MHC class ii protein complexes

Abstract

The present disclosure provides Multimeric Antigen-Presenting Polypeptides (MAPPs) for the presentation of epitopes in the context of a class II MHC receptor. The present disclosure provides nucleic acids comprising nucleotide sequences encoding those MAPPs, as well as cells genetically modified with the nucleic acids encoding the MAPPs. MAPPs are useful for selectively modulating activity of T cells having T cell receptors that recognize the antigens. Thus, the present disclosure provides compositions and methods for modulating the activity of T cells, as well as compositions and methods for treating persons who have diseases and / or disorders including metabolic disorders, cancers, autoimmune diseases, allergies, T1D, and / or celiac disease.

Description

MHC CLASS II PROTEIN COMPLEXESCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 601,905, filed November 22, 2023. Incorporation of Sequence Listing

[0002] The sequence listing in ST.26 XML format entitled 2910-22_ST26_2024-11-21. xml, created on November 21, 2024, comprising 1,001,829 bytes, prepared according to 37 CFR 1.822 to 1.824, and submitted concurrently with the filing of this application, is incorporated herein by reference in its entirety.I. Introduction

[0003] Major Histocompatibility Complex (MHC) proteins, also referred to in humans as Human Leukocyte Antigen (HLA) proteins, play a critical role in mammalian immune systems and are central to the adaptive immune response. The classic human MHC class II group of molecules is comprised of three different protein isotypes, HLA-DR, -DP, and -DQ, each of which is highly polymorphic. MHC molecules present fragments of larger molecules to T Cell Receptors (TCR) through a complex formed between an antigen-presenting cell displaying an MHC protein and a T cell displaying a TCR. MHC / HLA proteins have been of interest as immunological tools and potential therapeutics since they were first described and have continued to move toward the forefront of immunotherapy applications. While it has been possible to express Class I MHC / HLA molecules in quantities suitable for their use as therapeutics, Class II MHC / HLA proteins have proven more difficult to produce in amounts suitable for their use as therapeutics. The different isotypes require, for example, different chaperones (see, e.g., J. Biol. Chem. 285(52): 40800-40808 (2010)). Difficulties expressing Class II HLA proteins in amounts suitable for their widespread adoption as therapeutics are particularly pronounced for HLA-DQ gene products such as HLA DQ 2.5, which is a heteroduplex of the o and |3 subunits expressed by the HLA DQA1*0501 and HLA DQB1*0201 alleles.

[0004] Although the immune system is designed to avoid the development of immune responses to proteins and other potentially antigenic materials of the body, in some instances the immune system develops T cells with specificity for an epitope of an autoantigen (self-antigen) leading to autoimmune diseases. The immune system may also fail to respond to certain self- and non-self-antigens, for example, Type 1 diabetes (T1D) and celiac disease non-self-antigens, allowing cancer cells to grow unchecked by the immune system.IL Summary

[0005] The present disclosure provides multimeric antigen-presenting polypeptide complexes ("MAPP” singular and "MAPPs” plural) that can be expressed in cell culture, can display stability at temperatures well above that of the normal human body (37 °C), and can withstand multiple freeze-thaw cycles without substantial loss of protein to aggregation or denaturation.

[0006] MAPPs are at least heterodimeric and include at least one framework polypeptide and at least one dimerization polypeptide. Framework polypeptides comprise one or more polypeptide dimerization sequences that permit specific binding with other polypeptides (dimerization polypeptides) having a counterpart dimerization sequence, thereby forming at least a heterodimer (see FIG. 1). Framework polypeptides also comprise a multimerization sequence(s) that permits two or more framework polypeptides to associate, thereby forming a higher order structure (e.g., a duplex of the two or more heterodimers, a "duplex MAPP”; see, e.g., FIG. 1). Neither thedimerization sequence nor the multimerization sequence of the framework polypeptide (or the counterpart dimerization sequence) comprises an MHC class II (e.g., HLA) a chain or p chain polypeptide sequence, and as such, interactions brought about by those sequences are not considered dimerization or multimerization of framework and / or dimerization peptides. Accordingly, the framework polypeptides provide a structure upon which other polypeptides (e.g., immunomodulatory and / or MHC polypeptides) can be organized by interactions at the dimerization sequences, and which can interact with other framework polypeptides by way of multimerization sequences.

[0007] The framework and dimerization peptide containing MAPPs, duplex MAPPs, and MAPPs of higher order (e.g., triplex MAPPs) described herein provide a means by which peptide epitopes may be presented in the context of an MHC (e.g., HLA) to a target T cell displaying a TCR specific for the presented epitope, while at the same time permitting for the flexible presentation of one or more immunomodulatory polypeptides (MODs). Suitable peptide epitopes in MAPPs may include peptide epitopes associated with, for example, an autoimmune disease such as a peptide epitope of a T1D-associated antigen or a peptide epitope of a celiac disease-associated antigen. Those peptide epitopes may be referred to by the diseases they are associated with (e.g., T1D-associated peptide epitopes and celiac disease-associated peptide epitopes). The MAPPs, duplex MAPPs, and higher order MAPPs thereby permit delivery of one or more MODs in an epitope selective (e.g., dependent / specific) manner that permits (I) formation of an active immune synapse with a target T cell selective for the epitope, and (ii) modulation (e.g., control / regulation) of the target T cell's response to the epitope.

[0008] Peptide epitope presentation to a target T cell by a MAPP is accomplished via a moiety that comprises MHC Class II polypeptides and the peptide epitope. Such moieties may be either (I) a single polypeptide chain or (ii) a complex comprising two or more polypeptide chains.

[0009] Where the peptide epitope, MHC Class II polypeptides, and optionally one or more MODs are provided in a single polypeptide chain, it is termed a "presenting sequence.” See, e.g., FIG. 23. The presenting sequence(s) may be integrated into a MAPP as part of a framework polypeptide or a dimerization polypeptide. The MAPPs described herein typically contain one or two presenting sequences. Duplex MAPPs thus typically comprise two, three or four presenting sequences, but also may comprise one presenting sequence (e.g., if one of the MAPPs does not comprise a presenting sequence). MAPPs and duplex MAPPs may comprise more presenting sequences depending on, for example, the number of dimerization sequences in the framework polypeptide. The presenting sequences may be integrated into a MAPP as part of a framework polypeptide, a dimerization polypeptide, or both. Compare, for example, FIG. 17, structures A-D, and FIG. 18, structures A-D.

[0010] The present disclosure provides modifications to MHC class II sequences that form part of the MAPP presenting sequences and stabilize the expressed MAPPs. The MHC modifications include formatting the molecules as a single polypeptide comprising sequences of the peptide epitope, a linker (L1 linker), and both MHC Class II a and p chain (subunit) sequences. The single chain presenting sequence polypeptides may be stabilized by one or more (e.g., two or more) disulfide bonds. A first type of stabilizing disulfide bond, sometimes referred to herein as a "body disulfide,” is formed between a cysteine in the N-terminal portion of the p1 domain polypeptide sequence and a cysteine in the C-terminal portion of the a1 domain polypeptide sequence. This disulfide bond may decrease proteinbreakdown (degradation). A body disulfide is shown schematically as a dashed line in the expanded presenting sequence of FIG.1 , and also is provided in, for example, FIG. 23, constructs A and B.

[0011] A second type of stabilizing disulfide bond that may be present is sometimes referred to herein as a "linker disulfide.” A linker disulfide may stabilize the construct and / or constrain the peptide epitope, localizing it to the vicinity of the MHC binding cleft, thereby increasing the relative amount of time (residence time) the epitope spends in the binding cleft formed between a and p chain polypeptide sequences. A linker disulfide is formed between a cysteine present or introduced into the linker joining the peptide epitope and the MHC polypeptides (the L1 linker) and a cysteine in the MHC a subunit sequence, typically a cysteine in the C-terminal portion of the a1 domain. A linker disulfide is shown schematically as a dashed line in the presenting sequences of FIG. 23, constructs C and D.

[0012] MAPPs may also comprise a number of substitutions in the MHC (e.g., HLA) a1 domain sequence that improve hydrogen bonding between the a and p subunit sequences, and / or substitutions that enhance epitope- peptide HLA binding interactions. The modifications to the proteins permit MAPPs to be expressed in cell culture, for example reaching levels of at least 50 mg / liter (mg / l), and in some instances can reach about 100 mg / l, 150 mg / l, 200 mg / l, 250 mg / l or more. In addition to being expressed at such levels, embodiments of MAPPs and their higher order complexes (e.g., duplexes) disclosed herein exhibit substantial stability when subjected to heat and freeze-thaw. The molecules are capable of withstanding exposure to temperatures well above that of the normal human body (37 °C), often in excess of 60 °C, while also withstanding multiple freeze-thaw cycles without substantial loss of protein to aggregation or denaturation. The disulfide bonds and amino acid (aa) substitutions, either singly or in combination, can act to stabilize the MAPPs to thermal stress (e.g., freeze-thaw and / or temperatures above 37 °C and often in excess of 60 °C). The disulfide bond substitutions also reduce protein denaturation and / or nonspecific aggregation during cellular expression (e.g., in culture) or under conditions where the protein is subject to thermal stress. Such substitutions in the a1 domain may act together with linker and / or body disulfide bonds to stabilize the protein.

[0013] MAPPs, and accordingly their higher order complexes (duplexes, triplexes etc.), comprising MHC Class II polypeptide sequences and a peptide epitope for presentation to a TCR may present peptides to T cells (e.g., CD4+ T cells) that have a TCR specific for the epitope. If a TCR engaged by a MAPP is specific for the MAPP's epitope, it may result in CD69 expression and / or signaling by the lek protein tyrosine kinase associated with CD4, resulting in the recruitment and activation of ZAP-70 protein kinase and downstream signaling events. Once engaged with the TCR of a T cell, the effect of a MAPP on the T cell also depends on which MODs, if any, are present as part of the MAPP.

[0014] MOD-containing MAPPs of the present disclosure (e.g., those comprising T 1 D-associated or celiac- associated peptide epitope sequences) can function as a means of selectively delivering the MODs to T cells specific for the MAPP epitope, thereby resulting in MOD-driven responses to those MAPPs (e.g., the reduction in number and / or suppression of CD4+ effector T cells reactive with the MAPP's epitope). Depending on the chosen MOD, the incorporation of one or more MODs with increased affinity for their cognate receptor on T cells (co-MOD) may reduce the specificity of MAPPs and duplex MAPPs for epitope specific T cells where MOD-co-MOD binding interactions significantly compete with MHC / epitope binding to target cell TCRs. Conversely, and again depending on the chosen MOD, the inclusion of MODs with reduced affinity for their co-MOD(s), and the affinity of the epitope for a TCR, may provide for enhanced selectivity of MAPPs and duplex MAPPs, while retaining the desired activity of the MODs.Where a MOD already possesses a relatively low affinity for its cognate receptor, mutations that reduce the affinity may be unnecessary and / or undesirable.

[0015] The ability of MAPPs (e.g., duplex MAPPs) to modulate T cells provides methods of modulating T cell activity in vitro and in vivo, and accordingly the use of MAPPs as therapeutics useful in methods of treating a variety of diseases and disorders including cancers, autoimmune diseases, and allergies. In cases where a MAPP contains a MOD, e.g., one that stimulates Treg function, such as IL-2 and / or TGF-p (e.g., a masked TGF-p), it can suppress immune responses through the action of the Treg in an epitope specific manner. In such instances, any Ig Fc polypeptide sequences employed would not bring about Antibody-Dependent Cellular Cytotoxicity (ADCC) and / or Complement-Dependent Cytotoxicity (CDC).

[0016] In cases where a MAPP contains, or is linked to, a polypeptide that can direct targeted cell killing (e.g., Fc polypeptide sequences that bring about ADCC and / or CDC), the MAPP may be used to remove CD4+ T cells whose TCR recognizes the epitope presented by the MAPP. In such cases, the MAPP may not include a MOD since the goal of such MAPPs is to eliminate CD4+ T cells as opposed to modulating them.

[0017] In contrast, where a MAPP contains a MOD that stimulates Treg function, such as IL-2 and / or TGF-p (e.g., a masked TGF-p), it can suppress immune responses through the action of the Treg in an epitope specific manner. In such instances, the effector function of any Ig Fc polypeptide sequences employed could be suppressed or eliminated such that they have limited or no ability to bring about ADCC, ADCP (antibody-dependent cellular phagocytosis), or CDC.

[0018] Although the disclosure provides MAPPs comprising one or more MODs it should be understood that the MAPPs can lack MODs (a MODIess MAPP). MODIess MAPPs my have intact wt. effector functions. Alternatively, MODIess MAPPs may include Fc sequences with an enhanced ability to bring about one or more effector function related outcomes (ADCC, ADCP, and / or CDC) or may have suppressed / eliminated ability to bring about effector function related outcomes.

[0019] The present disclosure provides nucleic acids comprising nucleotide sequences and vectors encoding individual MAPP polypeptides and MAPPs (e.g., all polypeptides of a MAPP), as well as cells genetically modified with the nucleic acids and vectors for producing individual MAPP polypeptides and / or MAPP proteins (e.g., duplex MAPPs). The present disclosure also provides methods of producing MAPPs, duplex MAPPs, and higher order MAPPs utilizing such cells. ill Brief Description Of The Drawings

[0020] FIG. 1 is provided to illustrate the terminology used to describe MAPPs and duplex MAPPs with presenting sequences. The peptides are oriented from N-terminus (left) to C-terminus (right). The figure shows first and second framework polypeptides, which in this case are different and, in this instance, have specific multimerization sequences comprising a knob and counterpart hole. Such "knob-in-hole” configurations may include knob-in-hole configurations without a stabilizing disulfide bond (herein “KiH") or with a stabilizing disulfide bond (herein “KiHs-s”). Also shown in the figure are first and second dimerization polypeptides, each comprising an epitope and MHC sequences N-terminal to their counterpart dimerization sequences. The dashed numbered circles indicate five potential locations for the addition of polypeptide sequences, including MODs (discussed below). The figure depicts the formation of first and second heterodimer MAPPs, each comprising a framework polypeptide and a dimerizationpolypeptide. The heterodimers may interact through the multimerization sequence to form a multimer (a duplex MAPP as shown). The use of knob-in-hole sequences permits the assembly of an asymmetric interspecific duplex MAPP where, for example, different MOD sequences are provided at positions 1 and T and / or positions 3 and 3'. While interactions between polypeptide chains through peptide interaction sequences may initially be non-covalent in nature, interchain disulfide bond formation reactions may occur, thereby providing covalently linked polypeptides at, for example, either dimerization sequences or multimerization sequences. Throughout the figures, lines connecting various elements of MAPP polypeptides are optional aas serving as linkers (e.g., peptide linkers). The word "sequence” may be abbreviated by "seq.”

[0021] FIGs. 2A-2H provide aa sequences from immunoglobulin polypeptides including their heavy chain constant regions (“Ig Fc” or"Fc”, e.g., the CH2-CH3 domain of lgG1) (SEQ ID NOs:1-13).

[0022] FIG. 2I provides a sequence from an Ig CH1 domain (SEQ ID NO:14).

[0023] FIG. 2J provides a sequence from a human Ig-J chain (SEQ ID NO: 15).

[0024] FIG.3A provides a sequence from an immunoglobulin kappa Ig K light chain aa sequence (kappa chain, lgCi_ K) constant region (SEQ ID NO: 16).

[0025] FIG. 3B provides a sequence of an immunoglobulin lambda Ig A chain light chain aa sequence (lambda chain, Ig C A) constant region (SEQ ID NO:17).

[0026] FIG. 4 provides a sequence from Homo sapiens MHC DRA protein DRA*01 :02 GenBank NP_061984.2 (SEQ ID NO:18). Aas 1-84 = a1 domain; 85-178 = o2 domain (italicized and underlined); 179-191 = membrane proximal region connecting peptide (bolded); and 192-214 = transmembrane domain (underlined). Positions A37, R44, G49, and I72 are bolded and underlined. The sequence "TKR” for linker disulfide cysteine substitution at aas 74-76 and the sequence “TPI” for body disulfide cysteine substitution at aas 80-82 are bolded and underlined. DRA*01 :01 contains a Vai residue at position 217 of the intracellular domain in place of the Leu in DRA*01 :02 (SEQ ID NO: 19).

[0027] FIG. 5 provides sequences from selected alleles of Homo sapiens MHC (HLA) DRB1 protein. The Swiss- Prot / UniProt reference (“sp”) and other database references for some of the alleles are as follows: DRB1-1 (DRB1*01:01) P04229.2, EMBL / EBI HLA00664 (SEQ ID NQ:20); DRB1*01:02, EMBL / EBI HLA00665 (SEQ ID NO:21); DRB1*01:03 (SEQ ID NO:22); DRB1-3 (DRB1*03:01) sp P01912.2 (SEQ ID NO:23); DRB1*03:02 (SEQ ID NO:24); DRB1*03:04 (SEQ ID NO:25); DRB1-4 (DRB1*04:01) sp P13760.1 (SEQ ID NO:26); DRB1*04:02 (SEQ ID NO:27); DRB1*04:03 (SEQ ID NO:28); DRB1*04:04 (SEQ ID NO:29); DRB1*04:05 (SEQ ID NQ:30); DRB1*04:06 (SEQ ID NO:31); DRB1*04:08 (SEQ ID NO:32); DRB1-7 (DRB1*07:01) sp P13761.1 (SEQ ID NO:33); DRB1-8 (DRB1*08:01) sp Q30134.2 (SEQ ID NO:34); DRB1*08:02 (SEQ ID NO:35); DRB1*08:03 (SEQ ID NO:36); DRB1-9 (DRB1*09:01) sp Q9TQE0.1 (SEQ ID NO:37); DRB1-10 (DRB1*10:01) sp Q30167.2 (SEQ ID NO:38); DRB1-11 (DRB1*11 :01) sp P20039.1 (SEQ ID NO:39); DRB1*11:03 (SEQ ID NQ:40); DRB1*11:04 (SEQ ID NO:41); DRB1-12 (DRB1*12:01) sp Q95IE3.1 (SEQ ID NO:42); DRB1-13 (DRB1*13:01) sp Q5Y7A7.1 (SEQ ID NO:43); DRB1*13:03 (SEQ ID NO:44); DRB1-14 (DRB1*14:01) sp Q9GIY3.1 (SEQ ID NO:45); DRB1*14:02 (SEQ ID NO:46); DRB1*14:05 (SEQ ID NO:47); DRB1*14:06 (SEQ ID NO:48); DRB1-15 (DRB1*15:01) sp P01911 (SEQ ID NO:49); DRB1*15:02 (SEQ ID NQ:50); DRB1*15:03 (SEQ ID NO:51); DRB1*15:04 (SEQ ID NO:52); DRB1*15:05 (SEQ ID NO:53); DRB1*15:06 (SEQ ID NO:54); DRB1*15:07 (SEQ ID NO:55); and DRB1-16 (DRB1*16:01) sp Q29974.1 (SEQ ID NO:56).

[0028] FIGs. 6-8 provide sequences from selected Homo sapiens MHC (HLA) DRB3, DRB4 and DRB5 proteins (SEQ ID NO:57 to SEQ ID NO:62, respectively). In each of FIGs. 6 through 8, aas 1-95 = p1 domain; aas 96-188 = p2 domain; aas 189-198 = membrane proximal region (underlined and italicized); and some locations for body disulfide cysteine substitution (aas 4-8) are bolded and underlined. References for the DR3 alleles in FIG. 6 include: DRB3*01:01, GenBank NP_072049.1 (SEQ ID NO:57), DRB3*02:01, GenBank CAA23781.1 (SEQ ID NO:58); and DRB3*03:01, GenBank AAN15205.1 (SEQ ID NO:59). References for the DR4 alleles in FIG. 7 include: DRB4*01:01, GenBank AAA36296.1 and ImMunoGeneTics ("IMGT”) / HLA Acc No: HLA00905 (SEQ ID NQ:60) and DRB4*01:03, GenBank NP_068818.4 and IMGT / HLA Acc No: HLA00908 (SEQ ID NO:61). References for the DRB5*01:01 allele in FIG. 8 include GenBank NP_002116.2 and IMGT / HLA Acc No: HLA00915 (SEQ ID NO:62).

[0029] FIG. 9 provides sequences from selected Homo sapiens MHC DQA1 proteins (SEQ ID NO:63 to SEQ ID NO:73). Because some DQA1 alleles omit one aa at position 55 relative to DQA1*01 :01, two different lengths are reported for aa positions beyond aa 55. As indicated in the figure aas 1-85 or 86 = a1 domain; 86 or 87 - 180 or 181 = o2 domain (italicized and underlined); 181 or 182 - 193 or 194 = membrane proximal region connecting peptide (bolded); and 194 or 195 - 216 or 217 = transmembrane domain (underlined). Positions 40 (e.g., E40), 47 (e.g., C47), 52 (e.g., S52 or H52), and 74 or 75 (S75 or I75) are bolded and underlined. The sequence “IKR” for linker disulfide cysteine substitution at aas 76-78 or 77-79, and the sequence "TAA” for body disulfide cysteine substitution at aas 82-84 or 83-85 are bolded and underlined. For DQA1 allele sequence information see: DQA1*01:01, IMGT / HLA Acc No. HLA00601, GenBank: AAK11577.1 (SEQ ID NO:63); DQA1*01:02, IMGT / HLA Acc No:HLA00603, GenBank NP_002113.2 (SEQ ID NO:64); DQA1*01:03, GenBank AAU88031.1 (SEQ ID NO:65); DQA1*01:04, GenBank: AAU88004.1 (SEQ ID NO:66); DQA1*02:01, IMGT / HLA Acc No:HLA00607, NCBI PDB 6PX6_A (SEQ ID NO:67); DQA1*03:01, IMGT / HLA Acc No:HLA00609, GenBank: AAA59756.1 (SEQ ID NO:68); DQA1*03:02, GenBank: AAU88001.1 (SEQ ID NO:69); DQA1*04:01, IMGT / HLA Acc No:HLA00612, GenBank: AAA36267.1 (SEQ ID NQ:70); DQA1*05:01, IMGT / HLA Acc No:HLA00613, UniProtKB / Swiss-Prot: P01909 (SEQ ID NO:71); DQA1*05:050, IMGT / HLA Acc No:HLA00619, GenBank: AAU87975.1 (SEQ ID NO:72); and DQA1*06:01, IMGT / HLA Acc No:HLA00620, GenBank: QCY59255.1 (SEQ ID NO:73).

[0030] FIG. 10 provides a sequence from Homo sapiens MHC DQA2 protein HLA DQA2*01:01, GenBank NP_064440.1 (SEQ ID NO:74), as the mature protein lacking its signal sequence. Aas 1-86 = a1 domain; 87-181 = o2 domain (italicized and underlined); 182-194 = membrane proximal region connecting peptide (bolded); and 195- 217 = transmembrane domain (underlined). Positions 40 (E40), 47 (Q47), 52 (S52), and 75 (F75) are bolded and underlined. The sequence "MQR” for linker disulfide cysteine substitution at aas 77-79 and the sequence TAA for body disulfide cysteine substitution at aas 83-85 are bolded and underlined.

[0031] FIG. 11 provides sequences from selected Homo sapiens MHC DQB1 proteins (SEQ ID NO:75 to SEQ ID NO:85). Aas 1-94 = p1 domain; aas 95-188 = p2 domain; aas 189-198 = membrane proximal region (underlined and italicized); and some locations for body disulfide cysteine substitution (aas 4-8) are bolded and underlined.References for the DQB1 alleles in FIG. 11 include: DQB1*02:01, IMGT / HLA Acc No: HLA00646, NCBI Accession No. NP_001230891.1 (SEQ ID NO:75); DQB1*02:02, IMGT / HLA Acc No: HLA00623, NCBI Accession No. 6PX6_B (SEQ ID NO:76); DQB1*03:01, IMGT / HLA Acc No: HLA00625, NCBI Accession No. P01920.2 (SEQ ID NO:77); DQB1*03:02, IMGT / HLA Acc No: HLA00627, NCBI Accession No. AAA98746.1 (SEQ ID NO:78); DQB1*03:03,IMGT / HLA Acc No: HLA00629, NCBI Accession No. AAA59755.1 (SEQ ID NO:79); DQB1*03:04, IMGT / HLA Acc No: HLA00630, NCBI Accession No. ATY52316.1 (SEQ ID NO:80); DQB1*04:01, IMGT / HLA Acc No: HLA00636, NCBI Accession No. CAC8953441.1 (SEQ ID NO:81); DQB1*04:02, IMGT / HLA Acc No: HLA00637, NCBI Accession No. AAA36270.1 (SEQ ID NO:82); DQB1*05:01, IMGT / HLA Acc No: HLA00638, NCBI Accession No. AAA59765.1 (SEQ ID NO:83); DQB1*06:01, IMGT / HLA Acc No: HLA00643, NCBI Accession No. AXU93762.1 (SEQ ID NO:84); and DQB1*06:02, IMGT / HLA Acc No: HLA00646, NCBI Accession No. NP_002114.3 (SEQ ID NO:85).

[0032] FIG. 12 provides sequences from selected Homo sapiens MHC DQB2 proteins (SEQ ID NO:86 and SEQ ID NO:87). Aas 1-94 = p1 domain; aas 95-187 = p2 domain; aas 188-197 = membrane proximal region (underlined and italicized); and some locations for body disulfide cysteine substitution (aas 4-8) are bolded and underlined. For DQB2 Isoform I (DQB2-ISO-1) allele sequence information, see GenBank NP_001287719.1 and / or UniProtKB - P05538-1 (SEQ ID NO:86), and for DQB2 Isoform 2, see GenBank NP_001185787.1 and / or UniProtKB - P05538-2 (SEQ ID NO:87).

[0033] FIG. 13 provides the sequence of Homo sapiens MHC DPA proteins DPA1*01:03 and DPA1*02:01 (SEQ ID NOs:88 and 89). Aas 1-87 = a1 domain; 88-181 = o2 domain (italicized and underlined); 182-194 = membrane proximal region connecting peptide (bolded); and 195-216 = transmembrane domain (underlined). Positions 40 (D40), 47 (H47), 52 (G52), and 75 (T75) are bolded and underlined. The sequence “IQR” for linker disulfide cysteine substitution at aas 77-79 and the sequence "TQA” for body disulfide cysteine substitution at aas 83-85 are bolded and underlined. For DPA1 allele sequences, see: DPA1*01:03, GenBank NP_001229453.1 and IMGT / HLA Acc No: HLA00499 (SEQ ID NO:88); and DPA1*02:01, GenBank: AAH09956.1 and IMGT / HLA Acc No: HLA00504 (SEQ ID NO:89).

[0034] Fig. 14 provides the sequence of selected Homo sapiens MHC DPB1 proteins (SEQ ID NQ:90 to SEQ ID NO: 102, respectively). Aas 1-92 = p1 domain; aas 93-186 = p2 domain; aas 187-196 = membrane proximal region (underlined and italicized); and some locations for body disulfide cysteine substitution (aas 4-8) are bolded and underlined. References for the DPB alleles in FIG. 14 include DPB1*01 :01 , IMGT / HLA Acc No: HLA00510 (SEQ ID NQ:90); DPB1*02:01, IMGT / HLA Acc No: HLA00517 (SEQ ID NO:91); DPB1*03:01, IMGT / HLA Acc No: HLA00520 (SEQ ID NO:92); DPB1 *04:01, IMGT / HLA Acc No: HLA00521, GenBank NP_002112.3 (SEQ ID NO:93); DPB1*04:02, IMGT / HLA Acc No: HLA00522, GenBank BBD34228.1 (SEQ ID NO:94); DPB1*06:01, IMGT / HLA Acc No: HLA00524 (SEQ ID NO:95); DPB1*09:01 (SEQ ID NO:96); DPB1*11 :01 , IMGT / HLA Acc No: HLA00528 (SEQ ID NO:97); DPB1*13:01 (SEQ ID NO:98); DPB1*35:01 (SEQ ID NO:99); DPB1*71 :01 , IMGT / HLA Acc No: HLA00590 (SEQ ID NQ:100); DPB1*104:01, p chain aa sequence, IMGT / HLA Acc No: HLA02046 (SEQ ID NQ:101); and DPB1*141:01, p chain aa sequence, IMGT / HLA Acc No: HLA10364 (SEQ ID NQ:102).

[0035] FIG. 15 shows an alignment of several MHC (HLA) gene products from the DQA1, DQA2, DRA and DPA1 a subunit genes permitting corresponding aas between the different gene products to be identified. From top to bottom, they are SEQ ID NO: 103 to SEQ ID NO: 108.

[0036] FIG. 16 shows an alignment of several MHC (HLA) gene products from the DQB1*02:01, DQB2-ISO-1, DRB1*01 :01, DRB1*04:01, DRB3 DRB1*01 :01, DRB4 DRB1*01:01, DRB5 DRB1*01 :01, and DPB1 DRB1*01:01 p subunit genes permitting corresponding aas between the different gene products to be identified. From top to bottom, they are SEQ ID NOs:75, 86, 20, 26, 57, 60, 62, and 90.

[0037] FIG. 17 provides a series of duplex MAPP structures based on framework polypeptides having both (I) a multimerization sequence, and (ii) first and second dimerization sequences that may be the same or different. The structure is shown generically in A with locations 1-5 and T-5' indicating locations for additional peptide sequences or MOD sequences. The MHC / epitope moiety is illustrated generically in FIGs. 17-22. Locations 4 and 4' are shown at the N-terminus of a presenting sequence, and locations 5 and 5' are shown at the C-termini of those polypeptides. Locations 1 and 1' are shown at the N-terminus of the framework peptide and locations 3 and 3' at the C-terminus of the framework polypeptide. In A and C, the framework polypeptides are multimerized to form a duplex of heterodimers via non-covalent binding between the multimerization sequences. In B and D, the framework polypeptides are multimerized to form a duplex of heterodimers using an immunoglobulin Fc region knob-in-hole motif, although other methods of non-covalently or covalently bonding the multimerization sequences may be used. In C the duplexes contain heterodimers in which two different asymmetric interspecific dimerization sequences bind together the framework peptides and their associated dimerization peptides. In D the framework peptides are joined together by a knob-in-hole Fc motif and the dimerization peptide and framework peptide are joined together by different dimerization sequences to form a duplex of heterodimers. Structures E-H parallel those of A-D respectively except that the presenting sequences are at the N-terminal portion of the framework polypeptide.

[0038] FIG. 18 provides in A to D a series of MAPP structures as in FIG. 17, with the addition of presenting sequences at the N-terminus of the framework peptides. Positions 4 and 4' may still serve as locations for peptide addition (e.g., MOD addition).

