form a dimer through interactions between the first and second scaffold sequences (e.g., IgFc), and (v) Each existing connector sequence is selected independently. (See example) Figure 5B (AE in the text.) In this construct, the first and second peptide sequences form a dimer through interactions between first and second scaffold sequences, which can be Ig or non-Ig aa sequences. The scaffold sequences of the first and second peptides can be, for example, a pair of heavy chain constant region aa sequences (CH1-CH2) that dimerize to form an IgFc structure. The scaffold sequences forming the dimer can also be a pair of interspecies Ig sequences, such as KiH or KiHs-s variants of IgG. Alternatively, the scaffold sequence can be a light chain constant region (Cλ or Cκ) aa sequence paired with a heavy chain CH1 region aa sequence to form a dimer.

[0087] b) ATP-dependent immune cell function agonists containing NBD and ICB and / or AD, and also containing TSB.

[0088] Constructs containing NBD and ICB and / or AD may also contain one or more sequences (e.g., TSB) that allow the construct to be targeted to cells or tissues expressing a homologous binding partner expressing the targeting aa sequence. When the target is TAA, TSB allows for direct targeting of tumor cells. Figure 5D The illustrated construct depicts a construct containing NBD, which includes TSBs associated with one or more ICBs and / or one or more ADs. See, for example... Figure 5DStructures BF, H, and I, when replaced as in the building block series 1-2, 1-5, 2-8, 2-2, 2-5, 3-8, 3-2, 3-5, 3-8, 4-2, 4-5, and 4-8, wherein "ICB or TSB" is selected as TSB. For example, in Figure 5E and Figure 5F The document also provides concrete instances of this construct.

[0089] 3. Constructs containing NBD, including TSB and optional scaffolds, exhibit significantly reduced effector function. The NBD-containing constructs described herein may contain one or more NBD aa sequences, one or more TSB aa sequences, and optionally one or more scaffold aa sequences, wherein one or more Ig-mediated effector functions are significantly attenuated (e.g., completely eliminated). At the ATP levels found in the TME, the size and affinity of the TAA bound to the TSA in this construct vary with the dimer (see, for example, AD in Figure 4B) or higher-order complexes (see, for example...). Figure 4A The formation of C in the TME increases. Changes in size and affinity may lead to accumulation in the TME. Such constructs may function by blocking checkpoint proteins such as CTLA-4 (e.g., TSB is based on the variable region of ipilimumab, which antagonizes CTLA-4 function). Alternatively, the construct may block ligand-receptor binding or prevent receptors on tumor cells from performing functional cell signaling (e.g., trastuzumab prevents EGF-dependent tumor cells from responding to EGF by binding to HER2). See Zhao et al. Antibodies (Basel) 10(1): 7 (2021).

[0090] A construct containing one or more NBDs, one or more TSBs, and optionally one or more scaffold aa sequences as elements can have those elements arranged in any order. When "ICB or TSB" is a TSB, Figure 5DThe structural AI describes a series of possible permutations of such constructs when substituted in construct series 1-10 to 1-12, 2-10 to 2-12, 3-10 to 3-12, or 4-10 to 4-12. Some positions where NBD and TSB may be located are indicated by numbered circles and listed in a table below the structure. Constructs are grouped in the table such that the first number of the construct series indicates the location of the H-NBD. Constructs may contain additional NBDs or TSBs, resulting in constructs having two or more NBDs and / or two or more TSBs. Each TSB sequence in any construct is independently selected. For stability, the H-NBD present in the construct, when located at the N-terminal position, may contain disulfide bonds and CAP (not shown). Elements of the construct can be linked by one or more independently selected optional adapter sequences, as indicated by the lines between elements in the figure. For the purposes of the constructs in this figure, TSBs can be monovalent (e.g., scFv), bivalent (e.g., in IgG), or bispecific (for two different TAAs).

[0091] For the purpose of including NBD-containing constructs with TSB, Figure 5D In the table, a "-" usually indicates that a position is not filled (i.e., it is a bond or connector aa sequence). However, it's important to note that positions marked "---" can be filled with one or more ICB or AD sequences (AD), which can be tandem. Additional elements can be included at the N-terminus, C-terminus, or between any two elements, except in the case of tandem ICB / AD placement, where AD sequences are directly connected or separated by aa connector sequences. The scaffold aa sequence is optional and may be absent (structure G); if present, it can be homodimerized or nondimerized (structure A), or heterodimerized (as shown in structures CF, H, and I).

[0092] 4. NBD-containing constructs formed by NBD heterodimerization in the presence of ATP. TSB and ICB / AD can be incorporated into NBD-containing constructs that, in the presence of ATP concentrations (e.g., those seen in TME), can form heterodimers or other higher-order complexes (e.g., trimers) via a pair of homologous binding partners (e.g., TAP1 and TAP2). The formation of heterodimers can be driven by incorporation of a first (1st) heterodimerized NBD into the first member (first subconstruct or first polypeptide) of the construct pair and a second (2nd) heterodimerized NBD into the second member (second subconstruct or second polypeptide) of the construct pair. The 1st and 2nd heterodimerized NBDs act as homologous binding partners. The member pairs of the constructs can be divided into six groups, where each polypeptide or subconstruct (e.g., composed of peptides dimerized through its scaffold) contains the elements listed in Table 1.

[0093] Table 1. Organization of some heterodimeric constructs The first and second NBDs form a heterodimer pair (e.g., the first NBD comes from TAP1 and the second NBD comes from TAP2).

[0094] The scaffolds present in the constructs that constitute ICB and / or AD are generally unable to stimulate Ig-mediated immune cell effector functions, such as ADCC, ADCP, and / or CDC.

[0095] The scaffold present in the constructs constituting the TSB can optionally stimulate Ig-mediated immune cell effector functions such as ADCC, ADCP, and / or CDC, and can be accompanied by one or more substitutions to enhance one or more of these functions.

[0096] When more than one ICB, AD, and / or TSB is present in a heterodimeric construct, they are each selected independently. For constructs with two or more ADs, the active heterodimeric AD (e.g., IL-12, IL-23, IL-27, and IL-35 from the IL-12 superfamily) may be recombined by locating one subunit of the heterodimer in a first polypeptide or subconstruct and locating the second subunit of the heterodimer in a second polypeptide or subconstruct.

[0097] To illustrate the types of NBD-containing construct pairs covered in this disclosure (some of which are summarized in Table 1), Figure 3 A series of non-limiting examples of such NBD-containing construct pairs are listed. In vivo, the dimerized constructs can guide immune cells bound to ICB / AD to interact with target tumor cells recognized by TSB, thereby triggering an immune response against the target tumor cells. This construct allows selective targeting of tumor cells in the TME, as they require elevated ATP levels in the TME to form the complex, thus limiting off-target binding and stimulation of immune cells. Paired constructs containing TSB and ICB can be enhanced by incorporating one or more ADs (which may be the same or different), which may require dimerization through the interaction of the first and second NBD-containing constructs to stimulate target immune cells (e.g., in the case of IL-12 superfamily members).

[0098] Figure 3Entries A and B provide a pair of constructs, wherein the first NBD-containing construct contains (e.g., from the N-terminus to the C-terminus or from the C-terminus to the N-terminus) one or more first heterodimerized NBDs linked to the TSB, and the second NBD-containing construct contains (e.g., from the N-terminus to the C-terminus) a homologous second heterodimerized NBD and an AD. The elements of the constructs are linked by independently selected optional linker sequences. This construct allows AD to target tumor cells in the TME, as they require elevated ATP levels in the TME to heterodimerize, thereby limiting off-target binding and avoiding systemic stimulation of immune cells by AD. Furthermore, mixtures containing more than one construct containing a second heterodimerized NBD and non-identical AD can be used to construct... Figure 3 The construct shown in B, after ATP-mediated complex formation, can present more than one AD to immune cells. In, for example... Figure 3 Using more than one subunit of the second heterodimerized NBD and the heterodimerized AD in the D construct (e.g., in the IL-12 superfamily) can also lead to reconstructed AD activity (e.g., IL-12 activity). Although Figure 3 A or B is not shown, but one or both heterodimerized constructs of A or B may also contain a scaffold aa sequence (see example). Figure 3 Entry E in the text.

[0099] Figure 3 A pair of exemplary constructs of the type shown in entries A or B may include, for example: (i) a first NBD-containing construct containing one or more TAP1 NBDs linked to TSBs (e.g., mesothelial (MSLN), EpCAM, and CTLA-4) with affinity for TAAs; and (ii) a second NBD-containing construct containing TAP2 NBDs, which acts as a homologous binding partner of TAP1 NBDs in the presence of ATP and IL-2 sequences. Such constructs heterodimerize upon exposure to levels of ATP present in the TME and can stimulate T cells present in the TME.

[0100] Figure 3 Entries C and D each provide a pair of heterodimerized constructs, wherein the first NBD-containing construct comprises (e.g., from the N-terminus to the C-terminus) one or more first heterodimerized NBDs linked to TSB, and the second NBD-containing construct comprises (e.g., from the N-terminus to the C-terminus) a homologous second heterodimerized NBD and ICB. In the presence of ATP, the constructs form BiTE-like constructs. The elements of the constructs are linked by independently selected optional linker sequences. Either or both of the first and second constructs of the heterodimerized pair may contain a scaffold sequence (see, for example...). Figure 3(F to I in the text). Constructs containing TSB and / or ICB may also contain AD as shown in entries D, E, H, and I. If an AD is present in a construct and the construct also contains ICB, ADs that need to be presented to T cells in the form of dimers or higher-order complexes to stimulate immune cells can be selected (e.g., TNF family members such as 4-1BBL) to prevent off-target stimulation.

[0101] Figure 3 The pair of constructs shown in entry C includes, for example,: (i) a first NBD-containing construct comprising (a) a TAP1 NBD linked to (b) a TSB; and (ii) a second NBD-containing construct comprising (a) a TAP2 NBD that acts as a homologous binding partner of the TAP1 NBD in the presence of ATP, and (b) a monovalent ICB (e.g., an anti-CD3 antibody fragment or a single-chain antibody construct, such as a nanobody or scFv) that binds to T cells. The TSB can be an antibody, an antibody fragment, or a single-chain antibody construct (such as scFv or nanobody) that has affinity for TAAs (e.g., mesothelial (MSLN), EpCAM, and CTLA-4) expressed on the surface of tumor cells.

[0102] Figure 3 Entries A through I appear as combinations of two molecular constructs. Because the first construct containing TSB in those combinations is separate from the second construct containing AD and / or ICB, it is possible to form groups (mixtures) containing more than one type of first or second construct. For example, it is possible to form Figure 3 A mixture of the first and second constructs described in any of entries A through I, wherein the mixture comprises two or more first constructs, each having a TSB targeting a different TAA. It can form Figure 3 A mixture of the first and second constructs described in either entry A or B, wherein the mixture comprises two or more second constructs, each second construct having one or more ADs targeting different immune cell receptors. Similarly, it is possible to form Figure 3 A mixture of the first and second constructs described in any of the entries C through I, wherein the mixture comprises two or more second constructs, each having a different ICB targeting a different immune cell surface antigen. Except for a population in which only one of the ICB, TSB, or AD is changed, the mixture of the first and second constructs may comprise two or more first and two or more second constructs that are changed in two or more elements selected from ICB, TSB, and AD.

[0103] Figure 3Entries J through M each provide a pair of constructs, wherein the first NBD-containing construct contains (e.g., from the N-terminus to the C-terminus) one or more first heterodimerized NBDs linked to ICB, and the second NBD-containing construct contains (e.g., from the N-terminus to the C-terminus) a homologous second heterodimerized NBD and AD. The elements of the constructs are linked by independently selected optional linker sequences. This construct allows for the activation of immune cells within the TME while avoiding any substantial activation outside the TME, as they require elevated ATP for heterodimerization, thus limiting off-target binding and avoiding systemic stimulation of immune cells by AD. Furthermore, the ADs in the constructs listed in K or M may be the same or different, and may require dimerization to function on target immune cells.

[0104] Figure 3 The exemplary construct pair of heterodimers shown in J and K includes: (i) a first NBD-containing construct comprising one or more TAP1 NBDs linked to an ICB (e.g., an antibody, antibody fragment, or single-chain antibody construct, such as VHH or nanobodies) having affinity for CD28 expressed on the surface of T cells; and (ii) a second NBD-containing construct comprising a TAP2 NBD that acts as a homologous binding partner of the TAP1 NBD in the presence of ATP and IL-2 sequences. When such constructs are synthesized into heterodimers via ATP at levels present in the TME, T cells present in that environment can be stimulated.

[0105] Constructs and compositions comprising mixtures of constructs each containing one or more TSBs (e.g., one or two), or constructs containing two or more TSBs targeting different TAAs, are available for therapeutic applications, wherein using more than one TSB targeting a different TAA (or a different epitope of a TAA) can prevent tumors from evading treatment targeting a single TAA. Similarly, using more than one ICB and / or AD can recruit more than one type of immune cell and provide a stronger immune response to tumor cells, thereby achieving a stronger immune response against the tumor than monotherapy using a single ICB and / or AD.

[0106] 5. Formation of NBD-containing constructs containing two or more NBD molecules and complexes of NBD-containing molecular constructs. The NBD-containing constructs described herein may contain two or more NBDs, enabling these constructs to efficiently polymerize into a complex in the presence of ATP. As described elsewhere, constructs may be arranged such that two NBDs within any given molecule do not interact to form an intramolecular dimer (they are not cis-bound), but rather interact with NBDs on other molecules (trans-bound). Two or more NBDs may be arranged in tandem pairs. When elements such as NBDs are placed in tandem, they are adjacent to each other in the construct, and no element in the construct separates them except for the intermediate linker aa sequence (e.g., in the case of NBDs, there is no intermediate AD, TSB, or ICB between the tandem NBDs). NBDs may also be located within the construct so that they are not placed in tandem. NBD-containing constructs containing two or more NBDs (e.g., in tandem) may also be formed from constructs containing ICB / AD or TSB by adding one or more NBDs.

[0107] A complex containing at least two NBD-containing molecular constructs can be formed by placing the construct in an environment such as a TME, where the ATP level is high enough for ATP to occupy the binding site. Figure 3 Such complexes of N and O in and Figure 4A Examples of the complexes in the examples are provided. Figure 4A In the case shown, more than one NBD aa sequence is incorporated into the construct disclosed herein, which can reversibly polymerize in the presence of ATP to form a complex. The complex formed in the presence of ATP can be a dimer, trimer, tetramer, or even a higher-order structure. See, for example... Figure 4A Incorporating more than one NBD into the construct can maximize potency and efficacy and may affect other properties of the construct. For example, constructs that form a complex in the presence of ATP, where the element expressed on the target cell surface is multivalent (e.g., multivalent TSB or ICB / AD), can exhibit increased effective affinity for the target cell and form a larger complex that can be retained in the TME for a longer period of time, thereby effectively prolonging the in vivo half-life, etc.

[0108] The complex may contain at least two NBDs arranged in tandem, with at most one connector aa sequence (e.g., a rigid connector) separating the NBD aa sequences (see [link]). Figure 4A The NBD consists of structures A, B, and E, and an AD, ICB, and / or TSB. Alternatively, the NBD can be connected to either the TSB or the ICB individually (see [link to NBD documentation]). Figure 4A (Structures C and D). The NBD-containing constructs used to form the complex may contain homodimerized or heterodimerized NBD. Although Figure 4ANot shown in the figure, but by constructing molecules that prevent the formation of effective ATP-binding interfaces between domains, self-association of homodimeric NBDs within the same construct molecule can be prevented. For example, this can be achieved by placing NBDs far apart on the protein (e.g., with ATP-binding sites on opposite faces of the construct), limiting the distance between NBDs on the construct, and / or using rigid peptide linkers between NBDs.

[0109] Constructs containing at least two NBDs and one AD, including NBD-containing constructs. Figure 4A Examples of structures A and B are provided below. Structure A uses NBD to dimerize in the presence of ATP and can form dimers and / or larger constructs, such as... Figure 4A Structure A is shown. Conversely, structure B contains structures that form heterodimers (not shown) or polymers in the presence of ATP. When exposed to the levels of ATP present in the TME, these constructs may form large complexes that present as AD (e.g., as AD homodimers or heterodimers). Such NBD-containing construct complexes can stimulate targeted immune cells in the TME, such as NK cells and / or T cells. The AD present in the complex can be the same (e.g., Figure 4A Structure A) or different (e.g., Figure 4A (Structure B). For example, the aa sequences of the first AD and the second AD present in the complex may be identical, leading to activation of immune cell responses when presented as a dimer or higher-order complex. Alternatively, the first AD and the second AD present in the complex may originate from different molecules (e.g., different interleukins), eliciting immune cell activation when they are present together. The aa sequences of the first AD and the second AD present in the complex may also be different (not identical) and combine together to form the active fragment of a heterodimeric immunomodulator (such as IL-12, IL-23, IL-27, or IL-35), where each immunomodulator consists of two subunits. NBD-containing constructs, each containing a single NBD, can be assembled in the presence of ATP to form a complex containing two different (not identical) ADs. Alternatively, an active AD consisting of two subunits can be assembled in the presence of ATP using two constructs, each containing a single NBD (see example...). Figure 2 (Structures A and B).

[0110] In addition to the aforementioned NBD-containing constructs, this disclosure provides NBD-containing constructs that include at least two NBDs and one such as... Figure 4A The TSB shown in structure C, and / or as Figure 4AThe ICB aa sequence is shown in structure d. This construct optionally includes one or more independently selected scaffold aa sequences and / or one or more independently selected adapter sequences. TSBs include, but are not limited to, antibodies (e.g., scFv and / or nanobodies) that bind to TAAs (e.g., HER-2, mesothelial cell line (MSLN), EpCAM, CTLA-4, etc.). ICBs include molecules, including but not limited to antibodies that bind to immune cell surface antigens (e.g., CD3, CD8, or CD4 on αβ T cells, TRGV9 on δγ T cells, CD16 on NK cells, etc.). Unless otherwise stated, ICBs are monovalent to avoid stimulating immune cells other than the TME, and their sequences are modified to significantly limit or prevent such responses, such as ADCC or CDC.

[0111] Once present in the TME, ATP levels allow NBD domains to form higher-order complexes (e.g., dimers). Constructs containing at least two NBDs and a TSB and / or ICB form large complexes that can lead to immune stimulation. Constructs containing at least two NBDs, an IgFc scaffold, and a TSB can form complexes in the TME (see example...). Figure 4A (structure C), the complex can, for example, lead to ADCC or CDC. Constructs containing at least two NBDs and one ICB can form a complex in the TME (see, for example...). Figure 4A (Structure D) This complex can, for example, lead to stimulation of targeted immune cells due to cross-linking of target antigens (e.g., CD3 on CD8+ T cells or CD16 on NK cells) on its surface.

[0112] An NBD-containing construct that includes at least two NBDs and one TSB (e.g., Figure 4A Structure C) and NBD-containing constructs containing at least two NBDs and one ICB (e.g., Figure 4A Combining structures (D) can produce a complex containing TSB and ICB. A first heterodimerized NBD is linked to TSB, and a second heterodimerized NBD is linked to ICB, ensuring that the complex formed in the presence of ATP contains both ICB and TSB, and can stimulate the immune cell response to target tumor cells. This is illustrated by a mixture of constructs containing at least two NBDs and one TSB and constructs containing at least two NBDs and one ICB, which form large complexes in the TME (see example). Figure 4A The structures in E and F are shown in the diagram (where NBD is tandemly represented). In the presence of sufficient ATP, the complex formed by this structure can bind immune cells to tumor targets via TSB, while simultaneously activating immune cells by cross-linking receptors on the surface of immune cells (e.g., via the monomeric ICB now present in the multivalent complex).

[0113] The TSB and / or ICB aa sequences present in each construct used to form the complex do not need to be the same. Therefore, Figure 3 The complex, with structures A to I, and Figure 4A The complex, structure C to I, may contain constructs having more than one TSB targeting different TAAs (e.g., two or more TSBs) and more than one ICB (when present). Using two or more different TSBs targeting different TAAs in a single complex not only makes the complex formed by constructs consisting of two or more NBDs active against tumors expressing different tumor antigen targets, but also limits tumor evasion through loss of tumor antigen expression. Using different ICBs in a single complex can also stimulate immune responses from more than one type of immune cell (e.g., CD8+ T cells and NK cells). It is also possible to incorporate AD into complexes formed in TMEs, including incorporating AD into complexes containing TSB constructs. For example, this can be achieved by combining one or more constructs containing AD and NBDs (e.g., two or more NBDs) into a complex, such as... Figure 4A As shown in H; or, by using one or more constructs containing AD, NBD (e.g., two or more NBDs) and TSB (e.g., as shown in H); Figure 4A (As shown in I) are combined into a complex to achieve this.

[0114] Because TSB, ICB, 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 different constructs (e.g., before or during administration to a patient). Therefore, a single construct can be used for a variety of different treatment combinations.

[0115] D. Components of a building block containing NBD 1. NBD and its interaction with ATP The NBD incorporated into the constructs described herein can be selected based on several criteria. If the construct is intended for human therapeutic use, the NBD is preferably extracted from human ATP-binding proteins to limit its immunogenicity, and preferably capable of solution-phase dimerization or formation of higher-order complexes upon exposure to sufficient ATP. If an immunogenic non-human protein is used, the immunogenicity can be partially humanized by modifying the immunogenic region to more closely or completely match the human homolog. Solution-phase formation of the dimers or higher-order complexes used in the constructs described herein occurs at ATP concentrations found in the target tissue microenvironment (TME), but not in normal tissues. Therefore, the ATP dissociation constant of the NBD used in the constructs provided herein may, for example, be an order of magnitude (10-fold higher) higher than the upper limit of ATP concentrations found in the extracellular space of normal tissues (i.e., about 1 μM or higher), up to the ATP concentration level found in the TME. In some cases, the affinity of the NBD will be from about 1 μM to about 200 μM. For example, the ATP affinity of NBD can be in the range of about 1 μM to about 5 μM, or in the range of about 5 μM to about 25 μM. The ATP affinity of NBD can be in the range of about 10 μM to about 25 μM, or in the range of about 25 μM to about 200 μM. The ATP affinity of NBD can be in the range of about 25 μM to about 50 μM. In other instances, the affinity of NBD can be in the range of about 50 μM to about 100 μM or about 100 μM to about 200 μM. For example, the ATP dissociation constant of NBD used in the constructs provided herein can be greater than about 5 μM or greater than about 10 μM.

[0116] Some proteins form homodimers in the presence of sufficient ATP, while others form heterodimers. Various aspects of the polypeptide constructs described herein can utilize both homodimerized and heterodimerized NBDs. If homodimerization of the NBD is desired when sufficient ATP is present in solution, the amino acid residues at the dimer interface can be modified to remove or replace amino acid residues that interfere with the formation of the interface between the two NBD molecules. Alternatively, amino acids that contribute to the interaction leading to homodimer formation may be replaced with amino acids that do not contribute to the interaction between the two NBD molecules. The sequences TAP1 and TAP2 are known to form heterodimers and can be used in the heterodimer-forming constructs described herein. Sequences from ABCG5 and ABCG8 are also known to form heterodimers and can be used in the constructs described herein to achieve heterodimer formation. See, for example, Alam et al., Annu. Rev. Biophys. 2023. 52:275–300.

[0117] a) ATP-binding cassette as NBD Many proteins contain NBDs, making them suitable as NBDs for the constructs described herein. Among these proteins, ATP-binding cassette (ABC) transporters are suitable candidates, partly because they are an abundant, highly conserved family of transporters, thus unlikely to be immunogenic. ABC transporters constitute a large superfamily of membrane proteins with multiple functions, utilizing energy from ATP hydrolysis to facilitate substrate movement in and out of the cytoplasm. See, for example, Locher. Philos Trans R Soc Lond B Biol Sci ., 364(1514): 239–245 (2009), and the references cited therein. The human genome is thought to encode 48 or 49 different ABC transporters. See above. ABC transporters are divided into seven distinct families: ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White. Representative members of each family, including TAP1, are described in Figure 6 A comparison was made. The NBDs of the ATP-binding cassette family are largely conserved, consisting of two subdomains. See above. The first is similar to the RecA protein, containing a P-loop (Walker-A motif). The second subdomain, called the "helical subdomain," contains the LSGGQ motif (SEQ ID NO: 10). See above. The NBDs of transport proteins assemble into dimers in a head-to-tail orientation, forming two ATP-binding and hydrolysis sites at their interface (see Figure 5). In this orientation, the LSGGQ motif (SEQ ID NO: 10) in each of the two domains is adjacent to the opposing Walker-A sequence at the ATP-sandwich interface. For example, Loo et al. (2002) J Biol Chem. , 277(44): 41303-41306 and Smith et al. (2002) Mol Cell. , 10(1): 139–149 Figure 3 Such interfaces are described in A. When there is no nucleotide (ATP) at the binding site, a gap appears at the domain interface, making dimer formation difficult. When ATP binds, the NBD dimers and hydrolyzes the bound ATP, after which the dimer dissociates (see, for example, Locher 2009, ibid.). By introducing amino acid substitutions, the NBD domain sequence can be made ATP-defective or significantly ATP-defective (e.g., reducing the ATP hydrolysis rate by one or more orders of magnitude), thereby forming stable dimers (see, for example, Vakkasoglu et al., “D-helix affects dimerization of ATP-binding cassette (ABC) transporters associated with the antigen processing 1 (TAP1) nucleotide-binding domain”, (2107). PloS ONE,12(5): e0178238, available at doi). Org / 10.1371 / journal. Pone.0178238). For example, hydrolyzed Asp or Glu residues found in NBD proteins can be replaced by Asn or Gln residues (see, for example) Figure 3 ).

