Antigen Presenting Polypeptide Complexes and Methods of Use Thereof

Abstract

The present disclosure provides Multimeric Antigen Presenting Polypeptides (MAPPs) for the presentation of T1D 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 those MAPPs. MAPPs of the present disclosure are useful for selectively modulating activity of T cells having T cell receptors that recognize the T1D epitopes presented by the MAPP. 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 T1D.

Description

I. INTRODUCTION

An adaptive immune response involves the engagement of the T cell receptor (TCR), present on the surface of a T cell, with a small peptide antigen non-covalently presented on the surface of an antigen presenting cell (APC) by a major histocompatibility complex (MHC; also referred to in humans as a human leukocyte antigen (HLA) complex). This engagement represents the immune system's targeting mechanism and is a requisite molecular interaction for T cell modulation (activation or inhibition) and effector function. In addition to epitope-specific cell targeting, T cells may be targeted by immunomodulatory proteins found in, for example, APCs, that affect various functions of the target cells (e.g., activation or inhibition of various T cell functions) through their counterpart costimulatory proteins (e.g., receptors) on the T cells. Both signals—epitope-TCR binding, and engagement of APC costimulatory proteins with T cell costimulatory proteins—are required to drive T cell specificity and activation or inhibition. The TCR is specific for a given epitope; however, costimulatory proteins are not epitope specific, and instead are generally expressed on all T cells or on subsets of T cells.

APCs generally serve to capture and break proteins from foreign organisms, or abnormal proteins, into smaller fragments suitable as signals for scrutiny by the larger immune system, including T cells. In particular, APCs break down proteins into small peptide fragments, which are then paired with proteins of the MHC and displayed on the cell surface. Cell surface display of an MHC together with a peptide fragment, also known as a T cell epitope, provides the underlying scaffold surveilled by T cells, allowing for specific recognition. The peptide fragments can be pathogen-derived (infectious agent-derived), tumor-derived, or derived from natural host proteins (self-proteins). Moreover, APCs can recognize other foreign components, such as bacterial toxins, viral proteins, viral DNA, viral RNA, etc., whose presence denotes an escalated threat level. The APCs relay this information to T cells through additional costimulatory signals in order to generate a more effective response.

T cells recognize peptide-major histocompatibility complex (pMHC) complexes through a specialized cell surface receptor, the TCR. The TCR is unique to each T cell; as a consequence, each T cell is highly specific for a particular pMHC target. In order to adequately address the universe of potential threats, a very large number (˜10,000,000) of distinct T cells with distinct TCRs exist in the human body. Further, any given T cell, specific for a particular T cell peptide, is initially a very small fraction of the total T cell population. Although normally dormant and in limited numbers, T cells bearing specific TCRs can be readily activated and amplified by APCs to generate highly potent T cell responses that involve many millions of T cells. Such activated T cell responses are capable of attacking and clearing viral infections, bacterial infections, and other cellular threats including tumors. Conversely, the broad, non-specific activation of overly active T cell responses against self-antigens or shared antigens, such as those targeted in Type 1 Diabetes (T1D), can give rise to T cells that inappropriately attack and destroy healthy tissues or cells.

HLA proteins are divided into two major classes, Class I and Class II proteins, which are encoded by separate loci. Unless expressly stated otherwise, for the purpose of this disclosure, references to MHC or HLA proteins are directed to Class II MHC or HLA proteins. HLA Class II proteins each comprise alpha and beta polypeptide chains encoded by separate loci. HLA Class II gene loci include HLA-DM (HLA-DMA and HLA-DMB that encode HLA-DM α chain and HLA-DM β chain, respectively), HLA-DO (HLA-DOA and HLA-DOB that encode HLA-DO α chain and HLA-DO β chain, respectively), HLA-DP (HLA-DPA and HLA-DPB that encode HLA-DP α chain and HLA-DP β chain, respectively), HLA-DQ (HLA-DQA and HLA-DQB that encode HLA-DQ α chain and HLA-DQ β chain, respectively), and HLA-DR (HLA-DRA and HLA-DRB that encode HLA-DR α chain and HLA-DR β chain, respectively).

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 self-antigens leading to autoimmune diseases, and. in particular Type 1 diabetes (T1D).

II. SUMMARY

The present disclosure provides multimeric antigen-presenting polypeptide complexes (“MAPP” singular and “MAPPs” plural) that 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. 1A). 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., FIGS. 1A and 1B). 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) α chain or β 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.

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 epitope-presenting peptides (“peptide epitopes” or simply “epitopes”) may be presented in the context of an MHC (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 immunomodulatory polypeptides (“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 (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.

The presentation by a MAPP of a peptide epitope to a target T cell 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.

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. 15.

As an alternative to utilizing a single polypeptide to present an epitope, the MHC components (e.g., α1, α2, β1 and β2 domain sequences) and the epitope may be divided among two separate polypeptide sequences, which together are denoted herein as a “presenting complex.” See, e.g., FIGS. 17 and 18. A presenting complex is integrated into a MAPP by having a presenting complex first amino acid sequence (“presenting complex 1^st sequence”) as part of a framework or dimerization polypeptide. The remaining MHC sequence(s) are part of a polypeptide termed the presenting complex second amino acid sequence (“presenting complex 2^nd sequence”). The peptide epitope and any independently selected MODs that are present may be part of the polypeptide comprising either the presenting complex 1^st sequence or the presenting complex 2^nd sequence. The presenting complex 1^st sequence and presenting complex 2^nd sequence generally associate through non-covalent interactions between the α chain and β chain polypeptide sequences, and may be stabilized by disulfide bonds between either the MHC sequences or peptide/polypeptide linkers attached to the N- or C-terminus of the MHC sequences. The presenting complex 1^st sequence and presenting complex 2^nd sequence may also associate through dimerization or interspecific dimerization sequences if present in those polypeptides.

Although an individual MAPP may not comprise a presenting sequence or presenting complex, for the purpose of this disclosure the MAPPs are, unless stated otherwise, understood to comprise at least one presenting sequence or presenting complex.

MAPPs that comprise a presenting sequence 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. 9, structures A-D, and FIG. 10, structures A-D.

Likewise, MAPPs with presenting complexes typically contain one or two presenting complexes, and accordingly, duplex MAPPs with presenting complexes typically comprise two, three or four presenting complexes, but also may comprise one presenting complex (e.g., if one of the MAPPs does not comprise a presenting complex). As discussed above, MAPPs and duplex MAPPs may comprise more presenting complexes depending on, for example, the number of dimerization sequences in the framework polypeptide.

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. Once engaged with the TCR of a T cell, the effect of a MAPP on the T cell depends on which MODs, if any, are present as part of the MAPP.

MOD-containing MAPPs of the present disclosure comprising T1D-associated peptide epitope sequences can function as a means of selectively delivering the MODs to T cells specific for the T1D-associated 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 T1D-associated epitopes). 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 TCR. 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.

The ability of MAPPs (e.g., duplex MAPPs) of the present disclosure 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 an autoimmune disease, such as T1D.

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.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A 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 are first and second dimerization polypeptides having an N-terminal epitope and counterpart dimerization sequences. The dashed 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 dimerization polypeptide. 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 1′ and/or positions 3 and 3′. While interactions between polypeptide chains through peptide sequences that interact 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 amino acids serving as linkers (e.g., peptide linkers).

FIG. 1B parallels FIG. 1A and is provided to illustrate the terminology used to describe MAPPs and duplex MAPPs with presenting complexes. The word “sequence” may be abbreviated by “seq.”

FIGS. 2A-2H provide amino acid sequences of immunoglobulin polypeptides including their heavy chain constant regions (“Ig Fc” or “Fc”, e.g., the CH2-CH3 domain of IgG1) (SEQ ID NOs:1-13).

FIG. 2I provides the sequence of an Ig CH1 domain (SEQ ID NO:14).

FIG. 2J provides the sequence of a human Ig J chain (SEQ ID NO:86).

FIG. 3A provides the sequence of an Ig κ chain (kappa chain) constant region (SEQ ID NO:15).

FIG. 3B provides the sequence of an Ig λ chain (lambda chain) constant region (SEQ ID NO:16).

FIG. 4 provides an amino acid sequence of an HLA Class II DRA (sometimes referred to as DRA1) α chain (SEQ ID NO:17).

FIG. 5 provides amino acid sequences of HLA Class II DRB1 β chains (SEQ ID NOs:18-54).

FIG. 6 provides amino acid sequences of HLA Class II DRB3 β chains (SEQ ID NOs:55-58).

FIG. 7 provides an amino acid sequence of two HLA Class II DRB4 β chain (SEQ ID NOs:59-60).

FIG. 8 provides an amino acid sequence of an HLA Class II DRB5 β chain (SEQ ID NO:61).

FIG. 9 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 1′-5′ indicating locations for additional peptide sequences (e.g., MOD polypeptide sequences). The MHC/epitope moiety is illustrated generically, and can be either a presenting sequence (see, e.g., FIGS. 15-16), or a presenting complex (see FIGS. 17-22). Locations 4 and 4′ are shown at the N-terminus of a presenting sequence or the N-terminus of a presenting complex polypeptide, 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.

FIG. 10 provides in A to D a series of MAPP structures as in FIG. 9, with the addition of presenting sequences or presenting complexes at the N-terminus of the framework peptides. Positions 4 and 4′ may still serve as locations for peptide addition (e.g., MOD polypeptide addition).

FIG. 11 provides in A to D a series of MAPP structures as in FIG. 9, 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).

FIG. 12 provides a series of MAPP structures as in FIG. 11, with the addition of presenting sequences or presenting complexes at the N-terminus of the framework peptides. Positions 4 and 4′ may still serve as locations for peptide addition (e.g., MOD polypeptide addition).

FIG. 13 provides in A to J a series of MAPP structures as in FIG. 11. In each instance, a presentation sequence lacking a MOD sequence is present on the dimerization peptide (marked as a single chain MHC 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 variants of IL-2, PD-L1, and CD80, although other MODs may be used, e.g., wt. and/or variant TGF-β or 4-1BBL. Positions 3 and 3′ are shown for orientation in A to G. In H to J the 3 and 3′ locations are unfiled, e.g., for illustration purposes wt. and/or variant TGF-β or 4-1BBL MODs may be located there, although other MODs may be used, e.g., wt. and/or variants of IL-2, PD-L1, and CD80.

FIG. 14 shows four MAPP heterodimer constructs as structures A-D that can form duplex MAPPs. The polypeptide sequences of structures A to D are provided in FIGS. 24-27.

FIG. 15 shows in A to C three different MHC Class II presenting sequences (from the epitope at the N-terminus to the C-terminus). The sequences optionally comprise one or more independently selected MODs (including two or more MODs in tandem) at the indicated locations.

FIG. 16 shows in A to H different embodiments of MHC Class II presenting sequences (from left to right N-terminus to C-terminus).

FIGS. 17 to 22 show a series of MHC Class II presenting complexes from left to right N- to C-terminus. The sequence bearing the symbol “-//-” is the presenting complex 1^st sequence. The other sequence is its associated presenting complex 2^nd sequence. The symbol “-//-” denotes the point of attachment of the complex to the remainder of the dimerization or framework peptide. In FIGS. 20A-L, the presenting complex 1^st sequence and its associated presenting complex 2^nd sequence include dimerization sequences to unite the peptides (shown as an Ig Fc region associated with the Ig light chain constant region Cκ (kappa chain), although other sequences could be utilized). In FIGS. 21A-F and 22A-F, the presenting complex 1^st sequence and its associated presenting complex 2^nd sequence include dimerization sequences to unite the peptides (shown as a leucine zipper pair, although other sequences could be utilized).

FIG. 23 provides a table showing examples of HLA Class II alleles, MODs, and T1D epitopes to be incorporated into a MAPP for T1D therapy.

FIG. 24 provides the polypeptide sequences (SEQ ID NOs:62-63) and annotation for an example of the structure presented in FIG. 14, structure A.

FIG. 25 provides the polypeptide sequences (SEQ ID NOs 64-65) and annotation for an example of the structure presented in FIG. 14, structure B.

FIG. 26 provides the polypeptide sequences (SEQ ID NOs 66-67) and annotation for an example of the structure presented in FIG. 14, structure C.

FIG. 27 provides the polypeptide sequences (SEQ ID NOs 68-69) and annotation for an example of the structure presented in FIG. 14, structure D.

IV. DETAILED DESCRIPTION

A. Definitions

The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is 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.

The terms “polypeptide” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids, 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 include 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 amino acid at that position in the wt. polypeptide (i.e. 172 or K75). To the extent that the sequence of the wt. polypeptide is altered, either by addition or deletion of one or more amino acids, the specific residue or residue number will refer to the same specific amino acid in the altered polypeptide (e.g., the addition of one amino acid at the N-terminus of a peptide referenced as position 172 will be understood to indicate the amino acid, Ile, that is now position 73). Substitution of an amino acid at a specific position is denoted by an abbreviation comprising, in order, the original amino acid, the position number, and the substituted amino acid, e.g., substituting the Ile at position 72 with a cysteine is denoted as I72C.

A nucleic acid or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids is the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including blast.ncbi.nlm.nih.gov/Blast.cgi for BLAST+2.10.0, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, and mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol., 215:403-10. Unless otherwise indicated, the percent sequence identities described herein are those determined using the BLAST program. In the event of a conflict between the results produced by different release versions of BLAST, BLAST+ 2.10.0 released Dec. 23, 2019, is employed as the basis for determining sequence identity.

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: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan). Amino acid also includes 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.

As used herein, the term “in vivo” refers to any process or procedure occurring inside of the body, e.g., of a T1D patient.

As used herein, “in vitro” refers to any process or procedure occurring outside of the body.

The term “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. Exemplary conservative aa substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

The term “binding” refers to a direct association between molecules and/or atoms, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. Non-covalent interactions/binding refers to a direct association between two molecules, due to, for example, electrostatic, hydrophobic, ionic, and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. Non-covalent binding interactions are generally characterized by a dissociation constant (K_D) of less than 10⁻⁶ M, less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M, less than 10⁻¹² M, less than 10⁻¹³ M, less than 10⁻¹⁴ M, or less than 10⁻¹⁵ M. “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 the TCR.

“Affinity” as used herein generally refers to the strength of non-covalent binding, increased binding affinity being correlated with a lower K_D. As used herein, the term “affinity” may be described by the dissociation constant (K_D) for the reversible binding of two agents (e.g., an antibody and an antigen). Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 40-fold greater, at least 60-fold greater, at least 80-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of an antibody or receptor for an unrelated aa sequence (e.g., ligand). Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.

“T cell” includes all types of immune cells expressing CD3, including T-helper cells, cytotoxic T cells (CD8′ cells), T-regulatory cells (Treg), and NK-T cells.

The term “immunomodulatory polypeptide” (also referred to as a “costimulatory polypeptide” or, as noted above, “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”) 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 MODs, 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 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. A MOD can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A MOD also encompasses, inter alia, an antibody or antibody fragment that specifically binds with and activates a cognate co-stimulatory molecule (co-MOD) present on a T cell such as, but not limited to, antibodies against the receptors for any of IL-2, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-DC, B7-H2, B7-H3, and CD83.

“Heterologous,” as used herein, means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

“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.

The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are 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.

The terms “treatment,” “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease or symptom in a mammal and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i.e., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, 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, and in some cases after the symptomatic stage of the disease.

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.

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.

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.

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. Purifying, as used herein, may refer to a range of different resultant purities, e.g., wherein the purified substance makes up more than 80% of all the substance in the solution, including more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, and the like. 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.

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 within the disclosure along with any other stated or intervening value in the range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges that are also encompassed within the disclosure subject to any specifically excluded limit in the stated range. Where the stated range has a value (e.g., an upper or lower limit), ranges excluding those values are also included.

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

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. As such, 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, or use of a “negative” limitation.

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. Description

1. MAPP Structure and the Role of Framework and Dimerization Peptides

The present disclosure provides MAPPs for use in the treatment of T1D. 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 sequences that permit specific binding with other polypeptides (dimerization polypeptides) having a counterpart dimerization sequence thereby forming at least a heterodimer (see FIGS. 1A and 1B). 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., FIGS. 1A and 1B). Neither the dimerization sequence nor the multimerization sequence of the framework polypeptide (or the counterpart dimerization sequence) comprises an MHC Class II (HLA) α chain or β chain polypeptide sequence; and as such, interactions brought about by those MHC Class II sequences are not considered 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 at 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 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 is intended (e.g., by reference to duplex MAPPs etc.).

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/regulation 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 4-1BBL), 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/or PD-L1) they decrease T cell activation, proliferation, differentiation and/or effector functions in an epitope selective manner.

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 complexes or epitope presenting sequences. Duplex MAPP and higher order MAPP architecture can be particularly useful when both the MODs and the epitope presenting complexes (or 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 complex or presenting sequence 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 complex or presenting sequence 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 and presenting complex or presenting sequence 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 complex or 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 complex or presenting sequence 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 complex or presenting sequence may be located at the N-terminus of different framework and/or dimerization polypeptide sequences.

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 sequence 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 specifically/selectively pair with each other that 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. 9-13). Interspecific sequences 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, duplex MAPPs where each of the multimerization and dimerization sequences are different and provide separate peptide pairings.

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 λ or κ constant region sequences) (see, for example, FIGS. 11 and 12, structures A to D). In another 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 selected to be different (e.g., a dimerization sequence pair comprising an Ig CH1 paired with light chain λ or κ sequence and a dimerization sequence comprising a leucine zipper pair; see, for example, FIG. 13, structures E to H). For example, in a duplex MAPP the multimerization sequences may be knob-in-hole Ig sequences, 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 λ domain pair.

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 to T cells in some cases may be enhanced relative to the situation encountered in antigen presenting cells (APC) where epitope can diffuse away from the MHC (e.g., HLA) complex and any MODs the APC is presenting. This may not occur with a MAPP, however, where the epitope and MOD 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 may be further limited where the polypeptide(s) of the MAPP (e.g., framework, dimerization sequence, and if present, the presenting complex 2^nd sequence) 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.

Incorporation of one or more MODs with affinity for their cognate receptor on T cells (“co-MOD”) 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 polypeptide sequences. Wild-type and variant MODs are described further below.

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 T1D diabetes resulting from immune dysregulation/disfunction.

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.

Each presenting sequence or presenting complex present in a MAPP comprises MHC Class II α and β 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.

As indicated in the description of the drawings, MAPPs may comprise one or more independently selected peptide sequences or (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 interpolypeptide disulfide bond. Various linkers are described in more detail below.

2. Exemplary MAPP Architectures

MAPPs of the present disclosure 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 typically will further comprise either one or more epitope presenting sequences or one or more epitope presenting complexes. Exemplary structures for such MAPPs appear in FIGS. 1A, 1B, and 9-13. The structures depicted in FIG. 13 represent MAPPs with multimerizing framework polypeptides that have epitope presenting sequences (the “Single Chain MHC” with the “Epitope”). In FIGS. 9-12, the structures represent MAPPs with multimerizing framework polypeptides where the epitope MHC combination represents either epitope presenting sequences or epitope presenting complexes.

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. 4-8). In embodiments, MAPPs comprise at least one, or at least two, dimerization peptides that comprise an epitope presenting sequence. See, e.g., FIG. 1A.

One group of MAPPs, those having epitope presenting sequences, comprise a multimerizing framework polypeptide having, from N-terminus to C-terminus, a dimerization sequence and a 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 framework polypeptide comprises a presenting sequence located on the N-terminal side of their dimerization or counterpart dimerization sequences. In such a MAPP the presenting sequence may comprise a peptide epitope and one or more MHC polypeptide sequences, with the peptide epitope sequence located: (i) at or within 10 aa, 15 aa, 20 aa, 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 comprises 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 that comprise MHC Class II α1, α2, β1, and β2 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. 4-8.

Another group of MAPPs, those having epitope presenting complexes, comprise a multimerizing framework polypeptide having, from N-terminus to C-terminus, a dimerization sequence and a 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 the framework and dimerization polypeptides and/or at least one (e.g., one or both) of the framework and dimerization polypeptides comprise a presenting complex 1^st sequence located on the N-terminal side of their dimerization sequence. A presenting complex 2^nd sequence is associated with the presenting complex 1^st sequence (e.g., non-covalently or covalently such as by one or two interchain disulfide bonds) to form a presenting complex. In such a MAPP each of the presenting complex 1^st sequence and its associated presenting complex 2^nd sequence is comprised of one or more MHC polypeptide sequences, with one of the sequences further comprising the peptide epitope. The peptide epitope may be located (i) at or within 10 aa, 15 aa, 20 aa, or 25 aa of the N-terminus of the presenting complex 1^st sequence or presenting complex 2^nd sequence, or (ii) in a polypeptide located at the N-terminus of the presenting complex 1^st sequence or presenting complex 2^nd sequence, with the polypeptide 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, or the peptides of a presenting complex comprises one or more independently selected MODs located at their N-terminus or C-terminus (or on the N-terminal or C-terminal side of the dimerization sequences).

MAPPs of the present disclosure 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. 4-8.

As discussed above, a dimerization sequence of a framework polypeptide may interact with dimerization peptides to form heterodimers. A 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 Components

1. Framework Polypeptides and Dimerization Polypeptides

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.

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, framework polypeptides 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.

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 sequences, epitope presenting sequences, and/or a presenting complex 1^st sequence (one polypeptide of an epitope presenting complex, see FIG. 1B). When placed at the N- and/or C-termini of a framework polypeptide, such polypeptide elements are part of the framework polypeptide (e.g., a single translation product formed in a cell).

Within a MAPP, all of the dimerization sequences may be non-interspecific (such as leucine zipper pairs) while the multimerization sequence is either interspecific or non-interspecific (see, e.g., structures A & B of FIGS. 9 and 10). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be a non-interspecific (e.g., IgFc (e.g., CH2, CH3 domains) or leucine zippers) pair 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 a non-interspecific leucine zipper polypeptides. Where an Fc polypeptide is employed it may be, for example, from an IgA, IgD, IgE, IgG, or IgM, which may be a human polypeptide sequence, a humanized polypeptide sequence, a Fc region polypeptide of a synthetic heavy chain constant region, or a consensus heavy chain constant region.

Within a MAPP, all of the dimerization sequences may be interspecific, while the multimerization sequences are not interspecific (see, e.g., FIG. 13A). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be an IgFc sequence, with a ZW1 sequence or its counterpart employed as the dimerization sequence of the first framework polypeptide and an Ig CH1 domain or its counterpart Ig C_L K sequence as the dimerization sequence of the second framework polypeptide.