[0039] FIG. 19 provides in A to H a series of MAPP structures as in FIG. 17, where the dimerization sequences are Ig CH1 sequences (CH1) that pair with Ig light chain sequences (CL). The framework peptides are multimerized (dimers in this instance) through the interaction of Ig Fc (e.g., CH2 and CH3) regions, with the structures in B and D having knob-in-hole motifs to permit heteroduplexes to be formed. The peptides are also joined by disulfide bonds (e.g., those that form between Ig Fc region peptides).

[0040] FIG. 20 provides a series of MAPP structures as in FIG. 19, with the addition of presenting sequences at the N-terminus of the framework peptides. Positions 4 and 4' may still serve as locations for peptide addition (e.g., MOD addition).

[0041] FIG. 21 provides in A to J a series of MAPP structures as in FIG. 19. In each instance, a presenting sequence lacking a MOD sequence is present on the dimerization peptide (marked as a single chain MHO and epitope). Locations 2, 2', 4, 4', 5 and 5'are unfilled and not shown. Locations 1 and 1' are substituted with one or more MODs, e.g., for illustration purposes wild-type (wt.) and / or variant MODs of IL-2, PD-L1, and CD80, although other MODs may be used, e.g., wt. and / or variant TGF-p or 4-1 BBL. Positions 3 and 3' are shown for orientation in A to G. In H to J the 3 and 3' locations are unfilled, e.g., for illustration purposes wt. and / or variant TGF-p (which may be masked) or 4-1 BBL MODs may be located there, although other MODs may be used, e.g., wt. and / or variant MODs of IL-2, PD- L1, and CD80. Structures K and L show masked TGF-p MODs at the 3 and / or 3' position.

[0042] FIG. 22 shows four MAPP heterodimer constructs as structures A to D that can form duplex MAPPs. The dashed lines between the a1 and |31 domain elements represent a body disulfide between those elements, although a linker disulfide may be employed in addition to the body disulfide or in place of the body disulfide. In the polypeptide sequences of structures A to D, the MOD can be, for example, PDL1 .

[0043] FIG. 23 shows in A to D four different MHC Class II presenting sequences optionally containing a MOD sequence as in B and D. The presenting sequences are shown from the epitope at the N-terminus to their C- terminus linkage point indicated by the symbol " -II- , where they may join to, for example, a framework or dimerization peptide either directly or via a linker (e.g., an L3 linker). In A and B the presenting sequences are shown having a body disulfide bond. In C and D the presenting sequences are shown having a linker disulfide bond. The lines connecting the individual elements of the presenting sequences are optional peptide linkers. Each of the polypeptide linkers [L1 (between the epitope and |31 domain), L2 (between the |32 and a1 domain), L3 (C-terminal and proximate to the o2 domain), La (between the a1 and a2 domains), L|3 (between the p1 and p2 domains), and L4 (C-terminal to the multimerization sequence, such as between the multimerization sequence and a MOD at position 3)] may be present or absent, and are independently selectable. At E the figure shows the presenting sequence of A attached by an L3 linker to other MAPP sequences (e.g., a framework or dimerization polypeptide). Linker positions L1-L4, La, and Lp are indicated between the elements of the presenting sequences. The 2 above L1 indicates the G2C position in the linker, the 5 above the p1 domain represents aa 5 at which a cysteine may be substituted and a body disulfide bond may be formed. Positions 40, 47, 52, 74, 77, and 83 above the a1 domain represent aa where substitutions may be made, including some at which a cysteine may be located and used to form a body or linker disulfide bond.

[0044] FIG. 24 provides a table showing examples of HLA class II alleles, MODs, and T1D-haplotypes with risk of an autoimmune disease. The table also provides epitopes to be incorporated into autoantigens (self-epitopes) associated with a MAPP for T1D therapy.

[0045] FIG. 25 provides a table showing associations of HLA class II alleles and haplotypes with risk of an autoimmune disease. The table also provides epitopes of autoantigens (self-epitopes) associated with a number of the diseases listed.

[0046] FIG. 26 provides at A structures I and II, which are MAPP constructs that form duplexes. Production of exemplary samples of such constructs is described in Example 1 and SDS PAGE gels of the purified exemplary protein constructs under reducing and non-reducing conditions is provided at B.

[0047] FIG. 27 provides the sequences of the protein constructs from Example 1. For the sake of brevity the membrane proximal regions, when present, are listed as part of the a2 and |32 domains. Some linker sequences are bolded and italicized.

[0048] FIG. 28A provides the sequences of three different isoforms of Homo sapiens TGF-p (TGF-|31, TGF-|32, and TGF-|33), SEQ ID NO:141, SEQ ID NO: 142, and SEQ ID NO: 143, respectively, as preproproteins and the mature form of TGF-|33 (SEQ ID NO: 144) along with the C77S mutant of the mature protein (SEQ ID NO: 145).

[0049] FIG. 28B provides an alignment of TGF-p isoforms 1-3 with the residues corresponding to the mature form of TGF-|32 bolded, except aa residues Lys 25, Cys 77, lie 92, and Lys 94 of TGF-|32 and their corresponding residues in TGF-p isoforms 1 and 3 that are underlined and italicized but not bolded. References for the isoforms include TGF- |31 (NP_000651.3) SEQ ID NO:141, TGF-|31 (P01137 with P10L substitution) SEQ ID NO:146, TGF-|32 (AAA50405.1) SEQ ID NO: 142, and TGF-p-3 isoform 1 (NP_0013168.1) SEQ ID NO: 143.

[0050] FIG. 29A provides the sequences of a type 1 TGF-p receptor (T|3RI) and its ectodomain (SEQ ID NO:147 and SEQ ID NO: 148, respectively).

[0051] FIG. 29B provides the sequences of a type 2 TGF-p receptor (TpRII), its ectodomain, and fragments of the ectodomain. The locations indicated in bold and underlining in the isoform B are aas F30, D32, S52, E55 and D118 of the mature polypeptide, any of which may be substituted with an aa other than that occurring in the aa sequence provided. The ectodomain fragments are based upon NCBI Ref. Seq. NP_003233.4, and UniProtKB Ref. P37173; with the ectodomain sequence corresponding to aas 49 to 159 of those sequences. The substitution at aspartic acid "D119” of the mature protein with an alanine “ ” (bolded, italicized, and underlined) is marked as a "D118A” substitution for consistency with the literature describing that substitution when the signal peptide is understood to be 23 aas in length as opposed to 22 aas in the NCBI record. The aa D119 numbering assignment is based on the mature protein, and accordingly, it is D141 of the precursor protein when the 22 aa signal sequence is included. The location of D32, sometimes substituted with asparagine (D32N), corresponds to D55 in the precursor protein. The corresponding aas in mature isoform A lacking its signal sequence are F55, D57, S77, E80, and D143 (see, e.g., SEQ ID NO:149 to SEQ ID NO:156).

[0052] FIG. 29C provides the sequences of type-3 TGF-p receptor (TpRIII) isoforms A and B (SEQ ID NO:157 and SEQ ID NO: 158, respectively.IV< Detailed DescriptionA. Definitions

[0053] The terms "polynucleotide” and "nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0054] The terms "polypeptide” and "protein” are used interchangeably herein, and refer to a polymeric form of aas, which unless stated otherwise are the naturally occurring proteinogenic L-amino acids that are incorporated biosynthetically into proteins during translation in a mammalian cell. Furthermore, as used herein, a "polypeptide" and a "protein” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods. References to a specific residue or residue number in a known polypeptide, e.g., position 72 or 75 of human DRA MHC class II polypeptide, are understood to refer to the aa at that position in the wt. polypeptide (i.e. I72 or K75). To the extent that the sequence of the wt. polypeptide is altered, either by addition or deletion of one or more aas, the specific residue or residue number will refer to the same specific aa in the altered polypeptide (e.g., in the addition of one aa at the N-terminus of a peptide, reference to position I72 will be understood to indicate the aa, lie, that is now position 73). Substitution of an aa at a specific position is denoted by an abbreviation comprising, in order, the original aa, the position number, and the substituted aa, e.g., substituting the lie at position 72 with a cysteine is denoted as I72C.

[0055] A nucleic acid or polypeptide has a certain percent "sequence identity” to another nucleic acid or polypeptide, meaning that, when aligned, that percentage of nucleotides or aas are the same, and in the same relative position,when comparing the two sequences. Unless stated otherwise, to determine sequence identity the sequences are aligned using the computer program BLAST (BLAST+2.10.0 using default parameters), which is available over the world wide web at sites including blast.ncbi.nlm.nih.gov / Blast.cgi for BLAST+2.10.0. Unless stated otherwise, for determining positions of corresponding aas (e.g., when making specific substitutions), sequence comparisons are conducted using Clustal Omega Version 1.2.2 (using default parameters) available on the internet at www.ebi.ac.uk / Tools / msa / clustalo / . Where a polypeptide sequence comprises fewer aas or more aas than a reference sequence having a SEQ ID NO, the percent sequence identity of the polypeptide sequence to the reference SEQ ID NO sequence is determined by aligning and comparing the aas of the polypeptide sequence in the same relative position as the aas in the reference SEQ ID NO, without reference to the additional aas in the reference SEQ ID NO (where the reference SEQ ID NO has more aas than the polypeptide sequence) or the additional aas in the polypeptide sequence (where the polypeptide sequence has more aas than the reference SEQ ID NO). For example, as discussed below, a DRA a1 domain sequence may have a percent sequence identity to DQA1 SEQ ID NO:63 The percent sequence identity of the DRA a1 domain sequence to SEQ ID NO:63 is determined by aligning and comparing the aas in the DRA a1 domain sequence with their corresponding aas in SEQ ID NO:63, i.e., the aas of the DRA a1 domain sequence in the same relative position as the aas in the reference SEQ ID NO:63. If the DRA a1 domain sequence has more aas than SEQ ID NO:63, then only the aas in the DRA a1 domain sequence that have the same relative position as the aas in SEQ ID NO:63 are considered in determining percent sequence identity and the additional aas in the DRA a1 domain sequence are not included in determining the percent identity of the DRA a1 domain sequence to SEQ ID NO:63. Similarly, if SEQ ID NO:63 has more aas than the DRA a1 domain sequence, then only the aas in SEQ ID NO:63 that have the same relative position as the aas in the DRA a1 domain sequence are considered in determining the percent identity of the DRA a1 domain sequence to SEQ ID NO:63 and the additional aas in SEQ ID NO:63 are not included in determining the percent identity.

[0056] As used herein, amino acid (“aa” singular or "aas" plural) means the naturally occurring proteogenic amino acids incorporated into polypeptides and proteins in mammalian cell translation. Unless stated otherwise, these are: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Vai, 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 (Gin, glutamine), I (lie, 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 appear in some proteins found in mammalian cells; however, unless their presence is expressly indicated they are not understood to be included.

[0057] A "conservative amino acid substitution” refers to the interchangeability in proteins of aa residues having similar side chains. For example, a group of aas having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of aas having aliphatic-hydroxyl side chains consists of serine and threonine; a group of aas having amide containing side chains consists of asparagine and glutamine; a group of aas having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of aas having basic side chains consists of lysine, arginine, and histidine; a group of aas having acidic side chains consists of glutamate and aspartate; and a group of aas having sulfur containing side chains consists of cysteine and methionine. Exemplaryconservative aa substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine-glycine, and asparagine-glutamine.

[0058] As used herein the term "in vivo” refers to any process or procedure occurring inside of a living body, e.g., the body of a patient.

[0059] As used herein, "in vitro” refers to any process or procedure occurring outside of a living body, e.g., the body of a patient.

[0060] The term "binding” refers to a direct association between molecules and / or atoms, due to, for example, covalent, electrostatic, hydrophobic, ionic, and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. "Covalent bonding” or "covalent binding” as used herein refers to the formation of one or more covalent chemical bonds between two different molecules. The term "binding,” as used with reference to the interaction between a MAPP and a T cell receptor (TCR) on a T cell, refers to a non-covalent interaction between the MAPP and TCR.

[0061] "Affinity” as used herein generally refers to the strength of non-covalent binding, increased binding affinity being correlated with a lower KD or Kd. As used herein, the term "affinity” may be described by the dissociation constant (KD or Kd) for the reversible binding of two agents (e.g., an antibody and an antigen). As used herein, the term "avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0062] "T cell” includes all types of immune cells expressing CD3, including T-helper cells (CD4+T helper cells), cytotoxic T cells (CD8+cells), regulatory T cells (Tregs), and NK-T cells.

[0063] The term "immunomodulatory polypeptide” (also referred to as a "MOD”), as used herein, includes a wt. or variant of a polypeptide or portion thereof that can specifically bind a cognate co-immunomodulatory polypeptide ("co-MOD” e.g., a receptor upon which it may act as an agonist or antagonist) present on a T cell, and provide a modulatory signal to the T cell when the TCR of the T cell is engaged with an MHC-epitope moiety that is specific for the TCR. Unless stated otherwise the term "MOD” includes wt. and / or variant immunomodulatory polypeptides, and statements including reference to both wt. and variant MODs are made to emphasize that one, the other, or both are being referenced. The signal provided by the MOD engaging its co-MOD along with other signals received by the T cell mediates (e.g., directs) 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.

[0064] "MAPP” as used herein can be singular or plural; where required, "MAPPs” refer to the plural. MAPP and MAPPs include higher order complexes of MAPPs including duplexes, triplexes, etc. MAPPs may be MOD-less or MOD-containing. MOD-less MAPPs do not comprise an aa sequence (polypeptide sequence) of a MOD. In contrast, MOD-containing MAPPs comprise all or part of the aa sequence (polypeptide sequence) of at least one (e.g., at least two) MOD.

[0065] As used herein "higher order complexes” of MAPPs include, but are not limited to, MAPP complexes comprising: two (duplexes), three (triplexes), four (quadraplexes), five (pentaplexes), six (hexaplexes) MAPPs, or more than six MAPPs. Recitations such as "MAPPs and higher order complexes thereof (e.g., duplexes)” do not change the scope of MAPP as used herein, but instead are made, for example, to emphasize that a singular MAPP or its higher order complexes are contemplated, and / or for antecedent basis.

[0066] "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 resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.

[0067] The terms "recombinant expression vector” and "DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the 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 DNA regulatory sequences.

[0068] The terms "treatment,” "treating” and the like are used herein to generally mean the use of a therapeutic agent for the purpose of obtaining one or more desired pharmacologic and / or physiologic effects. The effect may be prophylactic in terms of completely or partially preventing a disease, disorder, or symptom(s) thereof, and / or may be therapeutic in terms of a partial or complete cure for a disease or disorder and / or adverse effect attributable to the disease or disorder. "Treatment” as used herein covers any treatment of a disease, disorder, or symptom in a mammal, and includes: (a) preventing the disease, disorder, or symptom from occurring in a subject which may be predisposed to acquiring the disease, disorder, or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease, disorder, or symptom, i.e., arresting its development; and / or (c) relieving the disease or disorder, i.e., causing regression of the disease or disorder. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease or disorder, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease or disorder, and in some cases after the symptomatic stage of the disease or disorder.

[0069] 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, and in addition include rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), felines, canines, etc.

[0070] Unless indicated otherwise, the term "substantially” is intended to encompass both "wholly” and "largely but not wholly.” For example, an Ig Fc that "substantially does not induce cell lysis” means an Ig Fc that induces no cell lysis at all or that largely but not wholly induces no cell lysis.

[0071] As used herein, the term "about” used in connection with an amount indicates that the amount can vary by 10%. For example, "about 100” means an amount of from 90-110. Where "about” is used in the context of a range, the "about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and "about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0072] The terms "purifying,” "isolating,” and the like refer to the removal of a desired substance, e.g., a MAPP, from a solution containing undesired substances, e.g., contaminates, or the removal of undesired substances from a solution containing a desired substance, leaving behind essentially only the desired substance. In some instances, a purified substance may be essentially free of other substances, e.g., contaminates. 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.

[0073] Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range to a tenth of the lower limit of the range is encompassed by the disclosure along with any other stated or intervening value in the range.

[0074] Unless defined otherwise, 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.

[0075] It must 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, reference to "a Treg” includes a plurality of such Tregs and reference to "the MHC Class II alpha chain” includes reference to one or more MHC Class II alpha chains and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. This statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,” "only,” and the like in connection with the recitation of claim elements, as well as use of a "negative” limitation.

[0076] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.B. Description1. MAPP Structure and the Role of Framework and Dimerization Peptides

[0077] The expression of MHC Class II proteins generally results in the poor yield of proteins and / or protein that is aggregated or relatively unstable. Among the most problematic Class II proteins to express in an active form at favorable levels are some of the DQ alleles associated with immune diseases. MHC Class II proteins such as the HLA DQ 2.5 heterodimer (comprised of the HLA a and p subunits DQA1*05:01 and DQB 1 *02:01 ) that appear to have weak interactions between the a and p subunits and / or weak associations with peptide epitopes are particularly problematic. The presence of such weak interactions, either alone or in combination, results in events such as collapsing of the binding pocket (e.g., around the P1 binding region), and the subsequent irreversible aggregation and / or breakdown of the protein during cellular expression. Even with relatively stable Class II molecules that form compact Class II heterodimers of the a and p subunit sequences, such as some DR heterodimers that have relatively compact epitope binding pockets, Class II molecules are stabilized significantly by hydrogen bonds with the epitope(e.g., at P1 , P4, P6 / 7, and P9). However, the residue interactions near the P1 pocket disproportionately stabilize the DR heterodimer. As a result, expression of DR protein constructs is highly dependent on the aa sequence of the peptide epitopes (e.g., fused to one of the HLA subunits) that can occupy and stabilize the heterodimer.

[0078] The present disclosure enables expression of MHC (HLA) Class II protein constructs at increased levels by introducing a combination of features that stabilize the MHC heterodimer. The MAPPs described herein utilize a single chain format to force a subunit (a1 and a2 domains) and p subunit (p1 and p2 domains) folding and pairing with the p subunit placed N-terminal to the a subunit. The specific ordering of the MHC domains generally is p1, p2, a1 and o2, with optional linkers located between the domains. The epitope to be bound in the MAPP binding pocket and presented to a TCR is fused to the Class II construct by an L1 linker attached to the p1 domain of the p subunit's sequence. The paired a and p subunits are stabilized by at least one disulfide bond formed between the C-terminal portion of the a subunit's a1 domain and either the N-terminus of the p1 domain or the L1 linker attached to it (see, e.g., FIG. 1 and FIG. 23). Additional stabilization may be obtained by introducing aa substitutions that improve hydrogen bonding between the a and p subunit sequences, and / or substitutions that enhance peptide MHC (HLA) binding interactions. The increased peptide-MHC (HLA) interactions effectively increase the affinity between the peptide and the binding pocket sequences, causing the peptide to have an increased residence time in the binding pocket, relative to the affinity and residence time observed in the absence of the substitutions. Additionally, embodiments of the MAPP constructs described herein display resistance to denaturation (thermal stability) at elevated temperatures and upon freeze-thaw testing.

[0079] FIG. 1 illustrates the architecture of the MAPPs of the present disclosure with the N-terminus at the left. The presenting sequence(s) indicated as Epitope-MHC in FIG. 1 are shown in more detail in FIG. 23 along with the locations of the linkers La, Lp, L1 through L3, and a MOD located C-terminal to the a2 domain in panels B and D. For reference, FIG 23, panel E, shows a presenting sequence with various aas in the L1 linker, the p1 domain, and the a1 domain numbered. Although the structure in panel E shows a body disulfide between the p1 domain (e.g., aa 5) and the a1 domain (e.g., aa 83), a linker disulfide between an aa of the L1 linker (e.g., position 2) and an aa in the a1 domain may be employed (instead of, or in addition to, the body disulfide) for the formation of a stabilizing disulfide as illustrated in FIG 23, panels C and D. The other numbers appearing above the construct in panel E are given for orientation and represent locations (aa positions) in the MAPP presenting sequence where disulfide bonds or substitutions (e.g., that benefit expression levels and / or stability (such as thermal stability) and / or resistance to nonspecific aggregation) may be made. Each of the polypeptide linkers [L1 (between the epitope and p1 domain), L2 (between the p2 and a1 domain), L3 (C-terminal and proximate to the a2 domain), La (between the a1 and a2 domains), Lp (between the p1 and p2 domains), and L4 (C-terminal to the multimerization sequence of the framework polypeptide) may be present or absent, and are independently selectable.

[0080] Throughout the disclosure, positions in the MHC a chain a1 and a2 domain sequences are referred to by numbering starting at 1 from the N-terminus of the a1 domain through the C-terminus of the a2 domain. Similarly, positions in the MHC p chain p1 and p2 domain sequences are referred to by numbering the p chain sequences starting with the N-terminus of the p1 domain at 1 and going through to the C-terminus of the p2 domain. Linker sequences, MOD sequences, and the sequences of any additional peptides that are present in a MAPP are similarly numbered starting at their N-terminal aa. The numbering as shown in FIG. 23, panel E, is presented forexemplification and is keyed to a construct prepared from HLA DQA1*05:01 and DQB1*02:01, which make up HLA DQ 2.5. Positions 40, 47, 52, and 74 of HLA DQ 2.5, and their corresponding locations in other alleles, represent locations where aa substitutions enhancing expression and stability may be made. Position 5 in the p1 domain and position 83 in the a1 domain exemplify specific aas in DQ 2.5 constructs where cysteine substitutions for formation of a body disulfide bond (shown as a dashed line in FIG. 23 at A and B) may be made. Position 2 in the L1 linker and position 77 in the a1 domain exemplify specific aas where cysteine substitutions for formation of a linker disulfide bond (shown as a dashed line in FIG 23 at C and D) may be made. The positions for aa substitutions and disulfide bond formation in other Class II alleles corresponding to those in DQ 2.5 may be identified by aa sequence alignment (e.g., with the Clustal Omega Version 1.2.2 available on the internet at www.ebi.ac.uk / Tools / msa / clustalo / ) using FIGs. 15 and 16 to align across the a and p subunits (chains) of the individual MHC alleles of different Class II gene products (e.g., DQ, DR, and DP gene products). Corresponding positions in some a and p subunits (chains) are provided in the aa sequences aligned in FIGs. 15 and 16.

[0081] The present disclosure provides MAPPs for, among other things, use in the treatment of autoimmune diseases (e.g., T1 D and celiac disease), cancers, and allergies. As discussed above, the MAPPs include at least one framework polypeptide and at least one dimerization polypeptide. Framework polypeptides comprise one or more polypeptide dimerization sequence that permits specific binding with other polypeptides (dimerization polypeptides) having a counterpart dimerization sequence, thereby forming at least a heterodimer (see FIG. 1). Framework polypeptides also comprise a multimerization sequence(s) that permits two or more framework polypeptides to associate, thereby forming a higher order structure (e.g., a duplex of the two or more heterodimers, a duplex MAPP (see, e.g., FIG. 1). Neither the dimerization sequence nor the multimerization sequence of the framework polypeptide (or the counterpart dimerization sequence) comprises an MHC class II (e.g., HLA) a chain or p chain polypeptide sequence, and as such, interactions brought about by those sequences are not considered to be dimerization or multimerization of framework and / or dimerization peptides. Accordingly, the framework polypeptides provide a structure upon which other polypeptides can be organized by interactions with the dimerization sequences, and which can interact with other framework polypeptides by way of multimerization sequences. The terms MAPP and MAPPs as used herein will be understood to refer in different contexts to the heterodimer comprising a framework and dimerization peptide structure as well as to higher order complexes of those MAPP heterodimers, such as duplexes (duplex MAPPs). It will be clear to the skilled artisan when specific reference to only higher order structures are intended (e.g., by reference to duplex MAPPs, etc.).

[0082] As discussed above, the framework and dimerization peptide containing MAPPs, duplex MAPPs, and MAPPs of higher order (e.g., triplex MAPPs) described herein provide a means by which peptide epitopes may be delivered in the context of MHC polypeptides (e.g., HLA) to a target T cell displaying a TCR specific for the epitope, while at the same time permitting for the flexible presentation of one or more MODs. The MAPPs, duplex MAPPs, and higher order MAPPs thereby permit delivery of one or more MODs in an epitope selective (e.g., dependent / specific) manner that permits formation of an active immune synapse with a target T cell selective for the epitope, and control / regul ation of the target T cell's response to the epitope. Accordingly, where MAPPs comprise stimulatory or activating MODs (e.g., IL-2, CD80, CD86, and / or 4-1 BBL), they increase T cell proliferation and / or effector functions in an epitope selective manner. In contrast, where MAPPs comprise suppressive / inhibitory MODs (e.g., FasL and / orPD-L1), they decrease T cell activation, proliferation, differentiation, and / or effector functions in an epitope selective manner.

[0083] The framework / dimerization polypeptide architecture of MAPPs and their higher order structures may also be understood to provide flexibility in locating MODs and epitope presenting sequences. Duplex MAPP and higher order MAPP architecture can be particularly useful when both the MOD and the epitope presenting sequences are positioned so as to provide the desired biological activity as well as other desired properties of the MAPP, e.g., thermal stability and manufacturability. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting sequence(s) are positioned at the N-terminus of a polypeptide, e.g., each may be located at the N-terminus of different framework and / or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting sequence(s) are positioned at the C- terminus of a polypeptide, e.g., each may be located at the C-terminus of different framework and / or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD presenting sequence(s) are positioned at the N-terminus and C-terminus of a polypeptide, respectively, e.g., the MOD may be located at the N-terminus and the presenting sequence may be located at the C-terminus of different framework and / or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting sequence(s) are positioned at the C-terminus and N-terminus of a polypeptide, respectively, e.g., the MOD may be located at the C-terminus and the presenting sequence may be located at the N-terminus of different framework and / or dimerization polypeptide sequences.

[0084] The structure of MAPPs, and particularly higher order MAPPs such as duplexes, may be specified by the use of pairs of polypeptides having different sequences that specifically pair with each other. Multimerization of framework polypeptides results from interactions between multimerization sequences, and dimerization (the interaction of a framework polypeptide and a dimerization polypeptide) results from the interaction of a dimerization sequence on the framework polypeptide and a counterpart dimerization on a dimerization polypeptide. For example, in a duplex MAPP the multimerization sequences may be Ig Fc heavy chain (e.g., CH2-CH3) sequences, and the dimerization sequence and counterpart dimerization sequences may be the same (e.g., all leucine zipper sequences). An additional degree of control may be obtained by utilizing non-identical peptide sequences that specifical ly / selecti vely pair with each other and are referred to herein generally as "interspecific sequences,” in the case of dimerization sequences, "interspecific dimerization sequences,” or in the case of multimerization sequences, "interspecific multimerization sequences,” and which give rise to asymmetric interspecific pairs of sequences. The structure of MAPPs thus permits diverse and effective placement of each polypeptide into the MAPP architecture (see, e.g., FIGs. 17-22). Interspecific sequences may include an Ig heavy chain Fc (e.g., CH2-CH3) region modified with, for example, knob-in-hole variations, and Fos peptide sequences paired with Jun peptide sequences. Accordingly, MAPP architectures include, but are not limited to, MAPPs where each, or some, of the dimerization sequences are different (permit different peptide pairings), for example, in duplex MAPPs where each of the multimerization and dimerization sequences is different and provides separate peptide pairings.

[0085] In an embodiment, the framework peptide multimerization sequence is an Ig Fc heavy chain region (optionally a knob-in hole Fc sequence pair) and the dimerization sequences are the same (e.g., Ig CH1 sequences paired with light chain A or K constant region sequences) (see, for example, FIGs. 17 and 18, structures A to D). In anotherembodiment, the framework peptide multimerization sequence is an Ig Fc heavy chain region (optionally a knob-in hole Fc sequence pair) and the dimerization sequences are selected to be different (e.g., a dimerization sequence pair comprising an Ig CH1 paired with light chain A or K sequence and a dimerization sequence comprising a leucine zipper pair, see for example, FIG. 21, structure I). For example, in a duplex MAPP the multimerization sequences may be a knob-in-hole Ig sequence, one dimerization sequence and its counterpart dimerization sequence may be leucine zipper sequences, and a second dimerization sequence and its counterpart dimerization sequence may be an Ig CH1 and Ig CL A domain pair.

[0086] MAPPs and accordingly their higher order complexes (duplexes, triplexes, etc.) comprise MHC Class II polypeptide sequences that bind an epitope for presentation to a TCR, and accordingly may present peptides to T cells (e.g., CD4+T cells). The effect of MAPPs on T cells with TCRs specific to the epitope depends on which, if any, MODs are present in the MAPP. As noted above, MAPPs, duplex MAPPS and higher order MAPPs comprising MOD(s) permit MOD delivery to T cells in an epitope selective manner and the MODs principally dictate the effect of MAPP-T cell engagement in light of the specific cell type stimulated and the environment. While not wishing to be bound by any particular theory, the effect of MAPP (e.g., duplex MAPP) presentation of MOD(s) and epitope(s) to T cells in some cases may be enhanced relative to the situation encountered in antigen-presenting cells (APO) where epitope can diffuse away from the MHC (e.g., HLA) complex and any MODs the APO is presenting. This may not occur with a MAPP, however, where the epitope and MOD(s) are part of the MAPP polypeptide(s) and cannot diffuse away even if the epitope's affinity for the MHC complex would normally permit it to leave the comparable cell complex. The ability of epitope to diffuse away from MHC and MOD components of a MAPP, duplex MAPP, or higher order MAPP is further limited where the polypeptide(s) of the MAPP (e.g., framework and dimerization sequences) are covalently attached to each other (e.g., by disulfide bonds). Consequently, MAPPs and their higher order structures may be able to prolong delivery of MOD(s) to T cells in an epitope selective manner relative to systems where epitopes can diffuse away from the presenting MHC.

[0087] Incorporation of one or more MODs with affinity for their cognate receptor (co-MOD) on T cells can reduce the specificity of MAPPs (e.g., duplex MAPPs) for epitope selective / specific T cells. The reduction in epitope selectivity / specificity of the MAPPs becomes more pronounced where MOD / co-MOD binding interactions increase in strength (binding energy) and significantly compete with MHC / epitope binding to target cell TCR. The inclusion of variant MODs with reduced affinity for their co-MOD(s) thus may provide a lower contribution of MOD binding energy, thereby permitting MHC-epitope interactions in which the TCR dominates the binding and provides 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 may be incorporated into MAPPs and their higher order complexes alone or in combination with wt. MOD sequences. Wt. and variant MODs are described further below. Where a MOD has a relatively low binding affinity for its co-MOD, however, a MAPP may have acceptable selectivity without a reduced-affinity MOD, and thus a variant MOD having reduced affinity may be unnecessary.