[0118] When sufficient ATP is present, if it is desired that the NBD of ABC proteins form homodimers in solution, residues at the dimer interface can be modified to remove or replace amino acid residues that interfere with interface formation, or to replace amino acids that do not participate in interface interactions with amino acids that contribute to interface interactions. For example, the NBD of rat TAP1 is known to homodimerize in solution, which can serve as a model for ATP sandwich interface formation, thereby achieving homodimerization in solution in the presence of ATP (see, for example, Vakkasoglu et al. 2017, ibid.). Aligning the NBD aa sequence with sequences known to undergo homodimerization (e.g., rat TAP1 NBD) can be used to guide changes in the target sequence. As mentioned earlier, sequence alignment can be performed using the National Center for Biotechnology Information (NCBI) BLAST program or the Clustal Omega program of the European Institute of Bioinformatics. As stated above, substitutions in the human TAP1 D-helix corresponding to the corresponding D-helix sequence in rat TAP1 enhance homodimerization of the human TAP1 sequence.

[0119] The NBD aa sequence of a protein containing ABC may include all or part of the NBD domain of a phospholipid transport ATPase (ABC or ABCA1 transporter) encoded by the ABCA1 gene, UniProtKB – O95477 (SEQ ID NO: 1). The NBD may contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 1. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 1.

[0120] The NBD aa sequence may contain 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 contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 2. The NBD may contain a sequence having at least about 96% or at least about 98% sequence identity with SEQ ID NO: 2.

[0121] The NBD aa sequence may contain all or part of the NBD domain of the multidrug resistance-associated protein (MRP, multidrug resistance-associated protein 1) encoded by the ABCC1 gene, UniProtKB – P33527 (SEQ ID NO: 3). The NBD may contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 3. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 3.

[0122] The NBD aa sequence may contain all or part of the NBD domain of a member 1 protein (ALD, adrenoleukodystrophy protein) encoded by the ABCD1 gene, UniProtKB / Swiss-Prot: P33897 (SEQ ID NO: 4). The NBD may contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 4. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 4.

[0123] The NBD aa sequence may contain all or part of the NBD domain of the ATP-binding box 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 contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 5. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 5.

[0124] The NBD aa sequence may contain all or part of the NBD domain of a protein (GCN20, TNFα-induced ATP-binding protein) encoded by the ABCF1 gene, UniProtKB – Q8NE71 (SEQ ID NO: 6). The NBD may contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 6. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 6.

[0125] The NBD aa sequence may contain all or part of the NBD domain of the ATP-binding box 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 contain a sequence having at least about 90% or at least about 95% sequence identity with SEQ ID NO: 7. Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 7.

[0126] (1) TAP1 and TAP2 ATP-binding sequences as NBD TAP1 and TAP2 are homologous binding partners that couple ATP binding and its heterodimerization to peptide transport across the intracellular membrane and are members of the ABC transporter superfamily, representing the source of NBD in the constructs provided herein. Rat TAP1 NBD is known to homodimerize in the presence of ATP. Human TAP1 NBD does not homodimerize in the presence of ATP, but homodimerization can be induced by previously defined substitutions. See, for example, Vakkasoglu et al. (2017), ibid. Substitutions leading to homodimerization of human TAP1 in the presence of ATP include one or more (e.g., each) of N676, S677, Q680, E682, and Q683, shown in bold and italics in SEQ ID NO: 8 (as shown below). For example, TAP1 substitutions leading to homodimerization may include one or more (e.g., all) of N676G, S677N, Q680R, E682Q, and Q683R. The portions of the TAP1 and TAP2 proteins used as NBDs (e.g., domains) may also include substitutions of one or more cysteine ​​residues present in the sequence (e.g., substitution with serine residues) to prevent the formation of unwanted disulfide bonds, which could lead to nonfunctional folding or unnecessary intermolecular disulfide bond formation. TAP1 and TAP2 NBDs may also contain His tags, such as at the C-terminus of the domains, for the purpose of purifying constructs containing these NBDs.

[0127] For example, human TAP1 can contain the following aa sequences: The nucleotide binding sequence contains aa 492-748; see, for example, NCBI reference sequence NP_000584.3 or UniProtKB – Q03518 (version 3, i.e., Q03518.3). The catalytic Asp residue at position 668 is shown in bold and italics, as are the homodimerization-related residues N676, S677, Q680, E682, and Q683.

[0128] Therefore, the NBD aa sequence from human TAP1 used for incorporation into the construct described herein may include, for example, an aa sequence having greater than about 90% or greater than about 95% sequence identity with the NCBI reference sequence: NP_000584.3 (aa 492-748) or UniProtKB – Q03518.3 (aa 492-748): Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 9. Any TAP1 NBD sequence used in the constructs of this disclosure may be cysteine-free, wherein C662 and C735 (renumbered aa C171 and C242 in SEQ ID NO: 9) are substituted with amino acids other than cysteine ​​(e.g., C662S and / or C735S substitutions). Residues N676, S677, Q680, E682, and Q683 of SEQ ID NO: 8 associated with homodimerization appear in SEQ ID NO: 9 as N185, S186, Q189, E191, and Q192, and are shown in bold and italics. D177 is also shown in bold and italics. For example, an NBD aa sequence from human TAP1 may contain at least 230 consecutive aa sequences of NBD having greater than about 97% or greater than about 98% sequence identity with the aa sequence of SEQ ID NO:9 provided in NCBI reference sequence NP_000584.2 (aa 492-748).

[0129] Deleting the C-terminal sequence of TAP1 (PADAPE (SEQ ID NO: 11)) can improve the melting point (thermal stability, see TAP1 NBD) of TAP1 NBD. Figure 17 The homodimerized TAP1 polypeptide suitable for the constructs of this disclosure may include the sequence of SEQ ID NO: 12. The homodimerized TAP1 polypeptide suitable for the constructs of this disclosure may include an aa sequence having greater than about 90% or greater than about 95% sequence identity with the NBD aa sequence of NCBI reference sequence NP_000584.3 (aa 492-748) or UniProtKB – Q03518.3 (aa 492-748). Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 12. Residues N676, S677, Q680, E682, and Q683 (see SEQ ID NO: 8) associated with homodimerization appear in SEQ ID NO: 12 as N185, S186, Q189, E191, and Q192, and are shown in bold and italics. For example, an NBD aa sequence from human TAP1 may contain at least 230 consecutive aa sequences of the NBD having greater than 97% or greater than 98% sequence identity with the aa sequence of SEQ ID NO: 12 provided in NCBI reference sequence NP_000584.2 (aa 492-748).

[0130] Homodimeric TAP1 peptides suitable for constructs of this disclosure may comprise the following sequences Alternatively, the NBD may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 291. Residues N676, S677, Q680, E682, and Q683 (see SEQ ID NO: 8) associated with homodimerization appear in SEQ ID NO: 291 as N185, S186, Q189, E191, and Q192, and are shown in bold and italics. For example, an NBD aa sequence derived from human TAP1 may contain an aa sequence that has greater than 97% or greater than 98% sequence identity with at least 230 consecutive aa sequences in the NBD region of the NCBI reference sequence NP_000584.2 (aa 492-748, as shown in SEQ ID NO: 291).

[0131] The NBD aa sequence from human TAP1 used for integration into the construct described herein may include substitutions of amino acid residues corresponding to C662 (e.g., C662S) and C735 (e.g., C735S) of SEQ ID NO: 8 to remove cysteine ​​residues; and substitutions of one or more corresponding amino acids from N676, S677, Q680, E682, and Q683 of SEQ ID NO: 8 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) to convert the D-helix of the human protein to the D-helix of rat TAP1. The NBD aa sequence from human TAP1 may also include substitutions at the position corresponding to D668 of SEQ ID NO: 8 (position D177), which result in an ATP-hydrolysis-deficient NBD (e.g., D668N, D668A, or D668Q substitution). SEQ ID NO: 13 shows the D668N substitution, but for mammalian cell expression, the D668A substitution can also be used. The substitutions in SEQ ID NO: 13 are shown in bold and italics. Therefore, homodimeric TAP1 peptides that can serve as NBD in the constructs of this disclosure may, for example, include the following aa sequence: The homodimerized TAP1 polypeptide that can be used as NBD in the constructs of this disclosure may include an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 13, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 230 or at least about 240 consecutive aa sequences provided in SEQ ID NO: 13, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and / or Q683.

[0132] At position D668 (hereinafter referred to as X177) corresponding to SEQ ID NO:8, there is a homodimerized TAP1 polypeptide substituted to make it ATP hydrolysis-deficient, which is suitable as the NBD in the construct of this disclosure and may, for example, contain the aa sequence of SEQ ID NO:13, wherein PADAPE is deleted: X can be any amino acid other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing an Asn(N)-linked glycosylation site. For example, X can be N, Q, or A, and when expressed in mammalian cells, X can be Q or A. In one embodiment, X is A.

[0133] The homodimerized TAP1 polypeptide that can be used as NBD in the constructs of this disclosure may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO:14, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 230 or at least about 240 consecutive aa sequences provided in SEQ ID NO:14, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and / or Q683.

[0134] At position D668 (hereinafter referred to as X177) corresponding to SEQ ID NO:8, there is a homodimerized TAP1 polypeptide substituted to make it ATP hydrolysis-deficient, which is suitable as the NBD in the construct of this disclosure and may, for example, include the aa sequence of SEQ ID NO:13: X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing an Asn(N)-linked glycosylation site. For example, X can be N, Q, or A; while when expressed in mammalian cells, X can be, for example, Q or A. In one embodiment, X is A.

[0135] The homodimerized TAP1 polypeptide that can be used as NBD in the constructs of this disclosure may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO:292, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 230 or at least about 240 consecutive aa sequences of the sequence shown in SEQ ID NO:292, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682, and / or Q683.

[0136] For example, the NBD aa sequence from human TAP1 may contain substitutions corresponding to C662 (e.g., C662S) and C735 (e.g., C735S) in SEQ ID NO: 8, appearing as s175 and S247. For example, the NBD aa sequence from human TAP1 may also contain substitutions corresponding to one or more amino acids from N676, S677, Q680, E682, and Q683 in SEQ ID NO: 8 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R), which are shown in bold and italics in the sequences below: The substitutions at C662, C735, D668, N676, S677, Q680, E682, and Q683 may be optionally retained. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 250 or at least about 260 consecutive aa sequences of the sequence shown in SEQ ID NO: 15, while the substitutions at C662, C735, D668, N676, S677, Q680, E682, and / or Q683 may be optionally retained.

[0137] Furthermore, the NBD aa sequence derived from human TAP1 may contain a substitution at the Asp residue corresponding to position D668 in SEQ ID NO: 8 (e.g., D668N, D668A, or D668Q substitution), which makes the NBD ATP-defective. Therefore, the NBD polypeptide aa sequence of TAP1 may, for example, contain the aa sequence: X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing an Asn(N)-linked glycosylation site. For example, X can be N, Q, or A, and when expressed in mammalian cells, X can be Q or A.

[0138] The homodimerized TAP1 polypeptide that can be used as NBD in the constructs of this disclosure may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 16, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682 and / or Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 240 or at least about 250 consecutive aa sequences of the sequence shown in SEQ ID NO: 16, while optionally retaining substitutions at C662, C735, D668, N676, S677, Q680, E682 and / or Q683. The C-terminal sequence PADAPE (SEQ ID NO: 11) may be deleted from SEQ ID NO: 16.

[0139] To stabilize the TAP NBD sequence, the coupled helical aa sequence ETEFFQQNQT (aa 2-11, SEQ ID NO: 17) of TAP1, separated by a linker containing the G4S aa sequence (SEQ ID NO: 40), and the coupled helical aa sequence GLQTVRSF (SEQ ID NO: 18) of TAP2, can be incorporated as an N-terminal “CAP” into the TAP aa sequence (e.g., the TAP1 sequence) used as the NBD in the construct disclosed herein. Therefore, the capped homodimerized human TAP1 NBD aa sequence may contain the aa sequence: The sequence contains substitutions at positions C662 (e.g., C662S), C735 (e.g., C735S), N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) in SEQ ID NO: 8, which are shown in bold and italics. Furthermore, the NBD aa sequence from human TAP1 may contain aa(D209) corresponding to D668 in SEQ ID NO: 8, making NBD ATP-defective. X can be any aa sequence other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing an Asn(N)-linked glycosylation site. For example, X can be N, Q, or A, and when expressed in mammalian cells, X can be Q or A. Capped isodimerized TAP1 peptides suitable as NBDs in the constructs disclosed herein may include an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 19 or SEQ ID NO: 20, while optionally retaining the CAP sequence and / or substitutions at C662, C735, D668, N676, S677, Q680, E682, and / or Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 230 or at least about 240 consecutive aa sequences of the sequence provided in SEQ ID NO: 19 or SEQ ID NO: 20, while optionally retaining the CAP sequence and / or substitutions located at C662, C735, D668, N676, S677, Q680, E682 and / or Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 270 or at least about 280 consecutive aa sequences of the sequence provided in SEQ ID NO: 19 or SEQ ID NO: 20.

[0140] Capped homodimerized human TAP1 NBD aa sequences may contain aa sequences: The sequence contains substitutions at positions C662 (e.g., C662S), C735 (e.g., C735S), N676, S677, Q680, E682, and Q683 (e.g., N676G, S677N, Q680R, E682Q, and / or Q683R) in SEQ ID NO: 8, which are shown in bold and italics. Furthermore, the NBD aa sequence from human TAP1 may contain aa(D209) corresponding to D668 in SEQ ID NO: 8, making NBD ATP-defective. X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing an Asn(N)-linked glycosylation site. For example, X can be N, Q, or A, and when expressed in mammalian cells, X can be Q or A.

[0141] In the constructs disclosed herein, the capped homodimerized TAP1 polypeptide suitable as NBD may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 293 or SEQ ID NO: 294, while optionally retaining the CAP sequence and / or substitutions at C662, C735, D668, N676, S677, Q680, E682 and / or Q683. Alternatively, the NBD aa sequence may have greater than 97% or greater than 98% sequence identity with at least about 240 consecutive aa sequences provided in SEQ ID NO: 293 or SEQ ID NO: 294, while optionally retaining the CAP sequence and / or substitutions at C662, C735, D668, N676, S677, Q680, E682 and / or Q683. Alternatively, the NBD aa sequence has a sequence identity of more than 97% or more than 98% with at least about 270 or at least about 280 consecutive aa sequences provided in SEQ ID NO: 293 or SEQ ID NO: 294.

[0142] In any construct covered by this disclosure, if a TAP1 NBD (e.g., containing any of the aa sequences in SEQ ID NO: 9, 12-16, 19, or 20) is present, the Asp (D) residue corresponding to D668 may be substituted to make the TAP1 polypeptide hydrolyzed. The position corresponding to D668 can be any aa other than D to make the sequence ATP hydrolysis-deficient. The position corresponding to D668 can be any aa other than D or N to make the sequence ATP hydrolysis-deficient and avoid introducing an Asn (N)-linked glycosylation site. The position corresponding to D668 can be N, Q, or A, for example, Q or A when expressed in mammalian cells. In one embodiment, the position corresponding to D668 can be A (Ala). Furthermore, to facilitate expression in mammalian cells, S542 (see SEQ ID NO: 8) or any other corresponding position in the TAP1 aa sequence (e.g., S51 in SEQ ID NO: 9 and 12) can be substituted with an amino acid other than serine or threonine, thereby removing degradation-related sites without introducing glycosylation sites. For example, S54 can be substituted with alanine to obtain S54A substitution.

[0143] Additional substitutions that enhance ATP binding or dimer stability at the interfaces between TAP monomers (i.e., dimer interfaces) may be incorporated into the NBD of the protein constructs described herein (including constructs containing NBDs of SEQ ID NO: 9, 12-16, 19, 20, and 291-294). Such substitutions in TAP1 include, but are not limited to, the Q701H substitution described by Proko et al. in Molecular Cell 24:51–62 (2006) and / or the E564R substitution described by Proko and Gaudet in Biochemistry 47(21):5699-708 (2008). Thus, the NBD present in the constructs of this disclosure may contain the TAP1 aa sequence with C662S, C735S, N676G, S677N, Q680R, E682Q, Q683R, and D668N substitutions and optionally the Q701H substitution.

[0144] As described below, intrachain disulfide bonds can be incorporated into the TAP1 NBD sequence to improve stability (e.g., thermal stability as assessed by melting point). Stable disulfide bonds include those formed by substitution of cysteine ​​residues at sites L493 and E573 of the TAP1 sequence, resulting in an L493C:E573C disulfide bond in SEQ ID NO: 8, or at the corresponding positions in constructs containing NBDs of SEQ ID NO: 9, 12-16, 19, and 20. Other stable disulfide bonds that can be incorporated into any of these NBD sequences include, but are not limited to: i) a disulfide bond formed between a cysteine ​​residue at C735 and a cysteine ​​residue substituted for E722 (C735:E722C disulfide bond); and ii) a disulfide bond formed between a cysteine ​​residue at C662 and a cysteine ​​residue substituted for R694 (C662:R694C disulfide bond). The NBD used in the constructs disclosed herein can be incorporated with substituents at the interface between the TAP monomer and the stable disulfide bond.

[0145] Some NBDs in the TAP protein do not homodimerize but instead heterodimerize with their homologous NBDs. These NBDs are used to prepare the construct pairs disclosed herein (see, for example...). Figure 2 B and D in the middle and Figure 3 ) or construct complex (see, for example) Figure 4AFor example, a pair of constructs containing human TAP1 NBD and human TAP2 NBD can be prepared. In the presence of sufficient ATP, the pair of constructs consisting of TAP1 and TAP2 NBDs forms a heterodimer through the interaction of TAP1 and TAP2. Using NBDs capable of heterodimerization, such as TAP1 and TAP2, different specific combinations of activating sequences can be combined for immune cell stimulation. For example, a construct containing IL-12A (p35), an optional linker aa sequence, and TAP1 can heterodimerize with a construct containing IL-12B (p40), an optional linker aa sequence, and TAP2 in the presence of sufficient ATP to form a pair of constructs exhibiting an active IL-12AB heterodimer. If desired, such constructs may also contain a scaffold aa sequence, such as an IgFc aa sequence.

[0146] Figure 7 The sequence of human TAP2 is given. In some cases, the NBD of the construct disclosed herein may contain an aa sequence from the NBD of human TAP2. For example, the NBD may contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with the NBD aa sequence of UniProtKB – Q03519 (TAP2_human): For example, the NBD of the construct disclosed herein may contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with the NBD of the sequence.

[0147] For example, the NBD in the construct described herein may include an aa sequence that has greater than about 90% or greater than about 95% sequence identity with the human TAP2 aa sequence: NBD may also contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 22.

[0148] The removal of the catalytic acidic amino acid residue corresponding to the D668N substitution in TAP1 results in a TAP2 NBD ATP hydrolysis defect, and the substitution can be incorporated into the TAP2 NBD aa sequence (e.g., E176N, E176A, or E176Q substitution).

[0149] (2) Modified TAP1 and TAP2 sequences as NBD The NBDs of TAP1 and / or TAP2 dimerize at ATP levels found in the TME. Developing therapeutics that leverage the benefits of dimerization can be achieved by enhancing dimerization in the presence of ATP (rather than ADP and / or other nucleotides) and / or by modifications that produce stable proteins (e.g., modifications that enhance the thermal stability of each NBD and / or NBD dimer). Such modifications may include, for example, the deletion, insertion, and / or substitution of amino acids, as well as the formation of disulfide bonds. To evaluate the effects of various modifications, a construct (construct 103) was prepared comprising, in the N-terminal to C-terminal direction: a CAP (specifically, a coupling helix from TAP1, a coupling helix from TAP2, see, for example, SEQ ID NO: 17 and 18), TAP1NBD, resulting in an ATP hydrolysis defect by catalytically substituting Asp at position 668 for Asn (D668N), and a histidine tag for purification. The overall structure of construct 103 (coupled helix 1-coupled helix 2-TAP1 NBD (D-ring modification without cysteine, D668N)-HIS) can be referenced. Figure 14A Let's understand it using B in the context. The sequence of constructor 103 is as follows: Figure 14B As shown.

[0150] The deletion of the C-terminus in the NBD of construct 103 indicates that deletion of up to 6 aa (up to position 742 of SEQ ID NO: 8) raises the melting point above 50°C, but further deletion leads to a decrease in the melting point. Figure 17 As shown, this can be seen by comparing the melting temperature I of construct 103 with that of constructs 175-179 and 211. These constructs include a C-terminal deletion of NBD. Construct 179 contains a deletion approximately up to the N-terminus of α-helix 9 (a9, CYWAMVQ (SEQ ID NO: 23)), and construct 211 contains a deletion approximately up to the N-terminus of α-helix 8 (a8, THQQLME (SEQ ID NO: 24)). Therefore, construct 179 substantially lacks the a9 helix to the NBD C-terminus, while construct 211 substantially lacks the a8 helix (including the a9 helix) to the NBD C-terminus.

[0151] The Tm value can be increased by introducing cysteine ​​residues (L493C:E573C disulfide bond) at the L493 and E573 positions to form a disulfide bond, such as... Figure 17The comparison of Tm measurements for constructs 103 and 182 is shown. Disulfide bonds formed at other locations may also stabilize the NBD of TAP1. For example, disulfide bonds may form between C735:E722C or C662:R694C in TAP1. Although these disulfide bonds do not cause substantial changes in Tm, they may stabilize or alter protein properties (e.g., reduce sensitivity to proteolysis or resistance to thermal denaturation, such as irreversible denaturation leading to precipitation or aggregation). Similar to the L493C:E573C disulfide bond discussed above, disulfide bonds formed between cysteine ​​residues (C662 or C735) present in the TAP1 sequence and cysteine ​​residues substituted by R721C or Q580C in the TAP1 sequence may result in a significant increase in Tm (e.g., relative to construct 103). For example, the C735:R721C disulfide bond in construct 272 and the C662:Q580C disulfide bond in construct 289 significantly increased the Tm of NBD to above 50 °C.

[0152] Substitutions that enhance the homodimerization ability of the TAP1 sequence include substitutions in the D-helix of the TAP1 NBD, including substitutions at the N676, S677, Q680, E682, and / or Q683 sites (see SEQ ID NO: 8). Substitutions at N676G, S677N, Q680R, E682Q, and Q683R in the human TAP1 D-helix (NSQLQVEQLL, SEQ ID NO: 25) convert the human D-helix sequence into the sequence in rat TAP1 (GNQLRVQRLL, SEQ ID NO: 26), which homodimerizes in the presence of sufficient ATP.

[0153] It has been found that introducing E587R of human TAP1 (see SEQ ID NO: 8) in the presence of ADP reduces nonspecific dimerization. Constructs with the TAP1 E587R substitution (or, for example, the corresponding substitution E552R in TAP2) exhibit more ATP-specific dimerization compared to TAP1 NBD constructs lacking this substitution.

[0154] In addition to the modifications described above, N-terminal CAP aa sequences, N-terminal AD sequences, or N-terminal targeting sequences can be added to NBD for various purposes. While N-terminal CAP addition is not necessary for the thermal stability of NBD, it can enhance its thermal stability compared to constructs with the "uncapped" natural TAP1 NBD sequence. It should be noted that construct 451, similar to construct 182, includes several stable substitutions but lacks CAP, exhibiting the highest single Tm (56.3 °C) among any NBD constructs identified in this paper. Figure 14B and 15 It provides the sequence and structure of the constructs.

[0155] For example, placing a CAP sequence or other sequences (AD and / or targeting sequences) at the N-terminus of an NBD sequence can produce higher expression levels than constructs with an "uncapped" N-terminus TAP1 NBD. The CAP structure can take various forms, including but not limited to aa sequences that form the TAP1 and TAP2 coupling helices and Ig Fc sequences, which can also function as scaffold sequences.

