Antigen-presenting polypeptide complexes with TGF-β and methods of use thereof

Abstract

The present disclosure provides multimeric antigen-presenting polypeptides (MAPPs) that include a peptide epitope and a reversibly masked TGF-β peptide capable of acting as an agonist of a cellular TGF-β receptor. MAPPs are capable of presenting peptide epitopes to T cells in the context of class II MHC receptors. The present disclosure provides nucleic acids that include nucleotide sequences encoding those MAPPs, as well as cells engineered with nucleic acids encoding MAPPs. MAPPs are useful for selectively modulating the activity of T cells that have a T cell receptor that recognizes an epitope presented by the MAPPs. Thus, the present disclosure provides compositions and methods for modulating the activity of T cells, as well as compositions and methods for treating individuals with diseases and / or disorders, including autoimmune diseases, graft-versus-host disease, host-versus-graft disease, and / or allergies.

Description

[Technical Field]

[0001] This application contains a Sequence Listing that has been submitted electronically via EFS-web, which serves as both a paper copy and a computer readable form (CRF), consisting of a file entitled "2910_28PCT_seqlist_ST25", created on April 20, 2022, 422,756 bytes in size, and incorporated herein by reference in its entirety.

[0002] I. Introduction The adaptive immune response involves the engagement of T cell receptors (TCRs) present on the surface of T cells with small antigen molecules noncovalently presented on the surface of antigen-presenting cells (APCs) by major histocompatibility complexes (MHCs, also referred to in humans as human leukocyte antigen ("HLA") complexes). This engagement represents the targeting mechanism of the immune system and is a necessary molecular interaction for T cell regulation (activation or inhibition) and effector function. In addition to epitope-specific cell targeting, target T cells are activated, for example, through the engagement of costimulatory proteins found on APCs with counterpart costimulatory proteins (e.g., receptors) on the T cell. Both signals, i.e., epitope / TCR binding and engagement of APC costimulatory proteins with T cell costimulatory proteins, are required to drive T cell specificity and activation or inhibition. While TCRs are specific for a given epitope, costimulatory proteins are not epitope-specific and instead are generally expressed on all T cells or subsets of T cells.

[0003] APCs generally capture proteins from foreign organisms or abnormal proteins (e.g., resulting from genetic mutations in cancer cells) and serve to degrade them into smaller fragments suitable as signals for scrutiny by the larger immune system, including T cells. Specifically, APCs degrade proteins into small peptide fragments, which are then paired with proteins of the major histocompatibility complex ("MHC") and presented on the cell surface. Cell surface presentation of MHC, along with peptide fragments also known as T cell epitopes, provides the basic scaffolding monitored by T cells, enabling specific recognition. Peptide fragments can be derived from pathogens (infectious agents), tumors, or natural host proteins (self proteins). Additionally, APCs can recognize other foreign components, such as bacterial toxins, viral proteins, DNA, and RNA, whose presence indicates an increased threat level. APCs relay this information to T cells through additional costimulatory signals to generate more effective responses.

[0004] T cells recognize peptide-major histocompatibility complex ("pMHC") complexes through specialized cell surface receptors, the T cell receptor ("TCR"). TCRs are unique to each T cell, and as a result, each T cell is highly specific for a particular pMHC target. To adequately address a multitude of potential threats, the human body contains a very large number (approximately 10,000,000) of distinct T cells with distinct TCRs. Furthermore, any given T cell specific for a particular T cell peptide initially represents only a small fraction of the total T cell population. While typically resting and limited in number, T cells with specific TCRs can be easily activated and amplified by APCs to generate highly potent T cell responses involving millions of T cells. Such activated T cell responses can attack and eliminate other cellular threats, including viral infections, bacterial infections, and tumors. Conversely, broad, non-specific activation of overly active T cell responses against self- or shared antigens can result in T cells inappropriately attacking and destroying healthy tissues or cells.

[0005] MHC proteins are referred to as human leukocyte antigens (HLA) in humans. HLA proteins are divided into two major classes, class I and class II proteins, which are encoded by separate genetic loci. Unless otherwise specified, for purposes of this disclosure, reference to MHC or HLA proteins refers to class II MHC or HLA proteins. HLA class II proteins each contain α and β polypeptide chains encoded by separate genetic loci. HLA class II loci include HLA-DM (HLA-DMA and HLA-DMB, which encode the HLA-DM α-chain and HLA-DM β-chain, respectively), HLA-DO (HLA-DOA and HLA-DOB, which encode the HLA-DO α-chain and HLA-DO β-chain, respectively), HLA-DP (HLA-DPA and HLA-DPB, which encode the HLA-DP α-chain and HLA-DP β-chain, respectively), HLA-DQ (HLA-DQA and HLA-DQB, which encode the HLA-DQ α-chain and HLA-DQ β-chain, respectively), and HLA-DR (HLA-DRA and HLA-DRB, which encode the HLA-DR α-chain and HLA-DR β-chain, respectively).

[0006] Although the immune system is designed to avoid generating immune responses against the body's proteins and other potentially antigenic substances, in some cases the immune system generates T cells with specificity for epitopes of autoantigens, leading to autoimmune diseases.

[0007] Transforming growth factor beta (TGF-β) is a cytokine belonging to the transforming growth factor superfamily, which includes three mammalian (human) isoforms: TGF-β1, TGF-β2, and TGF-β3. TGF-β is synthesized as a precursor molecule containing a propeptide region in addition to the TGF-β sequence, which homodimerizes to form the active form of TGF-β. TGF-β is secreted by macrophages and other cell types as a latent complex in association with two other polypeptides, latent TGF-β binding protein (LTBP) and latent-associated peptide (LAP). The latent TGF-β complex is stored in the extracellular matrix (ECM) and is bound to the cell surface by CD36, for example, via thrombospondin-1 (which can be activated by plasmin), or by latent transforming growth factor β binding proteins 1, 2, 3, and / or 4 (LTBP1-4).

[0008] The biological functions of TGF-β are observed after activation of latent TGF-β, which is tightly regulated in response to ECM perturbations. TGF-β can be activated by various cell- or tissue-specific pathways, or pathways observed in multiple cell or tissue types, although the complete mechanisms behind such activation pathways are not fully understood. Activators include, but are not limited to, proteases, integrins, pH, and reactive oxygen species (ROS). In reality, cell / tissue-bound latent TGF-β complexes function to sense and respond to environmental perturbations by releasing active TGF-β in a spatial and / or temporal manner. Depending on the context of its release, the released TGF-β acts to promote or inhibit cell proliferation. This also recruits stem / progenitor cells to participate in tissue regeneration / remodeling processes. Abnormalities in TGF-β ligand expression, bioavailability, activation, receptor function, or post-transcriptional modification can disrupt normal function and lead to pathological consequences associated with many diseases, such as through excessive precursor recruitment (e.g., in osteoarthritis or Kamurati-Engelman disease) or by transdifferentiation of resident cells into undesired lineages (e.g., in epithelial-mesenchymal transition during cancer metastasis or tissue / organ fibrosis). Xu et al., Bone Research, 6 (Article No. 2) (2018).

[0009] Several approaches to modulating TGF-β action at the protein level by capturing the protein and effectively neutralizing its action have been described in the literature, sometimes referred to as "TGF-β traps." For example, monoclonal antibodies such as methelimumab (CAT192) against TGF-β1 and fresolimumab against multiple isoforms of TGF-β have been developed to bind to, capture, and neutralize TGF-β in vivo. In addition, receptor traps have also been developed that tightly bind to and capture TGF-β, thereby capturing and neutralizing it (see, for example, Swagrtra, et al., Mol Cancer Ther; 11(7):1477-87 (2012) and U.S. Patent Publication No. 2018 / 0327477). Summary of the Invention

[0010] II. The present disclosure provides multimeric antigen-presenting polypeptide complexes (singular "MAPP" and plural "MAPPs") that are at least heterodimeric and comprise at least one framework polypeptide and at least one dimerization polypeptide. The framework polypeptide comprises one or more polypeptide dimerization sequences that enable specific binding with other polypeptides (dimerization polypeptides) having a complementary dimerization sequence, thereby forming at least a heterodimer (see, e.g., Figures 1A and 1B). The framework polypeptide also comprises a multimerization sequence(s) that associates two or more framework polypeptides, thereby forming a higher-order structure (e.g., two or more heterodimeric duplexes, "duplex MAPPs"; see, e.g., Figures 1A and 1B). Neither the dimerization sequence nor the multimerization sequence (or the counterpart dimerization sequence) of the framework polypeptide contains an MHC class II (e.g., HLA) α or β chain polypeptide sequence, and therefore, interactions mediated by those sequences are not considered dimerization or multimerization of the framework and / or dimerization peptide.

[0011] The present disclosure also provides immunomodulatory polypeptides ("MODs") comprising a TGF-β aa sequence ("masking sequence") that is reversibly masked by a peptide having affinity for the TGF-β sequence, collectively referred to as "masked TGF-β MODs." In addition to framework and dimerization polypeptides, the MAPPs described herein further comprise either or both of the TGF-β sequence and the masking sequence of a masked TGF-β MOD. Individual MAPPs may comprise a complete masked TGF-β MOD, in which both the TGF-β sequence and the masking sequence are present on the same polypeptide (i.e., located "cis"; see, e.g., Figure 1C (c) and (d)). Alternatively, an individual MAPP may comprise a complete masked TGF-β MOD, in which the TGF-β sequence and the masking sequence are located on separate polypeptides (e.g., framework and dimerization polypeptides) of the MAPP (e.g., in "trans"). When the masking sequence and the TGF-β sequence are arranged in trans, they may be partial polypeptides present in separate MAPPs (e.g., framework polypeptides of two different MAPPs), and when these polypeptides are combined in a higher-order MAPP complex (e.g., a double-chain MAPP, see e.g., Figure 1C(a)), a complete masked TGF-β MOD is formed. When the masking sequence and the TGF-β sequence are partial polypeptides in different MAPPs, pairing between the TGF-β sequence and the masking sequence in the higher-order MAPP (duplex) can be obtained by using an interspecies multimerization sequence (see e.g., Figure 1C(a) and (b)).

[0012] Unlike the TGF-β traps and related molecules discussed above, which are designed to bind to and capture TGF-β and act as antagonists of TGF-β action, masked TGF-β MODs deliver active TGF-β polypeptides (e.g., TGF-β signaling pathway agonists). The TGF-β polypeptide and masking polypeptide (e.g., a TGF-β receptor fragment) of masked TGF-β MODs interact with each other to reversibly mask the TGF-β polypeptide sequence, allowing it to interact with its cellular receptor. In addition, the masking sequence competes with cellular receptors that can remove TGF-β, such as non-signaling TβRIII, thereby enabling MAPP to effectively deliver active TGF-β agonists to target cells. MAPP constructs enable epitope-specific / selective presentation of reversibly masked TGF-β to target cells, while also providing sites for presentation of one or more additional MODs. The ability of the MAPP construct to include one or more additional MODs thereby allows for the combined presentation of TGF-β and the additional MOD(s) to induce target T cell responses in a substantially epitope-specific / selective manner.

[0013] Thus, the frameworks and dimerization peptides comprising the MAPPs, duplex MAPPs, and higher-order MAPPs (e.g., triplex MAPPs) described herein provide a means by which epitope-presenting peptides ("peptide epitopes" or simply "epitopes") can be presented to target T cells displaying a TCR specific for the epitope in the context of class II MHC (e.g., class II HLA), while simultaneously allowing for the flexible presentation of at least one masked TGF-β MOD, and optionally one or more additional MODs, to target T cells for modulation of the target T cells. The MAPPs and higher-order MAPP complexes thereby enable delivery of one or more masked TGF-β MODs in an epitope-selective (e.g., dependent / specific) manner that enables (i) formation of an active immune synapse with target T cells, such as CD4+ cells, that is selective for the epitope, and (ii) modulation (e.g., control / regulation) of the target T cell's response to the epitope.

[0014] The terms "MAPP" and "MAPPs" as used herein will be understood to refer in different contexts to heterodimers comprising framework and dimerization peptide structures, as well as higher order complexes of those MAPP heterodimers, such as duplexes (duplex MAPPs). Where both MAPP and higher order complexes are referred to, this is done for purposes of emphasis. It will be clear to one of skill in the art when specific reference to only higher order structures is intended (e.g., by reference to duplex MAPPs).

[0015] The frameworks and dimerization peptides comprising the MAPPs, duplex MAPPs, and higher-order MAPPs (e.g., triplex MAPPs) described herein provide a means by which epitope-presenting peptides ("peptide epitopes" or simply "epitopes") can be presented to target T cells displaying a TCR specific for the epitope in the context of class II MHC (e.g., class II HLA), while simultaneously allowing for the flexible presentation of at least one masked TGF-β MOD, and optionally, one or more additional MODs. The MAPPs and higher-order MAPP complexes thereby enable delivery of one or more masked TGF-β MODs in an epitope-selective (e.g., dependent / specific) manner that allows for (i) formation of an active immune synapse with target T cells, such as CD4+ cells, that are selective for the epitope, and (ii) modulation (e.g., control / regulation) of the target T cell's response to the epitope.

[0016] Presentation of peptide epitopes by MAPP to target T cells is achieved via an MHC class II polypeptide and a moiety containing the peptide epitope, which can be either (i) a single polypeptide chain or (ii) a complex containing two or more polypeptide chains.

[0017] When the peptide epitope, MHC class II polypeptide, and optionally one or more MODs are provided in a single polypeptide chain, it is referred to as a "presentation sequence." See, e.g., Figure 25. The presentation sequence can be incorporated into a MAPP as part of a framework polypeptide or a dimerization polypeptide. A MAPP can have a presentation sequence as part of either or both of the framework or dimerization polypeptides. For example, compare Structures A-D in Figure 19 with Structures A-D in Figure 20.

[0018] As an alternative to utilizing a single polypeptide to present the epitope, the MHC components (e.g., α1, α2, β1, and β2 domain sequences) and epitope can be split between two separate polypeptide sequences, which together are referred to herein as a "presentation complex." See, for example, Figures 27-30. A presentation complex is incorporated into a MAPP by having a first amino acid sequence of the presentation complex (the "first sequence of the presentation complex") as part of the framework or dimerization polypeptide. The remaining MHC sequence(s) are part of a polypeptide referred to as a second amino acid sequence of the presentation complex (the "second sequence of the presentation complex"). The peptide epitope and any independently selected MODs present can be part of a polypeptide comprising either the first sequence of the presentation complex or the second sequence of the presentation complex. The first sequence of the presentation complex and the second sequence of the presentation complex generally associate through non-covalent interactions between the α chain polypeptide sequence and the β chain polypeptide sequence, and may be stabilized by disulfide bonds between either the MHC sequence or a peptide / polypeptide linker attached to the N-terminus or C-terminus of the MHC sequence. The first sequence of the presentation complex and the second sequence of the presentation complex may also associate through a dimerization or interspecies dimerization sequence, if present in the polypeptides.

[0019] Although individual MAPPs may not contain a presentation sequence or presentation complex, for purposes of this disclosure, unless otherwise specified, MAPPs are understood to contain at least one presentation sequence or presentation complex.

[0020] MAPPs that contain a presentation sequence typically contain one or two presentation sequences. Thus, double-chained MAPPS typically contain two, three, or four presentation sequences, but can also contain one presentation sequence (e.g., when one of the MAPPS does not contain a presentation sequence). MAPPs and double-chained MAPPs can contain more presentation sequences, depending, for example, on the number of dimerization sequences in the framework polypeptide. The presentation sequence can be incorporated into the MAPP as part of the framework polypeptide, the dimerization polypeptide, or both. For example, compare Structures A-D in Figure 19 with Structures A-D in Figure 20.

[0021] Similarly, MAPPs having a display complex typically contain one or two display complexes, and therefore, double-chained MAPPs having a display complex typically contain two, three, or four display complexes, but may also contain one display complex (e.g., when one of the MAPPs does not contain a display complex). As discussed above, MAPPs and double-chained MAPPs may contain more display complexes, depending, for example, on the number of dimerization sequences in the framework polypeptide.

[0022] The MAPP of this disclosure is: a framework polypeptide comprising (e.g., from N-terminus to C-terminus) a dimerization sequence and a multimerization sequence; a dimerization polypeptide that comprises a counterpart dimerization sequence that is complementary to the dimerization sequence of the framework polypeptide and that dimerizes therewith through covalent (e.g., disulfide bond) and / or non-covalent interactions to form a heterodimer; and at least one (e.g., at least two) presentation sequences and / or presentation complexes, each presentation sequence comprises a peptide epitope and an MHC class II α1, α2, β1, and β2 domain polypeptide sequence; each presentation complex comprises a first presentation complex sequence and a second presentation complex sequence, both of which comprise a peptide epitope and an MHC class II α1, α2, β1, and β2 domain polypeptide sequence, wherein the peptide epitope is part of the first presentation complex sequence or the second presentation complex sequence together with at least one of the α1, α2, β1, or β2 domain polypeptide sequence; At least one or both of the dimerization polypeptide and / or framework polypeptide (e.g., either the framework polypeptide, the dimerization polypeptide, or both polypeptides) comprises a presentation sequence or a first sequence of a presentation complex (e.g., located N-terminal to the dimerization sequence of the framework polypeptide or N-terminal to the partner dimerization sequence of the dimerization polypeptide), at least one of the framework polypeptide, dimerization polypeptide, presentation sequence, or presentation complex comprises (i) a TGF-β sequence, (ii) a masking sequence, or (iii) at least one (e.g., at least two) masked TGF-β immunomodulatory polypeptides ("masked TGF-β MODs"), each masked TGF-β MOD comprising a masking sequence and a TGF-β sequence; the framework polypeptide, or dimerization peptide (including the present presentation sequence(s) or presentation complex(es)), optionally comprises at least one (e.g., at least two, at least three, or more) additional MODs (wt. and / or variant), or a pair of additional MODs in tandem (both wt., both variant, or one wt. and one variant) (e.g., located at the N-terminus or C-terminus of the dimerization polypeptide or framework polypeptide, and / or C-terminal to the dimerization sequence); The framework polypeptide, dimerization polypeptide, display sequence, first sequence of the display complex, and / or second sequence of the display complex can have a structure that optionally includes one or more independently selected linker sequences (see, e.g., Figures 1A and 1B). Such MAPPs can be complexed to form duplexes or higher-order MAPPs comprising at least a first MAPP heterodimer and a second MAPP heterodimer, (i) the first MAPP comprises a first framework polypeptide having a first multimerization sequence and a first dimerization sequence, and a first dimerization polypeptide having a first partner dimerization sequence that is complementary to the first dimerization sequence; (ii) the second MAPP comprises a second framework polypeptide having a second multimerization sequence and a second dimerization sequence, and a second dimerization polypeptide having a second partner dimerization sequence complementary to the second dimerization sequence; the first and second framework polypeptides are associated by binding interactions between the first and second multimerization sequences, optionally including one or more interchain covalent bonds (e.g., one or two disulfide bonds), and the multimerization sequence is not the same as (e.g., not the same type and / or identical to) and does not substantially associate or bind with the dimerization sequence or the counterpart dimerization sequence (see, e.g., the duplexes of Figures 19-23); The double-chain or higher-order MAPP comprises at least one masked TGF-β MOD, wherein the masking sequence and the TGF-β sequence are in cis or trans.

[0023] It is understood that the dimerization sequence and the multimerization sequence are different polypeptide sequences and do not substantially bind to each other, e.g., the framework polypeptides do not form hairpin structures, self-polymerize, or self-aggregate to a substantial extent. The MAPPs of the present disclosure may be subject to the proviso that neither the dimerization sequence nor the multimerization sequence of the framework polypeptide comprises an MHC-class II polypeptide sequence having at least 85% (e.g., 90%, 95%, or 98%) sequence identity with an MHC-class II polypeptide in any of Figures 4-18B (e.g., at least 20 (e.g., at least 30, 40, 50, 60, or 70) consecutive aas of an MHC-class II polypeptide in those Figures). It is also understood that none of the α1, α2, β1, and β2 domain polypeptide sequences comprises a transmembrane domain or portion thereof that anchors the MAPP in a cell membrane.

[0024] The term MAPP(s) as used in this disclosure refers to MAPPs comprising a framework polypeptide and a dimerization polypeptide heterodimer, however, the term MAPP and its plural form MAPPs also refer to higher order complexes thereof comprising two or more copies of the heterodimer. When specific forms of higher order complexes are referred to, e.g., heterodimeric duplexes, they are designated as duplexes, triplexes, etc. Thus, unless otherwise specified, when the term MAPP or MAPPs is used, the term includes higher order complexes such as duplexed MAPPs, particularly where therapeutic applications and treatments are involved.

[0025] MAPPs, and thus their higher order complexes (duplexes, triplexes, etc.), contain MHC class II polypeptide sequences and peptide epitopes for presentation to TCRs that can present peptides to T cells (e.g., CD4+ T cells) that have a TCR specific for the epitope. Upon engagement with the TCR of a T cell, the effect of TGF-β MOD-containing MAPPs on the T cell depends on which additional MODs, if any (e.g., IL-2 MOD polypeptides), are present as part of the MAPP.

[0026] The masked TGF-β MOD-containing MAPPs of the present disclosure can function as a means to generate TGF-β-driven T cell responses. For example, TGF-β itself can inhibit the development of effector cell functions in T cells, activate macrophages, and / or promote tissue repair after local immune and inflammatory effects have resolved. Thus, TGF-β MOD-containing MAPPs can be employed in vitro or in vivo, including as therapeutic agents, to induce any of these functions. TGF-β also regulates the differentiation of functionally distinct subsets of T cells. TGF-β in the presence of IL-1 and / or IL-6 promotes the development of cells of the Th17 lineage, particularly in the absence of either IL-2 or an IL-2 agonist (e.g., an antibody that binds to and acts as an IL-2 receptor agonist).

[0027] TGF-β MOD-containing MAPPs, particularly those containing one or more IL-2 MODs (e.g., variant MODs), or those co-administered with IL-2 or an IL-2 agonist, can induce CD4 T cell proliferation specific / selective for epitopes presented by the MAPP. + FOXP3 +This can lead to the induction and / or proliferation and / or maintenance (survival) of Treg cells. Contacting T cells in vitro or in vivo with a combination of MAPP and IL-2 (either as IL-2 MOD, an IL-2R agonist, or IL-2) potently inhibits the differentiation of effector T helper (Th) cells into cells of the Th1, Th2, and / or Th17 lineages. Thus, masked TGF-β MOD-containing MAPPs (e.g., those with IL-2 MOD) can suppress immune responses to epitopes contained in the MAPPs, for example, through the induction, proliferation, and / or maintenance of Treg cells induced / produced in response to the MAPPs, as well as any downstream effects of these Treg cells, including suppression of CD8+ T cells (activation and / or proliferation) and / or suppression of B cells (e.g., antibody production and / or proliferation). Thus, MAPPs (e.g., double-chain MAPPs) may provide methods for suppressing T cell and B cell activity in vitro and in vivo, as well as the use of MAPPs (e.g., double-chain MAPPs) as therapeutic agents in in vivo or in vitro methods of treatment. Thus, the present disclosure provides methods for modulating T cell and / or B cell activity in vitro and in vivo in disorders associated with immune dysregulation / dysfunction, including allergies and autoimmune diseases, and metabolic disorders. MAPPs are also used in the prevention and / or treatment of graft rejection, either in the context of host-versus-graft rejection / disease ("HVGD") or graft-versus-host rejection / disease ("GVHD").

[0028] In addition to the foregoing, MOD-containing MAPPs, including masked TGF-β MODs, can serve as a means to selectively deliver MODs to T cells specific for MAPP-associated epitopes, thereby resulting in a MOD-driven response to those MAPPs (e.g., reduction and / or suppression of the number of CD4+ effector T cells reactive with the MAPP-associated epitope). Depending on the MOD selected, incorporation of one or more MODs with increased affinity for their cognate receptor on T cells ("co-MODs") can reduce the specificity of MAPPs and dual-chain MAPPs for epitope-specific T cells, where the MOD-co-MOD binding interaction significantly competes with MHC / epitope binding to the target cell TCR. Conversely, again depending on the MOD selected, inclusion of MODs with reduced affinity for their co-MOD(s) and affinity of the epitope for the TCR can provide enhanced selectivity of MAPPs and dual-chain MAPPs while retaining the desired activity of the MODs. If a MOD already possesses a relatively low affinity for its cognate receptor, mutations that reduce affinity may be unnecessary and / or undesirable for its incorporation into a MAPP.

[0029] The ability of MAPPs (e.g., dual-chain MAPPs) to regulate T cells provides a method for modulating T cell activity in vitro and in vivo, and thus MAPPs (e.g., dual-chain MAPPs) are useful as therapeutic agents in methods for treating a variety of diseases and conditions, including autoimmune diseases, GVHD, HVGD, and allergies, as well as metabolic disorders.

[0030] The present disclosure provides nucleic acids comprising nucleotide sequences encoding individual MAPP polypeptides and MAPPs (e.g., all polypeptides of a MAPP), as well as cells engineered with the nucleic acids and vectors for producing MAPP polypeptides and / or MAPP proteins (e.g., double-chain MAPPs). The present disclosure also provides methods for producing MAPPs, double-chain MAPPs, and higher-order MAPPs using such cells. [Brief explanation of the drawings]

[0031] III. [Figure 1A] The diagram is provided to illustrate the terminology used to describe MAPPs and dual-chain MAPPs with presentation sequences. The peptides are oriented from the N-terminus (left) to the C-terminus (right). The diagram shows first and second framework polypeptides, which differ in this case and have specific multimerization sequences, in this case, including a knob and a mating hole. Such "knob-in-hole" configurations can include knob-in-hole configurations that do not include a stabilizing disulfide bond (herein "KiH") or that include a stabilizing disulfide bond (herein "KiHs-s"). Also shown are first and second dimerization polypeptides with an N-terminal peptide epitope and a mating dimerization sequence. Dashed circles indicate five potential positions for the addition of polypeptide sequences, including a MOD sequence (discussed below). The diagram shows the formation of first and second heterodimeric MAPPs, each including a framework polypeptide and a dimerization polypeptide. Heterodimers can interact through multimerization sequences to form multimers (duplex MAPPs as shown). The use of knobs-in-holes sequences allows for the assembly of asymmetric interspecies duplex MAPPs, for example, where different MOD sequences are provided at positions 1 and 1' and / or 3 and 3'. Interactions between polypeptide chains through peptide interaction sequences can initially be noncovalent in nature, but interchain disulfide bond formation reactions can occur, thereby providing, for example, covalently linked polypeptides in either the dimerization or multimerization sequences. Throughout the figures, lines connecting various elements of the MAPP polypeptides are optional amino acids that function as linkers (e.g., peptide linkers).

[0032] [Figure 1B] Parallel to Figure 1A, provided to illustrate the terminology used to describe MAPPs and double-chain MAPPs with presentation complexes (epitopes not shown). The word "sequence" can be abbreviated by "seq."

[0033] [Figure 1C] 1 shows MAPPs having an interspecies multimerization sequence, an epitope-presenting sequence, and at least one masked TGF-β MOD. In (a) and (b), the masked polypeptide sequence of the masked TGF-β MOD and the TGF-β sequence are arranged in trans at positions 3 and 3' of the MAPP. In (c) and (d), the MAPP has two masked TGF-β MODs at positions 3 and 3' of the MAPP, with the masked polypeptide sequence of each masked TGF-β MOD and the TGF-β sequence arranged in cis. In (a) and (c), the masked TGF-β MODs are shown in the "closed" position. In (b) and (d), the masked TGF-β MODs are shown in the "open" position. Additional MODs, such as IL-2, can be arranged in other positions, such as positions 1 and 1'.

[0034] [Figure 1D] Parallel to Figure 1C, but each MAPP shown has an epitope-presenting complex instead of a presentation sequence. The word "sequence" can be abbreviated by "seq."

[0035] [Figures 2A-H] The amino acid sequences of immunoglobulin polypeptides including their heavy chain constant regions (“Ig Fc” or “Fc”, e.g., the CH2-CH3 domains of IgG1) (SEQ ID NOs: 1-13) are provided.

[0036] [Figure 2I] The sequence of the Ig CH1 domain (SEQ ID NO: 14) is provided.

[0037] [Figure 2J] The sequence of the human Ig-J chain (SEQ ID NO: 122) is provided.

[0038] [Figure 3A] The sequence of the Ig kappa chain constant region (SEQ ID NO: 15) is provided.

[0039] [Figure 3B] The sequence of the Ig λ chain constant region (SEQ ID NO: 16) is provided.

[0040] [Figure 4] 1 provides the amino acid sequence of the HLA class II DRA (sometimes referred to as DRA1) alpha chain (SEQ ID NO: 17).

[0041] [Figure 5] The amino acid sequences of the HLA class II DRB1 β chain (SEQ ID NOs: 18 to 54) are provided.

[0042] [Figure 6] The amino acid sequence of the HLA class II DRB3 β chain (SEQ ID NOs: 55 to 58) is provided.

[0043] [Figure 7] The amino acid sequence of the HLA class II DRB4 β chain (SEQ ID NOs: 59-60) is provided.

[0044] [Figure 8] 1 provides the amino acid sequence of the HLA class II DRB5 β chain (SEQ ID NO: 61).

[0045] [Figure 9] 1 provides the amino acid sequence of the HLA class II DMA alpha chain (SEQ ID NO: 62).

[0046] [Figure 10] 1 provides the amino acid sequence of the HLA class II DMB β chain (SEQ ID NO: 63).

[0047] [Figure 11] 1 provides the amino acid sequence of the HLA class II DOA alpha chain (SEQ ID NO: 64).

[0048] [Figure 12] 1 provides the amino acid sequence of the HLA class II DOB β chain (SEQ ID NO: 65).

[0049] [Figure 13] The amino acid sequence of the HLA class II DPA1 α chain (SEQ ID NOs: 66-67) is provided.

[0050] [Figure 14] The amino acid sequence of the HLA class II DPB1 β chain (SEQ ID NOs: 68-79) is provided.

[0051] [Figure 15] The amino acid sequence of the HLA class II DQA1 α chain (SEQ ID NOs: 80-90) is provided.

[0052] [Figure 16] 1 provides the amino acid sequence of the HLA class II DQA2 alpha chain (SEQ ID NO: 91).

[0053] [Figure 17] The amino acid sequence of the HLA class II DQB1 β chain (SEQ ID NOs: 92-103) is provided.

[0054] [Figure 18A-B] The amino acid sequence of the HLA class II DQB2 β chain (SEQ ID NOs: 104-105) is provided.

[0055] [Figure 19]A series of duplex MAPP structures based on a framework polypeptide are provided, each having (i) a multimerization sequence and (ii) both a first and a second dimerization sequence, which may be the same or different. The structures are generally shown in A, with positions 1-5 and 1'-5' indicating the location of an additional peptide sequence (e.g., a MOD polypeptide sequence). The MHC / epitope portion is generally depicted and may be either a presentation sequence (see, e.g., Figures 25-26) or a presentation complex (see, e.g., Figures 27-32). Positions 4 and 4' are shown at the N-terminus of the presentation sequence or the N-terminus of the presentation complex polypeptide, and positions 5 and 5' are shown at the C-terminus of those polypeptides. Positions 1 and 1' are shown at the N-terminus of the framework peptide, and positions 3 and 3' are shown at the C-terminus of the framework polypeptide. In A and C, the framework polypeptides multimerize to form heterodimeric duplexes via non-covalent bonds between the multimerization sequences. In B and D, the framework polypeptides are multimerized to form heterodimeric duplexes using an immunoglobulin Fc region knob-in-hole motif, although other methods of covalently linking multimerization sequences may be used. In C, the duplex comprises a heterodimer in which two different asymmetric interspecies dimerization sequences link together the framework peptide and its associated dimerization peptide. In D, the framework peptides are linked together by a knob-in-hole Fc motif, and the dimerization peptide and framework peptide are linked together by different dimerization sequences to form the heterodimeric duplex.