All of the dimerization sequences, or all of the dimerization and multimerization sequences, in a MAPP may differ in that they bind only specific binding partners present in the MAPP (e.g., each are 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 IgFc sequences, with a ZW1 sequence or its counterpart employed as the dimerization sequence of the first framework polypeptide, and an Ig CH1 or its counterpart Ig C_L sequence as the dimerization sequence of the second framework polypeptide.

2. Multimerization and Dimerization Polypeptide Sequences

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 duplexes 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 framework peptides, in which case they are multimerization sequences; or they bring together a dimerization and multimerization sequence, in which case they are designated as dimerization sequences.

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) as shown in FIG. 9, structure A. 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., IgFc) 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 C_L 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., KiH 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.

Interspecific binding sequences may in some instances form some amount of homodimers, but preferentially dimerize by binding more strongly with their counterpart interspecific binding sequences. 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 70%, 80%, 90%, 95%, 98% or 99% heterodimers when an equimolar mixture of the polypeptides is combined (for example in PBS 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. 9, structure B, with an interspecific multimerization sequence and structure C with two different interspecific dimerization sequences. Moreover, because interspecific sequences are selective for their counterpart sequences, 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.

Sequences are considered orthogonal to other sequences when they do not form complexes (bind) with each other's counterpart sequences. See FIG. 9, structure D, 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).

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., such as 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 are less than 300 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 200 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 100 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 75 aa. In another embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 50 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 30 aa.

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-like 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 CL sequences (Ig CH1/CL pairs such as an Ig CH1 sequence paired with an Ig C_L κ or λ light chain constant region sequence).

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 IgFc multimerization sequence, and a CH1 dimerization sequence it may comprise all or part a native or variant immunoglobulin sequence set forth in any of FIGS. 2A to 2H that comprise 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 CH3 domains, may have at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to an aa sequence of an Fc region depicted in 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. 4A-4H in which a C-terminal lysine occurs.

Such immunoglobulin sequences can covalently link the polypeptides of MAPP complex together by forming one or two interchain disulfide bonds, thereby stabilizing MAPPs, particularly where a pair of interspecific Ig sequence such as knob-in-hole polypeptide pairs are employed. Where an Fc polypeptide sequence, 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, which may be a human polypeptide sequence, a humanized polypeptide sequence, a 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 complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC). 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.

Framework and/or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity 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 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 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 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 contiguous aas (at least 150, at least 175, or at least 200 contiguous aas), or all aas, of the IgE Fc sequence depicted in FIG. 2C (SEQ ID NO:3).

A MAPP may comprise one or more IgG Fc sequences as dimerization and/or multimerization sequences. The Fc polypeptide of a MAPP can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc sequence has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to an aa sequence of an Fc region depicted in FIG. 2D-2G. Framework and/or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity 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. IgG1 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 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (e.g., at least 150, at least 175, at least 200, or at least 225) contiguous aas, or all aas, of the IgG2 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 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (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 IgG3 Fc 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 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (e.g., at least 150, at least 175, at least 200, or at least 220) contiguous aas, or all aas, of the IgG4 Fc sequence depicted in FIG. 2G (SEQ ID NO:11 or 12).

Framework and/or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity 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 polypeptide sequence depicted in FIG. 2H (SEQ ID NO:13).

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.

A framework or dimerization polypeptide may comprise an aa sequence having 100% aa sequence identity to the wt. human IgG1 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 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 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 antibody sequences with reduced complement component 1q (“C1q”) binding compared to the wt. protein, and accordingly a reduction in complement dependent cytotoxicity.

L234 and other aas in the lower hinge region (e.g., aas 234 to 239, which correspond to aas 14-19 of SEQ ID NO:4) of IgG are involved in binding to the Fc lambda receptor (FcγR), and accordingly, mutations at that location reduce binding to the receptor (relative to the wt. protein). 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 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 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.

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 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 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).

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 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 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 complement dependent cytotoxicity. In one embodiment, the substitution is a P331S substitution. In another embodiment, the substitution is a P331A substitution.

A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide 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.

A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide 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 IgG1 Fc) having L234F, L235E, and P331S substitutions (corresponding to aa positions 14, 15, and 111 of the aa sequence depicted in FIG. 2D).

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 and 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 IgG1, 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 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 the table that follows, with cross reference to the numbering of the aa positions as they appear in the wt. IgG1 sequence (SEQ ID NO:4) set forth in FIG. 2D shown in brackets “{ }”.

TABLE 1
Interspecific immunoglobulin sequences and their cognate counterpart interspecific sequences
	Substitutions in the first	Substitutions in the second
Interspecific	interspecific polypeptide	(counterpart) interspecific
Pair Name	sequence	polypeptide sequence	Comments
KiH	T366W	T366S/L368A/Y407V	Hydrophobic/steric
	{T146W}	{T146S/L148A/Y187V}	complementarity
KiHs-s	T366W/S354C*	T366S/L368A/Y407V/Y349C	KiH + inter-CH3
	{T146W/S134C*}	{T146S/L148A/Y187V/Y129C}	domain S-S bond
HA-TF	S364H/F405A	Y349T/T394F	Hydrophobic/steric
	{S144H/F185A}	{Y129T/T174F}	complementarity
ZW1	T350V/L351Y/F405A/Y407V	T350V/T366L/K392L/T394W	Hydrophobic/steric
	{T130V/L131Y/F185A/Y187V}	{T130V/T146L/K172L/T174W}	complementarity
7.8.60	K360D/D399M/Y407A	E345R/Q347R/T366V/K409V	Hydrophobic/steric
	{K140D/D179M/Y187A}	{E125R/Q127R/T146V/K189V}	complementarity +
			electrostatic
			complementarity
DD-KK	K409D/K392D	D399K/E356K	Electrostatic
	{K189D/K172D}	{D179K/E136K}	complementarity
EW-RVT	K360E/K409W	Q347R/D399V/F405T	Hydrophobic/steric
	{K140E/K189W}	{Q127R/D179V/F185T}	complementarity &
			long-range electro-
			static interaction
EW-RVTs-s	K360E/K409W/Y349C*	Q347R/D399V/F405T/S354C	EW-RVT + inter-CH3
	{K140E/K189W/Y129C*}	{Q127R/D179V/F185T/S134C}	domain S-S bond
A107	K370E/K409W	E357N/D399V/F405T	Hydrophobic/steric
	{K150E/K189W}	{E137N/D179V/F185T}	complementarity +
			hydrogen bonding
			complementarity
Table 1 modified from Ha et al., Frontiers in Immunol. 7: 1-16 (2016).
*aa forms a stabilizing disulfide bond.

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 IgG1 CH3 domain of the interspecific sequence, and 57 residues derived from IgG1 in the IgA CH3 in its counterpart interspecific sequence. See Ha et al., Frontiers in Immunol. 7:1-16 (2016).

In an embodiment, 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.

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 KiH or KiHs-s sequence with a T146W sequence substitution, and its counterpart interspecific KiH or KiHs-s binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V sequence substitutions, where the framework and/or dimerization polypeptides comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D. One or both of the framework, or both of dimerization polypeptides optionally comprising 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., L131K); T146 (e.g., T146S); P175 (e.g., P175V); F185 (e.g., F185R); Y187 (e.g., Y187A); and K189 (e.g., K189Y) in the wt. IgG1 sequence of FIG. 2D.

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a T146W KiH sequence substitution, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V KiH sequence substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a T146W and S134C KiHs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, Y187V and Y129C KiHs-s substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a S144H and F185A HA-TF substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having Y129T and T174F HA-TF substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V, T146L, K172L, and T174W ZW1 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a K140D, D179M, and Y187A 7.8.60 substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V E125R, Q127R, T146V, and K189V 7.8.60 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2Ds

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a K189D, and K172D DD-KK substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V D179K and E136K DD-KK substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

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

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

In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a K150E and K189W A107 substitutions, its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V E137N, D179V, and F185T A107 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%. at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of FIG. 2D; where one or both of the framework and/or dimerization polypeptide sequences may comprise additional substitutions such as L14 and/or L15 substitutions (e.g., “LALA” substitutions L234A and L235A), and/or N77 (N297 e.g., N297A or N297G).

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.

In an embodiment, a MAPP framework or dimerization polypeptide comprises 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, where the framework or dimerization polypeptide comprise a sequence having at least 80%, 85%, 90%. 95%, 98%, 99%, or 100% sequence identity to at least 70, at least 80, at least 90, at least 100, or at least 110 contiguous aas of SEQ ID NOs: 14 and/or 15 respectively. See FIGS. 21 and 3A. 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 a dimerization or multimerization sequences. Among the substitutions that increase the stability of CH1-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 to each of the IgCH1 and Ig K sequences. The Ig CH1 sequence is modified to contain S64E and S66V substitutions (S70E and S72V of the sequence shown in FIG. 2I). The Ig K sequence is modified to contain S69L and T71S substitutions (S68L and T70S of the sequence shown in FIG. 3A).

In another embodiment, a framework or dimerization polypeptide of a MAPP comprises an Ig CH1 domain (e.g., the polypeptide of FIG. 2I SEQ ID NO:14), and its counterpart sequence comprises an Ig α chain constant region sequence such as is shown in FIG. 3B (SEQ ID NO:16), 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 sequences shown in FIG. 3B.

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 dimer (e.g., homodimer). Non-limiting examples of leucine-zipper polypeptides include a peptide comprising any one of the following aa sequences: RMKQIEDKIEEILSKIYHIENEIA RIKKLIGER (SEQ ID NO:70); LSSIEKKQEEQTSWLIWISNELTLIRNELAQS (SEQ ID NO:71); LSSIEKKLEEITSQLIQISNELTLIRNELAQ (SEQ ID NO:72; LSSIEKKLEEITSQLIQIRNELTLIRN ELAQ (SEQ ID NO:73); LSSIEKKLEEITSQLQQIRNELTLIRNELAQ (SEQ ID NO:74); LSSLEKKL EELTSQLIQLRNELTLLRNELAQ (SEQ ID NO:75); ISSLEKKIEELTSQIQQLRNEITLLRNEIAQ (SEQ ID NO:76). In some cases, a leucine zipper polypeptide comprises the following aa sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:77). Additional leucine-zipper polypeptides are known in the art, a number of which are suitable for use as multimerization or dimerization sequences.

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: LKSVENRLAVVENQLKTVIEELKTVKDLLSN (SEQ ID NO:78); LARIEEKLKTIKAQLSEIASTLNMIREQLAQ (SEQ ID NO:79); VSRLEEKVKT-LKSQVTELASTVSLLREQVAQ (SEQ ID NO:80); IQSEKKIEDISSLIGQIQSEITLIRNEIAQ (SEQ ID NO:81); and LMSLEKKLEELTQTLMQLQNELSMLKNELAQ (SEQ ID NO:82).

A MAPP may comprise a pair of two framework polypeptides and/or a framework and 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 bond between the cysteine residues. Examples of such aa sequences include: VDLEGSTSN-GRQCAGIRL (SEQ ID NO:83); EDDVTTTEELAPALVPPPKGTCAGWMA (SEQ ID NO:84); and GHDQETTTQGPGVLLPLPKGACTGQMA (SEQ ID NO:85).

Some aa sequences suitable as multimerization (oligomerization) sequences permit formation of MAPPs capable of forming structures greater than duplexes of a heterodimers comprising a framework and dimerization polypeptide. In some instances, triplexes, tetraplexes, pentaplexes may be formed. Such aa sequences include, but are not limited to, IgM constant regions (See, e.g., FIG. 2H) which forms hexamer, 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:86). 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 appear or are 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:

(SEQ ID NO: 87)
VTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFF
IRVRDGWKKLQLGELIPIPADSPPPPALSSNP.

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 and 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 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 and Presenting Complexes

As discussed in more detail below, Class II MHC polypeptides, include two types of polypeptide chains, α-chain and β-chain. More specifically, MHC Class II α-chain polypeptides include α1 and α2 domains, and β-chain polypeptides include β1 and β2 domains. Presenting sequences and presenting complexes comprise MHC Class II polypeptides sufficient to bind and present an epitope to a TCR. Presenting sequences and complexes may also comprise additional protein (peptide) elements including one or more independently selected MODs and/or one or more independently selected linkers (e.g., linkers placed between various domains). As discussed herein, unless stated otherwise, neither presenting sequences nor presenting complexes comprise a 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.

Conceptually, each of the presenting sequences and presenting complexes may be considered a “soluble MHC” that is fully capable of binding and presenting a peptide epitope. Unless stated otherwise, in presenting sequences all of the MHC α1, α2, β1, and β2 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., FIGS. 15 and 16.

Where the MHC α1, α2, β1, and β2 domain sequences are divided among two or more polypeptide chains, the “soluble MHC” is termed a presenting complex. The presenting complex has one chain that is part of a framework peptide or dimerization peptide, referred to as a “presenting complex 1^st sequence.” The second chain of the presenting complex is termed the “presenting complex 2^nd sequence.” The presenting complex 2^nd sequence may be associated non-covalently with the MHC components present in the presenting complex 1^st sequence (through binding interactions between MHC-Class II α1, α2, β1, and β2 domain components as in FIGS. 17 to 19), in addition, one or more disulfide bonds between the presenting complex 1^st sequence and the presenting complex 2^nd sequence. Alternatively, the presenting complex 2^nd sequence may be associated non-covalently with the MHC components present in the presenting complex 1^st sequence through binding interactions between MHC-Class II α1, α2, β1, and β2 domain components, and/or through binding sequences (e.g., such as interspecific binding sequences) as in FIGS. 20, 21 structures A-E, and 22, in the presence or absence of one or more disulfide bonds between the presenting complex 1^st sequence and the presenting complex 2^nd sequence.

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 α1, α2, β1, and β2 domain and epitope in a single polypeptide sequence In some cases MAPPs comprise presenting complexes with all of the Class II components required for binding and presenting the epitope of interest to a TCR, and the peptide epitope is part of the presenting complex 1^st sequence or the presenting complex 2^nd sequence.

As noted above, presenting sequences and complexes typically will comprise a peptide epitope that is part of a polypeptide chain. It is possible, however, to make MAPPS 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

As noted above, the epitope containing MAPPs include MHC Class II polypeptides of various species, including human MHC polypeptides (HLA polypeptides), rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides' of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., equines, bovines, ovines, caprines, etc.)), and the like.

For the purpose of this disclosure the term “MHC polypeptide” is meant to include Class II MHC polypeptides, including the α- and β-chains or portions thereof. More specifically, MHC Class II polypeptides include the α1 and α2 domains of Class II MHC α chains, and the β1 and β2 domains of Class II MHC β chains, which represent all or most of the extracellular Class II protein required for presentation of an epitope. In an embodiment, both the α and β Class II MHC polypeptide sequences in a MAPP are of human origin.

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). Unless expressly stated otherwise, as noted above, the MAPPs described herein are not intended to include membrane anchoring domains (such as transmembrane regions of MHC Class II α and β chains) or a part thereof sufficient to anchor MAPP molecules (e.g., more than 50% of the MAPP molecules), or a peptide thereof, 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 MAPPs described herein do not include the leader and/or intracellular portions (e.g., cytoplasmic tails) that may be present in some naturally-occurring MHC Class II proteins.

a. T1D and its Risk-Associated Alleles and Haplotypes

Certain alleles and haplotypes of MHC Class II have been associated with disease, e.g., increased risk of developing T1D. 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.

(i) MHC Class II Polypeptides in Type 1 Diabetes Mellitus (T1D)

Alleles/isoforms showing increased association with T1D represent suitable sources of MHC II α1, α2, β1, and β2 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 the HLA-DQ8, (e.g., HLA-DQB1*03:02) and alleles of the HLA-DQ2 serotype. Some high and moderate risk haplotypes and their association with various DR serotypes are shown in Table 2 adopted from Kantirova and Buc, Physiol. Res. 56: 255-266 (2007).

TABLE 2
DR serotype	DRB allele	DQ serotype	DQA allele	DQB allele
High risk T1D haplotypes
DR3	DRB1*0301	DQ 2.5	DQA1*0501	DQB1*0201
DR4	DRB1*0401	DQ 8.1	DQA1*0301	DQB1*0302
DR4	DRB1*0402	DQ 8.1	DQA1*0301	DQB1*0302
DR4	DRB1*0405	DQ 8.1	DQA1*0301	DQB1*0302
Moderate risk T1D haplotypes
DR1	DRB1*01	DQ 5	DQA1*0101	DQB1*0501
DR8	DRB1*0801		DQA1*0401	DQB1*0402
DR9	DRB1*0901		DQA1*0301	DQB1*0303

The serotypically defined DR3 and DR4 protein isoforms/haplotypes of the DRB1 gene are associated with increased risk that an individual expressing such alleles will develop T1D. 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 (e.g., HLA-DRB1*0401 and HLA-DRB1*0405) predispose individuals to T1D, whereas HLA-DRB1*04:03 allele/isoform may afford protection. DRB1*16:01 also show 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 α1, α2, β1, and β2 polypeptide sequences.

The above-mentioned alleles associated with an increased risk of T1D represent suitable candidates from which the α1, α2, β1, and/or β2 polypeptide sequences present in a MAPP may be taken.

The Table in FIG. 23 shows examples of HLA Class II alleles, MODs, and T1D epitopes that may be incorporated into MAPPs for T1D therapy.

b. MHC Class II α and β Polypeptides

As discussed above, MAPPs comprise Class II MHC polypeptides. Naturally occurring Class II MHC polypeptides comprise an α chain and a β chain (e.g., HLA α- and β-chains). While MHC Class II polypeptides include MHC Class II DP α and β polypeptides, DM α and β polypeptides, DO α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides. The polypeptides of central interest for the treatment of T1D are DQ α and β polypeptides and DR α and β polypeptides. As used herein, the term “Class II MHC polypeptide” refers to a Class II MHC α chain polypeptide, a Class II MHC β chain polypeptide, or only a portion of a Class II MHC α and/or β chain polypeptide, or combinations of the foregoing. For example, the term “Class II MHC polypeptide” as used herein can be a polypeptide that includes: i) only the al domain of a Class II MHC α chain; ii) only the α2 domain of a Class II MHC α chain; iii) only the al domain and an α2 domain of a Class II MHC α chain; iv) only the β1 domain of a Class II MHC β chain; v) only the β2 domain of a Class II MHC β chain; vi) only the β1 domain and the β2 domain of a Class II MHC β chain; vii) the al domain of a Class II MHC α chain, the β1 domain of a Class II MHC β chain, and the β2 domain of a Class II MHC; and the like.

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 DRA alleles, DRB1 alleles, DRB3 alleles, DRB4 alleles, DRB5 alleles, DRB6 alleles, DRB7 alleles, DRB9 alleles, DQA1 alleles, DQB1 alleles, DPA1, DPB1 alleles, DMA alleles, DMB alleles, DOA alleles and DOB alleles.

In some cases, a MAPP comprises a Class II MHC α chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring Class II MHC α chain. Thus, in some cases, a MAPP comprises only the α1 and α2 portions of a Class II MHC α chain; and does not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring Class II MHC α chain.

In some cases, a MAPP comprises a Class II MHC β chain, without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring Class II MHC β chain. Thus, in some cases, a MAPP comprises only the β1 and β2 portions of a Class II MHC R chain; and does not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring Class II MHC β chain.

(i) MHC Class II Alpha Chains

MHC Class II alpha chains comprise an al domain and an α2 domain. In some cases, the α1 and α2 domains present in an antigen-presenting cell are from the same MHC Class II α chain polypeptide. In some cases, the α1 and α2 domains present in an antigen-presenting cell are from two different MHC Class II α chain polypeptides.

MHC Class II alpha chains suitable for inclusion in a presenting sequence or complex of a MAPP may lack a signal peptide. An MHC Class II alpha chain suitable for inclusion in a MAPP can have a length of from about 60 aas to about 200 aas; for example, an MHC Class II alpha chain suitable for inclusion in a MAPP can have a length of from about from about 60 amino acids to about 80 amino acids, 80 aas to about 100 aas, from about 100 aas to about 140 aas, from about 140 aas to about 170 aas, from about 170 aas to about 200 aas. An MHC Class II al domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 95 aas; for example, an MHC Class II al domain suitable for inclusion in a MAPP of the present disclosure can have a length of from about 30 aas to about 50 aas, from about 50 aas to about 70 aas, or from about 70 aas to about 95 aas. In an embodiment a MHC Class II al domain of a MAPP is from about 70 aas to about 95 aas. An MHC Class II α2 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 95 aas; for example, an MHC Class II α2 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 50 aas, from about 50 aas to about 70 aas, or from about 70 aas to about 95 aas. In an embodiment, an MHC Class II α2 domain of a MAPP is from about 70 aas to about 95 aas.

(a) DRA Polypeptides

A suitable MHC Class II DRA polypeptide for inclusion in a MAPP may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 150, at least 160, or at least 170 contiguous amino acids of the aa sequence from aa 26 to aa 203 of the DRA*01:02 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 (e.g., 175, 176, 177, 178, 179, or 180 aas). A suitable MHC Class II DRA polypeptide for inclusion in a MAPP may at least 90%, or at least 95% aa sequence identity with at least 170 contiguous amino acids of the aa sequence from aa 26 to aa 203 of the DRA*01:02 aa sequence depicted in FIG. 4.

As used herein, the term “DRA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRA polypeptide comprises aas 26-203 of DRA*01:02:01 (see 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 242 (see FIG. 4).

A suitable DRA for inclusion in a MAPP polypeptide can have at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity with at least 160, at least 170, or at least 180 contiguous aas of the sequence from aa 26 to aa 216 of the DRA*01:02 sequence depicted in FIG. 4. A “DRA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.

Thus, in some cases, a suitable DRA polypeptide comprises the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITNV PPEVTVLTNS PVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:88, amino acids 26-203 of DRA*01:02, see FIG. 4), or an allelic variant thereof. In some cases, the allelic variant is the DRA*01:01 allelic variant that differs from DRA*01:02 polypeptide by having a valine in place of the leucine at position 242 of the sequence in FIG. 4. In some cases, a DRA polypeptide suitable for inclusion in a MAPP comprises an amino acid substitution, relative to a wt. DRA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys (e.g., for forming a disulfide bond stabilizing the MAPP).