[0088] The ability of MAPPs to modulate T cells in an epitope selective / specific manner thus provides methods of modulating activity of a T cell in vitro and in vivo, and accordingly, methods of treating autoimmune diseases (e.g.,T1D or celiac disease) resulting from immune dysregulation / dysfunction as well as diseases such as cancers, infections, allergies and autoimmune diseases.

[0089] The present disclosure provides nucleic acids comprising nucleotide sequences encoding MAPP polypeptides, cells genetically modified with the nucleic acids and capable of producing the MAPP, and methods of producing MAPPs and their higher order complexes utilizing such cells.

[0090] Each presenting sequence present in a MAPP comprises MHC class II a and p chain polypeptide sequences (e.g., human MHC class II sequences) sufficient to bind a peptide epitope and present it to a TCR. MHC Class II peptides may include sequence variations that are designed to stabilize the MHC, stabilize the MHC-peptide epitope complex, and / or stabilize the MAPP. Sequence variations may also serve to enhance cellular expression of MAPPs prepared in cell-based systems as well as the stability (e.g., thermal stability) of MAPPs and their higher order complexes such as duplex MAPPs. Some MHC class II sequences suitable for use in MAPPs are described below.

[0091] As indicated in the description of the drawings, MAPPs may comprise one or more independently selected peptide sequences (one or more "linker” or "linkers”) between any two or more components of the MAPP, which in the figures may be shown as a line between peptide and / or polypeptide elements of the MAPPs. The same sequences used as linkers may also be located at the N- and / or C-termini of the MAPP peptides to prevent, for example, proteolytic degradation. Linker sequences include but are not limited to polypeptides comprising: glycine; glycine and serine; glycine and alanine; alanine and serine; and glycine, alanine and serine; any one of which may comprise a cysteine for formation of an intra- or inter-polypeptide disulfide bond. Various linkers are described in more detail below.2. Exemplary MAPP Architectures

[0092] MAPPs comprise (I) framework polypeptides with a multimerization sequence and at least one dimerization sequence, and (II) dimerization polypeptides with a counterpart dimerization sequence that binds with the framework polypeptide's dimerization sequence. As discussed above, MAPPs further comprise one or more epitope presenting sequences. Exemplary structures for such MAPPs or their duplexes appear in FIG. 1 and FIGs. 17-22. The structures depicted in FIG. 21 represent MAPPs with multimerizing framework polypeptides and epitope presenting sequences (the "Single Chain MHC” with the "Epitope”).

[0093] Interactions of MHC (e.g., HLA) sequences are not considered herein to result in multimerization and / or dimerization. In an embodiment, neither the dimerization sequence nor the multimerization sequence of the framework polypeptide, nor the counterpart dimerization sequence of the dimerization polypeptide, comprises a Class II MHC polypeptide 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) contiguous aas of an MHC Class II polypeptide (e.g., a polypeptide in any of FIGs. 4 to 16). In embodiments, MAPPs comprise at least one, or at least two, dimerization peptide(s) that comprise(s) an epitope presenting sequence. See, e.g., FIG. 1.

[0094] One group of MAPPs, those having epitope presenting sequences, comprises: a multimerizing framework polypeptide having, from N-terminus to C-terminus, a dimerization sequence and multimerization sequence; and a dimerization polypeptide comprising a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide and dimerizing therewith through covalent and / or non-covalent interactions to form a heterodimer, wherein at least one (e.g., one, or both) of a dimerization polypeptide and the frameworkpolypeptide comprises a presenting sequence located on the N-terminal side of their dimerization or counterpart dimerization sequences. In such a MAPP the presenting sequence comprises a peptide epitope and one or more MHC polypeptide sequences, with the peptide epitope sequence located: (I) at or within 10 aas, 15 aas, 20 aas, or 25 aa of the N-terminus of the presenting sequence or (ii) in a polypeptide located at the N-terminus of the presenting 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 presenting sequence comprising one or more independently selected MODs located at their N-terminus and / or C-terminus (or on the N-terminal or C-terminal side of the dimerization or counterpart dimerization sequences), wherein the MHC polypeptide sequences are MHC class II polypeptide sequences and they comprise MHC class II o1, o2, p1, and p2 polypeptide sequences (e.g., human MHC class II sequences). In an embodiment, neither the dimerization sequence 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) contiguous aas of an MHC class II polypeptide in any of FIGs. 4 to 16.

[0095] MAPPs may be constructed such that neither the dimerization sequence 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) contiguous aas of an MHC class II polypeptide in any of FIGs. 4 to 16.

[0096] As discussed above, a dimerization sequence of a framework polypeptide may interact with dimerization peptides to form heterodimers. The multimerization sequence of the framework polypeptide may associate with another framework polypeptide multimerization sequence, forming a duplex (or higher order structure, such as a triplex, quadraplex or pentaplex) of the heterodimers. Where the multimerization sequences are interspecific (e.g., a knob-in-hole Fc peptide pair), and at least one heterodimer comprises an interspecific dimerization and counterpart dimerization pair, two different heterodimers may be formed. When the different heterodimers are combined to form a duplex MAPP, any one or more component (e.g., MODs) may differ (e.g., in type or location) between the two heterodimers.C. MAPP Components1. Framework Polypeptides and Dimerization Polypeptides

[0097] As may be understood from the preceding sections, framework polypeptides serve as the structural basis or skeleton of MAPPs, permitting the organization of other elements in the MAPP complex. Framework peptides interact with other peptides through binding interactions, principally at dimerization and multimerization sequences.Interactions at dimerization sequences permit association of non-framework peptides (e.g., dimerization peptides) with framework peptides. In contrast, multimerization sequences are involved in the interaction of two or more framework peptides.

[0098] 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 (e.g., not both leucine zipper variants) as the multimerization sequence. By utilizing different types of sequences for the interactions at multimerization and dimerization sequences, it becomes possible to control the interactions of the framework polypeptide with other framework polypeptides and with dimerization polypeptides. In an embodiment, frameworkpolypeptides comprise one multimerization sequence and one dimerization sequence. In an embodiment, framework polypeptides comprise at least one multimerization sequence and at least two independently selected dimerization sequences. Framework peptides may contain peptide sequences (e.g., linker sequences and / or MOD sequences) between any of the elements of the framework polypeptide or at the ends of the framework polypeptide including the multimerization sequences and dimerization sequences.

[0099] In addition to providing for the structural organization of MAPPs through their multimerization and dimerization sequences, framework peptides, and particularly their N- and C-termini, may also serve as locations for placement of elements such as MOD sequence(s), and / or epitope presenting sequences. 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).

[0100] Within a MAPP, all of the dimerization sequences may be non-interspecific (such as leucine zipper pairs) while the multimerization sequences are either interspecific or non-interspecific (see, e.g., structures A and B of FIGs. 17 and 18). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be a non-interspecific polypeptide (e.g., Ig Fc (e.g., CH2, CH3 domains) or leucine zippers) or the multimerization sequences may be an interspecific knob-in-hole sequence pair, with the dimerization sequences of the first and second framework polypeptides as non-interspecific leucine zipper polypeptides. Where an Fc polypeptide is employed it may be, for example, from an IgA, IgD, IgE, IgG, or IgM sequence, which may 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.

[0101] Within a MAPP, all of the dimerization sequences may be interspecific, while the multimerization sequences are not interspecific (see, e.g., FIG. 21 A). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be Ig Fc sequences, with the ZW1 sequence or its counterpart employed as the dimerization sequence of the first framework polypeptide and an Ig CH1 domain or its counterpart Ig CL K sequence as the dimerization sequence of the second framework polypeptide.

[0102] All of the dimerization sequences or all of the dimerization and multimerization sequences in a MAPP may differ in that they bind only to specific binding partners present in the MAPP (e.g., each is part of a different interspecific sequence pair). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be a pair of knob-in-hole Ig Fc sequences, with the ZW1 sequence or its counterpart employed as the dimerization sequence of the first framework polypeptide, and an Ig CH1 or its counterpart Ig CL sequence as the dimerization sequence of the second framework polypeptide.2. Multimerization and Dimerization Polypeptide Sequences

[0103] Amino acid sequences that permit polypeptides to interact may be utilized as dimerization sequences or counterpart dimerization sequences when they are involved in the formation of dimers between a framework polypeptide and a dimerization polypeptide. The same type of aa sequences may be utilized as multimerization sequences when they are used to form duplex or higher order structures (trimers, tetramers, pentamer, etc.) between framework polypeptides. In any given MAPP, sequences that can interact with each other are not utilized as both dimerization and multimerization sequences. Stated another way, the same aa sequence pair may serve as either dimerization or multimerization sequences depending on whether they: bring together two or more frameworkpeptides, in which case they are multimerization sequences, or they bring together dimerization and counterpart dimerization sequences, in which case they are designated as dimerization sequences.

[0104] Where dimerization or multimerization sequences employ identical sequences that pair or multimerize (e.g., some leucine zipper sequences), they can form symmetrical pairs or multimers (e.g., homodimers). In contrast, where dimerization or multimerization sequences that pair are not identical and require a specific complementary counterpart sequence to form a dimer, they are interspecific binding sequences and can form asymmetric pairs. Both immunoglobulin (e.g., Ig Fc) and non-immunoglobulin polypeptides can be interspecific or non-interspecific in nature. For example, both Fos / Jun binding pairs and Ig CH1 polypeptide sequences and light chain constant region CL sequences form interspecific binding pairs. Natural Ig Fc regions tend to be non-interspecific, but, as discussed below, can be made to form interspecific pairs (e.g., Ki H and KiHs-s pairs). Coiled-coil sequences, including leucine zipper sequences, can be either interspecific leucine zipper or non-interspecific leucine zipper sequences. See, e.g., Zeng et al., (1997) PNAS (USA) 94:3673-3678, and Li et al., (2012), Nature Comms. 3:662.

[0105] Interspecific binding sequences may in some instances form some amount of homodimers, but preferentially dimerize by binding more strongly with their counterpart interspecific binding sequence. Accordingly, specific heterodimers tend to be formed when an interspecific dimerization sequence and its counterpart interspecific binding sequence are incorporated into a pair of polypeptides. By way of example, where an interspecific dimerization sequence and its counterpart are incorporated into a pair of polypeptides, they may selectively form greater than 80%, 90%, 95%, 98% or 99% heterodimers when an equimolar mixture of the polypeptides is combined (for example in PBS or PBS plus saline buffer at 20° C). The remainder of the polypeptides may be present as monomers or homodimers, which may be separated from the heterodimers. See, for example, FIG. 17, structure B, with an interspecific multimerization sequence, and structure C, with two different interspecific dimerization sequences (indicated by the different arrow-like projections between the dimerization sequence and counterpart dimerization sequence). Moreover, because interspecific sequences are selective for their counterpart sequence, they can limit the interaction with other proteins expressed by cells (e.g., in culture or in a subject) particularly where the interspecific sequences are not naturally occurring or are variants of naturally occurring protein sequences.

[0106] Sequences are considered orthogonal to other sequences when they do not form complexes (bind) with each other's counterpart sequences. See FIG. 19, structure D, or FIG. 21, structure I, where the MAPP comprises an interspecific multimerization sequence and two independently selected interspecific dimerization sequences, all of which are orthogonal to each other. Any of the MAPPS described herein may have two or more (e.g., three, four or more) orthogonal dimerization sequences. In an embodiment, MAPPs with multimerizing framework peptides may have orthogonal multimerization and dimerization domains (where the dimerization domains may or may not be orthogonal to each other).

[0107] Some sequences permitting 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 may be of relatively compact size (e.g., less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, 60, 50, 40, or 30 aa). In an embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 300 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 200 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and / ormultimerization sequences is less than 100 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 75 aa. In another embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 50 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and / or multimerization sequences is less than 30 aa.

[0108] Dimerization / multimerization sequences include but are not limited to: immunoglobulin heavy chain constant region (Ig Fc) polypeptide sequences (e.g., sequences comprising CH2-CH3 regions of immunoglobulins such as those provided in FIGs. 2A-2H and SEQ ID NOs:1 to 13); polypeptides of the collectin family (e.g., ACRP30 or ACRP30-II ke proteins) that contain collagen domains consisting of collagen repeats, Gly-Xaa-Yaa and / or Gly-Xaa- Pro (which may be repeated from 10-40 times); coiled-coil domains; leucine-zipper domains; interspecific 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 C sequences (Ig CH1 / CL pairs such as an Ig CH1 sequence paired with an Ig CL K or A light chain constant region sequence).

[0109] Framework and / or dimerization polypeptides of a MAPP may comprise an immunoglobulin heavy chain constant region (e.g., CH2-CH3 domains) polypeptide sequence that functions as a dimerization or multimerization sequence. Where the framework polypeptide comprises an Ig Fc multimerization sequence and a CH1 dimerization sequence, it may comprise all or part of a wild-type or variant immunoglobulin sequence set forth in any of FIGs. 2A to 2H that comprises the CH1, CH2 and CH3 domains and any hinge sequences that may be present. An Ig Fc sequence, or any one or more of the CH1, CH2, and / or CH3 domains, may have at least about 85% or at least about 90% (e.g., at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to an aa sequence of an Fc region depicted in FIGs. 2A-2H. In one instance they may have at least about 85% or at least about 90% aa sequence identity to an Fc sequence depicted in any of FIGs. 2A-2H. In one instance they may have at least about 95% or at least about 98% aa sequence identity to an Fc sequence depicted in any of FIGs. 2A-2H. In one instance they may have at least about 99% or 100% aa sequence identity to an Fc sequence depicted in any of FIGs. 2A-2H. In particular, the terminal lysine provided in some of the sequences provided in FIGs. 2A-2H (e.g., the IgG sequences in FIGs. 2D, 2E, 2F, and 2G) may be removed during cellular processing of the MAPPs and may not be present on some or all of the MAPP molecules as expressed. See, e.g., van den Bremer et al. (2015) mAbs 7:4, and Sissolak et al. (2019) J. Industrial Microbiol. & Biotechnol. 46:1167. Alternatively, in preparing MAPPs, the nucleotides encoding a C-terminal lysine may simply be omitted from any of the sequences provided in FIGs. 2A-2H in which a C-terminal lysine occurs.

[0110] Immunoglobulin sequences may covalently link the polypeptides of a MAPP complex together by forming one or two interchain disulfide bonds, thereby stabilizing MAPPs, particularly where a pair of interspecific Ig sequences, such as knob-in-hole polypeptide pairs, is employed. Where an Fc polypeptide sequence (e.g., comprising CH2 and CH3 sequences), alone or in combination with a CH1 polypeptide sequence, is employed as a multimerization or dimerization sequence, it may be, for example, from an IgA, IgD, IgE, IgG, or IgM sequence, which may 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. As discussed below, the Ig Fc region can further contain substitutions that can substantially remove the ability of the Ig Fc to effect GDC, ADCP, or ADCC, orin the alternative, to enhance ADCC, ADCP or CDC. Accordingly, framework and / or dimerization polypeptides, and in particular Ig Fc sequences used as multimerization or dimerization sequences, may comprise substitutions that reduce or substantially eliminate ADCC and / or CDC responses.

[0111] Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, or at least about 99%) or 100% aa sequence identity to 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), or all aas, of the IgA Fc sequence depicted in FIG. 2A (SEQ ID NO:1). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domains) of the IgA Fc sequence depicted in FIG. 2A (SEQ ID NO:1). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to 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), or all aas, of the IgD Fc sequence depicted in FIG. 2B (SEQ ID NO:2). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to the IgD Fc sequence depicted in FIG. 2B (SEQ ID NO:2). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domains) of the IgD Fc sequence depicted in FIG. 2B (SEQ ID NO:2). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., 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 contiguous aas, including, e.g., at least 150, at least 175, at least 200, or at least 220 contiguous aas, or all aas, of the IgE Fc sequence depicted in FIG. 2C (SEQ ID NO:3). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to the IgE Fc sequence depicted in FIG. 2B (SEQ ID NO:3). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the IgE Fc sequence depicted in FIG. 2C (SEQ ID NO:3).

[0112] A MAPP may comprise one or more IgG Fc sequences as dimerization and / or multimerization sequences. The Fc polypeptide of a MAPP can be human lgG1 Fc, human lgG2 Fc, human lgG3 Fc, human lgG4 Fc, etc. In some cases, the Fc sequence has at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to an aa sequence of an Fc region depicted in any of FIGs. 2D-2G. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domains) of the Ig sequences depicted in any of FIGs. 2D-2G (SEQ ID NOs:4-12). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to an Ig sequence depicted in any of FIGs. 2D-2G (SEQ ID NOs:4-12). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to an Ig sequence depicted in any of FIGs. 2D-2G (SEQ ID NOs:4-12). Framework and / or dimerization polypeptides of aMAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to at least 125 contiguous aas (e.g., at least 150, at least 175, at least 200, or at least 220 contiguous aas), or all aas, of the wt. lgG1 Fc polypeptide sequence depicted in FIG. 2D (SEQ ID NO:4). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG1 Fc sequence depicted in FIG. 2D (SEQ ID NO:4). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO:4. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO:4. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., 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 (e.g., at least 150, at least 175, at least 200, or at least 225) contiguous aas, or all aas, of the lgG2 Fc polypeptide sequence depicted in FIG. 2E (SEQ ID NO:9). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG2 Fc sequence depicted in FIG. 2E (SEQ ID NO:9). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO:9. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO:9. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., 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 (e.g., at least 150, at least 175, at least 200, at least 225, or at least 240) contiguous aas, or all aas, of the lgG3 Fc polypeptide sequence depicted in FIG. 2F (SEQ ID NO: 10). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG3 Fc sequence depicted in FIG. 2F (SEQ ID NQ:10). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO: 10. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NQ:10. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., 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 (e.g., at least 150, at least 175, at least 200, or at least 220) contiguous aas, or all aas, of the lgG4 Fc polypeptide sequence depicted in FIG. 2G (SEQ ID NOs:11 or 12). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG4 Fc sequence depicted in FIG. 2G (SEQ ID NON 1). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO:11 . Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO: 11 .

[0113] Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or100%) aa sequence identity to 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 (CH2, CH3, CH4) polypeptide sequence depicted in FIG. 2H (SEQ ID NO: 13). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO: 13. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO:13.

[0114] Framework and / or dimerization polypeptides of a MAPP comprising immunoglobulin sequences (e.g., depicted in FIGs. 2A-2H) can be covalently linked together by formation of at least one or at least two interchain disulfide bonds between cysteines that are adjacent to the immunoglobulin hinge regions. Such disulfide bonds can stabilize the interaction of framework and dimerization polypeptide heterodimers or, for example, duplexes of such heterodimers when the disulfide bonds are between framework multimerization sequences.

[0115] A framework or dimerization polypeptide may comprise an aa sequence having 100% aa sequence identity to the wt. human lgG1 Fc polypeptide depicted in FIG. 2D. A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to the wt. human lgG1 Fc polypeptide depicted in FIG. 2D that includes a substitution of N297 (N77 as numbered in FIG. 2D, SEQ ID NO:7) with an aa other than asparagine. In one case, N297 is substituted by alanine (N297A). Substitutions at N297 lead to the removal of carbohydrate modifications and result in antibody sequences with reduced complement component 1q (“C1 q") binding compared to the wt. protein, and accordingly a reduction in CDC. K322 substitutions (e.g., K322A) show a substantial reduction in FcyR binding affinity and ADCC, with the C1q binding and CDC functions substantially or completely eliminated. Hezareh et al., (2001) J. Virol. 75:12161-168. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG1 Fc sequence depicted in FIG. 2D (SEQ ID NO:7). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO:7. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO:7.

[0116] Amino acid L234 and other aas in the lower hinge region (e.g., aas 234 to 239, such as L235, G236, G237, P238, S239), which correspond to aas 14-19 of SEQ ID NO:4) of IgG are involved in binding to the Fc gamma receptor (FcyR), and accordingly, mutations at that location reduce binding to the receptor (relative to the wt. protein) and result in a reduction in ADCC). Hezareh et al. (2001), have demonstrated that the double mutant (L234A, L235A) does not effectively bind either FcyR or C 1 q, and both ADCC and CDC functions were substantially or completely abolished. A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence having at least about 85% or at least about 90% (e.g., 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 contiguous aas (e.g., at least 150, at least 175, at least 200, or at least 210 contiguous aas), or all aas, of the wt. human lgG1 Fc polypeptide depicted in FIG. 2D that includes a substitution of L234 (L14 of the aa sequence depicted in FIG. 2D) with an aa other than leucine. Such a framework and / or dimerization polypeptide may comprise a sequence that has at least about 95% orat least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG1 Fc sequence depicted in FIG. 2D (SEQ ID NO:4).

[0117] Substitutions that can enhance one or more antibody directed effector functions (e.g., ADCC, ADCP, and / or GDC responses) of lgG1 heavy chain sequences (e.g., used in framework or dimerization peptides) relative to their corresponding wt. sequence include substitutions at S239D and / or I332E (e.g., the double substitutions S239D / I332E), and the triple substitutions S239D / I332E / A330L in human lgG1. See, e.g., Lazar et al. (2006) PNAS: 103(11), 4005-4010. Those subsitutions in the wt. human lgG1 Fc sequence (SEQ ID NO:4) correspond to S19D, I112E, and A110L. In an embodiment, the substitutions are the double and triple substitutions S239D / I332E or S239D / I332E / A330L. In an embodiment, the lgG1 Fc substitutions may be the triple substitutions S239D / I332E / A330L, which permit enhancement of ADCC without substantial alteration of CDC function (see Lazar et al., 2006).

[0118] Additional substitutions that may enhance at least one antibody related effector function of an IgGI Fc sequence include, but are not limited to: S298A / E333A / K334A; S239D / A330L / I332E; S239D / I332E;G236A / S239D / A330L / I332E; G236A; S239D / I332E / G236A; L234Y / G236W / S298A; F243L / R292P / Y300L / V305I / P396L; K326W / E333S; K326A / E333S; K326M / E333S; C221D / D222C; S267E / H268F / S324T; H268F / S324T; and E345R. See Saunders (2019) Front. Immunol. 10: 1296.doi: 10.3389 / fimmu.2019.01296. The corresponding locations in the lgG1 Fc sequence of SEQ ID NO:4 can be obtained by subtracting 220 from the indicated positions. In an embodiment at least ADCC or ADCP is enhanced by the substitutions relative to the wt. unsubsituted lgG1 aa sequence. In an embodiment at least CDC or complement fixation is enhanced by the substitutions relative to the wt. unsubsituted lgG1 aa sequence. Substitutions that result in enhanced ADCC, ADCP, and / or CDC may be combined with substitutions that lead to interspecific pairing between IgG Fc sequences including KiH and KiHs-s substitutions.

[0119] A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to the wt. human lgG1 Fc polypeptide depicted in FIG. 2D that includes a substitution of L235 (L15 of the aa sequence depicted in FIG. 2D) with an aa other than leucine. In some cases, the framework and / or dimerization polypeptide present in a MAPP with substitutions in the lower hinge region includes L234A and L235A ("LALA”) substitutions (the positions corresponding to positions 14 and 15 of the wt. aa sequence depicted in FIG. 2D; see, e.g., SEQ ID NO:8). Such a framework and / or dimerization polypeptide may comprise a sequence that has at least about 95% or at least about 98% aa sequence identity to at least about 200 contiguous aas (e.g., the CH2 and CH3 domain) of the lgG1 Fc sequence depicted in FIG. 2D (SEQ ID NO:8). Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 85% or at least about 90% aa sequence identity to SEQ ID NO:8. Framework and / or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 90% or at least about 95% aa sequence identity to SEQ ID NO:8.

[0120] A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to the wt. humanlgG1 Fc polypeptide depicted in FIG. 2D that includes a substitution of P331 (P111 of the aa sequence depicted in FIG. 2D) with an aa other than proline. Substitutions at P331, like those at N297, lead to reduced binding to C1q relative to the wt. protein, and thus a reduction in GDC. In one embodiment, substitutions of D270, K322, and / or P329 (corresponding to D50, K102, and P109 of SEQ ID NO:4 in FIG. 2D), for example with alanine, may be utilized individually or in any combination with or without a P331 substitution to reduce binding to C1q. In another embodiment, the substitution may comprise, for example, a P331S or a P331A substitution.

[0121] A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to the wt. human lgG1 Fc polypeptide depicted in FIG. 2D, and include substitutions of D270, K322, and / or P329 (corresponding to D50, K102, and P109 of SEQ ID NO:4 in FIG. 2D) that reduce binding to C1q protein relative to the wt. protein. The substitutions may comprise, for example, a P331S or a P331A substitution.

[0122] A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 85% or at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) aa sequence identity to the wt. human lgG1 Fc polypeptide depicted in FIG. 2D, including substitutions at L234 and / or L235 (L14 and / or L15 of the aa sequence depicted in FIG. 2D) with aas other than leucine such as L234A and L235A, and a substitution of P331 (P111 of the aa sequence depicted in FIG. 2D) with an aa other than proline such as P331S. In one instance, a framework or dimerization polypeptide present in a MAPP comprises the "Triple Mutant” aa sequence (SEQ ID NO:6) depicted in FIG. 2D (human lgG1 Fc) having L234F, L235E, and P331S substitutions (corresponding to aa positions 14, 15, and 111 of the aa sequence depicted in FIG. 2D).

[0123] Where an asymmetric pairing between two polypeptides of a MAPP is desired, a framework or dimerization polypeptide present in a MAPP may comprise, consist essentially of, or consist of an interspecific binding sequence. Interspecific binding sequences favor formation of heterodimers with their cognate polypeptide sequence (i.e., the interspecific sequence and its counterpart interspecific sequence), particularly those based on immunoglobulin Fc (Ig Fc) sequence variants. Such interspecific polypeptide sequences include KiH, KiHs-s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. One interspecific binding pair comprises a T366Y and Y407T mutant pair in the CH3 domain interface of lgG1, or the corresponding residues of other immunoglobulins. See Ridgway et al., Protein Engineering, 9:7, 617-621 (1996). A second interspecific binding pair involves the formation of a knob by a T366W substitution, and a hole 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 interspecific binding pair has a first Ig Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide with S354C and T366W substitutions (disulfide bonds can form between the Y349C and the S354C). See, e.g., Brinkmann and Konthermann, mAbs, 9:2, 182-212 (2015). Ig Fc polypeptide sequences, either with or without knob-in-hole modifications, can be stabilized by the formation of disulfide bonds between the Ig Fc polypeptides (e.g., the hinge region disulfide bonds). Several interspecific binding sequences based upon immunoglobulin sequences are summarized in Table 1 which follows, with cross reference to the numbering of the aa positions as they appear in the wt. lgG1 sequence (SEQ ID NO:4) set forth in FIG. 2D shown in bracketsTable 1 : Pairs of interspecific immunoglobulin sequences and their cognate counterpart interspecific sequencesTable 1 modified from Ha et al., Frontiers in Immunol., 7:1-16 (2016).Asterisks appearing in Table 1 indicate the aas form a stabilizing disulfide bond.

[0124] In addition to the interspecific pairs of sequences in Table 1, framework and / or dimerization polypeptides may include interspecific "SEED” sequences having 45 residues derived from IgA in an lgG1 CH3 domain of the interspecific sequence, and 57 residues derived from lgG1 in the IgA CH3 in its counterpart interspecific sequence. See Ha et al., Frontiers in Immunol. 7:1-16 (2016).

[0125] A framework or dimerization polypeptide found in a MAPP may comprise an interspecific binding sequence or its counterpart interspecific 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.

[0126] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 KiH or KiHs-s sequence with a T146W sequence substitution and its counterpart interspecific KiH or KiHs-s binding partner polypeptide comprises an lgG1 sequence having T146S, L148A, and Y187V sequence substitutions. Where a KIH or KIHs-s sequence is present, the framework and / or dimerization polypeptides may comprise a sequence having atleast 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. One or both of the framework or dimerization peptides, or both of the dimerization polypeptides optionally may comprise substitutions at one of more of: 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). Those 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., L131 K), T146 (e.g., T146S), P175 (e.g., P175V), F185 (e.g., F185R), Y187 (e.g., Y187A), and K189 (e.g., K189Y) in the wt. lgG1 sequence of FIG. 2D.

[0127] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with a T146W KiH sequence substitution, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T146S, L148A, and Y187V KiH sequence substitutions. Where a KIH or KIHs-s sequence is present, the framework and / or dimerization polypeptides may comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0128] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with T146 and S134C KiHs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T146S, L148A, Y187V and Y129C KiHs-s substitutions. Where a KIHs-s sequence is present, the framework and / or dimerization polypeptides may comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0129] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with S144H and F185A HA-TF substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having Y129T and T174F HA-TF substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptide sequences may comprise additional substitutionssuch as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G).

[0130] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, T146L, K172L, and T174W ZW1 substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0131] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with K140D, D179M, and Y187A 7.8.60 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, E125R, Q127R, T146V, and K189V 7.8.60 substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0132] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with K189D and K172D DD-KK substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, D179K and E136K substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0133] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with K140E and K189W EW-RVT substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, Q127R, D179V, and F185T EW-RVT substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aasequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0134] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with K140E, K189W, and Y129C EW-RVTs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0135] A MAPP may comprise a framework or dimerization polypeptide comprising an lgG1 sequence with K150E and K189W A107 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 sequence having T130V, E137N, D179V, and F185T A107 substitutions, with the framework and / or dimerization polypeptides comprising a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 150 (e.g., at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. The framework and / or dimerization polypeptides may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 (e.g., at least 220) or all 227 contiguous aas of the wt. lgG1 of FIG. 2D. 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 an N77 substitution (N297, e.g., N297A or N297G).

[0136] 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 be paired with Ig CH1 sequences (see FIG. 2I) as multimerization or dimerization sequences and their counterpart sequences of a framework polypeptide.

[0137] A MAPP framework or dimerization polypeptide may comprise an Ig CH1 domain (e.g., the polypeptide of FIG. 2I), and the sequence with which it will form a complex (its counterpart binding partner) comprises an Ig K chain constant region sequence, with the framework and / or dimerization polypeptide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to at least 80, at least 90, at least 100, or all contiguous aas of SEQ ID NOs:14 and / or 16. A MAPP framework or dimerization polypeptide may comprise an Ig CH1 domain and the sequence with which it will form a complex (its counterpart binding partner) comprises an Ig K chain constant region sequence, with the framework and / or dimerization polypeptide comprising a sequence having at least 95%, or at least 98% sequence identity to at least 90, or at least 100 contiguous aas of SEQ ID NOs:14 and / or 16. See FIG. 2I and FIG. 3 at A. The framework or dimerization polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 90 or at least100 contiguous aas of SEQ ID NOs:14 and / or 16. The Ig CH1 and Ig K sequences may be modified to increase their affinity for each other and, accordingly, the stability of any heterodimer formed utilizing them as dimerization or multimerization sequences. Among the substitutions that increase the stability of CH 1 -Ig K heterodimers are 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 IgCH 1 and Ig K sequences. The Ig CH1 sequence in MD13 is modified to contain S64E and S66V substitutions (S70E and S72V of the sequence shown in FIG. 2I). The Ig K sequence in MD13 is modified to contain S69L and T71S substitutions (S68L and T70S of the sequence shown in FIG. 3A).