[0056] [Figure 20] In A-D, a series of MAPP structures, such as those in Figure 19, are provided, which include the addition of a presentation sequence or presentation complex to the N-terminus of the framework peptide. Positions 4 and 4' can still serve as positions for peptide addition (e.g., MOD polypeptide addition).

[0057] [Figure 21]In A-D, we provide a series of MAPP structures, such as those in Figure 19, in which the dimerization sequence is an Ig CH1 sequence (CH1) paired with an Ig light chain sequence (CL). The framework peptides are multimerized (in this case, dimers) through interactions of the Ig Fc (e.g., CH2 and CH3) regions, and the structures in B and D have knobs-in-holes motifs to allow heteroduplex formation. The peptides are also linked by disulfide bonds (e.g., those formed between Ig Fc region peptides).

[0058] [Figure 22] A series of MAPP structures are provided, such as Figure 21, which include the addition of a presentation sequence or presentation complex to the N-terminus of the framework peptide. Positions 4 and 4' can still serve as positions for peptide addition (e.g., MOD polypeptide addition).

[0059] [Figure 23]A series of MAPP structures, such as those in Figure 21, are provided in A-H. In each case, a presentation sequence lacking the MOD sequence is present on the dimerization peptide (marked as single-chain MHC and epitope). Positions 2, 2', 4, 4', 5, and 5' are not filed and are not numbered. Positions 1 and 1' are replaced with one or more MODs, e.g., wild-type (wt.) and / or variants of IL-2, PD-L1, and 4-1BBL for illustrative purposes, although other MODs, e.g., wild-type and / or variant CD80 or CD86, may also be used. In A-D, positions 3 and 3' are used to present a masked TGF-β MOD with the masking sequence and TGF-β polypeptide sequence in trans (on different polypeptides of the MAPP duplex). Positions 3 and 3' in E-H parallel those in A-D, respectively, except that positions 3 and 3' in E-H each have a masked TGF-β with the masking sequence and TGF-β sequence in cis. The Fc CH2, CH3 sequence in E-H can be replaced by an interspecies sequence, such as the KiH Fc sequence shown in A-D. In each case, the masked TGF-β MOD is in a closed position, with the mask engaged with the TGF-β polypeptide sequence such that it cannot effectively act as an agonist of the cellular TGF-β receptor.

[0060] [Figure 24] Four MAPP heterodimer constructs are shown as structures A through D that can form double-chain MAPPs. The polypeptide sequences for structures A through D, in which the "MOD" is PDL1, are provided in Figures 34 through 37, although other MODs, such as IL-2, may be utilized. Masking sequences and TGF-β sequences in cis or trans can be incorporated, for example, into the carboxy terminus (3 or 3' position) of the framework peptide (the Fc sequence in A through C or the CH1 sequence in D).

[0061] [Figure 25]In A-C, three different MHC class II presentation sequences from the epitope (from N- to C-terminus) are shown. The sequences optionally contain one or more independently selected MODs (including two or more MODs in tandem) at the positions indicated.

[0062] [Figure 26] In A-I, nine different embodiments of MHC class II presentation sequences are shown (from left to right, N-terminus to C-terminus).

[0063] [Figure 27-32] A series of MHC class II presentation complexes are shown from left to right, from N-terminus to C-terminus. The sequence with the symbol "- / / -" is the first sequence of the presentation complex. The other sequence is the second sequence of its related presentation complex. The symbol "- / / -" indicates the dimerization or attachment point of the complex to the remainder of the framework peptide. In Figures 30A-L, the first sequence of the presentation complex and its related second sequence of the presentation complex contain dimerization sequences for docking the peptide (shown as an Ig Fc region associated with an Ig light chain constant region Cκ (kappa chain), although other sequences can be used). In Figures 31A-F and 32A-F, the first sequence of the presentation complex and its related second sequence of the presentation complex contain dimerization sequences for docking the peptide (shown as a leucine zipper pair, although other sequences can be used).

[0064] [Figure 33] A table is provided showing the association of certain HLA class II alleles and haplotypes with risk of autoimmune disease. The table also provides some epitopes of autoantigens (self-epitopes) associated with some of the diseases listed.

[0065] [Figure 34] The sequences of the three different isoforms of TGF-β as preproproteins and the mature form of TGF-β3 are provided, along with the C77S mutant of the mature protein.

[0066] [Figure 35] An alignment of TGF-β isoforms 1 to 3 is provided, in which the residues corresponding to the mature form of TGF-β2, except for aa residues Lys25, Cys77, Ile92, and Lys94 of TGF-β2, are shown in bold, and their corresponding residues in the other forms of TGF-β isoforms 1 and 3 are underlined and italicized rather than bold: TGF-β1 (NP_000651.3 and P01137), TGF-β2 (AAA50405.1), and TGF-β-3 isoform 1 (NP_0013168.1).

[0067] [Figure 36A] The sequence of the type 1 TGF-β receptor (TβRI) and its ectodomain is provided.

[0068] [Figure 36B] The sequences of the type 2 TGF-β receptor (TβRII), its ectodomain, and a fragment of the ectodomain are provided. In isoform B, the positions shown in bold and underlined are aaas F30, D32, S52, E55, and D118 of the mature polypeptide, any of which may be substituted with aaa other than the naturally occurring aa. The ectodomain fragment is based on NCBI reference sequence NP_003233.4 and UniProtKB reference P37173, and the ectodomain sequence corresponds to aaas 49-159 of those sequences. The substitution of alanine "A" (bold, italicized, and underlined) at aspartic acid "D119" in the mature protein is marked as the "D118A" substitution for consistency with the literature describing the substitution when the signal peptide is understood to be 23 aas in length, as opposed to 22 aas in the NCBI record. The numbering assignment for aa D119 is based on the mature protein, and is therefore D141 of the precursor protein when the 22-aa signal sequence is included. Position D32, which may be substituted with asparagine (D32N), corresponds to D55 in the precursor protein. The corresponding aas in mature isoform A, which lacks its signal sequence, are F55, D57, S77, E80, and D143 (see, e.g., SEQ ID NO: 283).

[0069] [Figure 36C] The sequence of the type 3 TGF-β receptor (TβRIII) is provided.

[0070] [Figure 37] In (a), structures of the MAPPs of Example 1 are provided, each having a masking sequence and a TGF-β polypeptide at the C-terminus (positions 3 and 3') of the framework polypeptide. The masked TGF-β is shown in the closed position, where the mask engages the TGF-β polypeptide. The structure could also be shown in the open position, where TGF-β is available to interact with the TGF-β receptor. In (b), the diagram shows an SDS-PAGE gel of eight different MAPPs (in lanes 1-8) and four control proteins (lanes 9-12) after expression and purification. The optional disulfide bond that holds the epitope (circle in the class II pHLA structure) within the MHC / HLA binding cleft (e.g., between the linker connected to the epitope and the MHC α chain) is not shown.

[0071] [Figure 38] 1 provides the amino acid sequences of the polypeptides that form the eight MAPPs and four control proteins of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0072] IV. A.Definition The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0073] The terms "polypeptide" and "protein" are used interchangeably herein and, unless otherwise specified, refer to polymeric forms of amino acids, which are naturally occurring proteinogenic L-amino acids that are biosynthetically incorporated into proteins during translation in mammalian cells. Furthermore, as used herein, "polypeptide" and "protein" include modifications to the native sequence (generally conservative in nature, as known to those of skill in the art) such as deletions, additions, and substitutions, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts producing the protein or errors resulting from polymerase chain reaction (PCR) amplification or other recombinant DNA methods. Reference to a specific residue or residue number in a known polypeptide, e.g., position 72 or 75 of the human DRA MHC class II polypeptide, is understood to refer to the amino acid at that position in the wild-type polypeptide (i.e., I72 or K75). To the extent that the sequence of the wild-type polypeptide is altered, either by the addition or deletion of one or more amino acids, the specific residue or residue number will refer to the same specific amino acid in the altered polypeptide (e.g., the addition of one amino acid at the N-terminus of a peptide referenced as position I72 would now be understood to refer to the amino acid He, which is position 73). Amino acid substitutions at specific positions are indicated by an abbreviation that includes, in order, the original amino acid, the position number, and the substituted amino acid; for example, substituting He at position 72 with cysteine ​​is designated as I72C.

[0074] A nucleic acid or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when the two sequences are aligned, the same percentage of bases or amino acids are in the same relative positions. Sequence identity can be determined in several different ways. To measure sequence identity, sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available on the World Wide Web at sites including blast.ncbi.nlm.nih.gov / Blast.cgi, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / for BLAST+2.10.0. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise indicated, the percent sequence identity set forth herein is determined using the BLAST program.

[0075] As used herein, amino acid (singular "aa" or plural "aas") refers to the naturally occurring proteinogenic amino acids that are incorporated into polypeptides and proteins during mammalian cell translation. Unless otherwise specified, L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan). Amino acids also include the amino acids hydroxyproline and selenocysteine, which occur in some proteins found in mammalian cells, but are not understood to be included unless their presence is explicitly indicated.

[0076] As used herein, the term "in vivo" refers to, for example, any process or procedure that occurs inside a patient's body.

[0077] As used herein, "in vitro" refers to any process or procedure that occurs outside the body.

[0078] The term "conservative amino acid substitution" refers to the interchangeability of AAs residues with similar side chains in proteins. For example, the group of AAs with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of AAs with aliphatic hydroxyl side chains consists of serine and threonine; the group of AAs with amide-containing side chains consists of asparagine and glutamine; the group of AAs with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of AAs with basic side chains consists of lysine, arginine, and histidine; the group of AAs with acidic side chains consists of glutamate and aspartate; and the group of AAs with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative AAs substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0079] The term "binding" refers to a direct association between molecules and / or atoms due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges. As used herein, "covalent bonding" or "covalent binding" refers to the formation of one or more covalent chemical bonds between two different molecules. The term "binding" when used in reference to the interaction between MAPP and the T cell receptor (TCR) on a T cell refers to a non-covalent interaction between MAPP and the TCR.

[0080] "Affinity" as used herein generally refers to the strength of non-covalent binding, with increased binding affinity being indicated by a lower K D As used herein, the term "affinity" refers to the dissociation constant (K) of the reversible binding of two agents (e.g., an antibody and an antigen). D As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0081] "T cells" are T helper cells (CD4 + T helper cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including immune cells (IL-1), T regulatory cells (Tregs), and NK-T cells.

[0082] The term "immunomodulatory polypeptide" (also referred to as a "co-stimulatory polypeptide," or "MOD," as discussed above) as used herein includes wild-type or variant polypeptides, or portions thereof, that specifically bind to a cognate co-immunomodulatory polypeptide ("co-MOD") present on a T cell and can provide a regulatory signal to a T cell when the TCR of the T cell engages an MHC-epitope portion specific for the TCR. Unless otherwise specified, the term "MOD" includes wild-type and / or variant MODs, and statements including reference to both wild-type and variant MODs are made to emphasize that one, the other, or both are being referred to. The signal provided by a MOD engaging its co-MOD mediates (e.g., induces) a T cell response. Such responses include, but are not limited to, proliferation, activation, differentiation, suppression / inhibition of proliferation, activation and / or differentiation, and the like.

[0083] "Heterologous" as used herein means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

[0084] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from the endogenous nucleic acid found in a natural system. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in a cell or a cell-free transcription and translation system.

[0085] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically generated for the purpose of expressing and / or propagating insert(s) or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

[0086] The terms "treatment," "treating," and equivalents are generally used herein to mean obtaining a desired pharmaceutical and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing a disease or condition from occurring in a subject who may be predisposed to, but has not yet been diagnosed with, the disease or condition; (b) inhibiting a disease or condition, i.e., arresting its progression; and / or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease, in which treatment stabilizes or alleviates undesirable clinical symptoms in the patient, is particularly advantageous. Such treatment is desirably performed before complete loss of function of the affected tissue. The subject treatments will desirably be administered during, and optionally after, the symptomatic stage of the disease.

[0087] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include humans and non-human primates, as well as rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), cats, dogs, and the like.

[0088] Unless otherwise indicated, the term "substantially" is intended to encompass both "entirely" and "largely, but not entirely." For example, an Ig Fc that "does not substantially induce cytolysis" means an Ig Fc that does not induce cytolysis at all or that does not induce cytolysis largely, but not entirely.

[0089] As used herein, the term "about" when used in relation to an amount indicates that the amount may vary by 10%. For example, "about 100" means an amount of 90 to 110. When "about" is used in relation to a range, "about" when used in relation to a smaller amount in the range means that the smaller amount includes an amount 10% less than the smaller amount in the range, and "about" when used in relation to a larger amount in the range means that the larger amount includes an amount 10% more than the larger amount in the range. For example, about 100 to about 1000 means that the range extends to 90 to 1100.

[0090] The terms "purifying," "isolating," or equivalents refer to the removal of a desired substance, e.g., MAPP, from a solution containing undesired substances, e.g., contaminants, or the removal of undesired substances from a solution containing the desired substance, leaving essentially only the desired substance. In some cases, a purified substance may be essentially free of other substances, e.g., contaminants. As will be understood by those of skill in the art, generally, components of the solution itself, e.g., water or buffer, or salts, are not considered when determining the purity of a substance.

[0091] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, down to one-tenth of the lower limit of the range, is encompassed within the disclosure, along with any other stated or intervening value within the range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes values ​​(e.g., upper or lower limits), ranges excluding those values ​​are also included.

[0092] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the cited publications.

[0093] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a Treg" includes a plurality of such Tregs; a reference to "MHC class II α chain" includes a reference to one or more MHC class II α chains and equivalents thereof known to those skilled in the art; and so forth. Furthermore, it should be noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0094] It should be understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every subcombination were individually and explicitly disclosed herein.

[0095] B. Description 1. MAPP structure and the role of framework and dimerization peptides The present disclosure provides MAPPs for use in treating diseases and disorders, including, inter alia, autoimmune diseases, GVHD, HVGD, and allergies, as well as metabolic disorders. As discussed above, MAPPs comprise at least one framework polypeptide and at least one dimerization polypeptide. The framework polypeptide comprises one or more polypeptide dimerization sequences that enable specific binding with other polypeptides (dimerization polypeptides) having a complementary dimerization sequence, thereby forming at least a heterodimer (see Figures 1A and 1B). The framework polypeptide also comprises a multimerization sequence(s) that enable two or more framework polypeptides to associate, thereby forming a higher-order structure (e.g., two or more heterodimeric duplexes as "duplex MAPPs"; see, e.g., Figures 1A and 1B). Neither the dimerization nor multimerization sequences (or counterpart dimerization sequences) of the framework polypeptides contain MHC (e.g., HLA) class II α or β chain polypeptide sequences, and therefore interactions mediated by those sequences are not considered to be dimerization or multimerization of the framework and / or dimerization peptides. Thus, the framework polypeptides provide a structure that can organize other polypeptides through interactions at the dimerization sequences and can interact with other framework polypeptides through the multimerization sequences.

[0096] Figures 1C and 1D show, by way of example, a series of double-chain MAPPs with masked TGF-β MODs in either the closed or open position. In (a) and (b), the masked polypeptide sequence of the masked TGF-β MOD and the TGF-β sequence are arranged in trans at positions 3 and 3' of the MAPP. In (c) and (d), a MAPP has two masked TGF-β MODs at positions 3 and 3' of the MAPP, with the masked polypeptide sequence of each masked TGF-β MOD and the TGF-β sequence arranged in cis. Because the masking sequence and TGF-β sequence of each MOD in (c) and (d) are arranged in cis, the first and second multimerization sequences of the framework polypeptides do not need to be interspecies pairs. In (a) and (c), the masked TGF-β MODs are shown in the "closed" position, in which the TGF-β sequence is engaged by the masking polypeptide and is not available to interact with a cellular receptor. In (b) and (d), the masked TGF-β MODs are shown in the "open" position, where the TGF-β sequence is available to interact with the cellular receptor. Additional MODs, such as IL-2, can be placed in other positions, such as positions 1 and 1'.

[0097] The MAPPs, dual-chain MAPPs, and higher-order MAPPs (e.g., triplex MAPPs) discussed above provide a means by which peptide epitopes can be presented to target T cells exhibiting epitope-specific TCRs in the context of MHC (e.g., HLA) and masked TGF-β MOD(s), while simultaneously allowing for the flexible presentation of one or more MODs in addition to the masked TGF-β MOD(s). MAPPs, dual-chain MAPPs, and higher-order MAPPs thereby enable the delivery of one or more MODs in an epitope-selective (e.g., dependent / specific) manner that allows for the formation of an active immune synapse with target T cells that is selective for the epitope and the control / modulation of the target T cell's response to the epitope. The response of target T cells to a MAPP depends on the MODs and epitopes presented by the MAPP. Thus, when MAPPs contain stimulatory or activating MODs (e.g., IL-2, CD80, CD86, and / or 4-1BBL), they increase T cell proliferation and / or effector function in an epitope-selective manner. In contrast, when MAPPs contain suppressive / inhibitory MODs (e.g., FasL and / or PD-L1), they generally decrease T cell activation, proliferation, and / or effector function in an epitope-selective manner. MAPPs, particularly when they contain one or more masked TGF-β MOD and one or more IL-2 MOD polypeptide sequences, can function to increase the induction or proliferation of Tregs in an epitope-selective manner. MAPPs of the present disclosure bearing at least one masked TGF-β MOD alone or in combination with one or more IL-2 MOD polypeptide sequences can also be combined with additional MODs, such as PD-L1 or 4-1BBL, to provide additional regulatory signals.

[0098] The framework / dimerization polypeptide architecture of MAPPs and their higher-order structures can also be understood to provide flexibility in the positioning of MODs and epitope-presenting complexes or epitope-presenting sequences. Dual-chain MAPPs and higher-order MAPP structures can be particularly useful when both the MOD and epitope-presenting complex (or epitope-presenting sequence) are positioned to provide the desired biological activity and other desirable properties of MAPPs, such as thermal stability and manufacturability. In some cases, an acceptable combination of properties can be obtained when the MOD and presentation complex or presentation sequence are positioned at the N-terminus of a polypeptide, e.g., each can be positioned at the N-terminus of a different framework and / or dimerization polypeptide sequence. In some cases, an acceptable combination of properties can be obtained when the MOD and presentation complex or presentation sequence are positioned at the C-terminus of a polypeptide, e.g., each can be positioned at the C-terminus of a different framework and / or dimerization polypeptide sequence. In some cases, an acceptable combination of properties can be obtained when the MOD and the presentation complex or presentation sequence are located at the N-terminus and C-terminus of a polypeptide, respectively, e.g., the MOD can be located at the N-terminus of a different framework and / or dimerization polypeptide sequence, and the presentation complex or presentation sequence can be located at the C-terminus.In some cases, an acceptable combination of properties can be obtained when the MOD and the presentation complex or presentation sequence are located at the N-terminus and C-terminus of a polypeptide, respectively, e.g., the MOD can be located at the C-terminus of a different framework and / or dimerization polypeptide sequence, and the presentation complex or presentation sequence can be located at the N-terminus.

[0099] Examples of masked TGF-β MOD configurations include positioning a masked TGF-β MOD comprising a masking sequence and a TGF-β sequence in cis at the carboxy terminus of a MAPP framework polypeptide or dimerization polypeptide (see, e.g., positions 3, 3', 5, or 5' in Figures 1A and 1B). Masked TGF-β MODs comprising a masking sequence and a TGF-β sequence in cis can also be located, for example, at the amino terminus of a MAPP framework polypeptide or dimerization polypeptide (see, e.g., positions 1, 1', 4, or 4' in Figure 1). Other positions on a MAPP may also be utilized, including the N-terminus or C-terminus of the second sequence of the presentation complex.

[0100] The arrangement of TGF-β MODs in which the masking sequence and TGF-β sequence are in trans (on different polypeptides of a MAPP) is exemplified by having the masking sequence and the TGF-β sequence both at the carboxyl terminus or both at the N-terminus of the MAPP polypeptide. As an example, in a double-chain MAPP, the masking sequence and the TGF-β sequence can both be at the carboxyl terminus of the framework peptide (see, e.g., structures (a) and (b) in Figures 1C and 1D). Masked TGF-β MODs containing the masking sequence and the TGF-β sequence in trans can also be located, for example, at the amino terminus (positions 1 and 1') of a MAPP framework polypeptide.

[0101] The structure of MAPPs, particularly higher-order MAPPs such as duplexes, can be specified by using pairs of polypeptides with different sequences that specifically pair with each other. Multimerization of framework polypeptides results from the interaction between the multimerization sequences, and dimerization (interaction of framework and dimerization polypeptides) results from the interaction of the dimerization sequence on the framework polypeptide with the counterpart dimerization sequence on the dimerization polypeptide. For example, in a duplex MAPP, the multimerization sequence can be an Ig Fc heavy chain (e.g., CH2-CH3) sequence, and the dimerization sequence and the counterpart dimerization sequence can be the same (e.g., all leucine zipper sequences). An additional degree of control can be obtained by utilizing non-identical peptide sequences that specifically / selectively pair with each other, resulting in an asymmetric interspecies pair of sequences, commonly referred to herein as "interspecies sequences," in the case of a dimerization sequence "interspecies dimerization sequence" or "interspecies multimerization sequence." Thus, the structure of MAPPs allows for diverse and effective placement of each polypeptide within the MAPP structure (see, for example, Figures 19-23). ​​Interspecies sequences include, for example, an Ig heavy chain Fc (e.g., CH2-CH3) region modified with knob-in-hole variations and a Fos peptide sequence paired with a Jun peptide sequence. Thus, MAPP structures include, but are not limited to, MAPPs in which each or some of the dimerization sequences differ (allowing for different peptide pairings). For example, double-chain MAPPs in which each multimerization and dimerization sequence differs, providing distinct peptide pairs.

[0102] In one embodiment, the framework peptide multimerization sequences are Fc heavy chain regions (and optionally interspecies Fc sequences such as knobs-in-holes Fc sequences) and the dimerization sequences are the same (e.g., an Ig CH1 sequence paired with a light chain lambda or kappa constant region sequence) (see, e.g., Figures 21 and 22, structures A-D). In another embodiment, the framework peptide multimerization sequences are Fc heavy chain regions (and optionally interspecies Fc sequences such as knobs-in-holes Fc sequences) and the dimerization sequences are selected to be different (e.g., a pair of dimerization sequences comprising an Ig CH1 paired with a light chain lambda or kappa sequence, and a dimerization sequence comprising a leucine zipper pair; see, e.g., Figure 23, structures E-H). For example, in a double-chain MAPP, the multimerization sequence can be a knobs-in-holes Ig sequence, one dimerization sequence and its partner dimerization sequence can be a leucine zipper sequence, and the second dimerization sequence and its partner dimerization sequence can be an Ig CH1 and Ig CLλ domain pair.

[0103] MAPPs, and thus their higher order complexes (duplexes, triplexes, etc.), contain MHC class II polypeptide sequences that bind epitopes for presentation to TCRs and thus to T cells (e.g., CD4 +MAPPs can present peptides to T cells (e.g., T cells) with epitope-specific TCRs. The effect of MAPPs on T cells with TCRs specific for the epitope depends on which, if any, MODs are present in addition to the masked TGF-β MOD(s) present in the MAPP. As discussed above, MAPPs containing MOD(s), dual-chain MAPPs, and higher-order MAPPs enable MOD delivery to T cells in an epitope-selective manner, with the MODs primarily directing the effect of MAPP-T cell engagement in light of the specific cell type and environment being stimulated. While not wishing to be bound by any particular theory, the effect of MAPP (e.g., dual-chain MAPP) presentation of MOD(s) and epitopes to T cells may, in some cases, be enhanced for situations encountered on antigen-presenting cells (APCs), where the epitope can diffuse away from the MHC (e.g., HLA) complex and any MODs the APC is presenting. This is not possible with MAPPs, where the epitope and MOD(s) are part of a MAPP polypeptide(s) and cannot diffuse away from the MHC complex, even though the affinity of the epitope for the MHC complex normally allows it to diffuse away from the corresponding cellular complex. The inability of an epitope to diffuse away from the MHC and MOD components of a MAPP or its higher-order MAPP complex may be further limited if the polypeptide(s) of the MAPP (e.g., framework, dimerization sequence, and, if present, secondary sequence of the presentation complex) are covalently linked to each other (e.g., by disulfide bonds). Consequently, MAPPs and their higher-order structures may be capable of extending the delivery of MOD(s) to T cells in an epitope-selective manner relative to systems in which the epitope can diffuse away from the presenting MHC.

[0104] Incorporation of one or more MODs ("co-MODs") with affinity for their cognate receptors on T cells can reduce the specificity of MAPPs (e.g., dual-chain MAPPs) for epitope-selective / specific T cells. Reduction in epitope selectivity / specificity of MAPPs becomes more pronounced when the MOD / co-MOD binding interaction increases in strength (binding energy) and significantly competes with MHC / epitope binding to the target cell TCR. Thus, inclusion of variant MODs, including TGF-β MODs with reduced affinity for their co-MOD(s), may provide a lower contribution of MOD binding energy, thereby allowing the TCR to dominate binding and MHC-epitope interactions that provide epitope-selective interactions with T cells while retaining the activity of the MODs. Variant MODs with one or more substitutions (or deletions or insertions) that reduce the affinity of the MOD for their co-MOD can be incorporated into MAPPs and their higher-order complexes, either alone or in combination with wild-type MOD polypeptide sequences. Wild-type and variant MODs are further described below. Inclusion of a masking sequence that tightly binds to a TGF-β polypeptide sequence effectively reduces the apparent affinity of the TGF-β polypeptide sequence for a cellular receptor, thereby reducing the contribution of the TGF-β polypeptide to cellular TβR binding upon MAPP engagement with a T cell, allowing MHC-epitope interactions with the TCR to dominate T cell binding interactions, resulting in epitope-specific / selective T cell interaction and epitope-specific / selective delivery of the masked TGF-β MOD and any other MODs on the MAPP to the target T cell.

[0105] Thus, the ability of MAPPs to regulate T cells in an epitope-selective / specific manner provides a method for modulating T cell activity in vitro and in vivo, and therefore for treating diseases such as GVHD, HVGD, and disorders associated with immune dysregulation / dysfunction, including allergies and autoimmune diseases.

[0106] The present disclosure provides nucleic acids comprising nucleotide sequences encoding MAPP polypeptides, cells genetically engineered with the nucleic acids and capable of producing MAPP, and methods of using such cells to produce MAPPs and their higher order complexes.

[0107] Each presentation sequence or presentation complex present in a MAPP contains sufficient MHC class II α and β chain polypeptide sequences (e.g., human MHC class II sequences) to bind and present a peptide epitope to a TCR. MHC class II peptides can contain sequence variations designed to stabilize the MHC, stabilize the MHC-peptide epitope complex, and / or stabilize the MAPP. Sequence variations can also serve to enhance cellular expression of MAPPs prepared in cell-based systems, as well as the stability (e.g., thermostability) of MAPPs and their higher-order complexes, such as double-chain MAPPs. Several MHC class II sequences suitable for use in MAPPs are described below.

[0108] As indicated in the figure legends, MAPPs may contain one or more independently selected peptide sequences or (one or more "linkers" or "linkers") between any two or more components of the MAPP, which may be shown in the figures as lines between the peptide and / or polypeptide elements of the MAPPs. The same sequences used as linkers may also be located at the N-terminus and / or C-terminus of the MAPP peptide, for example, to prevent proteolysis. Linker sequences include, but are not limited to, glycine, glycine and serine, glycine and alanine, alanine and serine, and polypeptides containing glycine, alanine and serine, as well as any one that may contain cysteine ​​for intra- or inter-polypeptide disulfide bond formation. Various linkers are described in more detail below.

[0109] 2. Exemplary MAPP Structure MAPPs of the present disclosure comprise (i) a framework polypeptide having a multimerization sequence and at least one dimerization sequence, and (ii) a dimerization polypeptide having a counterpart dimerization sequence that binds to the dimerization sequence of the framework polypeptide. As discussed above, MAPPs of the present disclosure further comprise one, two, or more masked TGF-β MODs and either one or more epitope-presenting sequences or one or more epitope-presenting complexes. Exemplary structures of such MAPPs appear in Figures 1A-1D, and in some cases in Figures 19-23, at least one MOD is shown as a masked TGF-β MOD. The structure shown in Figure 23 represents a MAPP having at least one masked TGF-β MOD, a multimerization framework polypeptide, and an epitope-presenting sequence (a "single-chain MHC" bearing an "epitope"). In Figure 1A and Figures 19-22, the structures represent MAPPs with multimerizing framework polypeptides in which epitope-MHC combinations represent either epitope-presenting sequences or epitope-presenting complexes.

[0110] Interactions of MHC (e.g., HLA) sequences are not considered herein to result in multimerization and / or dimerization. In one embodiment, neither the dimerization sequence nor the multimerization sequence of a framework polypeptide nor the partner dimerization sequence of a dimerization polypeptide comprises a class II MHC polypeptide sequence having at least 90% (e.g., 95% or 98%) sequence identity with at least 15 (e.g., at least 20, 30, 40, 50, 60, or 70) consecutive aas of an MHC class II polypeptide (e.g., a polypeptide of any of Figures 4-18B). In embodiments, MAPPs comprise at least one or at least two dimerization peptides comprising an epitope-presenting sequence. See, e.g., Figure 1A.

[0111] One group of masked TGF-β MOD-containing MAPPs with epitope-presenting sequences comprises, in addition to the masked TGF-β MOD, a multimerization framework polypeptide having, from N- to C-terminus, a dimerization sequence and a multimerization sequence, and a dimerization polypeptide that comprises a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide and dimerizes therewith through covalent and / or non-covalent interactions to form a heterodimer, wherein at least one (e.g., one or both) of the dimerization polypeptide and the framework polypeptide comprises a presentation sequence located N-terminal to the dimerization sequence or counterpart dimerization sequence. In such MAPPs, the presentation sequence can comprise a peptide epitope and one or more MHC polypeptide sequences, wherein the peptide epitope sequence is located (i) at or within the N-terminal 10 aa, 15 aa, 20 aa, or 25 aa of the presentation sequence, or (ii) within a polypeptide located at the N-terminus of the presentation sequence, comprising, from N-terminus to C-terminus, a MOD, one or more optional linkers, and the peptide epitope; optionally, at least one (e.g., one, two, or each) of the framework polypeptide, dimerization peptide, and presentation sequence comprises one or more independently selected MODs located at their N-terminus and / or C-terminus (or N- or C-terminal to the dimerization or partner dimerization sequence); and the MHC polypeptide sequences are MHC class II polypeptide sequences, including MHC class II α1, α2, β1, and β2 polypeptide sequences (e.g., human MHC class II sequences). In one embodiment, neither the dimerization nor the multimerization sequence of the framework polypeptide comprises a class II MHC peptide sequence having at least 90% (e.g., 95% or 98%) sequence identity to at least 15 (e.g., at least 20, 30, 40, 50, 60, or 70) consecutive aas of an MHC class II polypeptide of any of Figures 4-18B.