In some cases, a MAPP comprises a variant DRA polypeptide that comprises a non-naturally occurring Cys residue (e.g., for forming a disulfide bond stabilizing the MAPP). For example, in some cases, a MAPP comprises a variant DRA polypeptide that comprises an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, 172C, K75C, T80C, P81C, 182C, T93C, N94C, and S95C.

A suitable DRA al domain for inclusion in a MAPP polypeptide, including naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO:89); and can have a length of about 84 aas (e.g., 80, 81, 82, 83, 84, 85, or 86 aas).

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

(b) DQA Polypeptides

A suitable MHC Class II α DQA polypeptide for inclusion in a MAPP may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with the α1, α2, β1, and/or the β2 domain(s) of DQA1*0101, DQA1*0301, or DQA1*0401. In some cases, the DQA polypeptide has a length of about 178 aas (e.g., 175, 176, 177, 178, 179, or 180 aas). The sequences of those HLA polypeptides are available, for example on the world wide web at hla.alleles.org/nomenclature/index.html, which is run by the Anthony Nolan Research Institute, and at www.ncbi.nlm.nih.gov (National Center for Biotechnical Information or “NCBI”) operated by the U.S. National Library of Medicine.

(ii) MHC Class II Beta Chains

MHC Class II beta chains comprise a β1 domain and a β2 domain. In some cases, the β1 and β2 domains present in an antigen-presenting cell are from the same MHC Class II β chain polypeptide. In some cases, the β1 and β2 domains present in an antigen-presenting cell are from two different MHC Class II β chain polypeptides.

MHC Class II beta chains suitable for inclusion in a MAPP (e.g., a higher order MAPP construct such as a duplex MAPP) lack a signal peptide. An MHC Class II beta chain suitable for inclusion in a MAPP can have a length of from about 60 aas to about 210 aas; for example, an MHC Class II beta chain suitable for inclusion in a MAPP can have a length of from about 60 aas to about 90 aas, from about 90 aas to about 120 aas, from about 120 aas to about 150 aas, from about 150 aas to about 180 aas, from about 180 aas to 210 aas. An MHC Class II 1 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 105 aas; for example, an MHC Class II β1 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 50 aas, from about 50 aas to about 70 aas, from about 70 aas to about 90 aas, from about 90 aas to about 105 aas. An MHC Class II β2 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 105 aas; for example, an MHC Class II β2 domain suitable for inclusion in a MAPP can have a length of from about 30 aas to about 50 aas, from about 50 aas to about 70 aas, from about 70 aas to about 90 aas, from about 90 aas to about 105 aas.

In some cases, MHC Class II β chain polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. MHC Class II β chain polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond stabilizing the MAPP). For example, in some cases, the MHC Class II β chain polypeptide is a variant DRB1 MHC Class II polypeptide that comprises an aa substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC Class II β chain polypeptide is a variant DRB1 polypeptide comprising an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, aa sequence identity to the following mature DRB1 aa sequence lacking the signal peptide: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:91), and comprising an cysteine substitution at one or more (e.g., two or more) aas selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC Class II β chain polypeptide is a variant of a mature DRB3 polypeptide, mature DRB4 polypeptide, or mature DRB5 polypeptide (lacking their signal sequences) comprising a cysteine substitution at one or more (e.g., two or more) of positions 5, 7, 10, 19, 20, 33, 151, 152, and 153 (e.g., P5C, F7C, Q10C, N19C, G20C, N33C, G151C, D152C, and/or W153C substitutions).

(a) DRB Polypeptides

(1) DRB1 Polypeptides

In some cases, a suitable MHC Class II β chain polypeptide is a DRB1 polypeptide. In an embodiment, a DRB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity with at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of any DRB1 aa sequence depicted in FIG. 5, including naturally occurring allelic variants. FIG. 5 displays the DRB1 precursor proteins in which aas 1-29 are the signal sequence (underlined), 30-124 form the β1 region (bolded), 125-227 form the β2 region (bolded and underlined), and 228-250 the transmembrane region. In some cases, a DRB1 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB1 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond stabilizing the MAPP).

In some cases, a suitable MHC Class II β chain polypeptide is a DRB1 polypeptide. In an embodiment, a DRB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of a DRB1 sequence provided in FIG. 5, including one of the following DRB1 polypeptides:

• • (i) the DRB1-1 (DRB1*01:01) beta chain aa sequence Swiss-Prot/UniProt reference (“sp”) P04229.2 in FIG. 5;
  • (ii) the DRB1-3 (DRB1*03:01) beta chain aa sequence sp P01912.2 in FIG. 5;
  • (iii) the DRB1-4 (DRB1*04:01) beta chain aa sequence sp P13760.1 in FIG. 5;
  • (iv) the DRB1-7 (DRB1*07:01) beta chain aa sequence sp P13761.1 in FIG. 5;
  • (v) the DRB1-8 (DRB1*08:01) beta chain aa sequence sp Q30134.2 in FIG. 5;
  • (vi) the DRB1-9 (DRB1*09:01) beta chain aa sequence sp Q9TQE0.1 in FIG. 5;
  • (vii) the DRB1-10 (DRB1*10:01) beta chain aa sequence sp Q30167.2 in FIG. 5;
  • (viii) the DRB1-11 (DRB1*11:01) beta chain aa sequence sp P20039.1 in FIG. 5;
  • (ix) the DRB1-12 (DRB1*12:01) beta chain aa sequence sp Q951E3.1 in FIG. 5;
  • (x) the DRB1-13 (DRB1*13:01) beta chain aa sequence sp Q5Y7A7.1 in FIG. 5;
  • (xi) the DRB1-14 (DRB1*14:01) beta chain aa sequence sp Q9GIY3.1 in FIG. 5;
  • (xii) the DRB1-15 (DRB1*15:01) beta chain aa sequence sp P01911 in FIG. 5; and
  • (xiii) the DRB1-16 (DRB1*16:01) beta chain aa sequence sp Q29974.1 in FIG. 5.In some cases, the DRB1 β chain polypeptide has a length of about 198 aas, including, e.g., 195, 196, 197, 198, 199, 200, 201, or 202 aas.

A suitable “DRB1 polypeptide” for incorporation into a MAPP includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB1 polypeptide comprises aas 31-227 of DRB1*04:01 (DRB1-4) provided in FIG. 5 (SEQ ID NO:24) or an allelic variant thereof.

Another suitable DRB1 polypeptide may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 170, at least 180, or at least 190 contiguous aas of the following DRB1*04:01 aa sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPAS IEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRA RSESAQSKM (SEQ ID NO:95), which may bear one or more cysteine substitutions. In an embodiment the cysteine substitution is a P5C substitution. In an embodiment the cysteine substitution is a G151C substitution. In an embodiment the cysteine substitution is a W153C substitution.

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

A suitable DRB1 β1 domain can comprise the following amino acid sequence: GDTRCRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO:96), where P5 is substituted with a Cys (shown in bold and italics text).

A suitable DRB1 β2 domain, including-naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLV MLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO:97); and can have a length of about 103 aas, including, e.g., 100, 101, 102, 103, 104, 105, or 106 aas.

A suitable DRB1 β2 domain can comprise the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNGDCTFQTLV MLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO:98), where W153 is substituted with a Cys (shown in bold and italics text).

(2) DRB3 Polypeptides

In some cases, a suitable MHC Class II β chain polypeptide is a DRB3 polypeptide. In an embodiment, a DRB3 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-227 of any DRB3 aa sequence depicted in FIG. 6, which displays the DRB3 precursor proteins in which aas 1-29 are the signal sequence (underlined), 30-124 form the β1 region (shown bolded), 125-227 form the β2 region, and 228-250, the transmembrane region. In embodiments, a DRB3 β chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-227 of one of the following DRB3 polypeptides:

• • (i) the DRB1-3 (DRB3*01:01) beta chain aa sequence GenBank NP_072049.1 in FIG. 6;
  • (ii) the DRB1-3 beta chain aa sequence in GenBank accession EAX03632.1 in FIG. 6;
  • (iii) the DRB1-3 (DRB3*02:01) beta chain aa sequence GenBank CAA23781.1 in FIG. 6; and
  • (iv) the DRB1-3 (DRB3*03:01) beta chain aa sequence GenBank AAN15205.1 in FIG. 6.In some cases, a DRB3 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB3 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond stabilizing the MAPP).

A suitable “DRB3 polypeptide” for incorporation into a MAPP may include allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB3 polypeptide comprises aas 30 to 227 of DRB3*01:01 provided in FIG. 6 (SEQ ID NO:55), or an allelic variant thereof. Thus, in some cases, a suitable DRB3 polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to at least 170, at least 180, or at least 190 contiguous aas of the following sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRVHPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO:99), or an allelic variant thereof. In some cases, a DRB3 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB3 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys. Thus, e.g., in some cases, the MHC Class II β chain polypeptide is a variant DRB3 MHC Class II polypeptide that comprises a non-naturally occurring Cys at an aa selected from the group consisting of P5C, F7C, L10C, N19C, G20C, N33C, G151C, D152C, and W153C (of a mature DRB3 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO:92) depicted in FIG. 6).

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

(3) DRB4 Polypeptides

In some cases, a suitable MHC Class II β chain polypeptide is a DRB4 polypeptide. A DRB4 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-227 of a DRB4 aa sequence depicted in FIG. 7. In some cases, the DRB4 polypeptide has a length of about 198 aas, including, e.g., 195, 196, 197, 198, 199, 200, 201, or 202 aas. In some cases, a DRB4 polypeptide suitable for inclusion in a MAPP comprises an amino acid substitution, relative to a wt. DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.

A suitable “DRB4 polypeptide” for inclusion in a MAPP may include allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB4 polypeptide comprises aas 30 to 227 of DRB4*01:03 (SEQ ID NO:60) provided in FIG. 7, or an allelic variant thereof. In some cases, a DRB4 polypeptide suitable for inclusion in a MAPP comprises an amino acid substitution, relative to a wt. DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys. Thus, e.g., in some cases, the MHC Class II chain polypeptide is a variant DRB4 MHC Class II polypeptide that comprises a non-naturally occurring Cys residue; e.g., where the variant DRB4 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C of a mature DRB4 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO:93) depicted in FIG. 7).

A suitable DRB4 β1 domain, including-naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV QRRV (SEQ ID NO:104); and can have a length of about 95 aas (e.g., 93, 94, 95, 96, 97, or 98 aas).

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

(4) DRB5 Polypeptides

In some cases, a suitable MHC Class II β chain polypeptide is a DRB5 polypeptide. A DRB5 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with aas 30-227 of the DRB5 aa sequence depicted in FIG. 8. In some cases, the DRB5 polypeptide has a length of about 198 aas (including, e.g., 195, 196, 197, 198, 199, 200, 201, or 202 aas). In some cases, a DRB5 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB5 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys (e.g., for forming a disulfide bond stabilizing the MAPP).

A “DRB5 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRB4 polypeptide comprises aas 30 to 227 of DRB5*01:01 (SEQ ID NO:61) provided in FIG. 8, or an allelic variant thereof.

A suitable DRB5 β1 domain, including-naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: M VLSSPLALAG DTRPRFLQQD KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRV (SEQ ID NO:106); and can have a length of about 95 aas (e.g., 93, 94, 95, 96, 97, or 98 aas).

A suitable DRB5 β2 domain, including-naturally occurring allelic variants thereof, may comprise an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following aa sequence: EPKVTV YPARTQTLQH HNLLVCSVNG FYPGSIEVRW FRNSQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SPLTVEWRAQ SESAQS (SEQ ID NO:107); and can have a length of about 103 aas (e.g., 100, 101, 102, 103, 104, or 105 aas).

(b) DQB Polypeptides

A suitable MHC Class II α DQB polypeptide for inclusion in a MAPP may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with the α1, α2, β1, and/or the β2 domain(s) of DQB1*0201, DQB1*0302, DQB1*0303, DQB1*0402, or DQB1*0501. In some cases, the DQA polypeptide has a length of about 178 aas (e.g., 175, 176, 177, 178, 179, or 180 aas). The sequences of those HLA polypeptides are available, for example on the world wide web at hla.alleles.org/nomenclature/index.html, which is run by the Anthony Nolan Research Institute, and at www.ncbi.nlm.nih.gov (National Center for Biotechnical Information or “NCBI”) operated by the U.S. National Library of Medicine.

c. Individual Disease Risk-Associated Alleles

(i) DRB1

(a) DRB1*01:01

A MAPP may comprise a DRB1*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*01:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*01:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*01:01 aa sequence depicted in FIG. 5.

(b) DRB1*01:02

A MAPP may comprise a DRB1*01:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*01:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*01:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*01:02 aa sequence depicted in FIG. 5.

(c) DRB1*01:03

A MAPP may comprise a DRB1*01:03 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*01:03 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:03 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*01:03 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*01:03 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*01:03 aa depicted in FIG. 5.

(d) DRB1*03:01

DRB1*0301 (“DRB1*03:01” in FIG. 5) is associated with increased risk of developing T1D. Thus, a MAPP may comprise a DRB1*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*03:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*03:01 aa sequence depicted in FIG. 5. In some cases, a MAPP comprises a DRB1*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*03:01 aa sequence depicted in FIG. 5.

(e) DRB1*03:02

A MAPP may comprise a DRB1*03:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*03:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*03:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aa 30-124 of the DRB1*03:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*03:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*03:02 aa sequence depicted in FIG. 5.

(f) DRB1*03:04:

A MAPP may comprise a DRB1*03:04 polypeptide comprising an aa sequence having at least 70%, at least 80% at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*03:04 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*03:04 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*03:04 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*03:04 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*03:04 aa sequence depicted in FIG. 5.

(g) DRB1*04:01

A MAPP may comprise a DRB1*04:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*04:01 aa sequence depicted in FIG. 5. In some cases, a MAPP comprises a DRB1*04:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*04:01 aa sequence depicted in FIG. 5. In some cases, a MAPP comprises a DRB1*04:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*04:01 aa sequence depicted in FIG. 5.

(h) DRB1*04:02

A MAPP may comprise a DRB1*04:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*04:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*04:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*04:02 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*04:02 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*04:02 aa sequence provided in FIG. 5.

(i) DRB1*08:01

A MAPP may comprise a DRB1*08:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*08:01 aa sequence provided in FIG. 5 In some cases, a MAPP comprises a DRB1*08:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*08:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*08:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*08:01 aa sequence depicted in FIG. 5.

(j) DRB1*09:01

A MAPP may comprise a DRB1*09:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB1*09:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*09:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB1*09:01 aa sequence provided in FIG. 5. In some cases, a MAPP comprises a DRB1*09:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB1*09:01 aa sequence depicted in FIG. 5.

(ii) DRB3

In some cases, a MAPP comprises an MHC Class II β chain polypeptide of a DRB3 allele.

(a) DRB3*03:01

A MAPP may comprise a DRB3*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB3*03:01 aa sequence provided in FIG. 6. In some cases, a MAPP comprises a DRB3*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB3*03:01 aa sequence provided in FIG. 6. In some cases, a MAPP comprises a DRB3*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB3*03:01 aa acid sequence depicted in FIG. 6.

(iii) DRB4

In some cases, a MAPP comprises an MHC Class II β chain polypeptide of a DRB4 allele.

A MAPP may comprise a DRB4*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB4*01:01 aa sequence provided in FIG. 7. In some cases, a MAPP comprises a DRB4*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB4*01:01 aa sequence provided in FIG. 7. In some cases, a MAPP comprises a DRB3*03:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 125-227 of the DRB4*01:01 aa sequence provided in FIG. 7.

(iv) DRB5

A MAPP may comprise a MHC Class II β chain polypeptide of a DRB5 allele. In some cases, the DRB5 polypeptide has a length of about 198 aas (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 aas). In some cases, a DRB5 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB5 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys.

(a) DRB5*01:01

A MAPP may comprise a DRB5*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB5*01:01 aa sequence provided in FIG. 8. In some cases, a DRB5 polypeptide suitable for inclusion in a MAPP comprises an aa substitution, relative to a wt. DRB5 polypeptide, where the aa substitution replaces an aa (other than a Cys) with a Cys. Thus, e.g., in some cases, the MHC Class II β chain polypeptide is a variant DRB5 MHC Class II polypeptide that comprises a non-naturally occurring Cys residue; e.g., where the variant DRB5 MHC Class II polypeptide comprises an aa substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C (of a mature DRB5 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSYMAKLTVTL (SEQ ID NO:94) depicted in FIG. 8), or a naturally-occurring allelic variant thereof.

A MAPP may comprise a DRB5*01:01 β1 domain polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 30-124 of the DRB5*01:01 aa sequence provided in FIG. 8. In some cases, a MAPP comprises a DRB5*01:01 polypeptide comprising an aa sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to at least 170, at least 180, or at least 190, contiguous aas of the sequence from aa 30 to aa 227 of the DRB5*01:01 β2 domain aa sequence depicted in FIG. 8, or a naturally-occurring allelic variant thereof.

d. Disulfide Bonds and the Presenting Sequences and Presenting Complexes

Disulfide bonds involving an MHC peptide sequence may be included in a presenting sequence or complex of a MAPP. The disulfide bonds may increase the stability (e.g., thermal stability) and/or assist in positioning a peptide epitope in the binding pocket/groove of the MHC formed by its a and R chain sequences. The disulfide bonds may be between two MHC peptide sequences (e.g., a cysteine located in an α chain and a cysteine located in a β chain sequence). Disulfide bonds, and particularly disulfide bonds made to position a peptide epitope may be between two MHC peptide sequences or, alternatively, between a MHC peptide sequence and a linker attaching the peptide epitope and an MHC sequence (e.g., the linker between the epitope and β1 domain sequence in FIG. 15 structures A and B). Disulfide bonds for the stabilization and/or positioning epitope may be made using cysteines found within the MHC sequences and/or cysteines that have been added/engineered into one or more MHC sequences using the techniques of molecular biology. As discussed above, the α chain may include cysteines at positions 3, 4, 12, 28, 29, 72, 75, 80, 81, 82, 93, 94, and 95 of the mature α chain (lacking its signal sequence). For the DRA polypeptides cysteines substitutions include those at E3C, E4C, F12C, G28C, D29C, 172C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C (see FIG. 4). The β chain may include cysteine at positions 5, 7, 10, 19, 20, 33, 151, 152, and 153 of the mature β chain (lacking its signal sequence). For the DB1 polypeptides cysteines substitutions include those at positions P5C, F7C, Q10C (may be Y30C or E10C for some DRB1 alleles), N19C, G20C, H33C (may be N33C for some DRB1 alleles), G151C, D152C, and W153C.

Stabilizing disulfide bonds between α and β chain sequences include those between the α and β chain positions set forth in Table 3, which also provides the specific cysteine substitutions for HLA DRA*01:02 and DRB*0401 sequences. The stabilizing disulfide bonds between the MHC (e.g., HLA) a and β chains may be incorporated into any of the MAPP structures described herein. For example, such disulfide bonds may be incorporated into presenting sequences such as those shown in FIG. 15 and the presenting sequences shown in the MAPPs of FIG. 14. Stabilizing disulfide bonds may, for example be incorporated into a presenting sequence having, in order from the N- to C-terminus β1, β2, α1, and α2 domains (see e.g., FIG. 15 structure B).

	TABLE 3
			DRA*01:02	DRB*0401
	α chain	β chain	α chain	β chain
	position	position	substitution	substitution
	3	19	E3C	N19C
	3	20	E3C	G20C
	4	19	E4C	N19C
	4	20	E4C	G20C
	28	151	G28C	G151C
	28	152	G28C	D152C
	28	153	G28C	W153
	29	151	D29C	G151C
	29	152	D29C	D152C
	29	153	D29C	W153
	80	33	T80C	H33C
	81	33	P81C	H33C
	82	33	I82C	H33C
	93	156	T93C	Q156C
	93	153	T93C	W153C
	94	156	N94C	Q156C
	94	120	N94C	N120C
	95	156	S95C	Q156C
	95	120	S95C	N120C

Disulfide bonds between the MHC α and β chain sequences that assist in positioning the peptide epitope and/or stabilizing the structure of the presenting sequence or complex are formed between a first aa and second aa of the MAPP. The first aa is either (i) an aa position proximate to the point where a peptide epitope (or a peptide epitope and linker) are attached to an MHC peptide sequence or (ii) an aa (a cysteine) in a linker attached to the peptide epitope, while the second aa is position elsewhere in the MHC peptide sequence. By way of example, where a presenting sequence comprises from N-terminus to C-terminus a peptide epitope, β1 domain, β2 domain, al domain, and α2 domain aa sequences, a cysteine substituted within the first ten amino acids (e.g., aas 5-10) of the β1 domain can serve as a first aa and provide a point to anchor the peptide epitope and/or stabilize the MAPP when bonded to a with second cysteine located in, for example, the al domain, or α2 domain of the presenting sequence. Some examples of disulfide bonds between the MHC α and β chain sequences that assist in positioning the peptide epitope and/or stabilizing the structure of a presenting sequence or complex include those set forth in Table 4.

	TABLE 4
			DRA*01:02	DRB*0401
	α chain	β chain	α chain	β chain
	position	position	substitution	substitution
	12	7	F12C	F7C
	12	10	F12C	Q10C
	80	5	T80C	P5C
	80	7	T80C	F7C
	81	5	P81C	P5C
	81	7	P81C	F7C
	82	5	I82C	P5C
	82	7	I82C	F7C

Thus, for example, when a presenting sequence of complex comprises in the N-terminal to C-terminal direction a peptide epitope bound to a β1 domain, then a disulfide bond between a cysteine substituted at one of position 5-7 of the β chain, and a cysteine at one of aa positions 80-82 of the α chain may be use for positioning the peptide epitope or stabilizing the structure of a presenting sequence. By way of example a disulfide bond between a β chain P5C substitution and an α chain P81C substitution may be used for positioning of the peptide epitope and or stabilization of a presenting sequence. The same type of disulfide bonding is applicable to presenting complexes, and both presenting complexes and presenting sequences may have additional disulfide bonds (e.g., as in Table 3) for stabilization.