[0156] Since the coupling helixes of TAP1 and TAP2 are known to interact, integrating them into the bispecific ATP-dependent agonist disclosed herein as part of a CAP structure can provide a self-assembling structure that stabilizes adjacent portions of the molecule (e.g., the NBD of TAP1). In one embodiment, the CAP structure located at the N-terminus of the TAP NBD (e.g., the TAP1 NBD) comprises, in the N-to-C-terminal direction: (i) a TAP1 coupling helix, (ii) an optional independently selected CAP internal linker, (iii) a TAP2 coupling helix, and (iv) an optional independently selected linker (CAP-NBD linker) between the TAP2 coupling helix and the TAP NBD aa sequence. More specifically, the CAP may comprise: (i) a TAP1 coupling helix comprising SEQ ID NO: 17, an optional independently selected CAP internal linker, a TAP2 coupling helix comprising SEQ ID NO: 18, and an optional independently selected CAP-NBD linker.

[0157] The internal linker between coupling helix 1 and coupling helix 2 is optional. When the internal linker is present, its length can be, for example, from 1 aa to about 10 aa, or from 1 aa to 5 aa. Alternatively, the length of the internal linker may be from 6 aa to about 10 aa. The internal linker may contain, for example, a single aa, such as a G or A residue. The internal linker may contain a GG, GGG, or GGGS (SEQ ID NO: 30) aa sequence. The internal linker may contain one or two G4S (SEQ ID NO: 40-41) aa sequences.

[0158] The CAP-NBD linker between coupling helix 2 and NBD is optional. When the CAP-NBD linker is absent, coupling helix 2 fuses directly to the N-terminus of the NBD aa sequence. Constructs without the CAP-NBD linker may produce fewer degradation (proteolytic) products when expressed in *E. coli* compared to constructs with the CAP-NBD linker. When the CAP-NBD linker is present, its length may be from 1 aa to approximately 10 aa, or from 1 aa to 5 aa. The CAP-NBD linker may contain, for example, a single aa, such as a G or A residue. The CAP-NBD linker may contain a GG, GGG, or GGGS (SEQ ID NO: 30) aa sequence. The CAP-NBD linker may contain one or two G4S (SEQ ID NO: 40-41) aa sequences.

[0159] Both CAP internal joints and CAP-NBD joints can be rigid joints, for example, in polymers composed of Ala and Pro residues, such as polymers composed of repeating Ala-Pro sequences.

[0160] (3) CFTR ATP binding cassette as NBD In some cases, the NBD of the present disclosure construct may include an aa sequence from the NBD of human cystic fibrosis transmembrane conduction regulator (CFTR), or an aa sequence having greater than about 90% or greater than about 95% sequence identity with human CFTR. For example, the NBD may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with the NBD aa sequence GenPep Accession 6MSM_A.

[0161] For example, an NBD may contain the aa sequence of a human CFTR aa sequence. The NBD may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 28. Alternatively, the NBD may, for example, contain an aa sequence having greater than 97% or greater than 99% sequence identity with SEQ ID NO: 28. The NBD may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 28. Any CFTR NBD sequence embodiment may contain substitutions that cause hydrolytic defects.

[0162] For example, NBD may contain an aa sequence that has 80% or more sequence identity with the human CFTR aa sequence Ser-NBD1 [387–646(D405–436)], as described by Atwell et al. (2010) in Protein Engineering, Design & Selection, 23(5) 375–384.

[0163] The X at position 2 may be absent or be Leu; the X at position 229 may be absent or be G. Alternatively, the NBD may, for example, contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 29. The NBD may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 29. The NBD may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with aa 1-216 of SEQ ID NO: 29, wherein the X at position 2 may be absent or be Leu.

[0164] (4) ABCG5 and ABCG8 ATP-binding sequences serve as NBD ABCG5 and ABCG8 are homologous binding partners that couple ATP binding and its heterodimerization with sterol transmembrane transport and represent the source of NBD used in the constructs provided herein. Because ABCG5 and ABCG8 are capable of heterodimerization, their NBDs can be used to prepare heterodimeric constructs like TAP1 and TAP2.

[0165] ABCG5 has an aa sequence: MGDLSSLTPGGSMGLQVNRGSQSSLEGAPATAPEPHSLGILHASYSVSHRVRPWWDITSCRQQWTRQILKDVSLYVESGQIMCILGSSGSGKTTLLDAMSGRLGRAGTFLGEVYVNGRALRREQFQDCFSYVLQSDTLLSSLTVRETLHYTALLAIRRGNPGSFQKKVEAVMAELSLSHVADRLIGNYSLGGISTGERRRVSIAAQLLQDPKVMLFDEPTTGLDCMTANQIVVLLVELARRNRIVVLTIHQPRSELFQLFDKIAILSFGELIFCGTPAEMLDFFNDCGYPCPEHSNPFDFYMDLTSVDTQSKEREIETSKRVQMIESAYKKSAICHKTLKNIERMKHLKTLPMVPFKTKDSPGVFSKLGVLLRRVTRNLVRNKLAVITRLLQNLIMGLFLLFFVLRVRSNVLKGAIQDRVGLLYQFVGATPYTGMLNAVNLFPVLRAVSDQESQDGLYQKWQMMLAYALHVLPFSVVATMIFSSVCYWTLGLHPEVARFGYFSAALLAPHLIGEFLTLVLLGIVQNPNIVNSVVALLSIAGVLVGSGFLRNIQEMPIPFKIISYFTFQKYCSEILVVNEFYGLNFTCGSSNVSVTTNPMCAFTQGIQFIEKTCPGATSRFTMNFLILYSFIPALVILGIVVFKIRDHLISR; SEQ ID NO: 285 Uniprot entry Q9H222-1 (canonical sequence).

[0166] The NBD aa sequence from ABCG5 can form a heterodimer with the ABCG8 NBD sequence and can be used to prepare a construct that forms a heterodimer in the presence of ATP, with the following sequence: RPWWDITSCR QQWTRQILKD VSLYVESGQIMCILGSSGSG KTTLLDAMSG RLGRAGTFLG EVYVNGRALR REQFQDCFSY VLQSDTLLSS LTVRETLHYTALLAIRRGNP GSFQKKVEAV MAELSLSHVA DRLIGNYSLG GISTGERRRV SIAAQLLQDP KVMLFDEPTTGLDCMTANQI VVLLVELARR NRIVVLTIHQ PRSELFQLFD KIAILSFGEL IFCGTPAEML DFFNDCGYPCPE (SEQ ID NO: 286). Alternatively, the ABCG5 NBD aa sequence that can be used in the construct of this disclosure has at least about 96% or at least about 98% sequence identity with SEQ ID NO: 286. For example, an NBD aa sequence from human ABCG5 may contain at least 230 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence shown in SEQ ID NO:286.

[0167] The hydrolysis defect ABCG5 NBD corresponding to SEQ ID NO:286 may contain a sequence that can be used to prepare the construct disclosed herein: RPWWDITSCR QQWTRQILKD VSLYVESGQI MCILGSSGSG KTTLLDAMSG RLGRAGTFLGEVYVNGRALR REQFQDCFSY VLQSDTLLSS LTVRETLHYT ALLAIRRGNP GSFQKKVEAV MAELSLSHVADRLIGNYSLG GISTGERRRV SIAAQLLQDP KVMLFDXPTT GLDCMTANQI VVLLVELARR NRIVVLTIHQPRSELFQLFD KIAILSFGEL IFCGTPAEML DFFNDCGYPC PE (SEQ ID NO:295), where X can be any aa other than E or D to make the sequence ATP hydrolysis defective, or N can be excluded to avoid introducing Asn(N) linked glycosylation sites.

[0168] Alternatively, the ABCG5 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 295. For example, the NBD aa sequence from human ABCG5 may comprise at least 230 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence listed in SEQ ID NO: 295.

[0169] The NBD aa sequence from ABCG5 can heterodimerize with the ABCG8 NBD sequence and can be used to prepare heterodimerized constructs in the presence of ATP. It may also contain the following sequences: gssgs gkttlldams grlgragtfl gevyvngralrreqfqdcfs yvlqsdtllssltvretlhy tallairrgn pgsfqkkvea vmaelslshv adrlignysl ggistgerrr vsiaaqllqdpkvmlfdept tgldcmtanq ivvllvelarrnrivvltih (SEQ ID NO: 287).

[0170] Alternatively, the ABCG5 NBD aa sequence that may be used in the construct of this disclosure has at least about 96% or at least about 98% sequence identity with the sequence of SEQ ID NO: 287. For example, the NBD aa sequence from human ABCG5 may comprise at least 150 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence listed in SEQ ID NO: 287.

[0171] The hydrolysis defect ABCG5 NBD corresponding to SEQ ID NO: 287 may contain the following sequences: gssgs gkttlldams grlgragtfl gevyvngral rreqfqdcfs yvlqsdtllssltvretlhy tallairrgn pgsfqkkvea vmaelslshv adrlignysl ggistgerrr vsiaaqllqdpkvmlfdxpt tgldcmtanq ivvllvelar rnrivvltih (SEQ ID NO: 296), where X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing Asn(N)-linked glycosylation sites.

[0172] Alternatively, the ABCG5 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 296. For example, the NBD aa sequence from human ABCG5 may comprise at least 150 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence listed in SEQ ID NO: 296.

[0173] ABCG8 has an aa sequence: ;(SEQ ID NO: 288) Uniprot entry Q9H221-1 (canonical sequence).

[0174] The NBD aa sequence from ABCG8 can form a heterodimer with the NBD sequence of ABCG5 and can be used to prepare a construct that forms a heterodimer in the presence of ATP. The sequence may be: LEVRDLNYQV DLASQVPWFE QLAQFKMPWTSPSCQNSCEL GIQNLSFKVR SGQMLAIIGS SGCGRASLLD VITGRGHGGK IKSGQIWING QPSSPQLVRKCVAHVRQHNQ LLPNLTVRET LAFIAQMRLP RTFSQAQRDK RVEDVIAELR LRQCADTRVG NMYVRGLSGGERRRVSIGVQ LLWNPGILIL DEPTSGLDSF TAHNLVKTLS RLAKGNRLVL ISLHQPRSDI FRLFDLVLLMTSGTPIYLGA AQHMVQYFTA IGYPCPR; (SEQ ID NO: 289).

[0175] Alternatively, the ABCG8 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 289. For example, the NBD aa sequence from human ABCG8 may comprise at least 230 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence shown in SEQ ID NO: 289.

[0176] The hydrolysis defect ABCG8 NBD corresponding to SEQ ID NO: 289 may contain the following sequences: LEVRDLNYQV DLASQVPWFE QLAQFKMPWT SPSCQNSCEL GIQNLSFKVR SGQMLAIIGSSGCGRASLLD VITGRGHGGK IKSGQIWING QPSSPQLVRK CVAHVRQHNQ LLPNLTVRET LAFIAQMRLPRTFSQAQRDK RVEDVIAELR LRQCADTRVG NMYVRGLSGG ERRRVSIGVQ LLWNPGILIL DXPTSGLDSFTAHNLVKTLS RLAKGNRLVL ISLHQPRSDI FRLFDLVLLM TSGTPIYLGA AQHMVQYFTA IGYPCPR;(SEQ ID NO: 297); where X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing Asn(N)-linked glycosylation sites.

[0177] Alternatively, the ABCG8 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 297. For example, the NBD aa sequence from human ABCG8 may comprise at least 230 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence shown in SEQ ID NO: 297.

[0178] The NBD aa sequence from ABCG8 can heterodimerize with the ABCG5 NBD sequence and can be used to prepare heterodimerized constructs in the presence of ATP. It may also contain the following sequences: laiigssgcg raslldvitg rghggkiksg qiwingqpss pqlvrkcvah vrqhnqllpnltvretlafi aqmrlprtfs qaqrdkrved viaelrlrqc adtrvgnmyv rglsggerrr vsigvqllwnpgilildept sgldsftahn lvktlsrlak gnrlvlislh qprsdifrlf dlvllmtsgt piylgaaqhm (SEQ ID NO: 290).

[0179] Alternatively, the ABCG8 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 290. For example, the NBD aa sequence from human ABCG8 may comprise at least 190 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence shown in SEQ ID NO: 290.

[0180] The hydrolysis defect ABCG8 NBD corresponding to SEQ ID NO: 290 may contain the following sequences: laiigssgcg raslldvitg rghggkiksg qiwingqpss pqlvrkcvah vrqhnqllpnltvretlafi aqmrlprtfs qaqrdkrved viaelrlrqc adtrvgnmyv rglsggerrr vsigvqllwnpgilildxpt sgldsftahn lvktlsrlak gnrlvlislh qprsdifrlf dlvllmtsgt piylgaaqhm(SEQ ID NO:298); where X can be any aa other than E or D to make the sequence ATP-defective, or N can be excluded to avoid introducing Asn(N)-linked glycosylation sites.

[0181] Alternatively, the ABCG8 NBD aa sequence used in the constructs of this disclosure may have at least about 96% or at least about 98% sequence identity with SEQ ID NO: 298. For example, the NBD aa sequence from human ABCG8 may comprise at least 190 consecutive aa sequences having greater than 97% or greater than 98% sequence identity with the NBD aa sequence shown in SEQ ID NO: 298.

[0182] (5) Substitution in the NBD sequence and ATP-mediated dimer formation Substitutions that affect ATP-mediated dimerization or the formation of higher-order complexes may alter the concentration of homodimers or heterodimers of the peptides constituting NBD, which is reflected in the dimerized EC50. Substitutions affecting ATP-mediated dimerized EC50 can be incorporated into the NBD domains described herein.

[0183] It has been demonstrated that substitutions at the S542 (e.g., S542A), Q586 (e.g., Q586A), C662 (e.g., C662S), D667 (e.g., D667A), and C735 (e.g., C735S) positions alter the EC50 of ATP-induced dimerization or higher-order complexes formed from TAP1 NBD constructs. Furthermore, disulfide bonds formed between substituted cysteine ​​residues at L493 and E573 (e.g., L493C:E573C in construct 571) also affect EC50 alterations. See SEQ ID NO: 8, Example 7, comprising constructs 571, 647, and 648, and... Figure 21Although the amino acid positions provided are based on the TAP1 sequence provided in SEQ ID NO: 8, the corresponding positions in other TAP1 sequences (e.g., SEQ ID NO: 9, 12-14, 291, and 292), TAP2 sequences, and other ATP-binding cassette (ABC) transporters can be obtained by comparison with SEQ ID NO: 8 (see, for example...). Figure 6 The corresponding positions in TAP2 are S507, Q551, R626, and D631. There is no aa in TAP2 that directly corresponds to C735 in TAP1. The cysteine-substituted aa (L493C:E573C) in TAP1, which is linked by a disulfide bond, corresponds to T458 and E538 in TAP2, where cysteine ​​may be substituted and form a disulfide bond.

[0184] Based on the foregoing, the NBDs of TAP1 and / or TAP2 may contain substitutions at one or more of the aforementioned positions, resulting in alterations to the EC50 value of ATP-mediated dimerization or the formation of higher-order complexes. They may also contain one or more other substitutions (e.g., substitutions that render the NBD ATP-degradable). The NBDs of other ABC transporters may similarly contain conserved or non-conserved substitutions at corresponding positions in the sequence, as well as one or more other substitutions. The NBD of TAP1 may contain a substitution at D667 (e.g., D667A); the NBDs of TAP2 and other ABC transporters may contain substitutions at the corresponding amino acid position (e.g., alanine) (see, for example, construct 647). The NBD of TAP1 may contain a substitution at Q586 (e.g., Q586A); while the NBDs of TAP2 and other ABC transporters may contain substitutions at the corresponding amino acid site (e.g., alanine) (see, for example, construct 648). The NBD of TAP1 may contain substitutions of L493C and E573C to form disulfide bonds; and substitutions of S542 (e.g., S542A), C662 (e.g., C662S), and C735 (e.g., C735S); the NBDs of TAP2 and other ABC transporters may contain substitutions at the corresponding amino acid positions (see, for example, construct 571).

[0185] TAP1, TAP2, and other ABC transporters may contain substitutions of the Q-ring amino acids that affect ATP-mediated dimerization or the EC50 of higher-order ABC transporter complexes. For example, TAP1 may contain substitutions of Q586 and / or E587, while TAP2 may contain substitutions of Q551 and / or E552. The NBD aa sequences of TAP1 and TAP2 may contain non-conserved substitutions of the magnesium-binding amino acid Q586 in TAP1 or Q551 in TAP2, such as substitutions to alanine or other non-electrified amino acids (e.g., glycine, leucine, or isoleucine). Corresponding substitutions may be made in the NBD aa sequences of other ABC transporters. Any substitution can be conserved or non-conserved, and other substitutions in the NBD aa sequences are not excluded.

[0186] TAP1, TAP2, and other ABC transporters may contain substitutions in EC50 that can affect ATP-mediated dimerization or the formation of higher-order complexes in the Walker region aa. For example, TAP1 may contain substitutions of one or more of N540, G541, S542, G543, K544, S545, and / or T546, while TAP2 may contain substitutions of one or more of N505, G506, S507, G508, K509, S510 (Mg), and / or T511. The TAP1 and / or TAP2 NBD aa sequences may contain substitutions of the magnesium-binding aa S545 in TAP1 or S510 in TAP2, for example, with alanine or another uncharged aa (e.g., Gly, Leu, or Ile). Corresponding substitutions may be made in the NBD aa sequences of other ABC transporters. Any substitution can be conservative or non-conservative, and other substitutions in the NBD aa sequence are not excluded.

[0187] TAP1, TAP2, and other ABC transporters may contain substitutions in the EC50 that can affect the dimerization of ATP-mediated ABC transporters outside the Walker region aa or the formation of higher-order complexes. For example, TAP1 may contain substitutions of one or more of Q586 and D668, while TAP2 may contain substitutions of one or more of Q551 and E632, each of which interacts with water and / or magnesium in the nucleotide-binding pocket. The TAP1 and / or TAP2 NBD aa sequences may contain these amino acid substitutions, for example, with alanine or other uncharged amino acids (e.g., glycine, leucine, or isoleucine). Corresponding substitutions may be made in the NBD aa sequences of other ABC transporters. Any substitution may be conserved or non-conserved, and other substitutions in the NBD aa sequences are not excluded.

[0188] TAP1, TAP2, and other ABC transporters may contain substitutions of adenine or ribose amino acids that bind to ATP, which can affect the EC50 of ATP-mediated dimerization of ABC transporters or the formation of higher-order complexes. For example, TAP1 may have substitutions at one or more sites in Y412 (binding to adenine) and V520 (binding to ribose), while TAP2 may have substitutions at one or more sites in Y477 (binding to adenine) and V585 (binding to ribose). The TAP1 and / or TAP2 NBD aa sequences may contain these amino acid substitutions, such as substitution with alanine or other uncharged amino acids (e.g., glycine, leucine, or isoleucine). Corresponding substitutions may be made in the NBD aa sequences of other ABC transporters. Any substitution can be conserved or non-conserved, and other substitutions in the NBD aa sequences are not excluded.

[0189] 2. Connector The constructs of this disclosure optionally have independently selected linkers located between any two elements. To improve stability and prevent protein hydrolysis, independently selected linker sequences may also be placed at the ends of the aa sequences of the constructs of this disclosure. The linkers can be located between any two of the NBD aa sequence, the scaffold aa sequence, and the AD. The linkers typically consist of aa sequences of 4 to 50 amino acids in length (e.g., 4 to 25 amino acids or 25 to 50 amino acids) and can be selected based on their rigidity, etc. Each linker is independently selected.

[0190] In some cases, the linker is a flexible amino acid (AA) sequence composed of glycine, serine, and / or alanine residues. In other cases, the linker is an AA sequence composed of glycine and serine. For example, the sequence GGGS (SEQ ID NO: 30) or the sequence GGGGS (also represented as G4S or G4S, SEQ ID NO: 40) may appear or be repeated 1-10 times in the linker, respectively (GGGS). 1-10 (SEQ ID NO: 30-39) or (GGGGS) 1-10 (SEQ ID NO: 40-49). In some cases where the connector contains GGGS or GGGGS sequences, these sequences may appear 1-5 times or 5-10 times.

[0191] Rigid linkers can be used when it is necessary to maintain a substantially fixed distance or spatial spacing between domains to reduce or substantially eliminate unfavorable interactions between construct domains (e.g., between NBDs within the same construct). For example, rigid linkers can be used to prevent self-association of a pair of NBDs present in a single molecule. Exemplary rigid peptide or polypeptide linkers include linkers consisting of the sequence EAAAK (SEQ ID NO: 50, 198-216), which 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). In some cases, the EAAAK sequence may be repeated 1-5 times or 5-10 times. In some cases, the EAAAK sequence may be repeated 11-15 times or 16-20 times. Some rigid linkers may contain more than 30% or more than 40% proline residues (on a molar basis). Other rigid linkers may contain 30% to 40% or 40% to 60% proline residues (calculated in molar ratio). Rigid peptide linkers may include, but are not limited to, linkers of length 4 to 40 (e.g., 4-20 or 20-40) ge aa, which consist of the following repeating sequences: (Lys-Pro) (SEQ ID NO: 51, generally SEQ ID NO: 217-235 according to length), (Glu-Pro) (SEQ ID NO: 52, generally SEQ ID NO: 236-254 according to length), and (Thr-Pro-Arg) (SEQ ID NO: 53, generally SEQ ID NO: 255-274 according to length). Rigid peptide linkers may comprise Ala-Pro repeat sequences of 4 to 40 amino acids in length (generally SEQ ID NO: 54, specifically SEQ ID NO: 55, 56, 57, 58 and 275-284 according to length), including but not limited to 4 amino acids (AP2) (SEQ ID NO: 55), 6 amino acids (AP3) (SEQ ID NO: 56), 8 amino acids (AP4) (SEQ ID NO: 57) or 10 amino acids (AP5) (SEQ ID NO: 58). Other rigid linkers include linkers consisting of dipeptide sequences KP or EP repeated 2 to 10 times or 10 to 20 times (SEQ ID NO: 51 is the general case according to the number of repetitions, specifically SEQ ID NO: 217-235; SEQ ID NO: 52 is the general case according to the length, specifically SEQ ID NO: 236-254). Placing a rigid peptide linker at the C-terminus of any sequence of TAP1 NBD in SEQ ID NO: 9, 12-16, 19 and 20 can replace the PADAPE (SEQ ID NO: 11) sequence.

[0192] When the constructs of this disclosure contain a payload (e.g., a chemotherapeutic agent or a marker), any connector that attaches the payload to the construct in vivo, particularly in the TME, can be either cutterable or non-cutterable (stable). A stable connector ensures that a smaller amount of the payload (e.g., a cytotoxic payload) dissociates from the construct before reaching tumor cells, thereby improving safety (e.g., limiting patient side effects due to non-tumor targeting) and limiting the dose required to achieve therapeutic efficacy.

[0193] If it is desired that the payload be separable from the construct, they can be linked by cleavable (unstable) linkers, such as those based on chemical motifs (including disulfide bonds, hydrazones, and peptides) or acid-sensitive cleavable linkers (e.g., carbonate linkers) (see, for example, the polyethylene glycol acid-sensitive cleavable carbonate linker for Sacituzumab govitecan). Linkers containing amino acid sequences readily cleaved by one or more (e.g., two or more) proteases found in the tumor tissue environment (TME) can be used as cleavable linkers. The selection of the linker's aa sequence can make it susceptible to cleavage by proteases expressed at levels higher in the TME than in one or more non-tumor tissues, or select proteases expressed at levels higher than in peritumoral tissues. For example, amino acid sequences (including, but not limited to, sequences cleavable by legumain, matriptase-1, matriptase-2, cathepsin-S, MMP1, MMP2, MMP3, MMP7, MMP8, MMP12, MMP13, MMP14, or furin) can serve as cleavable linkers because the proteases are expressed in one or more tumors and have sequence-specific cleavage. Following these are some protease-sensitive sequences that can be incorporated into linkers cleaved by these proteases.

[0194] Legumain exhibits strict specificity for cleavage following aspartic acid and asparagine residues, and its expression levels in normal cells are significantly lower than in tumor or tumor-associated macrophages (TAMs). Therefore, sequences containing one or more Asn and / or Asp sequences are suitable for use as cleavable legumain linkers.

[0195] The substrates of Matriptase-1 and Matriptase-2 contain Arg or Lys at the P1 position and preferentially contain amino acids with smaller side chains, such as Ala and Gly, at the P2 position.

[0196] Cathepsin S can cleave substrates containing the -Val-Val-Arg- sequence, especially when the sequence is embedded in a polypeptide with at least two amino acid residues flanking the cleavage site.