[0112] Another group of MAPPs having epitope-presenting complexes includes a multimerization framework polypeptide having, from N- to C-terminus, a dimerization sequence and a multimerization sequence, and a dimerization polypeptide that comprises a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide and dimerizes with it through covalent and / or non-covalent interactions to form a heterodimer, wherein at least one (e.g., one or both) of the dimerization polypeptides and / or at least one (e.g., one or both) of the framework polypeptides comprises a first sequence of a presentation complex located N-terminally of the dimerization sequence. The second sequence of a presentation complex is associated with the first sequence of a presentation complex (e.g., non-covalently or covalently, such as by one or two interchain disulfide bonds) to form a presentation complex. In such MAPPs, the first sequence of a presentation complex and its associated second sequence of a presentation complex each comprise one or more MHC polypeptide sequences, and one of the sequences further comprises a peptide epitope. The peptide epitope can be located (i) within or 10 aa, 15 aa, 20 aa, or 25 aa from the N-terminus of the first sequence of the display complex or the second sequence of the display complex, or (ii) within a polypeptide located at the N-terminus of the first sequence of the display complex or the second sequence of the display complex, wherein the polypeptide comprises, from N-terminus to C-terminus, a MOD, one or more optional linkers, and the peptide epitope. Optionally, at least one (e.g., one, two, or each) of the framework polypeptide, dimerization peptide, or peptide of the display complex comprises one or more independently selected MODs located at their N-terminus or C-terminus (or N-terminus or C-terminal side of the dimerization sequence).

[0113] MAPPs can be constructed such that neither the dimerization nor the multimerization sequences of the framework polypeptides contain a class II MHC peptide sequence that has at least 90% (e.g., 95% or 98%) sequence identity with at least 15 (e.g., at least 20, 30, 40, 50, 60, or 70) consecutive aas of an MHC class II polypeptide of any of Figures 4-18B.

[0114] As discussed above, a dimerization sequence of a framework polypeptide can interact with a dimerization peptide to form a heterodimer. A multimerization sequence of a framework polypeptide can associate with another framework polypeptide multimerization sequence to form a heterodimeric duplex (or higher-order structure such as a triplex, quadruplex, or pentadplex). When the multimerization sequence is interspecies (e.g., a knobs-in-holes Fc peptide pair) and at least one heterodimer contains an interspecies dimerization pair and a counterpart dimerization pair, two different heterodimers can be formed. When different heterodimers combine to form a duplex MAPP, any one or more components (e.g., MODs) can differ (e.g., in type or position) between the two heterodimers.

[0115] C.MAPP component 1. Framework Polypeptides and Dimerization Polypeptides As can be understood from the preceding paragraphs, framework polypeptides serve as the structural basis or scaffold of MAPPs, enabling the organization of other elements in the MAPP complex. Framework peptides interact with other peptides primarily through binding interactions in dimerization and multimerization sequences. Interactions in dimerization sequences allow association of non-framework peptides (e.g., dimerization peptides) with framework peptides. In contrast, multimerization sequences are responsible for the interaction of two or more framework peptides.

[0116] The framework polypeptide(s) of MAPPs comprise at least one multimerization sequence and at least one independently selected dimerization sequence that is not identical to or of the same type as the multimerization sequence (e.g., both are not leucine zipper variants). Utilizing different types of sequences for interaction in the multimerization and dimerization sequences allows for control of the interaction of the framework polypeptide with other framework polypeptides and dimerization polypeptides. In one embodiment, a framework polypeptide comprises one multimerization sequence and one dimerization sequence. In one embodiment, a framework polypeptide comprises at least one multimerization sequence and at least two independently selected dimerization sequences. The framework polypeptide may comprise peptide sequences (e.g., linker sequences and / or MOD sequences) between any of the elements of the framework polypeptide or at the termini of the framework polypeptide comprising the multimerization and dimerization sequences.

[0117] In addition to providing structural organization of MAPPS through their multimerization and dimerization sequences, framework peptides, particularly their N- and C-termini, can also serve as locations for the placement of elements such as MOD sequences, epitopes, presentation sequences, and / or the first sequence of a complex (one polypeptide of an epitope-presenting complex, see FIG. 1B). When placed at the N- and / or C-termini of a framework polypeptide, such polypeptide elements are part of the framework polypeptide (e.g., a single translation product formed in a cell).

[0118] Within a MAPP, all of the dimerization sequences can be non-interspecies (such as leucine zipper pairs), while the multimerization sequences are either interspecies or non-interspecies (see, e.g., Structures A and B in Figures 19 and 20). For example, in a dual-chain MAPP having first and second framework polypeptides, the multimerization sequences can be non-interspecies (e.g., IgFc sequences such as CH2, CH3 domain sequences, or leucine zippers), or the multimerization sequences can be interspecies knob-in-hole sequence pairs, with the dimerization sequences of the first and second framework polypeptides as non-interspecies leucine zipper polypeptides. When an Fc polypeptide is employed, it can be derived from IgA, IgD, IgE, IgG, or IgM, for example, which can be a human polypeptide sequence, a humanized polypeptide sequence, an Fc region polypeptide of a synthetic heavy chain constant region, or a consensus heavy chain constant region.

[0119] Within MAPP, all of the dimerization sequences may be interspecies, while the multimerization sequences are not (see, e.g., Figure 23A). For example, in a dual-chain MAPP having a first and second framework polypeptide, the multimerization sequence may be an IgFc sequence, with the ZW1 sequence or its counterpart employed as the dimerization sequence for the first framework polypeptide, and the Ig CH1 domain or its counterpart Ig C domain. L The kappa sequence is employed as the dimerization sequence of the second framework polypeptide.

[0120] All of the dimerization sequences, or all of the dimerization and multimerization sequences in a MAPP, can differ in that they bind only to a specific binding partner present in the MAPP (e.g., each is part of a different interspecies sequence pair). For example, in a dual-chain MAPP having a first and second framework polypeptide, the multimerization sequences can be a pair of knobs-in-holes IgFc sequences, with the ZW1 sequence or its counterpart employed as the dimerization sequence for the first framework polypeptide and the Ig CH1 or its counterpart Ig C. L The sequence is adopted as the dimerization sequence for the second framework polypeptide.

[0121] 2. Multimerizing and Dimerizing Polypeptide Sequences An amino acid sequence that allows polypeptides to interact can be used as a dimerization sequence or a partner dimerization sequence when involved in the formation of a dimer between a framework polypeptide and a dimerization polypeptide. The same type of aa sequence can be used as a multimerization sequence when used to form a duplex or higher-order structure (trimer, tetramer, pentamer, etc.) between framework polypeptides. In any given MAPP, sequences that can interact with each other are not used as both a dimerization sequence and a multimerization sequence. In other words, the same pair of aa sequences can function as either a dimerization sequence or a multimerization sequence, depending on whether they bring two or more framework peptides together (in which case they are multimerization sequences) or bring together a dimerization and multimerization sequence (in which case they are designated as dimerization sequences).

[0122] When dimerization or multimerization sequences employ identical pairing or multimerizing sequences (e.g., some leucine zipper sequences), they can form symmetric pairs or multimers (e.g., homodimers), as shown in Structure A of Figure 19. In contrast, when pairing dimerization or multimerization sequences are not identical and require specific, complementary partner sequences to form dimers, they are interspecies binding sequences and can form asymmetric pairs. Both immunoglobulin (e.g., IgFc) and non-immunoglobulin polypeptides can be interspecies or non-interspecies in nature. For example, the Fos / Jun binding pair and the Ig CH1 polypeptide sequence and light chain constant region C LBoth sequences form interspecies binding pairs. Natural Ig Fc regions tend to be non-interspecies, but as discussed below, they can be engineered to form interspecies pairs (e.g., KiH pairs). Coiled-coil sequences containing leucine zipper sequences can be either interspecies leucine zipper or non-interspecies leucine zipper sequences. See, for example, Zeng et al., (1997) PNAS (USA) 94:3673-3678, and Li et al., (2012), Nature Comms. 3:662.

[0123] Although interspecies binding sequences may, in some cases, form some homodimers, they preferentially dimerize by binding more strongly with their counterpart interspecies binding sequences. Thus, specific heterodimers tend to form when an interspecies dimerization sequence and its counterpart interspecies binding sequence are incorporated into a pair of polypeptides. As an example, when an interspecies dimerization sequence and its counterpart are incorporated into a pair of polypeptides, they may selectively form greater than 70%, 80%, 90%, 95%, 98%, or 99% heterodimers when an equimolar mixture of the polypeptides is combined (e.g., in PBS buffer at 20°C). The remainder of the polypeptide may exist as a monomer or homodimer, which can be separated from the heterodimer. See, for example, Structure B in Figure 19, which contains an interspecies dimerization sequence, and Structure C, which contains two different interspecies dimerization sequences. Furthermore, because interspecies sequences are selective for their counterpart sequences, they may limit interactions with other proteins expressed by cells (e.g., in culture or in a subject), particularly if the interspecies sequences are not naturally occurring or are variants of naturally occurring protein sequences.

[0124] Sequences are considered orthogonal to other sequences when they do not complex (bind) with each other's counterpart sequences. See structure D in Figure 19, where a MAPP comprises an interspecies multimerization sequence and two independently selected interspecies dimerization sequences, all of which are orthogonal to each other. Any of the MAPPS described herein can have two or more (e.g., three, four, or more) orthogonal dimerization sequences. In one embodiment, MAPPs having a multimerization framework peptide can have an orthogonal multimerization domain and a dimerization domain (the dimerization domains may or may not be orthogonal to each other).

[0125] Several sequences that allow polypeptides to interact with sufficient affinity to be used as dimerization and / or multimerization sequences are provided, for example, in U.S. Patent Publication No. 2003 / 0138440. The sequences can be relatively compact in size (e.g., less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, 60, 50, 40, or 30 aa). In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 300 aa. In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 200 aa. In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 100 aa. In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 75 aa. In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 50 aa. In one embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 30 aa.

[0126] Dimerization / multimerization sequences include immunoglobulin heavy chain constant region (Ig Fc) polypeptide sequences (e.g., sequences comprising the CH2-CH3 regions of immunoglobulins such as those provided in Figures 2A-2H and SEQ ID NOs: 1-13), collectin family polypeptides (e.g., ACRP30 or ACRP30-like proteins) containing a collagen domain consisting of collagen repeats Gly-Xaa-Yaa and / or Gly-Xaa-Pro (which may be repeated 10-40 times), coiled-coil domains, leucine zipper domains, interspecies Ig Fc heavy chain constant regions (such as knob-in-hole sequences described in more detail below), Fos / Jun binding pairs, immunoglobulin heavy chain constant region (CH2-CH3) sequences, and Ig CH1 and light chain constant region CL sequences (Ig C). L Examples of Ig constant region sequences include, but are not limited to, an Ig CH1 / CL pair, such as an Ig CH1 sequence paired with a kappa or lambda light chain constant region sequence.

[0127] The framework and / or dimerization polypeptides of MAPP may comprise immunoglobulin heavy chain constant region (e.g., CH2-CH3 domain) polypeptide sequences that function as dimerization or multimerization sequences. Where the framework polypeptide comprises an IgFc multimerization sequence and a CH1 dimerization sequence, it may comprise all or a portion of a native or variant immunoglobulin sequence shown in any of Figures 2A-2H, including the CH1, CH2, and CH3 domains, and any hinge sequence that may be present. Any one or more of the Ig Fc sequence, or the CH1, CH2, and CH3 domains, may have at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with the aa sequence of the Fc region shown in Figures 2A-2H. In particular, the C-terminal lysine provided in some of the sequences provided in Figures 2A-2H (e.g., the IgG sequences in Figures 2D, 2E, 2F, and 2G) may be removed during cellular processing of MAPPs and may not be present in some or all of the expressed MAPP molecules. See, e.g., van den Bremer et al. (2015) mAbs 7:4 and Sissolak et al. (2019) J. Industrial Microbiol. & Biotechnol. 46:1167.

[0128] Such immunoglobulin sequences can covalently link the polypeptides of the MAPP complex together by forming one or two interchain disulfide bonds, thereby stabilizing MAPPs, particularly when a pair of interspecies Ig sequences, such as a knob-in-hole polypeptide pair, is employed. When an Fc polypeptide sequence is employed as a multimerization or dimerization sequence, alone or in combination with a CH1 polypeptide sequence, it can be derived from IgA, IgD, IgE, IgG, or IgM, for example, a human polypeptide sequence, a humanized polypeptide sequence, a synthetic heavy chain constant region Fc region polypeptide, or a consensus heavy chain constant region. As discussed below, the Ig Fc region can further include substitutions that can substantially eliminate the ability of the Ig Fc to effect complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

[0129] The framework and / or dimerization polypeptide of MAPP may comprise at least 150 contiguous aas (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous aas) of the IgA Fc sequence shown in Figure 2A (SEQ ID NO: 1), or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with all aas. The framework and / or dimerization polypeptide of MAPP may comprise at least 150 contiguous aas (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous aas) of the IgD Fc sequence shown in Figure 2B (SEQ ID NO: 2), or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with all aas. The framework and / or dimerization polypeptide of MAPP may comprise at least 125 contiguous aas (at least 150, at least 175, or at least 200 contiguous aas) of the IgE Fc sequence shown in Figure 2C (SEQ ID NO: 3), or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with all aas.

[0130] MAPPs can include one or more IgG Fc sequences as dimerization and / or multimerization sequences. The Fc polypeptide of MAPP can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc sequence has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with the aa sequence of the Fc region shown in Figures 2D-2G. The framework and / or dimerization polypeptides of MAPP may comprise a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with at least 125 contiguous aas (at least 150, at least 175, at least 200, or at least 220 contiguous aas) or all aas of the IgG1 Fc sequence shown in Figure 2D (SEQ ID NO:4).The framework and / or dimerization polypeptides of MAPP may comprise at least 125 (at least 150, at least 175, at least 200, or at least 225) contiguous aas or all aas of the IgG2 Fc sequence shown in Figure 2E (SEQ ID NO:9). The framework and / or dimerization polypeptide of MAPP may comprise a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (at least 150, at least 175, at least 200, at least 225, or at least 240) contiguous aas, or all aas, of the IgG3 Fc sequence shown in Figure 2F (SEQ ID NO: 10).The framework and / or dimerization polypeptide of MAPP may comprise a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (at least 150, at least 175, at least 200, or at least 220) contiguous aas, or all aas, of the IgG4 Fc sequence shown in Figure 2G (SEQ ID NO: 11 or 12).

[0131] The framework and / or dimerization polypeptide of MAPP may comprise a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 80 (at least 90, at least 100, at least 110, or at least all 112) contiguous aas, or all aas, of the IgG1 CH1 sequence provided in Figure 2I.

[0132] The framework and / or dimerization polypeptide of MAPP may comprise a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity with at least 125 (at least 150, at least 175, at least 200, at least 225, or at least 250) contiguous aas, or all aas, of the IgM Fc sequence shown in Figure 2H (SEQ ID NO: 13).

[0133] Framework and / or dimerization polypeptides of MAPPs comprising immunoglobulin sequences (e.g., as shown in Figures 2A-2H) can be covalently linked together by the formation of at least one or at least two interchain disulfide bonds between cysteines flanking the immunoglobulin hinge region. Such disulfide bonds can stabilize the interaction of framework and dimerization polypeptide heterodimers, or, for example, the duplex chains of such heterodimers, when the disulfide bonds are between framework multimerization sequences.

[0134] The framework or dimerization polypeptide may comprise an aa sequence having 100% aa sequence identity with the wt. human IgG1 Fc polypeptide shown in Figure 2D. The framework or dimerization polypeptide may comprise (e.g., as a multimerization sequence) an aa sequence having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity with the wt. human IgG1 Fc polypeptide shown in Figure 2D, including substitution of N297 with an aa other than asparagine (N77 numbered in Figure 2D, SEQ ID NO: 7). In some cases, N297 is substituted with alanine (N297A). The substitution at N297 leads to removal of the carbohydrate modification, resulting in an antibody sequence with reduced complement component 1q ("C1q") binding compared to the wt. protein, and therefore reduced complement-dependent cytotoxicity ("CDC"). K322 (e.g., K322A) substitutions show substantial reductions in FcγR binding affinity and ADCC, with substantial or complete elimination of C1q binding and CDC function. Hezareh et al., (2001) J. Virol. 75:12161-168.

[0135] Amino acid L234 and other aas within the lower hinge region of IgG (e.g., aas 234-239, such as L235, G236, G237, P238, and S239, corresponding to aas 14-19 of SEQ ID NO: 8) are involved in binding to the Fc gamma receptor (FcγR); therefore, mutations at that position (relative to the wt. protein) reduce receptor binding and result in reduced antibody-dependent cellular cytotoxicity (or alternatively, antibody-dependent cell-mediated cytotoxicity, "ADCC"). Hezareh et al. (2001) demonstrated that a double mutant (L234A, L235A) did not effectively bind to either FcγR or C1q, and both ADCC and CDC functions were substantially or completely abolished. A framework or dimerization polypeptide having a substitution in the lower hinge region may comprise an aa sequence having at least 125 contiguous aas (e.g., at least 150, at least 175, at least 200, or at least 210 contiguous aas) of the wt. human IgG1 Fc polypeptide shown in Figure 2D, or at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity with all aas, of the wt. human IgG1 Fc polypeptide shown in Figure 2D, including a substitution of L234 (L14 in the aa sequence shown in Figure 2D) with an aa other than leucine.

[0136] In view of the foregoing, framework or dimerization polypeptides, particularly Ig Fc sequences used as multimerization or dimerization sequences, may contain substitutions that reduce or substantially eliminate ADCC and / or CDC responses. Framework or dimerization polypeptides, particularly Ig Fc sequences used as multimerization or dimerization sequences, may also contain substitutions that reduce or substantially eliminate ADCC and / or CDC responses.

[0137] A framework or dimerization polypeptide having a substitution in the lower hinge region may comprise (e.g., as a multimerization sequence) an aa sequence having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide shown in Figure 2D, comprising a substitution of L235 (L15 in the aa sequence shown in Figure 2D) with an aa other than leucine. In some cases, the framework and / or dimerization polypeptide present in a MAPP having a substitution in the lower hinge region comprises L234A and L235A ("LALA") substitutions (positions corresponding to positions 14 and 15 of the wt. aa sequence shown in Figure 2D, see e.g., SEQ ID NO: 8).

[0138] A framework or dimerization polypeptide having a substitution within the lower hinge region may comprise (e.g., as a multimerization sequence) an aa sequence having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity with the wt. human IgG1 Fc polypeptide shown in FIG. 2D , including a substitution of P331 (P111 of the aa sequence shown in FIG. 2D ) with an aa other than proline. The substitution at P331, like the substitution at N297, leads to reduced binding to C1q for the wt. protein, and thus reduced complement-dependent cytotoxicity (CDC). In one embodiment, the substitution is a P331S substitution. In another embodiment, the substitution is a P331A substitution.

[0139] The framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide shown in Figure 2D, and may include substitutions of D270, K322, and / or P329 (corresponding to D50, K102, and P109 of SEQ ID NO: 4 in Figure 2D) that reduce binding to C1q protein relative to the wt. protein.

[0140] The framework or dimerization polypeptide may comprise (e.g., as a multimerization sequence) an aa sequence having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity with the wt. human IgG1 Fc polypeptide shown in Figure 2D, including substitutions at L234 and / or L235 with aa residues other than leucine, such as L234A and L235A (L14 and / or L15 of the aa sequence shown in Figure 2D), and substitutions of P331 with aa residues other than proline, such as P331S (P111 of the aa sequence shown in Figure 2D). In one case, the framework or dimerization polypeptide present in the MAPP comprises the "triple mutant" aa sequence (SEQ ID NO: 6) shown in Figure 2D (human IgG1 Fc), with L234F, L235E, and P331S substitutions (corresponding to aa positions 14, 15, and 111 of the aa sequence shown in Figure 2D).

[0141] When asymmetric pairing between two polypeptides of MAPP is desired, the framework or dimerization polypeptide present in MAPP can comprise, consist essentially of, or consist of an interspecies binding sequence. Interspecies binding sequences preferentially form heterodimers with their cognate polypeptide sequences (i.e., an interspecies sequence and its partner interspecies sequence), particularly those based on immunoglobulin Fc (Ig Fc) sequence variants. Such interspecies polypeptide sequences include KiH, KiHs-s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. One interspecies binding pair involves the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1 or corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second interspecies binding pair involves a knob formed by a T366W substitution and a hole formed by the triple substitutions T366S, L368A, and Y407V on the complementary Ig Fc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecies binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide with S354C and T366W substitutions (a disulfide bond can form between Y349C and S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Ig Fc polypeptide sequences, with or without knob-in-hole modifications, can be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between Ig Fc polypeptides. Some interspecies bond sequences, based on immunoglobulin sequences, are summarized in the table below, with cross-references to the numbering of the aa positions as they appear in the wt. IgG1 sequence (SEQ ID NO: 4) shown in Figure 2D, shown in brackets "{}". [Table 1]

[0142] In addition to the interspecies pairs of sequences in Table 1, the framework and / or dimerization polypeptides can include an interspecies "SEED" sequence having 45 residues derived from IgA within the IgG1 CH3 domain of the interspecies sequence and 57 residues derived from IgG1 within the IgA CH3 domain of its counterpart interspecies sequence. See Ha et al., Frontiers in Immunol. 7:1-16 (2016).

[0143] The framework or dimerization polypeptide found in MAPP may comprise an interspecies binding sequence or its counterpart interspecies binding sequence selected from the group consisting of KiH, KiHs-s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, A107, or SEED sequences.

[0144] A MAPP can comprise a framework or dimerization polypeptide comprising an IgG1 KiH or KiHs-s sequence with the T146W sequence substitution, and its counterpart interspecies KiH or KiHs-s binding partner polypeptide comprises an IgG1 sequence with the T146S, L148A, and Y187V sequence substitutions, wherein the framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of the wt. IgG1 of Figure 2D. Either or both of the framework or both dimerization polypeptides optionally include 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). These substitutions appear at 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) in the wt. IgG1 sequence in Figure 2D.

[0145] MAPP can comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a T146W KiH sequence substitution, and its counterpart interspecies binding partner polypeptide is T146S, L148A, and Y187V. IgG1 sequences having KiH sequence substitutions, wherein the framework and / or dimerization polypeptides comprise sequences having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of the wt. IgG1 of Figure 2D, and one or both of the framework and / or dimerization polypeptide sequences may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0146] MAPP can comprise a framework or dimerization polypeptide comprising an IgG1 sequence with T146W and S134C KiHs-s substitutions, and its counterpart interspecies binding partner polypeptide contains T146S, L148A, and Y129C. IgG1 sequence having a KiHs-s substitution, wherein the framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of the wt. IgG1 of Figure 2D, and one or both of the framework and / or dimerization polypeptide sequences may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0147] MAPP may include a framework or dimerization polypeptide comprising an IgG1 sequence with S144H and F185A HA-TF substitutions, and its partner interspecies binding partner polypeptide comprises an IgG1 sequence with Y129T and T174F HA-TF substitutions. The framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of wt.IgG1 in FIG. 2D. One or both of the framework and / or dimerization polypeptide sequences may include additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0148] MAPP may include a framework or dimerization polypeptide comprising an IgG1 sequence with T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its partner interspecies binding partner polypeptide comprises an IgG1 sequence with T130V, T146L, K172L, and T174W ZW1 substitutions. The framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of wt.IgG1 in FIG. 2D. One or both of the framework and / or dimerization polypeptide sequences may include additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0149] MAPP may include a framework or dimerization polypeptide comprising an IgG1 sequence having K140D, D179M, and Y187A 7.8.60 substitutions, and its counter-inter-species binding partner polypeptide comprises an IgG1 sequence having T130V E125R, Q127R, T146V, and K189V 7.8.60 substitutions. The framework and / or dimerization polypeptide includes a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., all of at least 125, 150, 170, 180, 190, 200, 210, 220, or 227) consecutive aas of wt.IgG1 in FIG. 2D.

[0150] MAPP may include a framework or dimerization polypeptide comprising an IgG1 sequence having K189D and K172D DD-KK substitutions, and its counter-inter-species binding partner polypeptide comprises an IgG1 sequence having T130V D179K and E136K DD-KK substitutions. The framework and / or dimerization polypeptide includes a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., all of at least 125, 150, 170, 180, 190, 200, 210, 220, or 227) consecutive aas of wt.IgG1 in FIG. 2D, and one or both of the framework and / or dimerization polypeptide sequences may include additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0151] MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with K140E and K189W EW-RVT substitutions, and its partner interspecies binding partner polypeptide comprises an IgG1 sequence with T130V Q127R, D179V, and F185T EW-RVT substitutions, and the framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., all of at least 125, 150, 170, 180, 190, 200, 210, 220, or 227) contiguous aas of the wt.IgG1 of FIG. 2D, and one or both of the framework and / or dimerization polypeptide sequences may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0152] MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with K140E, K189W, and Y129C EW-RVTs-s substitutions, and its partner interspecies binding partner polypeptide comprises an IgG1 sequence with T130V Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, and the framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., all of at least 125, 150, 170, 180, 190, 200, 210, 220, or 227) contiguous aas of the wt.IgG1 of FIG. 2D. One or both of the framework and / or dimerization polypeptide sequences may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0153] MAPP can include a framework or dimerization polypeptide comprising an IgG1 sequence having K150E and K189W A107 substitutions, and its partner interspecies binding partner polypeptide comprises an IgG1 sequence having T130V E137N, D179V, and F185T A107 substitutions. The framework and / or dimerization polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity with at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive aas of the wt.IgG1 of FIG. 2D. One or both of the framework and / or dimerization polypeptide sequences can include additional substitutions such as L14 and / or L15 substitutions (e.g., the "LALA" substitutions L234A and L235A), and / or N77 (N297, e.g., N297A or N@97G).

[0154] [[ID=@]]As an alternative to the use of immunoglobulin CH2 and CH3 heavy chain constant regions as dimerization or multimerization sequences, immunoglobulin light chain constant regions (see FIGS. 3A and 3B) can pair with Ig CH1 sequences (see FIG. 2I) and their partner sequences of framework polypeptides as multimerization or dimerization sequences.

[0155] The MAPP framework or dimerization polypeptide can include an Ig CH1 domain (e.g., the polypeptide of Figure 2I), and the sequence that forms a complex with it (its counterpart binding partner) can include an Ig κ chain constant region sequence, where the framework or dimerization polypeptide includes a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 70, at least 80, at least 90, at least 100, or at least 110 consecutive aas of SEQ ID NOs: 14 and / or 15, respectively. See Figures 2I and 3A. The Ig CH1 and Ig κ sequences can be modified to increase their affinity for each other and, therefore, the stability of any heterodimers formed using them as dimerization or multimerization sequences. Substitutions that increase the stability of CH1-Ig κ heterodimers include those identified as the MD13 combination in Chen et al., MAbs, 8(4):761-774 (2016). In the MD13 combination, two substitutions are introduced into each of the Ig CH1 and Ig κ sequences: the Ig CH1 sequence is modified to contain S64E and S66V substitutions (S70E and S72V in the sequence shown in Figure 2I), and the Ig κ sequence is modified to contain S69L and T71S substitutions (S68L and T70S in the sequence shown in Figure 3A).

[0156] The framework or dimerization polypeptide of MAPP can comprise an Ig CH1 domain (e.g., the polypeptide of SEQ ID NO: 14 in Figure 2I), and its counterpart sequence comprises an Ig λ chain constant region sequence as shown in Figure 3B (SEQ ID NO: 16), wherein the framework or dimerization polypeptide, respectively, comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 70 (e.g., at least 80, at least 90, at least 100, or at least 100) consecutive aas of the sequence shown in Figure 3B.

[0157] The framework and / or dimerization polypeptides of MAPP can each contain a leucine zipper polypeptide as a dimerization or multimerization sequence that binds to each other to form dimers (e.g., homodimers). Non-limiting examples of leucine zipper polypeptides include peptides having any one of the following aa sequences: RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 106), LSSIEKKQEEQTSWLIWISNELTLIRNELAQS (SEQ ID NO: 107), LSSIEKKLEEITSQLIQISNELTLIRNELAQ (SEQ ID NO: 108), LSSIEKKLEEITSQLIQIRNELTLIRNELAQ (SEQ ID NO: 109), LSSIEKKLEEITSQLQQIRNELTLIRNELAQ (SEQ ID NO: 110), LSSLEKKLEELTSQLIQLRNELTLLRNELAQ (SEQ ID NO: 111), ISSLEKKIEELTSQIQQLRNEITLLRNEIAQ (SEQ ID NO: 112). In some cases, the leucine zipper polypeptide comprises the following aa sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO: 113). Additional leucine zipper polypeptides are known in the art, several of which are suitable for use as multimerization or dimerization sequences.

[0158] The framework and / or dimerization polypeptides of MAPP may comprise a dimer-forming coiled-coil peptide. Non-limiting examples of coiled-coil polypeptides include, for example, peptides of any one of the following aa sequences: LKSVENRLAVVENQLKTVIEELKTVKDLLSN (SEQ ID NO: 114), LARIEEKLKTIKAQLSEIASTLNMIREQLAQ (SEQ ID NO: 115), VSRLEEKVKTLKSQVTELASTVSLLREQVAQ (SEQ ID NO: 116), IQSEKKIEDISSLIGQIQSEITLIRNEIAQ (SEQ ID NO: 117), and LMSLEKKLEELTQTLMQLQNELSMLKNELAQ (SEQ ID NO: 118).

[0159] MAPPs may comprise two framework polypeptides and / or pairs of framework and dimerization polypeptides, each having an aa sequence containing at least one cysteine ​​residue capable of forming a disulfide bond that allows homo- or heterodimerization of the polypeptides stabilized by a disulfide bond between the cysteine ​​residues. Examples of such aa sequences include VDLEGSTSNGRQCAGIRL (SEQ ID NO: 119), EDDVTTTEELAPALVPPPKGTCAGWMA (SEQ ID NO: 120), and GHDQETTTQGPGVLLPLPKGACTGQMA (SEQ ID NO: 121).

[0160] Some aa sequences suitable as oligomerization sequences allow the formation of MAPPs capable of forming structures larger than a heterodimeric duplex containing a framework and a dimerization polypeptide. In some cases, triplexes, quadruplexes, and pentamers may be formed. Such aa sequences include, but are not limited to, IgM constant regions that form hexamers (see, e.g., Figure 2H) or pentamers (especially when combined with a mature j-chain peptide lacking a signal sequence, such as that provided in Figure 2J (SEQ ID NO: 122)). Trimer-forming collagen domains can also be employed. The collagen domain can include the 3-aa sequence Gly-Xaa-Xaa and / or GlyXaaYaa, where Xaa and Yaa are independently any aa, and the sequence appears or is repeated multiple times (e.g., 10-40 times, e.g., 10-20, 20-30, or 30-40 times). In such sequences, Xaa and Yaa are frequently proline and hydroxyproline, respectively, in more than 25%, 50%, 75%, 80%, 90%, or 95% of the occurrences of Gly-Xaa-Yaa, or in each occurrence of Gly-Xaa-Yaa. In some cases, the collagen domain contains the sequence Gly-Xaa-Pro repeated 10-40 times, such as 10-20, 20-30, or 30-40 times. Collagen oligomerization peptides can include the following aa sequence: VTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPADSPPPPALSSNP (SEQ ID NO: 123).

[0161] Suitable framework polypeptides (e.g., those having an Ig Fc multimerization sequence) will, in some cases, be half-life extending polypeptides. Thus, in some cases, suitable framework polypeptides increase the in vivo half-life (e.g., serum half-life) of MAPPs compared to a control MAPP having a framework polypeptide with a different aa sequence. For example, in some cases, the framework polypeptide increases the in vivo half-life (e.g., serum half-life in a mammal, such as a human) of a MAPP compared to a control MAPP having a framework polypeptide with a different aa sequence. The half-life may be extended by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 100%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold. As an example, in some cases, an Ig Fc polypeptide sequence (e.g., utilized as a multimerization sequence to form a MAPP heterodimeric duplex comprising a framework and a dimerization polypeptide) increases the stability and / or in vivo half-life (e.g., serum half-life) of a MAPP duplex by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 100%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, relative to a control MAPP duplex lacking the Ig Fc polypeptide sequence.