Where a cysteine residue in a linker attached to the peptide epitope is employed to position the peptide epitope and/or stabilize the structure of a presenting sequence or complex, the cysteine is typically located at an aa proximate to the point where the linker and peptide epitope meet. For example, where the MAPP comprises an epitope place on the N-terminal side of a linker peptide sequence the cysteine may be within about 6 aas of the position were the linker and peptide epitope meet, that is to say at one of amino acids 1-5 (aa1, aa2, aa3, aa4, or aa5) of a MAPP comprising the construct epitope-aa1-aa-aa3-aa4-aa5-(remainder of the linker/MAPP). Where the linker comprises repeats of the sequence GGGGS (SEQ ID NO:133), aa1 to aa5 are G1, G2, G3, G4, and S5, and the linker substitutions may be referred to as, for example a “G2C.” This is exemplified by SEQ ID NO:159, that has four repeats of GGGGS in which the aa at position 2 of the linker (aa2), is a glycine substituted by a cysteine: GCGGSGGGGSGGGGSGGGGS. Examples of cysteine containing linkers suitable for forming disulfide bonds with a cysteine in an MHC peptide (e.g., a DRA peptide) in a presenting sequence or complex comprising an epitope placed on the N-terminal side of a linker bound to a DRB aa polypeptide (i.e., the MAPP comprises the structure epitope-aa1-aa2-aa3-aa4-aa5-remainder of linker if present]-DRB peptide such as a β1 domain) are set forth in Table 5. Also provided in Table 5 is the location for a cysteine substituted in a DRA peptide (see e.g., FIG. 4) that will form the disulfide bond for positioning the peptide epitope and/or stabilizing the structure of a presenting sequence or complex.

TABLE 5
Exemplary Linker disulfide bonds for  MAPPS comprising the structure
epitope-aa1-aa2-aa3-aa4-aa5-(remainder of linker if present)-DRB peptide
Cysteine	Cysteine		a chain position,
posi-	substitution in the	Cysteine substitution in a	(DRA Cysteine
tion	general structure	GGGGS containing linker	substitution)
aa1	epitope-C-aa2-aa3-aa4-aa5-	epitope-CGGGS-(remainder of	72 (I72C)
	(remainder of linker)-DRB	linker)-DRB
		(i.e., a linker G1C substitution)
aa2	epitope-aa1-C-aa3-aa4-aa5-	epitope-GCGGS-(remainder of	72 (I72C)
	(remainder of linker)-DRB	linker)-DRB
		(i.e., a linker G2C substitution)
aa3	epitope-aa1-aa2-C-aa4-aa5-	epitope-GGCGS-(remainder of	72 (I72C)
	(remainder of linker)-DRB	linker)-DRB
		(i.e., a linker G3C substitution)
aa4	epitope-aa1-aa2-aa3-C-aa5-	epitope-GGGCS-(remainder of	72 (I72C)
	(remainder of linker)-DRB	linker)-DRB
		(i.e., a linker G4C substitution)
aa5	epitope-aa1-aa2-aa3-aa4-C-	epitope-GCGGGC-(remainder	72 (I72C)
	(remainder of linker)-DRB	of linker)-DRB
		(i.e., a linker S5C substitution)
aa1	epitope-C-aa2-aa3-aa4-aa5-	epitope-CGGGS-(remainder of	75 (K75C)
	(remainder of linker)-DRB	linker)-DRB
aa2	epitope-aa1-C-aa3-aa4-aa5-	epitope-GCGGS-(remainder of	75 (K75C)
	(remainder of linker)-DRB	linker)-DRB
aa3	epitope-aa1-aa2-C-aa4-aa5-	epitope-GGCGS-(remainder of	75 (K75C)
	(remainder of linker)-DRB	linker)-DRB
aa4	epitope-aa1-aa2-aa3-C-aa5-	epitope-GGGCS-(remainder of	75 (K75C)
	(remainder of linker)-DRB	linker)-DRB
aa5	epitope-aa1-aa2-aa3-aa4-C-	epitope-GCGGGC-(remainder	75 (K75C)
	(remainder of linker)-DRB	of linker)-DRB

MAPPs with presenting sequences or complexes comprising an epitope-linker-DRB structure recited in Table 5 (see, e.g.: FIG. 14; FIG. 15 structures A and B; FIG. 16 structures A, D, F and H; FIG. 17; FIG. 18, and FIG. 19 A-F) may have for example a disulfide bond for positioning the peptide epitope and/or stabilizing the structure of a presenting sequence or complex. The disulfide may be formed between linker aa2 (e.g., a G2C) and a cysteine at DRA aa 72 (e.g., 172C). The disulfide may be formed between linker aa2 (e.g., a G2C) and a cysteine at DRA aa 72 (e.g., K75C).

Where a disulfide bond is formed between the linker and an MHC polypeptide of a presenting sequence or presenting complex, the presenting sequence or presenting complexes may have additional disulfide bonds (e.g., as in Table 3) for stabilization.

5. Immunomodulatory Polypeptides (“MODs”)

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), JAGI (CD339), lymphotoxin beta receptor, 3/TR6, OX40L (CD252), PD-L1, PD-L2, TGF-β1, TGF-β2, TGF-β3, 4-1BBL, and fragments of any thereof, such as ectodomain fragments, capable of engaging and signaling through their cognate receptor. Some MOD polypeptides suitable for inclusion in a MAPP of the present disclosure, and their “co-MODS (“co-immunomodulatory polypeptides” or cognate costimulatory receptors) include polypeptide sequences with T cell modulatory activity from the protein pairs recited in the following table:

Exemplary Pairs of MODs and Co-MODs

a) 4-1BBL (MOD) and 4-1BB (Co-MOD);
b) PD-L1 (MOD) and PD1 (Co-MOD);
c) IL-2 (MOD) and IL-2 receptor (Co-MOD);
d) CD80 (MOD) and CD28 (Co-MOD);
e) CD86 (MOD) and CD28 (Co-MOD);
f) OX40L (CD252) (MOD) and OX40 (CD134) (Co-MOD);
g) Fas ligand (MOD) and Fas (Co-MOD);
h) ICOS-L (MOD) and ICOS (Co-MOD);
i) ICAM (MOD) and LFA-1 (Co-MOD);
j) CD30L (MOD) and CD30 (Co-MOD);
k) CD40 (MOD) and CD40L (Co-MOD);
l) CD83 (MOD) and CD83L (Co-MOD);
m) HVEM (CD270) (MOD) and CD160 (Co-MOD);
n) JAG1 (CD339) (MOD) and Notch (Co-MOD);
o) JAG1 (CD339) (MOD) and CD46 (Co-MOD);
p) CD70 (MOD) and CD27 (Co-MOD);
q) CD80 (MOD) and CTLA4 (Co-MOD);
r) CD86 (MOD) and CTLA4 (Co-MOD);
s) PD-L1(MOD) and CD-80 (Co-MOD); and
t) TGF-β1, TGF-β2, and/or TGF-β3 (MODs) and TGF-β
Receptor (e.g., TGFBR1 and/or TGFBR2) (Co-MOD)

In some cases, the MOD is selected from an IL-2 polypeptide, a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGFβ polypeptide, and a PD-L2 polypeptide. In some cases, the MAPP or duplex MAPP comprises two different MODs, such as an IL-2 MOD or IL-2 variant MOD polypeptide and either a CD80 or CD86 MOD polypeptide. In another instance, the MAPP or duplex MAPP comprises an IL-2 MOD or IL-2 variant MOD polypeptide and a PD-L1 MOD polypeptide. 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 MHC sequences or epitope sequences intervene). The MOD polypeptide 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 stated otherwise, 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.

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 polypeptide sequence that alter affinity may be identified by scanning (making aa substitution e.g., alanine substitutions or “alanine scanning” or charged residue changes) along the length of a peptide and testing its affinity. 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 with Reduced Affinity

A MOD can comprise a wt. amino acid sequence, or can comprise one or more amino acid substitutions, insertions, and/or deletions relative to a wt. amino acid sequence. The immunomodulatory polypeptide can 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 amino acid sequence of a naturally-occurring MOD polypeptide.

Variant MODs comprise at least one amino acid substitution, addition and/or deletion as compared to the amino acid 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 amino acid substitution(s) present in the variant) for the co-MOD.

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

Suitable immunomodulatory domains that exhibit reduced affinity for a co-immunomodulatory domain can have from 1 aa to 20 aa differences from a wt. immunomodulatory domain. 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.

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. In some cases, a variant MOD present in a MAPP has a binding affinity for a cognate co-MOD that is from 100 nM to 100 μM. For example, in some cases, a variant MOD present in a MAPP has a binding affinity for a cognate co-MOD that is from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 M.

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

b. IL-2 and its Variants

As one non-limiting example, a MOD or variant MOD present in a MAPP is an IL-2 or variant IL-2 polypeptide. In some cases, a variant MOD present in a MAPP is a variant IL-2 polypeptide. Wild-type IL-2 binds to an IL-2 receptor (IL-2R). A wt. IL-2 aa sequence can be as follows: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (aa 21-153 of UniProt P60568, SEQ ID NO:108).

Wild-type IL2 binds to an IL2 receptor (IL2R) on the surface of a cell. An IL2 receptor is in some cases a heterotrimeric polypeptide comprising an alpha chain (IL-2Rα; also referred to as CD25), a beta chain (IL-2RD; also referred to as CD122) and a gamma chain (IL-2Rγ; also referred to as CD132). Amino acid sequences of human IL-2Rα, IL2Rβ, and IL-2Rγ are provided in the accompanying sequence listing as SEQ ID NO:109, SEQ ID NO:110 and SEQ ID NO:111 respectively, and are also provided in, for example, U.S. Patent Pub. No. 20200407416.

In some cases, a variant IL-2 polypeptide exhibits reduced binding affinity to one or more of the IL-2Rα, IL2Rβ, and/or IL-2Rγ chains of human IL-2R, compared to the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO:108. For example, in some cases, a variant IL-2 polypeptide binds to one or more of the IL-2Rα, IL2Rβ, and/or IL-2Rγ chains of human IL-2R with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO:108 for the α, β, and/or γ chains of IL-2R (e.g., an IL-2R comprising polypeptides comprising the aa sequence set forth in SEQ ID NOs:109-111), when assayed under the same conditions.

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

In some cases, a variant IL-2 polypeptide has a single aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO:108. In some cases, a variant IL-2 polypeptide has from 2 to 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:108. In some cases, a variant IL-2 polypeptide has 2, 3, 4, 5, 6, 7, 8, 9 or 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:108. In some cases, a variant IL-2 polypeptide has 2 or 3 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:108.

Suitable variant IL-2 polypeptide sequences include polypeptide sequences comprising an aa sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 80 (e.g., 90, 100, 110, 120, 130 or 133) contiguous aas of SEQ ID NO:108. Potential amino acids where substitutions may be introduced include one or more of the following positions:

• • (i) position 15, where the aa is other than E (e.g., A);
  • (ii) position 16, where the aa is other than H (e.g., A, T, N, C, Q, M, V or W);
  • (iii) position 20 is an aa other than D (e.g., A);
  • (iv) position 42, where the aa is other than F (e.g., A, M, P, S, T, Y, V or H);
  • (v) position 45, where the aa is other than Y (e.g., A);
  • (vi) position 88, where the aa is other than N (e.g., A or R);
  • (vii) position 126, where the aa is other than Q (e.g., A);

Combinations of the above substitutions include (H16X, F42X), (D20X, F42X), (E15X, D20X, F42X), (an H16X, D20X, F42X), (H16X, F42X, R88X), (H16X, F42X, Q126X), (D20X, F42X, Q126X), (D20X, F42X, and Y4X), (H16X, D20X, F42X, and Y45X), (D20X, F42X, Y45X, Q126X), (H16X, D20X, F42X, Y45X, Q126X), where X is the substituted aa, optionally chosen from the following: positions 15, 20, 45, 126—A; position 16—A or T, or also N, C, Q, M, V or W; position 42—A, or also M, P, S, T, Y, V or H; position 88—A or R.

IL-2 variants include polypeptides having at least 90% or at least 95% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) contiguous aas of SEQ ID NO:108, wherein the aa at position 16 is an aa other than H. In one case, the position of H16 is substituted by Asn, Cys, Gln, Met, Val, or Trp. In one case, the position of H16 is substituted by Ala. In another case, the position of H16 is substituted by Thr. Additionally, or alternatively, IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) contiguous aas of SEQ ID NO:108, wherein the aa at position 42 is an aa other than F. In one case, the position of F42 is substituted by Met, Pro, Ser, Thr, Trp, Tyr, Val, or His. In one case, the position of F42 is substituted by Ala.

IL-2 variants include polypeptides comprising an aa sequence comprising all or part of human IL-2 polypeptide having a substitution at position H16 and/or F42 (e.g., H16A and/or F42A substitutions).

IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 100, 110, 120, or 130) contiguous aas of SEQ ID NO:108, wherein the aa at position 16 is an aa other than H and the aa at position 42 is other than F. In one case, the position of H16 is substituted by Ala or Thr and the position of F42 is substituted by Ala or Thr. In one case, the position of H16 is substituted by Ala and the position of F42 is substituted by Ala (an H16A and F42A variant). In a second case, the position of H16 is substituted by Thr and the position of F42 is substituted by Ala (an H16T and F42A variant). In a third case, the position of H16 is substituted by Ala and the position of F42 is substituted by Thr (an H16A and F42T variant). In a fourth case, the position of H16 is substituted by Thr and the position of F42 is substituted Thr Ala (an H16T and F42T variant). As noted above, such variants will exhibit reduced binding to both the human IL-2Rα chain and IL2Rβ chain.

In any of the wt. or variant IL-2 sequences provided herein, the cysteine at position 125 may be substituted with an aa other than cysteine, such as alanine (a C125A substitution). In addition to any stability provided by the substitution, it may be employed where, for example, an additional peptide is to be conjugated to a cysteine residue elsewhere in a MAPP, thereby avoiding competition from the C125 of the IL-2 MOD sequence.

c. Fas Ligand (FasL) and its Variants

In some cases, a wt. and/or a variant Fas Ligand (FasL) polypeptide sequence is present as a MOD in a MAPP. FasL is a homomeric type-II transmembrane protein in the tumor necrosis factor (TNF) family. FasL signals by trimerization of the Fas receptor in a target cell, which forms a death-inducing complex leading to apoptosis of the target cell. Soluble FasL results from matrix metalloproteinase-7 (MMP-7) cleavage of membrane-bound FasL at a conserved site.

In an embodiment, a wt. Homo sapiens FasL protein has the sequence MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L, (SEQ ID NO:112, NCBI Ref. Seq. NP_000630.1, UniProtKB—P48023 where residues 1-80 are cytoplasmic, 81-102 are the transmembrane domain and aas 103-281 are extracellular (ectodomain). In some cases, a FasL polypeptide suitable for inclusion in a MAPP comprises an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to a contiguous stretch of at least 150 aas, at least 170, at least 180 aas, at least 200 aas, at least 225 aas, at least 250 aas, at least 270 aas, at least 280, or all aas of the aa sequence of SEQ ID NO:112.

A Fas receptor can have the sequence MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV, SEQ ID NO:114, NCBI Reference Sequence: NP_000034.1, UniProtKB—P25445, where aas 26-173 form the ectodomain (extracellular domain), aas 174-190 form the transmembrane domain, and 191-335 the cytoplasmic domain. The ectodomain may be used to determine binding affinity with FasL.

In some cases, a FasL polypeptide suitable for inclusion in a MAPP comprises an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to a contiguous stretch of at least 50 aas, at least 160 aas, at least 170, at least 175, or all of the aas the following aa sequence: QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO:113). Suitable variant FasL polypeptide sequences include polypeptide sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 140 contiguous aa (e.g., at least 150, at least 160, at least 170, or at least 175 contiguous aa) of SEQ ID NO:113) (e.g., which have at least one aa substitution, deletion or insertion).

In some cases, a variant FasL polypeptide (e.g., comprising a variant of SEQ ID NO:113) exhibits reduced binding affinity to a mature Fas receptor sequence (e.g., a FasL receptor comprising all or part of the polypeptide set forth in SEQ ID NO:114, such as its ectodomain), compared to the binding affinity of an FasL polypeptide comprising the aa sequence set forth in SEQ ID NO:113. For example, in some cases, a variant FasL polypeptide (e.g., comprising a variant of SEQ ID NO:113) binds an Fas receptor (e.g., comprising all or part of the polypeptides set forth in SEQ ID NO:114, such as its ectodomains), with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of an FasL polypeptide comprising the aa sequence set forth in SEQ ID NO:113.

A FasL polypeptide suitable for inclusion in a MAPP may comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK (SEQ ID NO:115); and has a length of about 150 aas, including 148, 149, 150, 151, or 152 aas.

d. PD-L1 and its Variants

As one non-limiting example, a MOD or variant MOD present in a MAPP is a PD-L1 or variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

A wt. human PD-L1 polypeptide can comprise the following aa sequence: MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST (SEQ ID NO:116); where aas 1-18 form the signal sequence, aas 19-127 form the Ig-like V-type or IgV domain, and 133-225 for the Ig-like C2 type domain.

A wt. human PD-L1 ectodomain aa sequence can comprise the following aa sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:117); where aas 1-109 form the Ig-like V-type or “IgV” domain, and aas 115-207 for the Ig-like C2 type domain.

A wt. human PD-L1 ectodomain aa sequence can also comprise the following aa sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNER LNVSIKI (SEQ ID NO:123); where aas 1-109 form the Ig-like V-type or “IgV” domain, and aas 115-207 for the Ig-like C2 type domain. See e.g., NCBI Accession and version 3BIK_A, which includes an N-terminal alanine as its first aa.

A wt. PD-L1 IgV domain, suitable for use as a MOD may comprise aa 18 and aas IgV aas 19-127 of SEQ ID NO:116, and a carboxyl terminal stabilization sequences, such as for instance the last seven aas (bolded and italicized) of the sequence: A FTVTVPKDLY VVEYGSNMTI ECKFPVEKQL DLAALIVYWE MEDKNIIQFV HGEEDLKTQH SSYRQRARLL KDQLSLGNAA LQITDVKLQD AGVYRCMISY GGADYKRITV KVNAPYAAAL HEH (SEQ ID NO:118). Where the carboxyl stabilizing sequence comprises a histidine (e.g., a histidine approximately 5 residues to the C-terminal side of the Tyr (Y) appearing as aa 117 of SEQ ID NO:118) to about aa 122, the histidine may form a stabilizing electrostatic bond with the backbone amide at aas 82 and 83 (bolded and italicized in SEQ ID NO:116 (Q107 and L106 of SEQ ID NO:116). As an alternative, a stabilizing disulfide bond may be formed by substituting one of aas 82 or 83) (Q107 and L106 of SEQ ID NO:116) and one of aa residues 121, 122, or 123 (equivalent to aa positions 139-141 of SEQ ID NO:116).

A wt. PD-1 polypeptide can comprise the following aa sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO:119).

In some cases, a variant PD-L1 polypeptide (e.g. a variant of SEQ ID NO:117 or PD-L1's IgV domain) exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO:119), compared to the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO:116 or SEQ ID NO:117. For example, in some cases, a variant PD-L1 polypeptide binds PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO:119) with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO:116 or SEQ ID NO:117.

e. TGF-β and its Variants

In some cases, at least one of the one or more MOD polypeptides present in a MAPP comprises the aa sequence of a wt. TGF-β polypeptide. In other instances, at least one of the one or more MOD polypeptides present in a MAPP is a variant TGF-β polypeptide. Wild-type TGF-β and variant TGF-β polypeptides bind to TGF receptor.

As noted above, in some cases, the MOD polypeptide present in a MAPP is a TGF-β polypeptide. The aa sequences of TGF-β polypeptides are known in the art. In some cases, the MOD polypeptide present in a MAPP is a TGF-β1 polypeptide. MOD polypeptide present in a MAPP is a TGF-β2 polypeptide. MOD polypeptide present in a MAPP is a TGF-β3 polypeptide. A suitable TGF-β polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the mature form of a human TGF-β1 polypeptide, a human TGF-β2 polypeptide, or a human TGF-β3 polypeptide. A suitable TGF-β polypeptide can have a length of from about 100 aas to about 125 aas; for example, a suitable TGF-β polypeptide can have a length of from about 100 aas to about 105 aas, from about 105 aas to about 110 aas, from about 110 aas to about 115 aas, from about 115 aas to about 120 aas, or from about 120 aas to about 125 aas.

A suitable TGF-β 1 polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following TGF-β1 aa sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO:120); or the foregoing sequence comprising a C77S substitution; where the TGF-β1 polypeptide has a length of about 112 aas.

A suitable TGF-β2 polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following TGF-β2 aa sequence: ALDAAYCF RNVQDNCCLR PLYIDFKRDL GWKWIHEPKG YNANFCAGAC PYLWSSDTQH SRVLSLYNTI NPEASASPCC VSQDLEPLTI LYYIGKTPKI EQLSNMIVKS CKCS (SEQ ID NO:121); or the foregoing sequence comprising a C79S substitution, where the TGF-β2 polypeptide has a length of about 112 aas.

A suitable TGF-β3 polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to the following TGF-β3 aa sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS (SEQ ID NO:122); or the foregoing sequence comprising a C77S substitution, where the TGF-β3 polypeptide has a length of about 112 aas.

6. Linkers

As noted above, a MAPP can include a linker sequence (aa, peptide, or polypeptide linker sequence) or “linker” interposed between any two elements of a MAPP, e.g., an epitope and an MHC polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between a first MHC polypeptide and a second MHC polypeptide; etc. Although termed “linkers,” sequences employed for linkers may also be placed at the N- and/or C-terminus of a MAPP polypeptide to, for example, stabilize the MAPP polypeptide or protect it from proteolytic degradation.

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

Polypeptide linkers in the MAPP may include, for example, polypeptides that comprise, consist essentially of, or consists of: i) Gly and Ser; ii) Ala and Ser; iii) Gly, Ala, and Ser; iv) Gly, Ser, and Cys (e.g., a single Cys residue); v) Ala, Ser, and Cys (e.g., a single Cys residue); and vi) Gly, Ala, Ser, and Cys (e.g., a single Cys residue). Exemplary linkers may comprise glycine polymers, glycine-serine polymers, glycine-alanine polymers; alanine-serine polymers (including, for example polymers comprising the sequences GSGGS (SEQ ID NO:124) or GGGS (SEQ ID NO:125), any of which may be repeated from 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times); and other flexible linkers known in the art. Glycine and glycine-serine polymers can both be used; both Gly and Ser are relatively unstructured and therefore can serve as a neutral tether between components. Glycine polymers access significantly more phi-psi space than even alanine, and are much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may also comprise an aa sequence comprising, but not limited to, GGSG (SEQ ID NO:126), GGSGG (SEQ ID NO:127), GSGSG (SEQ ID NO:128), GSGGG (SEQ ID NO:129), GGGSG (SEQ ID NO:130), GSSSG (SEQ ID NO:131), any which may be repeated from 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), or combinations thereof, and the like. Linkers can also comprise the sequence Gly(Ser)₄ (SEQ ID NO:132) or (Gly)₄Ser (SEQ ID NO:133), either of which may be repeated from 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In one embodiment the linker comprises the aa sequence AAAGG (SEQ ID NO:134), which may be repeated from 1 to 10 times.