[0138] A framework or dimerization polypeptide of a MAPP may comprise an Ig CH1 domain (e.g., the polypeptide of FIG. 2I, SEQ ID NO: 14), and its counterpart sequence comprises an Ig A chain constant region sequence such as is shown in FIG. 3 at B (SEQ ID NO: 17), where the framework or dimerization polypeptide 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, or at least 100) contiguous aas of the sequence shown in FIG. 2I. Alterntively, the framework or dimerization polypeptide comprises a sequence having at least 95% or 98% sequence identity to at least 100 or at least 110) contiguous aas of SEQ ID NO:14. The framework or dimerization polypeptide comprises a sequence having at least 90% or 95% sequence identity to SEQ ID NO:14 and its counterpart at least 90% or 95% sequence identity to SEQ ID NO:17.

[0139] Framework and / or dimerization polypeptides of a MAPP may each comprise a leucine zipper polypeptide as a dimerization or multimerization sequence. The leucine zipper polypeptides bind to one another to form dimers (e.g., homodimers). Non-limiting examples of leucine-zipper polypeptides include a peptide comprising any one of the following aa sequences: RMKQIEDKIEEILSKIYHIENEIARIKKLI GER (SEQ ID NO:159); LSSIEKKQEEQTSWLIWIS NELTLIRNELAQS (SEQ ID NQ:160); LSSIEKKLE EITSQLIQISNELTLIRNELAQ (SEQ ID NO:161); LSSIEKKLEEIT SQLIQIRNELTLIRNELAQ (SEQ ID NO:162); LSSIEKKLEEITSQLQQIRNELTLIRNELAQ (SEQ ID NO:163); LSSLEKKLEELTSQLIQLRN ELTLLRNELAQ (SEQ ID NO:164); ISSLEKKIEELTSQIQQLRN EITLLRNEIAQ (SEQ ID NO:165). In some cases, a leucine zipper polypeptide comprises the following aa sequence: LEIEAAFLERENTALET RVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:166). Additional leucine-zipper polypeptides are known in the art, a number of which are suitable for use as multimerization or dimerization sequences.

[0140] The framework and / or dimerization polypeptides of a MAPP may comprise a coiled-coil polypeptide that forms a dimer. Non-limiting examples of coiled-coil polypeptides include, for example, a peptide of any one of the following aa sequences: LKSVENRLAWENQLKTVIEELKTVKDLLSN (SEQ ID NO:167); LARIEEKLKTIKAQLSEIAS TLNMIREQLAQ (SEQ ID NO:168); VSRLEEKVKTLKSQVTELASTVSLLREQVAQ (SEQ ID NO:169); IQSEKKIEDIS SLIGQIQSEITLIRNEIAQ (SEQ ID NQ:170); and LMSLEKKLEELTQTLMQLQNELSMLKNELAQ (SEQ ID NO:171).

[0141] A MAPP may comprise a pair of two framework polypeptides and / or a framework and a dimerization polypeptide that each have an aa sequence comprising at least one cysteine residue that can form a disulfide bond permitting homodimerization or heterodimerization of those polypeptides stabilized by disulfide bonds between the cysteine residues. Examples of such aa sequences include: VDLEGSTSNGRQCAGIRL (SEQ ID NO:172); EDDVTTTEELAPALVPPPKGTCAGWMA (SEQ ID NO:173); and GHDQETTTQGPGVLLPLPKGACTGQMA (SEQ ID NO:174).

[0142] Some aa sequences suitable as multimerization (oligomerization) sequences permit formation of MAPPs capable of forming structures greater than duplexes of heterodimers comprising a framework polypeptide and a dimerization polypeptide. In some instances, triplexes, tetraplexes, and / or pentaplexes may be formed. Such aa sequences include, but are not limited to, IgM constant regions (see, e.g., FIG. 2H) which form hexamers or pentamers (particularly when combined with a mature j-chain peptide lacking a signal sequence such as that provided in FIG. 2J, SEQ ID NO:15). Collagen domains, which form trimers, can also be employed. Collagen domains may comprise the three aa sequence Gly-Xaa-Xaa and / or GlyXaaYaa, where Xaa and Yaa are independently any aa, with the sequence appearing or being repeated multiple times (e.g., from 10 to 40 times, such as 10-20, 20-30, or 30-40 times). In such sequences, Xaa and Yaa are frequently proline and hydroxyproline, respectively, in greater than 25%, 50%, 75%, 80%, 90% or 95% of the Gly-Xaa-Yaa occurrences, or in each of the Gly-Xaa-Yaa occurrences. In some cases, a collagen domain comprises the sequence Gly-Xaa-Pro repeated from 10 to 40 times, such as 10-20, 20-30, or 30-40 times. A collagen oligomerization peptide can comprise the following aa sequence: VTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPADSPPPPALSSNP (SEQ ID NO: 175).

[0143] Suitable framework polypeptides (e.g., those with an Ig Fc multimerization sequence) will, in some cases, be half-life extending polypeptides. Thus, in some cases, a suitable framework polypeptide increases the in vivo half-life (e.g., the serum half-life) of the MAPPs, compared to a control MAPP having a framework polypeptide with a different aa sequence. For example, in some cases, a framework polypeptide increases the in vivo half-life (e.g., the serum half-life in a mammal such as a human) of the 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 duplex of MAPP heterodimers comprising a framework polypeptide and a dimerization polypeptide) increases the stability and / or in vivo half-life (e.g., the serum half-life) of a MAPP duplex compared to a control MAPP duplex lacking the Ig Fc polypeptide sequence 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.3. Presenting Sequence

[0144] As discussed in more detail below, class II MHC polypeptides include two types of polypeptide chains, i.e., a-chains and p-chains. More specifically, MHC class II o-chain polypeptides include a1 and o2 domains, and p-chain polypeptides include p1 and p2 domains. Presenting sequences comprise MHC class II polypeptides sufficient to bind and present an epitope to a TCR. Presenting sequences 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 placed between various domains). As discussed herein, unless stated otherwise, presenting sequences do not comprise an MHC transmembrane domain (or intracellular domain such as a cytoplasmic tail) sufficient to anchor MAPP molecules (e.g., more than 50% of the MAPP molecules) in a mammalian cell membrane (e.g., a CHO cell membrane) when expressed therein.

[0145] Conceptually, each of the presenting sequences may be considered a "soluble MHC” that is fully capable of binding a peptide epitope and presenting the peptide epitope to a TCR. Unless stated otherwise, in presenting sequences all of the MHC a1 , a2, p1, and p2 domain sequences, as well as the epitope polypeptide, are present in a single polypeptide chain (single linear sequence of aas produced by translation). See, e.g., FIG. 23.

[0146] In some cases, a MAPP comprises one or more presenting sequence each having all of the Class II components required for binding and presenting the epitope of interest to a TCR, e.g., the a1 , a2, p1, and p2 domains and epitopes in a single polypeptide sequence.

[0147] As noted above, presenting sequences of the present disclosure comprise a peptide epitope that is part of a polypeptide chain. It is possible, however, to make constructs that comprise the MHC components, but which do not comprise a peptide epitope that is part of a polypeptide chain. In such embodiments, the epitope, which is non- covalently loaded into the MHC pocket, may be a separate peptide (e.g., phosphopeptide, lipopeptide, glycosylated peptide, etc.) or non-peptide epitope, and may be subject to dissociation from the MAPPs.4. MHC Class II Polypeptides

[0148] The MAPPs as described herein comprise MHC Class II sequences from any of a number of various species, including human MHC polypeptides (HLA polypeptides), rodent (e.g., mouse, rat, etc.) MHC polypeptides, MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., equines, bovines, ovines, caprines, etc.)), and the like. Typically, the MAPPs described herein comprise human MHC Class II sequences. The MAPPs as described herein may comprise human MHC Class II polypeptide sequences. The MAPPs as described herein may comprise mouse MHC Class II polypeptide sequences.

[0149] As used herein, the term "Class II MHC polypeptide” refers to a Class II MHC a subunit (chain) polypeptide, a Class II MHC p subunit (chain) polypeptide, or only a portion of a Class II MHC a and / or p chain polypeptide, or combinations of the foregoing. For example, the term "Class II MHC polypeptide” as used herein can refer to a polypeptide that includes: i) only the a1 domain of a Class II MHC a chain; ii) only the o2 domain of a Class II MHC a chain; iii) only the a1 domain and the o2 domain of a Class II MHC a chain; iv) only the p1 domain of a Class II MHC p chain; v) only the p2 domain of a Class II MHC p chain; vi) only the p1 domain and the p2 domain of a Class II MHC p chain; vii) the a1 domain of a Class II MHC a chain, the p1 domain of a Class II MHC p chain, and the p2 domain of a Class II MHC; and the like. MAPPs typically include the a1 and o2 domains of Class II MHC polypeptide a chains, and the p1 and p2 domains of Class II MHC polypeptide p chains, which represent all or most of the extracellular class II protein required for presentation of an epitope. The a1 and o2 domain sequences may be followed by an a chain membrane proximal region. Similarly, the p1 and p2 domain sequences may be followed by a p chain membrane proximal region. Both the a and p Class II MHC polypeptide sequences may be of human origin.

[0150] As discussed above, where the MAPPs and their higher order complexes (e.g., duplex MAPPs) are intended to be soluble in aqueous media under physiological conditions (e.g., soluble in human blood plasma at therapeutic levels), they are not intended to include membrane anchoring domains (such as transmembrane regions of MHC Class II a or p chains) or a part thereof sufficient to anchor the MAPP molecules (e.g., more than 50% of the MAPP molecules) in the membrane of a cell (e.g., a eukaryotic cell such as a mammalian cell such as a Chinese Hamster Ovary or "CHO” cell) in which the MAPP is expressed. Similarly, unless expressly stated otherwise, the MAPPsdescribed herein are mature proteins that do not include the leader and / or intracellular portions (e.g., cytoplasmic tails) that may be present in some MHC Class II proteins.

[0151] For the purpose of this disclosure, otherwise soluble MAPPs may be converted into membrane proteins by the addition of Membrane Association Sequences (MAS). MAS sequences may permit post translational modifications, such as lipid addition, that lead to association of the MAPPs with lipid bilayers. For example, aa sequences that result in direct or indirect covalent attachment to a lipid or prenyl group or glycosylphosphatidylinositol may be added to MAPPs. In an embodiment, a farnesyltransferase or geranylgeranyl transferase motif may be located at the COOH-terminus of proteins. The MAS may be in the form of a single transmembrane domain sequence, multiple transmembrane domain sequences that cross a cell membrane more than one time, or an amphipathic a helix that partitions into a monolayer of a lipid bilayer (a monotopic membrane interaction). For example, a MAS can be added to the C-terminus of one or more framework polypeptides present in a MAPP or higher order MAPP complex (e.g., at the 3 or 3' positions). MAS sequences are discussed under Additional Polypeptide Sequences.

[0152] Class II MHC aa sequences that may appear in MAPPs include aa sequences from MHC Class II DP a (DPA) and p (DPB) subunits, DQ a (DQA) and p (DQB) subunits, and DR a (DRA) and p (DRB) subunits. The human MHC or HLA locus is highly polymorphic in nature and thus, as used herein the term "Class II MHC polypeptide” includes allelic forms of any known Class II MHC polypeptide. See, e.g., the HLA Nomenclature site run 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 alleles of DRA, DRB1, DRB3, DRB4, DRB5, DRB6, DRB7, DRB9, DQA1, DQB1, DPA1, and DPB1.

[0153] Unless stated otherwise a MAPP may comprise Class II MHC a and p chain sequences, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails). Thus, a MAPP may comprise the o1, o2, p1, and p2 domains, and optionally the membrane proximal portions of Class II MHC a and p chains, but does not, unless stated otherwise, include any one or more of the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a Class II MHC a chain. The Class II MHC a chain sequences of a MAPP, and particularly the o1 and p1 domains, may include a variety of advantageous aa substitutions. A linker sequence denoted Lo may be interposed between the o1 and o2 domains (see, e.g., FIG. 1 and FIG. 23). Similarly, a linker sequence denoted "Lp” may be interposed between the p1 and p2 domains (see, e.g., FIG. 1 and FIG. 23).

[0154] When addressing corresponding substitutions in, for example, different o1 or p1 domains of MHC sequences (e.g., different alleles), corresponding aas and aa positions in the sequences are determined by aligning the sequences. For MHC a subunit alignments the combined o1 and o2 domain sequences are aligned for the MHC o subunit comparisons. For MHC p subunit alignments the combined p1 and p2 domain sequences are aligned for MHC p subunit comparisons. Unless stated otherwise, sequence comparisons for determining corresponding substitutions are conducted using Clustal Omega Version 1.2.2, which is available on the World Wide Web at www. ebi . ac. uk / T ools / msa / clustalo / . a) MHC Class II alpha chains

[0155] MHC Class II alpha subunits (chains) comprise an o1 domain and an o2 domain. In some cases, the o1 and o2 domain sequences present in an antigen-presenting cell (APC) are from the same MHC Class II a chainpolypeptide (the sequence of the same allele). In some cases, the a1 and o2 domain sequences present in an APC are from two different MHC Class II a chain polypeptides (alleles). FIGs. 4, 9, 10, 13, and 15 present DR, DP and DQ alpha chain a1 and o2 domain sequences along with their membrane proximal sequences, transmembrane domain sequences and intracellular domain sequences, but lacking their signal / leader sequence. Unless stated otherwise, the MHC Class II a chain sequences are numbered starting with the first aa of the a1 domain, i.e., the first aa following the signal sequence. In some instances, from 1 to 3 aas may be removed from the N-terminus of an MHC (e.g., HLA) Class II a1 domain as it appears in a MAPP. In such instances the numbering of the remaining aas of the a chain sequences does not change and can be determined by alignment with the corresponding unmodified MHC allele.

[0156] An MHC Class II alpha chain sequence comprising the a1 and o2 domain sequences suitable for inclusion in a MAPP may have a length of from about 165 aas to about 210 aas (including any La linkers interposed between the a1 and a2 domains but excluding membrane proximal sequences). For example, an MHC Class II alpha chain suitable for inclusion in a MAPP may have a length of from about 170 to about 190 aas or from about 175 to about 185 aas in length. An MHC Class II a1 domain suitable for inclusion in a MAPP may have a length of from about 75 aas to about 95 aas; for example, an MHC Class II a1 domain suitable for inclusion in a MAPP may have a length of from about 80 aas to about 90 aas, or from about 83 aas to about 88 aas. An MHC Class II a2 domain suitable for inclusion in a MAPP may have a length of from about 85 aas to about 105 aas; for example, an MHC Class II a2 domain suitable for inclusion in a MAPP may have a length of from about 90 aas to about 100 aas, or from about 92 aas to about 98 aas.

[0157] Where a La linker is present in a MAPP, only the aa sequences of the a1 and a2 domains are used when determining percent sequence identity. Accordingly, the percent sequence identity between the a1 domain sequence or the a2 domain sequence present in the MAPP and the a1 domain sequence or the a2 domain sequence present in a specific allele may be assessed over a span of contiguous a1 or a2 domain sequence aas in the MAPP that do not include the La linker aa sequence. Likewise, the collective percent sequence identity between the a1 and a2 domain sequences present in the MAPP and the a1 and a2 domain sequences present in a specific allele is determined without reference to the La linker aa sequence.

[0158] An MHC class II a chain polypeptide suitable for inclusion in a MAPP may comprise a substitution of an aa in the last 11 aas, including e.g., the last 10 aas, of the MHC a subunit a1 domain sequence for forming a linker disulfide or body disulfide bond for stabilizing the MAPP.

[0159] Some cysteine residues in MAPPs (e.g., MHC sequences of MAPPs) that are not part of a disulfide bond stabilizing a MAPP structure (unpaired cysteines), such as C47 of some DQA a1 domain sequences (e.g., DQA*05:01), can be linked to difficulties in expression. The presence of such unpaired cysteines may result in poor expression levels or the production of misfolded protein products. Substitution of such cysteines with an aa other than a cysteine (e.g., an aa other than cysteine or proline, such as serine, arginine, or lysine) can lead to higher levels of protein expression in the native state (e.g., non-denatured or non-aggregated) relative to the levels observed with the unpaired cysteine-containing molecule. Accordingly, unpaired cysteines appearing at, for example, aas 43 through 48 of the a chain polypeptide sequence (a1 and a2 domain sequence) may be substituted by an aa other than cysteine, or by an aa other than cysteine or proline. The cysteine substitutions may be made with serine,arginine, or lysine, or with serine, which is the aa closest to cysteine in size and other characteristics, but lacking the nucleophilicity of the cysteine thiol group.(1) DRA Polypeptides(a) DRA1 Polypeptides

[0160] A suitable MHC Class II DR a subunit (DRA) polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 150, at least 160, at least 165, or at least 170 contiguous aas of the a1 and o2 domain regions of a DRA aa sequence depicted in FIG. 4 or a naturally occurring allelic variant thereof. In some cases, the DRA polypeptide has a length of about 178 aas, including, e.g., 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 aas. A suitable MHC Class II DR a subunit polypeptide may have at least 90% or at least 95% aa sequence identity with the a1 and o2 domain regions of a DRA aa sequence depicted in FIG. 4 or a naturally occurring allelic variant thereof.

[0161] 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 1-178 of DRA*01:02 (see FIG. 4) or an allelic variant thereof. In some cases, the allelic variant is the DRA*01 :01 polypeptide (e.g., from the DRA*01 :01 :01 :01 allele) that differs from DRA*01:02 by having a valine in place of the leucine at position 217 (see FIG. 4).

[0162] A suitable DRA aa sequence for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 160, at least 165, at least 170, or at least 175 contiguous aas of the a1 and o2 domain sequences of DRA*01:02 depicted in FIG. 4. A suitable DRA aa sequence for inclusion in a MAPP may have at least 90% or 100% aa sequence identity to at least 165 contiguous aas of the DRA o1 and o2 domain sequences of DRA1*01:01 or DRA*01:02. A suitable DRA aa sequence for inclusion in a MAPP may have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of the DRA a1 and o2 domain sequences of DRA1*01:01 or DRA*01:02.

[0163] Thus, a suitable DRA polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to at least 165 contiguous aas of the DRA*01:02 a1 and o2 domain sequences: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITNV PPEVTVLTNS PVELREPNVL ICFIDKFTPP WNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:107, aas 1-178, see FIG. 4) or an allelic variant thereof. In some cases, a DRA polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRA polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond that stabilizes the MAPP). A suitable DRA aa sequence have at least 90% or at least 95% aa sequence identity to SEQ ID NO: 107. A suitable DRA aa sequence have at least 98% or at least 99% aa sequence identity to SEQ ID NO: 107.

[0164] A MAPP may comprise a variant DRA polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DRA polypeptide comprising a Cys substituted for an aa at any of positions 74-76 for formation of a linker disulfide bond (e.g., a Cys substitution selected from T74C, K75C, or R76C (see, e.g., FIG. 4). A MAPP may comprise a variant DRA polypeptide comprising a Cys substituted for an aa at any of positions 80-82 for formation of a body disulfide bond (e.g., a Cys substitution selected from T80C, P81C, or I82C (see, e.g., FIG. 4).

[0165] Separate from, or in addition to, substitutions introducing Cys residues for the formation of body or linker disulfide bonds, MAPPs containing DRA polypeptide sequences may comprise substitutions in the a1 domain sequence at one or more of positions 37, 44, 49, or 72 of HLA DRA*01 :02, or the corresponding positions in other DRA alleles based upon sequence alignment. Position 37 of the a1 domain may be substituted by an acidic residue such as E or D (e.g., an A37E substitution in DRA*01:02), position 49 may be substituted by an H (e.g., a G49H substitution in DRA*01:02), position 72, which is an I in the wt. sequence, may remain an I or may be substituted by another aliphatic aa such as L or V, and position 44 (I) is an aa other than cysteine, or (ii) when aa position 44 is an arginine it may be substituted by a serine or lysine (e.g., an R44S or R44K substitution in DRA*01:02).

[0166] A suitable DRA a1 domain sequence for inclusion in a MAPP may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to the aa sequence: IKEEVIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO: 176), and optionally having a length of about 84 aas, including, e.g., 80, 81, 82, 83, 84, 85, or 86 aas. A suitable DRA a1 domain sequence may also have at least 90% or at least 95% aa sequence identity to SEQ ID NO:176.

[0167] A suitable DRA o2 domain sequence for inclusion in a MAPP may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to the aa sequence: V PPEVTVLTNS PVELREPNVL ICFIDKFTPP WNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO: 177), and optionally having a length of about 94 aas, including, e.g., 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRA o2 domain sequence may also have at least 90% or at least 95% aa sequence identity to SEQ ID NO: 177.(2) DQA Polypeptides(a) DQA1 Polypeptides

[0168] A suitable MHC Class II DQA1 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 150, at least 160, at least 165, or at least 170 contiguous aas of the a1 and o2 domain regions of a DQA1 aa sequence depicted in FIG. 9 or a naturally occurring allelic variant thereof. In some cases, the DQA1 polypeptide has a length of about 181 aas, including, e.g., 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 aas. A suitable MHC Class II DQ a subunit polypeptide may have at least 90% or at least 95% aa sequence identity with the a1 and o2 domain regions of a DQA1 aa sequence depicted in FIG. 9 or FIG. 15, or naturally occurring allelic variants thereof.

[0169] As used herein, the term "DQA1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA1 polypeptide comprises aas 1-181 of DQA1*01 :01 (see FIG. 9), or an allelic variant thereof. In some cases, the allelic variant is the DQA1*05:01 (see FIG. 9).

[0170] A suitable DQA1 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 160, at least 165, at least 170, or at least 175 contiguous aas of the a1 and o2 domain sequences of the DQA1*01:01 or DQA1*05:01 sequences depicted in FIG. 9. A suitable DQA1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 165 contiguous aas of the DQA1 a1 and o2 domain sequences ofDQA1 *01 :01 or DQA1*05:01 depicted in FIG. 9. A suitable DQA1 aa sequence for inclusion in a MAPP polypeptidemay have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of the DQA1 a1 and o2 domain sequences of DQA1*01:01 or DQA1*05:01 depicted in FIG. 9. Thus, a suitable DQA1 polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to at least 165 contiguous aas of the DQA1*01:01 a1 and o2 domain sequence (see, e.g., FIG. 9, aas 1 through181). Alternatively, a suitable DQA1 polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to at least 165 contiguous aas of the DQA1*05:01 a1 and o2 domain sequences (e.g., FIG. 9). A suitable DQA1 aa sequence may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to the DQA1*01 :01 sequence of SEQ ID NO: 103, or the DQA1*02:01 sequence of SEQ ID NO:104. A suitable DQA1 aa sequence may comprise an aa sequence having at least 98% or 100% aa sequence identity to SEQ ID NOs:103 or 104. A suitable DQA1 aa sequence may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to the DQA1*05:01 sequence of SEQ ID NO: 105. A suitable DQA1 aa sequence may comprise an aa sequence having at least 98% or 100% aa sequence identity to SEQ ID NQ:105.

[0171] A MAPP may comprise a variant DQA1 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DQA1 polypeptide comprising a Cys substitution for formation of a linker disulfide bond at any one of the aas in the sequence IKR (see positions 76-78 or 77-79 depending on the allele depicted in FIG. 9, e.g., a Cys substitution selected from I77C, K78C, or R79C (see, e.g., FIG. 9). A MAPP may comprise a variant DQA1 polypeptide comprising a Cys substitution for forming a body disulfide at any one of the aas in the sequence TAA (see positions 82-84 or 83-85 depending on the allele depicted in FIG. 9 (e.g., a Cys substitution selected from T82C, A83C, or A84C in FIG. 9)).

[0172] Separate from, or in addition to, substitutions introducing Cys residues for the formation of body or linker disulfide bonds, MAPPs containing DQA1 polypeptide sequences may comprise substitutions in the a1 domain sequence at one or more of positions 40, 47, 52, or 75 of HLA DQA1*01:01, or the corresponding positions in other DQA1 alleles based upon sequence alignment. Position 40 of the a1 domain may be substituted by an acidic residue such as E or D (e.g., a G40E substitution in DQA1*05:01), position 52 may be substituted by an H (e.g., an R52H substitution in DQA1*05:01), position 75 may be substituted by an aliphatic aa such as I, L, or V (e.g., an S75I substitution in DQA1*05:01), and position 47 is (i) an aa other than cysteine, or (ii) when aa position 47 is a Cys, it may be substituted by an S or K (e.g., a C47S or C47K substitution in DQA1*05:01).

[0173] A suitable DQA1 a1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-85 of any of the DQA1 alleles provided in FIG 9, and optionally having a length of about 86 aas, including, e.g., 84, 85, 86, 87, 88, or 89 aas. A suitable DQA1 a1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-85 of any of the DQA1 alleles provided in FIG 9, and optionally having a length of about 86 aas, including, e.g., 84, 85, 86, 87, 88, or 89 aas. A suitable DQA1 a1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-85 of DQA1*01:01, and optionally having a length of about 86 aas, including, e.g., 84, 85, 86, 87, 88, or 89 aas. A suitable DQA1 a1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having atleast 90% or at least 95% aa sequence identity to aas 1-85 of DQA1*05:01, and optionally having a length of about 85 aas, including, e.g., 83, 84, 85, 86, 87, or 88 aas.

[0174] A suitable DQA1 o2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 87-180 of any of the DQA1 alleles provided in FIG 9, and optionally having a length of about 93 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas. A suitable DQA1 o2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 87-180 of any of the DQA1 alleles provided in FIG 9, and optionally having a length of about 93 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas. A suitable DQA1 o2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 87-180 of DQA1*01:01, and optionally having a length of about 93 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas. A suitable DQA1 o2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 87-180 of DQA1*05:01, and optionally having a length of about 93 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas.(b) DQA2 Polypeptides

[0175] A suitable MHC Class II DQA2 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 150, at least 160, at least 165, or at least 170 contiguous aas of the a1 and o2 domain regions of a DQA2 aa sequence depicted in FIG. 10 or a naturally occurring allelic variant thereof. In some cases, the DQA2 polypeptide has a length of about 181 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, or 185 aas). As used herein, the term "DQA2 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA2 polypeptide comprises aas 1-181 of DQA2*01:01 (see FIG. 10).

[0176] A suitable DQA2 aa sequence for inclusion in a MAPP polypeptide may have at least 85% or at least 90% aa sequence identity with at least 165, at least 170, or at least 175 contiguous aas of the a1 and o2 domain sequences of the DQA2*01:01 sequence depicted in FIG. 10. A suitable DQA2 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 165 contiguous aas of the DQA2 a1 and o2 domain sequences of DQA2*01 :01 . A suitable DQA2 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of the DQA2 a1 and o2 domain sequences of DQA2*01 :01 .

[0177] Thus, a suitable DQA2 polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to at least 165 contiguous aas of the DQA2*01:01 a1 and o2 domain sequences: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PWNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO: 178, aas 1-181, see FIG. 10), or an allelic variant thereof. In some cases, a DQA2 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DQA2 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond that stabilizes the MAPP). A suitable MHC Class II DQA2 polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to SEQ ID NO:178 or 106. A suitable MHC Class II DQA2polypeptide may comprise an aa sequence having at least 98% or 100%, aa sequence identity to SEQ ID NO:178 or 106.

[0178] A MAPP may comprise a variant DQA2 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DQA2 polypeptide comprising a Cys substituted for an aa at any of positions 77-79 for formation of a linker disulfide bond at any one of the aas in the sequence "MRQ” in FIG. 10 (e.g., a Cys substitution selected from M77C, R78C, or Q79C). A MAPP may comprise a variant DQA2 polypeptide comprising a Cys substituted for an aa in the sequence TAA (see positions 83, 84, and 85 in FIG. 10) for formation of a body disulfide bond (e.g., a Cys substitution selected from T83C, A84C, or A85C).

[0179] Separate from, or in addition to, substitutions introducing Cys residues for the formation of body or linker disulfide bonds, MAPPs containing DQA2 polypeptide sequences may comprise substitutions in the a1 domain sequence at one or more of positions 40, 47, 52, or 75 of HLA DQA2*01:01, or the corresponding positions in other DQA2 alleles based upon sequence alignment. Position 40 of the a1 domain may be substituted by an acidic residue such as E or D (e.g., an A40E substitution in DQA2*01:01), position 52 may be substituted by an H (e.g., an S52H substitution in DQA2*01:01), position 75 may be substituted by an aliphatic aa such as I, L, or V (e.g., an F75I substitution in DQA2*01:01), and position 47 is (I) an aa other than cysteine, or (ii) when aa position 47 is a Cys, it may be substituted by an S or K (e.g., a C47S or C47K substitution in DQA2*01:01).

[0180] A suitable DQA2 a1 domain sequence, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, or at least about 99%) or 100% aa sequence identity to aas 1-86 of HLA DQA2*01:01, and optionally having a length of about 86 aas, including, e.g., 84, 85, 86, 87, 88, or 89 aas. A suitable DQA2 a1 domain sequence may also have at least 90% or at least 95% aa sequence identity to aas 1-86 of HLA DQA2*01 :01 .

[0181] A suitable DQA2 o2 domain sequence, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least about 90% (e.g., at least about 90%, at least about 95%, at least about 98%, or at least about 99%) or 100% aa sequence identity to aas 87-181 of HLA DQA2*01:01, and optionally having a length of about 94 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas). A suitable DQA2 o2 domain sequence may also have at least 90% or at least 95% aa sequence identity to aas 87-181 of HLA DQA2*01 :01 .(3) DPA Polypeptides(a) DPA1 Polypeptides

[0182] A suitable MHC Class II DPA polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 150, at least 160, at least 165, or at least 170 contiguous aas of the a1 and o2 domain regions of a DPA aa sequence depicted in FIG. 13 or a naturally occurring allelic variant thereof. In some cases, the DPA polypeptide has a length of about 181 aas, including, e.g., 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 aas).

[0183] As used herein, the term "DPA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DPA polypeptide comprises aas 1-181 of DPA*01:03 (see FIG. 13), or an allelic variant thereof. In some cases, the allelic variant is DPA*02:01 (see FIG. 13).