[0162] 3. Presentation Sequence and Presentation Complex As discussed in more detail below, class II MHC polypeptides comprise two types of polypeptide chains: α chains and β chains. More specifically, MHC class II α chain polypeptides comprise α1 and α2 domains, and β chain polypeptides comprise β1 and β2 domains. Presentation sequences and presentation complexes comprise sufficient MHC class II polypeptides to bind to an epitope and present the epitope to a TCR. Presentation sequences and complexes may also comprise additional protein (peptide) elements, including one or more independently selected MODs and / or one or more independently selected linkers (e.g., linkers disposed between various domains). As discussed herein, unless otherwise specified, neither the presentation sequence nor the presentation complex comprises sufficient MHC transmembrane domains (or intracellular domains, such as cytoplasmic tails) to anchor MAPP molecules (e.g., more than 50% of MAPP molecules) therein when expressed in a mammalian cell membrane (e.g., a CHO cell membrane).

[0163] Conceptually, each presentation sequence and presentation complex can be considered a "soluble MHC" that is fully capable of binding and presenting peptide epitopes. Unless otherwise specified, in a presentation sequence, all of the MHC α1, α2, β1, and β2 domain sequences, as well as the epitope polypeptide, are present in a single polypeptide chain (a single linear sequence of aas produced by translation). See, e.g., Figures 25 and 26.

[0164] When the MHC α1, α2, β1, and β2 domain sequences are split into two or more polypeptide chains, the "soluble MHC" is referred to as a presentation complex. The presentation complex has one chain that is part of a framework peptide or dimerization peptide, referred to as the "first sequence of the presentation complex." The second chain of the presentation complex is referred to as the "second sequence of the presentation complex." The second sequence of the presentation complex can be non-covalently associated with the MHC components present in the first sequence of the presentation complex (through binding interactions between the MHC class II α1, α2, β1, and β2 domain components, as shown in Figures 27-29), as well as one or more disulfide bonds between the first sequence of the presentation complex and the second sequence of the presentation complex. Alternatively, the second sequence of the presentation complex can be non-covalently associated with the MHC component present in the first sequence of the presentation complex through binding interactions between the MHC-class II α1, α2, β1, and β2 domain components and through a binding sequence (e.g., interspecies binding sequences such as those in Figure 30, structures A-E in Figure 31, and Figure 32), in the presence or absence of one or more disulfide bonds between the first sequence of the presentation complex and the second sequence of the presentation complex.

[0165] In some cases, one or more presentation sequences of a MAPP comprise all of the class II components required to bind and present an epitope of interest to a TCR, e.g., the α1, α2, β1, and β2 domains and the epitope in a single polypeptide sequence. In MAPPs comprising a presentation complex, the peptide epitope can be part of the first sequence of the presentation complex or the second sequence of the presentation complex.

[0166] As discussed above, presentation sequences and complexes will typically include a peptide epitope that is part of the MAPP polypeptide chain. However, it is possible to generate MAPPS that include an MHC component but do not include a peptide epitope that is part of the MAPP polypeptide chain. In such embodiments, the epitope noncovalently loaded into the MHC pocket can be a separate peptide (e.g., a phosphopeptide, a lipopeptide, a glycosylated peptide, etc.) or a nonpeptide epitope that is subject to dissociation from the MAPPs.

[0167] 4. MHC class II polypeptides As described above, epitope containing MAPPs include MHC class II polypeptides of various species, including human MHC polypeptides (HLA polypeptides), rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, dogs, cats, ungulates (e.g., horses, cattle, sheep, goats, etc.)), and equivalents.

[0168] For purposes of this disclosure, the term "MHC polypeptide" is intended to include class II MHC polypeptides, including α and β chains or portions thereof. More specifically, MHC class II polypeptides include the α1 and α2 domains of the class II MHC α chain and the β1 and β2 domains of the class II MHC β chain, which represent all or most of the extracellular class II protein required for epitope presentation. In one embodiment, both the α and β class II MHC polypeptide sequences in the MAPP are human.

[0169] MAPPs and their higher-order complexes (e.g., double-chain MAPPs) are intended to be soluble in aqueous media under physiological conditions (e.g., soluble in human plasma at therapeutic levels). Unless otherwise specified, as noted above, the MAPPs described herein are not intended to include membrane anchoring domains (e.g., transmembrane regions of MHC class II α and β chains) or portions thereof sufficient to anchor the MAPP molecule (e.g., more than 50% of the MAPP molecule) or its peptides within the membrane of a cell in which the MAPP is expressed (e.g., a eukaryotic cell, such as a mammalian cell, such as a Chinese hamster ovary cell or "CHO" cell). Similarly, unless otherwise specified, the MAPPs described herein do not include leader and / or intracellular portions (e.g., cytoplasmic tails) that may be present in some naturally occurring MHC class II proteins.

[0170] The MAPPs of the present disclosure include class II MHC polypeptides. Naturally occurring class II MHC polypeptides include α and β chains (e.g., HLA α and β chains). MHC class II polypeptides include MHC class II DP α and β polypeptides, DM α and β polypeptides, DO α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides. As used herein, the term "class II MHC polypeptide" refers to a class II MHC α chain polypeptide, a class II MHC β chain polypeptide, or a portion of a class II MHC α and / or β chain polypeptide alone, or a combination of the foregoing. For example, the term "class II MHC polypeptide" as used herein can be a polypeptide comprising i) only the α1 domain of the class II MHC α chain, ii) only the α2 domain of the class II MHC α chain, iii) only the α1 and α2 domains of the class II MHC α chain, iv) only the β1 domain of the class II MHC β chain, v) only the β2 domain of the class II MHC β chain, vi) only the β1 and β2 domains of the class II MHC β chain, vii) the α1 domain of the class II MHC α chain, the β1 domain of the class II MHC β chain, and the β2 domain of the class II MHC, and the like.

[0171] Human MHC or HLA loci are highly polymorphic in nature, and therefore, as used herein, the term "Class II MHC polypeptide" includes allelic forms of any known Class II MHC polypeptide. See, for example, the HLA nomenclature site maintained by the Anthony Nolan Research Institute, available on the World Wide Web at hla.alleles.org / nomenclature / index.html, which indicates that there are numerous DRA alleles, namely, DRB1 allele, DRB3 allele, DRB4 allele, DRB5 allele, DRB6 allele, DRB7 allele, DRB9 allele, DQA1 allele, DQB1 allele, DPA1, DPB1 allele, DMA allele, DMB allele, DOA allele, and DOB allele.

[0172] In some cases, the MAPP comprises a class II MHC α chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC α chain. Thus, in some cases, the MAPP comprises only the α1 and α2 portions of the class II MHC α chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC α chain.

[0173] In some cases, the MAPPs comprise a class II MHC β chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC β chain. Thus, in some cases, the MAPPs comprise only the β1 and β2 portions of a class II MHC β chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC β chain.

[0174] (i) MHC class II α chain The MHC class II α chain comprises an α1 domain and an α2 domain. In some cases, the α1 and α2 domains present on an antigen-presenting cell are derived from the same MHC class II α chain polypeptide. In some cases, the α1 and α2 domains present on an antigen-presenting cell are derived from two different MHC class II α chain polypeptides.

[0175] MHC class II α chains suitable for inclusion in MAPP presentation sequences or complexes can lack a signal peptide. MHC class II α chains suitable for inclusion in MAPP can have a length of about 60 to about 200 aas. For example, MHC class II α chains suitable for inclusion in MAPP can have a length of about 60 to about 80 amino acids, 80 to about 100 aas, about 100 to about 140 aas, about 140 to about 170 aas, or about 170 to about 200 aas. An MHC class II α1 chain suitable for inclusion in a MAPP can be about 30 amino acids to about 95 amino acids in length; for example, an MHC class II α1 chain suitable for inclusion in a MAPP can be about 30 amino acids to about 50 amino acids, about 50 amino acids to about 70 amino acids, or about 70 amino acids to about 95 amino acids in length. In one embodiment, the MHC class II α1 domain of a MAPP is about 70 amino acids to about 95 amino acids in length. An MHC class II α2 chain suitable for inclusion in a MAPP can be about 30 amino acids to about 95 amino acids in length; for example, an MHC class II α2 chain suitable for inclusion in a MAPP can be about 30 amino acids to about 50 amino acids, about 50 amino acids to about 70 amino acids, or about 70 amino acids to about 95 amino acids in length. In one embodiment, the MHC class II α2 domain of the MAPP is from about 70 aas to about 95 aas.

[0176] (a) DRA polypeptide Suitable MHC Class II DRA polypeptides for inclusion in MAPP can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 150, at least 160, or at least 170 contiguous amino acids of the aa sequence from aa 26 to aa 203 (α1 and α2 domain regions) of the DRA aa sequence shown in Figure 4, or a naturally occurring allelic variant thereof. In some cases, the DRA polypeptide has a length of about 178 aas (e.g., 175, 176, 177, 178, 179, or 180 aas).

[0177] As used herein, the term "DRA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRA polypeptide comprises aas 26-203 of DRA*01:02:01 (see Figure 4), or an allelic variant thereof. In some cases, the allelic variant is a DRA*01:01 polypeptide (e.g., derived from the DRA*01:01:01:01 allele), which differs from DRA*01:02 by having a valine instead of a leucine at position 242 (see Figure 4).

[0178] A suitable DRA for inclusion in a MAPP polypeptide can have at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% aa sequence identity with at least 160, at least 170, or at least 180 contiguous aas of the sequence from aa 26 to aa 216 of the DRA*01:02 sequence shown in Figure 4. "DRA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants.

[0179] Thus, in some cases, a suitable DRA polypeptide comprises the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITNV PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLPSTEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO: 125, amino acids 26-203 of DRA*01:02; see Figure 4), or an allelic variant thereof. In some cases, the allelic variant is a DRA*01:01 allelic variant, which differs from the DRA*01:02 polypeptide by having a valine instead of a leucine at position 242 of the sequence in Figure 4. In some cases, a DRA polypeptide suitable for inclusion in a MAPP contains an amino acid substitution relative to a wild-type DRA polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys (e.g., to form a disulfide bond that stabilizes the MAPP).

[0180] In some cases, a MAPP comprises a variant DRA polypeptide that includes a non-naturally occurring Cys residue (e.g., for forming a disulfide bond that stabilizes the MAPP). For example, in some cases, a MAPP comprises a variant DRA polypeptide that includes at least one aa substitution selected from E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C (see, e.g., SEQ ID NO: 17 in Figure 4).

[0181] Suitable DRA α1 domains for inclusion in MAPP polypeptides, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO: 124), and may have a length of about 84 aas (e.g., 80, 81, 82, 83, 84, 85, or 86 aas).

[0182] Suitable DRA α2 domains for inclusion in MAPP polypeptides, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: V PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO: 126), and can have a length of about 94 aas (e.g., 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas).

[0183] (b) DMA polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DMA polypeptide. The DMA polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 27-217 of the DMA aa sequence shown in Figure 9, including naturally occurring allelic variants thereof. In some cases, the DMA polypeptide has a length of about 191 aas (e.g., 188, 189, 190, 191, 192, or 193 aas).

[0184] "DMA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DMA polypeptide includes AAS 27-217 (α1 and α2 domain regions) of DMA*01:01:01 (see Figure 9) or an allelic variant thereof.

[0185] Suitable DMA α1 domains, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: VPEA PTPMWPDDLQ NHTFLHTVYC QDGSPSVGLS EAYDEDQLFF FDFSQNTRVP RLPEFADWAQ EQGDAPAILF DKEFCEWMIQ QIGPKLDGKI PVSR (SEQ ID NO: 127) and may have a length of about 98 aas (e.g., 94, 95, 96, 97, 98, 99, 100, or 101 aas).

[0186] Suitable DMA α2 domains, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: GFPIAE VFTLKPLEFG KPNTLVCFVS NLFPPMLTVN WQHHSVPVEG FGPTFVSAVD GLSFQAFSYL NFTPEPSDIF SCIVTHEIDR YTAIAYW (SEQ ID NO: 128), and can have a length of about 93 aas (e.g., 90, 91, 92, 93, 94, 95, 96, or 97 aas).

[0187] (c) DOA polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DOA polypeptide. The DOA polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 26-204 (α1 and α2 domain regions) of the DOA aa sequence shown in Figure 11. In some cases, the DOA polypeptide has a length of about 179 aas (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 aas).

[0188] "DOA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DOA polypeptide comprises aas 26-204 of DOA*01:01:01:01 (see FIG. 11) or an allelic variant thereof. In some cases, an allelic variant can be DOA*01:02 by having arginine instead of cysteine ​​at position 80 (R80C), or DOA*01:03 by having valine instead of leucine at position 74 (L74V) relative to DOA*01:01:01:01.

[0189] A suitable DOA α1 domain, including naturally occurring allelic variants thereof, has the following aa sequence: [ka] (SEQ ID NO: 129) and can have a length of about 85 aas (e.g., 83, 84, 85, 86, 87, or 88 aas). Suitable α1 domain sequences can incorporate the L74V and / or R80C substitutions found in DOA*01:02 and DOA*01:03 (aas corresponding to L74 and R80 are shown in italics and bold).

[0190] A suitable DOA α2 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: VPPRVTVLPK SRVELGQPNI LICIVDNIFP PVINITWLRN GQTVTEGVAQ TSFYSQPDHL FRKFHYLPFV PSAEDVYDCQ VEHWGLDAPL LRHW (SEQ ID NO: 130) and may have a length of about 94 aa (e.g., 91, 92, 93, 94, 95, 96, or 97 aa).

[0191] (d) DPA1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DPA1 polypeptide. The DPA1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 29-209 (the α1 and α2 domain regions) of the DPA1 aa sequence shown in Figure 13. In some cases, the DPA1 polypeptide has a length of about 181 aas (e.g., 178, 179, 180, 181, 182, 183, or 184 aas).

[0192] "DPA1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DPA1 polypeptide includes aas 29-209 of DPA1*01:03:01:01 (see Figure 13), or an allelic variant thereof.

[0193] A suitable DPA1α1 domain, including its naturally occurring allelic variants, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATN (SEQ ID NO: 131), and may have a length of approximately 87 aa (e.g., 84, 85, 86, 87, 88, or 89 aa).

[0194] A suitable DPA1α2 domain, including its naturally occurring allelic variants, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO: 132), and may have a length of approximately 97 aas (e.g., 91, 92, 93, 94, 95, 96, or 97 aas).

[0195] Another DPA1 polypeptide comprises aas 29-209 of DPA1*02:01:01:01 (see FIG. 13 ) or variants thereof, having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity. A suitable DPA1α1 domain, including naturally occurring allelic variants thereof, can comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 29-115 of DPA1*02:01:01:01, SEQ ID NO: 67, and can have a length of about 87 aas (e.g., 84, 85, 86, 87, 88, or 89 aas). A suitable DPA1α2 domain, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to DPA1*02:01:01:01, aas 116-209 of SEQ ID NO: 67, and may have a length of approximately 97 aas (e.g., 91, 92, 93, 94, 95, 96, or 97 aas).

[0196] (e) DQA1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DQA1 polypeptide. A suitable DQA1 polypeptide, including naturally occurring allelic variants thereof, can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 24-204 (α1 and α2 domain regions) of any of the DQA1 aa sequences shown in Figure 15. In some cases, the DQA1 polypeptide has a length of about 181 aas (e.g., 177, 178, 179, 180, 181, 182, or 183 aas). In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*01:01 α chain aa sequence of Figure 15, aas 24-204 of ImMunoGeneTics ("IMGT") / HLA Acc No. HLA00601. In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*01:02 α chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc No. HLA00603, GenBank NP_002113. In one embodiment, the DQA1 alpha chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*02:01 alpha chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc number HLA00607.In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*03:01 α chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc No. HLA00609. In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*04:01 α chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc No. HLA00612. In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*05:01 α chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc No. HLA00613. In one embodiment, the DQA1 α chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the DQA1*06:01 α chain aa sequence of Figure 15, aas 24-204 of IMGT / HLA Acc No. HLA00620.

[0197] "DQA1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA1 polypeptide includes the following aa sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO: 133), or an allelic variant thereof.

[0198] A suitable DQA1 α1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN (SEQ ID NO: 134), and may have a length of about 87 aas (e.g., 84, 85, 86, 87, 88, or 89 aas).

[0199] A suitable DQA1 α2 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO: 135), and can have a length of about 94 aa (e.g., 91, 92, 93, 94, 95, 96, or 97 aa).

[0200] (f) DQA2 polypeptide In some instances, a suitable MHC class II α chain polypeptide is a DQA2 polypeptide. The DQA2 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 24 to 204 (α1 and α2 domain regions) of the DQA2 aa sequence shown in Figure 16. In some instances, the DQA2 polypeptide has a length of about 181 aas (e.g., 177, 178, 179, 180, 181, 182, or 183 aas).

[0201] "DQA2 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA2 polypeptide includes the following aa sequence: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO: 136), or an allelic variant thereof.

[0202] A suitable DQA2 α1 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN (SEQ ID NO: 137), and can have a length of about 87 aa (e.g., 84, 85, 86, 87, 88, or 89 aa).

[0203] A suitable DQA2 α2 domain, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO: 138), and may have a length of about 94 aas (e.g., 91, 92, 93, 94, 95, 96, or 97 aas).

[0204] (ii) MHC class II β chain An MHC class II β chain comprises a β1 domain and a β2 domain. In some cases, the β1 and β2 domains present on an antigen-presenting cell are derived from the same MHC class II β chain polypeptide. In some cases, the β1 and β2 domains present on an antigen-presenting cell are derived from two different MHC class II β chain polypeptides.

[0205] MHC class II β chains suitable for inclusion in MAPPs (e.g., higher-order MAPP constructs such as double-chain MAPPs) lack a signal peptide. MHC class II β chains suitable for inclusion in MAPPs can have a length of about 60 to about 210 aas. For example, MHC class II β chains suitable for inclusion in MAPPs can have a length of about 60 to about 90 aas, about 90 to about 120 aas, about 120 to about 150 aas, about 150 to about 180 aas, or about 180 to 210 aas. An MHC class II β1 domain suitable for inclusion in a MAPP can have a length of about 30 to about 10 aas. For example, an MHC class II β1 domain suitable for inclusion in a MAPP can have a length of about 30 to about 50 aas, about 50 to about 70 aas, about 70 to about 90 aas, or about 90 to about 10 aas. An MHC class II β2 domain suitable for inclusion in a MAPP can have a length of about 30 to about 10 aas. For example, an MHC class II β2 domain suitable for inclusion in a MAPP can have a length of about 30 to about 50 aas, about 50 to about 70 aas, about 70 to about 90 aas, or about 90 to about 10 aas.

[0206] MHC class II β chain polypeptides suitable for inclusion in MAPPs can include aa substitutions relative to wild-type MHC class II β chain polypeptides, where the aa substitution replaces an aa (other than Cys) with Cys (e.g., to form a disulfide bond that stabilizes MAPPs). For example, in some cases, the MHC class II β chain polypeptide is a variant DRB1 MHC class II polypeptide that includes an aa substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC class II β chain polypeptide has the following mature DRB1 aa sequence lacking the signal peptide: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR A variant DRB1 polypeptide comprising an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to ARSESAQSKM (SEQ ID NO: 139), and comprising cysteine ​​substitutions in one or more (e.g., two or more) aas selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC class II β chain polypeptide is a variant of a mature DRB3 polypeptide, a mature DRB4 polypeptide, or a mature DRB5 polypeptide (lacking their signal sequences) that includes cysteine ​​substitutions at one or more (e.g., two or more) of positions 5, 7, 10, 19, 20, 33, 151, 152, and 153 (e.g., P5C, F7C, Q10C, N19C, G20C, N33C, G151C, D152C, and / or W153C substitutions).

[0207] (a) DRB1 polypeptide In some cases, a suitable MHC class II α-chain polypeptide is a DRB1 polypeptide. In one embodiment, the DRB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 170, at least 180, or at least 190 contiguous aas of aa 30 to aa 227 of any of the DRB1 aa sequences shown in Figure 5, including naturally occurring allelic variants. Figure 5 shows the DRB1 precursor protein, in which aas 1 to 29 are the signal sequence (underlined), aa 30 to 124 are the β1 region (bold), aa 125 to 227 are the β2 region (bold and underlined), and aa 228 to 250 are the transmembrane region. In some cases, a DRB1 polypeptide suitable for inclusion in a MAPP comprises an aa substitution relative to a wild-type DRB1 polypeptide, where the aa substitution replaces an aa (other than Cys) with Cys.

[0208] An MHC class II β chain polypeptide suitable for incorporation into MAPP can be a DRB1 polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 170, at least 180, or at least 190 contiguous aas of aa 30-227 (β1 and β2 domain regions) of the DRB1 sequence provided in FIG. 5, and including one of the following DRB1 polypeptides: (i) DRB1-1 (DRB1*01:01) β-strand aa sequence Swiss-Prot / UniProt reference (“sp”) P04229.2 in Figure 5 ; (ii) DRB1-3 (DRB1*03:01) β-strand aa sequence sp P01912.2 in Figure 5 ; (iii) DRB1-4 (DRB1*04:01) β-strand aa sequence sp P13760.1 in Figure 5 ; (iv) DRB1-7 (DRB1*07:01) β-strand aa sequence sp P13761.1 in Figure 5 ; (v) DRB1-8 (DRB1*08:01) β-strand aa sequence sp Q30134.2 in Figure 5 ; (vi) DRB1-9 (DRB1*09:01) β-strand aa sequence sp Q9TQE0.1 in Figure 5 ; (vii) DRB1-10 (DRB1*10:01) β-strand aa sequence sp Q30167.2 in Figure 5 ; (viii) DRB1-11 (DRB1*11:01) β-strand aa sequence sp P20039.1 in Figure 5 ; (ix) DRB1-12 (DRB1*12:01) β-chain aa sequence sp Q95IE3.1 in Figure 5; (x) DRB1-13 (DRB1*13:01) β-chain aa sequence sp Q5Y7A7.1 in Figure 5 ; (xi) DRB1-14 (DRB1*14:01) β-chain aa sequence sp Q9GIY3.1 in Figure 5 ; (xii) DRB1-15 (DRB1*15:01) β-chain aa sequence sp P01911 in Figure 5, and (xiii) DRB1-16 (DRB1*16:01) β-strand aa sequence sp Q29974.1 in Figure 5 .

[0209] As used herein, "DRB1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB1 polypeptide includes AAS 31-227 of DRB1*04:01 (DRB1-4) or an allelic variant thereof, as provided in Figure 5 (SEQ ID NO: 24).

[0210] Another suitable DRB1 polypeptide may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 170, at least 180, or at least 190 consecutive aas of the following DRB1*04:01 aa sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO: 139), which may have one or more cysteine ​​substitutions. In one embodiment, the cysteine ​​substitution is a P5C substitution. In one embodiment, the cysteine ​​substitution is a G151C substitution. In one embodiment, the cysteine ​​substitution is a W153C substitution.

[0211] A suitable DRB1 β1 domain, including naturally occurring allelic variants thereof, can comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO: 140), and can have a length of about 95 aas (e.g., including 92, 93, 94, 95, 96, 97, or 98 aas).

[0212] A suitable DRB1 β1 domain has the following amino acid sequence: [ka] (SEQ ID NO: 141), where P5 is substituted with Cys (indicated in bold and italic text).

[0213] A suitable DRB1 β2 domain, including naturally occurring allelic variants thereof, can comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO: 142), and can have a length of about 103 aas (e.g., including 100, 101, 102, 103, 104, 105, or 106 aas).

[0214] A suitable DRB1 β2 domain has the following amino acid sequence: [ka] (SEQ ID NO: 143), where W153 is replaced with Cys (shown in bold and italic text).

[0215] (b) DRB3 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DRB3 polypeptide. In one embodiment, the DRB3 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-227 of any of the DRB3 aa sequences shown in Figure 6, which represents a DRB3 precursor protein in which aas 1-29 are a signal sequence (underlined), aas 30-124 form the β1 region (shown in bold), aas 125-227 form the β2 region, and aas 228-250 form the transmembrane region. DRB3 β chain polypeptides suitable for incorporation into MAPP may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aa 30-227 (β1 and β2 domain regions) of one of the following DRB3 polypeptides: (i) DRB1-3 (DRB3 01:01) β-strand aa sequence GenBank NP_072049.1 in Figure 6; (ii) DRB1-3 β-chain aa sequence in GenBank accession EAX03632.1 in Figure 6 ; (iii) the DRB1-3 (DRB3*02:01) β-strand aa sequence GenBank CAA23781.1 in Figure 6; and (iv) DRB1-3 (DRB3*03:01) β-strand aa sequence GenBank AAN15205.1 in Figure 6 . DRB3 polypeptides suitable for inclusion in MAPP may include aa substitutions relative to wild-type DRB3 polypeptides, which replace an aa (other than Cys) with Cys (e.g., to form a disulfide bond that stabilizes MAPP).

[0216] As used herein, the term "DRB3 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB3 polypeptide includes AAS 30-227 of DRB3*01:01 or an allelic variant thereof, as provided in Figure 6 (SEQ ID NO: 55). Thus, in some cases, a suitable DRB3 polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 170, at least 180, or at least 190 contiguous aas of the following sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRVHPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 144) or an allelic variant thereof. In some cases, a DRB3 polypeptide suitable for inclusion in a MAPP comprises an aa substitution relative to a wild-type DRB3 polypeptide, where the aa substitution replaces an aa (other than Cys) with Cys. Thus, for example, in some cases, the MHC class II β chain polypeptide is a variant DRB3 MHC class II polypeptide comprising a non-naturally occurring Cys at an aa selected from the group consisting of P5C, F7C, L10C, N19C, G20C, N33C, G151C, D152C, and W153C (of the mature DRB3 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO: 145) shown in FIG. 6)).

[0217] A suitable DRB3 β1 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV (SEQ ID NO: 146), and can have a length of about 95 AAS (e.g., 93, 94, 95, 96, 97, or 98 AAS). A suitable DRB3 β1 domain may comprise the following aa sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV (SEQ ID NO: 146), or a naturally occurring allelic variant. A suitable DRB3 β2 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 147), and can have a length of about 10 AAS (e.g., 100, 101, 102, 103, 104, or 105 AAS). A suitable DRB3 β2 domain can comprise the following aa sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 147), or a naturally occurring allelic variant thereof.

[0218] (c) DRB4 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DRB4 polypeptide. The DRB4 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aa 30 to 227 (β1 and β2 domain regions) of the DRB4 aa sequence shown in Figure 7. In some cases, the DRB4 polypeptide has a length of about 198 aa (e.g., including 195, 196, 197, 198, 199, 200, 201, or 202 aa). In some cases, a DRB4 polypeptide suitable for inclusion in a MAPP contains an amino acid substitution relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys.

[0219] As used herein, the term "DRB4 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB4 polypeptide includes aas 30-227 of DRB4*01:03 (SEQ ID NO: 60) as provided in Figure 7, or an allelic variant thereof. In some cases, a suitable DRB4 polypeptide for inclusion in a MAPP includes an amino acid substitution relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys. Thus, for example, in some cases, the MHC class II β chain polypeptide is a variant DRB4 MHC class II polypeptide that includes a non-naturally occurring Cys residue, e.g., the variant DRB4 MHC class II polypeptide includes an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C of the mature DRB4 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO: 148) shown in Figure 7).

[0220] A suitable DRB4 β1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV QRRV (SEQ ID NO: 149) and may have a length of about 95 aas (e.g., 93, 94, 95, 96, 97, or 98 aas).

[0221] A suitable DRB4 β2 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: QPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO: 150) and can have a length of about 103 aas (e.g., 100, 101, 102, 103, 104, or 105 aas).

[0222] (d) DRB5 polypeptide A suitable MHC class II β chain polypeptide for inclusion in a MAPP is a DRB5 polypeptide. The DRB5 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aa 30 to 227 (β1 and β2 domain regions) of the DRB5 aa sequence shown in Figure 8. In some cases, the DRB5 polypeptide has a length of about 198 aa (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 aa). In some cases, the DRB5 polypeptide suitable for inclusion in a MAPP contains an aa substitution relative to the wild-type DRB5 polypeptide, where an aa (other than Cys) is replaced with Cys (e.g., to form a disulfide bond that stabilizes the MAPP).

[0223] As used herein, the term "DRB5 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB5 polypeptide includes aas 30 to 227 of DRB5*01:01 (SEQ ID NO: 61) or an allelic variant thereof, as provided in Figure 8.

[0224] A suitable DRB5 β1 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: M VLSSPLALAG DTRPRFLQQD KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRV (SEQ ID NO: 151), and can have a length of about 95 AAS (e.g., 93, 94, 95, 96, 97, or 98 AAS).

[0225] A suitable DRB5 β2 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: EPKVTV YPARTQTLQH HNLLVCSVNG FYPGSIEVRW FRNSQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SPLTVEWRAQ SESAQS (SEQ ID NO: 152) and may have a length of about 103 aas (e.g., 100, 101, 102, 103, 104, or 105 aas).

[0226] (e) DMB polypeptide In some instances, a suitable MHC class II β chain polypeptide is a DMB polypeptide. The DMB polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 19-207 (β1 and β2 domain regions) of the DMB aa sequence shown in Figure 10. In some instances, the DMB polypeptide has a length of about 189 aas (e.g., including 187, 188, 189, 190, or 191 aas).

[0227] As used herein, the term "DMB polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DMB polypeptide includes aas 19-207 of DMB*01:03 (SEQ ID NO: 63) or an allelic variant thereof, as provided in Figure 10.

[0228] A suitable DMB β1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: GG FVAHVESTCL LDDAGTPKDF TYCISFNKDL LTCWDPEENK MAPCEFGVLN SLANVLSQHL NQKDTLMQRL RNGLQNCATH TQPFWGSLTN RT (SEQ ID NO: 153), and may have a length of about 94 aa (e.g., including 92, 93, 94, 95, 96, or 97 aa).

[0229] A suitable DMB β2 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: RPPSVQVA KTTPFNTREP VMLACYVWGF YPAEVTITWR KNGKLVMPHS SAHKTAQPNG DWTYQTLSHL ALTPSYGDTY TCVVEHTGAP EPILRDW (SEQ ID NO: 154), and may have a length of about 95 aa (e.g., including 93, 94, 95, 96, 97, or 98 aa).

[0230] (f) DOB polypeptide In some cases, a suitable MHC class II β chain polypeptide is a DOB polypeptide. The DOB polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 27-214 of the DOB aa sequence shown in Figure 12. In some cases, the DOB polypeptide has a length of about 188 aas (e.g., 186, 187, 188, 189, or 190 aas).

[0231] As used herein, the term "DOB polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DOB polypeptide includes AAS 27-214 (the B1 and B2 domain regions) of DOB*01:01 (SEQ ID NO: 65) as provided in Figure 12 or an allelic variant thereof.

[0232] A suitable DOB β1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: TDSP EDFVIQAKAD CYFTNGTEKV QFVVRFIFNL EEYVRFDSDV GMFVALTKLG QPDAEQWNSR LDLLERSRQA VDGVCRHNYR LGAPFTVGRK (SEQ ID NO: 155), and may have a length of about 94 AAS (e.g., including 92, 93, 94, 95, 96, or 97 aas).

[0233] A suitable DOB β2 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: VQPEVTVYPE RTPLLHQHNL LHCSVTGFYP GDIKIKWFLN GQEERAGVMS TGPIRNGDWT FQTVVMLEMT PELGHVYTCL VDHSSLLSPV SVEW (SEQ ID NO: 156), and may have a length of about 94 AAS (e.g., including 92, 93, 94, 95, 96, or 97 aas).