Rigid polypeptide linkers comprise a sequence of amino acids that effectively separates protein domains by maintaining a substantially fixed distance/spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid polypeptide linkers thus may be employed where it is desired to minimize the interaction between the domains of the MAPP. Rigid peptide linkers include peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an α-helical structure. Examples of rigid peptide linkers include, e.g., (EAAAK)n (SEQ ID NO:135), A(EAAAK)nA (SEQ ID NO:136), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO:137), (Lys-Pro)n, (Glu-Pro)n, (Thr-Pro-Arg)n, and (Ala-Pro)n where n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid linkers comprising EAAAK (SEQ ID NO:138) include EAAAK (SEQ ID NO:138), (EAAAK)₂ (SEQ ID NO:139), (EAAAK)₃ (SEQ ID NO:140), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO:141), and AEAAAKEAAAKA (SEQ ID NO:142). Non-limiting examples of suitable rigid linkers comprising (AP)n include PAPAP (SEQ ID NO:143; also referred to herein as “(AP)2”); APAPAPAP (SEQ ID NO:144; also referred to herein as “(AP)4”); APAPAPAPAPAP (SEQ ID NO:145; also referred to herein as “(AP)6”); APAPAPAPAPAPAPAP (SEQ ID NO:146; also referred to herein as “(AP)8”); and APAPAPAPAPAPAPAPAPAP (SEQ ID NO:147; also referred to herein as “(AP)10”). Non-limiting examples of suitable rigid linkers comprising (KP)n include KPKP (SEQ ID NO:148; also referred to herein as “(KP)2”); KPKPKPKP (SEQ ID NO:149; also referred to herein as “(KP)4”); KPKPKPKPKPKP (SEQ ID NO:150; also referred to herein as “(KP)6”); KPKPKPKPKPKPKPKP (SEQ ID NO:151; also referred to herein as “(KP)8”); and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:152; also referred to herein as “(KP)10”). Non-limiting examples of suitable rigid linkers comprising (EP)n include EPEP (SEQ ID NO:153; also referred to herein as “(EP)2”); EPEPEPEP (SEQ ID NO:154; also referred to herein as “(EP)4”); EPEPEPEPEPEP (SEQ ID NO:155; also referred to herein as “(EP)6”); EPEPEPEPEPEPEPEP (SEQ ID NO:156; also referred to herein as “(EP)8”); and EPEPEPEPEPEPEPEPEPEP (SEQ ID NO:157; also referred to herein as “(EP)10”).

As with other linker sequences, rigid peptide linkers may be interposed between any two elements of a MAPP. Rigid peptide linkers find particular use in joining MOD polypeptide sequences to other elements of a MAPP. In particular, rigid peptide linkers may be employed to link a MOD polypeptide sequence to the carboxy terminus of frame work polypeptides (position 3 and/or 3′) or a dimerization polypeptide (positions 5 and/or 5′) of a duplex MAPP. For example, a MOD polypeptide comprising an immunoglobulin CH2CH3 multimerization sequence may comprise a rigid peptide linker and a MOD (e.g., a wt. or variant IL-2 or PD-L1 MOD) at position 3 and/or 3′ (See, e.g., FIGS. 9-13). Rigid peptide linkers may also be used to link a MOD polypeptide to the N-terminus of a MAPP polypeptide (e.g., the N-terminus of a dimerization and/or framework polypeptide at positions 1 and/or 1′).

In some cases, a linker polypeptide, present in a polypeptide of a MAPP includes a cysteine residue that can form a disulfide bond with a cysteine residue present in another polypeptide of the MAPP. In some cases, for example, the linker comprises an aa sequence selected from CGGGS (SEQ ID NO:189), GCGGS (SEQ ID NO:190), GGCGS (SEQ ID NO:191), GGGCS (SEQ ID NO:192), and GGGGC (SEQ ID NO:193) with the rest of the linker comprised of Gly and Ser residues (e.g., GGGGS units that may be repeated from 1 to 10 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Cysteine containing linkers may also be selected from the sequences GCGASGGGGSGGGGS (SEQ ID NO:158), GCGGSGGGGSGGGGSGGGGS (SEQ ID NO:159), and GCGGSGGGGSGGGGS (SEQ ID NO:160).

Accordingly, the linker to which an epitope is attached may be from about 5 to about 50 aas in length. The linker to which an epitope may be attached may, for example be from about 5 to about 50 aas in length and comprise more than 50% Gly and Ser residues with one cysteine residue. The linker to which an epitope may be attached may be from about 5 to about 50 aas in length and comprise more than 50% (Gly)₄S repeats with one optional cysteine residue. The linker to which an epitope may be attached may be a (Gly)₄S sequence repeated from 3 to 8 (e.g., 3 to 7) times, optionally having one aa replaced by a cysteine residue.

7. Epitopes

A variety of peptide epitopes (also referred to herein as “epitopes”) may be present in a MAPP or higher order complexes of MAPPs (such as duplex MAPPs of the present disclosure), and presentable to a TCR on the surface of a T cell.

A peptide epitope present in a MAPP (e.g., a duplex MAPP) is designed to be specifically bound by a target T cell that has a T cell receptor (“TCR”) that is specific for the epitope and which specifically binds the peptide epitope of the MAPP. An epitope-specific T cell thus binds a peptide epitope having a reference aa sequence, but substantially does not bind an epitope that differs from the reference aa sequence. For example, an epitope-specific T cell binds a peptide epitope having a reference aa sequence, and binds an epitope that differs from the reference aa sequence, if at all, with an affinity that is less than 10⁻⁶ M, less than 10⁻⁵ M, or less than 10⁻⁴ M. An epitope-specific T cell can bind a peptide epitope for which it is specific with an affinity of at least 10⁻⁷ M, at least 10⁻⁸ M, at least 10⁻⁹ M, or at least 10⁻¹⁰ M.

a. Peptide Epitopes in MAPPs with Class II MHC Presenting Sequences and Presenting Complexes

Among the epitopes that may be bound and presented to a TCR by a MAPP with Class II MHC presenting sequences or Class II MHC presenting complexes are T1D-associated epitopes derived from self antigens associated with T1D (“T1D-associated antigens”). A Type 1 Diabetes-associated epitope (also referred to herein as a “T1D peptide epitope” or “T1D epitope”) present in a MAPP presents a T1D-associated epitope to a TCR on the surface of a T cell.

A T1D peptide epitope can have a length of from about 4 aas to about 25 aas (aa), e.g., the epitope can have a length of from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, or from 20 aa to 25 aa. For example, a T1D epitope present in a MAPP can have a length of 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, a T1D peptide epitope present in a MAPP has a length of from 10 aa to 20 aa, e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa and 20 aa.

Antigens associated with type 1 diabetes (T1D) include, e.g., preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, proinsulin C peptide, 65 kDa isoform of glutamic acid decarboxylase (GAD65), 67 kDa isoform of glutamic acid decarboxylase (GAD67), tyrosine phosphatase (IA-2), heat-shock protein HSP65, islet-specific glucose6-phosphatase catalytic subunit related protein (IGRP), islet antigen 2 (IA2), and zinc transporter (ZnT8). See, e.g., Mallone et al. (2011) Clin. Dev. Immunol. 2011:513210; and U.S. Patent Publication No. 2017/0045529. An antigen “associated with” a particular autoimmune disorder is an antigen that is a target of autoantibodies and/or autoreactive T cells present in individuals with that autoimmune disorder, T1D in this instance, where such autoantibodies and/or autoreactive T cells mediate a pathological state associated with the autoimmune disorder. A suitable T1D peptide epitope for inclusion in a MAPP can be a peptide epitope of from 4 aas to about 25 aas in length of any one of the aforementioned T1D-associated antigens.

As one non-limiting example, a T1D peptide epitope is proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO:161). As another non-limiting example, a T1D peptide epitope is the following insulin (InsA (1-15) peptide: GIVDQCCTSICSLYQ (SEQ ID NO:162). As another non-limiting example, a T1D peptide epitope is the following insulin (InsA(1-15; D4E) peptide: GIVEQCCTSICSLYQ (SEQ ID NO:163). As another non-limiting example, a T1D peptide epitope is the following GAD65 (555-567) peptide: NFFRMVISNPAAT (SEQ ID NO:164). As another non-limiting example, a T1D peptide epitope is the following GAD65 (555-567; F557I) peptide: NFIRMVISNPAAT (SEQ ID NO:165). As another non-limiting example, a T1D peptide epitope is the following islet antigen 2 (IA2) peptide: SFYLKNVQTQETRTLTQFHF (SEQ ID NO:166). As another non-limiting example, a T1D peptide epitope is the following proinsulin peptide: SLQPLALEGSLQSRG (SEQ ID NO:167). As another non-limiting example, a T1D peptide epitope is the following proinsulin peptide GSLQPLALEGSLQSRGIV (SEQ ID NO:168; proIns 75-92(K88S)).

A suitable T1D-epitope may comprise from about 4 to about 25 (about 5 to about 25 contiguous aas) of an aa sequence having at least about 75%, at least about 80%, at least about 90%, at least about 90%, at least about 95%, or 100%, aa sequence identity to aas 25-110 of the following human preproinsulin aa sequence (wherein italicized aas 1-24 form the signal peptide): MALWMRLLPL LALLALWGPD PAAA FVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN (SEQ ID NO:169); where the T1D peptide epitope has a length of 4 aas, 5 aas, 6 aas, 7, aas, 8 aas, 9 aas, 10 aas, 11 aas, 12 aas, 13 aas, 14 aas, 15 aas, 16 aas, 17 aas, 18 aas, 19 aas, 20 aas, 21 aas, 22 aas, 23 aas, 24 aas, or 25 aas. A suitable T1D-epitope may comprise from about 8 to about 25 (about 10 to about 25 contiguous aas) of an aa sequence having at least about 85%, or at least about 90%, to aas 25-110 of SEQ ID NO:169. In some cases, the T1D peptide epitope has the aa sequence: GAGSLQPLALEGS LQKRG (SEQ ID NO:170) (proIns 73-90). In some cases, the T1D peptide epitope has the aa sequence: SLQPLALEGSLQKRG (SEQ ID NO:171) (proIns 76-90). In some cases, the T1D peptide epitope has the aa sequence: SLQPLALEGSLQSRG (SEQ ID NO:172) (proIns 76-90; K88S). In some cases, the T1D peptide epitope has the aa sequence: QPLALEGSLQKRG (SEQ ID NO:173). In some cases, the T1D peptide epitope has the aa sequence: QPLALEGSLQSRG (SEQ ID NO:174).

8. Additional Polypeptides

A polypeptide chain of a MAPP (e.g., a dimerization or framework polypeptide) may include one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides can be included at the N-terminus of a polypeptide chain of a MAPP of the present disclosure, at the C-terminus of a polypeptide chain of a MAPP of the present disclosure, or within (internal to) a polypeptide chain of a MAPP.

a. Affinity Tags, Epitope Tags and Affinity Domains

Suitable affinity/epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:175); FLAG (e.g., DYKDDDDK (SEQ ID NO:176); c-myc (e.g., EQKLISEEDL; SEQ ID NO:177), and the like.

Affinity domains include peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification. DNA sequences encoding multiple consecutive single aas, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose. Exemplary affinity domains include HisX5 (HHHHH) (SEQ ID NO:178), HisX6 (HHHHHH) (SEQ ID NO:179), C-myc (EQKLISEEDL) (SEQ ID NO:177), Flag (DYKDDDDK) (SEQ ID NO:176), StrepTag (WSHPQFEK) (SEQ ID NO:180, hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:175), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:181), Phe-His-His-Thr (SEQ ID NO:182), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:183), metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

D. Nucleic Acids

The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding one or more polypeptides of a MAPP bearing a T1D associated epitope. In some cases, the nucleic acid is a recombinant expression vector; thus, the present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a MAPP.

1. Nucleic Acids Encoding a MAPP or MAPP Forming a Higher Order Complex, Such as a Duplex MAPP, that Comprises at Least One Dimerization Sequence and a Multimerization Sequence

The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a MAPP having a framework polypeptide that comprises at least one dimerization sequence and at least one multimerization sequence that permits two molecules of the framework polypeptide to form dimers or higher order complexes. The nucleic acids may additionally comprise a nucleotide sequence encoding a dimerization peptide. Where the MAPP comprises a presenting sequence, the nucleic acids encoding either or both of the framework polypeptide and/or dimerization peptide may include a sequence encoding a presenting sequence. Where the MAPP comprises a presenting complex, the nucleic acids encoding either or both of the framework polypeptide and/or dimerization peptide may further comprise sequences encoding a presenting complex 1^st sequence and/or a presenting complex 2^nd sequence. The nucleotide sequence(s) comprising any of the MAPP polypeptides can be operably linked to a transcription control element(s), e.g., a promoter. It will be apparent that individual polypeptides of a MAPP (e.g., a framework polypeptide and dimerization polypeptide) may be encoded on a single nucleic acid (e.g., under the control of separate promoters), or alternatively, may be located on two or more separate nucleic acids (e.g., plasmids).

2. Recombinant Expression Vectors

The present disclosure provides recombinant expression vectors comprising nucleic acids encoding one or more polypeptides of a MAPP or its higher order complexes. In some cases, the recombinant expression vector is a non-viral vector. In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, etc.

Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther, 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol., 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available.

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

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

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

E. Genetically Modified Host Cells

The present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid(s) that encode, or encode and express, MAPP proteins or higher order complexes of MAPPs (e.g., duplex MAPPs).

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

Genetically modified host cells can be used to produce a MAPP and higher order complexes of MAPPs. For example, a genetically modified host cell can be used to produce a duplex MAPP. For example, an expression vector(s) comprising nucleotide sequences encoding the MAPP polypeptide(s) is/are introduced into a host cell, generating a genetically modified host cell, which genetically modified host cell produces the polypeptide(s) (e.g., as an excreted soluble protein).

F. Methods of Producing MAPPs

The present disclosure provides methods of producing the MAPP presenting a T1D peptide epitope described herein. The methods generally involve culturing, in a culture medium, a host cell that is genetically modified with a recombinant expression vector(s) comprising a nucleotide sequence(s) encoding the MAPP (e.g., a genetically modified host cell of the present disclosure); and isolating the MAPP from the genetically modified host cell and/or the culture medium. As noted above, in some cases, the individual polypeptide chains of a MAPP are encoded in separate nucleic acids (e.g., recombinant expression vectors). In some cases, all polypeptide chains of a MAPP are encoded in a single recombinant expression vector.

Isolation of the MAPP from the host cell employed for expression (e.g., from a lysate of the expression host cell) and/or the culture medium in which the host cell is cultured, can be carried out using standard methods of protein purification. For example, a lysate of the host cell may be prepared, and the MAPP purified from the lysate using high performance liquid chromatography (HPLC), exclusion chromatography (e.g., size exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification technique. Alternatively, where the MAPP is secreted from the expression host cell into the culture medium, the MAPP can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the MAPP is purified, e.g., a composition is generated that comprises at least 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, of the MAPP in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.

In some cases, e.g., where the expressed MAPP comprises an affinity tag or affinity domain, the MAPP can be purified using an immobilized binding partner of the affinity tag. For example, where a MAPP comprises an Ig Fc polypeptide, the MAPP can be isolated from genetically modified mammalian host cell and/or from culture medium comprising the MAPP by affinity chromatography, e.g., on a Protein A column, a Protein G column, or the like. An example of a suitable mammalian cell is a CHO cell; e.g., an ExpiCHO-S™ cell (e.g., ThermoFisher Scientific, Catalog #A29127).

The polypeptides of the MAPP will self-assemble into heterodimers, and where applicable, spontaneously form disulfide bonds between, for example, framework polypeptides, or framework and dimerization polypeptides. As also noted above, when both framework polypeptides include Ig Fc polypeptides, disulfide bonds will spontaneously form between the respective Ig Fc polypeptides to covalently link the two heterodimers of framework and dimerization polypeptides to one another to form a covalently linked duplex MAPP.

G. Compositions

1. Compositions Comprising a MAPP

The present disclosure provides compositions, including pharmaceutical compositions, comprising a MAPP and/or higher order complexes of MAPPs (e.g., duplex MAPPs). Pharmaceutical composition can comprise, in addition to a MAPP, one or more known carriers, excipients, diluents, buffers, salts, surfactants (e.g., non-ionic surfactants), amino acids (e.g., arginine), etc., a variety of which are known in the art and need not be discussed in detail herein. For example, see “Remington: The Science and Practice of Pharmacy”, 19^th Ed. (1995), or latest edition, Mack Publishing Co.

In some cases, a subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile and/or substantially free of pyrogens. For example, in some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is substantially free of detectable pyrogens and/or other toxins, or such detectable pyrogens and/or other toxins are below a permissible limit.

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

Where a MAPP or higher order MAPP complex (e.g., duplex MAPP) is administered as an injectable (e.g. subcutaneously, intraperitoneally, intramuscularly, intralymphatically, and/or intravenously) directly into a tissue, a formulation can be provided as a ready-to-use dosage form, or as non-aqueous form (e.g. a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients. MAPPs may also be provided so as to enhance serum half-life of the subject protein following administration. For example, the protein may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may also be provided in controlled release or slow-release forms.

In some cases, a MAPP composition comprises: a) a MAPP higher order MAPP complex (e.g., a duplex MAPP) of the present disclosure; and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile and/or substantially pyrogen free. In some cases, the composition is suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins. Thus, the present disclosure provides a composition comprising: a) a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure; and b) saline (e.g., 0.9% NaCl), where the composition is sterile and is substantially free of detectable pyrogens and/or other toxins, or such detectable pyrogens and/or other toxins are below a permissible limit.

Other examples of components suitable for inclusion in formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. A pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

The concentration of a MAPP in a formulation can vary widely. For example, a MAPP or higher order MAPP complex (e.g., duplex MAPP) may be present from less than about 0.1% (usually at least about 2%) to as much as 20% to 50% or more by weight (e.g., from 1% to 10%, 5% to 15%, 10% to 20% by weight, or 20-50% by weight) by weight. The concentration will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.

The present disclosure provides a container comprising a composition of the present disclosure, e.g., a liquid composition. The container can be, e.g., a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

H. Methods

MAPPs and higher order MAPP complexes (e.g., duplex MAPP) are useful for modulating an activity of a T cell. Thus, the present disclosure provides methods of modulating an activity of a T cell, the methods generally involving contacting a target T cell with a MAPP or a higher order MAPP complex (e.g., duplex MAPP).

1. Methods of Modulating T Cell Activity

The present disclosure provides a method of selectively modulating the activity of a T cell that is specific or selective for an epitope presented by a T1D peptide, the method comprising contacting, in vitro or in vivo the T cell with a MAPP or higher order MAPP, such as a duplex MAPP, presenting a T1D peptide epitope, where contacting the T cell with the MAPP selectively modulates the activity of the epitope-specific T cell. The contacting may occur in vivo, typically in a human, but potentially also in another animal such as a rat, mouse, dog, cat, pig, horse, or primate. In some cases, the contacting occurs in vitro.

Where a MAPP comprises a T1D associated peptide epitope and a MOD such as a wt. or variant PD-L1 or FasL polypeptide that modulates target T cells to which the MAPP binds, such as by suppressing/inhibiting the target T cells, binding of the MAPP may reduce, directly or indirectly, the number and/or activity of autoreactive T cells (e.g., effector cells) and/or autoreactive B cells directed against the T1D-associated epitope. Suppression of the T cell activity may be assessed, for example, by the suppression of cytokine release (e.g.: IFN-γ from Th1 cells; IL-17 and/or IL-22 from Th17 cells; or IL-21 from Tfh cells).

In some cases, e.g., where a MAPP comprises a MOD such as a wt. or variant IL-2, and/or a TGF-β MOD polypeptide, the MAPP may increase the number and/or activity of target CD4⁺, FOXP3⁺ Treg cells (e.g., nTregs) and/or CD4⁺, FOXP3⁺, CD25⁺ Treg cells (e.g., iTreg cells). Such MAPPs may reduce, directly or indirectly (e.g., through bystander suppression), the number and/or activity of autoreactive T cells (e.g., T effector cells such as Th1, Th2, and/or Th17 cells) and/or autoreactive B cells directed against cells expressing T1D-associated epitopes. The activity of Treg cells may be assess by known means including assessment of the levels of IL-10 and/or TGF-β produced by those cells. MAPPs of the present disclosure, including those comprising a PD-L1 or PD-L2 MOD, need not contain an IL-2 MOD, and/or a TGF-β MOD provided there is sufficient levels of those molecules in the in vitro or in vivo environment in which the MAPPs are contacted with the target T cells. Contacting FoxP3⁺ Tregs (e.g., Tbet+FoxP3+ iTreg cells), with MAPPs in the presence of IL-2 (or an IL-2 MOD) and/or TGF-β (or a TGF-β MOD) may result in activation and/or proliferation of those Tregs.

In view of the foregoing, the present disclosure provides a method of decreasing the number and/or activity of autoreactive T cells (e.g., auto reactive Th1 and/or Th17 cells) and/or autoreactive B cells directed against cells expressing T1D-associated epitopes in an individual, the method comprising administering to the individual a pharmaceutical composition comprising a MAPP or higher order MAPP, such as a duplex MAPP, (e.g., one that comprises a MOD such as a wt. or variant of PD-L1 or FasL), where the administering results in a decrease in the number and/or activity of autoreactive T cells and/or B cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold.