[0197] Matrix metalloproteinases (MMPs) are a class of calcium-dependent zinc-containing endopeptidases that cleave tetrapeptide sequences containing a leucine residue at the carboxyl terminus. Table 2 below provides the tripeptide sequences of several MMPs before binding to the cleaved leucine residue. The bond to the leucine residue that has been specifically proteased is indicated by "~". These peptides are ordered according to their cleavage rate for each protease.

[0198] Table 2 Based on the research findings of Eckhard et al. Matrix Biol. (2016) 49, 37–60.

[0199] The expression level of furin in tumors is higher than that in adjacent normal tissues. See, for example, Azevedo et al., Cancers (Basel) Aug 1, 2023; 15(15):3909, doi: 10.3390 / cancers15153909. Frin exhibits sequence selectivity for the motif Arg-Xaa-Lys-Arg or Arg-Xaa-Arg-Arg, where Xaa is any amino acid. In some cases, Xaa can be selected from D, E, F, G, H, K, L, P, Q, R, S, T, and V, or from H, K, and R. See, for example, Hosaka et al., J. Bio. Chem., 266(19) 12127-12130 (1991).

[0200] Sequences sensitive to many other proteases can be used for cleavable adaptors, including but not limited to members of the cysteine-aspartic protease (cystasparagase) family, such as cystasparagase 7; enterokinase, granzyme tobacco etch virus protease (TEV protease), and ADAM protease (a deintegrin and metalloproteinase) family members.

[0201] 3. Bracket Scaffold aa sequences can be incorporated into the constructs described herein as a means of structural organization to increase the half-life of molecules in vivo and, where they can induce ADCC, ADCP, and / or CDC, serve as the basis for those effector functions. These scaffolds may be capable of forming dimers and higher-order complexes (e.g., trimers). In some cases, scaffolds can be formed from aa sequences and their non-identical homologous aa sequences combined with their pairing pairs (interspecific sequences), which selectively interact to selectively form specific heterodimers (interspecific heterodimers). Alternatively, scaffolds can be non-dimerizable (non-dimerizable), in which case they do not form dimers or higher-order complexes. Scaffolds incorporated into the constructs described herein may, for example, contain non-Ig aa sequences (e.g., XTEN peptides, leucine zippers, or albumin peptides), or be dimerizable, heterodimerizable (i.e., forming interspecific pairs), or retain monomeric Ig aa sequences (e.g., monomeric Fc (mFc) and monomeric CH3 (mCH3)). See, for example, Liu et al., Front Immunol. 2017; 8: 38. Scaffolds containing Ig CH2-CH3 region sequences (e.g., scFc sequences) can also function as monomeric scaffolds, even if they consist of individual polypeptides (e.g., a pair of interspecific Fc sequences, such as KiH sequence pairs), especially when the individual polypeptides are linked by disulfide bonds (e.g., such as KiHs-s sequence pairs).

[0202] Scaffolds, particularly IgFc scaffolds, can provide a variety of functions, including stabilizing molecules, prolonging their circulating half-life in the blood, and providing other immune system functions related to phagocytosis and cytotoxicity (e.g., ADCC, ADCP, and / or CDC). Scaffold sequences positioned at the N-terminus of the NBD (e.g., Ig scaffold sequences) can serve as CAP structures.

[0203] The scaffold polypeptide sequence may include Ig heavy chain constant region polypeptide sequences, which can function as dimerizing or multimerizing sequences. Immunoglobulin scaffolds, such as the heavy chain constant region, may contain cysteine ​​residues that spontaneously form disulfide bonds between scaffold sequences during cellular expression of the construct. If the scaffold contains an Iggaa sequence, it may also contain an Ig heavy chain constant region (CH2-CH3) polypeptide aa sequence (e.g., an IgFc aa sequence), which can dimerize with a second Ig CH2-CH3 aa sequence. One or more cysteine ​​residues involved in stabilizing the interchain disulfide bonds of the Ig heavy chain dimer may be substituted (e.g., with alanine or serine), preventing the Ig heavy chain sequence from dimerizing or heterodimerizing and forming interchain disulfide bonds.

[0204] The scaffold peptide may contain a wt. Homo sapiens IgG1 Fc peptide sequence: (See NCBI GenPept ACCESSION 7LBL_A), during expression, homodimers stable by interchain disulfide bonds may form. The scaffold may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 59. The scaffold may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 59.

[0205] The IgG Fc aa sequence may contain CH2 and / or CH3 domains, which are modified to prevent dimerization, resulting in a sequence that exists as a monomer. The IgG1 heavy chain constant region aa sequence may contain a set of substitutions that can significantly or completely prevent dimerization with another IgG1 aa sequence, including L351S, T366R, L368H, and P395K (corresponding to L131S, T146R, L348H, and P175K in SEQ ID NO: 59). See Ying et al. (2014) mAbs 6(5):1201-1210. The IgG1 Fc sequence with these substitutions still exists as a soluble monomer, capable of binding with high affinity to FcγRI (Kd approximately 10 nM) and to the neonatal Fc receptor (FcRn), thereby preventing lysosomal degradation of the antibody and prolonging its in vivo half-life. See above. Furthermore, monomeric IgG1 sequences with these substitutions fail to provide effector functions, including Fc-mediated CDC and ADCC (natural killer cell-mediated CDC and ADCC), consistent with their inability to bind to the FcγRIIIa receptor. See above.

[0206] In the molecules described herein, IgG1 sequences that include aa sequences may be suitable for sequence substitution as a monomeric (non-dimerized) Fc sequence (mFc) scaffold.

[0207] The stent may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO:60, while retaining substitutions that cause it to retain monomers. The stent may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO:60, while retaining substitutions that cause it to retain monomers.

[0208] A stable disulfide bond can be introduced into the sequence of SEQ ID NO: 60, located between cysteine ​​residues substituting L242 and K334 (denoted as C* in SEQ ID NO: 61 and SEQ ID NO: 62), and / or between cysteine ​​residues substituting P343 and A431 (denoted as C** in SEQ ID NO: 62). Therefore, the monomeric IgG1 Fc scaffold sequence can contain the aa sequence. The stent may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 61 or SEQ ID NO: 62, while retaining substitutions that cause it to retain monomers. The stent may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 61 or SEQ ID NO: 62, while retaining substitutions that cause it to retain monomers.

[0209] Non-dimerizing scaffolds can also be used to prepare adult single-chain Fc (scFc) dimers. See, for example, Zhou et al., Biomaterials 117:24-31 (2017). Such scFc dimers may be IgG1-based and have the following sequence The G4S connector sequence, shown in bold and italics, connects two IgFc sequences. For example, the scaffold may contain an aa sequence with greater than about 90% or greater than about 95% sequence identity to SEQ ID NO: 63. The scaffold construct of SEQ ID NO: 63 may also contain an aa sequence with greater than 96% or greater than 98% sequence identity to SEQ ID NO: 63. The scaffold construct of SEQ ID NO: 63 is capable of inducing ADCC, ADCP, and CDC, but substitutions including “LALA” and / or other substitutions as described below may be introduced to selectively alter the ability of scFc to inhibit or induce effector functions.

[0210] The scaffold peptide may comprise wt. Homo sapiens IgG2 Fc peptide of SEQ ID NO: 64 (see GenBank AAN76044, aa 99-32: It may be optionally modified to prevent dimerization. Alternatively, the stent may, for example, contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 64. The stent may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 64.

[0211] The scaffold peptide may comprise wt. Homo sapiens IgG3 Fc peptide of SEQ ID NO: 65 (see, for example, GenBank AAW65947, aa 19-24: It may be optionally modified to prevent dimerization. Alternatively, the stent may, for example, contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 65. The stent may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 65.

[0212] The scaffold peptide may contain wt. Homo sapiens IgG4 Fc peptide of SEQ ID NO: 66. Optional modifications can be made to prevent dimerization. Alternatively, the stent may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 66. The stent may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 66.

[0213] Other non-dimerizing (non-multimerizing) scaffold domains include, but are not limited to, human serum albumin (i) fusion proteins (see, for example, Mandrup et al., nature.com / articles / s42003-021-01790-2, available online), anti-HSA binding domains (including but not limited to anti-HSA peptides, see, for example, helical-cyclic-helical peptides in pubs.acs.org / doi / 10.1021 / acs.molpharmaceut.2c00106), antibodies and antibody fragments (e.g., scFv, FAB, etc.), and VHH domains (see, for example, semanticscholar.org / paper / Serum-albumin%E2%80%90binding-VHHs-with-variable-pH-enable-Faassen-Ryan / d34256a0d39a0ab92db9195210fa0fc7430758b6, available online). The Ig κ or Ig λ light chain constant region or the Ig heavy chain CH1 sequence can also be used as a scaffold. Any of the above compounds can be PEGylated or lipidized to prolong their half-life. (See pubs.acs.org / doi / 10.1021 / acsmedchemlett.8b00226 on the World Wide Web).

[0214] Any one or more cysteine ​​residues in the Ig heavy chain aa sequence used as a scaffold can be substituted (e.g., replaced with alanine or serine) to prevent the Ig heavy chain sequence from dimerizing (e.g., homodimerizing) and to form interchain disulfide bonds. Alternatively, if stable disulfide bonds are required between the Ig heavy chain constant region aa sequences, cysteine ​​residues can be introduced into the sequence, or one or more hinge region disulfide bond sequences can be utilized.

[0215] a) Interspecies immunoglobulin Fc scaffold peptide In the constructs disclosed herein, if there is an asymmetric pairing between two scaffold sequences, an aa sequence capable of selectively forming a heterodimer with a specific homologous counterpart can be used. Such homologous sequence pairs or “interspecific” sequences can be variants of Ig Fc polypeptide sequences. Many such interspecific polypeptide sequences have been described, including but not limited to: buttonhole structures without stable disulfide bonds (KiH) or buttonhole structures with stable disulfide bonds (KiHs-s), HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences.

[0216] An interspecific binding pair based on the IgG1 Fc sequence, including a T366Y substitution in the first sequence and a Y407T substitution in the second sequence (or the corresponding residue of other IgG), affects the CH3 domain interface. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996).

[0217] The buttonhole-in-pore structure, or KiH interspecific binding pair, involves the formation of a button via a T366W substitution in the first aa sequence, and the formation of a pore in the complementary Ig Fc sequence via a triple substitution of T366S, L368A, and Y407V. See Xu et al. mAbs 7:1, 231–242 (2015). The associated KiHs-s interspecific binding pairs, including Cys residues, can form stable interchain disulfide bonds, including a first Ig Fc polypeptide with substitutions of Y349C, T366S, L368A, and Y407V, and a second Ig Fc polypeptide with substitutions of S354C and T366W. A stable disulfide bond may form between Y349C and S354C when the sequences are co-expressed. See, for example, Brinkmann and Konthermann, mAbs 9:2, 182–212 (2015). Immunoglobulin Fc polypeptide sequences, whether or not modified with knob-in-pore structures, can be stabilized by forming disulfide bonds (e.g., hinge region disulfide bonds) between immunoglobulin Fc polypeptides.

[0218] Table 3 summarizes KiH, KiHs-s, and other interspecies binding sequence pairs based on Ig sequences. This table includes cross-references to the numbering of the wt. IgG1 Fc sequence shown in SEQ ID NO:59 (provided in brackets “{}”).

[0219] Table 3. Interspecific immunoglobulin sequences and their homologous interspecific sequences Table 3 is adapted from Ha et al. Frontiers in Immunol. Volume 7, Chapter 394, pp. 1-16 (2016), doi: 10.3389 / fimmu.2016.00394 * aa forms a stable disulfide bond.

[0220] Suitable scaffold peptides also include interspecies “SEED” sequences, where 45 residues in the IgG1 CH3 domain of the interspecies sequence are derived from IgA, while 57 residues in the IgA CH3 domain of the corresponding interspecies sequence are derived from IgG1. See, for example, Ha et al., Frontiers in Immunol. Vol. 7, No. 394, pp. 1-16 (2016), doi: 10.3389 / fimmu.2016.00394 and its citations.

[0221] Scaffold peptides suitable for use in the constructs described herein may include an aa sequence of an interspecies binding sequence and / or its corresponding interspecies binding sequence selected from the following: KiH; KiHs-s; HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW-RVTs-s; A107; or a SEED sequence. IgFc-based scaffolds may contain substitutions that inhibit or enhance effector functions (e.g., ADCC, ADCP, and / or CDC) relative to the effects observed with wt. sequences under otherwise identical conditions. The affected effector function may be ADCC or ADCP. The affected effector function may be CDC.

[0222] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having a T146W KiH sequence substitution, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V KiH sequence substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences in wt. IgG1 of SEQ ID NO: 59. The scaffold polypeptide may comprise a KiH substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences in wt. IgG1 of SEQ ID NO: 59. Such scaffold peptides may optionally be substituted at one or more of the following sites: L234 and L235 (e.g., L234A / L235A “LALA” or L234F / L235E); N297 (e.g., N297A); P331 (e.g., P331S); L351 (e.g., L351K); T366 (e.g., T366S); P395 (e.g., P395V); F405 (e.g., F405R); Y407 (e.g., Y407A); and K409 (e.g., K409Y), which, among other things, may inhibit effector function. Those substitutions appearing in the wt. IgG1 sequence of SEQ ID NO:59 include L14 and L15 (e.g., L14A / L15A “LALA” or L14F / L15E); N77 (e.g., N77A); P111 (e.g., P111S); L131 (e.g., L131K); T146 (e.g., T146S); P175 (e.g., P175V); F185 (e.g., F185R); Y187 (e.g., Y187A); and K189 (e.g., K189Y). Alternatively, IgFc-based scaffolds may contain substitutions that enhance effector function.

[0223] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having a T146W KiH sequence substitution, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V KiH sequence substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences in wt. IgG1 of SEQ ID NO: 59. The scaffold polypeptide may comprise a KiH substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences in wt. IgG1 of SEQ ID NO: 59. Such scaffold polypeptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substituted for L234A and L235A) and / or N77 substitutions (e.g., N297, N297A or N297G).

[0224] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having T146W and S134C KiHs-s substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T146S, L148A, Y187V, and Y129C KiHs-s substitutions; wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. The scaffold polypeptide may comprise KiHs-s substitutions and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0225] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having S144H and F185A HA-TF substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having Y129T and T174F HA-TF substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 of SEQ ID NO: 59. The scaffold polypeptide may comprise an HA-TF substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 of SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0226] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T130V, T146L, K172L, and T174W ZW1 substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. The scaffold polypeptide may comprise a ZW1 substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0227] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having substitutions at K140D, D179M, and Y187A 7.8.60 sites, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having substitutions at T130V, E125R, Q127R, T146V, and K189V 7.8.60 sites, wherein the scaffold polypeptide comprises at least 210 (e.g., at least 220 or all 227) consecutive aa sequences of at least 90% or at least 95% sequence identity with the wt. IgG1 sequence of SEQ ID NO: 59. The scaffold polypeptide may comprise a 7.8.60 substitution and have at least 96% or at least 97% sequence identity with the at least 210 (e.g., at least 220 or all 227) consecutive aa sequences of the wt. IgG1 sequence of SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0228] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having K189D and K172D DD-KK substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T130V, D179K, and E136K DD-KK substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 of SEQ ID NO: 59. The scaffold polypeptide may comprise a DD-KK substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 of SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0229] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having K140E and K189W EW-RVT substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T130V, Q127R, D179V, and F185T EW-RVT substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences of wt. IgG1 in SEQ ID NO: 59. The scaffold polypeptide may comprise an EW-RTV substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa sequences of wt. IgG1 in SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0230] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having K140E, K189W, and Y129C EW-RVTs-s substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T130V, Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, wherein the scaffold polypeptide comprises at least 210 (e.g., at least 220 or all 227) consecutive aa sequences having at least 90% or at least 95% sequence identity with the wt. IgG1 sequence of SEQ ID NO: 59. The scaffold polypeptide may comprise EW-RTVs-s substitutions and have at least 96% or at least 97% sequence identity with the at least 210 (e.g., at least 220 or all 227) consecutive aa sequences of SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0231] The scaffold polypeptide suitable for the constructs described herein may comprise an IgG1 Fc sequence having K150E and K189W A107 substitutions, and its corresponding interspecies binding mating polypeptide comprises an IgG1 sequence having T130V, E137N, D179V, and F185T A107 substitutions, wherein the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. The scaffold polypeptide may comprise an A10-7 substitution and have at least 96% or at least 97% sequence identity with at least 210 (e.g., at least 220 or all 227) consecutive aa of wt. IgG1 in SEQ ID NO: 59. Such scaffold peptide sequences may contain additional substitutions, such as L14 and / or L15 substitutions (e.g., “LALA” substitutions for L234A and L235A), and / or N77 substitutions (N297, such as N297A or N297G), or substitutions that can enhance one or more effector functions.

[0232] As an alternative to using the Ig CH2 and CH3 heavy chain constant regions as interspecies scaffold sequences, the immunoglobulin Igκ or Igλ light chain constant regions can be used together with the Ig CH1 sequence as interspecies sequence pairs. The scaffold sequence may contain the Igκ chain constant region of SEQ ID NO: 67 or the Igλ chain constant region of EQ D NO: 68.

[0233] Igκ chain constant region (SEQ ID NO: 67); or Igλ chain constant region (SEQ ID NO: 68).

[0234] The light chain constant region scaffold sequence may also contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 67 or 68. The CH1 sequence paired with the light chain constant region may constitute an aa sequence: The CH1 sequence used as a scaffold may also contain a sequence that has at least 90% or at least 95% sequence identity with SEQ ID NO: 69.

[0235] The Ig CH1 and Ig κ sequences can be modified to increase their affinity, thereby improving the stability of any heterodimer formed using them. Substitutions that increase the affinity and stability of the CH1-Ig κ heterodimer include the MD13 combination identified by Chen et al. in MAbs, 8(4):761-774 (2016). In the MD13 variant, each of the CH1 and Ig κ aa sequences contains two substitutions. The Ig CH1 sequence is modified to contain S64E and S66V substitutions (S70E and S72V of SEQ ID NO: 69). The Ig κ sequence is modified to contain S69L and T71S substitutions (S68L and T70S of SEQ ID NO: 69).

[0236] b) Modification of the effector function of immunoglobulin Fc scaffold Interactions between IgG antibodies can lead to effector functions, including ADCC and ADCP, which occur through the binding of the Fc region to members of the Fcγ receptor family (FcγR). The human protein family consists of FcγRI (CD64), FcγRII (CD32, including subtypes such as FcγRIIa, FcγRIIb, and FcγRIIc), and FcγRIII (CD16, including subtypes such as FcγRIIIa and FcγRIIIb). See, for example, Lazar et al. (2006) PNAS:103 (11), 4005-4010. The occurrence of CDC may be due to the interaction of IgFc with complement C1q proteins.

[0237] Substitutions capable of altering the effector function of Ig sequences can be incorporated into the IgFc sequence used as a scaffold in the constructs described herein. Non-interspecies homodimerizing sequences such as wt. IgG (e.g., SEQ ID NO: 59 and 64-66) and interspecies Ig sequences may include substitutions that alter binding to one or both of FcγR or C1q and alter the effector function of ADCC, ADCP, and / or CDC.

[0238] (1) IgFc effector function is weakened or completely suppressed The Ig heavy chain constant region aa sequence used as a scaffold may also contain one or more substitutions that, relative to the wt. sequence, significantly attenuate the ability to stimulate one or more Ig-mediated effector functions (e.g., ADCP, CDC, and / or ADCC). For example, the scaffold may contain substituted IgG1 sequences at one or more of L234, L235, G236, G237, P238, S239, and / or P331 (appearing as L14, L15, G16, G17, P18, S19, and / or P111 in SEQ ID NO: 59, or at the corresponding positions in any of SEQ ID NO: 60-66).

[0239] The scaffold polypeptide may include the Homo sapiens IgG1 Fc aa sequence of SEQ ID NO: 70, which contains LALA substitutions (L234A, L235A substitutions). The presence of LALA substitutions in the constant region of the IgG1 heavy chain prevents efficient binding of FcγR or C1q, significantly weakening or completely inhibiting ADCC, ADCP, and CDC stimulation. The substitution positions in the wt. IgG1 sequence can be converted to the positions provided in SEQ ID NO: 59 or 70 by subtracting 220 aa from the indicated positions. Therefore, the L234A and L235A substitutions appear at positions 14 and 15 of SEQ ID NO: 70, respectively, in bold and underlined alanine residues. Optionally, the sequence shown in SEQ ID NO:70 can be modified to prevent dimerization by adding L351S, T366R, L368H, and P395K substitutions (corresponding to L131S, T146R, L348H, and P175K in SEQ ID NO:59). The scaffold may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO:70. The scaffold may also contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO:70. Any one or more cysteine ​​residues in SEQ ID NO:70 may be substituted (e.g., with alanine or serine) to prevent the Ig heavy chain sequence from dimerizing and forming interchain disulfide bonds.

[0240] Substitution at positions D270, K322, P329, and / or P331 (corresponding to D50, K102, P109, and P111 in SEQ ID NO: 59) results in reduced binding of IgG1 protein to C1q relative to wt. , thus reducing CDC. The IgG1 sequence in SEQ ID NO: 16 or 17 may contain one or more of these substitutions. In one embodiment, the IgG1 heavy chain constant region aa sequence used as a scaffold contains LALA substitutions (L234A, L235A) and P331S substitutions: Or a sequence that has at least 90% sequence identity with the sequence. Alternatively, the scaffold contains an aa sequence that has at least 95% or at least 98% sequence identity with SEQ ID NO: 71.

[0241] Other substitutions that can significantly weaken one or more antibody-related effector functions (e.g., ADCC, ADCP, and / or CDC responses) of IgG1 antibodies include, but are not limited to: Leu235Glu; Ser228Pro / Leu235Glu; Leu234Ala / Leu235Ala / Pro329Gly; Pro331Ser / Leu234Glu / Leu235Phe; Asp265Ala; Gly237Ala; Glu318Ala; Glu233Pro; and Gly236Arg / Leu328Arg. As described above, the corresponding positions of those substitutions in the IgG1 Fc sequence provided in SEQ ID NO: 59 can be obtained by subtracting 220 from the positions shown. In one embodiment, at least ADCC is significantly weakened by substitution. In one embodiment, at least ADCP is significantly weakened by substitution. In one embodiment, at least CDC is significantly weakened by substitution. In one embodiment, at least complement binding is significantly weakened by substitution. See, for example, Saunders 2019 and its citations.

[0242] Substitutions in the IgG2 and IgG4 antibody sequences can significantly weaken the function of one or more antibody-related effects, including but not limited to: H268N / V309L / A330S / P331S (IgG2m4); V234A / G237A / P238Sr / H268A / V309L / A330S / P331S (IgG2σ); L234A / L235 / G237A / P238S / H268A / A330S / P331S (IgG1σ); and S228P / F234A / L235A (IgG4PAA). See, for example, Saunders 2019 and its citations.

[0243] (2) Enhance the function of Ig Fc effect In human IgG1, substitutions to the scaffold sequence constituting the IgG1 aa sequence, compared to the corresponding wt. sequence, can enhance one or more antibody-related effector functions (e.g., ADCC, ADCP, and / or CDC responses), including single substitutions S239D and I332E, double substitutions S239D / I332E, and triple substitutions S239D / I332E / A330L. See Lazar et al., 2006. The substitutions for S239D, I332E, and A330L in the human IgG1 Fc sequence (SEQ ID NO: 59) are S19D, I112E, and A110L, respectively. In one embodiment, the substitutions are double and triple mutants of S239D / I332E or S239D / I332E / A330L. In one implementation, the IgG1 Fc substitution can be a triple substitution S239D / I332E / A330L, which allows for enhanced ADCC without substantially altering CDC function (see Lazar et al., 2006).

[0244] Other amino acid substitutions that can enhance the function of one or more antibody-related effects in IgG1 antibodies include, but are not limited to: S298A / E333A / K334A; S239D / A330L / I332E; S239D / I332E; G236A / S239d / A330L / I332E; G236A; S239D / I332E / G236A; L234Y / G236W / S298A; F243L / R292P / Y300L / V305I / P396L; K326W / E333S; K326A / E333S; K326M / E333S; C221D / D222C; S267E / H268F / S324T; H268F / S324T; and E345R. See Saunders (2019) Front. Immunol. 10:1296. doi: 10.3389 / fimmu.2019.01296. The corresponding position in the IgG1 Fc sequence of SEQ ID NO: 59 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 binding is enhanced by substitution.

[0245] 4. Immune cell binding domain and activation domain The protein constructs disclosed herein contain aa sequences that can bind to and potentially interact with immune cells (e.g., T cells and NK cells). These aa sequences can generally be described as immune cell binding domains (also known as “immune cell conjugates,” ICBs, or plural forms of ICBs), and subsets therein that bind to immune cells and trigger responses (e.g., induce particle-dependent or particle-independent cytotoxic responses), referred to as “immune cell activation domains” (ADs, or plural forms of ADs).