[0184] A suitable DPA aa sequence for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 160, at least 165, at least 170, or at least 175 contiguous aas of the a1 and o2 domain sequences of the DPA*01:03 or DPA1*02:01 sequences depicted in FIG. 13. A suitable DPA aa sequence for inclusion in a MAPP may have at least 90% or 100% aa sequence identity to at least 165 contiguous aas of the DPA a1 and o2 domain sequences of DPA1*01 :03 or DPA*02:01 . A suitable DPA aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of the DPA a1 and o2 domain sequences of DPA1*01:03 or DPA*02:01.

[0185] Thus, a suitable DPA polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to at least 165 contiguous aas of the DPA*01:03 a1 and o2 domain sequences: AG AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAI LNNN LNTLIQRSNH TQATNDPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO:108, aas 1-181, see FIG. 13), or an allelic variant thereof. A suitable MHC Class II DPA1 polypeptide may have at least 90% or at least 95% aa sequence identity to SEQ ID NO: 108. A suitable MHC Class II DPA2 polypeptide may have at least 98% or 100%, aa sequence identity to SEQ ID NQ:108.

[0186] A MAPP may comprise a variant DPA polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DPA polypeptide comprising a Cys substituted for an aa at any of positions 77-79 for formation of a linker disulfide bond at any one of the aas in the sequence IQR in FIG. 13, (e.g., a Cys substitution selected from I77C, Q78C, or R79C). A MAPP may comprise a variant DPA polypeptide comprising a Cys substituted for an aa in the sequence TQA (see positions 83-85 in FIG. 13) for formation of a body disulfide bond (e.g., a Cys substitution selected from T83C, Q84C, or A85C).

[0187] Separate from, or in addition to, substitutions introducing Cys residues for the formation of body or linker disulfide bonds, MAPPs containing DPA polypeptide sequences may comprise substitutions in the a1 domain sequence at one or more of positions 40, 47, 52, or 75 of HLA DPA1*01:03, or the corresponding positions in other DPA alleles based upon sequence alignment. Position 40 of the a1 domain may be substituted by an acidic residue such as E or D (e.g., an A37E substitution in DPA*01:03), position 52 may be substituted by an H (e.g., a G49H substitution in DPA*01 :03), position 75, may be substituted by an aliphatic aa such as I, L, or V (e.g., a T75I substitution in DPA*01 :03), and position 47 (i) is an aa other than cysteine, or (ii) when aa position 47 is a His, it may be substituted by a serine or lysine (e.g., an H47S or H47K substitution in DPA*01:03).

[0188] A suitable DPA a1 domain sequence for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to the aa sequence: AGAIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANI AILNNN LNTLIQRSNH TQATN (SEQ ID NO: 179), and optionally having a length of about 87 aas, including, e.g., 84, 85, 86, 87, 88, or 89 aas. A suitable DPA a1 domain sequence may also have at least 90% or at least 95% aa sequence identity to SEQ ID NO:179.

[0189] A suitable DPA o2 domain sequence for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to the aasequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO: 180), and optionally having a length of about 94 aas, including, e.g., 91, 92, 93, 94, 95, 96, or 97 aas. A suitable DPA o2 domain sequence may also have at least 90% or at least 95% aa sequence identity to SEQ ID NO:180. b) MHC Class II beta chains

[0190] MHC Class II beta subunits (chains) comprise a p1 domain and a p2 domain. In some cases, the p1 and p2 domain sequences present in an APC are from the same MHC Class II p chain polypeptide. In some cases, the p1 and p2 domain sequences present in an APC are from two different MHC Class II p chain polypeptides. FIGs. 5-8, 11-12, 14, and 16 present DR, DP, and DQ beta chain p1 and p2 domain sequences along with their membrane proximal sequences, but lacking their signal / leader, transmembrane domain, and intracellular domain sequences. Unless stated otherwise, the MHC Class II p chain sequences are numbered starting with the first aa of the p1 domain, i.e., the first aa following the signal sequence. In some instances, from 1 to 3 aas may be removed from the N-terminus of an MHC (e.g., HLA) Class II p1 domain as it appears in a MAPP. In such instances the numbering of the remaining aas of the p1 domain does not change and can be determined by alignment with the corresponding unmodified MHC allele.

[0191] An MHC Class II beta chain sequence comprising the p1 and p2 domain sequences suitable for inclusion in a MAPP may have a length of from about 165 aas to about 210 aas (including any Lp linkers interposed between the p1 and p2 domains but excluding membrane proximal sequences). For example, an MHC Class II beta chain suitable for inclusion in a MAPP may have a length of from about 170 aas to about 200 aas or from about 180 aas to about 195 aas in length. An MHC Class II p1 domain suitable for inclusion in a MAPP may have a length of from about 85 aas to about 105 aas; for example, an MHC Class II p1 domain suitable for inclusion in a MAPP may have a length of from about 90 aas to about 100 aas, or from about 93 aas to about 98 aas. An MHC Class II p2 domain suitable for inclusion in a MAPP may have a length of from about 80 aas to about 105 aas; for example, an MHC Class II p2 domain suitable for inclusion in a MAPP may have a length of from about 85 aas to about 100 aas, or from about 90 aas to about 98 aas.

[0192] Where an Lp linker is present in a MAPP, only the p1 and p2 domain sequences are used when determining percent sequence identity. Accordingly, the percent sequence identity between the p1 domain sequence or the p2 domain sequence present in the MAPP and the p1 domain sequence or the p2 domain sequence present in a specific allele may be assessed over a span of contiguous p1 or p2 domain sequence aas in the MAPP that do not include the Lp linker aa sequence. Likewise, the collective percent sequence identity between the p1 and p2 domain sequences present in the MAPP and the p1 and p2 domain sequences present in a specific allele is determined without reference to the Lo linker aa sequence.

[0193] An MHC Class II p chain polypeptide suitable for inclusion in a MAPP may comprise an aa substitution for forming a body disulfide bond, where the aa substitution replaces any one of aas 1-8 (e.g., aas 4-8 as shown in FIGs. 5-8, 11-12, 14, and 16) of the p1 domain with a Cys. For example, in some cases, the MHC class II p chain polypeptide is a variant DRB1 MAPP that comprises a P5C or F7C substitution.(1) DRB Polypeptides

[0194] MHC Class II DRB polypeptides for inclusion in a MAPP have both p1 and p2 domains. Some non-limiting examples of DRB1, DRB3, and DRB4 polypeptides are provided in FIGs. 5-8. The p1 and p2 domains of the DRB proteins shown in those figures are typically about 188 aas in length, with aas 1-95 making up the p1 domain and aas 96-188 making up the p2 domain. Aas 189-198 make up the membrane proximal region.(a) DRB1 Polypeptides

[0195] A suitable MHC Class II DRB1 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DRB1 aa sequence depicted in FIG. 5 or a naturally occurring allelic variant thereof. In some cases, the DRB1 polypeptide has a length of about 188 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. Suitable MHC Class II DRp subunit polypeptides may have at least 90% or at least 95% aa sequence identity with the p1 and p2 domain regions of a DRB1 aa sequence depicted in FIG. 5 or a naturally occurring allelic variant thereof. Suitable MHC Class II DRp subunit polypeptides may have at least 98% or 100% aa sequence identity with the p1 and p2 domain regions of a DRB1 aa sequence depicted in FIG. 5 or a naturally occurring allelic variant thereof. As used herein, the term "DRB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB1 polypeptide comprises a sequence that comprises aas 1-188 of DRB1*01 :01 (see FIG. 5) or an allelic variant thereof. In some cases, the allelic variant is the DRB1*04:01 (see FIG. 5).

[0196] A suitable DRB1 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DRB1*01:01 or DRB1*04:01 sequences depicted in FIG. 5. A suitable DRB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DRB1 p1 and p2 domain sequences of DRB1*01:01 or DRB1*04:01. A suitable DRB1 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 170 contiguous aas of the DRB1 p1 and p2 domain sequences of DRB1*01:01 or DRB1*04:01. Thus, a suitable DRB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB1*01 :01 p1 and p2 domain sequence (see, e.g., FIG. 5, aas 1-188). Alternatively, a suitable DRB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB1*04:01 p1 and p2 domain sequence (see, e.g., FIG. 5). A suitable MHC Class II DRB3 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NC:20 or 26. A suitable MHC Class II DRB3 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NC:20 or 26.

[0197] A MAPP may comprise a variant DRB1 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DRB1 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DRB1 sequences shown in FIG. 5. Alternatively, a MAPP may comprise a Cys substitution for formation of a body disulfide bond at any one of aas 5-8 of the DRB1 sequences shown in FIG. 5. Accordingly, a suitable DRB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least170 contiguous aas of the DRB1 p1 and p2 domain sequence provided in FIG. 5, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PRFL (SEQ ID NO: 181) (e.g., as a P5C or an F7C substitution). In one instance the DRB1 sequence is DRB1*01:01. In another instance, the DRB1 sequence is DRB1*04:01.

[0198] A suitable DRB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-95 of any of the DRB1 alleles provided in FIG. 5, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93,94, 95, 96, 97, or 98 aas. A suitable DRB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-95 of any of the DRB1 alleles provided in FIG. 5, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB1*01 :01 , and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB1*04:01, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0199] A suitable DRB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 96-188 of any of the DRB1 alleles provided in FIG. 5, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of any of the DRB1 alleles provided in FIG. 5, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB1*01 :01 , and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 aas. A suitable DRB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB1*04:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94,95, or 96 aas.(b) DRB3 Polypeptides

[0200] A suitable MHC Class II DRB3 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DRB3 aa sequence depicted in FIG. 6 or a naturally occurring allelic variant thereof. In some cases, the DRB3 polypeptide has a length of about 188 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DRB3 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB3 polypeptide comprises a sequence that comprises aas 1-188 of DRB3*01 :01 (see FIG. 6), or an allelic variant thereof. In some cases, the allelic variant is DRB3*02:01 or DRB3*03:01 (see FIG. 6). Suitable MHC Class II DRp subunit polypeptides may have at least 90% or at least 95% aa sequence identity with the p1 and p2 domain regionsof a DRB3 aa sequence depicted in FIG. 6 or a naturally occurring allelic variant thereof. Suitable MHC Class II DRp subunit polypeptides may have at least 98% or 100% aa sequence identity with the p1 and p2 domain regions of a DRB3 aa sequence depicted in FIG. 6 or a naturally occurring allelic variant thereof.

[0201] A suitable DRB3 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DRB3*01:01, DRB3*02:01, or DRB3*03:01 sequences depicted in FIG. 6. A suitable DRB3 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DRB3 p1 and p2 domain sequences of DRB3*01:01 or DRB3*02:01. A suitable DRB3 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 170 contiguous aas of the DRB3 p1 and p2 domain sequences of DRB3*01:01 or DRB3*02:01. Thus, a suitable DRB3 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of aas 1-188 of the DRB3*01:01 p1 and p2 domain sequence (see FIG. 6). Alternatively, a suitable DRB3 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB3*02:01 p1 and p2 domain sequence aas 1 through 188 (see FIG. 6). A suitable MHC Class II DRB3 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:57. A suitable MHC Class II DRB3 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:57. A suitable MHC Class II DRB3 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:58 or 59. A suitable MHC Class II DRB3 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:58 or 59.

[0202] A MAPP may comprise a variant DRB3 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DRB3 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DRB3 sequences shown in FIG. 6. Alternatively, a MAPP may comprise a Cys substitution for formation of a body disulfide bond at any one of aas 5-8 of the DRB3 sequences shown in FIG. 6. Accordingly, a suitable DRB3 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of DRB3 p1 and p2 domain sequences provided in FIG. 6, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PRFL (SEQ ID NO: 181 ) (e.g., as a P5C or an F7C substitution). In one instance the DRB3 sequence is DRB3*01:01. In another instance, the DRB3 sequence is DRB3*02:01 or DRB3*03:01.

[0203] A suitable DRB3 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-95 of any of the DRB3 alleles provided in FIG. 6, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB3 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-95 of any of the DRB3 alleles provided in FIG. 6, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB3 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB3*01:01, and optionally having a length of about 95 aas,including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB3 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB3*02:01, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB3 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB3*03:01, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0204] A suitable DRB3 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 96-188 of any of the DRB3 alleles provided in FIG. 6, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB3 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of any of the DRB3 alleles provided in FIG. 6, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB3 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB3*01:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 aas. A suitable DRB3 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB3*02:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB3 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB3*03:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas.(c) DRB4 Polypeptides

[0205] A suitable MHC Class II DRB4 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DRB4 aa sequence depicted in FIG. 7 or a naturally occurring allelic variant thereof. In some cases, the DRB4 polypeptide has a length of about 188 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DRB4 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB4 polypeptide comprises a sequence that comprises aas 1-188 of DRB4*01 :01 (see FIG. 7), or an allelic variant thereof. In some cases, the allelic variant is the DRB4*01 :03 (see FIG. 7). Suitable MHC Class II DRp subunit polypeptides may have at least 90% or at least 95% aa sequence identity with the p1 and p2 domain regions of a DRB4 aa sequence depicted in FIG. 7 or a naturally occurring allelic variant thereof. Suitable MHC Class II DRp subunit polypeptides may have at least 98% or 100% aa sequence identity with the p1 and p2 domain regions of a DRB4 aa sequence depicted in FIG. 7 or a naturally occurring allelic variant thereof.

[0206] A suitable DRB4 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DRB4*01:01 or DRB4*01:03 sequences depicted in FIG. 7. A suitable DRB4 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DRB4 p1 and p2 domain sequences ofDRB4*01 :01 or DRB4*01:03. A suitable DRB4 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 170 contiguous aas of the DRB4 p1 and p2 domain sequences of DRB4*01:01 or DRB4*01:03. Thus, a suitable DRB4 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB4*01:01 p1 and p2 domain sequences (see, e.g., FIG. 7, aas 1-188). Alternatively, a suitable DRB4 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB4*01:03 p1 and p2 domain sequences (see, e.g., FIG. 7 aas 1-188). A suitable MHC Class II DRB4 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:60 or 61 . A suitable MHC Class II DRB4 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NC:60 or 61 .

[0207] A MAPP may comprise a variant DRB4 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DRB4 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DRB4 sequences shown in FIG. 7. Alternatively, a MAPP may comprise a Cys substitution for formation of a body disulfide bond at any one of aas 5-8 of the DRB4 sequences shown in FIG. 7. Accordingly, a suitable DRB4 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of DRB4 p1 and p2 domain sequences provided in FIG. 7, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PRFL (SEQ ID NO:181) (e.g., as a P5C or an F7C substitution). In one instance the DRB4 sequence is DRB4*01:01. In another instance, the DRB4 sequence is DRB4*01:03.

[0208] A suitable DRB4 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-95 of any of the DRB4 alleles provided in FIG. 7, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB4 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-95 of any of the DRB4 alleles provided in FIG. 7, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB4 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB4*01:01, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DRB4 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB4*01:03, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0209] A suitable DRB4 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 96-188 of any of the DRB4 alleles provided in FIG. 7, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB4 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of any of the DRB4 alleles provided in FIG. 7, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB4 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having atleast 90% or at least 95% aa sequence identity to aas 96-188 of DRB4*01 :01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 aas. A suitable DRB4 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB4*01:03, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94,95, or 96 aas.(d) DRB5 Polypeptides

[0210] A suitable MHC Class II DRB5 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DRB5 aa sequence depicted in FIG. 8 or a naturally occurring allelic variant thereof. In some cases, the DRB5 polypeptide has a length of about 188 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DRB5 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB5 polypeptide comprises a sequence that comprises aas 1-188 of DRB5*01 :01 (see FIG. 8), or an allelic variant thereof.

[0211] A suitable DRB5 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DRB5*01:01 sequence depicted in FIG. 8. A suitable DRB5 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DRB5 p1 and p2 domain sequences of DRB5*01:01. A suitable DRB5 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DRB5*01:01 p1 and p2 domain sequence (see, e.g., FIG. 8, aas 1-188). A suitable MHC Class II DRB5 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:62. A suitable MHC Class II DRB5 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:62.

[0212] A MAPP may comprise a variant DRB5 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DRB5 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DRB5 sequences shown in FIG. 8. Alternatively, a MAPP may comprise a Cys substitution for formation of a body disulfide bond at any one of aas 5-8 of the DRB5 sequences shown in FIG. 8. Accordingly, a suitable DRB5 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of DRB5 p1 and p2 domain sequences provided in FIG. 8, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PRFL (SEQ ID NO:181) (e.g., as a P5C or an F7C substitution). In one instance the DRB5 sequence is DRB5*01:01.

[0213] A suitable DRB5 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-95 of the DRB5 alleles provided in FIG. 8, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95,96, 97, or 98 aas. A suitable DRB5 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequencehaving at least 90% or at least 95% aa sequence identity to aas 1-88 of DRB5*01 :01, and optionally having a length of about 95 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0214] A suitable DRB5 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 96-188 of the DRB5 alleles provided in FIG. 8, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DRB5 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 96-188 of DRB5*01:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas.(2) DQB Polypeptides

[0215] MHC Class II DQB polypeptides for inclusion in a MAPP have both p1 and p2 domains. Some non-limiting examples of DQB polypeptides are provided in FIGs. 11 and 12. The p1 and p2 domains of the DQB proteins shown in those figures are typically about 187 or 188 aas in length, with aas 1-94 making up the p1 domain and aas 95-187 or 95-188 making up the p2 domain. Aas 188-197 or 189-198 make up the membrane proximal region.(a) DQB1 Polypeptides

[0216] A suitable MHC Class II DQB1 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DQB1 aa sequence depicted in FIG. 11 or an allelic variant thereof. In some cases, the DQB1 polypeptide has a length of about 188 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DQB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB1 polypeptide comprises a sequence that comprises aas 1-188 of DQB1*02:01, DQB1*03:01, DQB1*04:01, DQB1*05:01, or DQB1*06:01 (see FIG. 11), or an allelic variant thereof. In some cases, the allelic variant is DQB1*02:01, DQB1*02:02 or DQB1*03:01 (see FIG. 11). Suitable MHC Class II DQp subunit polypeptides may have at least 90% or at least 95% aa sequence identity with the p1 and p2 domain regions of a DQB1 aa sequence depicted in FIG. 11 or a naturally occurring allelic variant thereof. Suitable MHC Class II DQp subunit polypeptides may have at least 98% or 100% aa sequence identity with the p1 and p2 domain regions of a DQB1 aa sequence depicted in FIG. 11 or a naturally occurring allelic variant thereof.

[0217] A suitable DQB1 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DQB1*02:01, DQB1*03:01, DQB1*04:01, DQB1*05:01, or DQB1*06:01 sequences depicted in FIG. 11. A suitable DQB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DQB1 p1 and p2 domain sequences of DQB1*02:01, DQB1*03:01, DQB1*04:01, DQB1*05:01, or DQB1*06:01. A suitable DQB1 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or 100% aa sequence identity to at least 170 contiguous aas of the DQB1 p1 and p2 domain sequences of DQB1*02:01, DQB1*03:01, DQB1*04:01, DQB1*05:01, or DQB1*06:01. Thus, a suitable DQB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB1*02:01, DQB1*03:01, DQB1*04:01, DQB1*05:01, or DQB1*06:01 p1 and p2 domain sequences (see, e.g.,FIG. 11, aas 1-188). A suitable DQB1 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB1*02:01 p1 and p2 domain sequences. A suitable DQB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB1*03:01 p1 and p2 domain sequences. A suitable DQB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB1*04:01 or DQB1*05:01 p1 and p2 domain sequences. A suitable DQB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB1*06:01 p1 and p2 domain sequence. A suitable MHC Class II DQB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with any one of SEQ ID NOs:75-85. A suitable MHC Class II DQB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with any one of SEQ ID NOs:75-85.

[0218] A MAPP may comprise a variant DQB1 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DQB1 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DQB1 sequences shown in FIG. 11 . Alternatively, a MAPP may comprise a variant DQB1 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 4-8 or 5-8 of the DQB1 sequences shown in FIG. 11. Accordingly, a suitable DQB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of the DQB1 p1 and p2 domain sequences provided in FIG. 11, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PEDF (SEQ ID NO:182) (e.g., a P4C, E5C, D6C or F7C substitution). In one instance the DQB1 sequence is DQB1*02:01. In one instance the DQB1 sequence is DQB1*03:01. A suitable MHC Class II DQB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:75 or 77. A suitable MHC Class II DQB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:75 or 77. In another instance, the DQB1 sequence is DQB1*04:01 or DQB1*05:01. A suitable MHC Class II DQB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:81 or 83. A suitable MHC Class II DQB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:81 or 83. In another instance, the DQB1 sequence is DQB1*06:01. A suitable MHC Class II DQB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:84 or 85. A suitable MHC Class II DQB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:84 or 85.

[0219] A suitable DQB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-94 of any of the DQB1 alleles provided in FIG. 11, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-94 of any of the DQB1 alleles provided in FIG. 11, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DQB1*02:01, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB1 p1 domain for inclusion in a MAPPpolypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DQB1*03:01, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DQB1*04:01 or DQB1*05:01, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DQB1*06:01, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93,94, 95, 96, 97, or 98 aas.

[0220] A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 95-188 of any of the DQB1 alleles provided in FIG. 11, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-188 of any of the DQB1 alleles provided in FIG. 11, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-188 of DQB1*02:01, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-188 of DQB1*03:01, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94,95, or 96 aas. A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-188 of DQB1*04:01 or DQB1*05:01, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DQB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-188 of DQB1*06:01, and optionally having a length of about 94 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas.(b) DQB2 Polypeptides

[0221] A suitable MHC Class II DQB2 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DQB2 aa sequence depicted in FIG. 12 or an allelic variant thereof. In some cases, the DQB2 polypeptide has a length of about 187 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DQB2 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB2 polypeptide comprises a sequence that comprises aas 1-187 of DQB2 isoform 1 (DQB2-lso-1) or DQB2 isoform 2 (DQB2-lso-2) (see FIG. 12), or an allelic variant thereof. In some cases, the allelic variant is the DQB2-lso-1 or DQB2-lso-2 (see FIG. 12). A suitable MHC Class II DQB2 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with any one of SEQ ID NOs: 86-87. A suitable MHC Class II DQB2 subunit polypeptide may have at least 98% or 100% aa sequence identity with any one of SEQ ID NOs: 86-87.

[0222] A suitable DQB2 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DQB2-lso-1 or DQB2-lso-2 sequences depicted in FIG. 12. A suitable DQB2 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DQB2 p1 and p2 domain sequences of DQB2-lso-1 or DQB2-lso-2. A suitable DQB2 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 170 contiguous aas of the DQB2 p1 and p2 domain sequences of DQB2-lso-1 or DQB2-lso-2. Thus, a suitable DQB2 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 180 contiguous aas of the DQB2-lso-1 or DQB2-lso-2 p1 and p2 domain sequences (see, e.g., FIG. 12, aas 1-187). A suitable DQB2 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB2-lso-1 p1 and p2 domain sequences. A suitable DQB2 polypeptide may comprise an aa sequence having at least 95% aa sequence identity to at least 170 or at least 180 contiguous aas of the DQB2-lso-2 p1 and p2 domain sequences. A suitable MHC Class II DQB2 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:86 or 87. A suitable MHC Class II DQB2 subunit polypeptide may have at least 98% or 100% aa sequence identity with any one of SEQ ID NO:86 or 87.

[0223] A MAPP may comprise a variant DQB2 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DQB2 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DQB2 sequences shown in FIG. 12. Alternatively, a MAPP may comprise a variant DQB2 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 4-8 or 5-8 of the DQB2 sequences shown in FIG. 12. Accordingly, a suitable DQB2 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of DQB2-lso-1 or DQB2-lso-2 p1 and p2 domain sequences provided in FIG. 12, wherein the p1 sequence comprises a cysteine as a substitution in the subsequence PKDFL (e.g., a P4C, K5C, D6C or F7C substitution, SEQ ID NO: 183). In one instance the DQB2 sequence is DQB2- lso-1. In another instance, the DQB2 sequence is DQB2-lso-2.

[0224] A suitable DQB2 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-94 of any of the DQB2 alleles provided in FIG. 12, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DQB2 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-94 of any of the DQB2 alleles (DQB2- lso-1 or DQB2-lso-2) provided in FIG. 12, and optionally having a length of about 94 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0225] A suitable DQB2 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 95-187 of any of the DQB2 alleles provided in FIG. 12, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DQB2 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 95-187 of any of the DQB2 alleles (DQB2-lso-1 or DQB2-lso-2) provided in FIG. 12, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas.(3) DPB Polypeptides

[0226] MHC Class II DPB polypeptides for inclusion in a MAPP have both p1 and p2 domains. Some non-limiting examples of DPB polypeptides are provided in FIG. 14. The p1 and p2 domains of the DPB proteins shown in those figures are typically about 186 aas in length, with aas 1-92 making up the p1 domain and aas 93-186 making up the p2 domain. Aas 187-196 make up the membrane proximal region.(a) DPB1 Polypeptides

[0227] A suitable MHC Class II DPB1 polypeptide for inclusion in a MAPP may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain regions of a DPB1 aa sequence depicted in FIG. 14 or a naturally occurring allelic variant thereof. In some cases, the DPB1 polypeptide has a length of about 186 aas, including, e.g., 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188 aas. As used herein, the term "DPB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DPB1 polypeptide comprises a sequence that comprises aas 1-186 of DPB1*01:01 (see FIG. 14), or an allelic variant thereof. In some cases, the allelic variant is DPB1*02:01 or DPB1*03:01 (see FIG. 14). A suitable MHC Class II DPB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with any one of SEQ ID NCs:90-102. A suitable MHC Class II DPB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with any one of SEQ ID NCs:90-102.

[0228] A suitable DPB1 aa sequence for inclusion in a MAPP polypeptide may have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 165, at least 170, at least 175, at least 180, or at least 185 contiguous aas of the p1 and p2 domain sequences of the DPB1*01 :01, DPB1*02:01, DPB1*03:01, or DPB1*11:01 sequences depicted in FIG. 14. A suitable DPB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of the DPB1 p1 and p2 domain sequences of DPB1*01 :01 , DPB1*02:01, DPB1*03:01, or DPB1*11 :01. A suitable DPB1 aa sequence for inclusion in a MAPP polypeptide may have at least 95% or at least 98% aa sequence identity to at least 170 contiguous aas of the DPB1 p1 and p2 domain sequences of DPB1*01 :01, DPB1*02:01, DPB1*03:01, or DPB1*11 :01 . Thus, a suitable DPB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 170 or at least 180 contiguous aas of the DPB1*01:01 p1 and p2 domain sequences (see, e.g., FIG. 14, aas 1-188). A suitable DPB1 polypeptide may comprise an aa sequence having at least 95% aa or at least 98% sequence identity to at least 180 contiguous aas of the DPB1*02:01 p1 and p2 domain sequence (aas 1 through 186, see FIG. 14). A suitable MHC Class II DPB1 subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NQ:90 or 91. A suitable MHC Class II DPB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NQ:90 or 91. A suitable DPB1 polypeptide may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 180 contiguous aas of the DPB1*03:01 p1 and p2 domain sequences (aas 1 through 186, see FIG. 14). A suitable DPB1 polypeptide may comprise an aa sequence having at least 95% aa or at least 98% sequence identity to at least 180 contiguous aas of the DPB1*11 :01 p1 and p2 domain sequence (aas 1 through 186, see FIG. 14). A suitable MHC Class II DPB1subunit polypeptide may have at least 90% or at least 95% aa sequence identity with SEQ ID NO:92 or 100. A suitable MHC Class II DPB1 subunit polypeptide may have at least 98% or 100% aa sequence identity with SEQ ID NO:92 or 100.

[0229] A MAPP may comprise a variant DPB1 polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a body disulfide bond that stabilizes the MAPP). For example, a MAPP may comprise a variant DPB1 polypeptide comprising a Cys substitution for formation of a body disulfide bond at any one of aas 1-8 of the DPB1 sequences shown in FIG. 14. Alternatively, a MAPP may comprise a Cys substitution for formation of a body disulfide bond at any one of aas 4-8 or 5-8 of the DPB1 sequences shown in FIG. 14. Accordingly, a suitable DPB1 aa sequence for inclusion in a MAPP polypeptide may have at least 90% or at least 95% aa sequence identity to at least 170 contiguous aas of DPB1 p1 and p2 domain sequences provided in FIG. 14, wherein the p1 sequence comprises a cysteine as a substitution for one of the aas in the subsequence PENY (SEQ ID NO: 109), PENYL (SEQ ID NO:184) or PENYV (SEQ ID NO:185) (e.g., a P4C, E5C, N6C or Y7C substitution). In one instance the DPB1 sequence is DPB1*01:01. In another instance, the DPB1 sequence is DPB1*02:01 or DPB1*03:01. In another instance, the DPB1 sequence is DPB1*11 :01 .

[0230] A suitable DPB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 1-92 of any of the DPB1 alleles provided in FIG. 14, and optionally having a length of about 92 aas, including, e.g., 89, 90, 91, 92, 93,94, 95, 96, 97, or 98 aas. A suitable DPB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-92 of any of the DPB1 alleles provided in FIG. 14, and optionally having a length of about 92 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DPB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DPB1*01:01, and optionally having a length of about 92 aas, including, e.g., 89, 90, 91 , 92, 93, 94, 95, 96, 97, or 98 aas. A suitable DPB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DPB1*02:01 or DPB1*03:01, and optionally having a length of about 92 aas, including, e.g., 89, 90, 91, 92, 93, 94,95, 96, 97, or 98 aas. A suitable DPB1 p1 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 1-88 of DPB1*11 :01, and optionally having a length of about 92 aas, including, e.g., 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98 aas.

[0231] A suitable DPB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%) or 100% aa sequence identity to aas 93-186 of any of the DPB1 alleles provided in FIG. 14, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DPB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 93-186 of any of the DPB1 alleles provided in FIG. 14, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DPB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 93-186 of DPB1*01 :01 , and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 aas. A suitable DPB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas93-186 of DPB1*02:01 or DPB1*03:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas. A suitable DPB1 p2 domain for inclusion in a MAPP polypeptide may comprise an aa sequence having at least 90% or at least 95% aa sequence identity to aas 93-186 of DPB1*11:01, and optionally having a length of about 93 aas, including, e.g., 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 aas.(4) Membrane proximal regions

[0232] In addition to the o1, o2, p1, and p2 domain sequences of the Class II MHC (HLA) subunits present in MAPPs, the MAPPs may comprise membrane proximal regions. For example, in addition to the MHC p subunit p1 and p2 domain sequences, the MHC p subunit sequences may comprise a membrane proximal region (e.g., an MHC p subunit membrane proximal region). Similarly, in addition to the MHC a subunit a1 and a2 domain sequences, the MHC o subunit sequences may comprise a membrane proximal region (e.g., an MHC a subunit membrane proximal region). The membrane proximal regions are typically located following (are located on the C-terminal side) the p2 and / or o2 domain sequences. A membrane proximal region following an MHC p subunit sequence may have at least 85%, at least 90%, or at least 95% aa sequence identity to the membrane proximal region associated with the MHC p subunit present in the MAPP (e.g., 1 or 2 aa substitutions).