The present disclosure also provides a method of increasing proliferation of Tregs in vitro or in vivo, the method comprising contacting Tregs with a pharmaceutical composition comprising a MAPP or higher order MAPP, such as a duplex MAPP, in the presence of TGF-β and/or IL-2 (e.g., a MAPP comprising a TGF-β MOD and/or IL-2 MOD), where the contacting increases proliferation of Tregs. TGF-β and/or IL-2 may be supplied by the environment or supplemented by addition or administration to the in vitro or in vivo environment where contacting occurs. The present disclosure provides a method of increasing the number and/or activity of Tregs in an individual, the method comprising administering to the individual a pharmaceutical composition comprising a MAPP or higher order MAPP, such as a duplex MAPP, where the administering results in an increase in the number or activity of Tregs specific for the epitope presented by the MAPP in the individual or in a tissue (e.g., blood) of an individual. For example, the number of Tregs can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold. The activity of Tregs specific for the epitope presented by the MAPP may be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold based on the amount of TGF-β and/or IL-10 produced by Tregs specific for the epitope presented by the MAPP in an individual or in a tissue (e.g., blood) of an individual.

In some cases, the cell being contacted is a helper T cell, where contacting the helper T cell with a MAPP results in activation or suppression of the helper T cell (e.g., Th2 cells). In some cases, activation of the helper T cell results in an increase in the activity and/or number of CD8⁺ cytotoxic T cells, e.g., CD8⁺ cytotoxic T cells that target and kill an autoreactive cell.

2. Treatment Methods

The present disclosure provides treatment methods, the methods comprising administering to the individual a composition comprising an amount of a MAPP or higher order MAPP such as duplex MAPP, effective to selectively modulate the activity of a T1D epitope-specific T cell in an individual and to treat the individual. In some cases, a treatment method comprises administering to an individual in need thereof a pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP such as duplex MAPP useful for treating type 1 diabetes (T1D) occurring in human patients and in experimental animal models (e.g., non-obese diabetic (NOD) mouse and the Biobreeding (BB) rat).

The present disclosure provides a method of selectively modulating the activity of a T1D epitope-specific T cell in an individual, the method comprising administering to the individual a pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP such as duplex MAPP, where the MAPP selectively modulates the activity of the epitope-specific T cell in the individual. Selectively modulating the activity of an epitope-specific T cell can treat T1D in the individual. Thus, the present disclosure provides a treatment method comprising administering to an individual in need thereof a pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP such as duplex MAPP for the purpose of treating T1D.

The present disclosure provides a method of treating T1D in an individual, the method comprising administering to the individual a pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP such as a duplex MAPP, where the MAPP comprises a T1D peptide epitope (as described above), and where the MAPP comprises an inhibitory MOD (e.g., FasL and/or PD-L1). In some cases, an “effective amount” of a MAPP or higher order MAPP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of self-reactive (i.e., reactive with a T1D-associated antigen) CD4+ and/or CD8+ T cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to number of self-reactive T cells in the individual before administration of the MAPP, or in the absence of administration with the MAPP. In some cases, an “effective amount” of a MAPP is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th1 cytokines (e.g., IL-2, IL-10, and TNF-alpha/beta) in the individual. In some cases, an “effective amount” of a MAPP is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th2 cytokines (e.g., IL-4, IL-5, and IL-13) in the individual. In some cases, an “effective amount” of a MAPP is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th17 cytokines (e.g., IL-17A, IL-17F, and IL-22) in the individual. In some cases, an “effective amount” of a MAPP is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with T1D in the individual. In some instances, the MAPP reduces the number of CD4+ self-reactive T cells (i.e., the number of CD4+ T cells reactive with a T1D-associated antigen), which in turn leads to a reduction in CD8+ self-reactive T cells. In some instances, the MAPP increases the number or activity (e.g., IL-10 and/or TGF-β production) of CD4+ Tregs specific for a T1D peptide epitope presented by the MAPP, which in turn may reduce the number or activity of CD4+ self-reactive T cells, B cells, and/or CD8+T self-reactive T cells specific for that epitope.

The present disclosure provides a method of reducing elevated blood sugar (e.g., glucose) in an individual (e.g., a mammal such as a human) having or suspected of having T1D, the method comprising administering to the individual an effective amount of a MAPP, or one or more nucleic acids comprising nucleotide sequences encoding the MAPP, where the MAPP comprises a T1D peptide epitope (as described above). The individual may be a human having a fasting blood sugar in excess of 130 or about 140 mg/dL, or postprandial blood sugar in excess of 180 or about 200 mg/dL. Wherein the treatment reduces fasting blood sugar (e.g., such as to a level below 130 mg/dL) or post prandial blood sugar (e.g., to less than 180 mg/dL) in the individual relative to the level prior to receiving the MAPP. The reduction in blood sugar may be maintained for a period of at least about a week, at least about two weeks, at least about a month (30 days), or more than one month.

The present disclosure provides a method of reducing prediabetic glycosylated hemoglobin referred to as hemoglobin A1C (also referred to as hemoglobin A1c or HbA1c) levels (in the range of 5.7% to 6.4%) or diabetic hemoglobin A1C levels (above 6.4%) in an individual (e.g., a mammal such as a human) having or suspected of having T1D, the method comprising administering to the individual an effective amount of a MAPP, or one or more nucleic acids comprising nucleotide sequences encoding the MAPP, where the MAPP comprises a T1D peptide epitope (as described above). The treatment reduces diabetic hemoglobin A1C (e.g., to less than 6.4%, and preferably to less than 5.7%), or prediabetic A1C (e.g., such as to less than 5.7% and into the normal range) in the individual relative to the level prior to receiving the MAPP. The reduction in hemoglobin A1C may be maintained for a period of at least about a week, at least about two weeks, at least about a month (30 days), or more than one month.

The present disclosure also provides treatment methods, the methods comprising administering to the individual a pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP such as a duplex MAPP bearing an immunoglobulin sequence that can support complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC) to selectively engage with an epitope-specific T cell in an individual in order to treat the individual, (e.g., by depleting epitope-specific T cells by inducing cell lysis through activation of CDC, and/or ADCC). MAPPs used to promote CDC and/or ADCC may have no MOD sequence or a MOD sequence that has limited affinity for its co-MOD, provided the MAPP is specific for the target T cell. Accordingly, a MAPP is useful for treating T1D by selectively engaging with an epitope-specific T cell in an individual and depleting epitope-specific T cells when the MAPP bears an Ig Fc polypeptide that induces cell lysis through activation of CDC, and/or elicits ADCC.

3. Methods of Selectively Delivering a MOD

The present disclosure provides a method of delivering a MOD polypeptide such as IL-2, 4-1BBL, Fas-L, PD-L1, TGF-β, or a reduced-affinity variant of any thereof (e.g., PD-L1 and/or an IL-2 variant disclosed herein) to a selected T cell or a selected T cell population, e.g., in a manner such that a TCR specific for a given epitope is targeted. The present disclosure thus provides a method of delivering a MOD polypeptide such as a PD-L1 polypeptide, or a reduced-affinity variant of a naturally occurring MOD polypeptide such as a PD-L1 variant, selectively to a target T cell bearing a TCR specific for the T1D peptide epitope sequence present in a MAPP or higher order MAPP complex (e.g., duplex MAPP). The method comprises contacting a population of T cells with a MAPP or higher order MAPP complex (e.g., duplex MAPP). The population of T cells can be a mixed population that comprises: i) the target T cell with a TCR specific to a target epitope; and ii) non-target T cells that are not specific for the target epitope presented by the T1D peptide epitope (e.g., T cells that are specific for an epitope(s) other than the epitope to which the epitope-specific T cell binds). The epitope-specific T cell is specific for the peptide epitope present in the MAPP or higher order MAPP complex, and binds to the peptide MHC complex provided by the MAPP or higher order MAPP complex. Contacting the population of T cells with the MAPP or higher order MAPP complex delivers the MOD polypeptide (e.g., PD-L1 or a reduced-affinity variant of PD-L1) selectively to the T cell(s) that are specific for the epitope present in the MAPP or higher order complex.

Thus, the present disclosure provides a method of delivering a MOD polypeptide such as PD-L1, or a reduced-affinity variant of a naturally occurring MOD polypeptide such as a PD-L1 variant disclosed herein, or a combination of both, selectively to a target T cell selective for the epitope presented by the T1D peptide epitope as presented by the MAPP. Similarly, the disclosure provides a method of delivering an IL-2, MOD polypeptide or a reduced-affinity variant of a naturally occurring IL-2 MOD polypeptide such as disclosed herein, or a combination of both, to a target T cell that is selective for the epitope presented by the T1D peptide epitope as presented by the MAPP. In some cases, the IL-2 MOD bears a substitution at position H16 and/or F42 (e.g., H16 and F42 such as H16A and F42A) (see supra SEQ ID NO:108).

For example, a MAPP or higher order MAPP complex (e.g., duplex MAPP) is contacted with a population of T cells comprising: i) target T cells that are specific for the epitope present in the MAPP or a higher order MAPP complex; and ii) non-target T cells, e.g., a T cells that are specific for a second epitope(s) that is not the epitope present in the MAPP or a higher order MAPP complex. Contacting the population results in substantially selective delivery of the MOD polypeptide(s) (e.g., naturally-occurring or variant MOD polypeptide) to the target T cell. Less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 4%, 3%, 2% or 1%, of the MAPP or higher order MAPP complex (e.g., duplex MAPP) may bind to non-target T cells and, as a result, the MOD polypeptide (e.g., PD-L1 or PD-L1 variant) is selectively delivered to target T cell (and accordingly, not effectively delivered to the non-target T cells).

The population of T cells to which a MOD and/or variant MOD is selectively delivered may be in vivo. In some cases, the population of T cells to which a MOD and/or variant MOD is selectively delivered is in vitro.

In some cases, the population of T cells to which a MOD and/or variant MOD is selectively delivered is in vitro. For example, a mixed population of T cells is obtained from an individual, and is contacted with a MAPP or higher order MAPP complex (e.g., duplex MAPP) in vitro. Such contacting, which can comprise single or multiple exposures of the T cells to a defined dose(s) and/or exposure schedule(s) in the context of in vitro cell culture, can be used to determine whether the mixed population of T cells includes T cells that are specific for the epitope presented by the MAPP or higher order MAPP complex. The presence of T cells that are specific for the epitope of the MAPP or higher order MAPP complex can be determined by assaying a sample comprising a mixed population of T cells, which population of T cells comprises T cells that are not specific for the epitope (non-target T cells) and may comprise T cells that are specific for the epitope (target T cells). Known assays can be used to detect the desired modulation of the target T cells, thereby providing an in vitro assay that can determine whether a particular MAPP or higher order MAPP complex possesses an epitope that binds to T cells present in the individual, and thus whether the MAPP or higher order complex has potential use as a therapeutic composition for that individual. Suitable known assays for detection of the desired modulation (e.g., activation/proliferation or inhibition/suppression) of target T cells include, e.g., flow cytometric characterization of T cell phenotype, numbers, and/or antigen specificity. Such an assay to detect the presence of epitope-specific T cells, e.g., a companion diagnostic, can further include additional assays (e.g. effector cytokine ELISpot assays) and/or appropriate controls (e.g. antigen-specific and antigen-nonspecific multimeric peptide-HLA staining reagents) to determine whether the MAPP or higher order MAPP complex is selectively binding, modulating (activating or inhibiting), and/or expanding the target T cells. Thus, for example, the present disclosure provides a method of detecting, in a mixed population of T cells obtained from an individual, the presence of a target T cell that binds an epitope of interest, the method comprising: a) contacting in vitro the mixed population of T cells with a MAPP or higher order MAPP complex comprising an epitope of the present disclosure; and b) detecting modulation (activation or inhibition) and/or proliferation of T cells in response to said contacting, wherein modulation of and/or proliferation of T cells indicates the presence of the target T cell. Alternatively, and/or in addition, if activation and/or expansion (proliferation) of the desired T cell population is obtained using a MAPP or higher order MAPP complex (e.g., a duplex MAPP), then all or a portion of the population of T cells comprising the activated/expanded T cells can be administered back to the individual as a therapy.

In some instances, the population of T cells is in vivo in an individual. In such instances, a method of the present disclosure for selectively delivering a MOD polypeptide (e.g., PD-L1 or a reduced-affinity PD-L1) to an epitope-specific T cell comprises administering the MAPP or higher order MAPP complex (e.g., duplex MAPP) to the individual.

In some instances, the epitope-specific T cell to which a MOD polypeptide sequence (e.g., wt. or reduced affinity IL-2 and/or PD-L1 MOD) is being selectively delivered is referred to herein is a target regulatory T cell (Treg) that may inhibit or suppresses activity of an autoreactive T cell.

I. Dosages

A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. A MAPP (whether as a single heterodimer or, as described above, as a higher order complex such as a duplex MAPP) may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose; for example from 0.1 μg/kg body weight to 1.0 mg/kg body weight, from 0.1 mg/kg body weight to 0.5 mg/kg body weight, from 0.5 mg/kg body weight to 1 mg/kg body weight, from 1.0 mg/kg body weight to 5 mg/kg body weight, from 5 mg/kg body weight to 10 mg/kg body weight, from 10 mg/kg body weight to 15 mg/kg body weight, and from 15 mg/kg body weight to 20 mg/kg body weight. Doses below 0.1 mg/kg body weight or above 20 mg/kg are envisioned, especially considering the aforementioned factors. Amounts thus include from about 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, and above about 20 mg/kg body weight.

Those of skill will readily appreciate that dose levels can vary as a function of the MAPP or higher order MAPP complex (e.g., duplex MAPP), the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

In some cases, multiple doses of a MAPP or higher order MAPP complex (e.g., duplex MAPP) are administered. The frequency of administration of a MAPP or higher order MAPP complex (e.g., duplex MAPP) can vary depending on any of a variety of factors, e.g., severity of the symptoms, patient response, etc. For example, in some cases, a MAPP or higher order MAPP complex (e.g., duplex MAPP) is administered once per month, less frequently than once per month, e.g., once every 6 weeks, once every two months, once every three months, or more frequently than once per month, e.g., twice per month, three times per month, every other week (qow), one every three weeks, once every four weeks, once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The duration of administration of a MAPP, e.g., the period of time over which a MAPP is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a MAPP can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more, including continued administration for the patient's life.

Where treatment is of a finite duration, following successful treatment, it may be desirable to have the patient undergo periodic maintenance therapy to prevent the recurrence of the T1D disease state, wherein a MAPP is administered in maintenance doses, ranging from those recited above, i.e., 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, and above about 20 mg/kg body weight. The periodic maintenance therapy can be once per month, once every two months, once every three months, once every four months, once every five months, once every six months, or less frequently than once every six months.

J. Routes of Administration

A MAPP or higher order MAPP complex is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and localized routes of administration. A MAPP or higher order MAPP complex can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated for use in a method include, but are not necessarily limited to, enteral, parenteral, and inhalational routes.

Conventional and pharmaceutically acceptable routes of administration include intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Of these, intravenous, intramuscular and subcutaneous may be more commonly employed. Routes of administration may be combined, if desired, or adjusted depending upon, for example, the MAPP or higher order MAPP complex (e.g., duplex MAPP) and/or the desired effect. A MAPP or higher order MAPP complex can be administered in a single dose or in multiple doses.

K. Subjects Suitable for Treatment

Subjects suitable for treatment with a method include individuals who have T1D, including individuals who have been diagnosed as having T1D, and individuals who have been treated for T1D but who failed to respond to the treatment. Suitable subjects also may include individuals who have been diagnosed as being likely to develop T1D or who have symptoms indicating the imminent onset of T1D.

Subjects suitable for treatment include those with T1D or a genetic disposition to develop T1D including a family history of T1D (e.g., a grandparent, parent, or sibling with T1D). As discussed above, a number of serotypes have been associated with a substantial risk of developing T1D (e.g., DR3, DR4, DQ2.5 and DQ 8.1), and others with a moderate risk of developing T1D (e.g., DR1, DR8, DR9 and DQ5.

Subjects suitable for treatment include those who have T1D, including individuals who have been diagnosed as having T1D, and individuals who have been treated for T1D but who failed to respond to the treatment who have fasting blood sugars (blood sugar after not eating or drinking for 8 hours) in excess of about 130 mg/dL (or about 140 mg dL), and/or a blood sugar higher than about 180 mg/dL (or about 200 m/dL) 2 hours after a meal (postprandial hyperglycemia). This includes individuals with (i) a genetic disposition to T1D such as a family history of T1D and/or a serotype associated with T1D (e.g., DR4), and (ii) either an elevated fasting blood sugar in excess of 130 or about 140 mg/dL, or postprandial blood sugar in excess of 180 or about 200 mg/dL. See, e.g., www.cdc.gov/diabetes/managing/managing-blood-sugar/bloodglucosemonitoring.html.

Subjects suitable for treatment include those who have T1D, including individuals who have been diagnosed as having T1D, and individuals who have been treated for T1D but who failed to respond to the treatment who have hemoglobin A1C levels from 5.7 to 6.4% (prediabetic levels) or hemoglobin A1C levels above 6.4% (diabetic levels). See, e.g., www.cdc.gov/diabetes/managing/managing-blood-sugar/a1c.html. This includes individuals with both prediabetic or diabetic A1C levels and a genetic disposition to T1D such as a family history of T1D and/or a serotype associated with T1D (e.g., DR4).

V. CERTAIN ASPECTS

Certain aspects, including embodiments/aspects of the present subject matter described above, may be beneficial alone or in combination, with one or more other aspects recited hereinbelow. In addition, while the present subject matter has been disclosed with reference to certain aspects recited below and, in the claims, numerous modifications, alterations, and changes to the described aspects/embodiments are possible without departing from the sphere and scope of the present disclosure. Accordingly, it is intended that the present disclosure not be limited to the described embodiments, aspects, and claims, but that it has the full scope defined by the language of this disclosure and equivalents thereof.