[0246] When an ATP-dependent complexing action (e.g., dimerization) of a construct brings together two or more ICBs and / or ADs targeting the same immune cell or the same immune cell surface (target) molecules, the effective affinity of the ATP-mediated complex is increased relative to the monomeric ICB / ADs due to the bivalent or multivalent interactions that occur between the complex and the cell.

[0247] ICBs that bind to but do not act as agonists of immune cell function are typically antibodies or antibody-associated molecules / aa sequences (e.g., antigen-binding fragments of antibodies, Fab, Fab', single-chain antibodies, scFv, peptide aptamers, or nanobodies). ICBs can function as cell lineage-specific binders when they exhibit affinity for molecules selectively expressed on one or more specific cell lineages. Unless otherwise stated, ICBs are generally considered monovalent to prevent individual molecules from cross-linking with their cellular target molecules (e.g., immune cell surface antigens), thereby avoiding untargeted stimulation of these target immune cells. Because they are monovalent, individual ICBs generally cannot act as agonists and stimulate the immune cells to which they bind, but they can act as antagonists of the natural ligands of their target molecules (e.g., a monovalent antibody aa sequence based on ipilimumab can bind to CTLA-4 and antagonize its interaction with CD28). When two or more (e.g., multiple) constructs carrying ICB aa sequences targeting the same target molecule form a complex through ATP-induced dimerization or polymerization (see, for example...), the complex is formed. Figure 4A (D or E in the text), or bound to the surface of tumor cells (e.g., using...) Figure 3 When the construct shown in C is used, the ICB aa sequence can function as an AD. Therefore, while AD has the ability to stimulate various immune cell responses when it binds to immune cells (e.g., in a bivalent or multivalent state), the ICB, lacking this ability, acts as an immune cell targeting sequence, binding to cells expressing its homologous binding partner (cell target molecule).

[0248] The ADs of the constructs disclosed herein comprise various molecules or molecular fragments (e.g., cytokine fragments) capable of binding and stimulating immune cell responses (e.g., anti-tumor cell immune responses), particularly when two or more ADs are present (e.g., as dimers or higher-order complexes). ADs include immunomodulatory protein aa sequences (e.g., aa sequences of various interleukins) and other molecules (e.g., antibodies such as Fab, Fab', single-chain antibodies, scFv, peptide aptamers, or nanobodies) that can bind and functionally interact with receptors on the surface of immune cells, thereby generating a response (e.g., inducing particle-dependent or particle-independent cytotoxic responses). When present as monomers, for example in a TME, ADs are generally ineffective in inducing immune cell responses (having limited potency and / or efficacy). When two or more (e.g., multiple) constructs containing ADs are localized in a complex via ATP-induced dimerization or polymerization (e.g., using... Figure 2 The construct shown in Figure 4A or B (either AE in the diagram or A or B in Figure 4) binds to the surface of immune cells in a dimer form, or binds to the surface of tumor cells (e.g., using a method such as...). Figure 3 A, B, or D in the middle Figure 4A The constructs shown as D or B in the diagram are more effective in inducing immune cell responses than AD constructs present in monomeric form (e.g., higher potency and / or efficacy). The presence of two or more ADs in an ATP-induced complex (e.g., a dimer, heterodimer, or multimer) promotes cross-linking of AD cell receptors and stimulates receptor-mediated responses. Some ADs require cross-linking of their cell receptors to maximize cell activation, including but not limited to CD40L, IFN-γ, IL-12, and CD28 ligands such as CD80 or CD86.

[0249] The constructs disclosed herein may elicit ATP-dependent dimerization or multimerization responses in immune cells, including but not limited to granule-dependent and / or granule-independent responses, such as stimulation from NK cells and / or T cells (e.g., CD8+ T cells). Some responses, including cytotoxic responses from T cells and NK cells, may result from the direct binding of the constructs disclosed herein to these immune cells. Other responses may be indirect results of the activation of other immune cells. For example, the recruitment and / or activation of macrophages by the constructs disclosed herein may be due to activation by NK cells releasing chemokines IFNγ or CCL5, or due to direct binding of the constructs disclosed herein (e.g., using chemokines as AD) to macrophages.

[0250] Table 4 lists examples of immune cells and ICBs, some of which may also function as AD (e.g., CD3). It is important to note that Ig scaffold portions capable of stimulating ADCC, ADCP, and / or CDC are considered separately. Sequences stimulating ADCC, ADCP, and / or CDC can be explicitly excluded from ICBs and / or ADs and may still be present, optionally present, or absent from immunoglobulin scaffold aa sequences.

[0251] a) Immune cell binding domain (ICB) ICBs can target a variety of different immune cells that are present or recruited in the TME. These immune cells and their corresponding ICB targets include those listed in Table 4 below.

[0252] Table 4 Some immune cells and their corresponding ICB targets and ICB Antibody-related molecules include, but are not limited to, antigen-binding fragments of antibodies, Fabs, Fab's, and polypeptides composed of single-chain antibodies, scFvs, aptamers, or nanobody aa sequences.

[0253] ICBs, such as those listed in Table 4, can be incorporated into a first NBD-containing construct that can undergo ATP-mediated formation of homodimers, heterodimers, and / or higher-order complexes. The NBD-containing construct containing an ICB can, for example, undergo ATP-mediated complexation with a second NBD-containing construct containing a TSB (TSB) targeting a TAA. This complex can be described as “BiTE-like”, capable of cross-targeting tumor cells, leading to tumor cell lysis (see, for example...). Figure 3 C, D, and FI in the middle, and Figure 4A (The complexes in E, F, H, and I). Incorporating antibody-associated molecules / aa sequences (including but not limited to anti-CD3, anti-TCR, and / or anti-CD16) as ICBs / ADs into such cross-targeting complexes can produce potent cytolytic activity. Cytolytic activity can be enhanced when using ICBs that interact with more than one type of immune cell. Some exemplary ICBs are provided below.

[0254] The ICB peptide may contain scFv, a human anti-CD3 antibody based on a UCHT-1 monoclonal antibody, with the following sequence: AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTDYTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIKGGG GSGGGGSGG GSGGGGSGG SEVQLVESGGGLVQPGGSLR LSCAASGYSF TGYTMNWVRQ APGKGLEWVA LINPYKGVTT YADSVKGRFT ISVDKSKNTAYLQMNSLRAE DTAVYYCARS GYYGDSDWYF DVWGQGTLVT VSS (SEQ ID NO:72). The ICB may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 72. In addition, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 72.

[0255] The ICB peptide may contain scFv, a human anti-CD3 antibody based on a UCHT-1 monoclonal antibody, with the following sequence: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKA PKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO:73). The ICB may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 73. In addition, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 73.

[0256] The ICB polypeptide may comprise an anti-human CD3 VHH having the following sequence: QVQLVESGGG LVQPGGSLRLSCAASGSIFS ANTMGWYRQA PGKQRELVAG MNTSGSTVYG DSVKGRFTIS RDNAKNIAYL QMNSLIPEDTAVYYCTLVQR GPNYWGQGTQ VTVSS (SEQ ID NO: 74). The ICB may also comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 74. Furthermore, the ICB may comprise an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 74.

[0257] ICB peptides may contain an anti-human CD3 scFv (vH-vL) based on an L2K antibody (see, e.g., Dreier et al., Int. J. Cancer. 2002; 100:690–697. doi: 10.1002 / ijc.10557), with the sequence: DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQGLEWIGY INPSRGYTNY ADSVKGRFTITTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSG GGGSGGGGGSG GGGSDIVLTQSPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW IYDTSKVASG VPARFSGSGS GTDYSLTINSLEAEDAATYY CQQWSSNPLT FGGGTKVEIK (SEQ ID) NO: 75). The ICB may also contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 75. In addition, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 75.

[0258] The ICB peptide may comprise a human anti-CD3 scFv (vH-vL) based on an L2K antibody having the sequence shown in SEQ ID NO: 76, which contains a disulfide bond formed between cysteine ​​substitution sites at Q43C and G99C (G233C as shown): DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGCGLEWIGYINPSRGYTNY ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSGGGGSGGGGSG GGGSDIVLTQ SPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW IYDTSKVASGVPARFSGSGS GTDYSLTINS LEAEDAATYY CQQWSSNPLT FGCGTKVEIK (SEQ ID NO: 76). The ICB may also contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 76. Furthermore, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 76.

[0259] The ICB peptide may comprise a human anti-CD3 scFv (vH-vL) based on an L2K antibody having the sequence shown in SEQ ID NO: 77, which contains a disulfide bond formed between cysteine ​​substitution sites at Q43C and G100C (G234C as shown): DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGCGLEWIGYINPSRGYTNY ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSGGGGSGGGGSG GGGSDIVLTQ SPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW IYDTSKVASGVPARFSGSGS GTDYSLTINS LEAEDAATYY CQQWSSNPLT FGGCTKVEIK (SEQ ID NO: 77). The ICB may also contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 77. Furthermore, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 77.

[0260] The ICB peptide may comprise a human anti-CD3 scFv (vH-vL) based on an L2K antibody having the sequence shown in SEQ ID NO: 78, which contains a disulfide bond formed between cysteine ​​substitution sites at G44C and G99C (G233C as shown): DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQCLEWIGYINPSRGYTNY ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYY DDHYCLDYWG QGTTVTVSSGGGGSGGGGSG GGGSDIVLTQ SPATLSLSPG ERATLSCRAS QSVSYMNWYQ QKPGKAPKRW IYDTSKVASGVPARFSGSGS GTDYSLTINS LEAEDAATYY CQQWSSNPLT FGCGTKVEIK (SEQ ID NO: 78). The ICB may also contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 78. Furthermore, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 78.

[0261] ICB peptides may include an L2K-based anti-human CD3 scFv (vH-vL) antibody having the sequence specified in SEQ ID NO:79, including a disulfide bond between cysteine ​​substitutions at G44C and G100C (G234C is shown in the figure): The ICB may also contain an aa sequence that has greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 79. Furthermore, the ICB may contain an aa sequence that has greater than 96% or greater than 98% sequence identity with SEQ ID NO: 79.

[0262] The repeated GGGS (SEQ ID NO: 30) and / or GGGGS (SEQ ID NO: 40) in any of SEQ ID NO: 72-73 or 75-79 are joint sequences between domains.

[0263] ICBs may contain, for example, an aa sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody aa sequence). Anti-CD16 VHH peptides that can be used as ICBs may contain one of the following: Alternatively, the ICB may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 80 or 81. The ICB may also contain a sequence having at least 96% or at least 98% sequence identity with at least 100 or at least 110 consecutive aa of SEQ ID NO: 80 or 81.

[0264] b) Immune cell activation domain (AD) Antigen-binding (AD) sequences can originate from various sources, including interleukins and antibodies that bind to interleukin receptors or other cellular proteins. AD includes, but is not limited to, antibodies (e.g., scFV or nanobodies) and antigen-binding fragments of antibodies, such as: anti-TCRα or anti-TCRβ chains, anti-CXCR1, anti-CD3γ, anti-CD3ε, anti-TCRδ chains or anti-TCRγ chains (e.g., TRGV9 / anti-TRGV9), anti-CD4, anti-CD13, anti-CD15, anti-CD16 (FcγRIII), anti-CD257 (anti-BAFF), anti-CD28, anti-CD152 (anti-CTLA-4), anti-CD33, anti-CD40, anti-CD40L, anti-CD34, anti-CD66b, anti-CD68, anti-CD84, anti-CD117, anti-CD137 (anti-41BB), anti-CD163, anti-CD193, anti-CD206, etc. Anti-FcεR1α. AD includes, but is not limited to, CD3, CD80, CD86, CD137L, 4-1BBL, IL-2, IL-7, IL-12, IL-15, IL23, IL27, IL-35, PD1, PDL1, TNF, lymphotoxin α, lymphotoxin αβ, interferon-γ (IFN-γ), and TNF peptides, which functionally interact with receptors on immune cells, thereby affecting their activity.

[0265] AD may contain aa sequences of TNF superfamily members, such as TNF, lymphotoxin α, lymphotoxin αβ, and BAFF (CD257). AD may also contain aa sequences of B7 superfamily members, such as CD80 and CD86. AD may also contain all or part of the aa sequences of IL-15, IL-12 (e.g., scIL12), IL-2, or IL-7 (e.g., all or part of the extracellular domain). AD may contain all or part of the extracellular portion of the p35 and p40 aa sequences of IL-12 (e.g., one or both subunits of human IL-12). AD may contain aa sequences with greater than 95% or greater than 98% sequence identity to the p35 and p40 extracellular domains of IL-12.

[0266] For example, at the ATP levels found in TME, it can be used to construct and cause homodimerization (see example...). Figure 2 (A and C in the text) or form homologous complexes (see, for example) Figure 4A Some ADs in A) include, but are not limited to, the following immunomodulatory aa sequences: IL-15, IL-12, or IL-7. Antibody-associated sequences binding PD1 (e.g., single-chain scFv or nanobody sequences) (e.g., anti-PD1 scFv or nanobody aa sequences) or aa sequences binding PDL1 (e.g., anti-PDL1 scFv or nanobody aa sequences) may be used as ADs. Similarly, ADs may contain aa sequences binding CD40 (e.g., anti-CD40 scFv or nanobody aa sequences or CD40L aa sequences) or aa sequences binding CD40L (e.g., anti-CD40L scFv or nanobody aa sequences, or CD40 aa sequences). ADs may contain aa sequences binding to CD137 / 4-1BB (e.g., anti-CD137 scFv or nanobody aa sequences, or CD137L / 4-1BBL aa sequences). AD may contain an IL-10-binding aa sequence (e.g., an anti-IL-10 scFv or nanobody aa sequence) or an IL-10R-binding aa sequence (e.g., an anti-IL-10R scFv or nanobody aa sequence).

[0267] AD may contain an aa sequence that binds to CD3 (e.g., an anti-CD3 scFv or nanobody aa sequence), which may also function as an ICB. AD may contain an aa sequence that binds to CTLA-4 (e.g., an anti-CTLA-4 scFv or nanobody aa sequence). AD may contain an aa sequence that binds to CD28 (e.g., an anti-CD28 scFv or nanobody aa sequence). AD may contain an interferon-γ (IFN-γ) aa sequence, or an aa sequence that binds to the IFN-γ receptor (e.g., an anti-IFN-γ receptor scFv or nanobody aa sequence). AD may contain an aa sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody aa sequence).

[0268] For example, the presence of homodimerization at ATP levels that can be used to detect it in the TME (see example...) Figure 2 The AD of the constructs (A and C) or forming homologous complexes (see, for example, A in Figure 4) includes, but is not limited to, immunomodulatory aa sequences of IL-15, IL-12, or IL-7. Antibody-associated sequences (e.g., single-chain scFv or nanobody sequences) with binding activity against PD1, PDL1, CD40, CD40L, CD137 / 4-1BB, IL-10, or IL10R may also be used.

[0269] In addition to the above, nucleic acids (including those containing CpG repeat sequences or one or more IMT504 sequences) can be used as adjuvants (ADs) and can be incorporated into constructs containing homodimerized or heterodimerized NBDs. However, nucleic acids need to be partially coupled to other protein constructs, such as protein constructs prepared through cell expression.

[0270] (1) IL-2 AD may contain an IL-2 aa sequence capable of binding to and stimulating signaling from its homologous IL-2 receptor (IL-2R). IL-2 binds to the heterotrimeric IL-2R, composed of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132) subunits. The sequences of human IL-2 and all three human IL-2R subunits are known. See, for example, the published PCT application WO 2020 / 132138 A1.

[0271] IL-2 AD may contain one or more insertions, deletions, or substitutions that result in reduced binding to IL-2Rα, thereby minimizing or significantly reducing Treg activation. Furthermore, in addition to altering IL-2 sequence changes that change IL-2Rα interactions, IL-2 MOD aa sequences may include insertions, deletions, or substitutions that result in reduced binding to IL-2Rβ and / or IL-2Rγ subunits, thereby reducing affinity for IL-2R. WO 2020 / 132138 A1, WO 2019 / 051091, and WO 2020 / 132297 disclose IL-2 sequences that bind to IL-2R and have altered affinities for IL-2Rα, IL-2Rβ, and / or IL-2Rγ.

[0272] The IL-2 AD applicable to the protein constructs described herein may contain an aa sequence having at least 90% or at least 95% sequence identity with the wt. IL-2 aa sequence: APTSSSTKKT QLQLE H LLLD LQMILNGINNYKNPKLTRML T FKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:105). Alternatively, the IL-2 MOD may contain an aa sequence having at least 96% or at least 98% aa sequence identity with the IL-2 aa sequence of SEQ ID NO: 105. A suitable IL-2 AD sequence may contain an aa sequence having 100% identity with SEQ ID NO: 105, or an aa sequence with at least one amino acid insertion, deletion, or substitution in the aa sequence of SEQ ID NO: 105. The IL-2 AD may contain substitutes with limited binding to IL-2Rα and / or IL-2Rβ, resulting in reduced binding to IL-2R. For example, such substitutions in IL-2 are described in WO 2020 / 132138 A1, WO 2019 / 051091, and WO 2020 / 132297. Examples of such substitutions include substitutions at H16 and F42, which are highlighted and italicized in SEQ ID NO: 105. Substitutions at these positions include replacing H16 and F42 with aa residues other than His and Phe, respectively. H16 and F42 can also be substituted with independently selected Ala or Thr residues, such as the H16A F42A variant that produces the IL-2 sequence (e.g., SEQ ID NO: 105). Other IL-2 variants include the following substitutions: H16A F42T; H16T F42A; or H16T F42T.

[0273] (2) CD80 AD may contain the CD80 polypeptide aa sequence: Alternatively, AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 106. AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 170 or 180 consecutive aa of SEQ ID NO: 106.

[0274] (3) CD86 AD may contain a CD86 polypeptide aa sequence, which includes a CD86 (IgV domain): Alternatively, AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 107 or 108. AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 100 or at least 110 consecutive aa of SEQ ID NO: 107 or 108.

[0275] (4) IL-12 AD may contain an aa sequence that binds to the IL-12 receptor as an agonist (e.g., an aa sequence of IL-12, anti-CD28 scFv, or nanobody). IL-12 and IL-12 superfamily members are heterodimers. AD may contain single-chain IL-12 (scIL-12), with the following sequence: It contains fragments of the p40 subunit, GGGGS linker repeats, and fragments of the p35 subunit. Alternatively, AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 109. AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 480 or at least 500 consecutive aa sequences of SEQ ID NO: 109.

[0276] As described above, the constructs of this disclosure can contain heterodimerized NBD in the presence of ATP. Therefore, the complex between the first and second constructs containing heterodimerized NBD can be used to assemble active IL-12 superfamily members as AD (see, for example...). Figure 2 (The B and D, where the first and second AD are subunits of this superfamily member). For example, in the presence of ATP, active IL-12 AD heterodimers can be formed in the following manner: (i) A first construct containing the p40 subunit (as the first immunoregulatory domain) comprising the following sequence: Or a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO:110; and (ii) A second construct containing the p35 subunit (as a second immunoregulatory domain) and comprising the t sequence: Or a sequence that has at least 90% or at least 95% sequence identity with SEQ ID NO: 111.

[0277] (5) PD1 AD may contain all or part of an aa sequence that binds to PD1 (e.g., an anti-PD1 scFv or nanobody aa sequence) or all or part of an aa sequence that binds to PDL1 (e.g., an anti-PDL1 scFv or nanobody aa sequence).

[0278] (6) CD40 AD may contain an aa sequence binding to CD40 (e.g., an anti-CD40 scFv or nanobody aa sequence or a CD40L aa sequence) or an aa sequence binding to CD40L (e.g., an anti-CD40L scFv or nanobody aa sequence, or a CD40 aa sequence). AD may contain a CD40L sequence: Or an aa sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 112. Alternatively, a polypeptide that can be used as an AD comprises a trimer of a CD40L aa sequence separated by a linker sequence (e.g., a linker sequence containing a GGGGS repeat sequence, SEQ ID NO: 40): Or an aa sequence that has at least 90% or at least 95% sequence identity with SEQ ID NO: 113.

[0279] (7) 4-1BBL AD may contain an aa sequence that binds to CD137 / 4-1BB (e.g., an anti-CD137 scFv or nanobody aa sequence, or a CD137L / 4-1BBL aa sequence). 4-1BBL peptides that can be used as AD may contain the following 4-1BBL sequences: The 4-1BBL AD may comprise an aa sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 114 or SEQ ID NO: 115. Alternatively, the polypeptide that can be used as an AD may comprise a trimer of 4-1BBL aa sequences separated by a linker sequence (e.g., a linker sequence comprising repeats of GGGGS (SEQ ID NO: 40)). The AD of 4-1BBL may contain an aa sequence that has at least 90% or at least 95% sequence identity with SEQ ID NO: 116.

[0280] (8) CD3 ADs may contain an aa sequence that binds to CD3 (e.g., an anti-CD3 scFv or aa sequence of nanobodies). As mentioned above, such ADs can function as ICBs.

[0281] The AD may comprise an anti-human CD3 scFv aa sequence based on a UCHT-1 monoclonal antibody, which comprises the aa sequence of SEQ ID NO: 72 or 73. The AD may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 72 or 73. Alternatively, the AD may comprise an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 72 or 73.

[0282] The AD may contain an anti-human CD3 VHH aa sequence, which includes the sequence of SEQ ID NO: 74. The AD may contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 74. Alternatively, the AD may contain an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 74. AD may comprise an anti-human CD3 aa sequence based on an anti-human CD3 L2K antibody. L2K-based AD may comprise an anti-human CD3 scFv (vH-vL) having the aa sequence of SEQ ID NO: 75 or 76. AD may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 75 or 76. Furthermore, AD may also comprise an aa sequence with greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 75 or 76. 76 Aa sequences with greater than 96% or greater than 98% sequence identity.

[0283] L2K-based adjuvants (ADs) may comprise anti-human CD3 scFv (vH-vL) having the aa sequence of SEQ ID NO: 77 or 78. The AD may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 75 or 76. Alternatively, the AD may comprise an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 75 or 76.

[0284] L2K-based adjuvant inhibitors (ADs) may comprise anti-human CD3 scFv (vH-vL) having the aa sequence of SEQ ID NO: 79. The AD may comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with SEQ ID NO: 79. Alternatively, the AD may comprise an aa sequence having greater than 96% or greater than 98% sequence identity with SEQ ID NO: 79.

[0285] (9) CD28 AD may contain an aa sequence that binds to CD28 (e.g., an anti-CD28 scFv or nanobody aa sequence). Anti-CD28 scFv that can be used as AD may contain the following sequences: Alternatively, AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 117. AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 220 or at least 230 consecutive aa of SEQ ID NO: 117.

[0286] (10) CD16 The AD may contain an aa sequence that binds to CD16 (e.g., an anti-CD16 scFv or nanobody aa sequence). An anti-CD16 VHH peptide that can be used as an AD may contain an aa sequence of SEQ ID NO: 80 or SEQ ID NO: 81. Alternatively, the AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 80 or 81. The AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 100 or at least 110 consecutive aa sequences of SEQ ID NO: 80 or 81.

[0287] (11) IFN-γ ADs may contain an interferon-γ (IFN-γ) polypeptide sequence or an aa sequence that binds to the IFN-γ receptor (e.g., an anti-IFN-γ receptor scFv or nanobody aa sequence). scIFN-γ polypeptides that can be used as IFN-γ ADs may contain the following sequences: Alternatively, AD may contain a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 82. AD may also contain a sequence having at least 96% or at least 98% sequence identity with at least 220 or at least 230 consecutive aa in SEQ ID NO: 82.

[0288] (12) TNF AD may contain a tumor necrosis factor α (TNFα) polypeptide sequence or an aa sequence that binds to a TNF receptor (e.g., TNFR2 expressed on immune system cells). AD may contain an anti-TNFR scFv or nanobody aa sequence, particularly an anti-TNFR2 scFv or nanobody aa sequence.

[0289] AD may contain single-chain TNF (scTNF), which contains the following sequence: It comprises three repeats of a TNF fragment separated by a linker, consisting of GGGS repeats. Alternatively, AD may comprise a sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 83. AD may also comprise a sequence having at least 96% or at least 98% sequence identity with at least 480 or at least 500 consecutive aa in SEQ ID NO: 83.

[0290] (13) Nucleic acid ADs are not necessarily composed of aa sequences; for example, they can be composed of nucleic acid sequences. Non-limiting examples of nucleic acid ADs include nucleic acid sequences having CpG repeat sequences (e.g., CpG oligodeoxynucleotide sequences) or IMT504 (TCATCATTTTGTCATTTTGTCATT (SEQ ID NO: 87; see, for example, Insula et al., 2007 Stem Cells, 25:1047–1054), optionally having 1, 2, or 3 nucleotide substitutions, deletions, or insertions.