[0233] The membrane proximal region following the p2 domain sequence (see FIG. 1 and FIG. 23) may be from the same allele as the p2 domain sequence present in the MAPP, and may be located so that the sequence from the N- terminus of the p2 domain to the C-terminus of the membrane proximal region corresponds to the sequence of the allele from which they were derived. For example, where a MAPP comprises an HLA DQB1*02:01 p2 domain, the membrane proximal region of the DQB1*02:01 allele may directly follow the p2 domain sequence.

[0234] Similar to the situation with MHC p subunit sequences, the membrane proximal region following an MHC a subunit sequence may have at least 85%, at least 90%, or at least 95% aa sequence identity to the membrane proximal region associated with an MHC a subunit (e.g., the membrane proximal region may have 1 or 2 aa substitutions). The membrane proximal region may be from the same allele as the o2 domain sequence present in the MAPP and may be located so that the sequence from the N-terminus of the o2 domain to the C-terminus of the membrane proximal region corresponds to the sequence of the allele from which they were derived. For example, where a MAPP comprises an HLA DQA1*05:01 o2 domain, the membrane proximal region of the DQA1*05:01 allele may directly follow the o2 domain sequence. c) MHC Class II disease risk-associated alleles and haplotypes

[0235] Certain alleles and haplotypes of MHC Class II have been associated with disease, e.g., increased risk of developing a particular disease. 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, 8th ed., Margaritte-Jeannin et al. (2004) Tissue Antigens 63:562, and Kurko et al. (2013) Clin. Rev. Allergy Immunol. 45:170. A number of those diseases and their associated alleles and / or haplotypes are described in WO 2020 / 181273 assigned to Cue Biopharma, Inc., and references cited therein. Some HLA haplotypes and alleles associated with increased risk that an individual expressing such HLA haplotypes and / or alleles will develop a given autoimmune disease are set forth in the table provided in FIGs. 24 and 25. The tables in FIG. 25 also provides a listing of some molecules associated with the disease (e.g., autoantigens such as proteins and peptides) that can function as epitopes or a source of epitopes. Some HLA haplotypes and alleles associated with increased risk that an individual expressing such HLAhaplotypes and / or alleles will develop Type 1 Diabetes are also set forth in Table 2. A MAPP of the present disclosure that is directed to the treatment of a specific disease can include any of the disease associated HLA haplotypes and / or alleles and the corresponding epitopes set out in FIGs. 24 or 25 or in Table 2. The peptide epitope can be, for example, a peptide of from 4 aas to about 25 aas in length of any of the autoantigens set out in Table 2 or in FIGs. 24 or 25.

[0236] The following are notes to the table provided in FIG. 25: 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 may be associated with DQA1*01 :01; 4) DR2 alleles include DRB1 *15:01 -15:04 and DRB1 *16:01 -16:06; 5) DR3 haplotypes include: DRB1*03:01, DRB1*03:02, DRB1*03:03, and DRB1*03:04; 6) DR4 haplotypes include: DRB1*04:01 through DRB1*04:13; AH = ancestral haplotype; 7) Simmonds et al., Am. J. Hum. Genet. 76:157-163, (2005), see the table in FIG. 17, HLAs with odds ratios greater than 1.5 include the following DRB1, DQB1 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; DQBV-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 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 topoisomerasel (truncated) (Unprot P11387); DNA topoisomerase 1 (full length) (NCBI Accession: NP_003277.1 and P11387); and U1-SnRNP 68 / 70 KDa (NCBI Accession: P08621.2).(1) Individual disease risk-associated alleles

[0237] The association of a number of HLA alleles with one or more autoimmune diseases is described in, for example, FIGs. 24 and 25, and in Table 2. The sequences of the disease-associated alleles are provided in the figures accompanying this disclosure (e.g., DRB1 alleles are provided in the figures). Where disease associations are made to groups of alleles (e.g., DRB1*03), the sequences of additional alleles may be obtained from standard references including those provided by the U.S. National Center for Biotechnology Information (NCBI) or online at hla.alleles.org / nomenclature / index.html.

[0238] An exemplary association between various disease states and particular HLA alleles includes the association of the alleles of the HLA-DR3 with early-age onset myasthenia gravis, Hashimoto's thyroiditis, autoimmune hepatitis, primary Sjogren's syndrome, and SLE. Other exemplary associations include: DRB1*0301 (DRB1*03:01 provided in the figures) association with an increased risk of developing early onset Grave's disease and / or type 1 autoimmune hepatitis; DRB1*04:01 association with an increased risk of developing multiple sclerosis and / or rheumatoid arthritis; DRB1*04:02 association with an increased risk of developing idiopathic pemphigus vulgaris and / or SLE (e.g., SLE-associated anti-cardiolipin, SLE-associated anti-|32 glycoprotein I); DRB1*0403 association with an increased risk of developing SLE (e.g., increased risk of developing SLE-associated anti-cardiolipin antibodies and / or SLE-associated anti-p2 glycoprotein I antibodies); DRB1*04:05 association with an increased risk of developing rheumatoid arthritis and / or autoimmune hepatitis; and DRB1*04:06 association with an increased risk of developing anti-caspase-8 autoantibodies (e.g., in silicosis-systemic sclerosis (SSc)-systemic lupus erythematosus (SLE)).

[0239] Certain DQB1 alleles are also associated with an increased risk that an individual expressing such an allele will develop an autoimmune disease. For example, DQB1*03:01, and DQB1*06:02 are associated with an increased risk of developing MS and / or a more severe MS phenotype (e.g., more severe inflammatory and neurodegenerative damage).(a) MHC Class II Polypeptides in T1D

[0240] Alleles / isoforms showing increased association with Type 1 Diabetes (T1D) represent suitable sources of MHC Class II a1 , a2, p1, and p2 polypeptide sequences for incorporation into MAPPs directed to the treatment of T1D. T1D is associated with alleles belonging to the HLA-DR3 and HLA-DR4 haplotypes / serotypes, with the strongest risk associated with alleles of the HLA-DQ8 (e.g., HLA-DQB1*03:02) and HLA-DQ2 serotypes. Some high and moderate risk haplotypes and their association with various DR serotypes are shown in Table 2, adopted from Kantarova and Buc, Physiol. Res. 56: 255-266 (2007).Table 2: High Risk T1D Haplotypes

[0241] The stereotypically defined DR3 and DR4 protein isoforms / haplotypes of the DRB1 gene are associated with increased risk that an individual expressing such alleles will develop T1 D. The DR3 serotype includes the alleles encoding the DRB1*03:01, *03:02, *03:03, and *03:04 proteins, with the HLA-DRB1*0301 allele often found associated with a predisposition to T1D. The DR4 serotype includes the alleles encoding the DRB1*04:01, *04:02, *04:03, *04:04, *04:05, *04:06, *04:07, *04:08, *04:09, *04:10, *04:11, *04:12, and *04:13 proteins. Certain HLA-DR4 proteins (e.g., HLA-DRB1*04:01 and HLA-DRB1*04:05) predispose individuals to T1D, whereas the HLA- DRB1*04:03 allele / isoform may afford protection. DRB1*16:01 also shows an increased frequency in diabetic children relative to healthy controls (Deja, et al., Mediators of Inflammation 2006:1-7 (2006)). Alleles / isoforms showing increased association with T1D represent suitable sources of MHC II a1 , a2, p1, and p2 polypeptide sequences.

[0242] DQ2 and DQ8 are serotypes within the HLA-DQ system that are determined by recognition of DQ p-chains. While T1 D is associated with DR3 and DR4 alleles as discussed above, among the strongest associated risk factors for T1 D are the presence of the HLA-DQ8 serotype (e.g., the HLA-DQB1*03:02 isoform), particularly the HLA-DQ8.1 serotype (HLA-DQA1*03:01 / DQBr03:02), and the alleles of the HLA-DQ2 serotype (e.g., DQB1*02 alleles such as DQB1*02:01, DQB1*02:02, or DQB1*02:03). Jones et al., Nat. Rev. Immunol. 2006, 6: 271-282. By contrast, individuals that carry the HLA-DQB1*06:02 allele appear to be protected against T1 D. Id.

[0243] DQ2 is most common in Western Europe, North Africa, and East Africa, with the highest frequencies observed in parts of Spain and Ireland. Although the HLA-DR associations with T1D are not as strong as those of HLA-DQ, insulin-reactive T cells derived from lymph nodes draining the pancreas of patients with T1 D appear to be HLA-DR4.1 restricted rather than HLA-DQ8 or HLA-DQ2 restricted (Kent et al., Nature 2005 435: 224-228). The crystal structure of HLA-DQ2 shows a distinctive P6 pocket with a large volume and polar character defined by the presence of Ser30p (see e.g., FIG. 11 Ser 30) rather than Tyr30p, which is typically found in other HLA-DQ molecules. This is a unique feature of HLA-DQ2, as is the presence of a positively charged lysine residue at 71 [3 (see FIG. 11 Lys 71) and, when combined with the polar nature of the P4 and P9 pockets, makes this MHC Class II peptide binding groove the most suitable for accommodating peptides with negatively charged anchor residues (see, e.g., Jones et al, Nat. Rev. Immunol. 2006, 6:271-282). This is a key factor in allowing HLA-DQ2 to present glutenderived peptides that are high in proline and glutamate residues (generated by deamidation of glutamines). Id. In an embodiment, Ser30p of DQ2 (e.g., DQB1*02:01) molecules can be replaced with a cysteine (S30C) to permit conjugation of a peptide epitope that is co-translated as part of a T cell modulatory antigen-presenting polypeptide to that position (e.g., utilizing a cysteine at position 6 of the peptide epitope).

[0244] The DQB1 locus alone has also been reported to be associated with T1 D when position [357 is a neutral residue such as Ala or Ser. Both the DQ2 and DQ8 serotypes, which are associated with TID, lack an Asp at the 57|3 position, and instead have an Ala in its place (see, e.g., Ala 57 in FIG. 11, HLA-DQB1*02:01 and HLA-DQB1*03:02), conferring T1 D susceptibility. In contrast, DQB1*06:02, which has an Asp at position [357 (position 57 in FIG. 11) was found to be associated with resistance to T1 D. Jones et al, Nat. Rev. Immunol. 2006, 6: 271-282. Position [357 of the molecule is a critical residue in the (P9) residue binding pocket of DQB1 , which is involved in antigen presentation and T cell receptor (TCR) interaction.

[0245] Individuals with the HLA haplotype DQA1*03:01-DRBr03:02, especially when combined with DQA1*05:01- DRB1*02:01, are highly susceptible (10-20-fold increase) to T1 D (see Notkins, A. L, J. Biol. Chem., 2002, 277(46): 43545-48). Among the stereotypically defined groups showing susceptibility to T1 D are HLA-DR4.1 (HLA- DRA1*01 :01 / DRB1*04:01), HLA-DR4.5 (HLA-DRA1*01 :01 / DRB1*04:05), HLA-DQ 2.5 (HLA-DQA1*05:01 / DQB1*02:01), and HLA-DQ8.1 (HLA-DQA1*03:01 / DQB1*03:02) (see, e.g., Jones et al., Nat. Rev. Immunol. 2006, 6: 271-282). The DRB1*04:05-DQBr04:01 / DRB 08:02-DQBr03:02 genotype has been shown to be associated with acute onset and slow progressive T1D. Fulminant diabetes has been associated with DRB1*04:05- DQB1*04:01 / DRBr04:05-DQBr04:01 genotype in a Japanese population study (Kawabata, et al., Diabetologia 2009, 52:2513-21)^

[0246] The above-mentioned alleles associated with an increased risk of T1 D represent suitable candidates from which the a1 , a2, [31, and / or |32 polypeptide sequences present in a MAPP may be taken. In an embodiment, theMAPP is DQ 2.5-like with the a1 and a2 polypeptides from DQA1*05:01, and the p1 and p2 polypeptides taken from DQB1*02:01. In an embodiment, the MAPP is DQ8.1 -like with the a1 and o2 polypeptides from DQA1*03:01, and the p1 and p2 polypeptides taken from DQB1*03:02.

[0247] The Table in FIG. 24 shows examples of HLA Class II alleles, MODs, and T1D-epitopes that may be incorporated into a MAPP for T1D therapy.

[0248] The above-mentioned alleles associated with an increased risk of T1 D represent suitable candidates from which the a1, a2, p1, and / or p2 polypeptide sequences present in a MAPP may be taken.(b) MHC Class II Polypeptides and Celiac disease

[0249] HLA haplotypes DQ2 and DQ8 are associated with increased risk that an individual expressing such HLA haplotypes will develop celiac disease. DQ2 represents the second highest risk factor for celiac disease; the highest risk factor is a close family member with the disease. It is estimated that approximately 95% of all celiac patients have at least one DQ2 allele, and of those individuals about 30% have two copies of a DQ2 allele. DQ2 isoforms vary in their association with celiac disease, the DQ 2.5 isoform (DQB1*02:01 / DQAr05:01) being strongly associated. DQB1*0201 is genetically linked to DQA1*05:01 forming the DQ 2.5 haplotype. DQ 2.5 is present in high levels in northern, islandic Europe, and the Basque region of Spain with the phenotype frequency exceeding 50% in parts of Ireland.

[0250] The immunodominant site for DQ 2.5 is on o2-gliadin, which has a protease resistant 33mer that has 6 overlapping DQ 2.5 restricted epitopes. The multiple epitopes produce strong binding of T cells to the DQ 2.5-33mer complexes. DQ 2.5 binds gliadin, but the binding is sensitive to deamidation caused by tissue transglutaminase, whose action produces most of the highest affinity sites / epitopes. All or part of the 33mer (LQLQPFPQPELPYPQPEL PYPQPELPYPQPQPF, SEQ ID NO:186) or a similarly described 19mer (LGQQQPFPPQQPYPQPQPF, SEQ ID NO:187) (e.g., 8 or more, 9, or more, 10 or more, 12 or more, 14 or more, or 16 or more contiguous aas) may be utilized as a peptide epitope. See, e.g., Bruun.et al. 2016, J. Diabetes Res. 2016, 2016:1-11, Article ID 2424306.

[0251] As noted above, T1D is associated with the DQ 2.5 phenotype, and there may be a link between Gluten- Sensitive Enteropathy (GSE) and early onset male T1D. Recent studies indicate a combination of DQ 2.5 and DQ8 (both acid peptide presenters) greatly increase the risk of adult onset T1 D. The presence of DQ2 with DR3 may decrease the age of onset and the severity of the disease.

[0252] While the DQ 2.5 haplotype confers the single highest known genetic risk for celiac disease, comparable risk can also come from very similar alleles of different haplotypes (e.g., other DQA1*05 and DQB1*02 alleles). The DQ2.2 phenotype has the form o2-p2 (e.g., DQA1*02:01:DQBr02:02) and is associated with the occurrence of some celiac disease. Because the HLA DQB1*02:02 and its linked DQA1* alleles of the DQ2.2 haplotype do not produce a DQA1*05 subunit (o5 e.g., DQA1*05:01), the heterodimer cannot effectively present a-2 gliadin; it can, however, present other gliadins. Accordingly, a MAPP comprising DQ 2.2 polypeptide sequences (e.g., DQA1*02:01:DQBr02:02) may be used to present non-o-2 gliadin peptides.

[0253] The DQ2.2 / DQ7.5 phenotype, also referred to as DQ 2.5trans, is also associated with celiac disease. The serotypically defined DQ7.5 phenotype has a DQA1*05:05:DQBr03:01 haplotype. When DQA1*05:05 or DQA1*05:01 gene products are processed to the cell surface they become the o5 subunit and can assemble an MHC Class II molecule with either of the DQ 2.2 alleles, DQB1*02:02 or DQB1*02:01. As a result, the isoformsproduced by the phenotype of two haplotypes, DQ2.2 / DQ7.5, include HLA DQ o5p2(DQ 2.5), o2p2(DQ2.2), o2p7(DQ7.2, e.g, DQA1*02:01:DQB1*03:01), and a5p7(DQ7.5).

[0254] DQ8 is typically involved in celiac disease in those individuals where DQ2 is not present. The DQ8.1 haplotype encodes the DQA1*03:01:DQBr03:02 haplotype. DQ8 is extremely high in Native Americans of Central America and tribes of Eastern American origin.

[0255] Two Class II HLA genotypes (DQA1*05:DQB1*02 {o5p2} and DQA1*03:DQB1* 03:02 {a3p3}) contribute substantially to the genetic risk of celiac disease in families, and have been suggested to be virtually required for celiac disease to occur in Caucasian individuals (see Murry et al., Clin. Gastroenterol. Hepatol. 2007, 5(12): 1406— 1412). Among the stereotypically defined groups showing susceptibility to T1D and celiac disease are HLA-DQ 2.5 (HLA-DQA1*05:01 / DQB1*02:01) and HLA-DQ8.1 (HLA-DQA1*03:01 / DQB1*03:02) (see, e.g., Jones et al., Nat. Rev. Immunol. 2006, 6: 271-282).

[0256] The alleles associated with an increased risk of celiac disease described above represent suitable candidates from which the a1 , a2, p1, and / or p2 polypeptide sequences of MAPPs may be taken. In an embodiment, the MAPP is DQ 2.5-like with the a1 and o2 polypeptides taken from DQA1*05:01, and the p1 and p2 polypeptides taken from DQB1*02:01. In an embodiment, the MAPP is DQ2.2-like with the a1 and o2 polypeptides taken from DQA1*02:01, and the p1 and p2 polypeptides taken from DQB1*02:01. In an embodiment, the MAPP is DQ8.1 -like with the a1 and o2 polypeptides taken from DQA1*03:01, and the p1 and p2 polypeptides taken from DQB1*03:02. In an embodiment, the MAPP comprises a1 , a2, p1, and p2 polypeptides taken from isoforms produced by the DQ2.2 / DQ7.5 haplotypes, including the HLA DQ o5p2(DQ 2.5), o2p2(DQ2.2), o2p7(DQ7.2, e.g., DQA1*02:01:DQB1*03:01), and o5p7(DQ7.5) molecules.DRB1*03:01

[0257] DRB1*03:01 (DRB1*0301, see FIG. 5) is associated with an increased risk of developing T1D. Thus, a MAPP may comprise a DRB1*03:01 polypeptide comprising an aa sequence having at least 90%, at least 95%, at least 98%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DRB1*03:01 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*03:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DRB1*03:01 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*03:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DRB1*03:01 aa sequence depicted in FIG. 5.DRB1*04:01

[0258] DRB1*04:01 (DRB1*0401, see FIG. 5 and FIG. 16, SEQ ID NO:26) is associated with an increased risk of developing T1D. Thus, a MAPP may comprise a DRB1*04:01 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DRB1*04:01 aa sequence depicted in FIG. 5 or FIG 16. A MAPP may comprise a DRB1*04:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DRB1*04:01 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*04:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DRB1*04:01 aa sequence depicted in FIG. 5.DRB1*04:02

[0259] DRB1*04:02 (DRB1*0402, see FIG. 5) is associated with an increased risk of developing T1D. Thus, a MAPP may comprise a DRB1*04:02 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DRB1*04:02 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*04:02 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DRB1*04:02 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*04:02 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DRB1*04:02 aa sequence depicted in FIG. 5. DRB1*04:05

[0260] DRB1*04:05 (“DRB1*0405” see FIG. 5) is associated with an increased risk of developing T1D. Thus, a MAPP may comprise a DRB1*04:05 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DRB1*04:05 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*04:05 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DRB1*04:05 aa sequence depicted in FIG. 5. A MAPP may comprise a DRB1*04:05 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DRB1*04:05 aa sequence depicted in FIG. 5.DQA1*05:01-DQB1*02:01 (DQ 2.5)

[0261] DQ 2.5 (DQA1*05:01-DQBr02:01) is associated with an increased risk of developing celiac disease. Thus, a MAPP may comprise a DQA1*05:01 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the a1 and o2 domains (aas 1-181) of the DQA1*05:01 aa sequence depicted in FIG. 9 (e.g., 90% or 95% sequence identity to FIG 15, SEQ ID NO:105). A MAPP may comprise a DQA1*05:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the a1 domain of the DQA1*05:01 aa sequence depicted in FIG. 9. A MAPP may comprise a DQA1*05:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the o2 domain of the DQA1*05:01 aa sequence depicted in FIG. 9.

[0262] A MAPP may comprise a DQB1*02:01 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DQB1*02:01 aa sequence set forth in FIG. 11 (e.g., 90% or 95% sequence identity to FIG 16, SEQ ID NO:75). A MAPP may comprise a DQB1*02:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DQB1*02:01 aa sequence set forth in FIG. 11 . A MAPP may comprise a DQB1*02:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DQB1*02:01 aa sequence depicted in FIG. 11.

[0263] A MAPP may comprise: i) a DQA1*05:01 a chain polypeptide, and ii) a DQB1*02:01 p chain polypeptide. A MAPP may comprise: i) an MHC a chain polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the a1 and o2 domains (aas 1-181) of the DQA1*05:01 sequence depicted in FIG. 9 (e.g., 90% or 95% sequence identity to SEQ ID NO: 105), and ii) an MHC p chain polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DQB1*02:01 sequence depicted inFIG. 11 . A MAPP may comprise: i) an MHC a chain polypeptide comprising an aa sequence having at least 95% or at least 98%, aa sequence identity to the a1 and o2 domains (aas 1-181) of the DQA1*05:01 sequence depicted in FIG. 9, and ii) an MHC p chain polypeptide comprising an aa sequence having at least 95%, or at least 98% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DQB1*02:01 sequence depicted in FIG. 11. DQA1*03:01-DQB1*03:02 (DQ8)

[0264] DQA1*03:01-DQBr03:02 (DQ8) is associated with an increased risk of developing celiac disease. Thus, a MAPP may comprise a DQA1*03:01 polypeptide comprising an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the a1 and o2 domains (aas 1-181) of the DQA1*03:01 aa sequence depicted in FIG. 9. A MAPP may comprise a DQA1*03:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the a1 domain of the DQA1*03:01 aa sequence depicted in FIG. 9. A MAPP may comprise a DQA1*03:01 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the o2 domain of the DQA1*03:01 aa sequence depicted in FIG. 9.

[0265] A MAPP may comprise a DQB1*03:02 polypeptide comprising an aa sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the p1 and p2 domains (aas 1-188) of the DQB1*03:02 aa sequence set forth in FIG. 11. A MAPP may comprise a DQB1*03:02 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p1 domain of the DQB1*03:02 aa sequence set forth in FIG. 11. A MAPP may comprise a DQB1*03:02 polypeptide comprising an aa sequence having at least 90% or at least 95% aa sequence identity to the p2 domain of the DQB1*03:02 aa sequence set forth depicted in FIG. 11.DRB1*04:01 and DRA1*01 :01

[0266] A MAPP may comprise: I) an MHC a chain polypeptide comprising an aa sequence having at least 90% or at least 95% (e.g., at least 96%, at least 98%, at least 99%, or 100%) aa sequence identity to the a1 and o2 domains (aas 1-181) of the DRA1*01 :01 aa sequence provided in FIG. 4 (e.g., 90% or 95% sequence identity to SEQ ID NO: 19) and ii) an MHC p chain polypeptide comprising an aa sequence having at least 90% or at least 95% (e.g., at least 96%, at least 98%, at least 99%, or 100%) aa sequence identity to the p1 and p2 domains (aas 1-188) of the DRB1*04:01 aa sequence depicted in FIG 5 (e.g., 90% or 95% sequence identity to SEQ ID NO:26). A MAPP may comprise: I) a DRA1*01:01 a chain polypeptide, and ii) a DRB1*04:01 p chain polypeptide.DQA1*03:01 and DQB1*03:02

[0267] A MAPP may comprise: I) an MHC a chain polypeptide comprising an aa sequence having at least 90% or at least 95% (e.g., at least 96%, at least 98%, at least 99%, or 100%) aa sequence identity to the a1 and o2 domains (aas 1-181) of the DQA1*03:01 sequence depicted in FIG. 9 and ii) an MHC p chain polypeptide comprising an aa sequence having at least 90% or at least 95% (e.g., at least 96%, at least 98%, at least 99%, or 100%) aa sequence identity to the p1 and p2 domains (aas 1-188) of the DQB1*03:02 sequence depicted in FIG. 11.

[0268] A MAPP may comprise an MHC Class II a- and / or p- chain allele sequence that is associated with increased risk of developing T 1 D and / or celiac disease, such as where the patient or subject to be treated with the MAPP expresses the MHC Class II a- and / or p- chain allele.5. Immunomodulatory polypeptides

[0269] A MAPP may comprise one or more immunomodulatory polypeptides or "MODs.” MODs that are suitable for inclusion in a MAPP include, but are not limited to, 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), lymphotoxin beta receptor, 3 / TR6, CX40L (CD252), PD-L1, PD-L2, TGF-p1, TGF-p2, TGF-p3, 4-1 BBL, and fragments of any thereof, such as ectodomain fragments, capable of engaging and signaling through their cognate receptor (“co-MOD”). MODs also may include antibodies or antibody fragments that are capable of interacting with receptors on T cells (e.g., other than the TOR itself). In some cases, the MODs induce responses such as proliferation, activation, and / or differentiation. In some cases, the MODs induce responses such as suppression / inhibition of proliferation, activation and / or differentiation. In some cases, the MODs can induce the formation, activation and / or proliferation of Tregs. Some MODs suitable for inclusion in MAPPs and their co-MODS include polypeptide sequences with T cell modulatory activity from the protein pairs recited in Table 3:Table 3: Exemplary Pairs of MODs and Co-MODs

[0270] In some cases, the MOD is selected from an IL-2 polypeptide, a 4-1 BBL 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 TGFp polypeptide, and a PD-L2 polypeptide. In some cases, the MAPP or duplex MAPP comprises two different MODs, such as an IL-2 MOD or IL-2 variant MOD and either a CD80 or CD86 MOD. In another instance, the MAPP or duplex MAPP comprises a wild-type (wt.) or variant IL-2 MOD and a TGF-p MOD. In another instance, the MAPP or duplex MAPP comprises an IL-2 MOD or IL-2 variant MOD and a PD-L1 MOD. In some case MODs, which may be the same or different, are present in a MAPP or duplex MAPP in tandem. When MODs are presented in tandem, their sequences are immediately adjacent to each other on a single polypeptide, either without any intervening sequence or separated by only a linker polypeptide (e.g., no MHO sequences or epitope sequences intervene). The MOD may comprise all or part of the extracellular portion of a full- length MOD. Thus, for example, the MOD can in some cases exclude one or more of a signal peptide, a transmembrane domain, and an intracellular domain normally found in a naturally occurring MOD. Unless statedotherwise, a MOD present in a MAPP or duplex MAPP does not comprise the signal peptide, intracellular domain, or a sufficient portion of the transmembrane domain to anchor a substantial amount (e.g., more than 5% or 10%) of a MAPP or duplex MAPP into a mammalian cell membrane.

[0271] In some cases, a MOD suitable for inclusion in a MAPP comprises all or a portion of (e.g., an extracellular portion of) the aa sequence of a naturally occurring MOD. In other instances, 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 instances, a variant MOD exhibits a binding affinity for a co-MOD that is lower than the affinity of a corresponding naturally occurring MOD (e.g., a MOD not comprising the aa substitution(s) present in the variant) for the co-MOD. Suitable variations in MOD sequences that alter affinity may be identified by scanning (making aa substitutions e.g., alanine substitutions or "alanine scanning,” or charged residue changes) along the length of a peptide, followed by testing the affinity of the resulting variants. Once key aa positions altering affinity are identified, those positions can be subject to a vertical scan in which the effect of one or more aa substitutions other than alanine are tested. a) MODs and Variant MODs

[0272] A MOD may comprise a wt. aa sequence, or it may be a variant MOD that comprises, e.g., one or more (e.g., 1-20) aa substitutions, insertions, and / or deletions relative to a wt. aa sequence. The MOD may comprise only the extracellular portion of a full-length immunomodulatory polypeptide. Alternatively, a MOD can comprise all or a portion of (e.g., an extracellular portion of) the aa sequence of a naturally occurring MOD. A variant MOD may comprise 1-5 or 5-20 aa substitutions, insertions, and / or deletions relative to its wt. MOD aa sequence (e.g., the sequence of the wt. MOD'S extracellular domain).

[0273] Variant MODs comprise at least one aa substitution, addition and / or deletion as compared to the aa sequence of a naturally occurring immunomodulatory polypeptide. As noted above, in some instances a variant MOD exhibits a binding affinity for a co-MOD that is lower than the affinity of a corresponding naturally occurring MOD (e.g., an immunomodulatory polypeptide not comprising the aa substitution(s) present in the variant) for the co-MOD.

[0274] MODs and variant MODs, including reduced affinity variants of proteins such as PD-L1, CD80, CD86, 4- 1 BBL and IL-2 are described in the published literature. For example, published PCT application WO 2020 / 132138 A1 describes MODs and specific variants of MODs, including PD-L1, CD80, CD86, 4-1 BBL, and IL-2 MODs described in paragraphs

[0260] -

[0455] , which are hereby incorporated by reference.

[0275] Suitable MODs that exhibit reduced affinity for a co-MOD can have from 1 aa to 20 aa differences from a wt. MOD. For example, in some cases, a variant MOD present in a MAPP may include a single aa substitution compared to a corresponding reference (e.g., wt.) MOD. A variant MOD present in a MAPP may include 2 aa substitutions compared to a corresponding reference (e.g., wt.) MOD. A variant MOD present in a MAPP may include 3 or 4 aa substitutions compared to a corresponding reference (e.g., wt.) MOD. A variant MOD present in a MAPP may include 5 or 6 aa substitutions compared to a corresponding reference (e.g., wt.) MOD. A variant MOD present in a MAPP may include 7, 8, 9, or 10 aa substitutions compared to a corresponding reference (e.g., wt.) MOD. A variant MOD present in a MAPP may include 11-15 or 15-20 aa substitutions compared to a corresponding reference (e.g., wt.) MOD.