[0234] (g) DPB1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DPB1 polypeptide. The DPB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 30-215 of any of the DPB1 aa sequences shown in Figure 14, including naturally occurring allelic variants. Figure 14 shows the DPB1 precursor protein, in which aas 1-29 are a signal sequence (underlined), aas 30-121 form the β1 region, and aas 122-215 form the β2 region. In some cases, a DPB1 polypeptide suitable for inclusion in a MAPP contains an aa substitution relative to the wild-type DPB1 polypeptide, replacing an aa (other than Cys) with Cys (e.g., to form a disulfide bond that stabilizes the MAPP).

[0235] MHC class II β chain polypeptides suitable for inclusion in MAPP include DPB1 polypeptides. In some cases, the DPB1 polypeptide has a length of about 186 aas (e.g., including 184, 185, 186, 187, or 188 aas). In one embodiment, DPB1 can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-215 (the β1 and β2 domain regions) of the DPB1 sequence provided in Figure 14, which includes one of the following DPB1 polypeptides: (i) DPB1*01:01 β-chain aa sequence of IMGT / HLA Acc number HLA00514 in Figure 14; (ii) DPB1*02:01 β-chain aa sequence of IMGT / HLA Acc number HLA00517 in Figure 14; (iii) DPB1*03:01 β-chain aa sequence of IMGT / HLA Acc number HLA00520 in Figure 14; (iv) DPB1*04:01 β-chain aa sequence in GenBank NP_002112.3, IMGT / HLA Acc number HLA00521 in Figure 14; (v) DPB1*06:01 β-chain aa sequence of IMGT / HLA Acc number HLA00524 in Figure 14; (vi) DPB1*11:01 β-chain aa sequence of IMGT / HLA Acc number HLA00528 in Figure 14; (vii) DPB1*71:01 β-chain aa sequence of IMGT / HLA Acc number HLA00590 in Figure 14; (viii) DPB1*104:01 β-chain aa sequence of IMGT / HLA Acc number HLA02046 in Figure 14; (ix) DPB1*141:01 β-chain aa sequence of IMGT / HLA Acc number HLA10364 in Figure 14 .

[0236] As used herein, "DPB1 polypeptide" includes allelic variants, for example, naturally occurring allelic variants. Thus, in some cases, a suitable DPB1 polypeptide includes the following aa sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR RVQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO: 157), or an allelic variant thereof.

[0237] A suitable DPB1 β1 domain, including its naturally occurring allelic variants, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR R (SEQ ID NO: 158) and may have a length of about 92 AAS (e.g., including 90, 91, 92, 93, or 94 aas).

[0238] A suitable DPB1 β2 domain, including its naturally occurring allelic variants, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: VQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO: 159), and may have a length of approximately 94 aa (e.g., including 92, 93, 94, 95, 96, or 97 aa).

[0239] (h) DQB1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DQB1 polypeptide. The DQB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to aas 33-220 of the DQB1 aa sequence shown in Figure 17. In some cases, the DQB1 polypeptide has a length of about 188 aas (e.g., 186, 187, 188, 190, 191, or 192 aas).

[0240] As used herein, "DQB1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB1 polypeptide includes aas 33-220 (β1 and β2 domain regions) of DQB1*06:02 (SEQ ID NO: 103) provided in Figure 17, or an allelic variant thereof.

[0241] A suitable DQB1 β1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity with the following aa sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRR (SEQ ID NO: 160), and may have a length of about 94 AAS (e.g., including 92, 93, 94, 95, or 96 aas).

[0242] A suitable DQB1 β2 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: VEPT VTISPSRTEA LNHHNLLVCS VTDFYPGQIK VRWFRNDQEE TAGVVSTPLI RNGDWTFQIL VMLEMTPQRG DVYTCHVEHP SLQSPITVEW (SEQ ID NO: 161), and may have a length of about 94 aa (e.g., including 92, 93, 94, 95, or 96 aa).

[0243] (i) DQB2 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DQB2 polypeptide. The DQB2 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 33 to 215 (β1 and β2 domain regions) of the DQB2 aa sequence shown in Figure 18A or Figure 18B. In some cases, the DQB2 polypeptide has a length of about 182 aas (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 aas).

[0244] As used herein, "DQB2 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB2 polypeptide includes the following aa sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTVTISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO: 162), or an allelic variant thereof.

[0245] A suitable DQB2 β1 domain, including naturally occurring allelic variants thereof, may comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTV (SEQ ID NO: 163), and may have a length of about 94 AAS (e.g., including 92, 93, 94, 95, 96, or 97 aas).

[0246] A suitable DQB2 β2 domain, including naturally occurring allelic variants thereof, can comprise an AA sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% AA sequence identity to the following aa sequence: TISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO: 164), and can have a length of about 94 aa (e.g., including 92, 93, 94, 95, 96, or 97 aa).

[0247] (iii) MHC class II disease risk-associated alleles and haplotypes Certain alleles and haplotypes of MHC class II are associated with an increased risk of developing diseases, e.g., certain diseases. See, e.g., Erlich et al. (2008) Diabetes 57:1084, Gough and Simmonds (2007) Curr. Genomics 8:453, Mitchell et al. (2007) Robbins Basic Pathology Philadelphia: Saunders, 8 thSee, e.g., Margaritte-Jeannin et al. (2004) Tissue Antigens 63:562, and Kurko et al. (2013) Clin. Rev. Allergy Immunol. 45:170. Several of these diseases and their associated alleles and / or haplotypes are described in WO 2020 / 181273, assigned to Cue Biopharma, and the references cited therein. Some HLA haplotypes and alleles associated with an increased risk of individuals expressing such HLA haplotypes and / or alleles developing a given autoimmune disease are shown in the table provided in FIG. 33. The table also provides a list of disease-associated molecules (e.g., autoantigens such as proteins and peptides) that can act as epitopes or epitope sources. A targeted treatment for a specific disease can include any of the disease-associated HLA haplotypes and / or alleles and corresponding epitopes shown in FIG. 33. Peptide epitopes can be, for example, peptides from 4 aas to about 25 aas in length of any of the autoantigens shown in the table.

[0248] The following are notes to the table provided in Figure 33: 1) AH8.1 (e.g., HLA A1-B8-DR3-DQ2 haplotype); 2) DQ3 alleles include DQB1*03 alleles, such as DQB1*03:01 to DQB1*03:05 proteins; 3) DQ5 alleles include DQB1*05 alleles, such as DQB1*05:01 to DQB1*05:04, and can be associated with DQA1*01:01; 4) DR2 alleles include 5) DR3 haplotype includes DRB1*03:01, DRB1*03:02, DRB1*03:03, and DRB1*03:04; 6) DR4 haplotype includes DRB1*04:01 to DRB1*04:13; AH = ancestral haplotype; 7) Simmonds et al. al., Am. J. Hum. Genet. 76:157-163, (2005), see Table 1. HLA with odds ratios greater than 1.5 include the following DRB1, DAB1, and DQA1 alleles: DRB1*: -03:01 to -03:05, -10:01, -08:01 to 11, -16:01 to 16:06, -11:01 to -11, 21, -01:01 to -01:04, -04:01 to -04:22, and -15:01 to -15:05, DQB1*: -02, -04, -03:01, -03:04, -05, -06:01 to 06:09, and -03:02, and HLA-DQA1*: -05:01 to -05:02, -06:01, -04:01, -01:01, -01:02, -01:04, -01:03, -03:11, and -03:12; 8) Li et al., Mol Med Rep.;17(5):6533-6541(2018), noting epitopes derived from autoantigens including: SMD1 (NCBI accession: CAE11897.1); SMD2 (NCBI accession: AAC13776.1); SMD3 (NCBI accession: AAA57034.1); proliferating cell nuclear antigen (PCNA) (NCBI accession: NP_872590.1); acidic ribosomal phosphoprotein (P1) (NCBI accession: AAA36471.1); acidic ribosomal phosphoprotein (P2) (NCBI accession: AAA36472.1); snRNP-B / B' (NCBI accession: P14678.2); U1-snRNP-C (NCBI accession: NP_003084.1); U1-snRNP-A (NCBI accession: NP_004587.1); nucleolin (NCBI accession: AAA59954.1); acidic ribosomal phosphoprotein (P0) (NCBI accession: AAA36470.1); DNA topoisomerase 1 (cleaved) (NCBI accession: NP_003277.1); DNA topoisomerase 1 (full length) (NCBI accession: NP_003277.1); and U1-SnRNP 68 / 70 kilodaltons (kDa) (NCBI accession: P08621.2).

[0249] (a) Individual disease-risk-associated alleles Associations of several HLA alleles with one or more autoimmune diseases are set forth, for example, in Figure 33. Sequences of disease-associated alleles are provided in figures accompanying this disclosure (e.g., DRB1 alleles are provided in Figure 5). Where disease associations are made to groups of alleles (e.g., DRB1*03), sequences of additional alleles can be obtained from standard references, including those provided by the US National Center for Biotechnology Information (NCBI) and at hla.alleles.org / nomenclature / index.html.

[0250] Exemplary associations between various disease states and specific HLA alleles include the association of HLA-DR3 alleles with early-onset myasthenia gravis, Hashimoto's thyroiditis, autoimmune hepatitis, primary Sjogren's syndrome, and SLE. Other exemplary associations include the association of DRB1*0301 ("DRB1*03:01" in Figure 5) with increased incidence of early-onset Graves' disease and / or type 1 autoimmune hepatitis, and the association of DRB1*04:01 with increased risk of developing multiple sclerosis and / or rheumatoid arthritis.

[0251] DRB1*04:02 association with increased risk of developing idiopathic pemphigus vulgaris and / or SLE (e.g., SLE-associated anticardiolipin, SLE-associated anti-β2-glycoprotein I).

[0252] DRB1*0403 association with increased risk of developing SLE (e.g., increased risk of developing SLE-associated anticardiolipin antibodies and / or SLE-associated anti-β2 glycoprotein I antibodies), DRB1*04:05 association with increased risk of developing rheumatoid arthritis and / or autoimmune hepatitis, and DRB1*04:06 association with increased risk of developing anti-caspase 8 autoantibodies (e.g., in silicosis-systemic sclerosis (SSc)-systemic lupus erythematosus (SLE)).

[0253] Certain DQB1 alleles are also associated with an increased risk of developing autoimmune diseases in individuals expressing such alleles, for example, DQB1*0301 and DQB1*0602 are associated with an increased risk of developing MS and / or more severe MS phenotypes (e.g., more severe inflammatory and neurodegenerative damage).

[0254] (iv) Disulfide bonds and presentation sequences and presentation complexes Disulfide bonds involving MHC peptide sequences can be included in the presentation sequence or complex of MAPP. Disulfide bonds can increase the stability (e.g., thermal stability) of and / or aid in the positioning of peptide epitopes within the binding pocket / groove of MHC formed by its α and β chain sequences. Disulfide bonds can be between two MHC peptide sequences (e.g., a cysteine ​​located in the α chain and a cysteine ​​located in the β chain sequence). Disulfide bonds, particularly those created to position peptide epitopes, can be between two MHC peptide sequences, or alternatively, between an MHC peptide sequence and a linker attaching the peptide epitope and the MHC sequence (e.g., the linker between the epitope and the β1 domain sequence in Structures A and B of Figure 15). Disulfide bonds for epitope stabilization and / or positioning can be created using cysteines found within the MHC sequence and / or cysteines provided in one or more MHC sequences using molecular biology and protein engineering techniques. As discussed above, the α chain can include, for example, a cysteine ​​at positions 3, 4, 12, 28, 29, 72, 75, 80, 81, 82, 93, 94, or 95 of the mature α chain (lacking its signal sequence). For DRA polypeptides, cysteine ​​substitutions include, for example, those at E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C (see Figure 4). The β chain can include, for example, a cysteine ​​at positions 5, 7, 10, 19, 20, 33, 151, 152, or 153 of the mature β chain (lacking its signal sequence). For DB1 polypeptides, possible cysteine ​​substitutions include those at positions P5C, F7C, Q10C (which may be Y10C or E10C for some DRB1 alleles), N19C, G20C, H33C (which may be N33C for some DRB1 alleles), G151C, D152C, and W153C.

[0255] Stabilizing disulfide bonds between the α and β chain sequences within the body of the MHC complex (body disulfides) include those between the α and β chain positions shown in Table 3, which also provides the specific cysteine ​​substitutions of the HLA DRA*01:02 and DRB*0401 sequences. Stabilizing disulfide bonds between MHC (e.g., HLA) α and β chains can be incorporated into any of the MAPP structures described herein. For example, such disulfide bonds can be incorporated into presentation sequences such as those shown in Figure 15 and the MAPPs in Figure 14. Stabilizing disulfide bonds can be incorporated into presentation sequences having, for example, β1, β2, α1, and α2 domains, in order from N- to C-terminus (see, e.g., structure B in Figure 15). [Table 2]

[0256] A disulfide bond between the MHC α and β chain sequences, which helps position the peptide epitope and / or stabilize the structure of the presentation sequence or complex, is formed between the first and second aa of MAPP, where the first aa is either (i) an aa position proximal to the point where the peptide epitope (or peptide epitope and linker) is attached to the MHC peptide sequence, or (ii) an aa (cysteine) in the linker attached to the peptide epitope, while the second aa is elsewhere in the MHC peptide sequence. As an example, if a presentation sequence includes, from N- to C-terminus, a peptide epitope, a β1 domain, a β2 domain, an α1 domain, and an α2 domain aa sequence, a substituted cysteine ​​within the first 10 amino acids of the β1 domain (e.g., aas 5-10) can function as the first aa and, when coupled to, for example, a second cysteine ​​located in the α1 or α2 domain of the presentation sequence, can provide a point for anchoring the peptide epitope and / or stabilizing MAPP. Some examples of disulfide bonds between MHC α and β chain sequences that assist in positioning peptide epitopes and / or stabilizing the structure of the presentation sequence or complex include those shown in Table 4. [Table 3]

[0257] Thus, for example, if the presentation sequence of a complex contains a peptide epitope linked to the β1 domain from the N-terminus to the C-terminus, a disulfide bond between a cysteine ​​substituted at one of positions 5-7 of the β chain and a cysteine ​​substituted at one of positions 80-82 of the α chain can be used to position the peptide epitope or stabilize the structure of the presentation sequence. As an example, a disulfide bond between a P5C substitution in the β chain and a P81C substitution in the α chain can be used to position the peptide epitope and / or stabilize the presentation sequence. The same type of disulfide bond is applicable to presentation complexes, and both the presentation complex and the presentation sequence can have additional disulfide bonds (e.g., as shown in Table 3) for stabilization.

[0258] When a cysteine ​​residue in a linker attached to a peptide epitope is employed to position the peptide epitope and / or stabilize the structure of a presentation sequence or complex, the cysteine ​​is typically located aa adjacent to the point where the linker and peptide epitope assemble. For example, if a MAPP contains an epitope located at the N-terminus of the linker peptide sequence, the cysteine ​​can be within approximately 6 aa of the position where the linker and peptide epitope assemble, i.e., at one of amino acids 1-5 (aa1, aa2, aa3, aa4, or aa5) of the MAPP, including construct epitope-aa1-aa2, aa3-aa4-aa5-(remainder of linker / MAPP). If the linker contains repeats of the sequence GGGGS (SEQ ID NO: 166), aa1-aa5 are G1, G2, G3, G4, and S5, and the linker substitution can be referred to, for example, as "G2C." This is exemplified by SEQ ID NO: 165, which has four repeats of GGGGS, where the aa at position 2 of the linker (aa2) is a glycine substituted by a cysteine: [ka] (SEQ ID NO: 165). Examples of cysteine-containing linkers suitable for forming disulfide bonds with cysteines in MHC peptides (e.g., α chain peptide sequences such as DRA peptides) in presentation sequences or complexes containing epitopes located at the N-terminus of the linker attached to an MHC β chain, such as a DRB polypeptide (i.e., MAPP is an MHC β1 domain, such as a DRB β1 domain, structural epitope-aa1-aa2-aa3-aa4-aa5-[remainder of linker, if present]). Table 5 also provides the locations of substituted cysteines in a DRA polypeptide (e.g., see Figure 4) that form disulfide bonds to position the peptide epitope and / or stabilize the structure of the presentation sequence or complex. [Table 4]

[0259] MAPPs having presentation sequences or complexes containing the epitope-linker-DRB structures listed in Table 5 (e.g., Figure 14, Structures A and B in Figure 15, Structures A, D, F, and H-I in Figure 16, Figure 17, Figure 18, and Figure 19A-F) may have disulfide bonds, for example, to position the peptide epitope and / or stabilize the structure of the presentation sequence or complex. A disulfide can be formed between linker aa2 (e.g., G2C) and a cysteine ​​at aa72 of DRA (e.g., I72C). A disulfide can be formed between linker aa2 (e.g., G2C) and a cysteine ​​at aa72 of DRA (e.g., K75C).

[0260] If a disulfide bond is formed between the linker and the MHC polypeptide of the presentation sequence or presentation complex, the presentation sequence or presentation complex may have additional disulfide bonds (e.g., as in Table 3) for stabilization.

[0261] 5. Immunomodulatory Polypeptides ("MODs") MAPPs may contain one or more immunomodulatory polypeptides or "MODs." MODs suitable for inclusion in MAPPs include IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, IL-23, CD7, CD30L, CD40, CD70, CD80, (B7-1), CD83, CD86 (B7-2), HVEM (CD270), ILT3 (immunoglobulin-like transcript 3), ILT4 (immunoglobulin-like transcript 4), Fas Ligand (FasL), ICAM (intercellular adhesion molecule), ICOS-L (inducible costimulatory ligand), JAG1 (CD339), lymphocytes, and the like. These include toxin beta receptor, 3 / TR6, OX40L (CD252), PD-L1, PD-L2, TGF-β1, TGF-β2, TGF-β3, 4-1BBL, and fragments of any of these, such as ectodomain fragments capable of engaging and signaling through their cognate receptors. Some MOD polypeptides, and their "co-MODS" ("co-immunomodulatory polypeptides" or cognate costimulatory receptors), suitable for inclusion in MAPPs include polypeptide sequences with T cell modulatory activity from the protein pairs listed in the table below. [Table 5]

[0262] In some cases, the MOD is selected from an IL-2 polypeptide, a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGFβ polypeptide, and a PD-L2 polypeptide. In some cases, the MAPP or dual-chain MAPP comprises two different MODs, such as an IL-2 MOD or an IL-2 variant MOD polypeptide, and either a CD80 or CD86 MOD polypeptide. In other cases, the MAPP or dual-chain MAPP comprises an IL-2 MOD or an IL-2 variant MOD polypeptide, and a PD-L1 MOD polypeptide. In some cases, the MODs, which may be the same or different, are present in tandem in the MAPP or dual-chain MAPP. When MODs are presented in tandem, their sequences are either directly adjacent to each other on a single polypeptide, without any intervening sequences, or separated only by a linker polypeptide (e.g., no intervening MHC or epitope sequences). A MOD polypeptide can include all or part of the extracellular portion of a full-length MOD. Thus, for example, a MOD can, in some cases, exclude one or more of the signal peptide, transmembrane domain, and intracellular domain typically found in naturally occurring MODs. Unless otherwise specified, a MOD present in a MAPP or double-chained MAPP does not include a sufficient portion of the signal peptide, intracellular domain, or transmembrane domain to anchor a significant amount (e.g., more than 5% or 10%) of the MAPP or double-chained MAPP within a mammalian cell membrane.

[0263] In some cases, a MOD suitable for inclusion in a MAPP comprises all or a portion (e.g., the extracellular portion) of the aa sequence of a naturally occurring MOD. In other cases, a MOD suitable for inclusion in a MAPP is a variant MOD that comprises at least one aa substitution compared to the aa sequence of a naturally occurring MOD. In some cases, the variant MOD exhibits a binding affinity for the co-MOD that is lower than the affinity of the corresponding naturally occurring MOD for the co-MOD (e.g., a MOD that does not include the aa substitution(s) present in the variant). Suitable variations of a MOD polypeptide sequence that modify affinity can be identified by scanning along the length of the peptide (making aa substitutions, e.g., alanine substitutions or "alanine scanning" or charged residue changes) and testing its affinity. Once key aa positions that modify affinity are identified, those positions can be subjected to a vertical scan, in which the effect of one or more aa substitutions other than alanine is tested.

[0264] a. Reduced affinity MODs and variant MODs A MOD can comprise a wild-type amino acid sequence or can comprise one or more amino acid substitutions, insertions, and / or deletions relative to the wild-type amino acid sequence. An immunomodulatory polypeptide can comprise only the extracellular portion of a full-length immunomodulatory polypeptide. Alternatively, a MOD can comprise all or a portion (e.g., the extracellular portion) of the amino acid sequence of a naturally occurring MOD polypeptide.

[0265] Variant MODs contain at least one amino acid substitution, addition, and / or deletion compared to the amino acid sequence of a naturally occurring immunomodulatory polypeptide. As noted above, in some cases, the variant MOD exhibits a binding affinity for the co-MOD that is lower than the affinity of the corresponding naturally occurring MOD (e.g., an immunomodulatory polypeptide that does not contain the amino acid substitution(s) present in the variant) for the co-MOD.

[0266] MOD polypeptides and variants, including affinity-reduced variants, of proteins such as PD-L1, CD80, CD86, 4-1BBL, and IL-2, are described in the published literature, for example, in published PCT application WO2020132138A1, the disclosure of which relates to immunomodulatory polypeptides, and the specific variant immunomodulatory polypeptides of PD-L1, CD80, CD86, 4-1BBL, and IL-2, are expressly incorporated herein by reference, specifically including paragraphs (00260)-(00455) of WO2020132138A1.

[0267] A suitable immunomodulatory domain exhibiting reduced affinity for a co-immunomodulatory domain may have a difference of 1 to 20 aa from the wild-type immunomodulatory domain. For example, in some cases, a variant MOD present in a MAPP may contain a single aa substitution compared to the corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may contain two aa substitutions compared to the corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may contain three or four aa substitutions compared to the corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may contain five or six aa substitutions compared to the corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may contain seven, eight, nine, or ten aa substitutions compared to the corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may contain 11 to 15 or 15 to 20 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD.

[0268] As discussed above, a variant MOD suitable for inclusion in a MAPP may exhibit reduced affinity for its cognate co-MOD compared to the affinity of the corresponding wild-type MOD for the cognate co-MOD.

[0269] The binding affinity between a MOD polypeptide sequence and its cognate co-MOD polypeptide can be determined by biolayer interferometry (BLI) using purified MOD polypeptide sequence and purified cognate co-MOD according to the procedures described in published PCT application WO2020 / 132138A1.

[0270] b. Masked TGF-β MODs As discussed above, MAPPs of the present disclosure include at least one TGF-β polypeptide reversibly masked by a polypeptide ("masking polypeptide") that binds to the TGF-β polypeptide, which together form a masked TGF-β MOD. The masking polypeptide can be, for example, a TGF-β receptor polypeptide or an antibody that functions to reversibly mask the TGF-β polypeptide present in the MAPP, and the TGF-β polypeptide can otherwise act as an agonist of a cellular TGF receptor. The masked TGF-β MODs provide an active TGF-β polypeptide (e.g., a TGF-β signaling pathway agonist). The TGF-β polypeptide and the masking polypeptide (e.g., a TGF-β receptor fragment) interact with each other to reversibly mask the TGF-β polypeptide, thereby allowing the TGF-β polypeptide to interact with its cellular receptor. Additionally, the masking sequence competes with cellular receptors that can remove TGF-β, such as non-signaling TβRIII, thereby allowing the TGF-β MOD (and thus the MAPP) to effectively deliver active TGF-β agonists to target cells. While the MAPP constructs discussed herein enable epitope-specific presentation of reversibly masked TGF-β to target T cells, they also provide sites for presentation of one or more additional MODs. Thus, the ability of MAPP constructs containing one or more additional MODs allows for the combined presentation of TGF-β and additional MOD(s) to induce target T cell responses in a substantially epitope-specific / selective manner to provide modulation of the target T cells. MAPPs thereby enable delivery of one or more masked TGF-β MODs in an epitope-selective (e.g., dependent / specific) manner that allows for (i) formation of an active immune synapse with target T cells, such as CD4+ cells, that are selective for the epitope, and (ii) modulation (e.g., control / regulation) of the target T cell's response to the epitope.Upon engagement with the TCR of a T cell, the effect of the masked TGF-β MOD-containing MAPP on the T cell will depend on whether any additional MODs are present as part of the MAPP, and if so, which additional MOD(s) are present.

[0271] Additionally, although the MAPPs of the present disclosure can contain both one or more masked TGF-β MODs and one or more additional MODs, such as wt. or variant IL-2, PD-L1, and / or 4-1BBL MOD (discussed above), if desired, the MAPPs of the present disclosure can contain only one or more masked TGF-β MODs. That is, one or more additional MODs, such as wt. or variant IL-2, PD-L1, and / or 4-1BBL MOD, need not be included in the MAPPs of the present disclosure. The masked TGF-β MOD-containing MAPPs of the present disclosure can function as a means of generating TGF-β-driven T cell responses. For example, TGF-β itself can inhibit the development of effector cell function in T cells, activate macrophages, and / or promote tissue repair after local immune and inflammatory effects have subsided.

[0272] Masked TGF-β MODs contain a masked TGF-β polypeptide, but the TGF-β polypeptide can still act as a TβR agonist because the TGF-β polypeptide-mask complex is reversible, "breathing" between an open state in which the TGF-β polypeptide is available to the cellular receptor and a closed state in which the mask engages the TGF-β polypeptide. Thus, the masking polypeptide functions to bind to the TGF-β polypeptide and prevent it from entering into a tight complex with, for example, ubiquitous non-signaling TβR3 molecules that could otherwise scavenge free TGF-β. Furthermore, because the active form of TGF-β is a dimer with higher affinity for TβR3, substitutions that limit dimerization (e.g., a C77S substitution of cysteine ​​at position 77 with serine) can be incorporated into the TGF-β sequence to avoid its receptor-mediated scavenge.

[0273] One effect of the masking sequence is to reduce the effective affinity of TGF-β1, TGF-β2, and TGF-β3 polypeptides for TβR. At the same time, the affinity of the masking polypeptide for the TGF-β polypeptide can be modified so that it dissociates more readily from the TGF-β polypeptide, making the TGF-β polypeptide more available to cellular TβR proteins. That is, if the affinity of the masking polypeptide for the TGF-β polypeptide is reduced, the masked TGF-β MOD will spend more time in an open state. Because the TβRII protein is generally the first peptide in the heteromeric TβR1 / TβR2 signaling complex that interacts with TGF-β, controlling the affinity of the TGF-β polypeptide for TβRII effectively controls the entry of TGF-β into the active signaling complex, while the TGF-β polypeptide is in an open state available to bind to the cellular receptor. For example, incorporation of substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 of TGF-β2 (or the corresponding positions in TGF-β1, TGF-β3) reduces affinity for TβRII polypeptides. Reduced affinity allows interaction between the target cell's TCR and the MAPPs MHC polypeptide and epitope, effectively controlling binding and allowing target cell-specific interaction.

[0274] When a TβRII polypeptide is used as a masking polypeptide, the possibility of direct interaction with cellular TβRI receptors and off-target signaling can be addressed by appropriate modification of the masking sequence. If it is desired to block / limit signaling by the masked TGF-β polypeptide through TβRI and / or modify (e.g., reduce) the affinity of the masked TβRII polypeptide for TGF-β, N-terminal deletions and / or aa substitutions can be incorporated into the masked TβRII polypeptide. Possible modifications include deletion of N-terminal amino acids (e.g., N-terminal Δ14 or Δ25 deletions) and / or substitutions at one or more of L27, F30, D32, S49, I50, T51, S52, I53, E55, V77, D118, and / or E119. Some specific TβRII modifications that result in reduced TβRI association with TβRII and reduced affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q.

[0275] The TGF-β polypeptides present in MAPP are optionally variant TGF-β polypeptides, including variant TGF-β polypeptides that have lower affinity for or are selective for at least one class of TGF-β receptor compared to wild-type TGF-β polypeptides.

[0276] Although TGF-β1, TGF-β2, or TGF-β3 polypeptides can be incorporated into MAPPs as part of a masked TGF-β polypeptide, various factors can influence the selection of a specific TGF-β polypeptide and the specific sequence and aa substitutions employed. For example, TGF-β1 and TGF-β3 polypeptides undergo "clipping" of their amino acid sequences when expressed in certain mammalian cell lines (e.g., CHO cells). In addition, dimerized TGF-β (e.g., TGF-β2) has a higher affinity for TβR3 (betaglycan receptor) than for the TβR2 receptor, which can result in off-target binding and loss of biologically active masked protein to the large in vivo pool of non-signaling TβR3 molecules. To minimize high-affinity off-target binding to TβR3, it may be desirable to replace residues that connect dimeric TGF-β molecules linked by disulfide bonds. Thus, cysteine ​​77 (C77) can be substituted with an amino acid other than cysteine ​​(eg, serine to form a C77S substitution).

[0277] The amino acid sequence of a TGF-β polypeptide is known in the art. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β1 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β2 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β3 polypeptide. Suitable TGF-β polypeptides can have from about 70 aas to about 125 aas, for example, suitable TGF-β polypeptides can have a length of from about 70 aas to about 80 aas, from about 80 aas to about 90 aas, from about 90 aas to about 100 aas, from about 100 aas to about 105 aas, from about 105 aas to about 110 aas, from about 110 aas to about 112 aas, from about 113 aas to about 120 aas, or from about 120 aas to about 125 aas. A suitable TGF-β polypeptide sequence can include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 80, at least 90, at least 100, or at least 110 consecutive aas of a mature form of a human TGF-β1 polypeptide, a human TGF-β2 polypeptide, or a human TGF-β3 polypeptide.

[0278] (i) TGF-β1 polypeptide A suitable TGF-β1 polypeptide has the following TGF-β1 amino acid sequence: [ka] (SEQ ID NO: 167, 112 aa in length), wherein the TGF-β1 polypeptide has a length of about 112 aas. A TGF-β1 preproprotein is provided in Figure 34 as SEQ ID NO: 279. Amino acids R25, C77, V92, and R94 are in bold and italic. See Figure 34.

[0279] In some cases, a suitable TGF-β1 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-β1 polypeptide has the following TGF-β1 amino acid sequence: [ka] (SEQ ID NO: 168), wherein amino acid 77 is Ser. Positions 25, 77, 92, and 94 are in bold and italic.

[0280] (ii) TGF-β2 polypeptide A suitable TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: [ka] (SEQ ID NO: 169), wherein the TGF-β2 polypeptide has a length of about 112 aas. A TGF-β2 preproprotein is provided in Figure 34 as SEQ ID NO: 280. Residues Lys25, Cys77, Ile92, and Lys94 are in bold and italic.

[0281] In some cases, a suitable TGF-β2 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: [ka] (SEQ ID NO: 170), wherein amino acid 77 is replaced by Ser, which is in bold and italic.

[0282] (iii) TGF-β3 polypeptide A suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: [ka] (SEQ ID NO: 171), and the TGF-β3 polypeptide has a length of about 112 aas. TGF-β3 isoform 1 preproprotein is provided in Figure 34 as SEQ ID NO: 281. Positions 25, 92, and 94 are in bold and italic.

[0283] In some cases, a suitable TGF-β3 polypeptide comprises a C77S substitution. In some cases, a suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: [ka] (SEQ ID NO: 172), wherein amino acid 77 is Ser. Positions 25, 92, and 94 are in bold and italic.

[0284] (iv) additional TGF-β polypeptide sequence variations In addition to sequence variations that alter TGF-β molecule dimerization (e.g., cysteine ​​77 substitutions such as C77S), TGF-β1, TGF-β2, and TGF-β3 polypeptides with sequence variations that affect affinity and other properties can be incorporated into masked TGF-β MODs. When a variant TGF-β (e.g., a TβR polypeptide such as a TβRII polypeptide) with reduced affinity for the masking polypeptide is present in a masked TGF-β MOD, these components dissociate more readily, making the TGF-β polypeptide more available to cellular TβR proteins. Because the TβRII protein is generally the first peptide in the heteromeric TβR signaling complex that interacts with TGF-β, interaction with TβRII effectively controls the entry of TGF-β into the active signaling complex. Therefore, variants that control the affinity of TGF-β for TβRII can effectively control the entry of masked TGF-β MODs into the active signaling complex.