• • 1. A multimeric antigen-presenting polypeptide complex (MAPP) comprising:
    • a framework polypeptide comprising (e.g., from N-terminus to C-terminus) a dimerization sequence and a multimerization sequence;
    • a dimerization polypeptide comprising a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide, and dimerizing therewith through covalent (e.g., disulfide bonds) and/or non-covalent interactions to form a MAPP heterodimer; and
    • at least one (e.g., at least two) presenting sequence and/or presenting complex,
    • wherein each presenting sequence comprises a T1D peptide epitope, and MHC Class II α1, α2, β1, and β2 domain polypeptide sequences;
    • wherein each presenting complex comprises a presenting complex 1^st sequence and a presenting complex 2^nd sequence that together comprise a T1D peptide epitope, and MHC Class II α1, α2, β1, and β2 domain polypeptide sequences, where the epitope is part of the presenting complex 1^st sequence or presenting complex 2^nd sequence along with at least one of the α1, α2, β1, and β2 polypeptide sequences;
    • wherein at least one or both of the dimerization polypeptide and/or the framework polypeptide (e.g., either the framework polypeptide, the dimerization polypeptide, or both polypeptides) comprises a presenting sequence or a presenting complex 1st sequence (e.g., located on the N-terminal side of the framework polypeptide's dimerization sequence, or the N-terminal side of the dimerization polypeptide' counterpart dimerization sequence);
    • wherein optionally at least one (e.g., one, two, or more) of the framework polypeptide or the dimerization peptide (including the presenting sequence(s) or presenting complex(es)) comprises one, two, three or more independently selected MOD and/or variant MOD polypeptide sequences (e.g., located at the N-terminus or C-terminus of the dimerization polypeptide or the framework polypeptide, and/or on the C-terminal side of the dimerization sequence(s)); and
    • wherein the framework polypeptide, dimerization polypeptide, presenting sequence, presenting complex 1^st sequence and/or presenting complex 2^nd sequence optionally comprise one or more independently selected linker sequences. (See, e.g., FIGS. 1A and 1B).It is understood that the dimerization sequence and multimerization sequence are different polypeptide sequences and do not bind in any substantial manner to each other, e.g., the framework polypeptides do not, to any substantial extent, form hair pin structures, self-polymerize, or self-aggregate. Similarly, this aspect may be subject to the proviso that neither the dimerization sequence nor the multimerization sequence of the framework polypeptide comprises an MHC-Class II polypeptide sequence having at least 85% (e.g. 90%, 95% or 98%) sequence identity to at least 20 (e.g., at least 30, 40, 50, 60 or 70) contiguous aas of an MHC-Class II polypeptide in any of FIGS. 4 through 8 (SEQ ID NOs:17-61). It is also understood that none of the α1, α2, β1 and β2 domain polypeptide sequences include a transmembrane domain, or a portion thereof, that will anchor the MAPP in a cell membrane.
  • 2. The MAPP of aspect 1, wherein the MHC Class II α1 and α2 domain polypeptide sequences comprise human Class II α1 and α2 domain polypeptide sequences selected from HLA DR alpha (DRA), DQ alpha 1 (DQA1), and DQ alpha 2 (DQA2) α1 and α2 domain polypeptide sequences.
  • 3. The MAPP of aspect 1 or 2, wherein the MHC Class II β1 and β2 domain polypeptide sequences comprise human class β1 and β2 domain polypeptide sequences selected from HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), DR beta 5 (DRB5), DQ beta 1 (DQB1), and DQ beta 2 (DQB2) β1 and β2 domain polypeptide sequences.
  • 4. The MAPP of any preceding aspect, wherein at least one presenting sequence or presenting complex (e.g., at least two, three, or all presenting sequences and/or complexes) comprises:
    • a. α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 50 (e.g., at least about 60, 70, 80, 85, or 90) contiguous aas of the HLA DR alpha (DRA) polypeptide sequence SEQ ID NO:17 (provided in FIG. 4); and
    • b. β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 50 (e.g., at least about 60, 70, 80, 85, or 90) contiguous aas of an HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), or DR beta 5 (DRB5) polypeptide sequence provided in any one of FIG. 5, 6, 7, or 8 (SEQ ID NOs:18-61).
  •  For example, a MAPP of aspect 4 may be a MAPP wherein at least one presenting sequence or presenting complex (e.g., at least two or all presenting sequences and/or complexes) comprises:
    • α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of an HLA DR alpha (DRA) polypeptide sequence provided in FIG. 4; and
    • β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of an HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), or DR beta 5 (DRB5) β1 polypeptide sequences provided in any one of FIG. 5, 6, 7, or 8.
  • 5. The MAPP of aspect 4, wherein:
    • the β1 and β2 domain polypeptide sequences each have at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 50 (e.g., at least about 60, 70, 80, 85, or 90) contiguous aas of an HLA DR beta 1 (DRB1) polypeptide sequence provided in FIG. 5 (e.g., DRB1*0301, DRB1*0401, DRB1*0402, DRB1*0405, DRB1*0801, or DRB1*0901).
  •  For example, a MAPP of aspect 5 may be a MAPP wherein the β1 and β2 domain polypeptide sequences each have at least 90% or at least 95% (e.g., 98% or 100%) sequence identity to a β1 or β2 domain of an HLA DR beta 1 (DRB1) polypeptide sequence provided in FIG. 5 (e.g., DRB1*0301, DRB1*0401, DRB1*0402, DRB1*0405, DRB1*0801, or DRB1*0901).
  • 6. The MAPP of aspect 5, wherein:
    • the β1 and β2 domain polypeptide sequences each have at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 50 (e.g., at least about 60, 70, 80, 85, or 90) contiguous aas of an HLA DR beta 1 (DRB1), DRB1*0301, DRB1*0401, DRB1*0402, or DRB1*0405 polypeptide sequence provided in FIG. 5.For example, a MAPP of aspect 6 may be a MAPP wherein the β1 and β2 domain polypeptide sequences each have at least 90% or at least 95% (e.g., 98% or 100%) sequence identity to a β1 or β2 domain of an HLA DR beta 1 (DRB1), DRB1*0301, DRB1*0401, DRB1*0402, or DRB1*0405 polypeptide sequence provided in FIG. 5 (e.g., DRB1*0301, DRB1*0401, DRB1*0402, DRB1*0405, DRB1*0801, or DRB1*0901).
  • 7. The MAPP of any of aspects 1-3, wherein at least one presenting sequence or presenting complex comprises:
    • a. α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of an HLA DQ alpha 1 or DQ alpha 2 (DQA1 or DQA2) polypeptide sequence; and
    • b. β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of an HLA DQ beta 1 or DQ beta 2 (DQB1 or DQB2) polypeptide sequence.
  •  For example, a MAPP of aspect 7 may be a MAPP wherein at least one presenting sequence or presenting complex (e.g., at least two or all presenting sequences and/or complexes) comprises:
    • α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of an HLA DQ alpha 1 or DQ alpha 2 (DQA1 or DQA2) polypeptide sequence; and
    • β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of an HLA DQ beta 1 (DQB1) polypeptide sequence.
  • 8. The MAPP of any of aspects 1-3, wherein at least one presenting sequence or presenting complex comprises either:
    • (i) α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of an HLA DQ alpha 1 (DQA1) sequence, and β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of HLA DQ beta 1 (DQB1) β1 or β2 domain polypeptide sequences; or
    • (ii) α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of an HLA DQ alpha 2 (DQA2) polypeptide sequence, and β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of an HLA DQ beta 2 (DQB2) polypeptide sequences.
  •  For example, a MAPP of aspect 8 may be a MAPP wherein at least one presenting sequence or presenting complex comprises either:
    • (i) α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of a DQA1 polypeptide sequence, and
      • β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of a DQB1 polypeptide sequence; or
    • (ii) α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of a DQA2 polypeptide sequence, and
      • β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of a DQB2 polypeptide sequence.
  • 9. The MAPP of any of aspects 1-3, wherein at least one presenting sequence or presenting complex comprises either:
    • (i) α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of a DQA1*0501 polypeptide sequence, and β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of a DQB1*0201 polypeptide sequence; or
    • (ii) α1 and α2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of a DQA1*0301 sequence, and β1 and β2 domain polypeptide sequences each having at least 85% (e.g., at least 90%, at least 95% or 98%) or 100% sequence identity to all or at least about 60 (e.g., at least about 70, 80, 85, or 90) contiguous aas of a DQB1*0302 polypeptide sequence.
  •  For example, a MAPP of aspect 9 may be a MAPP wherein at least one presenting sequence or presenting complex comprises either:
    • (i) α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of a DQA1*0501 polypeptide sequence, and 3β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of a DQB1*0201 polypeptide sequence; or
    • (ii) α1 and α2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to an al or α2 domain of a DQA1*0301 sequence, and
      • β1 and β2 domain polypeptide sequences each having at least 90% or at least 95% (e.g., at least 98% or 100%) sequence identity to a β1 or β2 domain of a DQB1*0302 polypeptide sequence.
  • 10. The MAPP of any preceding aspect, wherein the MAPP comprises at least one linker comprising:
    • (i) Gly (polyG or polyglycine), Gly and Ala (e.g., GA or AG), Ala and Ser (e.g., AS or SA), Gly and Ser (e.g., GS, GSGGS (SEQ ID NO:124), GGGS (SEQ ID NO:125), GGSG (SEQ ID NO:126), GGSGG (SEQ ID NO:127), GSGSG (SEQ ID NO:128), GSGGG (SEQ ID NO:129), GGGSG (SEQ ID NO:130), GSSSG (SEQ ID NO:131), GGGGS (SEQ ID NO:133)), or Ala and Gly (e.g., AAAGG SEQ ID NO:134), any of which may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times; or
    • (ii) a cysteine-containing linker sequence selected from CGGGS (SEQ ID NO:189), GCGGS (SEQ ID NO:190), GGCGS (SEQ ID NO:191), GGGCS (SEQ ID NO:192), and GGGGC (SEQ ID NO:193), with the remainder of the linker comprised of Gly and Ser residues (e.g., GGGGS (SEQ ID NO:133) units that may be repeated from 1 to 10 times).
  • 11. The MAPP of any preceding aspect, wherein the MAPP comprises one or more linker aa sequences independently selected from GCGASGGGGSGGGGS SEQ ID NO:158, GCGGSGGGGSGGGGSGGGGS SEQ ID NO:159, GCGGSGGGGSGGGGS SEQ ID NO:160, and GCGGS(G₄S) (SEQ ID NO:194) where the G₄S unit may be repeated from 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times); wherein the linker cysteine residue optionally forms a disulfide bond (e.g., with another peptide sequence of the MAPP).
  • 12. The MAPP of any preceding aspect, wherein the MAPP comprises a presenting sequence, wherein the presenting sequence comprises, in the N-terminal to C-terminal direction:
    • a) the T1D peptide epitope, the β1, α1, α2 and β2 domain polypeptide sequences (see, e.g., FIG. 15, structure A);
    • b) the T1D peptide epitope, the β1, β2, α1, and α2 domain polypeptide sequences (see, e.g., FIG. 15, structure B);
      • or
    • c) the T1D peptide epitope, the α1, α2, β1, and β2 domain polypeptide sequences (see, e.g., FIG. 15 structure C);
    • wherein the presenting sequence optionally comprises one or more MOD or variant MOD polypeptide sequences; and
    • wherein said presenting sequence optionally comprises one or more independently selected linker sequences (e.g., joining any one or more of the peptide epitope, α1, α2, β1, and/or β2 domains and/or at the N- or C-terminus).
  • 13. The MAPP of any preceding aspect, wherein the at least one (e.g., at least two) presenting sequence comprises one or more MOD polypeptide sequences.
  • 14. The MAPP of any preceding aspect, wherein the at least one (e.g., at least two) presenting sequence comprises one or more MOD polypeptide sequences, and wherein the presenting sequence has a structure selected from those set forth in FIGS. 15A-C or FIGS. 16A-H.
  • 15. The MAPP of any of aspects 1-11 comprising at least one (e.g., at least two) presenting complex, wherein the at least one presenting complex comprises a presenting complex 1^st sequence and a presenting complex 2^nd sequence, and wherein:
    • (i) the presenting complex 1^st sequence comprises the al domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the β1 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the β1) (see, e.g., FIG. 17, structures A, B, and D);
    • (ii) the presenting complex 1^st sequence comprises the α2 domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the β2 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the β2) (see, e.g., FIG. 17, structures A, B, and D);
    • (iii) the presenting complex 1^st sequence comprises the 31 domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the al domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the α1);
    • (iv) the presenting complex 1^st sequence comprises the β2 domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the α2);
    • (v) the presenting complex 1^st sequence comprises the al domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the β1 and/or β2);
    • (vi) the presenting complex 1^st sequence comprises the α2 domain polypeptide sequence, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the β1 and/or β2);
    • (vii) the presenting complex 1^st sequence comprises the α1 and/or α2 domain polypeptide sequences, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the β1 and/or β2) (see, e.g., FIG. 17, structures A, B, and D, FIG. 18, structures B, D, and F, and FIG. 19, structure B); or
    • (viii) the presenting complex 1^st sequence comprises the β1 and/or β2 domain polypeptide sequences, and its associated presenting complex 2^nd sequence comprises the T1D peptide epitope sequence and the α1 and α2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the α1 and/or α2) (see, e.g., FIG. 19, structures D, E, F, G, and H), and wherein the at least one presenting complex optionally comprises one or more, or two more MODs or variant MODs.
  • 16. The MAPP of any of aspects 1-11 comprising at least one (e.g., at least two) presenting complex, wherein the at least one presenting complex comprises a presenting complex 1^st sequence and a presenting complex 2^nd sequence, and wherein:
    • (i) the presenting complex 1^st sequence comprises the peptide epitope sequence and the al domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the al sequence), and its associated presenting complex 2^nd sequence comprises the β1 domain polypeptide sequence;
    • (ii) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the α2), and its associated presenting complex 2^nd sequence comprises the β2 domain polypeptide sequence (see, e.g., FIG. 19, structure C);
    • (iii) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the β1 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the β1), and its associated presenting complex 2^nd sequence comprises the al domain polypeptide sequence (see, e.g., FIG. 17, structure C, FIG. 18, structures A, C, and E, and FIG. 19, structure A);
    • (iv) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the β2 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the β2), and its associated presenting complex 2^nd sequence comprises the α2 domain polypeptide sequence (see, e.g., FIG. 17, structure C, FIG. 18, structures A, C, and E, and FIG. 19, structures A and G;
    • (v) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the al domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the al sequence), and its associated presenting complex 2^nd sequence comprises the β1 and β2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the β1 and/or β2);
    • (vi) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence (e.g., the T1D epitope is placed N-terminal to the α2 sequence), and its associated presenting complex 2^nd sequence comprises the β1 and β2 domain polypeptide sequences;
    • (vii) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the al and/or α2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the α1 and/or α2), and its associated presenting complex 2^nd sequence comprises the β1 and β2 domain polypeptide sequences; or
    • (viii) the presenting complex 1^st sequence comprises the T1D peptide epitope sequence and the β1 and/or β2 domain polypeptide sequences (e.g., the T1D epitope is placed N-terminal to the 31 and/or β2), and its associated presenting complex 2^nd sequence comprises the α1 and α2 domain polypeptide sequences (see, e.g., FIG. 17, structure C, FIG. 18, structures A, C, and E, and FIG. 19, structure A), and
      • wherein the at least one presenting complex optionally comprises one or more, or two more MODs or variant MODs.
  • 17. The MAPP of any of aspects 1-11 comprising at least one presenting complex that comprises one or more (e.g., two or more) MOD or variant MOD polypeptide sequences.
  • 18. The MAPP of any of aspects 1-11 comprising at least one (e.g., at least two) presenting complex, wherein the at least one presenting complex has a structure selected from those in FIGS. 17 to 22.
  • 19. The MAPP of any of aspects 1-11 comprising at least one (e.g., at least two) presenting complex, wherein the at least one presenting complex has a structure selected from those in FIGS. 20 to 22.
  • 20. The MAPP of any preceding aspect, wherein the at least one presenting sequence, presenting complex 1^st sequence, or presenting complex 2^nd sequence comprises its T1D peptide epitope sequence within 10 aa, 15 aa, 20 aa, or 25 aa of its N-terminus.
  • 21. The MAPP of any preceding aspect comprising a cysteine-containing linker, wherein the cysteine residue in the linker forms a disulfide bond between a presenting sequence and another polypeptide of the MAPP, or between a presenting complex 1^st sequence and another polypeptide of the MAPP (e.g., with a presenting complex 2^nd sequence).
  • 22. The MAPP of any preceding aspect, wherein the dimerization and multimerization sequences are independently selected from non-interspecific sequences or interspecific sequences.
  • 23. The MAPP of aspect 22, wherein the interspecific and non-interspecific sequences are selected from the group consisting of: immunoglobulin heavy chain constant regions (Ig Fc, e.g., CH2-CH3); collectin polypeptides, coiled-coil domains, leucine-zipper domains; Fos polypeptides; Jun polypeptides; Ig CH1; Ig C_L κ; Ig C_Lλ; knob-in-hole without disulfide (“KiH”); knob-in hole with a stabilizing disulfide bond (“KiHs-s”); HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW-RVTs-s; and A107 sequences.
  • 24. The MAPP of aspect 23, wherein the MAPP has the structure of construct sequences 3877 and 3881 (see FIG. 24 and FIG. 14, structure A) SEQ ID NOs:62 and 63, or construct sequences 3888 and 3878 (see FIG. 27 and FIG. 14, structure D) SEQ ID NOs:68 and 69.
  • 25. The MAPP of aspect 23, wherein the MAPP has the structure of construct sequences 3883 and 3884 (see FIG. 25 and FIG. 14, structure B) SEQ ID NOs:64 and 65, or construct sequences 3885 and 3886 (see FIG. 26 and FIG. 14, structure C) SEQ ID NOs:66 and 67.
  • 26. The MAPP of any preceding aspect complexed to form a higher order MAPP (e.g., a duplex or other higher order MAPP) comprising at least a first MAPP and a second MAPP of any of aspects 1-25, wherein:
    • (i) the first MAPP comprises a first framework polypeptide having a first multimerization sequence and a first dimerization sequence, and a first dimerization polypeptide having a first counterpart dimerization sequence complementary to the first dimerization sequence; and
    • (ii) the second MAPP comprises a second framework polypeptide having a second multimerization sequence and a second dimerization sequence, and a second dimerization polypeptide having a second 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 (e.g., one or two disulfide bonds), and the multimerization sequences are not the same as (e.g., not the same type and/or not identical to), and do not substantially associate with or bind to, the dimerization sequences or counterpart dimerization sequences. See, e.g., the duplexes in FIGS. 9 to 13.
  • 27. The duplex MAPP of aspect 26, wherein the first and second dimerization sequences are identical, and the first and second counterpart dimerization sequences are identical. See, e.g., FIG. 11, FIG. 12, and FIG. 13, structures A-H.
  • 28. The duplex MAPP of aspect 26, wherein the first and second dimerization sequences do not substantially associate with or bind to each other.
  • 29. The duplex MAPP of aspect 26, wherein the first and second multimerization sequences are interspecific multimerization sequences that form an interspecific pair, the first and second dimerization sequences are identical, and the first and second counterpart dimerization sequences are identical. See, e.g., FIG. 9, structure B and FIG. 11, structures B and D.
  • 30. The duplex MAPP of aspect 29, wherein the first and second dimerization sequences do not substantially associate with or bind to each other.
  • 31. The duplex MAPP of any of aspects 29 to 30, wherein the first or second framework polypeptide comprises at least one MOD (e.g., two or three MODs) that is/are not present on the other framework polypeptide, optionally wherein the at least one MOD comprises a polypeptide sequence that is a variant of a wt. MOD sequence.
  • 32. The duplex MAPP of aspect 26, wherein the first and second multimerization sequences are identical, the first dimerization sequence and the first counterpart dimerization sequence are interspecific dimerization sequences forming a first interspecific pair, and the second dimerization sequence and second counterpart dimerization sequence are interspecific dimerization sequences forming a second interspecific pair. See, e.g., FIG. 9, structure C.
  • 33. The duplex MAPP of aspect 32, wherein the first and second dimerization sequences are identical and the first and second counterpart dimerization sequences are identical. See, e.g., FIG. 11, structure A.
  • 34. The duplex MAPP of aspect 32, wherein the first and second dimerization sequences are not identical and do not substantially associate with or bind to each other.
  • 35. The duplex MAPP of aspect 32, wherein the polypeptides of the first interspecific pair are different from (not identical to) and do not bind or interact with the polypeptides of the second interspecific pair.
  • 36. The duplex MAPP of any of aspects 34 to 35, wherein the first or second dimerization polypeptide comprises at least one MOD (e.g., two or three MODs) that is/are not present on the other dimerization polypeptide, optionally wherein the at least one MOD comprises a polypeptide sequence that is a variant of a wt. MOD sequence.
  • 37. The duplex MAPP of aspect 26, wherein
  • the first and second multimerization sequences are interspecific multimerization sequences that form an interspecific multimerization pair,
  • the first dimerization sequence and the first counterpart dimerization sequence are interspecific dimerization sequences forming a first interspecific pair, and
  • the second dimerization sequence and second counterpart dimerization sequence are interspecific dimerization sequences forming a second interspecific pair. See, e.g., FIG. 9, structure D.
  • 38. The duplex MAPP of aspect 37, wherein the first and second dimerization sequences are identical and the first and second counterpart dimerization sequences are identical. See, e.g. FIG. 11, structure D.
  • 39. The duplex MAPP of aspect 37, wherein the first and second dimerization sequences do not substantially associate with or bind to each other.
  • 40. The duplex MAPP of aspect 37, wherein the polypeptides of the first interspecific pair of polypeptides are different from (i.e., not identical to) and do not bind or interact with the polypeptides of the second interspecific pair.
  • 41. The duplex MAPP of any of aspects 39 to 40, wherein the first or second dimerization polypeptide comprises at least one MOD (e.g., two or three MODs) that is/are not present on the other dimerization polypeptide, optionally wherein the at least one MOD comprises a polypeptide sequence that is a variant of a wt. MOD sequence.
  • 42. The duplex MAPP of any of aspects 37 to 41, wherein the first or second framework polypeptide comprises at least one MOD (e.g., two or three MODs and/or variant MODs) that is/are not present on the other framework polypeptide, optionally wherein the at least one MOD comprises a polypeptide sequence that is a variant of a wt. MOD sequence.
  • 43. The duplex MAPP of any of aspects 26 to 42, 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 (e.g., Ig CH2-CH3), 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, an Ig CH1 and Ig C_L κ or λ constant region polypeptide pair, a KiH pair, a 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.
  • 44. The duplex MAPP of aspect 43, wherein the multimerization sequences, the first dimerization sequence and its counterpart first dimerization sequence, and the second dimerization sequence and its counterpart second dimerization sequence are each selected from the group consisting of: immunoglobulin heavy chain constant regions (e.g., IgFc CH2-CH3), collectin family dimerization sequences, coiled-coil domains, and leucine-zipper domains, and
    • wherein the first and second dimerization sequences are selected independently and may be the same or different.
  • 45. The duplex MAPP of aspect 43, wherein the multimerization sequences are selected from the group consisting of immunoglobulin heavy chain constant regions (e.g., Ig CH2-CH3), collectin family dimerization sequences, coiled-coil domains, and leucine-zipper domains, and
    • wherein a pair comprising the first dimerization sequence and its counterpart dimerization sequence and a pair comprising the second dimerization sequence and its counterpart dimerization sequence are independently selected from the group consisting of a Fos and Jun polypeptide pair, an Ig CH1 and Ig C_L κ or λ constant region polypeptide pair, a KiH pair, a 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; and