[0276] As discussed above, a variant MOD suitable for inclusion in a MAPP may exhibit reduced affinity for a cognate co-MOD, compared to the affinity of a corresponding wt. MOD for the cognate co-MOD.

[0277] Binding affinity between a MOD sequence and its cognate co-MOD can be determined by bio-layer interferometry (BLI) using the purified MOD sequence and purified cognate co-MOD, following the procedure set forth in published PCT Application WO 2020 / 132138 A1. b) Masked TGF- and its variants

[0278] As discussed above, a MAPP may comprise at least one TGF-p polypeptide reversibly masked by a polypeptide (a "masking polypeptide”) that binds to the TGF-p polypeptide, which together form a masked TGF-p MOD. The masking polypeptide can be, for instance, a TGF-p receptor polypeptide or an antibody that functions to reversibly mask the TGF-p polypeptide present in the MAPP, where the TGF-p polypeptide is otherwise capable of acting as an agonist of a cellular TGF receptor. The masked TGF-p MODs provide active TGF-p polypeptides (e.g., TGF-p signaling pathway agonists). The TGF-p polypeptides and masking polypeptides (e.g., a TGF-p receptor fragment) interact with each other to reversibly mask the TGF-p polypeptide, thereby permitting the TGF-p polypeptide to interact with its cellular receptor. In addition, the masking sequence competes with cellular receptors that can scavenge TGF-p, such as the non-signaling TpRIII, thereby permitting the TGF-p MOD (and thus the MAPP) to effectively deliver active TGF-p agonist to target cells. While the MAPP constructs discussed herein permit epitope-specific presentation of a reversibly masked TGF-p to a target T cell, they also provide sites for the presentation of one or more additional MODs (e.g., IL-2). The ability of the MAPP construct to include one or more additional MODs thus permits the combined presentation of TGF-p and the additional MOD(s) to direct a target T cell's response in a substantially epitope-specific / selective manner in order to provide modulation of the target T cell. The MAPP thereby permits delivery of one or more masked TGF-p MODs in an epitope-selective (e.g., dependent / specific) manner that permits (I) formation of an active immune synapse with a target T cell, such as a CD4+ cell selective for the epitope, and (II) modulation (e.g., control / regulation) of the target T cell's response to the epitope. Once engaged with the TOR of a T cell, the effect of a masked TGF-p 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) is / are present.

[0279] As discussed herein, the masking polypeptide may be on the same polypeptide chain as the TGF-p polypeptide, in which case the TGF-p polypeptide and masking polypeptide together may be referred to as a "masked TGF-p construct” or present in a “cis" configuration (see FIG. 21, structure K), or on a separate polypeptide chain from the TGF-p polypeptide, in which case the TGF-p polypeptide and masking polypeptide together may be referred to as a "masked TGF-p complex” or present in a "trans” configuration (see FIG. 21, structure L). Such masked TGF-p constructs and complexes are described in US Patent No. 11,692,018, the disclosure of which as it pertains to masked TGF-p constructs and complexes is specifically incorporated herein by reference.

[0280] Further, although the MAPPs of this disclosure may comprise both one or more masked TGF-p MODs and one or more additional MODs (e.g., wt. or variant IL-2, PD-L1, IL-10, and / or 4-1 BB polypeptide aa sequences), if desired, the MAPPs of this disclosure may comprise only one or more masked TGF-p MODs. That is, the one or more additional MODs such as wt. or variant IL-2, PD-L1 and / or IL-10 MODs need not be included in a MAPP of this disclosure along with a masked TGF-p MOD. The masked TGF-p MOD-containing MAPPs can function as a meansof producing TGF-p-driven T cell responses. For example, TGF-p by itself can inhibit the development of effector cell functions of T cells, activate macrophages, and / or promote tissue repair after local immune and inflammatory actions subside.

[0281] Although masked TGF-p MODs comprise a TGF-p polypeptide that is masked, the TGF-p polypeptide can still act as a T|3R agonist because the TGF-p polypeptide-mask complex is reversible and "breathes” between an open state, where the TGF-p polypeptide is available to cellular receptors, and a closed state, where the mask engages the TGF-p polypeptide. The masking of the TGF-p polypeptide is reversible as a non-cleavable linker joins the mask to the TGF-p polypeptide or another peptide of the MAPP. That non-cleavable linker is not subject to site specific proteases (e.g., that give rise to a single cleavage in the linker) whose action on the linker would permit the mask to diffuse away from the TGF-p polypeptide. Accordingly, the masking polypeptide, which remains attached to the MAPP, functions to bind TGF-p polypeptide and prevent it from entering into tight complexes with, for example, ubiquitous non-signaling TpRIII molecules that can scavenge otherwise free TGF-p. Moreover, because the active forms of TGF-p are dimers that have higher affinity for TpRIII, substitutions that limit dimerization (e.g., a C77S substitution of the cysteine at position 77 with a serine) can be incorporated into TGF-p sequences in order to avoid scavenging by that receptor.

[0282] One effect of the masking sequence is to reduce the effective affinity of TGF-|31, TGF-|32, and TGF-|33 polypeptides for T|3Rs. At the same time, the affinity of the masking polypeptide for the TGF-p polypeptide can be altered so that it dissociates more readily from the TGF-p polypeptide, making the TGF-p polypeptide more available to cellular T|3R proteins. That is, where the affinity of a masking polypeptide for a TGF-p polypeptide is reduced, the masked TGF-p MOD will spend more time in the open state. Although the TGF-p polypeptide is available for binding to cellular receptors in the open state, the affinity for TGF-p for the heteromeric TpRI / TpRI I signaling complex is governed by TpRII interactions with TGF-p because TpRII generally interacts with TGF-p first, and therefore effectively controls entry of TGF-p into active signaling complexes. The incorporation of a substitution at, for example, one or more, two or more, or all three of Lys 25, lie 92, and / or Lys 94 of TGF-|32 (or the corresponding positions of TGF- 1, TGF-|33) reduces affinity for TpRII polypeptides. The reduced affinity permits interactions between the target cell's TCR and the MAPP's MHC polypeptides and peptide epitope to effectively control binding and allows for target cell-specific interactions.

[0283] When a TpRII polypeptide is used as the masking polypeptide, the possibility of direct interactions with cellular T|3RI receptors and off -target signaling can be addressed by appropriate modifications of the masking sequence. Where it is desirable to block / limit signaling by the masked TGF-p polypeptide through T|3RI and / or modify (e.g., reduce) the affinity of a masking TpRII polypeptide for TGF-p, it is possible to incorporate N-terminal deletions and / or aa substitutions in the masking TpRII polypeptide. Modifications that can be made include deletions of N- terminal aas (e.g., N-terminal A14 or A25 deletions), and / or substitutions at one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific T|3RI I modifications resulting in a reduction in T|3RI association with TpRII and reduced affinity for TGF-p include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q.

[0284] The TGF-p polypeptide present in a MAPP is in some cases a variant TGF-p polypeptide, including a variant TGF-p polypeptide that has a lower affinity for at least one class of TGF-p receptors, or is selective for at least one class of TGF-p receptors, compared to a wt. TGF-p polypeptide.

[0285] While a TGF-p1 polypeptide, a TGF-p2 polypeptide, or a TGF-p3 polypeptide can be incorporated into a MAPP as part of a masked TGF-p polypeptide, a variety of factors may influence the choice of the specific TGF-p polypeptide, and the specific sequence and aa substitutions that will be employed. For example, TGF-p1 and TGF-p3 polypeptides are subject to "clipping” of their aa sequences when expressed in certain mammalian cell lines (e.g., CHO cells). In addition, dimerized TGF-p (e.g., TGF-|32) has a higher affinity for the TpRIII (beta glycan receptor) than for the TpRII receptor, which could lead to off target binding and loss of biologically active masked protein to the large in vivo pool of non-signaling TpRIII molecules. To minimize high-affinity off target binding to TpRIII, it may be desirable to substitute the residues leading to dimeric TGF-p molecules, which are joined by a disulfide bond. Accordingly, cysteine 77 (C77) may be substituted by an aa other than cysteine (e.g., a serine forming a C77S substitution).

[0286] Amino acid sequences of TGF-p polypeptides are known in the art. In some cases, the TGF-p polypeptide present in a masked TGF-p polypeptide is a TGF-|31 polypeptide. In some cases, the TGF-p polypeptide present in a masked TGF-p polypeptide is a TGF-|32 polypeptide. In some cases, the TGF-p polypeptide present in a masked TGF-p polypeptide is a TGF-|33 polypeptide.

[0287] A suitable TGF-p polypeptide can have a length from about 70 aas to about 125 aas, for example, a suitable TGF-p polypeptide can have a length 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-p polypeptide can comprise an aa sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 80, at least 90, at least 100, or at least 110 contiguous aas of the mature form of a human TGF-|31 polypeptide, a human TGF-|32 polypeptide, or a human TGF-|33 polypeptide.(1) TGF- 1 polypeptides

[0288] A suitable TGF-|31 polypeptide may comprise an aa sequence having at least 85%, 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, at least 110, or 112 aas of the following TGF-|31 aa sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO: 188), where the TGF-|31 polypeptide has a length of about 112 aas. A suitable TGF-|31 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas (e.g., all aas) of SEQ ID NO:188. A suitable TGF-|31 polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO: 188. A suitable TGF-|31 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:188. A TGF-|31 preproprotein is provided in FIG. 28 as SEQ ID NO: 141. Aas R25, C77, V92 and R94 are bolded and italicized. See FIG. 28.

[0289] In some cases, a suitable TGF-|31 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-|31 polypeptide comprises an aa sequence having at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-p1 aa sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPSCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO: 189), where aa 77 is Ser. A suitable TGF-p1 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas of SEQ ID NO: 189. Positions 25, 77, 92 and 94 are bolded and italicized.(2) TGF- 2 polypeptides

[0290] A suitable TGF-p2 polypeptide can comprise an aa sequence having at least 85%, 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, at least 110, or 112 aas of the following TGF-p2 aa sequence: ALDAAYCFR NVQDNCCLRP LYIDFKRDLG WKWIHEPKGY NANFCAGACP YLWSSDTQHS RVLSLYNTIN PEASASPCCV SQDLEPLTIL YY / GKTPKIE QLSNMIVKSC KGS (SEQ ID NO: 190), where the TGF-p2 polypeptide has a length of about 112 aas. A suitable TGF-p2 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas (e.g., all aas) of SEQ ID NQ:190. A suitable TGF-p2 polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO: 190. A suitable TGF-p2 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NQ:190. A TGF-p2 preproprotein is provided in FIG. 28 as SEQ ID NO:142. Residues Lys 25, Cys 77, lie 92, and Lys 94, are bolded and italicized.

[0291] In some cases, a suitable TGF-p2 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-p2 polypeptide comprises an aa sequence having at least 85%, 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, at least 110, or 112 aas of the following TGF-p2 aa sequence: ALDAAYCFR NVQDNCCLRP LYIDFKRDLG WKWIHEPKGY NANFCAGACP YLWSSDTQHS RVLSLYNTIN PEASASPSCV SQDLEPLTIL YYIGKTPKIE QLSNMIVKSC KCS (SEQ ID NO: 191 ), which is SEQ ID NQ:190 in which Cys 77 is substituted by a Ser (C77S) that is bolded and italicized. A suitable TGF-p2 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas of SEQ ID NO: 191.(3) TGF- 3 polypeptides

[0292] A suitable TGF-p3 polypeptide can comprise an aa sequence having at least 85%, 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, at least 110, or 112 aas of the following TGF-p3 aa sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMWKSCK CS (SEQ ID NO: 144), where the TGF-p3 polypeptide has a length of about 112 aas. A suitable TGF- p3 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas (e.g., all aas) of SEQ ID NO:144. A suitable TGF-p3 polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO: 144. A suitable TGF-p3 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:144. A TGF-p3 isoform 1 preproprotein is provided in FIG. 28 as SEQ ID NO:143. Positions 25, 77, 92 and 94 are bolded and italicized.

[0293] In some cases, a suitable TGF-p3 polypeptide comprises a C77S substitution. In some cases, a suitable TGF-p3 polypeptide comprises an aa sequence having at least 85%, 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, at least 110, or 112 aas of the following TGF-p3 aa sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPSCVP QDLEPLTILY YVGRTPKVEQ LSNMWKSCK CS (SEQ ID NO:145), where aa 77 is Ser. Positions 25, 77, 92 and 94, are bolded and italicized. A suitable TGF-p3 polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 100 or at least 110 aas of SEQ ID NO: 145.(4) Additional TGF- polypeptide sequence variations

[0294] In addition to sequence variations that alter TGF-p molecule dimerization (e.g., cysteine 77 substitutions such as C77S), TGF-p1, TGF-p2, and TGF-p3 polypeptides having sequence variations that affect affinity and other properties may be incorporated into a masked TGF-p MOD. When a variant TGF-p with reduced affinity for the masking polypeptide (e.g., a TpR polypeptide such as a TpRII polypeptide) is present in the masked TGF-p MOD those components dissociate more readily, making the TGF-p polypeptide more available to cellular TpR proteins. Because the TpRII protein is generally the first peptide of the heteromeric TpR signaling complex to interact with TGF-p, interactions with TpRII effectively controls entry of TGF-p into active signaling complexes. Accordingly, variants controlling the affinity of TGF-p for TpRII may effectively control entry of masked TGF-p MODs into active signaling complexes.

[0295] The present disclosure includes and provides for masked TGF-p MODs comprising a variant masking TpR (e.g., TpRII) polypeptide sequence and / or a variant TGF-p polypeptide having altered (e.g., reduced) affinity for each other (relative to an otherwise identical masked TGF-p MOD without the sequence variation(s). Affinity between a TGF-p polypeptide and a TpR (e.g., TpRII) polypeptide may be determined using BLI as described above for MODs and their co-MODs.(a) Additional TGF- 2 sequence variants

[0296] The present disclosure includes and provides for masked TGF-p2 MODs comprising a masking TpR (e.g., TpRII) polypeptide sequence and either a wt. or a variant TGF-p2 polypeptide, where the variant polypeptide has a reduced affinity for the masking TpR (relative to an otherwise identical wt. TGF-p polypeptide sequence without the sequence variations).

[0297] The disclosure provides for masked TGF-p MODs that comprise a masking TpRII receptor sequence and a variant TGF-p2 polypeptide having at least 85% or at least 90%, (e.g., 95%, 98% or 99%) sequence identity to at least 100 contiguous aas of SEQ ID NO: 142, and comprising a substitution reducing the affinity of the variant TGF-p2 polypeptide for the TpRII receptor sequence. The TGF-p2 polypeptide may have at least 95% or at least 98% aa sequence identity to at least 110 aas of SEQ ID NO: 142.

[0298] In some cases, a masked TGF-p MOD comprises a masking TpRII polypeptide and a variant TGF-p (e.g., TGF-p2) polypeptide comprising a substitution at one or more, two or more, or all three of Lys 25, lie 92, and / or Lys 94 (see the mature form of TGF-p2 in SEQ ID NO: 190 for the location of the residues, and SEQ ID NO:190 in FIG. 28 for the corresponding residues in preprotein and the mature forms of TGF-p1 and TGF-p3). Those aa residues have been shown to affect the affinity of TGF-p2 for TpRII polypeptides (see De Crescenzo et al., J. Mol. Biol. 355:47-62 (2006)). The MAPP optionally comprises one or more independently selected MODs such as IL-2 or a variant thereof. In one instance, the masked TGF-p MOD comprises a masking TpRII polypeptide and a TGF-p2 polypeptide having an aa other than Lys or Arg at position 25 of SEQ ID NO: 190, with the MAPP optionally comprising one or more additional independently selected MODs (e.g., one or more IL-2 MODs or reduced affinity variants thereof). A masked TGF-p MOD with a masking TpRII polypeptide may comprise a TGF-p2 polypeptide having an aa other than lie or Vai at position 92 of SEQ ID NO:190 (or an aa other than lie, Vai, or Leu at position 92), with the MAPP optionally comprising one or more additional independently selected MODs (e.g., one or more IL-2 MODs or reduced affinity variants thereof). A masked TGF-p MOD with a masking TpRII polypeptide may comprise a TGF-p2 polypeptide having an aa other than Lys or Arg at position 94 of SEQ ID NO: 190, with the MAPP optionally comprising one or more additional independently selected MODs (e.g., one or more IL-2 MODs or reduced affinity variants thereof). A masked TGF-p MOD with a masking TpRII polypeptide may comprise a TGF-p2 polypeptide comprising a substitution at one or more, two or more or all three of Lys 25, lie 92, and / or Lys 94, with the MAPP optionally comprising one or more additional independently selected MODs. A masked TGF-p MOD with a masking TpRII polypeptide may comprise a TGF-p2 polypeptide comprising a substitution at one or more, two or more or all three of Lys 25, lie 92, and / or Lys 94, with the MAPP optionally comprising one or more independently selected IL-2 MODs or reduced affinity variants thereof. Any of those TGF-p2 MOD aa sequences may further comprise a C77S substitution.(b) Additional TGF- 1 and TGF- 3 sequence variants and placement in tandem

[0299] In some cases, a masked TGF-p MOD comprises a masking TpRII polypeptide and a variant TGF-p1 or TGF-p3 polypeptide comprising a substitution at one or more, two or more or all three aa positions corresponding to Lys 25, lie 92, and / or Lys 94 in the mature TGF-p2 polypeptide of SEQ ID NO:142 (SEQ ID NO:190). In the mature TGF-p1 or TGF-p3 polypeptides, the aa that corresponds to: Lys 25 is Arg 25, lie 92 is Vai 92, and Lys 94 is Arg 94, each of which is a conservative substitution. See, e.g., SEQ ID NOs:141, 146, 188, and 189 for TGF-p1 , and SEQ ID NOs:143, 144, and 145 for TGF-p3.

[0300] As noted above, the masked TGF-p MOD optionally comprises one or more independently selected MODs such as IL-2 or a variant thereof. In one instance, the masked TGF-p MOD with a masking TpRII polypeptide comprises a TGF-p1 or p3 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 MODs or reduced affinity variants thereof). In one instance, the masked TGF-p MOD with a masking TpRII polypeptide comprises a TGF-p1 or p3 polypeptide having an aa other than Vai or lie at position 92 (or an aa other than lie, Vai, or Leu at position 92), and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MODs or reduced affinity variants thereof). In another instance, the masked TGF-p MOD with a masking TpRII polypeptide comprises a TGF-p2 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 MODs or reduced affinity variants thereof). In one specific instance, a masked TGF-p MOD with a masking TpRII polypeptide comprises a TGF-p1 or p3 polypeptide comprising a substitution at one or more, two or more or all three of Arg 25, Vai 92, and / or Arg 94, and further comprises one or more independently selected MODs (e.g., IL-2 or variant IL-2 MODs). In another specific instance, a masked TGF-p MOD with a masking TpRIIpolypeptide comprises a TGF-p1 or p3 polypeptide comprising a substitution at one or more, two or more or all three of Arg 25, Vai 92, and / or Arg 94, and further comprises one or more independently selected IL-2 MODs, or reduced affinity variants thereof.(5) TGF-P receptor polypeptides and other polypeptides that bind and mask TGF-p

[0301] In any of the above-mentioned TGF-p polypeptides or polypeptide complexes the polypeptide that binds to and masks the TGF-p polypeptide (the "masking polypeptide”) can take a variety of forms, including fragments of TpRI, TpRII, TpRI 11 and anti-TGF-p antibodies or antibody-related molecules (e.g., antigen binding fragment of an antibody, Fab, Fab', single chain antibody, scFv, peptide aptamer, or nanobody).(a) TGF-P Receptor Polypeptides

[0302] The masking of TGF-p in masked TGF-p MODs may be accomplished by utilizing a TGF-p receptor fragment (e.g., the ectodomain sequences of TpRI, TpRII or TpRII I) that comprises polypeptide sequences sufficient to bind a TGF-p polypeptide (e.g., TGF-p1, TGF-p2 or TGF-p3). In an embodiment, the masking sequence comprises all or part of the TpRI, TpRII, or TpRI II ectodomain.(I) TGF-p Receptor I (TpRI)

[0303] The polypeptide sequence masking TGF-p in a masked TGF-p MODs may be derived from a TpRI (e.g., isoform 1, SEQ ID NO: 147, see FIG. 29A) and may comprise all or part of the TpRI ectodomain (aas 34-126). A suitable TpRI polypeptide for masking TGF-p may comprise an aa sequence having at least 85% or at least 90% (e.g., at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 70, at least 80, or least 90 aas of the following TpRI ectodomain aa sequence: LQCFCHL CTKDNFTCVT DGLCFVSVTE TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYC CNQDHCNKIE LPTTVKSSPG LGPVEL (SEQ ID NO: 148). A suitable TpRI polypeptide for masking TGF-p may comprise an aa sequence having at least 95% or 100% aa sequence identity to at least 80 or all aas of SEQ ID NO:148. A suitable TpRI polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO: 148. A suitable TpRI polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:148.(ii) TGF-p Receptor II (TpRII)

[0304] A polypeptide sequence masking TGF-p in a masked TGF-p MOD may be derived from a TpRII (e.g., isoform A, SEQ ID NO:149) and may comprise all or part of the TpRII ectodomain sequence (aas 24 to 177). A suitable TpRII isoform A polypeptide for masking TGF-p may comprise an aa sequence having at least 85% or at least 90% (e.g., 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, at least 110, at least 120, at least 130, at least 140, at least 150 or at least 154 aas of the following TpRII isoform A ectodomain aa sequence: IPPHVQK SDVEMEAQKD EIICPSCNRT AHPLRHINND MIVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEE (SEQ ID NQ:150). The location of the aspartic acid residue corresponding to D118 in the B isoform is bolded and italicized. A suitable TpRII polypeptide for masking TGF-p may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 140 contiguous aas or all aas of SEQ ID NQ:150. A suitable TpRII polypeptide may comprise an aa sequence havingat least 85% or at least 90% aa sequence identity to SEQ ID NO:150. A suitable TpRII polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO: 150.

[0305] A polypeptide sequence masking TGF-p in a masked TGF-p MOD may be derived from TpRII isoform B (SEQ ID NO: 151 ) and may comprise all or part of the TpRII ectodomain sequence (aas 24 to 166). A suitable TpRII isoform B polypeptide for masking TGF-p may comprise an aa sequence having at least 85% or at least 90% (e.g., 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, at least 110, at least 120, at least 130, at least 140, or 143 aas of the TpRII isoform B ectodomain aa sequence: IPPHVQKSVN NDMIVTDNNG AVKFPQLCKFCDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNI IFSEEY NTSNPDLLLV IFQ (SEQ ID NO:152). A suitable TpRII isoform B polypeptide for masking TGF-p may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 120, at least 130, at least 140, or 143 aas of SEQ ID NO: 152. A suitable TpRII isoform B polypeptide for masking TGF-p may comprise an aa sequence having at least 95% or at least 98% aa sequence identity to at least 130 or 140 aas of SEQ ID NO:152. A suitable TpRII polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO:152. A suitable TpRII polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:152. As discussed below, any one or more of F30, D32, S52, E55, and / or D118 (italicized and bolded) may be substituted by an aa other than the aa occurring at those positions in the sequence provided (e.g., alanine). A polypeptide sequence masking TGF-p may comprise the polypeptide of SEQ ID NO: 152 bearing a D118A or D118R substitution. Accordingly, a suitable TpRII polypeptide for masking TGF-p may comprise the sequence IPPHVQKSVN NDMIVTDNNG AVKFPQLCKFCDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSXEC NDNI IFSEEY NTSNPDLLLV IFQ, where X is A or R (SEQ ID NO:343). A sequence masking TGF-p may comprise a sequence having at least 95% or at least 98% sequence identity to SEQ ID NO:343. A sequence masking TGF-p may comprise the peptide of SEQ ID NO:343 bearing a D118A or D118R substitution and one or more of F30A, D32N, S52L and / or E55A substitutions.

[0306] Although TpRII's ectodomain may be utilized as a masking polypeptide, that region of the protein has charged and hydrophobic regions (e.g., patches) that can lead to unfavorable isoelectric points (pl values) and can be toxic to cells expressing the polypeptide. In addition, combining a TpRII ectodomain with an active TGF-p polypeptide can result in a complex that could combine with cell surface TpRI and cause activation of that signaling receptor (e.g., signaling through the Smad pathway). Modifying TpRII ectodomain sequences used to mask TGF-p by removing or altering sequences involved in TpRI association can avoid the unintentional stimulation of cells by the masked TGF-p except through their own cell surface heterodimeric TpRI / TpRI I complex. Modifications of TpRII may also alter (e.g., reduce) the affinity of the TpRII for TGF-p (e.g., TGF-p3), thereby permitting control of TGF-p unmasking and its availability as a signaling molecule. Masked TGF-p MODs comprising TpR (e.g., TpRII) peptides with the highest affinity for TGF-p (e.g., TGF-p3) most tightly mask the TGF-p sequence and require higher doses to achieve the same effect. In contrast, aa substitutions in TpRII that lower the affinity unmask the TGF-p polypeptide and are biologically effective at lower doses.

[0307] Accordingly, where it is desirable to block / limit signaling by the masked TGF-p polypeptide through TpRI and / or modify (e.g., reduce) the affinity of a masking TpRII polypeptide for TGF-p, a number of alterations to TpRIImay be incorporated into the TpRII polypeptide sequence. Modifications that can be made include the above- mentioned deletions of N-terminal aas, such as 14 or 25 N-terminal aas (from 1 to 14 aas or from 1 to 25 aas, A14, A25 modifications) and / or substitutions at one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific TpRII modifications resulting in a reduction in TpRI association with TpRII and reduced affinity for TGF-p 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:152. See, e.g, J. Groppe et al. Mol Cell 29, 157- 168 (2008), and De Crescenzo et al . J Mol Biol 355, 47-62 (2006), for the effects of those substitutions on TGF- p3- TpRI I and TpRI-TpRII complexes. Modifications of TpRII including an N-terminal A25 deletion and / or substitution at F24 (e.g., an F24A substitution) substantially or completely block the signal through the canonical SMAD signaling pathway. In one aspect, the aspartic acid at position 118 (D118) of the mature TpRII B isoform sequence (SEQ ID NO:152) is replaced by an aa other than Asp or Glu, such as Ala, giving rise to a D118A substitution or by an Arg giving rise to a D118R substitution. The Asp residues corresponding to D118 are indicated in FIG. 29B, SEQ ID NO:149 to SEQ ID NO: 156, with bold and underlining. N-terminal deletions of from 1 to 25 aas in length (e.g., a A25 deletion) and / or substitution at F24 (e.g., an F24A substitution) may be combined with D118 substitutions (e.g., D118A or D118R). N-terminal deletions of from 1 to 25 aas in length (e.g., a A25 deletion) and / or substitution at F24 (e.g., an F24A substitution) may also be combined with substitutions at any of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119 (e.g., D118A) substitutions, and particularly any of the specific substitutions recited for those locations in SEQ ID NO: 152 described above, to alter the affinity.

[0308] Deletions of the N-terminus of the TpRII polypeptides may also result in loss of TpRI interactions and prevent masked TGF-p MODs comprising a TpRII polypeptide from acting as a constitutively active complex that engages and activates TpRI signaling. A 14 aa deletion (A14) of the TpRII polypeptide substantively reduces the interaction of the protein with TpRI, and a A25 aa deletion of TpRII appears to completely abrogate the interaction with TpRI. N-terminal deletions also substantially alter the pl of the protein, with the A14 TpRII ectodomain mutant displaying a pl of about 4.5-5.0 (e.g., about 4.74). Accordingly, TGF-p MODs may comprise TpRII ectodomain polypeptides (e.g., polypeptides of SEQ ID NQ:150 or SEQ ID NO:152) with N-terminal deletions, such as from 14 to 25 aas, including, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aas. Modified ectodomain sequences, including those that limit interactions with TpRI, that may be utilized to mask TGF-p polypeptides in a masked TGF-p MOD are described in the paragraphs that follow.

[0309] In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 85% or at least 90% (e.g., at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 100, at least 110, at least 120, at least 125, or 129 aas of the TpRII isoform B ectodomain sequence: IPPHVQKSVN NDMIVTDNNG AVKFPQLCKFCDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO:153). In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 95% or at least 98% aa sequence identity to at least 110, at least 120, at least 125, or 129 aas of the TpRII isoform B ectodomain sequence of SEQ ID NO:153. A suitable TpRII polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO:153. A suitable TpRII polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:153. Any one or more of F30, D32, S52,E55, and / or D118 (italicized and bolded) may be substituted by an aa other than the aa occurring at those positions in the sequence provided (e.g., alanine). In an embodiment, the sequence masking TGF-p comprises the polypeptide of SEQ ID NO:153 bearing a D118A substitution. In an embodiment, the sequence masking TGF-p comprises the polypeptide of SEQ ID NO:153 bearing a D118A substitution and one or more of F30A, D32N, S52L and / or E55A substitutions.

[0310] N-terminal deletions of TpRII, such as from 14 to 25 aas, including, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aas, that block inadvertent cell signaling due to the masked TGF-p / TpRII complex interacting with TpRI may be combined with other TpRII ectodomain substitutions, including those at any one or more of F30, D32, S52, E55, and / or D118. The combination of deletions and substitutions ensures the masked TGF-p MOD does not cause cell signaling except through the cell's membrane bound TpRI and TpRII receptors.

[0311] In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 85% or at least 90% (e.g., at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 90, at least 100, at least 110, or 115 aas of the TpRII isoform B ectodomain sequence: VTDNNG AVKFPQLCKFCDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO: 192), which has aas 1-14 (A14) deleted. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 95% or at least 98% aa sequence identity to at least 110 or at least 114 aas of the TpRII isoform B ectodomain sequence of SEQ ID NO:192. A suitable TpRII polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO:192. A suitable TpRII polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:192. Any one or more of F30, D32, S52, E55, and / or D118 (italicized and bolded) may be substituted by an aa other than the aa occurring at those positions in the sequence provided (e.g., alanine). In an embodiment, the sequence masking TGF-p comprises the peptide of SEQ ID NO:192 bearing a D118A substitution (see, e.g., SEQ ID NO:154 in FIG. 29B). In an embodiment, the sequence masking TGF-p comprises the polypeptide of SEQ ID NO:192 bearing a D118A substitution and one or more of F30A, D32N, S52L and / or E55A substitutions.