[0285] The present disclosure encompasses and provides masked TGF-β MODs, including variant masking TβR (e.g., TβRII) polypeptide sequences and / or variant TGF-β polypeptides, that have altered (e.g., reduced) affinity for each other (relative to an otherwise identical masked TGF-β MOD lacking the sequence variation(s)). The affinity between a TGF-β polypeptide and a TβR (e.g., TβRII) polypeptide can be determined using BLI (blood level intensity) as described above for MODs and their co-MODs.

[0286] (a) Additional TGF-β2 sequence variants The present disclosure includes and provides masked TGF-β2 MODs comprising a masked TβR (e.g., TβRII) polypeptide sequence and either a wt. or variant TGF-β2 polypeptide, where the variant polypeptide has reduced affinity for the masked TβR (relative to an otherwise identical wt. TGF-β polypeptide sequence without sequence variation).

[0287] The present disclosure provides masked TGF-β MODs comprising a masked TβRII receptor sequence and a variant TGF-β2 polypeptide having greater than 85% (e.g., greater than 90%, 95%, 98%, or 99%) sequence identity to at least 100 contiguous aa of SEQ ID NO: 169 and comprising substitutions that reduce the affinity of the variant TGF-β2 polypeptide for the TβRII receptor sequence.

[0288] In some cases, the masked TGF-β MOD comprises a masking TβRII polypeptide and a variant TGF-β (e.g., TGF-β2) polypeptide comprising substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 (see SEQ ID NO: 169 for residue locations and Figure 35 for corresponding residues in TGF-β1 and TGF-β3). These aa residues have been shown to affect the affinity of TGF-β2 for TβRII polypeptides (see Crescenzo et al., J. Mol. Biol. 355:47-62 (2006)). MAPP optionally comprises one or more independently selected MODs, such as IL-2 or a variant thereof. In one case, the masked TGF-β MOD comprises a masking TβRII polypeptide and a TGF-β2 polypeptide having an aa other than Lys or Arg at position 25 of SEQ ID NO: 169, and the MAPP optionally comprises one or more additional independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). The masked TGF-β MOD with a masking TβRII polypeptide can comprise a TGF-β2 polypeptide having an aa other than Ile or Val at position 92 of SEQ ID NO: 169 (or an aa other than Ile, Val, or Leu at position 92), and the MAPP optionally comprises one or more additional independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants). A masked TGF-β MOD having a masking TβRII polypeptide may include a TGF-β2 polypeptide having an aa other than Lys or Arg at position 94 of SEQ ID NO: 169, and the MAPP optionally includes one or more additional independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). A masked TGF-β MOD having a masking TβRII polypeptide may include a TGF-β2 polypeptide including substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94, and the MAPP optionally includes one or more additional independently selected MODs.The masked TGF-β MOD having a masking TβRII polypeptide can include a TGF-β2 polypeptide containing substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94, and the MAPP optionally includes one or more additional independently selected IL-2 MODs or affinity-reduced variants thereof.

[0289] (b) Additional TGF-β1 and TGF-β3 sequence variants and their tandem arrangements In some cases, the masked TGF-β MOD comprises a masking TβRII polypeptide and a variant TGF-β1 or TGF-β3 polypeptide containing substitutions at one or more, two or more, or all three aa positions corresponding to Lys25, Ile92, and / or Lys94 in SEQ ID NO: 169 of TGF-β2. In TGF-β1 or TGF-β3, the corresponding aa positions are as follows: Lys25 is Arg25, Ile92 is Val92, and Lys94 is Arg94, each of which is a conservative substitution. See, e.g., SEQ ID NOs: 279 and 168 for TGF-β1, and SEQ ID NOs: 281 and 172 for TGF-β3.

[0290] As described above, the masked TGF-β MOD optionally comprises one or more independently selected MODs, such as IL-2 or variants thereof. In one example, the masked TGF-β MOD with a masking TβRII polypeptide comprises a TGF-β1 or β3 polypeptide having an aa other than Arg or Lys at position 25, and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one example, the masked TGF-β MOD with a masking TβRII polypeptide comprises a TGF-β1 or β3 polypeptide having an aa other than Val or Ile at position 92 (or an aa other than Ile, Val, or Leu at position 92), and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In another example, the masked TGF-β MOD having a masking TβRII polypeptide comprises a TGF-β2 polypeptide having an aa other than Arg or Lys, and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one specific example, the masked TGF-β MOD having a masking TβRII polypeptide comprises a TGF-β1 or β3 polypeptide comprising substitutions at one or more, two or more, or all three of Arg25, Val92, and / or Arg94, and further comprises one or more independently selected MODs (e.g., IL-2 or variant IL-2 MODs). In another specific example, the masked TGF-β MOD having a masking TβRII polypeptide comprises a TGF-β1 or β3 polypeptide comprising a substitution at one or more, two or more, or all three of Arg25, Val92, and / or Arg94, and further comprises one or more independently selected IL-2 MODs or affinity-reduced variants thereof.

[0291] (v) TGF-β receptor polypeptides and other polypeptides that bind to and mask TGF-β. In any of the TGF-β polypeptides or polypeptide complexes described above, the polypeptide that binds to and masks the TGF-β polypeptide (the "masking polypeptide") can take a variety of forms, including TβRI, TβRII, TβRIII, and anti-TGF-β antibodies, or fragments of antibody-related molecules (e.g., antigen-binding fragments of antibodies, Fab, Fab', single-chain antibodies, scFv, peptide aptamers, or nanobodies).

[0292] (a) TGF-β receptor polypeptide Masking of TGF-β in masked TGF-β MODs can be achieved by utilizing a TGF-β receptor fragment (e.g., the ectodomain sequence of TβRI, TβRII, or TβRIII) that contains a polypeptide sequence sufficient to bind to a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3). In one embodiment, the masking sequence comprises all or a portion of the TβRI, TβRII, or TβRIII ectodomain.

[0293] (1) TGF-β receptor I (TβRI) The polypeptide sequence that masks TGF-β in masked TGF-β MODs can be derived from TβRI (e.g., isoform 1, SEQ ID NO: 282) and can include all or a portion of the TβRI ectodomain (aas 34-126). A suitable TβRI polypeptide for masking TGF-β can include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 70, at least 80, at least 90, at least 100, or 103 aas of the following TβRI ectodomain aa sequence: LQCFCHL CTKDNFTCVT DGLCFVSVTE TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYC CNQDHCNKIE LPTTVKSSPG LGPVEL (SEQ ID NO: 173).

[0294] (2) TGF-β receptor II (TβRII) The polypeptide sequence that masks TGF-β in a masked TGF-β MOD can be derived from TβRII (e.g., isoform A, SEQ ID NO: 283) and can include all or part of the TβRII ectodomain sequence (aa 24-177). A suitable TβRII isoform A for masking TGF-β has the following TβRII ectodomain aa sequence: [ka] The amino acid sequence may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 154 aas of (SEQ ID NO: 174). The position of the aspartic acid residue corresponding to D118 in the B isoform is in bold and italics.

[0295] The polypeptide sequence that masks TGF-β in a masked TGF-β MOD may be derived from TβRII isoform B (SEQ ID NO: 284) and may include all or part of the TβRII ectodomain sequence (aa 24-166). A suitable TβRII isoform B for masking TGF-β has the TβRII isoform B ectodomain aa sequence: [ka] The TGF-β masking polypeptide sequence may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, at least 120, at least 130, at least 140, or 143 aa sequence identity with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the aa sequence of SEQ ID NO: 175. As discussed below, any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the naturally occurring aa at those positions (e.g., alanine). The TGF-β masking polypeptide sequence may comprise a polypeptide of SEQ ID NO: 175 with a D118A or D118R substitution. The sequence that masks TGF-β may comprise the peptide of SEQ ID NO: 175 with a D118A or D118R substitution and one or more of an F30A, D32N, S52L and / or E55A substitution.

[0296] The ectodomain of TβRII can be utilized as a masking polypeptide, but regions of the protein contain charged and hydrophobic patches that can lead to an unfavorable pI and be toxic to cells expressing the polypeptide. Additionally, combining the TβRII ectodomain with an active TGF-β polypeptide can result in a complex that can combine with cell-surface TβRI and activate its signaling receptor (e.g., signaling through the Smad pathway). Modifying the TβRII ectodomain sequence used to mask TGF-β by removing or altering sequences involved in TβRI association can avoid unintended stimulation of cells by masked TGF-β other than through the unique cell-surface heterodimeric TβRI / TβRII complex. Modification of TβRII can also alter (e.g., reduce) the affinity of TβRII for TGF-β (e.g., TGF-β3), thereby enabling unmasking of TGF-β and control of its availability as a signaling molecule. Masked TGF-β MODs containing TβR (e.g., TβRII) peptides with the highest affinity for TGF-β (e.g., TGF-β3) mask the TGF-β sequence most tightly and require higher doses to achieve the same effect. In contrast, aa substitutions in TβRII that reduce affinity unmask the TGF-β polypeptide and are biologically effective at lower doses.

[0297] Thus, if it is desirable to block / limit signaling by a masked TGF-β polypeptide through TβRI and / or modify (e.g., reduce) the affinity of the masked TβRII polypeptide for TGF-β, several modifications to TβRII can be incorporated into the TβRII polypeptide sequence. Possible modifications include deletion of the above-mentioned N-terminal amino acids, such as 14 or 25 N-terminal amino acids (1-14 aas or 1-25 aa, Δ14, Δ modification), and / or substitution at one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific TβRII modifications that result in reduced TβRI association with TβRII and reduced affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q, based on SEQ ID NO: 175. See, e.g., J. Groppe et al. Mol Cell 29, 157-168, (2008) and De Crescenzo et al. JMB 355, 47-62 (2006) for the effects of these substitutions on TGF-β3-TβRII and TβRI-TβRII complexes. Modifications of TβRII, including an N-terminal Δ25 deletion and / or a substitution at F24 (e.g., an F24A substitution), substantially or completely block signaling through the canonical SMAD signaling pathway. In one embodiment, the aspartic acid at position 118 (D118) of the mature TβRII B isoform (SEQ ID NO: 175) is replaced by an amino acid other than Asp or Glu, such as Ala, resulting in a "D118A" substitution, or by Arg, resulting in a D118R substitution. The Asp residue corresponding to D118 is shown in SEQ ID NOs: 174, 284, 175, 176, 177, 178, and 285 (bold and underlined in Figure 36B). N-terminal deletions of 1 to 25 aa in length (eg, a Δ25 deletion) and / or substitutions at F24 (eg, an F24A substitution) may be combined with a D118 substitution (eg, D118A or D118R).N-terminal deletions of 1 to 25 aa in length (e.g., a Δ25 deletion) and / or substitutions at F24 (e.g., an F24A substitution) may also be combined with substitutions at any of the L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119 substitutions (e.g., D118A), and in particular any of the specific substitutions listed for those positions in SEQ ID NO: 175 described above to modify affinity.

[0298] N-terminal deletion of the TβRII polypeptide also results in loss of TβRI interaction, preventing masked TGF-β MODs containing the TβRII polypeptide from acting as a constitutively active complex that participates in and activates TβRI signaling. A 14-aa deletion (Δ14) of the TβRII polypeptide substantially reduces the protein's interaction with TβRI, and a Δ25-aa deletion of TβRII appears to completely abolish the interaction with TβRI. N-terminal deletions also substantially alter the protein's pI, with the Δ14 TβRII ectodomain mutant exhibiting a pI of approximately 4.5-5.0 (e.g., approximately 4.74). Thus, TGF-β MODs can include a TβRII ectodomain polypeptide (e.g., the polypeptide of SEQ ID NO: 174 or 284) having an N-terminal deletion, such as 14 to 25 aas (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa). Modified ectodomain sequences, including those that limit interaction with TβRI, that can be utilized to mask the TGF-β polypeptide in a masked TGF-β MOD are described in the following paragraphs.

[0299] In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD is the TβRII isoform B ectodomain sequence: [ka] (SEQ ID NO: 176). Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the naturally occurring aa at that position (e.g., alanine). In one embodiment, the sequence that masks TGF-β comprises a peptide of SEQ ID NO: 176 with a D118A substitution. In one embodiment, the sequence that masks TGF-β comprises a polypeptide of SEQ ID NO: 176 with a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0300] Combinations of N-terminal deletions of TβRII, such as 14 to 25 aas (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa), that block inadvertent cell signaling resulting from a masked TGF-β / TβRII complex interacting with TβRI, can be combined with other TβRII ectodomain substitutions, including those at any one or more of F30, D32, S52, E55, and / or D118. The combination of deletions and substitutions ensures that the masked TGF-β MOD does not trigger cell signaling other than through the cellular membrane-bound TβRI and TβRII receptors.

[0301] In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD is the TβRII isoform B ectodomain sequence: [ka] The TGF-β masking sequence includes a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, or 114 aas of (SEQ ID NO: 177) (aas 1-14 (Δ14) deleted) with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity. Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the aa naturally occurring at those positions (e.g., alanine). In one embodiment, the TGF-β masking sequence includes a peptide of SEQ ID NO: 177 with a D118A substitution. In one embodiment, the TGF-β masking sequence includes a polypeptide of SEQ ID NO: 177 with a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0302] In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD is the TβRII isoform B ectodomain sequence: [ka] The TGF-β masking sequence includes a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, or 104 aas identity with at least 70, at least 80, at least 90, at least 100, or 104 aas of (SEQ ID NO: 178) (aas 1-25 (Δ25) deleted). Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the aa naturally occurring at that position (e.g., alanine). In one embodiment, the TGF-β masking sequence includes a polypeptide of SEQ ID NO: 178 with a D118A substitution (shown in Figure 36B as SEQ ID NO: 285). In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD comprises the peptide of SEQ ID NO: 178 with a D118A substitution and one or more of an F30A, D32N, S52L, and / or E55A substitution. In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD comprises the peptide of SEQ ID NO: 178 with a D118A and F30A substitution (see Figure 5B). In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD comprises the peptide of SEQ ID NO: 178 with a D118A and D32N substitution (see Figure 36B). In one embodiment, the sequence that masks TGF-β in a masked TGF-β MOD comprises the peptide of SEQ ID NO: 178 with a D118A and S52L substitution (see Figure 36B). In one embodiment, the sequence that masks TGF-β in the masked TGF-β MOD comprises the peptide of SEQ ID NO: 178 with D118A and E55A (see Figure 36B).

[0303] (3) TGF-β receptor III (TβRIII) In one embodiment, the polypeptide sequence that masks TGF-β in a masked TGF-β MOD can be derived from TβRIII (e.g., isoform A, SEQ ID NO: 286 and isoform B, 125) and can include all or a portion of the TβRIII ectodomain (aas 27-787 of the A isoform or 27-786 of the B isoform). In some cases, a suitable TβRIII polypeptide for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 120 aas of the ectodomain sequence of the TβRIII A or B isoform (e.g., provided in Figure 36C as SEQ ID NO: 286 or SEQ ID NO: 287).

[0304] (b) Antibody Although TGF-β receptor polypeptides (e.g., ectodomain sequences) can function to bind to and mask TGF-β polypeptides in masked TGF-β MODs, other polypeptide sequences (protein sequences) that bind to TGF-β sequences can also be employed as masking polypeptides. Suitable polypeptide or protein sequences that can be used to mask TGF-β include antibodies with affinity for TGF-β (e.g., antibodies specific for one or more of TGF-β1, TGF-β2, or TGF-β3), or antibody-related molecules such as anti-TGF-β antibody fragments, nanobodies with affinity for TGF-β polypeptides, and particularly single-chain anti-TGF-β antibodies (e.g., any of which may be humanized). Several antibodies, including scFV antibodies, that bind to and neutralize TGF-β have been described. See, e.g., US Pat. No. 9,090,685. Throughout the embodiments and / or aspects of the invention described in this disclosure, the TβR (e.g., TβRII) sequence used to mask the TGF-β polypeptide may be replaced with a masking antibody sequence (e.g., an scFV or nanobody) having affinity for the TGF-β polypeptide. For example, in each of the masked TGF-β MODs of FIG. 1 , in which the TGF-β receptor sequence is used to mask the TGF-β polypeptide, the receptor polypeptide may be replaced with a masking antibody polypeptide (e.g., an scFV or nanobody) having affinity for the TGF-β polypeptide.

[0305] One potential advantage of using an antibody (e.g., a single-chain antibody) as a masking polypeptide is its ability to limit the isoform(s) of the TGF-β polypeptide to be masked. As an example, a single-chain antibody sequence based on methelimumab (CAT192) against TGF-β1 (e.g., Lord et al., mAbs 10(3):444-452(2018)) can be used to mask that TGF-β isoform when present in TGF-β MODs. In another embodiment, a single-chain antibody sequence specific for TGF-β2 is used to mask that TGF-β isoform when present in TGF-β MODs. In another embodiment, a single-chain antibody sequence specific for TGF-β3 is used to mask that TGF-β isoform when present in TGF-β MODs. Single-chain antibodies can also be specific for combinations of TGF-β isoforms (e.g., ectodomain sequences present in masked TGF-β MODs selected from the group consisting of TGF-β1 & TGF-β2, TGF-β1 & TGF-β3, and TGF-β2 & TGF-β3). Single-chain antibodies can also be pan-specific for TGF-β1, TGF-β2, and TGF-β3 ectodomain sequences present in masked TGF-β MODs. See, e.g., WO 2014 / 164709. Antibodies and single-chain antibodies with desired specificity and affinity for TGF-β isoforms can be prepared by a variety of methods, including screening hybridomas and / or modifications (e.g., combinatorial modifications) of antibody variable region sequences for affinity to the target TGF-β polypeptide sequence.

[0306] In one embodiment, the masked TGF-β MOD comprises a single chain antibody to mask a TGF-β sequence (e.g., a TGF-β3 sequence). In one such embodiment, the single chain amino acid sequence is specific for TGF-β3 as set forth in SEQ ID NO: 171, which contains a C77S substitution (see SEQ ID NO: 281).

[0307] (vi) Location of TGF-β and TGF-β masking sequences in MAPPs The masking sequence (e.g., TGF-β receptor sequence) of a masked TGF-β MOD can be part of the same polypeptide as the TGF-β sequence, i.e., both the masking sequence and the TGF-β sequence are present in "cis." Alternatively, the masking sequence (e.g., TGF-β receptor sequence) and the TGF-β sequence can be part of different polypeptides, i.e., they are present in "trans."

[0308] When the masking sequence and TGF-β sequence of a masked TGF-β MOD are present (arranged in cis) in a single aa sequence (single polypeptide) of a MAPP, the aa sequences can be arranged N-terminally to C-terminally as either a) TGF-β receptor sequence(s) followed by TGF-β sequence(s), or b) TGF-β sequence(s) followed by TGF-β receptor sequence(s). Regardless of the N-terminal to C-terminal order, the masked TGF-β MOD polypeptide sequence can be linked at its N-terminus or C-terminus to any other MAPP polypeptide. Independently selected linker polypeptides (e.g., Gly4Ser repeats) can be used to link the masking sequence (e.g., TGF-β receptor sequence) and the TGF-β sequence, and to link the TGF-β MOD to a polypeptide (e.g., a framework or dimerization polypeptide) of a MAPP. As an example, a cis masked TGF-β MOD may be linked to the C-terminus of a MAPP polypeptide and have the following order from N-terminus to C-terminus: a) a TGF-β receptor sequence (e.g., a TβRII sequence), followed by a TGF-β sequence (e.g., TGF-β3). To further the example, a cis masked TGF-β MOD may be linked (e.g., at its C-terminus) to a framework polypeptide, and the cis masked TGF-β MOD may optionally be followed by another MOD, such as IL-2.

[0309] An example of a masked TGF-β MOD with TβR and TGF-β in cis (masked TGF-β MOD in cis) has the sequence: QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSAECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSGGGGSALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLR SADTTHSTVLGLYNTLNPEASASPSCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS (SEQ ID NO:296), where aas 1-111 are the human TβRII masking sequence with the N-terminal 25 aas deleted (Δ25) and a D118A substitution, aas 112-136 are a linker (five Gly4Ser repeats), and aas 137-248 are the human TGF-β3 sequence with a C77S substitution. Such a sequence can be linked, for example, by its N-terminus directly or indirectly to the C-terminus of a MAPP polypeptide (e.g., a framework polypeptide) via an independently selected linker. In addition, the cis-masked TGF-β MOD sequence can have another MOD sequence (e.g., a human IL-2 or variant IL-2 MOD polypeptide sequence) appended to it.

[0310] When the masking sequence (e.g., TGF-β receptor sequence) and TGF-β sequence of a masked TGF-β MOD are present as part of different MAPP polypeptides (arranged in trans), those polypeptide sequences bind to different (separate) interacting MAPP polypeptides, thereby pairing the TGF-β sequence with the masking polypeptide (e.g., TGF-β receptor sequence). The TGF-β sequence and masking sequence can be located at the N-terminus or C-terminus of a MAPP polypeptide (e.g., framework or dimerization polypeptide). Independently selected linker polypeptides (e.g., Gly4Ser repeats) can be used to link the masking sequence (e.g., TGF-β receptor sequence) or TGF-β sequence to other MAPP polypeptides. As an example, in a trans-masked TGF-β MOD, the TGF-β receptor sequence (e.g., TβRII) can be part of one framework polypeptide, the TGF-β sequence (e.g., TGF-β3) can be part of a second framework polypeptide, and the first and second framework polypeptides associate through an interspecies multimerization sequence. To further the example, the TGF-β sequence and the TGF-β receptor sequence can be located at the C-terminus of the framework polypeptide, optionally followed by another MOD, such as IL-2. As an example, a MAPP having first and second framework polypeptides with interspecies multimerization sequences can have a masking TβR sequence located at the C-terminus of the first framework polypeptide and a TGF-β polypeptide located at the C-terminus of the second framework polypeptide (positions 3 and 3' (see, e.g., Figures 1A and 1B). The masking TβR sequence can be, for example, a TβRII sequence lacking its N-terminal 25 aas (Δ25) and with a D118A substitution: SQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSAECNDNIIFSEEYNTSNPD (SEQ ID NO: 179).The TGF-β polypeptide can be a human TGF-β3 polypeptide with a C77S substitution: ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPSCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS (SEQ ID NO: 180). Independently selected linkers can be used to link the TGF-β and TβR sequences to the framework polypeptide. See, e.g., Example 1, Figures 37 and 38.

[0311] c. IL-2 and its variants As one non-limiting example, the MOD or variant MOD present in the MAPP is IL-2 or a variant IL-2 polypeptide. In some cases, the variant MOD present in the MAPP is a variant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R). The wild-type IL-2 aa sequence can be as follows: [ka] (aa 21-153 of UniProt P60568, SEQ ID NO: 181).

[0312] Wild-type IL2 binds to the IL2 receptor (IL2R) on the surface of cells. The IL2 receptor is a heterotrimeric polypeptide, sometimes comprising an α chain (IL-2Rα, also referred to as CD25), a β chain (IL-2Rβ, also referred to as CD122), and a γ chain (IL-2Rγ, also referred to as CD132). The amino acid sequences of human IL-2Rα, IL2Rβ, and IL-2Rγ are provided in the accompanying sequence listing as SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 184, respectively, and are also provided, for example, in U.S. Patent Publication No. 20200407416.

[0313] In some instances, the variant IL-2 polypeptide exhibits reduced binding affinity to one or more of the IL-2Rα, IL2Rβ, and / or IL-2Rγ chains of human IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO: 181. For example, in some instances, the variant IL-2 polypeptide binds to one or more of the IL-2Rα, IL2Rβ, and / or IL-2Rγ chains of human IL-2R with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO: 181 to the α, β, and / or γ chains of IL-2R (e.g., an IL-2R comprising a polypeptide comprising the aa sequence set forth in SEQ ID NOs: 82-184), when assayed under the same conditions.

[0314] For example, IL-2 variants with a phenylalanine substitution (e.g., with alanine) at position 42 exhibit substantially reduced binding to the IL-2Rα chain, in which case the variant may reduce Treg activation. IL-2 variants with a histidine substitution (e.g., with alanine) at position 16 exhibit reduced binding to the IL2Rβ chain, thereby reducing the likelihood of MAPP binding to non-target T cells due to off-target binding of IL-2 MOD. Some IL-2 variants, such as those with substitutions of amino acids F42 and H16, exhibit substantially reduced binding to the IL-2Rα chain and also reduced binding to the IL2Rβ chain. See, e.g., Quayle, et al., Clin Cancer Res; 26(8) April 15, 2020.

[0315] In some cases, the variant IL-2 polypeptide has a single aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 181. In some cases, the variant IL-2 polypeptide has 2 to 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO: 181. In some cases, the variant IL-2 polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO: 181. In some cases, the variant IL-2 polypeptide has 2 or 3 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO: 181.

[0316] Suitable variant IL-2 polypeptide sequences include polypeptide sequences comprising an aa sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity with at least 80 (e.g., 90, 100, 110, 120, 130, or 133) consecutive aas of SEQ ID NO: 181. Potential amino acid substitutions can be introduced at one or more of the following positions: (i) aa is other than E (e.g., A), position 15; (ii) position 16, where aa is other than H (e.g., A, T, N, C, Q, M, V, or W); (iii) position 20 is an aa other than D (e.g., A); (iv) position 42, where aa is other than F (e.g., A, M, P, S, T, Y, V, or H); (v) aa is other than Y (e.g., A), 45th place; (vi) position 88, where aa is other than N (e.g., A or R); (vii) aa is other than Q (e.g., A), position 126. Combinations of the above substitutions include (H16X, F42X), (D20X, F42X), (E15X, D20X, F42X), (H16X, D20X, F42X), (H16X, F42X, R88X), (H16X, F42X, Q126X), (D20X, F42X, Q126X), (D20X, F42X and Y4X), (H16X, D20X, F42X and Y45X). , (D20X, F42X, Y45X, Q126X), (H16X, D20X, F42X, Y45X, Q126X), where X is optionally a substitution aa selected from the following: positions 15, 20, 45, 126 - A, position 16 - A or T, or equivalently N, C, Q, M, V, or W, position 42 - A, or equivalently M, P, S, T, Y, V, or H, position 88 - A or R.

[0317] IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) consecutive aas of SEQ ID NO: 181, wherein the aa at position 16 is an aa other than H. In some cases, position H16 is substituted by Asn, Cys, Gln, Met, Val, or Trp. In some cases, position H16 is substituted by Ala. In other cases, position H16 is substituted by Thr. Additionally or alternatively, an IL-2 variant comprises a polypeptide having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) consecutive aas of SEQ ID NO: 181, wherein the aa at position 42 is an aa other than F. In some cases, position F42 is substituted by Met, Pro, Ser, Thr, Trp, Tyr, Val, or His. In some cases, position F42 is substituted by Ala.

[0318] IL-2 variants include polypeptides that include an aa sequence that includes all or part of a human IL-2 polypeptide with substitutions at positions H16 and / or F42 (eg, H16A and / or F42A substitutions).

[0319] IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 100, 110, 120, or 130) consecutive aas of SEQ ID NO: 181, wherein the aa at position 16 is an aa other than H and the aa at position 42 is other than F. In some cases, position H16 is substituted by Ala or Thr, and position F42 is substituted by Ala or Thr. In some cases, position H16 is substituted by Ala and position F42 is substituted by Ala (H16A and F42A variants). In a second case, position H16 is substituted by Thr, and position F42 is substituted by Ala (H16T and F42A variants). In the third case, position H16 is substituted by Ala and position F42 is substituted by Thr (H16A and F42T variants). In the fourth case, position H16 is substituted by Thr and position F42 is substituted by Thr Ala (H16T and F42T variants). As mentioned above, such variants will exhibit reduced binding to both the human IL-2R α and IL2R β chains.

[0320] In any of the wild-type or variant IL-2 sequences provided herein, the cysteine ​​at position 125 may be substituted with an aa other than cystine, such as alanine (C125A substitution). In addition to any stability provided by the substitution, this may be employed, for example, when an epitope-bearing peptide or additional peptide is conjugated to the cysteine ​​residue elsewhere in the MAPP, thereby avoiding competition from C125 of the IL-2 MOD sequence.

[0321] d.PD-L1 and its variants In one non-limiting example, the MOD or variant MOD present in the MAPP is PD-L1 or a variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

[0322] The wild-type human PD-L1 polypeptide has the following aa sequence: [ka] (SEQ ID NO: 185), where aas 1 to 18 form the signal sequence, aas 19 to 127 form the Ig-like V-type or IgV domain, and 133 to 225 form the Ig-like C2-type domain.

[0323] The wild-type human PD-L1 ectodomain aa sequence is the following aa sequence: [ka] (SEQ ID NO: 186), where aas 1-109 form an Ig-like V-type or "IgV" domain and aas 115-207 form an Ig-like C2-type domain.

[0324] The wild-type human PD-L1 ectodomain aa sequence also includes the following aa sequence: [ka] (SEQ ID NO: 187), where aas 1-109 form an Ig-like V-type or "IgV" domain and aas 115-207 form an Ig-like C2-type domain. See, for example, NCBI accession and version 3BIK_A, which includes an N-terminal alanine as its first aa.

[0325] A suitable wild-type PD-L1 IgV domain for use as a MOD comprises aa 18 and aas IgV aas 19-127 of SEQ ID NO: 185, as well as a carboxyl-terminal stabilizing sequence, such as the last 7 aas of the sequence (bold and italicized): [ka] (SEQ ID NO: 188). If the carboxyl stabilizing sequence includes a histidine up to about aa 122 (e.g., the histidine about 5 residues C-terminal to the Tyr (Y) that appears as aa 117 in SEQ ID NO: 188), the histidine can form a stabilizing electrostatic bond with the backbone amide at aa 82 and 83 (bold and italicized in SEQ ID NO: 185 (Q107 and L106 in SEQ ID NO: 185)). Alternatively, a stabilizing disulfide bond can be formed by substituting one of aa 82 or 83 (Q107 and L106 in SEQ ID NO: 185) and one of aa residues 121, 122, or 123 (equivalent to aa positions 139-141 in SEQ ID NO: 185).

[0326] A wild-type PD-1 polypeptide can comprise the following aa sequence: [ka] (SEQ ID NO: 189).

[0327] In some cases, the variant PD-L1 polypeptide (e.g., a variant of SEQ ID NO: 186 or the IgV domain of PD-L1) exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 189) compared to the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 185 or SEQ ID NO: 186. For example, in some cases, the variant PD-L1 polypeptide binds to PD-1 (e.g., a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 189) with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 185 or SEQ ID NO: 186.

[0328] e.4-1BBL and its variants In some cases, wild-type and / or variant 4-1BBL MOD polypeptide sequences are present as MODs in MAPPs. Wild-type 4-1BBL binds to 4-1BB (CD137). The wild type 4-1BBL aa sequence may be as follows: MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 190). NCBI Reference Sequence: NP_003802.1, aas 29-49 is the transmembrane region.

[0329] In some cases, the variant 4-1BBL polypeptide is a variant of the tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL. The wild-type aa sequence of the THD of human 4-1BBL can include one of SEQ ID NOs: 191-193, for example, as follows: PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 191), D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 192), and D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 193).

[0330] The wild-type 4-1BB aa sequence may be as follows: MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (SEQ ID NO: 194).