[0291] 5. Tumor-specific binding domain (TSB) The tumor-specific binding domain (also known as a tumor-specific binder, TSB in the singular and TSBs in the plural) of the construct described herein is a polypeptide aa sequence that functions as a targeting sequence, guiding the construct to tumor cells by binding to target molecules (e.g., tumor-associated antigens or neoantigens) expressed or associated with the surface of tumor cells. TSBs include polypeptides and other molecules, such as antibodies, including antigen-binding fragments of antibodies, Fab, Fab', single-chain antibodies, scFv, peptide aptamers, and nanobodies. In some cases, the TSB is selected from nanobodies, scFab, and scFv. Bispecific antibodies targeting two different TAAs or two different epitopes on TAAs can be used as TSBs. TSBs can be expressed as part of the peptide of the construct described herein. Alternatively, TSBs can be linked to the polypeptide of the construct via enzymatic or non-enzymatic (e.g., via a cross-linking agent) methods (see example...). Figure 5F In this case, the bispecific antibody is attached to the NBD via a linker; however, the antibody can also be attached to the linker via a cross-linking agent.

[0292] Molecules targeted by TSBs can be tumor-associated antigens (TAAs) whose cell surface expression is limited to or substantially limited to one or more tumor cell types. TAAs are a subset of cancer-associated antigens (CAAs) whose cell surface expression is limited to or substantially limited to one or more types of cancer cells found in tumors. Although the constructs described herein are primarily designed to function in TMEs, constructs targeting TAAs / CAAs found on the surface cells of non-solid tumors (e.g., leukemia) can be used to treat a patient's non-solid tumors alone or concurrently with the patient's solid tumor treatment. Besides molecules whose expression is limited to or substantially limited to tumor cells, other molecules present on the surface of tumor cells can also serve as targets, including but not limited to cellular proteins expressed on normal (non-transformed) cells that are upregulated on the surface of tumor cells. Checkpoint proteins (e.g., the V-domain Ig repressor of T cell activation, or "VISTA", the T-cell immunoglobulin and mucin domain 3, or "Tim-3", and programmed death ligand 1, or "PD-L1") are expressed on normal cells and many tumor cells (e.g., transformed tumor cells) and may also serve as target molecules for TSBs. Tumor cells often upregulate checkpoint proteins to evade immunity or treatment, and targeting checkpoint proteins provides both a target that may be highly expressed on the surface of tumor cells and checkpoint inhibition via TSBs. Proteins not limited to expression on transformed tumor cells can be considered targets of TSBs, especially when they are upregulated and expressed at high levels on tumor cells, because immune responses directed by NBD-containing constructs containing TSBs are confined to the TME, where ATP levels are high enough to allow TSBs to pair or complex with NBD-containing constructs, such as those containing AD, ICB, or scaffolds capable of inducing ADCC, ADCP, and / or CDC (see, e.g., Figure 3 , 4A (and 4B).

[0293] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to: carcinoembryonic antigen (CEA), mesothelin (MSLN), Erb-B2 receptor tyrosine kinase 2 (HER2 or ERBB2), epithelial cell adhesion molecule (EPCAM), vascular endothelial growth factor receptor (VEGF), prostate six-span membrane epithelial antigen (STEAP), epidermal growth factor receptor (EGFR), phosphatidylinositol proteoglycan-3 (GPC3), mucin 17 (MUC17), prostate-specific membrane antigen (PSMA), mucin 1 (MUC1), mucin 16 (MUC16), trophoblast cell surface antigen (TROP2), fibroblast growth factor receptor 2 (FGFR2b), tight junction protein 6 (CLDN6), CD276 (B7-H3), carbonic anhydrase (CA9), podocyte spike protein (PDPN), and placental-like alkaline phosphate. Enzymes (ALPP, e.g., ALPPL2), anthrax toxin receptor 1 (ANTXR1), tight junction protein 18 (CLDN18), folate hydrolase-1 (FOLH1), guanylate cyclase C (GUCY2C), interleukin-13 receptor 13 subunit α-2 (IL13RA2), foot glycocalyx protein (PODXL), prostate stem cell antigen (PSCA), protein tyrosine kinase 7 (PTK7), folate receptor 1 (FOLR1), V domain immunoglobulin T cell activation inhibitory factor (VISTA), T cell immunoglobulin and mucin domain protein 3 (TIM-3), PD-L1, CTLA-4, tissue factor (human), c-Met tyrosine kinase, CD22, CD79b, CD19, CD30, folate receptor α (FRα), conjugation-4, B7H3, cMET, and lymphocyte activation gene 3 (LAG-3).

[0294] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to: prostate six-transmembrane epithelial antigen (STEAP), prostate stem cell antigen (PSCA), and prostate-specific membrane antigen (PSMA).

[0295] Some TAAs associated with solid tumors that can be targeted by TSBs include, but are not limited to: mesothelial cell line (MSLN), EpCAM, and CTLA-4.

[0296] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to: tight junction protein 6 (CLDN6), tight junction protein 18 (CLDN18), foot glycocalyx protein (PODXL), and placental-like proteins (ALPPs, such as ALPPL2).

[0297] Some TAAs associated with solid tumors that can be targeted by TSB include, but are not limited to, MUC1, MUC16, or MUC17.

[0298] Some checkpoint proteins that may be targets of TSB include, but are not limited to, VISTA, TIM-3, PD-L1, CTLA-4, or LAG-3.

[0299] TSB peptides can be anti-human cortisol scFv peptides, associated with murine SS1 antibodies, and also used in Amatuximab. Their sequence is: QVQLQQSGPE LEKPGASVKI SCKASGYSFT GYTMNWVKQS HGKSLEWIGL ITPYNGASSYNQKFRGKATL TVDKSSSTAY MDLLSLTSED SAVYFCARGG YDGRGFDYWG SGTPVTVSS G XGGSGGGGGSG GGGS DIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW IYDTSKLASG VPGRFSGSGSGNSYSLTISS VEAEDDATYY CQQWSKHPLT FGSGTKVEIK, where X is V or G (SEQ ID NO: 88). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0300] TSB peptides may include an anti-human cortisol scFv (vH-vL) peptide associated with the monoclonal antibody YP218, with the following sequence: QEQLVESGGG LVQPGASLTL TCTASGIDFS RYYMCWVRQA PGKGLEGIAC IYIGGSGSTYYASWAKGRFT ISKASSTTVT LQMTSLTAAD TATYFCARGT NLNYIFRLWG PGTLVTVSS G XGGSGGGGGSG GGGS DVVMTQ TPASVSEPVG GTVTIKCQAS QRISSYLSWY QQKPGQRPKL LIFGASTLAS GVPSRFKGSGSGTEYTLTIS DLECADAATY YCQSYAYFDS NNWHAFGGGT EVVV, where X is V or G (SEQ ID NO: 89). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0301] TSB peptides may include an anti-human cortisol scFv (vL-vH) peptide associated with the monoclonal antibody YP218, with the following sequence: DVVMTQTPAS VSEPVGGTVT IKCQASQRIS SYLSWYQQKP GQRPKLLIFG ASTLASGVPSRFKGSGSGTE YTLTISDLEC ADAATYYCQS YAYFDSNNWH AFGGGTEVVV GXGGSGGGGS GGGGS QEQLVESGGGLVQPG ASLTLTCTAS GIDFSRYYMC WVRQAPGKGL EGIACIYIGG SGSTYYASWA KGRFTISKASSTTVTLQMTS LTAADTATYF CARGTNLNYI FRLWGPGTLV TVSS, where X is V or G (SEQ ID NO: 90). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0302] TSB peptides may include an anti-human cortisol scFv (vH-vL) peptide associated with monoclonal antibody 15B6, with the following sequence: EVQLQQSGPV LVKPGASVKI SCKASGYSFT GYYMHWVRQS NGKSLEWIGR INPYTGVPSYKHNFKDKASL TVDKSSSTAY MELHSLTSED SAVYYCAREL GGYWGQGTTL TVSS GXGGSG GGGSGGGGGS QAVVTQESALT TSPGETVTLT CRSSTGAVTT GNYPNWVQEK PDHLFTGLIA GTNNRAPGVP ARFSGSLIGDKAALTITGAQ TEDEAIYFCA LWFSSHWVFG GGTKLTVLG, where X is V or G (SEQ ID NO: 91). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0303] TSB peptides may include an anti-human cortisol scFv (vL-vH) peptide associated with monoclonal antibody 15B6, with the sequence: PDHLFTGLIA GTNNRAPGVP ARFSGSLIGD KAALTITGAQ TEDEAIYFCA LWFSSHWVFGGGTKLTVL GG XGGSGGGGGSG GGGSEVQLQQ SGPVLVKPGA SVKISCKASG YSFTGYYMHW VRQSNGKSLEWIGRINPYTG VPSYKHNFKD KASLTVDKSS STAYMELHSL TSEDSAVYYC ARELGGYWGQ GTTLTVSS, where X is V or G (SEQ ID NO: 92). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0304] The TSB peptide may contain an anti-human cortisol scFv (vL-vH) peptide associated with monoclonal antibody 15B6, which has a disulfide bond linking the vL and vH positions at G102C and K43C (or K168C as shown). Its sequence is: QAVVTQESALTTSPGETVTL TCRSSTGAVT TGNYPNWVQE KPDHLFTGLI AGTNNRAPGV PARFSGSLIG DKAALTITGAQTEDEAIYFC ALWFSSHWVF GCGTKLTVLG GGGGSGGGGS GGGGS EVQLQ QSGPVLVKPG ASVKISCKASGYSFTGYYMH WVRQSNGCSL EWIGRINPYT GVPSYKHNFK DKASLTVDKS SSTAYMELHS LTSEDSAVYYCARELGGYWG QGTTLTVSS (SEQ ID NO: 93). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0305] The TSB peptide may contain an anti-human cortisol scFv (vL-vH) peptide associated with monoclonal antibody 15B6, which has a disulfide bond linking G102C and S44C (S169C as shown) at the vL and vH positions, and has the following sequence: QAVVTQESAL TTSPGETVT LTCRSSTGA VTTGNYPNWV QEKPDHLFTG LIAGTNNRAP GVPARFSGSLIGDKAALTIT GAQTEDEAIY FCALWFSSHW VFGCGTKLTV LG GGGGSGGG GSGGGGSEVQ LQQSGPVLVKPGASVKISCK ASGYSFTGYY MHWVRQSNGK CLEWIGRINP YTGVPSYKHN FKDKASLTVD KSSSTAYMELHSLTSEDSAV YYCARELGGY WGQGTTLTVSS (SEQ ID NO: 94). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0306] The TSB peptide may contain an anti-human cortisol scFv (vL-vH) peptide associated with monoclonal antibody 15B6, which has a disulfide bond linking G103C and K43C (K168C as shown) at the vL and vH positions, and its sequence is: QAVVTQESALTTSPGETVTL TCRSSTGAVT TGNYPNWVQE KPDHLFTGLI AGTNNRAPGV PARFSGSLIG DKAALTITGAQTEDEAIYFC ALWFSSHWVF GGCTKLTVLG GGGGSGGGGS GGGGS EVQLQ QSGPVLVKPG ASVKISCKASGYSFTGYYMH WVRQSNGCSL EWIGRINPYT GVPSYKHNFK DKASLTVDKS SSTAYMELHS LTSEDSAVYYCARELGGYWG QGTTLTVSS (SEQ ID NO: 95). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0307] The TSB peptide may contain an anti-human cortisol scFv (vL-vH) peptide associated with monoclonal antibody 15B6, which has a disulfide bond linking G103C and S44C (as shown, S169C) at the vL and vH positions, and its sequence is: QAVVTQESALTTSPGETVTL TCRSSTGAVT TGNYPNWVQE KPDHLFTGLI AGTNNRAPGV PARFSGSLIG DKAALTITGAQTEDEAIYFC ALWFSSHWVF GGCTKLTVLG GGGGSGGGGS GGGGSEVQLQ QSGPVLVKPG ASVKISCKASGYSFTGYYMH WVRQSNGKCL EWIGRINPYT GVPSYKHNFK DKASLTVDKS SSTAYMELHS LTSEDSAVYYCARELGGYWG QGTTLTVSS (SEQ ID NO: 96). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0308] The TSB-binding peptide may comprise an anti-human cortisol (VHH) peptide associated with the monoclonal antibody SD1, with the sequence: QVQLVQSGGG LVQPGGSLRL SCAASDFDFA AYEMSWVRQA PGQGLEWVAI ISHDGIDKYY TDSVKGRFTISRDNSKNTLY LQMNTLRAED TATYYCLRLG AVGQGTLVTV SSS (SEQ ID NO: 97). The TSB peptide may also comprise an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB peptide may comprise an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0309] TSB peptides may include anti-human EpCAM scFv (vH-vL) peptides associated with monoclonal antibodies MT201 and Adecatumumab, with the following sequence: EVQLLESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAVISYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDM GWGSGWRPYY YYGMDVWGQGTTVTVSS GXG GSGGGGSGGG GS ELQMTQSP SSLSASVGDR VTITCRTSQS ISSYLNWYQQ KPGQPPKLLIYWASTRESGV PDRFSGSGSG TDFTLTISSL QPEDSATYYC QQSYDIPYTF GQGTKLEIKR TV, where X is V or G (SEQ ID NO: 98). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0310] TSB peptides may include anti-human EpCAM scFv (vL-vH) peptides associated with monoclonal antibodies MT201 and adecatumumab, with the following sequence: ELQMTQSPSS LSASVGDRVT ITCRTSQSIS SYLNWYQQKP GQPPKLLIYWASTRESGVPD RFSGSGSGTD FTLTISSLQP EDSATYYCQQ SYDIPYTFGQ GTKLEIKRTV GXGGSGGGGS GGGGS EVQLL ESGGGVVQPG RSLRLSCAAS GFTFSSYGMH WVRQAPGKGL EWVAVISYDG SNKYYADSVKGRFTISRDNS KNTLYLQMNS LRAEDTAVYY CAKDMGWGS GWRPYYYYGMD VWGQGTTVTV SS, where X is V or G (SEQ ID NO: 99). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0311] TSB peptides may include an anti-human CTLA-4 scFv (vL-vH) peptide associated with ipilimumab, with the following sequence: EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKR G GGGSGGGGGSG GGGS QVQLVESGGGVVQPGR SLRLSCAASG FTFSSYTMHW VRQAPGKGLE WVTFISYDGN NKYYADSVKG RFTISRDNSKNTLYLQMNSL RAEDTAIYYC ARTGWLGPFD YWGQGTLVTV SS (SEQ ID NO: 100). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0312] The TSB peptide may comprise an anti-human CTLA-4 scFv (vL-vH) peptide associated with ipilimumab, which has a disulfide bond linking the vL and vH positions at Q101C and K43C (K167C as shown) and has the following sequence: EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG CGTKVEIKR G GGGSGGGGGSG GGGS QVQLVESGGGVVQPGR SLRLSCAASG FTFSSYTMHW VRQAPGCGLE WVTFISYDGN NKYYADSVKG RFTISRDNSKNTLYLQMNSL RAEDTAIYYC ARTGWLGPFD YWGQGTLVTVS S (SEQ ID NO: 101). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0313] A TSB polypeptide may contain an anti-human CTLA-4 scFv (vL-vH) polypeptide associated with ipilimumab, which has a disulfide bond linking Q101C and G44C (G167C as shown) at the vL and vH positions, with the following sequence: IVLTQSPGTL SLSPGERATL SCRASQSVGS SYLAWYQQKP GQAPRLLIYG AFSRATGIEP DRFSGSGSGTDFTLTISRLE PEDFAVYYCQ QYGSSPWTFG CGTKVEIKR G GGGSGGGGGSG GGGS QVQLVE SGGGVVQPGRSLRLSCAASG FTFSSYTMHW VRQAPGKCLE WVTFISYDGN NKYYADSVKG RFTISRDNSK NTLYLQMNSLRAEDTAIYYC ARTGWLGPFD YWGQGTLVTV SS (SEQ ID NO: 102). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0314] The TSB peptide may contain an anti-human CTLA-4 scFv (vL-vH) peptide associated with ipilimumab, which has a disulfide bond linking the vL and vH positions at G102C and K43C (K167C as shown), with the following sequence: EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP DRFSGSGSGTDFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QCTKVEIKR G GGGSGGGGGSG GGGS QVQLVE SGGGVVQPGRSLRLSCAASG FTFSSYTMHW VRQAPGCGLE WVTFISYDGN NKYYADSVKG RFTISRDNSK NTLYLQMNSLRAEDTAIYYCA RTGWLGPFD YWGQGTLVTVS S (SEQ ID NO: 103). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0315] The TSB peptide may contain an anti-human CTLA-4 scFv (vL-vH) peptide associated with ipilimumab, which has a disulfide bond linking the vL and vH positions at G102C and G44C (G168C as shown), with the following sequence: EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP DRFSGSGSGTDFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QCTKVEIKR G GGGSGGGGGSG GGGS QVQLVE SGGGVVQPGRSLRLSCAASG FTFSSYTMHW VRQAPGKCLE WVTFISYDGN NKYYADSVKG RFTISRDNSK NTLYLQMNSLRAEDTAIYYC ARTGWLGPFD YWGQGTLVTVS S (SEQ ID NO: 104). The TSB polypeptide may also contain an aa sequence having greater than about 90% or greater than about 95% sequence identity with this sequence. Alternatively, the TSB polypeptide may contain an aa sequence having greater than 96% or greater than 98% sequence identity with this sequence.

[0316] The connector sequences in SEQ ID NO: 88-104 are shown in bold and italics.

[0317] 6. Payload / Marking Payloads and markers (e.g., drug molecules) can be attached to or incorporated into constructs to facilitate their use as therapeutic and / or diagnostic agents. Types of payloads / markers that can be incorporated into constructs of this disclosure include payloads / markers for detecting the location of marker constructs (e.g., for medical imaging, etc.) and payloads / markers with therapeutic effects: some suitable payloads / markers include: (i) radioactive markers; (ii) radioisotope markers and other contrast agents; (iii) optical markers (e.g., fluorescent markers, ultraviolet / visible markers, near-infrared markers, such as IRdye700DX (IR700)); (iv) photoacoustic markers (e.g., near-infrared photoacoustic imaging (NIR-PAI)); (v) MRI / NMR markers or contrast agents; (vi) SPECT markers (e.g., 123 I, 99m Tc, 201 Tl or 111 (i) Positron emission tomography (PET) markers; (ii) paramagnetic markers; (x) chemotherapeutic agents or cytotoxic agents, etc. Some markers, such as radionuclides used for diagnostic and therapeutic purposes, may fall into more than one of the above categories.

[0318] On average, the constructs disclosed herein may contain about 0.1 to about 0.5 moles or about 0.5 to 1.0 moles of independently selected payload (e.g., independently selected radiolabeled or chemotherapeutic agents) per mole of the construct. On average, the constructs disclosed herein may contain about 1.0 to about 2.0 moles or about 2.0 to 4.0 moles of independently selected payload per mole of the construct. On average, the constructs disclosed herein may contain about 4.0 to about 6.0 moles or about 6.0 to 8.0 moles of independently selected payload per mole of the construct. On average, the constructs disclosed herein may contain about 8.0 to about 10.0 moles or more than 10 moles of independently selected payload per mole of the construct.

[0319] As described above, the constructs described herein are capable of accumulating in tumor tissue, and thus can be administered in non-tumor tissues (e.g., plasma, blood, serum, and / or peritumoral tissue) at doses below therapeutic and / or toxic levels, while accumulating at levels in tumor tissue that are at or above therapeutic and / or diagnostic levels. Therefore, the constructs of this disclosure, including those with payloads and / or labels, can be administered in non-tumor tissues at subtherapeutic levels while accumulating within the tumor at levels equal to or above the minimum therapeutic / diagnostic levels required for procedures including, but not limited to, diagnostic (e.g., imaging) and / or therapeutic effects (e.g., by delivery of radionuclides).

[0320] Radioactive payloads (radiolabels) can take various forms, including but not limited to radiolabels chelated to the constructs of this disclosure via chelating groups, which are covalently or non-covalently attached to the construct. This form is sometimes referred to as indirect labeling via recombination or chelation and is typically used with metallic radionuclide ions. Radiolabeling can also be achieved by directly adding the nuclide to the constructs of this disclosure (e.g., labeling tyrosine with potassium iodide and NaI, or forming phosphotyrosine or phosphoserine), or by indirectly covalently attaching the radiolabeled portion to the construct. Finally, radiolabels can be incorporated into the constructs of this disclosure during the translation of all or part of the construct in a cellular or cell-free translation system.

[0321] The constructs disclosed herein can be modified to possess chelating functional groups, ultimately binding to a radionuclide, typically in the form of a divalent or trivalent cation of a radiometal. Various chelating agents can be conjugated to, but are not limited to, the following construct molecules disclosed herein: diethylenetriaminepentaacetic acid (DTPA); 2,2′,2′′,2′′′-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA); 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA); and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). For example, DOTA can be conjugated with… 67 Ga、 90 Y and 111 In forms complexes and can be used to radiolabel constructs with these nuclides. Similarly, NOA can be used to... 64 Cu is used for radioactive labeling of molecules. Deferroamine (DFO) is a zirconium chelating agent suitable for the formation of DFO. 89 Zirconium-labeled constructs. The chelating agent 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid tetrahydrochloride (CPTA) can be used with copper (e.g.) 64 Cu) chelation. DFO-MAL-Cys-MZHER2 can be used for chelation. 89 Zr (see, for example, Xu et al., EJNMMI Res. 2020; 10: 58). Deferrasiro can be used to form iron chelate conjugates of the construct, see, for example, Piolatto et al. Sci Rep 11, 12581 (2021). https: / / doi.org / 10.1038 / s41598-021-91983-w. (Iron bonding) can be used to form iron chelate conjugates of the construct, see, for example, Piolatto et al. Sci Rep11, 12581 (2021). https: / / doi.org / 10.1038 / s41598-021-91983-w. Other molecules containing chelating groups can also be used, such as porphyrins, polyamines, crown ethers, dithiocarbamates, polyoximes, etc. The chelating group can be coupled to the construct using standard chemical methods, for example, by linking N-hydroxysuccinimide to an amine (e.g., a lysine side chain), or by linking maleimide to the thiol group of cysteine.

[0322] Indirect radiolabeling of the constructs described herein can be achieved by adding a radiolabeled portion. For example, the radiolabeled portion can be attached to the construct using a reagent containing an N-succinimide or N-hydroxysuccinimide group that reacts with an amine, or a maleimide group that reacts with a thiol group of cysteine. For example, a construct with exposed lysine residues can be labeled with N-succinimide-3-[halo]benzoate labeled with any halogenated isotope (e.g., fluorine, iodine, or bromine isotopes). Other reagents for labeling the construct include, but are not limited to, those labeled with halogenated radioisotopes: N-succinimide-4-iodobenzoate (PIB); N-succinimide-3-iodobenzoate (SIB); N-succinimide-5-iodo-3-pyridinecarboxylate (SIPC); tetrafluorophenyl-4-fluoro-3-iodobenzoate (TFIB); Bolton-Hunter reagent; iodine-N-(2-aminoethyl)maleimide. (IBM); (4-Benzylammonium isothiocyanate)undecylchlorodiboronate (DABI); 2,3,5,6-Tetrafluorophenyl-3-(nitrocarboryl)propionate (TCP); N-succinimide-5-guanidinomethyl-3-iodobenzoate (iso-SGMIB); N-succinimide-4-guanidinomethyl-3-iodobenzoate (SGMIB); and 1-(3-[125l]iodophenyl)maleimide (IPM).

[0323] When translation is performed in cell culture or cell-free systems, the labeling of constructs typically involves the incorporation of radioisotopes of nitrogen, oxygen, sulfur, and / or hydrogen. Phosphorus labeling may be added as a post-translational modification. Incorporation of any of these elemental isotopes can be achieved by adding labeled amino acids to the translation system employed, or, for phosphorus, by adding an appropriately labeled phosphorus compound (labeled ATP). The same process can also be used to incorporate stable isotopes suitable for imaging (e.g., 13...). C MRI imaging).