[0312] In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 85% or at least 90% (e.g., at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 80, at least 90, at least 100, or 104 aas of the TpRII isoform B ectodomain sequence: QLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO: 193), which has aas 1-25 (A25) deleted. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 95% or at least 98% aa sequence identity to at least 90, or at least 100 aas of the TpRII isoform B ectodomain sequence of SEQ ID NO:193. A suitable TpRII polypeptide may comprise an aa sequence having at least 85% or at least 90% aa sequence identity to SEQ ID NO:193. A suitable TpRII polypeptide may comprise an aa sequence having at least 95% or 100% aa sequence identity to SEQ ID NO:193. Any one or more of F30, D32, S52, E55, and / or D118 (italicized and bolded) may be substituted by an aa other than the aa occurring at those positions in the sequence provided (e.g., alanine). In an embodiment, the sequence masking TGF-p comprises the polypeptide of SEQ ID NO:193 bearing a D118A substitution (shown as SEQ ID NO:155 in FIG. 29B). In an embodiment, thesequence masking TGF-p in a masked TGF-p MOD comprises the polypeptide of SEQ ID NO:193 bearing a D118A substitution and one or more of F30A, D32N, S52L and / or E55A substitutions. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises the polypeptide of SEQ ID NO:155 or SEQ ID NO:156 (see FIG. 29B) bearing D118A and F30A substitutions. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises the polypeptide of SEQ ID NO:155 or SEQ ID NO:156 (see FIG. 29B) bearing D118A and D32N substitutions. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises the polypeptide of SEQ ID NO:155 or SEQ ID NO:156 (see FIG. 29B) bearing D118A and S52L substitutions. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises the polypeptide of SEQ ID NO:155 or SEQ ID NO:156 (see FIG. 29B) bearing D118A and E55A substitutions.(iii) TGF-p Receptor III (TpRIII)

[0313] In an embodiment, the polypeptide sequence masking TGF-p in a masked TGF-p MOD may be derived from a TpRIII (e.g., isoform A, SEQ ID NO: 157, and isoform B, SEQ ID NO:158) and may comprise all or part of a TpRIII ectodomain (aasof the A isoform or 27-786 of the B isoform). In some cases, a suitable TpRIII polypeptide for masking TGF-p comprises an aa sequence having at least 85% or at least 90% (e.g., at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to the TpRIII isoform A or B ectodomain sequence. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 95% or at least 98% aa sequence identity to the TpRIII isoform A or B ectodomain sequence of SEQ ID NO:157 or 158. In an embodiment, the sequence masking TGF-p in a masked TGF-p MOD comprises an aa sequence having at least 95% or at least 98% aa sequence identity to at least 100, or 120 aas of a TpRIII A isoform or B isoform ectodomain sequence (e.g., provided in FIG. 29C as SEQ ID NO:157 or SEQ ID NO:158).(b) Antibodies

[0314] Although TGF-p receptor polypeptides (e.g., ectodomain sequences) can function to bind and mask TGF-p polypeptides in masked TGF-p MODs, other polypeptide sequences (protein sequences) that bind to TGF-p sequences can also be employed as masking polypeptides. Among the suitable polypeptide or protein sequences that can be used to mask TGF-p are antibodies with affinity for TGF-p (e.g., antibodies specific for one or more of TGF-p1, TGF-p2, or TGF-p3) or antibody-related molecules such as anti-TGF-p antibody fragments, nanobodies with affinity for TGF-p polypeptides, and particularly single chain anti-TGF-p antibodies (e.g., any of which may be humanized). Some antibodies, including scFV antibodies that bind and neutralize TGF-p, have been described. See, e.g., US 9,090,685. Throughout the embodiments and / or aspects of the invention described in the present disclosure, TpR (e.g., TpRII) sequences used to mask TGF-p polypeptides may be replaced with masking antibody sequences (e.g., an scFV or a nanobody) with affinity for the TGF-p polypeptide. For instance, in each of the masked TGF-p MODs in FIG. 21 (see structures K and L) where a TGF-p receptor sequence is used to mask a TGF-p polypeptide, the receptor polypeptide may be replaced with a masking antibody polypeptide (e.g., scFV or a nanobody) with affinity for the TGF-p polypeptide.

[0315] One potential advantage of using an antibody (e.g., a single chain antibody) as a masking polypeptide is the ability to limit it to the isoform of the TGF-p polypeptide(s) to be masked. By way of example, single chain antibody sequences based on Metelimumab (CAT192) directed against TGF-p1 (e.g., Lord et al., mAbs 10(3):444-452 (2018)) can be used to mask that TGF-p isoform when present in TGF-p MODs. In another embodiment, a single chainantibody sequence specific for TGF-p2 is used to mask that TGF-p isoform when present in TGF-p MODs. In another embodiment, a single chain antibody sequence specific for TGF-p3 is used to mask that TGF-p isoform when present in TGF-p MODs. Single chain antibodies can also be specific for a combination of TGF-p isoforms (e.g., ectodomain sequences appearing in masked TGF-p MODs selected from the group consisting of: TGF-p1 and TGF-p2; TGF-p1 and TGF-p3; and TGF-p2 and TGF-p3). The single chain antibodies may also be pan-specific for TGF-|31, TGF-|32, and TGF-p3 ectodomain sequences appearing in masked TGF-p MODs. See, e.g., WO 2014 / 164709. Antibodies and single chain antibodies that have the desired specificity and affinity for TGF-p isoforms can be prepared by a variety of methods, including screening hybridomas and / or modification (e.g., combinatorial modification) to the variable region sequence of antibodies that have affinity for a target TGF-p polypeptide sequence.

[0316] In an embodiment, a masked TGF-p MOD comprises a single chain antibody to mask a TGF-p sequence (e.g., a TGF-p3 sequence). In one such embodiment the single chain aa sequence is specific for the TGF-p3 set forth in SEQ ID NO:144 comprising a C77S substitution (see SEQ ID NO: 145). c) Placement of TGF- and TGF- masking sequence in MAPPs

[0317] The masking sequence (e.g., a TGF-p receptor sequence) of a masked TGF-p MOD may be part of the same polypeptide as the TGF-p sequence; that is, both the masking and TGF-p sequences are present in “cis." Alternatively, the masking sequence (e.g., a TGF-p receptor sequence) and the TGF-p sequence may be part of different polypeptides, which is to say they are present in “trans."

[0318] When the masking sequence and the TGF-p sequence of a masked TGF-p MOD are present in a single aa sequence (single polypeptide) of a MAPP (placed in cis, e.g., as in FIG. 21, structure K, which has two masked TGF- p MODs each in cis and shown in the closed state), the aa sequence may be arranged in the N-terminal to C-terminal direction as either: a) TGF-p receptor sequence(s) followed by TGF-p sequence(s), or b) TGF-p sequence(s) followed by TGF-p receptor sequence(s). Regardless of the order from N-terminus to C-terminus, the polypeptide sequence of a masked TGF-p MOD may be linked to any other MAPP polypeptide at its N-terminus or C- terminus. Independently selected linker polypeptide(s) (e.g., Gly4Ser repeats SEQ ID NO:218) may be used to join the masking sequence (e.g., a TGF-p receptor sequence) and the TGF-p sequence, and also to join the TGF-p MOD to a polypeptide of the MAPP (e.g., a framework polypeptide sequence). As an example, a cis-masked TGF-p MOD may be linked to the C terminus of a MAPP as a single aa sequence (polypeptide) and have the order from N- terminus to C-terminus a) TGF-p receptor sequence (e.g., a TpRII sequence) followed by TGF-p sequence (e.g., TGF-|33). To further that example, the c / s-masked TGF-p MOD may be linked to a framework polypeptide (e.g., C- terminal to the multimerization sequence; see, e.g., FIG. 21, structure K) and the c / s-masked TGF-p MOD may optionally be followed by another MOD such as IL-2.

[0319] One example of a masked TGF-p MOD with the T|3R and TGF-p in cis (a c / s-masked TGF-p MOD) is the sequence: QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSAECNDNII FSEEYNTSNP DGGGGSGGGG SGGGGSGGGG SGGGGSALDT NYCFRNLEEN CCVRPLYIDF RQDLGWKWVH EPKGYYANFC SGPCPYLRSA DTTHSTVLGL YNTLNPEASA SPSCVPQDLE PLTILYYVGR TPKVEQLSNM WKSCKCS (SEQ ID NO:194), where: aas 1-111 are a human TpRII masking sequence with the N-terminal 25 aas removed (A25) and a D118A substitution, aas 112-136 are a linker (five Gly4Ser repeats, see SEQ ID NO:218), and 137-248 is a human TGF-|33 sequence with a C77Ssubstitution. Such a sequence may be attached, for example, by its N-terminus, directly or indirectly via an independently selected linker to the C-terminus of a MAPP as a single aa sequence (polypeptide) (e.g., attached to a framework polypeptide C-terminal to the multimerization sequence). In addition, the cis masked TGF-p MOD sequence may have appended to it another MOD sequence (e.g., a human IL-2 or variant IL-2 MOD sequence).

[0320] When the masking sequence (e.g., TGF-p receptor sequence) and the TGF-p sequence of a masked TGF-p MOD are present as part of different MAPP polypeptides (placed in trans), those polypeptide sequences are attached to different (separate) MAPP polypeptides that interact, thereby pairing the TGF-p sequence with the masking polypeptide (e.g., a TGF-p receptor sequence). The TGF-p sequence and masking sequence may be located at the C-terminus of MAPP polypeptides (e.g., at the C-terminus of a framework sequence such as an Ig Fc framework; see FIG. 21, structure L, where the masked TGF-p MOD is shown in the open state). An independently selected linker polypeptide (e.g., Gly4Ser repeats, SEQ ID NO:218) may be used to join the masking sequence (e.g., TGF-p receptor sequence) or the TGF-p sequence to other MAPP polypeptides. As an example, in a trans-masked TGF-p MOD, a TGF-p receptor sequence (e.g., TpRII) may be located on one framework polypeptide of a duplex MAPP and the TGF-p sequence (e.g., TGF-|33) may be part of the second framework polypeptide, where the first and second framework polypeptides associate through interspecific multimerization sequences (see e.g., FIG. 21, structure L). To further that example, the TGF-p sequence and TGF-p receptor sequence may be located at the C-terminus framework polypeptide sequence (e.g., an Ig Fc sequence) and may optionally be followed by another MOD such as IL-2. By way of example, a duplex MAPP having first and second framework polypeptides with interspecific multimerization sequences (e.g., an interspecific Ig Fc sequence such as a KIH sequence pair) may have a masking T|3R sequence located at the C-terminus of a first framework polypeptide, and a TGF-p polypeptide (and optionally another MOD) located at the C-terminus of the second framework polypeptide sequence. The masking T|3R sequence of a TGF-p MOD of a MAPP may be a TpRII sequence lacking its N-terminal 25 aas (A25) and bearing a D118A substitution: QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFI LEDAASPKCIMKEKKKPGETFFMCSCSSAECNDNIIFSEEYNTSNPD (SEQ ID NO:156). The TGF-p polypeptide may be a human TGF-|33 polypeptide bearing a C77S substitution: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPSCVP QDLEPLTILY YVGRTPKVE...

Claims

CLAIMS1 . A multimeric antigen-presenting polypeptide complex (MAPP) comprising:(I) a framework polypeptide comprising a dimerization sequence and a multimerization sequence;(ii) a dimerization polypeptide comprising a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide, and dimerizing therewith through covalent and / or non-covalent interactions to form a MAPP heterodimer; and(ill) at least one presenting sequence; wherein each presenting sequence comprises as a single polypeptide from N-terminus to C-terminus:(a) an epitope aa sequence,(b) an L1 aa linker sequence,(c) an MHC Class II p chain polypeptide sequence comprising p1 and p2 domain sequences,(d) an optional L2 aa linker sequence,(e) an MHC Class II a chain polypeptide sequence comprising a1 and o2 domain sequences, and(f) an optional L3 aa linker sequence; and wherein(I) the at least one presenting sequence presenting sequence comprises a body disulfide bond between a cysteine substituted for one of the N-terminal 8 aas of the p1 domain and a cysteine substituted for one of the C-terminal 11 aas of the a1 domain, and / or a linker disulfide bond between the L1 linker and a cysteine substituted for one of the C-terminal 11 aas of the a1 domain;(ii) optionally, when the presenting sequence comprises a cysteine at aa 43 through aa 48 of the a chain polypeptide sequence (a1 and o2 domain sequence), it is substituted by an aa other than cysteine;(ill) at least one or both of the dimerization polypeptide and / or the framework polypeptide comprises a presenting sequence;(iv) optionally at least one of the framework polypeptide or dimerization polypeptide comprises one, two, three or more independently selected MOD and / or variant MOD sequences; and(v) the framework polypeptide, dimerization polypeptide, and / or presenting sequence optionally comprise one or more independently selected linker sequences.

2. The MAPP of claim 1 , wherein at least one presenting sequence comprises:(I) a1 and o2 domain polypeptide sequences each having at least 90% or at least 95% sequence identity to all or at least about 50 contiguous aas of an HLA DRA*01*01 or HLA DRA*01*02 polypeptide sequence; and(ii) p1 and p2 domain polypeptide sequences each having at least 90% or at least 95% sequence identity to at least about 60 contiguous aas of a DRB1*0301, DRB1*0401, DRB1*0402, DRB1*0405, DRB1*0801, or DRB1*0901 polypeptide sequence.

3. The MAPP of claim 2, wherein:(I) any one or more of positions 37, 49 and / or 72 of the DRA a1 domain are substituted, preferably wherein position 37 is an E, and / or position 49 is an H, and / or position 72 is an I (lie); and / or(ii) position 44 of the DRA a1 domain sequence(a) is an aa other than cysteine, or(b) when aa position 44 of the DRA a1 domain sequence is an arginine it is substituted by a serine or lysine.

4. The MAPP of claim 1 , wherein:I) the p1 and p2 domain sequences have at least 95% or at least 98% aa sequence identity to all or at least170 contiguous aas of a p1 and p2 domain sequence of DRB1*01 :01, DRB1*01 :02, DRB1*01 :03, DRB1*03:01, DRB1*03:02, DRB1*03:04, DRB1*04:01, DRB1*04:02, DRB1*04:03, DRB1*04:04, DRB1*04:05, DRB1*04:06, DRB1*04:08, DRB1*07:01, DRB1*08:01, DRB1*08:02, DRB1*08:03, DRB1*09:01, DRB1*10:01, DRB1*11 :01, DRB1*11 :03, DRB1*11:04, DRB1*12:01, DRB1*13:01, DRB1*13:03, DRB1*14:01, DRB1*14:02, DRB1*14:05, DRB1*14:06, DRB1*15:01, DRB1*15:02, DRB1*15:03, DRB1*15:04, DRB1*15:05, DRB1*15:06, DRB1*15:07, DRB1*16:01, DRB3*01:01, DRB3*02:01, DRB3*03:01, DRB4*01:01, DRB4*01:03, or DRB5*01 :01, and / or the a1 and o2 domain sequences have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of a DRA a1 and o2 domain sequence of DRA1*01:01 or DRA*01:02; and / orII) the p1 and p2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 80 or at least 90 contiguous aas of a DRB p1 or p2 domain sequences of DRB1*01 :01 , DRB1*01:02, DRB1*01:03, DRB1*03:01, DRB1*03:02, DRB1*03:04, DRB1*04:01, DRB1*04:02, DRB1*04:03, DRB1*04:04, DRB1*04:05, DRB1*04:06, DRB1*04:08, DRB1*07:01, DRB1*08:01, DRB1*08:02, DRB1*08:03, DRB1*09:01, DRB1*10:01, DRB1*11 :01, DRB1*11:03, DRB1*11:04, DRB1*12:01, DRB1*13:01, DRB1*13:03, DRB1*14:01, DRB1*14:02, DRB1*14:05, DRB1*14:06, DRB1*15:01, DRB1*15:02, DRB1*15:03, DRB1*15:04, DRB1*15:05, DRB1*15:06, DRB1*15:07, DRB1*16:01, DRB3*01:01, DRB3*02:01, DRB3*03:01, DRB4*01:01, DRB4*01:03, or DRB5*01 :01, and / or the a1 and o2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 70 or at least 80 contiguous aas of a DRA a1 or a2 domain sequence of DRA1*01:01 or DRA*01:02.

5. The MAPP of claim 1 or 2, wherein: the p1 and p2 domain sequences have at least 90% or 100% aa sequence identity to at least 170 contiguous aas of DRB p1 and p2 domain sequence of DRB1*01 :01, DRB1*03:01, DRB1*04:01, DRB1*04:02, DRB1*04:05, DRB1*08:01, DRB1*09:01, DRB3*01:01, DRB4*01:01, or DRB*05:01; or the MHC Class II p chain polypeptide sequence has at least 90% or at least 95% aa sequence identity to at least 80 or at least 90 contiguous aas of DRB p1 or p2 domain sequences of DRB1 *01 :01 , DRB1*03:01, DRB1*04:01, DRB1*04:02, DRB1*04:05, DRB1*08:01, DRB1*09:01, DRB3*01:01, DRB4*01:01, or DRB*05:01.

6. The MAPP of claim 4 wherein:(I) any one or more of positions 37, 49 and / or 72 of the DRA a1 domain sequence are substituted; and / or(II) position 44 of the DRA a1 domain sequence(a) is an aa other than cysteine, or(b) when aa position 44 of the DRA a1 domain sequence is an arginine, it is substituted by a serine or lysine.

7. The MAPP of claim 1 , wherein: the p1 and p2 domain sequences have at least 95% or at least 98% aa sequence identity to all or at least 170 contiguous aas of a DQB p1 and p2 domain sequence of DQB1*02:01, DQB1*02:02, DQB1*03:01, DQB1*03:02, DQB1*03:03, DQB1*03:04, DQB1*04:01, DQB1*04:02, DQB1*05:01, DQB1*06:01, DQB1*06:02, DQB2 isoform 1 or DQB2 isoform 2; and / or the a1 and o2 domain sequences have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of a DQA ol and o2 domain sequence of DQA1*01 :01, DQA1*01 :02, DQA1*01 :03, DQA1 *01 :04, DQA1 *02:01, DQA1 *03:01, DQA1 *03:02, DQA1 *04:01, DQA1 *05:01, DQA1 *05:05, DQA1 *06:01, or DQA2*01:01.

8. The MAPP of claim 1 , wherein:I) the p1 and p2 domain sequences have at least 95% or at least 98% aa sequence identity to all or at least170 contiguous aas of a DQB p1 and p2 domain sequence of DQB1*02:01, DQB1*02:02, DQB1*03:02, DQB1*04:01, DQB1*04:02, DQB1*05:01, or DQB1*06:02, and / or the a1 and o2 domain sequences have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of a DQA ol and o2 domain sequence of DQA1*01 :01, DQA1*01 :02, DQA1*02:01, DQA1 *03:01, DQA1 *03:02, DQA1 *04:01, or DQA1 *05:01; orII) the p1 and p2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 80 or at least 90 contiguous aas of a DQB p1 or p2 domain sequence of DQB1*02:01, DQB1*02:02, DQB1*03:02, DQB1*04:01, DQB1*04:02, DQB1*05:01, or DQB1*06:02; and / or the a1 and o2 domain sequences have at least 90% or 95% aa sequence identity to at least 70 or at least 80 contiguous aas of a DQA ol or o2 domain sequence of DQA1*01 :01, DQA1*01 :02, DQA1*02:01, DQA1 *03:01, DQA1 *03:02, DQA1 *04:01, or DQA1 *05:01.

9. The MAPP of claim 7, wherein at least one presenting sequence comprises: o1 and o2 domain polypeptide sequences each having at least 90% or at least 95% aa sequence identity to at least 70 contiguous aas of DQA1*0501 polypeptide sequence, and p1 and p2 domain polypeptide sequences each having at least 90% or at least 95% sequence identity to at least 80 contiguous aas of DQB1*0201 polypeptide sequence.

10. The MAPP of claim 7, wherein at least one presenting sequence comprises: a1 and o2 domain polypeptide sequences each having at least 90% or at least 95% sequence identity to at least 70 contiguous aas of DQA1*0301 polypeptide sequence, and p1 and p2 domain polypeptide sequences each having at least 90% or at least 95% sequence identity to at least 80 contiguous aas of DQB1*0302 polypeptide sequence.

11. The MAPP of claim 9 wherein:(I) any one or more of positions 40, 52, 74 and / or 75 of the DQA1 or DQA2 a1 domains are substituted, preferably wherein position 40 is an E, position 52 is an H, and / or position 74 or 75 is an I; and / or(II) aa position 47 of the DQA a1 domain sequence(a) is an aa other than cysteine; or(b) is a serine, lysine, or arginine.

12. The MAPP of claim 1 , wherein:I) the p1 and p2 domain sequences have at least 95% or at least 98% aa sequence identity to all or at least 165 contiguous aas of a DPB p1 and p2 domain sequence of DPB1 *01 :01 , DPB1*02:01, DPB1*03:01, DPB1*04:01, DPB1*06:01, DPB1*09:01, DPB1*11 :01 , DPB1*13:01, DPB1*35:01, DPB1*71 :01 , DPB1*104:01 , or DPB1*141 :01 ; and / or the a1 and o2 domain sequences have at least 90% or 100% aa sequence identity to at least 165 contiguous aas of a DPA ol and o2 domain sequence of DPA1*01 :03 or DPA1*02:01; and / orII) the MHC Class II p chain polypeptide sequence has at least 90% or at least 95% aa sequence identity to at least 80 or at least 90 contiguous aas of a DPB p1 or p2 domain sequence of DPB1 *01 :01 , DPB1*02:01, DPB1*03:01, DPB1*04:01, DPB1*06:01, DPB1*09:01, DPB1*11 :01 , DPB1*13:01 , DPB1*35:01, DPB1*71 :01 , DPB1*104:01, or DPB1*141 :01 ; and / or the a1 and o2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 70 or at least 80 contiguous aas of a DPA a1 or a2 domain sequence of DPA1*01 :03 or DPA1*02:01.

13. The MAPP of claim 12, wherein:I) the p1 and p2 domain sequences have at least 95% or at least 98% aa sequence identity to all or at least165 contiguous aas of a DPB p1 and p2 domain sequence of DPB1*01:01 or DPB1 *02:01 ; and / or the a1 and o2 domain sequences have at least 95% or at least 98% aa sequence identity to at least 165 contiguous aas of a DPA ol and o2 domain sequence of DPA1*01:03 or DPA1*02:01; orII) the p1 and p2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 80 or at least 90 contiguous aas of a DPB p1 or p2 domain sequence of DPB1*01 :01 or DPB1*02:01 ; and / or the o1 and o2 domain sequences have at least 90% or at least 95% aa sequence identity to at least 70 or at least 80 contiguous aas of aDPA ol or o2 domain sequenceof DPA1*01 :03 or DPA1*02:01.

14. The MAPP of claim 12 or 13, wherein:(I) any one or more of positions 40, 47, 52 and / or 75 of the DPA a1 domain are substituted, preferably wherein position 40 of the DPA a1 domain is a D or an E, and / or position 52 is an H, and / or position 75 is an I; and / or(II) aa position 47 of the DPA a1 domain sequence(a) is an aa other than cysteine; or(b) when aa position 47 of the DPA a1 domain sequence is a histidine, then position 47 is substituted by a serine, lysine, or arginine.

15. The MAPP of claim 1 , wherein the MAPP comprises at least one linker:(I) wherein the linker comprises a sequence selected from GGSS (SEQ ID NO:220), GSGGS (SEQ ID NO:221), GGGS (SEQ ID NO:219), GGSG (SEQ ID NO:222), GGSGG (SEQ ID NO:223), GSGSG (SEQ ID NO:224), GSGGG (SEQ ID NO:225), GGGSG (SEQ ID NO:226), GSSSG (SEQ ID NO:227), and GGGGS (SEQ ID NO:218), any of which may be present once, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 times; or(ii) wherein the linker comprises a cysteine-containing linker sequence selected from CGGGS (SEQ ID NO:252), GCGGS (SEQ ID NO:253), GGCGS (SEQ ID NO:254), GGGCS (SEQ ID NO:255), and GGGGC (SEQ ID NO:256), with the remainder of the linker comprised of Gly and Ser residues.

16. The MAPP of any of claims 1-15, wherein the at least one presenting sequence comprises, in the N-terminal to C-terminal direction:(I) the epitope, p1, p2, a1 , and o2 domain polypeptide sequences; or(II) the epitope, p1, p2, a1 and o2 domain polypeptide sequences, and a MOD sequence; wherein said presenting sequence comprises a body disulfide bond and optionally comprises one or more independently selected linker sequences.

17. The MAPP of any of claims 1-15, complexed to form a duplex or higher order MAPP comprising at least a first MAPP heterodimer and a second MAPP heterodimer, wherein:(I) the first MAPP heterodimer comprises a first framework polypeptide having a first multimerization sequence and a first dimerization sequence, and a first dimerization polypeptide having a first counterpart dimerization sequence complementary to the first dimerization sequence; and(ii) the second MAPP heterodimer comprises a second framework polypeptide having a second multimerization sequence and a second dimerization sequence, and a second dimerization polypeptide having a second counterpart dimerization sequence complementary to the second dimerization sequence; and wherein 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, and the multimerization sequences are not the same as, and do not substantially associate with or bind to, the dimerization sequences or counterpart dimerization sequences.

18. The duplex MAPP of claim 17, wherein:(I) when the multimerization sequences are not an interspecific multimerization pair, the multimerization sequences are selected from the group consisting of immunoglobulin heavy chain constant regions, collectin family dimerization sequences, coiled-coil domains, and leucine-zipper domains; and(ii) when the multimerization sequences are an interspecific multimerization pair, the multimerization sequences are selected from the group consisting of a Fos and Jun polypeptide pair, Ig CH1 and Ig C K or A constant region polypeptide pair, a knob-in-hole without disulfide (“Ki H") pair, a knob-in hole with a stabilizing disulfide bond (“KiHs-s”) pair, an HA-TF polypeptide pair, a ZW-1 polypeptide pair, a 7.8.60 polypeptide pair, a DD-KK polypeptide pair, an EW-RVT polypeptide pair, an EW-RVTs-s polypeptide pair, and an A107 polypeptide pair.

19. The duplex MAPP of claim 17, wherein the multimerization sequences comprise Ig Fc regions and the first and second dimerization sequences comprise independently selected Ig CH1, Ig CL K or A, leucine zipper, Fos or Jun domains.

20. The duplex MAPP of claim 17, wherein:(I) the multimerization sequences of the first and second framework polypeptides are covalently linked by at least one disulfide bond; or(ii) the first dimerization sequence and its counterpart dimerization sequence and / or the second dimerization sequence and its counterpart 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.

21. The duplex MAPP of claim 17, comprising at least one wt. or variant MOD sequence, wherein each MOD is selected independently from the group consisting of IL-2, IL-10, FasL, PD-L1, TGF-p, CD80, CD86 and 4-1 BBL polypeptide sequences.

22. The duplex MAPP of claim 21 , comprising at least one wt. or variant PD-L1 polypeptide sequence.

23. The duplex MAPP of claim 21 , comprising at least one wt. or variant IL-2 MOD sequence.

24. The duplex MAPP of claim 21, comprising at least one wt. or variant IL-2 MOD sequence having a substitution atH16 and / or F42.

25. The duplex MAPP of claim 21, comprising at least one wt. or variant IL-10 MOD sequence.

26. The duplex MAPP of claim 17, comprising:(I) a framework polypeptide comprising an aa sequence having greater than 90% or greater than 95% aa sequence identity to aas 13-780 of construct 4629 (SEQ ID NO:344); and(II) a dimerization peptide comprising an aa sequence having greater than 90% or greater than 95% aa sequence identity to construct 3890 (SEQ ID NO: 131 ) or 4016 (SEQ ID NO:133); wherein the first and / or second MAPP comprises a stabilizing body disulfide bond.

27. The duplex MAPP of claim 17, comprising:(I) a dimerization polypeptide comprising an aa sequence having greater than 90% or greater than 95% aa sequence identity to aas 13-564 of construct 4632 (SEQ ID NO:346); and(II) a framework peptide comprising an aa sequence having greater than 90% or greater than 95% aa sequence identity to construct 3886 (SEQ ID NQ:130) or 4015 (SEQ ID NO:132); wherein the first and / or second MAPP comprises a stabilizing body disulfide bond.

28. The duplex MAPP of claim 21, wherein the epitope aa sequence comprises an epitope of a cancer-associated antigen, an infectious agent, an autoantigen, an allergen, a T1D-associated antigen, or a celiac disease- associated antigen.

29. The duplex MAPP of claim 21, wherein the epitope aa sequence comprises an epitope of an autoantigen associated with Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune gastritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture's syndrome, glomerulonephritis, Grave's disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis, and vitiligo.

30. The duplex MAPP of claim 21, wherein the epitope aa sequence comprises an epitope of an autoantigen associated with autoimmune gastritis, autoimmune hemolytic anemia, Crohn's disease, Grave's disease, multiple sclerosis, rheumatoid arthritis, scleroderma, or systemic lupus erythematosus (SLE).31 . A method of treatment or prophylaxis comprising administering to a patient / subject an effective amount of one or duplex MAPPS of claim 17.

32. The method of claim 31, wherein the patient / subject suffers from: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune gastritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture's syndrome, glomerulonephritis, Grave's disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis, vitiligo, T1D or celiac disease.

33. The method of claim 31, wherein the patient / subject suffers from autoimmune gastritis, autoimmune hemolytic anemia, Crohn's disease, Grave's disease, multiple sclerosis, rheumatoid arthritis, scleroderma, or systemic lupus erythematosus (SLE).

34. One or more nucleic acid molecules comprising sequences encoding a MAPP or a duplex MAPP of claim 17.

35. A method of producing cells expressing a MAPP and / or duplex MAPP, the method comprising introducing one or more nucleic acid molecules according to claim 34 into the cells in vitro; selecting for cells that produce the MAPP or duplex MAPP; and optionally selecting for cells comprising all or part of the one or more nucleic acids either unintegrated or integrated into at least one cellular chromosome.

36. One or more cells transiently or stably expressing the MAPP or duplex MAPP prepared by the method of claim 35; optionally, wherein the cells express from about 25 to about 350 mg / liter or more of the MAPP or duplex MAPP without a substantial reduction in viability relative to otherwise identical cells not expressing the MAPP or duplex MAPP.

37. A method of selectively delivering one or more MODs and / or variant MODs to a cell or tissue of a patient or subject having or suspected of having a disease or disorder, the method comprising administering to the patient or subject one or more MAPPs or duplex MAPP of claim 17.

38. The method of claim 37, wherein the patient has, or is suspected of having, a disease or disorder selected from: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune gastritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture's syndrome, glomerulonephritis, Grave's disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis, vitiligo, T1D or celiac disease.

39. The method of claim 37, wherein the patient has or is suspected of having T1 D or celiac disease.

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