[0331] A variant 4-1BBL polypeptide exhibits reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising an aa sequence set forth in one of SEQ ID NOs: 191-193. For example, a variant 4-1BBL polypeptide may bind to 4-1BB with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a 4-1BBL polypeptide comprising an aa sequence set forth in one of SEQ ID NOs: 191-193 for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising an aa sequence set forth in SEQ ID NO: 194) when assayed under the same conditions.

[0332] 4-1BBL variants suitable for use as MODs in MAPP include polypeptides with at least one aa substitution that have at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to one of SEQ ID NOs: 191, 192, or 193.

[0333] Suitable 4-1BBL variants for inclusion in MAPP include those with at least one aa substitution (e.g., two, three, or four substitutions) and have at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with at least 140 (e.g., at least 160, 175, 180, or 181) consecutive aas of SEQ ID NO: 191.

[0334] 6. Linker As noted above, a MAPP can include a linker sequence (aa, peptide, or polypeptide linker sequence) or "linker" intervening between any two elements of the MAPP, e.g., between an epitope and an MHC polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first MHC polypeptide and a second MHC polypeptide, etc. Although referred to as a "linker," sequences employed in linkers can also be located at the N-terminus and / or C-terminus of the MAPP polypeptide, e.g., to stabilize the MAPP polypeptide or protect it from proteolysis.

[0335] Suitable polypeptide linkers (also referred to as "spacers") are known in the art and can be readily selected and can be any of several suitable lengths, e.g., from 2 aa to 50 aa in length, e.g., from 2 aa to 10 aa, 10 aa to 20 aa, 20 aa to 30 aa, 30 aa to 40 aa, 40 aa to 50 aa, or longer than 50 aa. In embodiments, suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 aa in length. Linkers can generally be classified into three groups: flexible, rigid, and cleavable. See, e.g., Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357, and Klein et al. (2014) Protein Engineering, Design & Selection 27:325. Unless otherwise specified, the linkers employed in the MAPPs of the present disclosure are not cleavable linkers, as are commonly known in the art.

[0336] Polypeptide linkers in MAPP can include, for example, polypeptides comprising, consisting essentially of, or consisting of i) Gly and Ser, ii) Ala and Ser, iii) Gly, Ala, and Ser, iv) Gly, Ser, and Cys (e.g., a single Cys residue), v) Ala, Ser, and Cys (e.g., a single Cys residue), and vi) Gly, Ala, Ser, and Cys (e.g., a single Cys residue). Exemplary linkers include glycine polymers, glycine serine polymers, glycine alanine polymers, alanine serine polymers (e.g., polymers containing the sequence GSGGS (SEQ ID NO: 195) or GGGS (SEQ ID NO: 196)), any of which may be repeated 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), and other flexible linkers known in the art. Both glycine and glycine serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers have significantly more access to phi-psi space than alanine, and are significantly less restricted than residues with longer side chains (Scheraga, Rev. Computational (See Chem. 11173-142 (1992)). Exemplary linkers can also include aa sequences, including, but not limited to, GGSG (SEQ ID NO: 197), GGSGG (SEQ ID NO: 198), GSGSG (SEQ ID NO: 199), GSGGG (SEQ ID NO: 200), GGGSG (SEQ ID NO: 201), GSSSG (SEQ ID NO: 202), or combinations thereof, and equivalents, any of which can be repeated 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Linkers can also include the sequence Gly(Ser)4 (SEQ ID NO: 203) or (Gly)4Ser (SEQ ID NO: 166), either of which can be repeated 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In one embodiment, the linker includes the aa sequence AAAGG (SEQ ID NO: 204), which can be repeated 1 to 10 times.

[0337] A rigid polypeptide linker comprises a sequence of amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating adverse interactions between such domains. Thus, a rigid polypeptide linker can be employed when it is desired to minimize interactions between domains of MAPPs. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers with non-flexible helical structures, such as alpha-helical structures. Examples of rigid peptide linkers include, for example, (EAAAK)n (SEQ ID NO: 205), A(EAAAK)nA (SEQ ID NO: 206), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO: 207), (Lys-Pro)n, (Glu-Pro)n, (Thr-Pro-Arg)n, and (Ala-Pro)n, where n is an integer between 1 and 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid linkers comprising EAAAK (SEQ ID NO:208) include EAAAK (SEQ ID NO:208), (EAAAK)2 (SEQ ID NO:209), (EAAAK)3 (SEQ ID NO:210), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:211), and AEAAAKEAAAKA (SEQ ID NO:212). Non-limiting examples of suitable rigid linkers comprising (AP)n include PAPAP (SEQ ID NO:213, also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO:214, also referred to herein as "(AP)4"), APAPAPAPAPAPAP (SEQ ID NO:215, also referred to herein as "(AP)6"), APAPAPAPAPAPAPAPAP (SEQ ID NO:216, also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:217, also referred to herein as "(AP)10").Non-limiting examples of suitable rigid linkers comprising (KP)n include KPKP (SEQ ID NO:218, also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:219, also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:220, also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:221, also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKPKP (SEQ ID NO:222, also referred to herein as "(KP)10"). Non-limiting examples of suitable rigid linkers comprising (EP)n include EPEP (SEQ ID NO:223, also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:224, also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:225, also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEP (SEQ ID NO:226, also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:227, also referred to herein as "(EP)10").

[0338] In some cases, a linker polypeptide present in a polypeptide of a MAPP contains a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in another polypeptide of a MAPP. In some cases, for example, the linker includes an aa sequence selected from (CGGGS), (GCGGS), (GGCGS), (GGGCS), and (GGGGC), with the remainder of the linker being made up of Gly and Ser residues (e.g., GGGGS units that can be repeated 1 to 10 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Cysteine-containing linkers can also be selected from the following sequences: [ka] (SEQ ID NO: 228), [ka] (SEQ ID NO: 165), and [ka] (SEQ ID NO: 229).

[0339] Thus, the linker to which the epitope is attached can be about 5 to about 50 aas in length. The linker to which the epitope can be attached can be, for example, about 5 to about 50 aas in length and contain more than 50% Gly and Ser residues with one cysteine ​​residue. The linker to which the epitope can be attached can be about 5 to about 50 aas in length and contain more than 50% (Gly)4S repeats with one optional cysteine ​​residue. The linker to which the epitope can be attached can be a (Gly)4S sequence repeated 3 to 8 times (e.g., 3 to 7 times), optionally with one aa replaced by a cysteine ​​residue.

[0340] 7. Epitope Various peptide epitopes (also referred to herein as "epitopes" or "epitope peptides") may be present in MAPPs or higher-order complexes of MAPPs (such as double-chain MAPPs) and may be presentable to TCRs on the surface of T cells.

[0341] Peptide epitopes present in a MAPP (e.g., a double-chain MAPP) are designed to be specifically bound by target T cells that are epitope-specific and have a T cell receptor ("TCR") that specifically binds to the peptide epitope of the MAPP. Thus, epitope-specific T cells bind to peptide epitopes having a reference aa sequence, but do not substantially bind to epitopes that differ from the reference aa sequence.

[0342] a. Peptide epitopes in MAPPs with class II MHC presentation sequences and presentation complexes Epitopes that can be bound by MAPPs having class II MHC presentation sequences or class II MHC presentation complexes and presented to TCRs include epitope-presenting peptides (or simply epitopes) derived from various self- and non-self-antigens, depending on the nature of the MAPP and its desired use. Self- and non-self-antigens that can be incorporated into MAPPs include, but are not limited to, autoantigens and allergens for the treatment or prevention of autoimmune diseases and allergies. Epitopes associated with graft-versus-host disease (GVHD) or hereditary graft-versus-host disease (HVGD) can also be incorporated into MAPPs for the treatment of these conditions.

[0343] A peptide epitope can have a length of about 4 aa to about 25 aa (aa), for example, an epitope can have a length of 5 aa to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa. For example, a peptide epitope present in a MAPP can have a length of 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, the peptide epitope present in the MAPP has a length of between 10 aa and 20 aa, for example, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, and 20 aa.

[0344] (i) self epitope In some cases, the peptide epitopes of MAPPs are epitopes associated with or present on "self" antigens (autoantigens). Antigens associated with autoimmune diseases include Addison's disease (autoimmune adrenalitis, Morbus Addison), alopecia areata, Addison's anemia (Morbus Addison), and the like. Biermer), autoimmune hemolytic anemia (AIHA), cold autoimmune hemolytic anemia (AIHA) (cold hemagglutinin disease, cold autoimmune hemolytic anemia (AIHA) (cold agglutinin disease, (CHAD)), warm autoimmune hemolytic anemia (AIHA) (warm AIHA, warm autoimmune hemolytic anemia (AIHA)), autoimmune hemolytic Donath-Landsteiner anemia (paroxysmal cold hemoglobinuria), antiphospholipid syndrome (APS), atherosclerosis, autoimmune arthritis, temporal arteritis, Takayasu's arteritis (Takayasu's disease, aortic arch disease), temporal arteritis / giant cell arteritis, autoimmune chronic gastritis, autoimmune infertility, autoimmune inner ear disease (AIED), Graves' disease (Morbus Basedow), Bechterew's disease (Morbus Bechterew, ankylosing spondylitis (spondylitis ankylosans), Behcet's syndrome (Morbus Behcet), bowel diseases including autoimmune enteropathy (including ulcerative colitis and Morbus Crohn or Crohn's disease), autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (DCM), chronic fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyarthritis (CIDP), chronic polyarthritis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, CREST syndrome (a syndrome associated with calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), Crohn's disease (Morbus Crohn), ulcerative colitis, dermatitis herpetiformis, autoimmune skin diseases, dermatomyositis, essential mixed cryoglobulinemia, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Goodpasture's syndrome (anti-GBM mediated glomerulonephritis), Guillain-Barré syndrome (GBM, polyradiculitis), autoimmune blood diseases, Hashimoto's thyroiditis, hemophilia, acquired hemophilia, autoimmune hepatitis, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura (MorbusWerlhof, ITP), IgA nephritis, autoimmune infertility, juvenile rheumatoid arthritis (Morbus Still, Still's syndrome), Lambert-Eaton syndrome, systemic lupus erythematosus (SLE), lupus erythematosus (disciform), Lyme disease arthritis (Lyme disease, Borrelia arthritis), Meniere's disease (Morbus The antigen may be an autoantigen associated with autoimmune diseases such as Meniere's disease, mixed connective tissue disease (MCTD), multiple sclerosis (MS, disseminated encephalomyelitis, Charcot's disease), myasthenia gravis (MG), myositis, polymyositis, neuroautoimmune diseases, pemphigus vulgaris, bullous pemphigoid, polyglandular (autoimmune) syndrome (PGA syndrome, Schmidt's syndrome), polymyalgia rheumatica, primary agammaglobulinemia, primary autoimmune cholangitis, progressive systemic sclerosis (PSS), rheumatoid arthritis (RA, chronic polyarthritis, rheumatic disease of the joints, rheumatic fever), sarcoidosis (Morbus-Boeck, Benier-Beck-Schaumann disease), stiff man syndrome, scleroderma, Sjögren's syndrome, autoimmune uveitis, and Wegener's disease (Morbus-Wegner, Wegener's granulomatosis).

[0345] In some cases, the peptide epitope present in the MAPP is a peptide associated with Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-related infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture's syndrome, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), Graves' disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus (e.g., pemphigus vulgaris), pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis, or vitiligo.

[0346] Examples of autoantigens include aggrecan, alanyl-tRNA synthetase (PL-12), αβ crystallin, α fodrin (Sptan 1), α-actinin, α1 antichymotrypsin, α1 antitrypsin, α1 microglobulin, aldolase, aminoacyl-tRNA synthetase, amyloid, annexin, apolipoprotein, aquaporin, bactericidal / permeability-increasing protein (BPI), β-globin precursor BP1, β-actin, β-lactoglobulin A, β-2-glycoprotein I, β2-microglobulin, blood group antigens, C-reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase, cathepsin B, centromere proteins, chondroitin sulfate, chromatin, collagen, complement components, cytochrome C, and cytochrome P450. 2D6, cytokeratin, decorin, dermatan sulfate, DNA topoisomerase I, elastin, Epstein-Barr nuclear antigen 1 (EBNA1), entactin, extractable nuclear antigen, factor I, factor P, factor B, factor D, factor H, factor X, fibrinogen, fibronectin, formiiminotransferase cyclodeaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen granule membrane glycoprotein), glutenin, glycoprotein gpIIb / IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, histone, histidine Dilu-tRNA synthetase (Jo-1), hordein, hyaluronidase, immunoglobulin, integrin, interstitial retinol-binding protein 3, intrinsic factor, Ku (p70 / p80), lactate dehydrogenase, laminin, liver cytosolic antigen type 1 (LC1), liver / kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation-associated protein 5 (MDAS), Mi-2 (chromodomain helicase DNA-binding protein 4), mitochondrial proteins, muscarinic receptors, myelin-associated glycoprotein, myosin, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase,Nicotinic acetylcholine receptor A chain, nucleolin, nucleoporin, nucleosome antigen, PM / Scl100, PM / Scl75, pancreatic β-cell antigen, pepsinogen, peroxiredoxin 1, phosphoglucose isomerase, phospholipid, phosphatidylinositol, platelet-derived growth factor, polymerase β (POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), proteinase-3, proteolipid protein, proteoglycan, prothrombin, recoverin, rhodopsin, ribonuclease, ribonucleoprotein, ribosome, Ribosomal phosphoproteins, RNA, Sm proteins, Sp100 nuclear proteins, SRP54 (signal recognition particle 54 kDa), selectins, smooth muscle proteins, sphingomyelin, streptococcal antigens, superoxide dismutase, synovial joint proteins, T1F1 gamma collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid-stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis factor α, topoisomerase, U1-dnRNP 68 / 70 kDa, U1-snRNP A, U1-snRNP C, U-snRNP B / B', ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin.

[0347] For purposes of this disclosure, epitopes forming part of MAPPs are not associated with celiac disease or type 1 diabetes (T1D). In other words, autoantigens (or the autoepitopes they present) associated with celiac disease or T1D are not included in the MAPPs of this disclosure. Epitopes associated with type 1 diabetes (T1D) include, for example, those derived from preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, the 65 kDa isoform of glutamic acid decarboxylase (GAD65), the 67 kDa isoform of glutamic acid decarboxylase (GAD67), tyrosine phosphatase (IA-2), heat shock protein HSP65, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), islet antigen 2 (IA2), zinc transporter (ZnT8), and antigenic peptides thereof. See, e.g., Mallone et al. (2011) Clin. Dev. Immunol. 2011:513210 and U.S. Patent Publication No. 2017 / 0045529. Epitopes / antigens associated with celiac disease include, for example, celiac-associated epitopes derived from tissue transglutaminase, gliadin, glutenin, secalin, hordein, and avenin. An example of a secalin is lysecalin. An example of a hordein is barley hordein. An example of a glutenin is wheat glutenin. See, e.g., US 2016 / 0279233. An antigen "associated with" a particular autoimmune disorder is an antigen that is the target of autoantibodies and / or autoreactive T cells present in individuals with that autoimmune disorder, and such autoantibodies and / or autoreactive T cells mediate the pathological condition associated with the autoimmune disorder. The present disclosure does not encompass protein constructs comprising celiac or T1D associated antigens / epitopes, methods of preparing compositions comprising such protein constructs or nucleic acids encoding such proteins, or methods of treating T1D and / or celiac disease.

[0348] Autoantigens associated with alopecia areata (autoimmune alopecia) include, for example, hair follicle keratinocyte polypeptides, melanogenesis-associated autoantigens, and melanocyte polypeptides. An example of a melanocyte autoantigen is tyrosinase. Autoimmune alopecia-associated autoantigens also include trichohyalin (Leung et al. (2010) J. Proteome Res. 9:5153) and keratin 16. Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of hair follicle keratinocyte polypeptides, melanocyte polypeptides, melanogenesis-associated polypeptides, tyrosinase, trichohyalin, or keratin 16, ranging in length from 4 aas to about 25 aas. Autoantigens associated with Addison's disease include, for example, 21-hydroxylase. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of 21-hydroxylase from 4 aas to about 25 aas in length.

[0349] Autoantigens associated with autoimmune thyroiditis (Hashimoto's thyroiditis) include, for example, thyroglobulin, thyroid peroxidase, thyroid-stimulating hormone receptor (TSH-receptor), thyroid iodide transporter Na+ / I-symporter (NIS), pendrin, and the like. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of 4 aas to about 25 aas in length of any one of the aforementioned Hashimoto's thyroiditis-associated polypeptides.

[0350] An example of an autoantigen associated with Crohn's disease is pancreatic secretory granule glycoprotein-2 (GP2). Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of GP2 ranging from 4 aas to about 25 aas in length.

[0351] Autoantigens associated with Goodpasture's disease include, for example, the α3 chain of type IV collagen, e.g., aas 135 to 145 of the α3 chain of type IV collagen. See Penades et al. (1995) Eur. J. Biochem. 229:754; Kalluri et al. (1994) Proc. Natl. Acad. Sci. USA 91:6201. Suitable epitope-presenting peptides for inclusion in MAPP may be epitope-presenting peptides of the α3 chain of type IV collagen with a length of 4 to approximately 25 aas.

[0352] Autoantigens associated with Graves' disease include, for example, thyroglobulin, thyroid peroxidase, and thyrotropin receptor (TSH-R). Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of any one of the aforementioned Graves' disease-associated antigens, ranging in length from 4 aas to about 25 aas.

[0353] Autoantigens associated with mixed connective tissue disease include, for example, the U1 ribonucleoprotein (U1-RNP) polypeptide (also known as snRNP70). Sato et al. (2010) Mol. Cell. Biochem. 106:55. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of the U1-RNP polypeptide from 4 aas to about 25 aas in length.

[0354] Autoantigens associated with multiple sclerosis include, for example, myelin basic protein, myelin oligodendrocyte glycoprotein, and myelin proteolipid protein. Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of any one of the aforementioned multiple sclerosis-associated antigens, ranging in length from 4 aas to about 25 aas. As one non-limiting example, the peptide epitope can comprise the aa sequence ENPVVHFFKNIVTPR (SEQ ID NO: 230). In some cases, the MAPPs comprise the DRB1*15:01 MHC class II β chain and a peptide epitope of the aa sequence ENPVVHFFKNIVTPR (SEQ ID NO: 230).

[0355] Autoantigens associated with myasthenia gravis include, for example, acetylcholine receptor (AchR; see, e.g., Lindstrom (2000) Muscle & Nerve 23:453), muscle-specific tyrosine kinase, and low-density lipoprotein receptor-related protein-4. Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of any one of the aforementioned myasthenia gravis-associated antigens, 4 aas to about 25 aas in length. In some cases, suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of AchR, 4 aas to about 25 aas in length.

[0356] An example of an autoantigen associated with Parkinson's disease is alpha-synuclein. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of alpha-synuclein from 4 aas to about 25 aas in length. For example, suitable epitope-presenting peptides for inclusion in MAPP include any one of the following 5 aas to full-length peptides: GKTKEGVLYVGSKTK (SEQ ID NO: 231), KTKEGVLYVGSKTKE (SEQ ID NO: 232), MPVDPDNEAYEMPSE (SEQ ID NO: 233), DNEAYEMPSEEGYQD (SEQ ID NO: 234), EMPSEEGYQDYEPE (SEQ ID NO: 235), and SEEGYQDYEPEA (SEQ ID NO: 236), where "S" represents phosphoserine.

[0357] Autoantigens associated with pemphigus (e.g., pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid) include pemphigus vulgaris immunogens such as the desmosomal cadherin desmogelin 3 (Dsg3), pemphigus foliaceus immunogens such as Dsg1, and bullous pemphigoid immunogens such as hemidesmosomal peptides including BP230 antigen, GPAG1a, and BPAG1b. See, e.g., Cirillo et al. (2007) Immunology 121:377. Autoantigens associated with bullous pemphigoid include bullous pemphigoid antigen 1 (also known as BPAG1, BP230, or dystonin), bullous pemphigoid antigen 2 (also known as BPAG2, BP180, or type XVII collagen), and human integrin alpha-5 and beta-4 subunits. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of any of the aforementioned pemphigus-associated antigens from 4 aas to about 25 aas in length.

[0358] Autoantigens associated with myositis (e.g., polymyositis, dermatomyositis) include, for example, histidyl-tRNA synthetase. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of histidyl-tRNA synthetase from 4 aas to about 25 aas in length.

[0359] Autoantigens associated with rheumatoid arthritis include, for example, collagen, vimentin, aggrecan, fibrinogen, cyclic citrullinated peptides, α-enolase, histone polypeptides, lactoferrin, catalase, actinin, and actin (cytoplasmic 1 and 2 (β / γ)). Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of 4 aas to about 25 aas in length of any one of the aforementioned rheumatoid arthritis-associated antigens.

[0360] Autoantigens associated with scleroderma include nuclear antigens, and suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of scleroderma-associated nuclear antigens ranging in length from 4 aas to about 25 aas.

[0361] Autoantigens associated with Sjögren's syndrome include, for example, the Ro / La ribonucleoprotein (RNP) complex, α-fodrin, β-fodrin, pancreatic islet cell autoantigens, poly(ADP) ribose polymerase (PARP), nuclear mitotic apparatus (NuMA), NOR-90, the Ro 60kDa autoantigen, the Ro 52 antigen, the La antigen (see, e.g., GenBank Accession No. NP_001281074.1), and the p27 antigen. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of 4 aas to about 25 aas in length of any one of the aforementioned Sjögren's syndrome-associated antigens.

[0362] Autoantigens associated with systemic lupus erythematosus (SLE) include, for example, Ro60 autoantigen, low-density lipoprotein, Sm antigens (B / B', D1, D2, D3, E, F, G) of the U-1 small nuclear ribonucleoprotein complex, α-actin 1, α-actin 4, annexin AI, C1q / tumor necrosis factor-related protein, catalase, defensin, chromatin, histone proteins, transketolase, hCAP18 / LL37, and ribonucleoproteins (RNPs). Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of 4 aas to about 25 aas in length of any one of the aforementioned SLE-associated antigens.

[0363] Autoantigens associated with thrombocytopenic purpura include ADAMTS13 (a disintegrin and metalloproteinase containing thrombospondin type 1 motif, member 13) and von Willebrand factor-cleaving protease (VWFCP). Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of ADAMTS13 or VWFCP polypeptides ranging from 4 aas to approximately 25 aas in length.

[0364] Autoantigens associated with vasculitis include proteinase-3, lysozyme C, lactoferrin, leukocyte elastase, cathepsin G, and azurocidin. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of any of the aforementioned vasculitis-associated antigens, from 4 aas to about 25 aas in length.

[0365] Autoantigens associated with vitiligo include SOX9, SOX10, PMEL (premelanosomal protein), tyrosinase, TYRP1 (tyrosine-related protein 1), DDT (D-dopachrome tautomerase), Rab38, and MCHR1 (melanin-concentrating receptor). Suitable epitope-presenting peptides for inclusion in MAPPs can be epitope-presenting peptides of 4 aas to about 25 aas in length of any one of the aforementioned vitiligo-associated polypeptides.

[0366] An example of an autoantigen associated with autoimmune uveitis is interphotoreceptor retinoid-binding protein (IRBP). A suitable epitope-presenting peptide for inclusion in MAPP may be an epitope-presenting peptide of IRBP having a length of 4 to about 25 aas. A suitable epitope-presenting peptide for inclusion in MAPP may be an epitope-presenting peptide of any one of the aforementioned antigens having a length of 4 to about 25 aas.

[0367] Autoantigens associated with autoimmune polyendocrine syndrome include, for example, 17-alpha hydroxylase, histidine decarboxylase, tryptophan hydroxylase, and tyrosine hydroxylase. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of any one of the aforementioned autoimmune polyendocrine syndrome-associated antigens, ranging in length from 4 aas to about 25 aas.

[0368] Autoantigens associated with psoriasis include ADAMTS15. See, e.g., Prinz (2017) Autoimmunity Reviews 16:970. Suitable epitope-presenting peptides for inclusion in MAPP can be epitope-presenting peptides of ADAMTS15 polypeptides ranging from 4 aas to about 25 aas in length.

[0369] (ii) allergens In some cases, the peptide presented in the context of a MAPP comprises a class II MHC presentation sequence(s) or complex(es) and is an allergen. Allergens are too numerous to list, but by way of example, allergens include, but are not limited to, peanuts and tree nuts, plant pollen, latex, and the like. Allergens also include proteins from Hymenoptera proteins (e.g., allergens in honeybee and wasp venoms, such as phospholipase A2, melittin, "antigen 5" found in bee venom, and hyaluronidase).

[0370] Peptide-presenting epitopes for peanut allergens, such as Ara h 1 to 13 proteins from seven protein families, include those in Ara h 1 (e.g., PGQFEDFF (SEQ ID NO: 237), YLQGFSRN (SEQ ID NO: 238), FNAEFNEIRR (SEQ ID NO: 239), QEERGQRR (SEQ ID NO: 240), DITNPINLRE (SEQ ID NO: 241), NNFGKLFEVK (SEQ ID NO: 242), GNLELV (SEQ ID NO: 243), RRYTARLKEG (SEQ ID NO: 244), ELHLLGFGIN (SEQ ID NO: 245), HRIFLAGDKD (SEQ ID NO: 246), IDQIEKQAKD (SEQ ID NO: 247), KDLAFPGSGE (SEQ ID NO: 248), KESHFVSARP (SEQ ID NO: 249), NEGVIVKVSKEHVEELTKHAKSVSK (SEQ ID NO: 250)), Ara h 2 (e.g., HASARQQWEL (SEQ ID NO: 251), QWELQGDRRC (SEQ ID NO: 252), DRRCQSQLER (SEQ ID NO: 253), LRPCEQHLMQ (SEQ ID NO: 254), KIQRDEDSYE (SEQ ID NO: 255), YERDPYSPSQ (SEQ ID NO: 256), SQDPYSPSPY (SEQ ID NO: 257), DRLQGRQQEQ (SEQ ID NO: 258), KRELRNLPQQ (SEQ ID NO: 259), QRCDLDVESG (SEQ ID NO: 260)), and Ara h 3 (e.g., IETWNPNNQEFECAG (SEQ ID NO: 261), GNIFSGFTPEFLAQA (SEQ ID NO: 262), VTVRGGLRILSPDRK (SEQ ID NO: 263), DEDEYEYDEEDRRRG (SEQ ID NO: 264)). See, for example, Zhou et al. (2013) Intl. J. of Food Sci. 2013:8 pages article ID 909140.

[0371] 8. Additional Polypeptides The polypeptide chains of MAPP (e.g., dimerization or framework polypeptides) can include one or more polypeptides in addition to those described above. Suitable additional polypeptides include affinity tags and affinity domains. The one or more additional polypeptides may be included at the N-terminus of the polypeptide chain of MAPP, at the C-terminus of the polypeptide chain of MAPP, or within (within) the polypeptide chain of MAPP.

[0372] Affinity tags and affinity domains Suitable affinity tags / polypeptide affinity domains include, but are not limited to, hemagglutinin (HA, e.g., YPYDVPDYA (SEQ ID NO: 265), FLAG (e.g., DYKDDDDK (SEQ ID NO: 266)), c-myc (e.g., EQKLISEEDL, SEQ ID NO: 267), and the like.

[0373] Affinity tags / domains include, for example, peptide sequences that can interact with binding partners, such as those immobilized on solid supports, useful for identification or purification. DNA sequences encoding multiple consecutive single aa, such as histidine, when fused to an expressed protein, can be used for one-step purification of recombinant proteins by high affinity binding to resin columns such as nickel sepharose. Exemplary affinity tags / domains include HisX5 (HHHHH) (SEQ ID NO: 268), HisX6 (HHHHHH) (SEQ ID NO: 269), C-myc (EQKLISEEDL) (SEQ ID NO: 267), Flag (DYKDDDDK) (SEQ ID NO: 266), StrepTag (WSHPQFEK) (SEQ ID NO: 295), hemagglutinin, e.g., HA tag (YPYDVPDYA) (SEQ ID NO: 265), glutathione-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 297), FHHT (SEQ ID NO: 270), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, W EAAAREACCRECCARA (SEQ ID NO: 271), a metal binding domain, e.g., a zinc binding domain or a calcium binding domain, e.g., from a calcium binding protein, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, flekenin, caltrasin, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, calretinin, intein, biotin, streptavidin, MyoD, leucine zipper sequence, and maltose binding protein.

[0374] b. Targeting sequence MAPPs may include a targeting polypeptide or "targeting sequence" as part of any one or more framework and / or any one or more dimerization polypeptides. The targeting sequence serves to bind or "localize" the MAPPs to cells and / or tissues that exhibit the protein (or other molecule) to which the targeting sequence binds. The targeting sequence may be located, for example, at or near the carboxyl terminus of the framework or dimerization peptide (e.g., in place of the C-terminal MOD in Figure 1A or 1B, or at positions 3, 3', 5, and / or 5' of the MAPP in any of Figures 1A, 1B, or 6-9). In one embodiment, the targeting sequence may be located at position 3 and / or 3'. The targeting sequence serves to bind or "localize" the MAPPs to cells and tissues that exhibit the protein (or other molecule) to which the targeting sequence binds. In some cases, the targeting sequence is an antibody or antigen-binding fragment / portion thereof (e.g., a nanobody, such as an scFv or a heavy chain nanobody or a light chain nanobody). In some cases, the targeting sequence is a single-chain T cell receptor (scTCR). The targeting sequence can be translated as part of the MAPP (e.g., part of the framework polypeptide) or incorporated by covalent attachment of the targeting sequence (e.g., using a cross-linking agent), effectively making the targeting sequence a payload-like molecule attached to the MAPP. The targeting sequence can also be non-covalently attached to the MAPP. For example, a MAPP having a biotin-labeled framework polypeptide can be non-covalently attached to an avidin-labeled targeting antibody or Fab against, for example, an autoantigen. A bispecific antibody (e.g., a bispecific IgG or humanized antibody) having a first antigen-binding site against a portion of the MAPP (e.g., a framework polypeptide) can also be employed to non-covalently attach the MAPP to a targeting sequence (second bispecific antibody-binding site) against a cell or tissue target (e.g., an autoantigen).

[0375] CD4 +As such, anti-CD4 antibodies and antibody-related molecules (e.g., antigen-binding fragments, single-chain antibodies, nanobodies, etc.) can target MAPPs bearing at least one masked TGF-β MOD (alone or in combination with one or more IL-2 MODs) to CD4 T cells. + These antibodies can be employed to target T cells. Several anti-CD4 antibodies are known, including, but not limited to, YTS177, priliximab, keliximab, clenoliximab, zanolimumab, tregalizumab, cedelizumab, and ibalizumab. See, e.g., Konig et al. See also Helling et al., Immunology and Cell Biology 93:396-405 (2015). These and other anti-CD4 antibodies can function as MAPP targeting polypeptides or sequences and can also provide sequences for the construction of antibody-related molecules and sequences that bind to and target CD4. The targeting polypeptide or targeting sequence can be ibalizumab or an antibody-related molecule based on ibalizumab (e.g., having the antigen-binding sequence of ibalizumab).

[0376] 9. Payload-Drug and Other Conjugates The polypeptide chains of MAPPs can comprise a payload such as a therapeutic agent (e.g., a small molecule drug or therapeutic agent), a label (e.g., a fluorescent or radiolabel), or other bioactive agent linked (e.g., covalently attached) to the polypeptide chain. For example, if a MAPP comprises an Fc polypeptide, the Fc polypeptide can comprise a covalently attached payload such as an agent that treats an autoimmune disease, an agent that enhances the action of MAPP, or an agent that alleviates a symptom of a disease.

[0377] The payload can be linked directly or indirectly to the polypeptide chain of the MAPP (e.g., to the Ig Fc polypeptide in the MAPP). Direct linkage can involve linkage to the aa side chain without an intervening linker. Indirect linkage can be linkage via a cross-linking agent, such as a bifunctional cross-linking agent. The payload can be linked to the MAPP by any acceptable chemical bond, including, but not limited to, a thioether bond, an amide bond, a carbamate bond, a disulfide bond, or an ether bond, including those formed by reaction with a cross-linking agent.