[0324] Radionuclides that can be incorporated into the construct include, but are not limited to: 11 C 14 C 13 N、 15 O、 32 P,33 P、 47 Sc、 51 Cr、 57 Co、 58 Co、 18 F、 59 Fe、 62 Cu、 67 Cu、 67 Ga、 67 Ga、 75 Br、 75 Se、 75 Se、 76 Br、 77 As、 77 Br、 80m Br、 89 Sr、 90 Y、 95 Ru、 97 Ru、 99 Mo、 94m Tc、 99m Tc、 103m Rh、 103 Ru、 105 Rh、 105 Ru、 107 Hg、 109 Pd、 109 Pt、 111 Ag、 111 In、 113m In、 119 Sb、 121m Te、 122m Te、 125m Te、 111 In、 123 I、 124 I、 125 I、 126 I、 131 I、 133 I、 142 Pr、 143 Pr、 149 Pm、 152 Dy、 153 Sm、 161 Ho、 161 Tb、 165 Tm、 166 Dy、 166 Ho、 167 Tm、 168 Tm、 169 Er、 169 Yb、 177 Lu、 186 Re、 188 Re、189m Os、 189 Re、 192 Ir、 194 Ir、 197 Pt, 198 Au、 199 Au、 199 Au、 201 Tl、 203 Hg, 211 At、 211 Bi、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 215 Po, 217At, 219 Rn、 221 Fr、 223 Ra、 224 Ac、 225 Ac、 255 Fm or Th 227 Nuclides that can be incorporated into the construct include: 11 C 14 C 62 Cu、 64 Cu、 13 N、 18 F, 35 S 、67 Ga、 68 Ga、 15 O、 94m Tc, 99m Tc, 111 In、 123 I, 124 I, 125 I, 131 I 、 and 90 Y。氚(3 (H) can also be incorporated into constructs, and labeled amino acids can be used during translation, either by adding trityl groups or through indirect labeling of amino acids, or even through exchange reactions. Although tritium is a weak beta-emitting substance, it has a half-life of approximately 12 years and can be introduced into multiple sites on a single amino acid, thereby increasing the amount of radiolabeling in the molecule or construct.

[0325] As described above, the stoichiometry of the payload to be constructed (e.g., labeled and / or conjugated chemotherapeutic agents) may be limited (e.g., within a range). For example, based on molar averages, one or more radionuclides or other payload atoms (e.g., chemotherapeutic agents or non-radiolabeled substances) may be added to each construct molecule (e.g., two or more radionuclides or other payload atoms may be added to each construct molecule). Furthermore, more than one type of radionuclide and / or other payload may be added to the construct, such that all payloads on the construct are distinct. For example, the construct may carry a radionuclide containing fluorine (e.g., fluorine). 18 F) and / or iodine (e.g. ... 125 I, 126 I, 131 I or 133 TFIB of type I) may also carry radiosensitizers or photosensitizers. See, for example, Petrov et al., Int. J. Mol. Sci. 2022, 23, 13789. Stable markers (e.g.) may also be included. 13 C and 125 I. Radioactive labeling) is incorporated into the same molecule, or even a stable label and two radionuclides are incorporated.

[0326] One or more independently selected radiosensitizers (also known as radiosensitizers) may be incorporated into or administered in combination with the constructs of this disclosure, particularly when the constructs are radiolabeled. Small molecule radiosensitizers that can be administered / utilized with the constructs described herein include cisplatin, apazolidinone, AQ4N, curcumin, dihydroartemisinin, docetaxel, doxorubicin, genistein, gemcitabine, 5-fluorouracil, misotronidazole, mitomycin C, nelfinavir, papaverine, paclitaxel (which arrests cells in the G2 / M phase, which is most sensitive to radiation damage), papaverine (papain hydrochloride), resveratrol, RRX-001, TH-302, and / or telapamine. See, for example, Gong et al., International Journal of Nanomedicine 2021:16 1083–1102.

[0327] Similar to radiosensitizers, the constructs of this invention can be labeled with one or more independently selected photosensitizers and molecules used in photodynamic therapy. A variety of molecules can be used as photosensitizers, including but not limited to various phthalocyanines and porphyrins (e.g., verteporfin).

[0328] Active therapeutic agents or chemotherapeutic agents can be incorporated into the construct as payloads to form NBD-containing construct-drug conjugates. Therapeutic agent payloads include, but are not limited to: microtubule inhibitors monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), metansine (also known as DM1), and metansine; DNA binding agent calichiomycin; and topoisomerase inhibitors delutecan, SN-38 (the active form of irinotecan), and esanotecan. Other therapeutic agent payloads include, but are not limited to, siRNA and cytotoxic agents (e.g., lavtansine (DM4) and pyrrolobenzodiazepine (PBD)).

[0329] The constructs disclosed herein may include one or more fluorescent, ultraviolet / visible (UV / Vis), and / or near-infrared payloads or labeling molecules attached to each construct. Fluorescent and UV / Vis labels include, but are not limited to, small molecules (e.g., fluorescein and rhodamine) that can be covalently attached directly or indirectly via linkers to the construct molecules disclosed herein. As an alternative to small molecule fluorescent labeling, fluorescent protein sequences (e.g., green fluorescent protein sequences) may be used as labels. Fluorescent sequences may be covalently linked via bifunctional crosslinking agents or fused to peptides of the construct to translate into fusion proteins.

[0330] Near-infrared payloads / labels include, but are not limited to, the NIR fluorophore IRDye800CW (LI-COR's 800CW) and Alexa Fluor 680 (NHS ester provided by ThermoFisher Scientific). See, for example, Chillers et al., Mol. Pharmaceutics 2017, 14, 162. 1633. Other infrared markers include IRdye700DX, a phthalocyanine dye that can be used as a photosensitizer and has been used in photoimmunotherapy. See, for example, Mitsunaga et al. 2012, NatMed.; 17(12): 1685–1691. doi:10.1038 / nm.2554 and Akalux® IV.

[0331] If the payload and tag are not translated as part of the construct, they can be directly covalently linked to the construct by forming bonds with the amino acids of the construct, or indirectly linked to the construct via a linker. The payload can be conjugated to the construct using the same type of chemical methods described for forming antibody-drug conjugates. For example, reagents containing N-hydroxysuccinimide, N-succinimide, and / or maleimide can be used to conjugate the payload to the construct. Isocyanates and isothiocyanates can also be used to form conjugates of these constructs. For example, acid anhydrides can form ester, amide, or thioester bonds with the construct. See, for example, Pelted et al., J Nanobiotechnology, 2019; 17:90. Enzyme-mediated conjugation also provides site-specific binding of the payload to the construct. Enzyme-mediated construct-payload conjugate preparation methods involve the use of enzymes such as sorting enzymes or transglutaminases, which recognize two complementary motifs, more specifically, a first motif on the construct and a second motif attached to the peptide / peptide of the payload. These enzymes can catalyze enzyme-mediated conjugation by linking these motifs.

[0332] The adapter used to attach the payload can be cleavable so that the payload / label can remain attached to the conjugate and be released in vivo (e.g., at an intratumoral location). The adapters suitable for attaching payloads (including therapeutic agents and labels) have been discussed above. Whether directly or indirectly attached via an adapter, the payload / label can be attached to a specific location (e.g., a specific amino acid) or a specific portion of the construct of this disclosure (e.g., attached to a scaffold). Amino acids in the NBD or scaffold sequence can serve as modifications or attachment points for the adapter or payload / label. If the construct contains an immunoglobulin scaffold (e.g., a heavy chain constant region such as IgFc), the scaffold can be used as a site for incorporating one or more payloads and / or one or more label molecules. For example, amino acid side chains (including lysine and / or cysteine) located in the solvent-accessible portion of the construct (e.g., the scaffold aa sequence) can serve as attachment points. If a suitable amino acid (e.g., a solvent-accessible lysine or cysteine, for example, for NHS or maleimide conjugation) is not available, the amino acid can be added to the construct of this disclosure using molecular biology tools. Gene amplification can also be used to incorporate amino acids not found in naturally occurring L-amino acids of proteogen to achieve site-specific introduction of one or more payload molecules. For example, the cyclopropene derivative CypK of lysine can be introduced via the reverse electron-demanding Diels-Alder reaction to couple a payload (see Oller-Salvia Angew Chem Int Ed Engl. 2018 Mar 5; 57(11): 2831–2834).

[0333] E. Preparation method This disclosure includes and provides methods for preparing the constructs disclosed herein by transcribing and translating nucleic acids encoding the constructs disclosed herein in mammalian cells (CHO cells), yeast cells (e.g., Pichia pastoris), beet armyworm cells, or prokaryotic cells (including bacterial cells, e.g., Escherichia coli cells). The constructs are purified from the culture medium used to culture the cells expressing the protein constructs. While these constructs can be expressed in eukaryotic and prokaryotic cells, they can be advantageously modified to avoid secondary modifications that reduce expression levels or make the protein susceptible to proteolytic degradation. One modification that may lead to poor expression in mammalian cells relates to substitutions used to create hydrolytically deficient NBDs. While replacing D668 of TAP1 NBD with an amino acid other than Asp may result in NBD hydrolytic defects or significant hydrolytic defects, substitution with Asn may also lead to N-linked glycosylation of the protein in mammalian cells, resulting in underexpression, proteolytic degradation, and / or misfolding. Therefore, D668 can be substituted with any amino acid other than Asp (e.g., D668N, D668A, or D668Q) for expression in prokaryotic cells such as *E. coli*. Constructs containing TAP-1 NBDs more suitable for expression in mammalian cells may contain D668 substituted with an amino acid other than Asp or Asn (e.g., D668A or D668Q). The amino acids in TAP2 and other ATP-binding cassette NBDs used in the constructs of this disclosure that correspond to the catalytic Asp (D668) of TAP1 can be substituted in the same manner as the NBDs of TAP1 to avoid introducing post-translational modification sequences. The constructs can be obtained by translation, which is accomplished by contacting a vector (e.g., a DNA expression plasmid, etc.) containing the nucleic acid encoding the construct of this disclosure with cells capable of expressing the encoded construct. After cell expression of the construct (which can be controlled using an inducible promoter), the construct (typically culture medium and purified construct) is collected from the culture. Example 8 illustrates one method of obtaining the construct using cell expression. In construct preparation methods, cells that transiently express the construct or cell lines that stably integrate one or more copies of the nucleic acid sequence encoding the construct can also be used. Cell-free translation or coupled transcription-translation can also be used to prepare constructs or portions of constructs, in lieu of cell-based expression.

[0334] The individual components of the constructs disclosed herein can also be prepared separately, for example, through cell expression, cell-free expression, chemical synthesis, etc., as discussed above, and then conjugated together using one or more chemical or enzymatic processes. In fact, enzymatic methods, autocatalytic protein splicing, or chemical cross-linking (e.g., using heterobifunctional cross-linking agents) can be used to couple AD to NBD, TSB to NBD, and / or NBD to ICB (e.g., covalent linkage). When the components to be coupled are all composed of aa sequences, enzymatic methods and autocatalytic protein splicing represent coupling options that achieve both stoichiometry and structure of the coupling reaction while significantly avoiding potential secondary coupling reactions. For example, sequences containing NBD can be attached to AD, TSB binders, or ICBs using sorting enzymes and cleaving introns. See, for example, Bhagawati et al., Proc Natl Acad Sci USA , 116(44):22164-22172 (2019). Alternatively, an enzyme-catalyzed SpyTag / SpyCatcher system (which may employ the CnaB2 domain of the FbaB protein from Streptococcus pyogenes) can be used to conjugate AD with peptides containing NBD. See, for example, Reddington and Howarth, Curr. Opin. in Chem. Biol. , 29: 94–99 (2015). Skilled technicians will recognize that the individual parts of a construct can be chemically modified so that they come into contact to form a complete construct, for example, by providing a solvent-accessible cysteine ​​on one part of the construct and attaching a reactive maleimide group to a second part. The maleimide and cysteine ​​can undergo a Michael reaction in aqueous solution, thereby forming a covalent bond.

[0335] For example, heterodimerized NBD (e.g., from TAP1 and TAP2) can be expressed and purified in cells (e.g., by affinity chromatography and / or size exclusion chromatography). Different aliquots of NBD can then be linked to antibodies as TSB and AD to produce a pair of molecules that can heterodimerize in the presence of ATP to form molecules capable of immunostimulation and targeting tumor cells (see, for example...). Figure 1 (B) in the example. Figure 1 B and Figure 3 The N / O example is shown in the text. In another embodiment, aliquots of heterodimerized NBD can be separately linked to TSB and ICB, which can heterodimerize in the presence of ATP to form immunostimulatory BiTE-like molecules (see, for example...). Figure 3 (C) More complex structures, including but not limited to Figure 1The structure shown in -5 can be formed by expressing or synthesizing the individual parts of the construct separately and linking them together chemically (e.g., using a heterobifunctional cross-linking agent) or enzymatically (e.g., using the enzyme-catalyzed reaction described above). The links formed by connecting the different parts of the construct can be covalent or non-covalent (e.g., biotin-avidin links).

[0336] In addition to enzymatic and chemical conjugation methods for coupling components of the constructs described herein, affinity-directed methods that combine the specificity of protein-protein interactions with chemical conjugation can also be employed. Affinity-directed chemical conjugation provides efficient, simple, and specific chemical conjugation reactions. One such method, peptide-directed photocrosslinking (PEDIP), was demonstrated by Park et al. (2018), Bioconjug. Chem., 29:3240-3244, and has been shown to couple peptides to the constant region of the antibody heavy chain. See also Kishomoto et al., (2019), Bioconjug Chem., 30(3):697-702.

[0337] If AD is not an aa sequence that can be translated in a biological system (e.g., it is a nucleic acid, such as a CpG oligodeoxynucleotide), it can be coupled to other parts of the molecule using a bifunctional cross-linking agent (e.g., homobifunctional or heterobifunctional cross-linking agent).

[0338] A variety of methods known in the art can be used to purify the construct or parts thereof. For example, if the construct contains an IgFc region as a scaffold, it can be purified by protein A or protein G chromatography. Constructs with affinity tags (such as His tags) can be purified by immobilized metal affinity chromatography (IMAC) on metal chelate affinity columns (such as nickel or cobalt affinity columns). Furthermore, 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 be used alone or in combination with affinity chromatography or IMAC to purify products.

[0339] F. Formulations and treatment methods 1. Formulation The NBD-containing constructs and complexes disclosed in this article (see, for example) Figure 1-5HThe present invention can be formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable components (e.g., excipients). The pharmaceutical composition may include: (i) one or more constructs of the present disclosure; and (ii) one or more pharmaceutically acceptable excipients, such as nonionic surfactants, stabilizers, buffers, etc., which are known in the art and therefore will not be discussed in detail herein. Pharmaceutically acceptable excipients are described in various publications, including but not limited to "Remington: The Science and Practice of Pharmacy", 19th edition (1995) or the latest edition, Mack Publishing Co; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc, and the latest editions mentioned above.

[0340] This disclosure also provides pharmaceutical compositions comprising nucleic acids or recombinant expression vectors that encode all or part of one or more constructs of this disclosure.

[0341] The pharmaceutical compositions disclosed herein are typically in the form of aqueous solutions or other solutions. This disclosure also considers pharmaceutical compositions in the form of powders, granules, tablets, pills, suppositories, capsules, sprays, etc. The compositions can be formulated according to the following routes of administration.

[0342] This disclosure provides a composition comprising at least one construct disclosed herein and physiological saline (e.g., 0.9% or about 0.9% NaCl), optionally buffered to a suitable pH (e.g., with phosphate buffer), the composition being sterile and free of detectable pyrogens and / or other toxins, or with detectable pyrogens and / or other toxins below permissible limits.

[0343] Therefore, this disclosure includes and provides the use of the NBD-containing constructs of this disclosure in the preparation of medicaments for therapeutic applications, including but not limited to treating cancer in patients (e.g., mammalian patients, including humans). This disclosure also includes and specifies the use of the NBD-containing constructs of this disclosure in treating mammalian patients (e.g., humans), including for treating cancer in these patients.

[0344] The composition can be formulated for parenteral and / or other administration. The construct or composition containing the construct can be administered via any suitable route, such as intravenous, intramuscular, subcutaneous, intratumoral, or intralymphatic administration. The construct can also be administered in combination with one or more additional agents in a treatment regimen, which may act synergistically or non-synergistically with the construct. The additional agents can be administered before, simultaneously (alone or in combination), or after the construct. For example, the construct of this disclosure (e.g., a construct having an IL-12 activation domain) can be administered together with an immune checkpoint inhibitor (e.g., pembrolizumab (Keytruda®, targeting PD-1), avelumab (Bavencio®, targeting PD-L1), or ipilimumab (Yervoy®, targeting CTLA-4)).

[0345] Because cancers may evade treatment of the presently disclosed construct by expressing CD39 (which hydrolyzes ATP to ADP) or CD38 (which initiates a cascade of reactions that cleave NAD to adenosine, thereby inhibiting T cell activity), inhibitors of one or both of these molecules may be administered in combination with any therapeutic use of the construct. The inhibitors may be administered before, concurrently (e.g., mixed or alone), and / or after the CD38 and / or CD39 inhibitors. CD38 inhibitors include, but are not limited to, daratumumab (Darzalex®), ixartuximab (Sarclisa®), and combinations thereof. CD39 inhibitors include, but are not limited to, ceritinib (Zykadia®) (see J Immunother Cancer Aug 2022; 10(8):e004660. doi:10.1136 / jitc-2022-004660).

[0346] 2. Treatment methods Any solid tumor containing a higher level of ATP compared to normal tissue can be treated using the constructs disclosed herein. While cancers such as lymphoma may not form solid tumors, they may aggregate or accumulate (e.g., within tissue) to form “liquid tumors.” Cancer cells that produce these aggregates may be treated with the constructs described herein if the aggregated cells produce locally elevated levels of extracellular ATP. In fact, even small aggregates or single cells that produce high concentrations of extracellular ATP can be treated with the constructs described herein. Treatable tumors include, but are not limited to, mesotheliomas (e.g., pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma), melanomas, sarcomas (e.g., synovial sarcoma, soft tissue sarcoma, osteosarcoma, and liposarcoma), carcinomas (e.g., adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), carcinosarcomas, lymphomas, 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, a tumor can be lung cancer, liver cancer, skin cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, or a tumor of the reproductive tissue (such as ovarian sarcoma).

[0347] Treatment methods may include, for example, administering one or more constructs of this disclosure to a patient or subject, or administering one or more molecules comprising one or more nucleic acid sequences encoding one or more constructs of this disclosure. NBD-containing constructs may be administered at a systemically safe level (e.g., a subtherapeutic level and / or showing at most limited side effects in a systemically stimulated cycle), but accumulate as a dimer in the TME to therapeutically effective amounts and / or levels due to ATP-driven dimerization and / or due to the targeting effect of TSB (when present). Depending on the specific elements present in the construct, they may act through a variety of mechanisms, including but not limited to inducing Ig-mediated effector function, activating immune cells (e.g., NK cells, T cells (such as effector T cells), neutrophils, and / or macrophages), inhibiting or depleting myeloid suppressor cells and / or T reg cells in the TME, and / or unblocking checkpoint protein-mediated immune cell function. In addition to the foregoing effects, NBD, alone or as part of a construct containing other elements, may reduce ATP in the TME through chelation, or NBD may have hydrolytic activity that may reduce ATP in the TME through degradation. A decrease in ATP leads to a reduction in purinergic signaling, which has been shown to mediate a variety of cancer-related processes, including cell migration, resistance to cytotoxic therapies, and immune regulation. This is consistent with the abundance of ATP-regulated purinergic receptors on cancer cells, stromal cells, and various immune cell types. See, for example, Draganov & Lee, Adv Exp Med. Biol. 2021;1270:73-87 (doi: 10.1007 / 978-3-030-47189-7_5).

[0348] While the constructs of this disclosure allow for therapy via an immune response targeting tumor cells, their use in conjunction with various payloads (e.g., radiolabels (radionucleases), radiosensitizers, photosensitizers, chemotherapeutic agents, etc.) enables additional therapeutic options and combination therapies. NBD-containing constructs, comprising one or more payloads, optionally including one or more scaffolds, and containing only one NBD, can accumulate in the TME as a dimer due to ATP-driven dimerization. Constructs of this disclosure include (i) one or more payloads and an NBD, or (ii) one or more payloads, an NBD, and a scaffold (e.g., an immunoglobulin scaffold or a non-immunoglobulin scaffold), but lacking TSB and ICB, which can be used to administer therapy as a tissue (e.g., tumor)-specific / selective payload delivery mechanism via dimerization in a high-ATP environment (e.g., the TME). While these constructs can be used on their own for therapeutic purposes on tumors (e.g., inducing ADCC, ADCP, or CDC), the presence of one or more payloads allows for one or more other therapies. Other therapies include, but are not limited to: chemotherapy when at least one payload is a chemotherapeutic agent; radiotherapy when at least one payload is a radionuclide or radiosensitizer; and photodynamic therapy when at least one payload is a photosensitizer. Adding an ICB or AD to form a construct comprising (i) an ICB or AD, one or more payloads, and an NBD, or (ii) an ICB or AD, one or more payloads, an NBD, and a scaffold, thereby allowing a combination of ICB or AD-mediated immunotherapy and payload delivery to achieve one or more second-additional therapies (e.g., chemotherapy, radiotherapy, photodynamic therapy, etc.). Adding a TSB to form a construct comprising (i) a TSB, one or more payloads, and an NBD, or (ii) a TSB, one or more payloads, an NBD, and a scaffold, not only increases tumor-selective targeting that enhances selective payload delivery, but also allows a combination of TSB-mediated immunotherapy and payload delivery to achieve one or more additional treatments (e.g., chemotherapy, radiotherapy, photodynamic therapy, etc.). The formation of constructs by adding TSB and ICB or AD to form constructs comprising (i) TSB, ICB or AD, one or more payloads and NBD, or (ii) TSB, ICB or AD, one or more payloads, NBD and a scaffold allows for the combination of one or more immunotherapies with payload delivery to achieve one or more additional treatments. When any construct contains a detectable marker (e.g., a radionuclide or a radioisotope), they can also be used for diagnostic purposes.This construct can be administered at a level that is safe for systemic stimulation in one or more non-tumor tissues (e.g., in plasma or peritumoral tissue), or even at a potentially subtherapeutic level in non-target tissues such as the TME, but accumulates to a therapeutic level in the TME.

[0349] One or more payloads may be used for the diagnosis or treatment of a patient. For example, a chemotherapy payload attached to a construct via a cleavable connector may be released (e.g., significantly or completely) within the TME. When one or more payloads contain one or more markers that allow detection of the construct (e.g., markers that allow MRI or CT detection), the construct may be used to determine the location and size of a tumor, thereby aiding in the assessment of the progress of treatment and / or surgical interventions. When one or more payloads contain radiolabels and / or radiosensitizers, the construct may be used for radioimmunotherapy. Similarly, if the construct contains photosensitizers, the construct may be used for photodynamic therapy. Constructs containing radiolabels as well as radiosensitizers and / or photosensitizers may be provided in one or more non-tumor tissues (e.g., plasma or peritumoral tissue) at a systemically stimulating, safe dose (potentially even below therapeutic levels), but accumulate to therapeutic levels within the TME, thereby providing (i) a radiation source that can be supplemented by an external radiation source, and (ii) a cleavable sensitizer attached to the construct. When a radiolabeled construct contains photosensitizers, radiotherapy and / or photodynamic therapy are possible. By placing a radionuclide and a sensitizer on the same isodimerizable molecule, delivery of one or more radiolabeled and sensitizer-enhancing agents can be achieved. Alternatively, a pair of heterodimerized NBD constructs can be used, wherein the first construct contains the radiolabel (radionuclide) and the second construct contains the sensitizer. The formation and accumulation of heterodimers in the tumor provide the TME with the radionuclide and sensitizer, thereby facilitating treatment, allowing the use of photodynamic therapy and / or radiotherapy, and potentially supplemented by external radiation (e.g., X-ray radiation).

[0350] NBD-containing constructs, comprising TSB and one or more payloads, accumulate in the tumor microenvironment (TME) as dimers or higher-order complexes and target tumor cells. These constructs can be administered at systemically stimulating levels (or even potentially subtherapeutic levels) in one or more non-tumor tissues (e.g., in plasma or peritumoral tissue), but accumulate in the TME to therapeutic levels. In addition to any effects induced by the binding of the construct to tumor cells via ATP-driven dimer or higher-order complexes (e.g., induction of apoptosis), the payload may produce additional effects that aid in patient diagnosis or treatment. For example, chemotherapy payloads can act on tumors, while markers capable of detecting tumors can help determine treatment regimens and / or surgical interventions. When the payload contains a radiolabel or radiosensitizer, TSB-containing constructs can be used for radioimmunotherapy. Similarly, if the construct contains a photosensitizer, it can be used for photodynamic therapy. Constructs containing TSB, a radiolabel, and additional radiosensitizers and / or photosensitizers can be delivered at a dose that is safe for systemic stimulation (even potentially at subtherapeutic levels) in one or more non-tumor tissues (e.g., in plasma or peritumoral tissue) but accumulates to therapeutic levels in the TME, providing (i) a radiation source that can be supplemented by an external radiation source, and (ii) a sensitizer that can be cleavably linked to the construct. When such a radiolabeled construct contains a photosensitizer, radiotherapy and / or photodynamic therapy are possible. Delivery of one or more radiolabels and sensitizers can be achieved by providing the radionuclide and sensitizer on the same construct molecule that can be dimericated. Alternatively, a pair of constructs containing heterodimeric NBDs can be used, wherein the first construct contains the radionuclide (radiolabel) and the second construct contains the sensitizer. The formation and accumulation of heterodimers in tumors provide radionuclides and sensitizers in the TME, thereby facilitating therapeutic treatment by allowing the use of photodynamic therapy and / or radiotherapy, which may be supplemented by the application of exogenous radiation.