[0378] Crosslinkers (crosslinking agents) include cleavable crosslinkers and non-cleavable crosslinkers. Crosslinkers can be homobifunctional or heterobifunctional crosslinkers. In some cases, the crosslinker is a protease-cleavable crosslinker. Suitable crosslinkers can include moieties such as peptides (e.g., 2-10 aas in length, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 aas in length), alkyl chains, poly(ethylene glycol), disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups.Non-limiting examples of suitable cross-linking agents include N-succinimidyl-[(N-maleimidopropionamido)-tetraethylene glycol] ester (NHS-PEG4-maleimide), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexyl LC-SMCC, κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionyl)propionate (SEQ ID NO: 1) N-succinimidyl 4-(p-maleimidophenyl)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl 4(2-pyridylthio)pentanoate (SPP), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(maleimidophenyl)butanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), the "long chain" analog of SMCC (LC-SMCC), 3-maleimidopropanoic acid N-succinimidyl ester (BMPS), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0379] A MAPP payload conjugate can be formed by reacting a MAPP polypeptide (e.g., an IgFc polypeptide) with a cross-linking reagent to introduce 1 to 10 reactive groups. The polypeptide is then reacted with a molecule to be conjugated (e.g., a thiol-containing payload drug, label, or agent) to produce the MAPP-payload conjugate. For example, if the MAPP contains an IgFc polypeptide, the conjugate can be of the form (A)-(L)-(C), where (A) is a polypeptide chain containing the IgFc polypeptide, (L), if present, is a cross-linker, and (C) is a payload. (L), if present, links (A) to (C). In some cases, the MAPP contains an IgFc polypeptide containing one or more (e.g., two, three, four, five, or more than five) molecules of payload. Introducing a payload into the MAPP using an excess of cross-linker can allow multiple molecules of payload to be incorporated into the MAPP.

[0380] Suitable payloads (e.g., drugs) include virtually any small molecule (e.g., molecular weight less than 2,000 daltons) approved by the US Food and Drug Administration and / or listed in the 2020 US Pharmacopeia or National Formulary. In one embodiment, the drugs are less than 2,000 molecular weight. Suitable drugs include nonsteroidal anti-inflammatory drugs and glucocorticoids, and the like.

[0381] D. Nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding one or more polypeptides of MAPP. In some cases, the nucleic acid is a recombinant expression vector, and thus the present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding MAPP.

[0382] 1. A nucleic acid encoding a MAPP or a MAPP that forms a higher-order complex, such as a double-chain MAPP, comprising at least one dimerization sequence and a multimerization sequence. The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a MAPP having a framework polypeptide comprising at least one dimerization sequence and at least one multimerization sequence that allows two molecules of the framework polypeptide to form a dimer or higher-order complex. The nucleic acid may additionally comprise a nucleotide sequence encoding a dimerization peptide. When a MAPP comprises a presentation sequence, the nucleic acid encoding either or both of the framework polypeptide and / or the dimerization peptide may comprise a sequence encoding the presentation sequence. When a MAPP comprises a presentation complex, the nucleic acid encoding either or both of the framework polypeptide and / or the dimerization peptide may further comprise a sequence encoding a first sequence of the presentation complex and / or a second sequence of the presentation complex. The nucleic acid sequence encoding MAPPs may also encode a peptide epitope. The nucleotide sequence(s) comprising any of the MAPP polypeptides may be operably linked to a transcriptional control element(s), for example, a promoter. It will be apparent that the individual polypeptides of MAPP (e.g., framework polypeptides and dimerization polypeptides) can be encoded on a single nucleic acid (e.g., under the control of separate promoters) or, alternatively, can be located on two or more separate nucleic acids (e.g., plasmids).

[0383] 2. Recombinant Expression Vector The present disclosure provides a recombinant expression vector comprising a nucleic acid encoding one or more polypeptides of MAPP or its higher-order complex.In some cases, the recombinant expression vector is a non-viral vector.In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, for example, U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, or a non-integral viral vector.

[0384] Suitable expression vectors include viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655), adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997,Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997,Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999, Ali et al., Hum Mol Genet 5:591 594,1996, Srivastava in WO 93 / 09239, Samulski et al., J. Vir(1989)63:3822-3828, Mendelson et al., Virol.(1988)166:154-165, and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (see, for example, Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al., J Virol.73:7812 7816, 1999), retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available.

[0385] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., can be used in the expression vector (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0386] In some cases, the nucleotide sequence encoding one or more polypeptides of MAPP is operably linked to a control element, e.g., a transcription control element such as a promoter. The transcription control element can be functional in either eukaryotic cells, e.g., mammalian cells such as human, hamster, or mouse cells, or prokaryotic cells (e.g., bacteria). In some cases, the nucleotide sequence encoding the DNA-targeting RNA and / or site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding the DNA-targeting RNA and / or site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.

[0387] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retrovirus-derived long terminal repeats (LTRs), and mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. Expression vectors may also contain a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also contain appropriate sequences for amplifying expression.

[0388] E. Genetically Modified Host Cells The present disclosure provides genetically engineered host cells, wherein the host cells are genetically engineered with nucleic acid(s) that encode, or encode and express, a MAPP protein or a higher-order complex of MAPPs (e.g., double-chained MAPPs).

[0389] Suitable host cells include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2™), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL-9618™, CCL-61™, CRL9096), 293 cells (e.g., ATCC No. CRL-1573™), Vero cells, NIH Examples of host cells include, but are not limited to, 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10, CCL-10™), PC12 cells (ATCC No. CRL1721, CRL-1721™), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC class II heavy chain (MHC-H).

[0390] Genetically engineered host cells can be used to produce MAPP or higher-order complexes of MAPPs. For example, genetically engineered host cells can be used to produce double-chain MAPP. For example, expression vector(s) containing a nucleotide sequence encoding a MAPP polypeptide(s) are introduced into the host cell to generate a genetically engineered host cell, which produces the polypeptide(s) (e.g., as an excreted, bioavailable protein).

[0391] F. Methods for Producing MAPPs The present disclosure provides methods for producing MAPPs (e.g., dual-chain MAPPs) having at least one masked TGF-β MOD. The methods generally include culturing in a medium a host cell (e.g., a recombinant host cell of the present disclosure) that has been genetically engineered with a recombinant expression vector(s) that includes a nucleotide sequence(s) encoding a MAPP, and isolating the MAPP from the genetically engineered host cell and / or the medium. As noted above, in some cases, individual polypeptide chains of a MAPP are encoded in separate nucleic acids (e.g., recombinant expression vectors). In some cases, all polypeptide chains of a MAPP are encoded in a single recombinant expression vector.

[0392] Isolation of MAPP from the host cells employed for expression (e.g., a lysate of the expression host cells) and / or the medium in which the host cells are cultured can be carried out using standard methods of protein purification. For example, a lysate of the host cells can be prepared, and the MAPP can be purified from the lysate using high-performance liquid chromatography (HPLC), exclusion chromatography (e.g., size-exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification techniques. Alternatively, if the MAPP is secreted from the expression host cells into the medium, the MAPP can be purified from the medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In some cases, the MAPP is purified to produce a composition comprising, for example, at least 80%, at least about 85%, at least about 95%, or at least about 99.5% by weight of MAPP, with respect to the method of preparation of the product and contaminants associated with its purification. Percentages can be based on total protein.

[0393] In some cases, for example, if the expressed MAPP contains an affinity tag or affinity domain, the MAPP can be purified using an immobilized binding partner of the affinity tag. For example, if the MAPP contains an Ig Fc polypeptide, the MAPP can be isolated from the recombinant mammalian host cells and / or from the medium containing the MAPP by affinity chromatography, for example, on a Protein A column, Protein G column, or the like. An example of a suitable mammalian cell is a CHO cell, for example, an Expi-CHO-S™ cell (e.g., ThermoFisher Scientific, catalog number A29127).

[0394] The polypeptides of MAPP self-assemble into heterodimers and spontaneously form disulfide bonds, if applicable, between, for example, the framework polypeptides or between the framework polypeptides and the dimerization polypeptides. Also, as described above, if both framework polypeptides comprise an Ig Fc polypeptide with suitable cysteine ​​residues, disulfide bonds will spontaneously form between the respective Ig Fc polypeptides, covalently linking the two heterodimers of framework and dimerization polypeptides to each other to form a covalently linked duplex MAPP.

[0395] G. Composition 1. Composition containing MAPP The present disclosure provides compositions, including pharmaceutical compositions, comprising MAPP and / or higher-order complexes of MAPPs (e.g., double-chain MAPPs). In addition to MAPPs, pharmaceutical compositions can include one or more known carriers, excipients, diluents, buffers, salts, surfactants (e.g., non-ionic surfactants), amino acids (e.g., arginine), etc., the types of which are known in the art and need not be discussed in detail herein. See, e.g., "Remington: The Science and Practice of Pharmacy," 1999. th Ed. (1995) or latest edition, Mack Publishing Co.

[0396] In some cases, the subject pharmaceutical compositions will be suitable for administration to a subject, e.g., sterile and / or substantially free of pyrogens. For example, in some embodiments, the subject pharmaceutical compositions are suitable for administration to a human subject, e.g., the compositions are sterile and substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are below acceptable limits.

[0397] The compositions may be in the form of, for example, aqueous or other solutions, powders, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The compositions may be formulated according to various routes of administration, as described below.

[0398] When MAPP or higher-order MAPP complexes (e.g., double-chain MAPP) are administered directly into tissues as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, intralymphaticly, and / or intravenously), the formulation can be provided as a ready-to-use dosage form or in a non-aqueous form (e.g., a reconstitutable, shelf-stable powder) or an aqueous form, such as a liquid comprised of pharmaceutically acceptable carriers and excipients. MAPPs can also be provided to enhance the serum half-life of the subject proteins after administration. For example, proteins can be provided in liposomal formulations prepared as colloids or other conventional techniques for extending serum half-life. Various methods are available for preparing liposomes, as described, for example, in Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467; U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. The preparations may also be provided in controlled or sustained release forms.

[0399] In some cases, the MAPP composition comprises a) a MAPP higher-order MAPP complex (e.g., double-chain MAPP); and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile and / or substantially free of pyrogens, or the amount of detectable pyrogens and / or toxins is below acceptable limits. In some cases, the composition is suitable for administration to a human subject, e.g., the composition is sterile and free of detectable pyrogens and / or other toxins, or the amount of detectable pyrogens and / or other toxins is below acceptable limits. Thus, the present disclosure provides a composition comprising a) a MAPP or higher-order MAPP complex (e.g., double-chain MAPP); and b) saline (e.g., 0.9% NaCl), wherein the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or the amount of such detectable pyrogens and / or toxins is below acceptable limits.

[0400] Other examples of ingredients suitable for inclusion in formulations suitable for parenteral administration include isotonic sterile injection solutions, antioxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The pharmaceutical composition can be in a sterile container, such as a syringe. The formulation can be provided in single-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient for injection, such as water, immediately before use. Extemporaneous injection solut...

Claims

**Claim 1** A double-stranded or higher-order multimeric antigen-presenting polypeptide (MAPP) comprising at least a first MAPP heterodimer and a second MAPP heterodimer, wherein (i) the first MAPP heterodimer comprises: (1) a first framework polypeptide having a first multimerization sequence and a first dimerization sequence; (2) a first dimerization polypeptide having a first partner dimerization sequence complementary to the first dimerization sequence, wherein the first dimerization sequence and the first partner dimerization sequence dimerize via covalent and / or non-covalent interactions to form a MAPP heterodimer; and (3) at least one presentation sequence and / or presentation complex (wherein one or both of the first dimerization polypeptide and / or the first framework polypeptide comprises a first sequence of the presentation sequence or presentation complex), and (ii) the second MAPP heterodimer comprises: (1) a second framework polypeptide having a second multimerization sequence and a second dimerization sequence; (2) a second dimerization polypeptide having a second partner dimerization sequence complementary to the second dimerization sequence, wherein the second dimerization sequence and the second partner dimerization sequence dimerize via covalent and / or non-covalent interactions to form a MAPP heterodimer; and (3) at least one presentation sequence and / or presentation complex (wherein one or both of the second dimerization polypeptide and / or the second framework polypeptide comprises a first sequence of the presentation sequence or presentation complex), where (a) each presentation sequence comprises, within a single polypeptide: (i) a peptide epitope; and (ii) MHC class II α1, α2, β1, and β2 domain polypeptide sequences, (b) each presentation complex comprises a first sequence of the presentation complex and a second sequence of the presentation complex, where the first sequence of the presentation complex or the second sequence of the presentation complex comprises at least one of the peptide epitope and the α1, α2, β1, and β2 polypeptide sequences, and the first sequence of the presentation complex and the second sequence of the presentation complex together comprise the peptide epitope and the MHC class II α1, α2, β1, and β2 domain polypeptide sequences. (c) the framework polypeptide and / or the dimerization polypeptide comprises (i) a TGF-β sequence, (ii) a masking sequence, or (iii) at least one masked TGF-β immunomodulatory polypeptide(s) (a “masked TGF-β MOD”), each masked TGF-β MOD comprising a masking sequence and a TGF-β sequence, wherein each masking sequence reversibly binds to the TGF-β polypeptide sequence to mask it, (d) at least one framework polypeptide, dimerization peptide, presentation sequence, or presentation complex optionally comprises one or more independently selected additional MODs and / or additional variant MOD polypeptide sequences, (e) the framework polypeptide, the dimerization polypeptide, the presentation sequence, the first sequence of the presentation complex, and / or the second sequence of the presentation complex optionally comprises one or more linker sequences independently selected, (f) the dimerization sequence and the multimerization sequence are independently selected non-interspecies sequences or interspecies sequences, and the first and second framework polypeptides are optionally associated by a binding interaction between the first and second multimerization sequences comprising one or more interchain covalent bonds, and the multimerization sequence is not the same as and does not substantially associate or bind to the dimerization sequence or the partner dimerization sequence, and multimerization and / or dimerization do not result from an interaction between MHC sequences, and (g) the double-stranded or higher-order MAPP comprises at least one masked TGF-β MOD having the masking sequence and the TGF-β sequence in cis or in trans, said double-stranded or higher-order multimeric antigen-presenting polypeptide.

2. the non-interspecies sequence is selected from the group consisting of an immunoglobulin heavy chain constant region, the collectin family, a coiled-coil domain, and a leucine zipper domain, and The interspecies sequence is a Fos polypeptide that pairs with a Jun polypeptide, Ig CH1 and Ig C Ｌ κ, Ig CH1 and Ig C Ｌ λ, a knob-in-hole without disulfide ("KiH"), a knob-in-hole with a stabilized disulfide bond ("KiHs-s"), HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and the double-stranded MAPP according to claim 1, selected from the group consisting of the A107 sequence.

3. (A) the TGF-β sequence comprises (i) a TGF-β1 polypeptide sequence optionally comprising a substitution at C77, (ii) a TGF-β2 polypeptide sequence optionally comprising a substitution at C77, or (iii) a TGF-β3 polypeptide sequence optionally comprising a substitution at C77, and (B) the masking sequence comprises (i) a TGF-β receptor (“TβR”) I or TβR I ectodomain polypeptide sequence, (ii) a TGF-β receptor type II ectodomain polypeptide sequence, or (iii) a TGF-β receptor type III ectodomain polypeptide sequence, the double-stranded MAPP according to claim 2. (Claim 4) Each of the TGF-β sequences (i) a TGF-β1 sequence 【Chemical 1】 (ii) a TGF-β2 sequence [Chemical Formula 2] and (iii) a TGF-β3 sequence [Chemical Formula 3] selected from, having at least 90% sequence identity with a TGF-β sequence, and each of the masking sequences (i) a TGF-β receptor type I sequence 【Chemical Formula 4】 (ii) a TGF-β receptor type II isoform A sequence 【Chemical Formula 5】 optionally containing one or more substitutions at F55, D57, S77, E80, and D143, said TGF-β receptor type II isoform A sequence, (iii) a TGF-β receptor type II isoform B sequence 【Chemical Formula 6】 optionally containing one or more substitutions at F30, D32, S52, E55, and D118, said TGF-β receptor type II isoform B sequence, (iv) a TGF-β receptor type II isoform B sequence 【Chemical Formula 7】 optionally containing one or more substitutions at F30, D32, S52, E55, and D118, said TGF-β receptor type II isoform B sequence, (v) a TGF-β receptor type II isoform BΔ14 sequence 【Chemical 8】 optionally containing one or more substitutions at F30, D32, S52, E55, and D118, said TGF-β receptor type II isoform BΔ14 sequence, and (vi) a TGF-β receptor type II isoform BΔ25 sequence 【Chemical Formula 9】 optionally containing one or more substitutions at F30, D32, S52, E55, and D118, said TGF-β receptor type II isoform BΔ25 sequence, having at least 90% sequence identity with a TGF-β receptor sequence selected from, the double-stranded MAPP according to claim 3 containing a TGF-β aa sequence. (Claim 5) The TGF-β sequence has at least 90% sequence identity with a TGF-β3 sequence 【Chemical 10】 the double-stranded MAPP according to claim 4. (Claim 6) The TGF-β sequence contains amino acid substitutions at arginine 25 (R25), valine 92 (V92), and / or arginine 94 (R94), the double-stranded MAPP according to claim 5. (Claim 7) The TGF-β sequence contains a C77S substitution, the double-stranded MAPP according to claim 6. (Claim 8) The masking sequence optionally contains one or more substitutions at F30, D32, S52, E55, and D118, a TGF-β receptor type II isoform BΔ25 sequence 【Chemical 11】 having at least 90% sequence identity with, the double-stranded MAPP according to claim 5 containing a TGF-β aa sequence. (Claim 9) The double-stranded MAPP according to claim 8, wherein the TβRII isoform BΔ25 sequence comprises a D118 substitution.

10. The double-stranded MAPP according to claim 9, wherein the D118 substitution is a D118A substitution or a D118R substitution.

11. At least one of the presentation sequences or the presentation complex is α1 and α2 domain polypeptide sequences having 90% to 100% sequence identity respectively with HLA DR alpha (DRA), DP alpha 1 (DPA1), DQ alpha 1 (DQA1), or DQ alpha 2 (DQA2) polypeptide sequences, and not including a transmembrane domain or a part thereof that anchors the MAPP within the cell membrane, the α1 and α2 domain polypeptide sequences, and β1 and β2 domain polypeptide sequences having 90% to 100% sequence identity respectively with HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), DR beta 5 (DRB5), DP beta 1 (DPB1), DQ beta 1 (DQB1), or DQ beta 2 (DQB2) polypeptide sequences, and not including a transmembrane domain or a part thereof that anchors the MAPP within the cell membrane, the β1 and β2 domain polypeptide sequences, The double-stranded MAPP according to claim 4, comprising

12. (i) the at least one masked TGF-β MOD is present "in cis" as part of a single polypeptide amino acid sequence, comprising a masking sequence and a TGF-β sequence; (ii) the masking sequence and the TGF-β sequence of the at least one masked TGF-β MOD are present "in trans" as the masking sequence part and the TGF-β sequence part of different MAPP polypeptides that interact by an interspecies multimerization sequence or an interspecies dimerization sequence; or (iii) the masking sequence and the TGF-β sequence of the at least one masked TGF-β MOD are present "in trans" as the masking sequence located at the C-terminus of the first framework polypeptide and the TGF-β sequence located at the C-terminus of the second framework polypeptide that associate by interaction between interspecies multimerization sequences. The double-stranded MAPP according to claim 11.

13. The interspecies multimerization sequence is (i) an Ig Fc region, and independently selected Ig CH1, Ig C Ｌ κ or λ, leucine zipper, Fos or Jun domain, said first and second dimerization sequences (ii) the Ig Fc CH2 CH3 region, and the first and second dimerization sequences comprising an independently selected Ig CH1 or Ig C Ｌ κ or λ domain, (iii) An Ig Fc region selected from the group consisting of IgA, IgD, IgE, IgG, and IgM Fc regions, having at least about 90%, or 100% amino acid sequence identity with the amino acid sequence of the CH2 and / or CH3 domain of the Fc region of SEQ ID NOs: 1 to 13. (iv) An IgG1, IgG2, IgG3, or IgG4 CH2-CH3 domain having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with the amino acid sequence of the CH2 and / or CH3 domain of the Fc region of SEQ ID NOs: 4 to 12. (v) An IgG1 CH2-CH3 domain having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with the amino acid sequence of the CH2 and / or CH3 domain of the Fc region of SEQ ID NOs: 4 to 12. (vi) An interspecies immunoglobulin sequence selected from the group consisting of KiH pair, KiHs-s pair, HA-TF polypeptide pair, ZW-1 polypeptide pair, 7.8.60 polypeptide pair, DD-KK polypeptide pair, EW-RVT polypeptide pair, EW-RVTs-s polypeptide pair, and A107 polypeptide pair. (vii) A pair of interspecies immunoglobulin sequences selected from the group consisting of KiH or KiHs-s pair, or (viii) the first and second dimerization sequences are a pair of interspecies immunoglobulin sequences independently selected from Ig CH1, Ig C Ｌ κ or λ, leucine zipper, Fos or Jun domain, wherein When the multimerization sequence contains an Ig Fc region, the Ig Fc region optionally contains one or more substitutions that limit complement-dependent cytotoxicity and / or antibody-dependent cytotoxicity. Optionally, the first dimerization sequence and its partner dimerization sequence and / or the second dimerization sequence and its partner dimerization sequence are covalently linked by at least one disulfide bond, and the multimerization sequences of the first and second framework polypeptides are covalently linked by at least one disulfide bond, optionally at least two disulfide bonds. The double-stranded MAPP according to claim 12.

14. (i) The additional MOD or additional variant MOD polypeptide sequence is a single MOD or at least a pair of tandem additional MODs independently selected from the group consisting of 4-1BBL, PD-L1 IL-2, and any variant thereof. (ii) the additional MOD or additional variant MOD polypeptide sequence is a single MOD or at least a pair of tandem additional MODs comprising at least one IL-2 MOD polypeptide sequence or at least a pair of tandem IL-2 MOD polypeptide sequences, (iii) the additional MOD or additional variant MOD polypeptide sequence is a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, each variant IL-2 MOD having a lower affinity for IL-2R as compared to wild-type IL-2, or, (iv) the at least one additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, each variant IL-2 MOD having substantially reduced binding to the IL-2α chain and also having reduced binding to the IL-2Rβ chain, the double-stranded MAPP of claim 13.

15. The double-stranded MAPP of claim 14, wherein the additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, and each variant IL-2 MOD has a lower affinity for IL-2R as compared to wild-type IL-2.

16. The double-stranded MAPP of claim 15, wherein the additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, each variant IL-2 MOD showing substantial reduction in binding to the IL-2α chain and also showing reduction in binding to the IL2Rβ chain.

17. The double-stranded MAPP of claim 16, wherein each variant IL-2 MOD has an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42, based on the numbering of SEQ ID NO:

181.

18. The double-stranded MAPP of claim 17, wherein each variant IL-2 MOD has Ala at position 16 and Ala at position 42.

19. The double-stranded MAPP according to claim 14, wherein the additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD.

20. The double-stranded MAPP according to claim 14, wherein the at least one additional MOD or additional variant MOD polypeptide sequence comprises a pair of additional variant IL-2 MODs in tandem.

21. The double-stranded MAPP according to any one of claims 1 to 20, wherein the peptide epitope is an epitope of a self-antigen, an allergen, or a tissue graft, and has a length of about 4 to about 25 amino acids, or a length of about 8 to about 20 amino acids.

22. A multimeric antigen-presenting polypeptide (MAPP), comprising: (i) a framework polypeptide comprising a dimerization sequence and a multimerization sequence; (ii) a dimerization polypeptide having a partner dimerization sequence complementary to the dimerization sequence of the framework polypeptide, wherein the dimerization sequence and the partner dimerization sequence dimerize via covalent and / or non-covalent interactions to form a MAPP heterodimer; and (iii) at least one presentation sequence and / or presentation complex, wherein: (a) each presentation sequence comprises (i) a peptide epitope and (ii) MHC class II α1, α2, β1, and β2 domain polypeptide sequences within a single polypeptide; (b) each presentation complex comprises a first sequence of the presentation complex and a second sequence of the presentation complex, wherein: the first sequence of the presentation complex or the second sequence of the presentation complex comprises at least one of the peptide epitope and the α1, α2, β1, and β2 polypeptide sequences; and the first sequence of the presentation complex and the second sequence of the presentation complex together comprise the peptide epitope and the MHC class II α1, α2, β1, and β2 domain polypeptide sequences; (c) one or both of the dimerization polypeptide and / or the framework polypeptide comprises a presentation sequence or a first sequence of the presentation complex. (d) The framework polypeptide or the dimerizing polypeptide comprises at least one masked TGF-β immunomodulatory polypeptide(s) (the "masked TGF-β MOD"), each masked TGF-β MOD comprising (i) a TGF-β polypeptide sequence, and (ii) a TGF-β receptor polypeptide sequence as a masking sequence that reversibly binds to and masks the TGF-β polypeptide sequence, and (e) at least one framework polypeptide, dimerizing peptide, presentation sequence, or presentation complex optionally comprises one or more independently selected additional MODs and / or additional variant MOD polypeptide sequences, said at least one presentation sequence and / or presentation complex, comprising, wherein the framework polypeptide, the dimerizing polypeptide, the presentation sequence, the first sequence of the presentation complex, and / or the second sequence of the presentation complex optionally comprises one or more linker sequences independently selected, and multimerization and / or dimerization does not result from interaction between MHC sequences, said MAPP.

23. A framework polypeptide comprising a dimerizing sequence and a multimerizing sequence from the N-terminus to the C-terminus, A dimerizing polypeptide comprising a partner dimerizing sequence complementary to the dimerizing sequence of the framework polypeptide and dimerizing therewith via covalent and / or non-covalent interactions to form a MAPP heterodimer, and (i) at least one presentation sequence, or (ii) at least one presentation complex, The MAPP according to claim 22, comprising.

24. The MAPP according to claim 23, comprising a TβR aa sequence having at least 90% sequence identity with a TβRII isoform B Δ25 sequence, wherein the masking sequence optionally comprises one or more substitutions at F30, D32, S52, E55, and D118. 【Chemical Formula 12】

25. The MAPP according to claim 24, wherein the TβRII isoform B Δ25 sequence comprises a D118 substitution.

26. The MAPP according to claim 25, wherein the D118 substitution is a D118A substitution or a D118R substitution.

27. (i) the additional MOD or additional variant MOD polypeptide sequence is a single MOD or at least a pair of tandem additional MODs independently selected from the group consisting of 4-1BBL, PD-L1 IL-2, and any variant thereof, (ii) the additional MOD or additional variant MOD polypeptide sequence comprises a single MOD or at least a pair of tandem additional MODs comprising at least one IL-2 MOD polypeptide sequence or at least a pair of tandem IL-2 MOD polypeptide sequences, (iii) the additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, wherein each variant IL-2 MOD has a lower affinity for IL-2R compared to wild-type IL-2, or (iv) the at least one additional MOD or additional variant MOD polypeptide sequence comprises a single variant IL-2 MOD or at least a pair of tandem additional variant IL-2 MODs, wherein each variant IL-2 MOD has substantially reduced binding to the IL-2α chain and also has reduced binding to the IL-2Rβ chain, the MAPP of claim 24.

28. The MAPP or double-stranded MAPP according to any one of claims 1 to 20 and 22 to 27 for use in a method of treating or preventing a disease or condition, wherein the disease or condition is an autoimmune disease other than celiac disease and / or T1D, or an autoimmune disease in addition thereto, in a mammalian patient or subject, said MAPP or double-stranded MAPP.

29. The MAPP or double-stranded MAPP according to claim 21 for use in a method of treating or preventing a disease or condition, wherein the disease or condition is an autoimmune disease other than celiac disease and / or T1D, or an autoimmune disease in addition thereto, in a mammalian patient or subject, said MAPP or double-stranded MAPP.

30. The disease or condition is an autoimmune disease, optionally selected from the group consisting of Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-related infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture syndrome, glomerulonephritis, Graves' disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), vasculitis, and vitiligo, the MAPP or double-stranded MAPP according to claim 28.

31. The disease or condition is an autoimmune disease, optionally selected from the group consisting of Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-related infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture syndrome, glomerulonephritis, Graves' disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), vasculitis, and vitiligo, the MAPP or double-stranded MAPP according to claim 29.

32. A pharmaceutical composition for the treatment of a disease or condition of a mammalian patient or subject, comprising the MAPP or double-stranded MAPP according to any one of claims 1 to 20, and 22 to 27.

33. A pharmaceutical composition for the treatment of a disease or condition of a mammalian patient or subject, comprising the MAPP or double-stranded MAPP according to claim 21.

34. Use of the MAPP or double-stranded MAPP according to any one of claims 1 to 20, and 22 to 27, in the manufacture of a medicament for use in the prevention of a disease or condition of a mammalian patient or subject.

35. Use of the MAPP or double-stranded MAPP according to claim 21, in the manufacture of a medicament for use in the prevention of a disease or condition of a mammalian patient or subject.

36. One or more nucleic acids encoding a framework polypeptide and / or a dimerization polypeptide of MAPP or double-stranded MAPP according to any one of claims 1 to 20 and 22 to 27, wherein the framework polypeptide and / or the dimerization polypeptide optionally comprises an additional polypeptide, said nucleic acid.

37. One or more nucleic acids encoding a framework polypeptide and / or a dimerization polypeptide of MAPP or double-stranded MAPP according to claim 21, wherein the framework polypeptide and / or the dimerization polypeptide optionally comprises an additional polypeptide, said nucleic acid.

38. A method for producing a cell expressing MAPP or double-stranded MAPP, comprising introducing in vitro one or more nucleic acids according to claim 36 into the cell, selecting a cell producing the MAPP or the double-stranded MAPP, and optionally selecting a cell comprising all or part of the one or more nucleic acids, which is either not integrated into at least one cell chromosome or is integrated therein, wherein the cell is optionally selected from the group consisting of HeLa cells, CHO cells, 293 cells, Vero cells, NIH 3T3 cells, Huh-7 cells, BHK cells, PC12, COS cells, COS-7 cells, Rat1 cells, mouse L cells, human embryonic kidney (HEK) cells, and HL HepG2 cells, said method.

39. A method for producing a cell expressing MAPP or double-stranded MAPP, comprising introducing in vitro one or more nucleic acids according to claim 37 into the cell, selecting a cell producing the MAPP or the double-stranded MAPP, and optionally selecting a cell comprising all or part of the one or more nucleic acids, which is either not integrated into at least one cell chromosome or is integrated therein, wherein the cell is optionally selected from the group consisting of HeLa cells, CHO cells, 293 cells, Vero cells, NIH 3T3 cells, Huh-7 cells, BHK cells, PC12, COS cells, COS-7 cells, Rat1 cells, mouse L cells, human embryonic kidney (HEK) cells, and HL HepG2 cells, said method.

40. A cell that transiently or stably expresses MAPP or double-stranded MAPP, prepared by the method according to claim 38, wherein, optionally, the cell expresses from about 25 to about 350 mg / liter or more of the MAPP or the double-stranded MAPP without substantially reducing cell viability as compared to the same cell except that it does not express the MAPP or the double-stranded MAPP.

41. A cell that transiently or stably expresses MAPP or double-stranded MAPP, prepared by the method according to claim 39, wherein, optionally, the cell expresses from about 25 to about 350 mg / liter or more of the MAPP or the double-stranded MAPP without substantially reducing cell viability as compared to the same cell except that it does not express the MAPP or the double-stranded MAPP.

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