[0351] NBD-containing constructs, comprising one or more ICB and / or one or more AD aa sequences and one or more payloads, accumulate in the tumor microenvironment (TME) as dimers or higher-order complexes and induce one or more immune responses that directly or indirectly affect tumor cells as bystanders. Such constructs can be administered at systemically stimuli-safe levels (or even potentially subtherapeutic levels) in one or more non-tumor tissues (e.g., in plasma or peritumoral tissue), but accumulate in the TME to therapeutic levels due to the formation of ATP-driven dimers and / or higher-order complexes. In addition to any immune responses (e.g., cytotoxic T cell activation) induced by ICB and / or AD in the ATP-driven dimers or higher-order complexes of the constructs, any present payload can deliver additional effects that aid in patient diagnosis or treatment. For example, chemotherapy payloads can act on tumors, while markers capable of detecting tumors help determine treatment regimens and / or surgical interventions. Constructs containing ICB and / or AD can be used for radioimmunotherapy when the payload contains a radiolabel or radiosensitizer. Similarly, if the construct contains a photosensitizer, it can be used for photodynamic therapy. Constructs comprising ICB and / or AD, a radiolabel, and additional radiosensitizers and / or photosensitizers can be delivered at doses that are safe for systemic stimulation (even potentially at subtherapeutic levels) in one or more non-tumor tissues (e.g., in plasma or peritumoral tissue) but accumulate to therapeutic levels in the TME, providing (i) a radiation source that can be supplemented by an external radiation source, and (ii) a sensitizer that is cleavably linked to the construct. If such a radiolabeled construct contains a photosensitizer, radiotherapy and / or photodynamic therapy are also possible in addition to any immunotherapeutic effects of the construct (e.g., cytotoxic T cell activation). Delivery of one or more radiolabels and sensitizers can be achieved by placing the radionuclide and the sensitizer on the same isodimerizable molecule. Alternatively, a pair of heterodimerized NBD constructs can be used, wherein the first construct contains a radionuclide (radiolabel) and the second construct contains a sensitizer. The formation and accumulation of heterodimers in tumors provide radionuclides and sensitizers in the TME, thereby facilitating therapeutic treatment by allowing the use of photodynamic therapy and / or radiotherapy, which may be supplemented by the application of exogenous radiation.

[0352] The therapeutic efficacy of the NBD-containing constructs described herein can be enhanced by using more than one type of treatment regimen, as measured, for example, by reductions in tumor size, tumor number, circulating tumor nucleic acid, etc. As mentioned above, NBD-containing constructs may induce one or more types of immune responses, leading to targeted killing of tumor cells. Other treatment regimens to enhance efficacy may include using one or more chemotherapeutic agents as a payload or in combination. Other treatment regimens to enhance efficacy also include using radiolabeling, radiosensitizers, and / or photosensitizers as payloads for the NBD-containing constructs of this disclosure. In addition to improved efficacy, the use of such payloads can also improve the safety of treatment (reducing side effects). This is especially true when using radiosensitizers and photosensitizers, as the area where the tumor is located can be irradiated with radiation and / or light at appropriate frequencies (e.g., near-infrared) and the dose of radiation and light can be limited.

[0353] Using two or more TSBs that target different TAAs expressed on the same tumor cells may also improve the therapeutic selectivity and safety of the NBD-containing constructs described herein. Alternatively, two or more TSBs may target at least two different epitopes (non-overlapping epitopes) expressed on the TAA.

[0354] To achieve selective action in target tissues (e.g., in a TME) using the constructs described herein, the constructs should dimerize or form higher-order complexes (dimerization / complex formation) at ATP concentrations found in the target tissue, but not at ATP concentrations found in non-target tissues. To determine whether a patient or subject may benefit from treatment using the constructs described herein, and / or to select a construct that dimerizes / complexes at ATP concentrations found in their target tissue, the methods and uses of the constructs described herein (including the capping or lattice-forming methods described below) may include measuring ATP concentrations in the target tissue, non-target tissue, or both. ATP concentrations in tissues surrounding the target tissue (e.g., peritumoral tissue) may also be measured, in lieu of or supplementing measurements of ATP concentrations in non-target tissues. Depending on whether the degree of ATP-mediated dimerization / complexing in the target tissue is acceptable and / or desirable (especially in a therapeutic setting), measurements in surrounding non-target tissues may be more or less informative in selecting a construct that dimerizes / complexes at appropriate ATP concentrations. For example, in cases where potential damage to one or more surrounding tissues during treatment is a critical concern, it may be more appropriate to select EC50 constructs that form dimer / complexes at ATP concentrations slightly higher than those in the target tissue. This would allow a significant portion of the constructs to dimerize / complexes in that tissue (even if less than half of the constructs dimerize in that tissue), since even fewer dimer / complexes would form in surrounding tissues with lower ATP concentrations.

[0355] Increasing the proportion of constructs in ATP-mediated dimers / complexes can improve their localization and residence time in target tissues, especially when they contain TSB elements, partly due to the increased effective binding energy, which translates into increased affinity for the corresponding TAA (tear-associated amino acid). Increasing the proportion of constructs in ATP-mediated dimers / complexes is particularly desirable for constructs carrying payloads (e.g., radiolabeled and chemotherapeutic agents) that can exert their therapeutic effect without external stimulation (e.g., radiation or light in photodynamic therapy). This is also beneficial for payloads that require external stimulation for treatment (e.g., photosensitizers or radiosensitizers), as damage to non-target tissues can be significantly reduced when used in combination with other therapeutic methods, especially when external stimulation can be precisely applied to target tissues (e.g., light or radiation from one or more external sources is focused onto the target tissue).

[0356] As described above, these ATP measurements can be used to help select constructs that dimerize at appropriate ATP concentrations to produce therapeutic effects associated with construct dimer / complex formation, while limiting side effects from acting on non-target tissues. ATP levels in non-target and / or target tissues can be measured before or during treatment with the NBD-containing constructs described herein. Measurements of ATP concentrations in both target and non-target tissues can also be performed after any single or multiple administrations of the construct. Concentrations can be achieved in target tissues (e.g., TME of solid tumors), near non-solid cancers (where cells are localized to a specific location, such as an organ or tissue), or in non-target tissues using methods known in the art (see, for example, Rajendran et al., Biol Bull. 2016; 231(1): 73–84). Methods for assessing ATP concentrations in vivo include, but are not limited to, magnetic resonance imaging (MRI) / magnetic resonance spectroscopy (MRS), including phosphorus MRI (see, for example, Gams et al. Curr. Oncol. 2021, 28, 5041–5053 and...). Cancer et al. 2021, 13, 3569. In addition, analysis can be performed using liquid chromatography-mass spectrometry, microelectrode measurements, and molecular probes (e.g., fluorescent or luminescent probes) (e.g., biopsy samples).

[0357] Since typical ATP concentrations in non-target tissues are typically 0.01 µM to 0.1 μM (100 nM), the EC50 for ATP-induced dimerization / complexation can be selected to be higher than the range of ATP concentrations in non-target tissues. Concentrations in target tissues (such as the TME) can range from about 50 to about 1,000 μM or even higher, and the EC50 for ATP-induced dimerization / complexation can be selected to be lower than the lower limit of the target tissue concentration range, or slightly higher than the concentrations found in the target tissue. Therefore, the range of EC50 values ​​for ATP-induced dimerization / complexation consistent with the selective use of the constructs or compositions described herein in target tissues includes the following ranges. The constructs or compositions for ATP-induced dimerization / complexation have EC50 values ​​in the range of about 0.1 μM to about 0.5 μM ATP, or in the range of about 0.5 μM to about 5.0 μM ATP. The construct or composition is directed against ATP-induced dimerization / complexation, and its EC50 value is in the range of about 5.0 μM to about 50 μM ATP, or in the range of about 50 μM to about 250 μM ATP. Furthermore, the construct's EC50 value for ATP-induced dimerization / complexation is also in the range of about 250 μM to about 1 mM or higher ATP concentrations. Typically, by screening for specific constructs, the highest target tissue specificity and lowest off-target effects can be achieved; such constructs have an EC50 value of approximately 0.2 to 1 times the ATP concentration in the target tissue (e.g., up to about 2 times the ATP concentration in the target tissue), or approximately 0.4 to 1 times the ATP concentration in the target tissue, and at least 10 times the ATP concentration in non-target tissues.

[0358] Demonstration of ATP-induced dimerization of the construct (or its NBD) can be performed by size exclusion chromatography in, for example, a buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 10% glycerol, optionally with the addition of 0.005% Tween (v / v) (e.g., using a Superdex 200 10 / 300 Increase or 16 / 600 column (depending on the total protein content)), at a temperature of 20 to 24 °C. By running the NBD-containing protein in the buffer (as described above), with or without ATP, and comparing the resulting chromatograms to observe the protein retention time, the monomeric and dimer forms can be determined.

[0359] The EC50 value of ATP-induced constructs (or their NBDs) dimerization can be measured using biolayer interferometry (BLI) with an Octet Red 96e instrument (Pall ForteBio LLC, Freemont, CA) at 20–24 °C by observing changes in signal (i.e., interference patterns of white light reflected from the biolayer constructed on the biosensor tip) at different ATP concentrations (e.g., by ATP titration) in 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2·6H2O hexahydrate, and 0.005% Tween (v / v) pH 7.0. For constructs containing TSBs, EC50 is evaluated by immobilizing the target TAA on the biosensor tip and observing changes in the signal of the construct binding to the probe due to increased affinity of the dimer form at different ATP concentrations. For other constructs, particularly when forming heterodimeric construct pairs, a portion of the construct (e.g., the first member of the heterodimer) can be immobilized on a surface probe (e.g., for IgFc-containing constructs, a streptavidin-coated probe can be used, or an antibody can be immobilized on a portion of the construct, preferably excluding the NBD). An additional construct (e.g., the second member of the heterodimer pair) is exposed to the construct immobilized on the surface probe, and its response (signal changes) at different ATP concentrations is observed. The midpoint of the ATP-induced response of the BLI probe represents the EC50.

[0360] As an alternative to measuring ATP to determine whether a patient or subject can benefit from treatment with the constructs of this disclosure, the concentration or expression level of CD73 in the target tissue can be assessed. The assessment can be performed relative to control non-target tissue or peritumoral target tissue. Elevated CD73 levels (e.g., relative to control tissue) indicate that a patient or subject may benefit from treatment with the constructs of this disclosure, as CD73 is thought to be associated with elevated extracellular adenosine / ATP levels. Therefore, any treatment method or method of use of the constructs disclosed herein may include assessment of CD73 in target tissue (tumor TME tissue), non-target tissue (such as peritumoral tissue), or both.

[0361] 3. Capping or lattice formation methods Capping (CAP formation) or lattice formation refers to the recombination of molecules on or inside the cell membrane from a dispersed state (e.g., widely distributed on the cell surface) into one or more aggregates on the cell surface. This disclosure includes and provides methods for inducing capping or lattice formation on the surface of immune cells (e.g., T cells, macrophages, NK cells, etc.) or cancer cells. These methods may include administering an NBD-containing construct or complex of this disclosure to a patient. The method may use a construct having a single NBD (e.g., a TAP1 or TAP2 NBD that can bind two ATP molecules) and a single ICB or a single TSB. Examples of such constructs include homodimerized polypeptides (fusion proteins / peptides) comprising an NBD, an ICB, and an optional scaffold sequence (e.g., an IgFc CH2-CH3 aa sequence) comprising a single NBD (e.g., homodimerized) and an ICB or AD. Examples of such constructs include homodimerized polypeptides (fusion proteins / peptides) comprising an NBD, a TSB, and an optional scaffold sequence (e.g., an IgFc CH2-CH3 aa sequence).

[0362] The ability of a construct to form a cap or lattice on a cell surface can be evaluated in vitro using labeled constructs and appropriate microscopy methods (e.g., fluorescently labeled constructs and an optical microscope equipped with a suitable excitation source and visualization optics).

[0363] Capping performed by the constructs disclosed herein, including intentional capping via a construct with a cell surface antigen as monovalent (even after becoming divalent upon exposure to ATP), can induce a response in the cell. For example, capping proteins on the surface of effector T cells may induce activation (e.g., granule-dependent or granule-independent responses). Furthermore, when the construct contains an IgFc sequence capable of inducing ADCC, ADCP, or CDC, the capping of the target protein and the corresponding binding of the construct to the cell surface can enhance one or more of ADCC, ADCP, or CDC. Therefore, a construct having a TSB and containing an IgFc sequence capable of inducing ADCC, ADCP, or CDC, inducing capping / lattice formation in the TME due to elevated ATP levels, can be used to achieve any of the above processes.

[0364] VI. Aspects 1. A construct comprising a polypeptide, said polypeptide comprising: (i) The amino acid (aa) sequence of the first nucleotide binding domain (NBD) and (ii) Scaffolds (e.g., immunoglobulin heavy chain constant regions, such as scFc or KiH) S-S IgFc)aa sequence They are directly linked or linked via a linker peptide aa sequence (e.g., as fusion proteins / peptides); in (i) When the scaffold is an immunoglobulin heavy chain constant region (e.g., IgFc such as scFc)aa sequence, it is capable of stimulating one or more immune cell effector functions (e.g., by binding to Fcγ receptors), and (ii) The NBD contains one or more adenosine triphosphate (ATP) binding sites and, in the presence of ATP, can homodimerize or heterodimerize with a homologous, non-identical second NBD that is optionally linked to a second IgFc aa sequence via an optional linker (e.g., as a fusion protein / peptide). (See, for example) Figure 5A (AE in the text). It should be understood that the existing NBD can be defective in ATP hydrolysis.

[0365] 2. A construct of aspect 1, wherein the construct has Figure 5A The structure shown in A or B.

[0366] 3. The construct of aspect 1, which also contains a second NBD.

[0367] 4. A construct of aspect 3, wherein the construct has Figure 5A The structure shown in any of the CE diagrams.

[0368] 5. A construct of any one of aspects 1-4, wherein the immunoglobulin heavy chain constant region comprises a single-chain IgFc, the single-chain IgFc comprising a first immunoglobulin heavy chain constant region aa sequence and a second immunoglobulin heavy chain constant region aa sequence (e.g., a fusion polypeptide comprising a pair of Ig CH2-CH3 aa sequences, the effector function of which is optionally attenuated (e.g., significantly or completely)), wherein the first and second heavy chain aa sequences are optionally linked by one or more disulfide bonds (e.g., corresponding to the lower hinge region in wt. IgG sequence).

[0369] 6. A construct comprising: (i) A first polypeptide comprising a first immunoglobulin heavy chain constant region aa sequence (e.g., containing an Ig CH2-CH3aa sequence whose effector function is optionally attenuated (e.g., significantly or completely)) and an NBD amino acid (aa) sequence optionally linked directly or via a linker aa sequence to the first immunoglobulin heavy chain constant region aa sequence, and (ii) A second polypeptide comprising a second immunoglobulin heavy chain constant region aa sequence (e.g., containing a CH2-CH3 aa sequence whose effector function is optionally weakened (e.g., significantly or completely)) and an NBD aa sequence optionally linked directly or via a linker aa sequence to the second immunoglobulin heavy chain constant region aa sequence; in (i) At least one of the first and second polypeptides contains NBD (first NBD), (ii) Each NBD amino acid sequence contains one or more ATP-binding sites and, in the presence of ATP, can homodimerize or heterodimerize with a homologous, non-identical second NBD.

[0370] (iii) The first and second immunoglobulin heavy chain constant region aa sequences dimerize to form CH2-CH3 dimers (e.g., IgFc or IgFc-like structures) optionally capable of stimulating one or more effector functions, and (iv) Each existing connector sequence is selected independently. (See example) Figure 5B (AE in the text.) It should be understood that existing NBDs can be defective in ATP hydrolysis.

[0371] 7. A construct of aspect 6, wherein the first polypeptide comprises a first NBD.

[0372] 8. A construct of aspect 6, wherein the second polypeptide comprises a first NBD.

[0373] 9. A construction of aspect 6, wherein the first or second construction further comprises a second NBD.

[0374] 10. A construct of aspect 9, wherein the first polypeptide comprises a second NBD. (The NBD is restricted such that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP. See, for example...) Figure 5B The structure AE is in the middle, where the first and second positions are NBD, and the structures F, H and J are in the middle.

[0375] 11. A construct of aspect 9, wherein the second polypeptide comprises a second NBD. (The NBD is restricted such that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP. See, for example...) Figure 5B The structure AE in the text, where the 3rd and 4th bits are NBD).

[0376] 12. A construct of any of aspects 6-7, wherein the first polypeptide comprises a first NBD and the second polypeptide comprises a second NBD. (The NBDs are restricted such that two NBDs within the same molecule cannot interact to form a complex in the presence of ATP. See, for example...) Figure 5B The structure AE, where one of the first and second positions and one of the third and fourth positions is NBD, and the structures G, I, K and L).

[0377] 13. A construct of any of aspects 5-8, wherein the first and second immunoglobulin heavy chain constant region aa sequences form a heterodimer (e.g., a pair of interspecies aa sequences).

[0378] 14. The construct of aspect 13, wherein the first and second immunoglobulin heavy chain constant region aa sequences comprise interspecies aa sequence pairs selected from KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 aa sequences.

[0379] 15. A construct of either aspect 13 or 14, wherein the first and second immunoglobulin heavy chain constant region aa sequences comprise KiH or KiHs-s aa sequence pairs.

[0380] 16. A construct of any of aspects 5-14, wherein the first and second immunoglobulin heavy chain constant region aa sequences are linked by one or more (e.g., two or more) disulfide bonds between these sequences.

[0381] 17. A construct of any of aspects 1-16, wherein each immunoglobulin heavy chain constant region aa sequence comprises an independently selected IgG1, IgG2, IgG3, IgG4 or IgM aa sequence (e.g., SEQ ID NO: 63-66, or a sequence having greater than 96% or greater than 98% sequence identity with any one of SEQ ID NO: 63-66).

[0382] 18. A construct of any one of aspects 1-17, wherein each immunoglobulin heavy chain constant region aa sequence comprises an IgG1 aa sequence.

[0383] 19. An embodiment of any of aspects 1-18, wherein the immunoglobulin heavy chain constant region aa sequence comprises one or more substitutions, the substitutions resulting in an enhancement of one or more effector functions relative to an immunoglobulin heavy chain constant region lacking said substitutions.

[0384] 20. A construct of aspect 19, wherein each immunoglobulin heavy chain constant region aa sequence comprises an IgG1 aa sequence.

[0385] 21. An embodiment of aspect 20, wherein one or more (e.g., each) immunoglobulin heavy chain constant region aa sequences contain substitutions that enhance the ability to enhance sequence ...

Claims

1. A construct comprising: (i) The first polypeptide, composed of the amino acid sequence of the constant region of the first immunoglobulin heavy chain, and (ii) A second polypeptide consisting of the amino acid sequence of the constant region of the second immunoglobulin heavy chain; in (i) At least one of the first and second polypeptides comprises an NBD amino acid sequence that is directly or indirectly linked to the constant region amino acid sequence of the first or second immunoglobulin heavy chain, an NBD amino acid sequence having at least 90% amino acid sequence identity with the capped NBD amino acid sequence of SEQ ID NO:294, or a sequence having at least 90% sequence identity with it, said sequence being capable of homodimerization in the presence of ATP. (ii) The amino acid sequences of the first and second immunoglobulin heavy chain constant regions form IgFc, whose effector function is optionally attenuated (e.g., significantly or completely), and (iii) Each existing connector sequence is selected independently.

2. The construct of claim 1, wherein the amino acid sequences of the first and second immunoglobulin heavy chain constant regions comprise interspecies amino acid sequence pairs selected from: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 amino acid sequences.

3. The construct according to claim 2, wherein the interspecific amino acid sequence pair is a KiH or KiHs-s sequence pair.

4. The construct of claim 3, wherein at least one of the first or second polypeptides of the construct comprises a payload or a tag, wherein: (i) The payload or marker contains one or more independently selected therapeutic or chemotherapeutic agents, and / or (ii) The payload or marker includes one or more independently selected radioactive payloads or markers.

5. The construct of claim 4, further comprising one or more independently selected tumor-specific binding amino acid sequences (TSBs).

6. The construct of claim 5, wherein the amino acid sequence of one or more TSBs is selected from: nanobody, scFv, and VH (heavy chain variable fragment only) amino acid sequences.

7. The construct of claim 5, wherein the TSB binds to one or more tumor-associated antigens (TAAs), said tumor-associated antigens being independently selected from mucin, mesothelial (MSLN), EpCAM, CTLA-4, VISTA, TIM-3, PD-L1, and LAG-3 proteins.

8. A construct comprising: (i) A first polypeptide comprising a first scaffold amino acid sequence including a first Ig heavy chain constant region amino acid sequence and a first and / or second NBD amino acid sequence directly or via a linker amino acid sequence linked to the first scaffold amino acid sequence, and (ii) A second polypeptide comprising a second scaffold amino acid sequence containing an amino acid sequence of the Ig heavy chain constant region and a first and / or second NBD amino acid sequence optionally directly or via a linker amino acid sequence linked to the second scaffold amino acid sequence; in (i) At least one of the first and second polypeptides contains their first NBD amino acid sequence, each of the first NBD amino acid sequences having at least 90% amino acid sequence identity with, or having at least 90% sequence identity with, the capped NBD amino acid sequence of SEQ ID NO: 294, said sequence being capable of homodimerization in the presence of ATP. (ii) Each NBD amino acid sequence contains one or more ATP-binding sites and can dimerize in the presence of ATP. (iii) At least one of the first and second polypeptides contains one or more ICB and / or one or more AD amino acid sequences. (iv) The first and second stent sequences form a dimer through the interaction between the first and second stent sequences, and (v) Each existing connector sequence is selected independently.

9. The construct of claim 8, wherein the only NBD amino acid sequence in the construct is the first NBD of the first polypeptide, and the second polypeptide contains only one ICB or AD amino acid sequence in the construct.

10. The construct of claim 9, wherein the amino acid sequences of the first and second immunoglobulin heavy chain constant regions comprise interspecies amino acid sequence pairs selected from KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 amino acid sequences.

11. The construct of claim 10, wherein at least one of the first or second polypeptides of the construct comprises a payload or a tag, wherein: (i) The payload or marker contains one or more independently selected therapeutic or chemotherapeutic agents, and / or (ii) The payload or marker includes one or more independently selected radioactive payloads or markers.

12. The construct of claim 11, wherein the interspecific amino acid sequence pair is a KiH or KiHs-s sequence pair.

13. The construct of claim 12, wherein the ICB and / or AD amino acid sequences have an affinity for proteins selected from the group consisting of: CD3, CD2, CD4, CD8, CD13, CD16, CD25, CD28, CD33, CD34, CD66, CD68, CD84, CD137 / 4-1BB, CD163, CD193, CD206, CXCR1, DR5, FcεR1α, αβTCR, TCRα chain, TCRβ chain, δγ TCR, TCRγ chain, TCRδ chain, and TRGV9.

14. The construct of claim 13, wherein the ICB and / or AD amino acid sequences have an affinity for CD28 or CD137 / 4-1BB.

15. The construct of claim 14, wherein the ICB and / or AD is an anti-CD28 antibody, an anti-CD137 antibody, the 4-1BBL sequence of SEQ ID NO: 114 or 115, the 4-1BBL trimer aa sequence of SEQ ID NO: 116, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 114-116.

16. A complex comprising the construct of any one of claims 1-15, and one or more ATP molecules.

17. A pharmaceutical composition comprising a construct of any one of claims 1-15, and at least one pharmaceutically acceptable excipient, wherein the composition is sterile and free of detectable pyrogens, or the pyrogens are below acceptable limits.

18. A method of treating a human patient with cancer, the method comprising administering the pharmaceutical composition of claim 17 to the patient.

19. The method of claim 18, wherein the cancer is selected from: mesothelioma, melanoma, sarcoma, carcinoma, carcinosarcoma, lymphoma, and germ cell tumor.

20. The method of claim 19, wherein the cancer comprises one or more solid tumors.

21. The method of claim 20, wherein one or more of the solid tumors are tumors of the lung, liver, skin, gastrointestinal tract, pancreas, brain, or reproductive tissue.

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