    • wherein the pairs may be the same or different.
  • 46. The duplex MAPP of aspect 43, wherein the multimerization sequences are selected from the group consisting of a Fos and Jun polypeptide pair, an Ig CH1 and Ig C_L κ or λ constant region polypeptide pair, a KiH pair, a 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, and
    • wherein the first and second dimerization sequences, which may be the same or different, and their counterpart dimerization sequences are independently selected from the group consisting of immunoglobulin heavy chain constant regions (e.g., Ig CH2-CH3), collectin family dimerization sequences, coiled-coil domains, and leucine-zipper domains.
  • 47. The duplex MAPP of aspect 43, wherein the multimerization sequences, the first dimerization sequence and its counterpart first dimerization sequence, and the second dimerization sequence and its counterpart dimerization sequence are each selected as a pair from the group consisting of: Fos and Jun polypeptide pairs, Ig CH1 and Ig C_L κ or λ constant region polypeptide pairs, KiH pairs, KiHs-s pairs, HA-TF polypeptide pairs, ZW-1 polypeptide pairs, 7.8.60 polypeptide pairs, DD-KK polypeptide pairs, EW-RVT polypeptide pairs, EW-RVTs-s polypeptide pairs, and A107 polypeptide pairs, and
    • wherein the pairs comprising the first and second dimerization sequences may be the same or different.
  • 48. The duplex MAPP of aspect 43, wherein the multimerization sequences comprise Ig Fc regions and the first and second dimerization sequences comprise independently selected Ig CH1, Ig C_L K or leucine zipper, Fos or Jun domains.
  • 49. The duplex MAPP of aspect 43, wherein the multimerization sequences comprise Ig Fc regions and the first and second dimerization sequences comprise independently selected Ig CH1 or Ig C_L κ or λ domains.
  • 50. The duplex MAPP of any of aspects 48 to 49, wherein the Ig CH2-CH3 domains are selected from the group consisting of IgA, IgD, IgE, IgG and IgM Fc regions.
  • 51. The duplex MAPP of any of aspects 48 to 50, wherein the Ig Fc regions are selected from IgG1, IgG2, IgG3, and IgG4 CH2-CH3 domains.
  • 52. The duplex MAPP of any of aspects 48 to 51, wherein the Ig Fc regions are IgG1 CH2-CH3 domains.
  • 53. The duplex MAPP of aspect 43, wherein the multimerization sequences are a pair of interspecific immunoglobulin sequences.
  • 54. The duplex MAPP of aspect 53, wherein the pair of interspecific immunoglobulin sequences are selected from the group consisting of KiH pairs, KiHs-s pairs, HA-TF polypeptide pairs, ZW-1 polypeptide pairs, 7.8.60 polypeptide pairs, DD-KK polypeptide pairs, EW-RVT polypeptide pairs, EW-RVTs-s polypeptide pairs, and A107 polypeptide pairs.
  • 55. The duplex MAPP of aspect 53 or 54, wherein the pair of interspecific immunoglobulin sequences is a KiH pair.
  • 56. The duplex MAPP of any of aspects 53 to 55, wherein the knob-in hole pair further comprises at least one stabilizing disulfide bond (e.g., a KiHs-s pair).
  • 57. The duplex MAPP of any of aspects 53 to 56, wherein the first and second dimerization sequences comprise independently selected Ig CH1, Ig C_L κ or λ, leucine zipper, Fos or Jun domains.
  • 58. The duplex MAPP of any of aspects 53 to 56, wherein the first and second dimerization sequences comprise Ig CH1 or Ig C_L κ or λ domains.
  • 59. The duplex MAPP of any of aspects 53 to 56, wherein the first and second dimerization sequences comprise Ig CH1 domains.
  • 60. The duplex MAPP of any of aspects 55 to 56, wherein the first and/or second dimerization sequences do not comprise Ig CH1 domains.
  • 61. The MAPP of any of aspects 1 to 25, wherein the dimerization sequence and its counterpart dimerization sequence are covalently linked by at least one (e.g., two) disulfide bond(s).
  • 62. The duplex MAPP of any of aspects 26-60, wherein the dimerization sequence and counterpart dimerization sequence of the first MAPP and/or the dimerization sequence and counterpart dimerization sequence of the second MAPP are covalently linked by at least one (e.g., two) disulfide bond(s).
  • 63. The duplex MAPP of any of aspects 26-60 and 62, wherein the multimerization sequences of the first and second framework polypeptides of the first and second MAPP are covalently linked by at least one (e.g., two) disulfide bond(s). See, e.g., FIGS. 11 to 13.
  • 64. The duplex MAPP of any of aspects 26-60 and 62-63, wherein 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 (e.g., two) disulfide bond(s), and the multimerization sequences of the first and second framework polypeptides of both the first and second MAPP are covalently linked by at least one (e.g., two) disulfide bond(s). See, e.g., FIGS. 12 and 13.
  • 65. The MAPP or duplex MAPP of any preceding aspect, wherein the MAPP comprises only one presenting sequence or one presenting complex, or the duplex MAPP comprises only two presenting sequences or two presenting complexes. See, e.g., FIGS. 1A and 1B.
  • 66. The MAPP or duplex MAPP of aspect 65, wherein the one presenting sequence or presenting complex of the MAPP, or the two presenting sequences or presenting complexes of the duplex MAPP, is/are provided on the dimerization polypeptide(s), and further, when the MAPP or duplex MAPP comprises a presenting complex, wherein the presenting complex 1^st sequence(s) is/are located on the dimerization polypeptide(s) (e.g., located on the N-terminal side of the counterpart dimerization sequence). See, e.g., FIGS. 1A and 1B.
  • 67. The MAPP or duplex MAPP of aspect 65, wherein the one presenting sequence or the presenting complex 1^st sequence of the MAPP, or the two presenting sequences or presenting complex 1st sequences of the duplex MAPP, is/are provided on the framework polypeptide(s) (e.g., located on the N-terminal side of the dimerization sequence).
  • 68. The duplex MAPP of any of aspects 26-60 and 62-64, wherein the duplex MAPP comprises only one presenting sequence or one presenting complex.
  • 69. The duplex MAPP of aspect 68, wherein the one presenting sequence or the presenting complex 1^st sequence of the one presenting complex is an aa sequence of the one dimerization polypeptide (e.g., the presenting sequence is part of the dimerization polypeptide and located on the N-terminal side of the counterpart dimerization sequence of the one dimerization polypeptide).
  • 70. The duplex MAPP of aspect 68, wherein the one presenting sequence or the presenting complex 1^st sequence of the one presenting complex is an aa sequence of the framework polypeptide (e.g., is part of the framework polypeptide and located on the N-terminal side of the dimerization sequence).
  • 71. The duplex MAPP of any of aspects 26-60 and 62-64, wherein the duplex MAPP comprises at least two presenting sequences or at least two presenting complexes. See, e.g., the duplex in FIGS. 1A and 1B.
  • 72. The duplex MAPP of aspect 71, wherein one of the at least two presenting sequences or presenting complex 1^st sequences of the at least two presenting complexes is part of the first dimerization polypeptide, and the second of the at least two presenting sequences or presenting complex 1^st sequences is part of the second dimerization polypeptide (e.g., located on the N-terminal side of their counterpart dimerization sequences). See, e.g., the duplex in FIGS. 1A and 1B.
  • 73. The duplex MAPP of aspect 71, wherein one of the at least two presenting sequences or each of the presenting complex 1^st sequences of the at least two presenting complexes is part of the first framework polypeptide, and the second of the at least two presenting sequences or presenting complex 1^st sequences is part of the second framework polypeptide (e.g., located on the N-terminal side of their dimerization sequence).
  • 74. The duplex MAPP of any of aspects 26-60 and 62-64, wherein the duplex MAPP comprises at least four presenting sequences or four presenting complexes. See, e.g., FIG. 10, structures A-D.
  • 75. The duplex MAPP of aspect 74, wherein each one of the four presenting sequences or each one of the presenting complex 1^st sequences of the four presenting complexes is part of a different one of the first dimerization polypeptide, second dimerization polypeptide, first framework polypeptide and second framework polypeptide (e.g., located on the N-terminal side of their dimerization sequence or counterpart dimerization sequence). See, e.g., FIG. 10, structures A-D.
  • 76. The MAPP or duplex MAPP of any preceding aspect, wherein when a framework or dimerization polypeptide of the MAPP or duplex MAPP comprises one or more IgFc regions, and wherein at least one of the one or more IgFc regions comprises one or more substitutions that limit complement activation.
  • 77. The MAPP or duplex MAPP of any preceding aspect, wherein when a framework or dimerization polypeptide of the MAPP or duplex MAPP comprises one or more IgFc regions, and wherein at least one of the one or more IgFc regions comprises one or more substitutions at L234, L235, G236, G237, P238, S239, D270, N297, K322, P329, and/or P331 (respectively, aas L14, L15, G16, G17, P18, S19, D50, N77, K102, P109, and Pill of the wt. IgG1 aa sequence in FIG. 2D) or the corresponding substitutions in other IgFc regions.
  • 78. The MAPP or the duplex MAPP of aspect 77, wherein when a framework or dimerization polypeptide comprises an IgFc region comprising a substitution at N297 (e.g., N297A).
  • 79. The MAPP or the duplex MAPP of aspect 77, wherein when a framework or dimerization polypeptide comprises an IgFc region comprising a substitution at L234 and/or L235 (e.g., L234A, and/or L235A).
  • 80. The MAPP or the duplex MAPP of aspect 77, wherein when a framework or dimerization polypeptide comprises an IgFc region comprising a substitution at P331 (e.g., P331A or P331S).
  • 81. The MAPP or the duplex MAPP of aspect 77, wherein when a framework or dimerization polypeptide comprises an Ig Fc region that comprises: (i) one or more substitutions selected from the group consisting of L234, L235, and P331 (e.g., L234F, L235E, and/or P331S substitution(s)); or (ii) any one or more of D270, K322, and P329 (e.g., D270, K322, and/or P329 substitution(s)).
  • 82. The MAPP or the duplex MAPP of aspect 77, wherein when a framework or dimerization polypeptide comprises an IgFc region that comprises either (i) one or more substitutions selected from the group consisting of L234, L235, and P331, or (ii) one or more substitutions selected from the group consisting of D270, K322, and P329.
  • 83. The MAPP or duplex MAPP of any preceding aspect, comprising: at least one MOD (wt. or variant), or at least one pair of MODs in tandem (both wt., both variant, or one wt. and one variant), wherein the at least one MOD or at least one pair of MODs is located at one or more of positions 1, 1′, 2, 2′, 3, 3′, 4, 4′, 4″, 4′″, 5, and/or 5′. (see FIGS. 1A and 1B).
  • 84. The MAPP or duplex MAPP of any preceding aspect, wherein:
    • (a) at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), is located:
      • (i) on the N-terminal side (e.g., at the N-terminus) of at least one framework polypeptide dimerization sequence (see, e.g., positions 1 and 1′ in FIGS. 9 and 11);
      • (ii) on the N-terminal side (e.g., at the N-terminus) of at least one framework polypeptide dimerization sequence and any MHC Class II polypeptide sequences that may be part of the framework polypeptide (see, e.g., positions 4″ and 4′″ in FIGS. 10 and 12); and/or
      • (iii) on the C-terminal side (e.g., at the C-terminus) of at least one framework polypeptide framework multimerization sequence (see, e.g., positions 3 and 3′ in any of FIGS. 1 and 9 to 13); and/or
    • (b) at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), is located:
      • (i) on the N-terminal side (e.g., at the N-terminus) of each framework polypeptide dimerization sequence (see, e.g., positions 1 and 1′ in FIGS. 9 and 11);
      • (ii) on the N-terminal side (e.g., at the N-terminus) of each framework polypeptide dimerization sequence and any MHC Class II polypeptide sequences that may be part of the framework polypeptide (see, e.g., positions 4″ and 4′″ in FIGS. 10 and 12); and/or
      • (iii) on the C-terminal side (e.g., at the C-terminus) of each framework polypeptide framework multimerization sequence (see, e.g., positions 3 and 3′ in any of FIGS. 1 and 9 to 13).
  • 85. The MAPP or duplex MAPP of aspect 83 or aspect 84 comprising:
    • (i) at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), located on the N-terminal side (e.g., at the N-terminus) of at least one (e.g., each) framework polypeptide dimerization sequence (see, e.g., positions 1 and 1′ in FIGS. 9 and 11), or on the N-terminal side (e.g., at the N-terminus) of at least one (e.g., each) framework polypeptide dimerization sequence and any MHC Class II polypeptide sequences that may be part of the framework polypeptide (see, e.g., positions 4″ and 4′″ in FIGS. 10 and 12); and/or
    • (ii) at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), located on the C-terminal side (e.g., at the C-terminus) of at least one (e.g., each) framework polypeptide framework multimerization sequence (see, e.g., positions 3 and 3′ in any of FIGS. 1 and 9 to 13).
  • 86. The MAPP or duplex MAPP of any preceding aspect comprising: at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), located:
    • (i) on the N-terminal side (e.g., at the N-terminus) of at least one (e.g., two or each) dimerization polypeptide counterpart dimerization sequence (see, e.g., positions 4 and 4′ in any of FIGS. 1 and 10 to 12); and/or
    • (ii) on the C-terminal side (e.g., at the C-terminus) of at least one (e.g., two or each) dimerization polypeptide counterpart dimerization sequence (see, e.g., positions 5 and 5′ in any of FIGS. 1 and 9 to 12).
  • 87. The MAPP or duplex MAPP of any of aspects 83-86, wherein, when the at least one (e.g., at least two or each) dimerization polypeptide comprises a presenting sequence, the at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), may be located:
    • (i) between the counterpart dimerization sequence and the α1, α2, β1, 32 sequences and T1D epitope sequences;
    • (ii) between any of the α1, α2, β1, (2 and T1D epitope sequences;
    • (iii) between the T1D epitope and either the α1 and α2 or the β1 and (2 sequence; and/or
    • (iv) at the N-terminus of the presenting sequence. See FIG. 15.
  • 88. The MAPP or duplex MAPP of any of aspects 83-86, wherein, when the dimerization polypeptide comprises a presenting complex, the at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), may be located:
    • (i) between the counterpart dimerization sequence and any of the α1, α2, β1, and/or (2 or T1D epitope sequences present in the presenting complex 1^st sequence;
    • (ii) between any of the α1, α2, β1, and/or (2 or T1D epitope sequences present in the presenting complex 1^st sequence;
    • (iii) at the N-terminus of the presenting complex 1^st sequence;
    • (iv) at the N-terminus of the presenting complex 2^st sequence;
    • (v) between any of the α1, α2, β1, and/or β2 and T1D epitope sequences present in the presenting complex 2^st sequence; and/or
    • (vi) at the C-terminus of the presenting complex 2^st sequence. See, e.g., FIGS. 17A-D, 18C-F, 19A-H, 20C-L, and 21A-F.
  • 89. The duplex MAPP of any of aspects 26 to 60 and 62-88 comprising: at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), at positions 1 and/or 1′.
  • 90. The duplex MAPP of any of aspects 26 to 60 and 62-88 comprising: at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), at positions 1 and/or 1′, and at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), at positions 3 and/or 3′.
  • 91. The duplex MAPP of any of aspects 26 to 60 and 62-88 comprising: at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), at positions 1 and/or 1′, and at least one MOD (wt. or variant), or pair of MODs in tandem (both wt., both variant, or one wt. and one variant), at positions 5 and/or 5′.
  • 92. The MAPP or duplex MAPP of any preceding aspect comprising at least one (e.g., at least two, or at least three) MOD (wt. or variant), wherein each MOD is selected independently from the group consisting of: 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), JAGI (CD339), lymphotoxin beta receptor, 3/TR6, OX40L (CD252), PD-L1, PD-L2, TGF-β1, TGF-β2, TGF-β3, and 4-1BBL polypeptide sequences.
  • 93. The MAPP or duplex MAPP of any preceding aspect comprising at least one (e.g., at least two, or at least three) MOD (wt. or variant), wherein each MOD is selected independently from the group consisting of: 4-1BBL, PD-L1, IL-2, CD80, CD86, OX40L (CD252), Fas ligand (FasL), ICOS-L, ICAM, CD30L, CD40, CD83, HVEM (CD270), JAGI (CD339), CD70, CD80, CD86, TGF-β1, TGF-β2, and TGF-β3 polypeptide sequences.
  • 94. The MAPP or duplex MAPP of any preceding aspect comprising at least one (e.g., at least two, or at least three) MOD (wt. or variant), wherein each MOD polypeptide sequence is selected independently from the group consisting of IL-2, FasL, PD-L1, TGF-β, CD80, CD86 and 4-1BBL polypeptide sequences. For example, the MAPP or duplex MAPP may comprise at least one IL-2 MOD (wt. or variant) polypeptide sequence, and at least one MOD (wt. or variant) independently selected from CD80 and CD86 polypeptide sequences.
  • 95. The MAPP or duplex MAPP of any preceding aspect comprising at least one IL-2 MOD or variant MOD polypeptide sequence, or at least one pair of IL-2 MOD or variant MOD polypeptide sequences in tandem (optionally located at position 1 or 1′), and/or at least one wt. or variant PD-L1 MOD polypeptide sequence.
  • 96. The MAPP or duplex MAPP of aspect 94, comprising at least one IL-2 and/or TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3) MOD or variant MOD polypeptide sequence optionally located at position 1 or 1′.
  • 97. The MAPP or duplex MAPP of aspect 94, comprising at least one wt. or variant PD-L1 MOD polypeptide sequence, optionally wherein the MOD is located at position 1 or 1′.
  • 98. The MAPP or duplex MAPP of aspect 94, comprising at least one wt. or variant FasL MOD polypeptide sequence, optionally wherein the MOD is located at position 1 or 1′.
  • 99. The MAPP or duplex MAPP of any preceding aspect further comprising an additional peptide covalently attached to one or more framework polypeptides and/or dimerization polypeptides.
  • 100. The MAPP or duplex MAPP of aspect 99, wherein the additional peptide is an epitope tag or an affinity domain.
  • 101. The MAPP of any of aspects 1-24, or a duplex thereof wherein the MAPP comprises:
    • a framework polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a T1D peptide epitope, (ii) human MHC class II β1 and β2 domains, (iii) human Class II α1 and α2 domains, (iv) a human or humanized IgG CH1, and (v) an Ig Fc region (e.g., CH2 CH3 regions with optional “LALA” substitutions), each of (i) to (v) optionally separated by linker peptide sequences that are selected independently; and
    • a dimerization polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a human PD-L1 extracellular domain polypeptide, and (ii) a human or humanized kappa light constant region, with (i) and (ii) optionally separated by a linker peptide sequence;
    • wherein the IgG CH1 and the kappa light constant region optionally comprise M13 stabilization substitutions. See, e.g., FIG. 14, structure A.
  • 102. The MAPP of any of aspects 1-23, or a duplex thereof wherein the MAPP comprises:
    • a framework polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a human PD-L1 extracellular domain polypeptide, (ii) a human or humanized CH3 KIH s-s hole, and (iii) a human IgG Fc sequence (e.g., IgG1 CH2 and CH3 regions with optional “LALA” substitutions), each of (i) to (iii) optionally separated by linker peptide sequences that are selected independently; and
    • a dimerization polypeptide comprising from N-terminus to C-terminus aa sequences of (i) a T1D peptide epitope, (ii) human MHC class II β1 and β2 domains, (iii) human Class II α1 and α2 domains, and (iv) a human or humanized IgG1 with CH3 KIHs-s knob, each of (i) to (iv) optionally separated by linker peptide sequences that are selected independently. See, e.g., FIG. 14, structure B.
  • 103. The MAPP of any of aspects 1-23, or a duplex thereof wherein the MAPP comprises:
    • a framework polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a human PD-L1 extracellular domain polypeptide, (ii) a human or humanized CH1 (optionally with M13 stabilization substitutions), and (iii) a human Ig Fc sequence (e.g., IgG1 CH2 and CH3 regions with optional “LALA” substitutions), each of (i) to (iii) optionally separated by linker peptide sequences that are selected independently; and
    • a dimerization polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a T1D peptide epitope, (ii) human MHC class II β1 and β2 domains, (iii) human Class II α1 and α2 domains, and (iv) a human or humanized kappa light chain sequence (optionally with MD13 stabilization substitutions), each of (i) to (iv) optionally separated by linker peptide sequences that are selected independently;
    • wherein the IgG CH1 and the kappa light constant region optionally comprise M13 stabilization substitutions. See, e.g., FIG. 14, structure C
  • 104. The MAPP of any of aspects 1-24, or a duplex thereof wherein the MAPP comprises:
    • a framework polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a T1D peptide epitope, (ii) human MHC Class II β1 and β2 domains, (iii) human Class II α1 and α2 domains, (iv) an IgG Fc region (e.g., CH2 and CH3 regions with optional “LALA” substitutions), and (v) a human or humanized IgG CH1, each of (i) to (v) optionally separated by linker peptide sequences that are selected independently; and
    • a dimerization polypeptide comprising, from N-terminus to C-terminus, aa sequences of (i) a human PD-L1 extracellular domain polypeptide, and (ii) a human or humanized kappa light constant region, with (i) and (ii) optionally separated by a linker peptide sequence;
    • wherein the IgG CH1 and the kappa light constant region optionally comprise M13 stabilization substitutions. See, e.g., FIG. 14, structure D.
  • 105. A duplex of the MAPPs of any of aspects 101-104, wherein the duplex is formed by interactions between the IgFc multimerization sequences.
  • 106. The MAPP or duplex MAPP of any of aspects 101-105, wherein the immunoglobulin (Ig) sequences are IgG sequences (e.g., Ig G1 sequences) 107. The MAPP or duplex MAPP of any of aspects 101-106, wherein the MHC Class II α1 and α2 are DRA α1 and α2 domains.
  • 108. The MAPP or duplex MAPP of any of aspects 101-107, wherein the MHC Class II α1 and α2 domains have greater than 95% aa sequence identity to DRA*0101 α1 and α2 domains.
  • 109. The MAPP or duplex MAPP of any of aspects 101-108, wherein the MHC Class II β1 and β2 are DRB1 β1 and β2 domains.
  • 110. The MAPP or duplex MAPP of any of aspects 101-108, wherein the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DRB1*01:01, DRB1*0301, DRB1*0401, DRB1*0402, DRB1*0405, DRB1*0801, or DRB1*0901 β1 and β2 domains.
  • 111. The MAPP or duplex MAPP of any of aspects 101-108, wherein the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DRB1*03:01 or DRB1*04:01.
  • 112. The MAPP or duplex MAPP of any of aspects 101-108, wherein the MHC Class II α1 and α2 domains have greater than 95% aa sequence identity to DRA*β1:01 and the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DRB1*03:01 or DRB1*04:01.
  • 113. The MAPP or duplex MAPP of any of aspects 101-106, wherein the MHC Class II α1 and α2 are DQA1 α1 and α2 domains.
  • 114. The MAPP or duplex MAPP of any of aspects 101-106 and 113, wherein the MHC Class II α1 and α2 domains have greater than 95% aa sequence identity to DQA*01:01, DQA*03:01, DQA*04:01, or DQA*05:01,
  • 115. The MAPP or duplex MAPP of any of aspects 101-106, and 113-114, wherein the MHC Class II β1 and β2 are DQB1 β1 and β2 domains.
  • 116. The MAPP or duplex MAPP of any of aspects 113-115, wherein the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DQB1*02:01, DQB1*03:02, DQB1*03:03, DQB1*04:02, or DQB1*05:01 β1 and β2 domains.
  • 117. The MAPP or duplex MAPP of any of aspects 113-116, wherein the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DQB1*02:01 or DQB1*03:02.
  • 118. The MAPP or duplex MAPP of any of aspects 113-116, wherein the MHC Class II α1 and α2 domains have greater than 95% aa sequence identity to DQA*03:01 or DQA*05:01, and the MHC Class II β1 and β2 domains have greater than 95% aa sequence identity to DQB1*02:01 or DQB1*03:02.
  • 119. The MAPP or higher order MAPP (e.g., duplex MAPP) of any preceding aspect, wherein the T1D-epitope is an epitope of a T1D disease-associated antigen selected from an epitope of: preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, 65 kDa isoform of glutamic acid decarboxylase (GAD65), 67 kDa isoform of glutamic acid decarboxylase (GAD67), tyrosine phosphatase (IA-2), heat-shock protein HSP65, islet-specific glucose6-phosphatase catalytic subunit related protein (IGRP), islet antigen 2 (IA2), and zinc transporter (ZnT8).
  • 120. The MAPP or higher order MAPP (e.g., duplex MAPP) of aspect 119, wherein the T1D peptide epitope is selected from the group consisting of proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO:161), insulin (InsA (1-15) peptide GIVDQCCTSICSLYQ (SEQ ID NO:162), insulin (InsA(1-15; D4E) peptide GIVEQCCTSICSLYQ (SEQ ID NO:163), GAD65 (555-567) peptide NFFRMVISNPAAT (SEQ ID NO:164), GAD65 (555-567; F557I) peptide NFIRMVISNPAAT (SEQ ID NO:165), islet antigen 2 (IA2) peptide SFYLKNVQTQETRTLTQFHF (SEQ ID NO:166), proinsulin peptide SLQPLALEGSLQSRG (SEQ ID NO:167), and proinsulin peptide GSLQPLALEGSLQSRGIV (SEQ ID NO:168; prolns 75-92(K88S)).
  • 121. The MAPP or higher order MAPP (e.g., duplex MAPP) of aspect 119, wherein the T1D peptide comprises from 4 to 25 contiguous aas of an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to aas 25-110 of the human preproinsulin aa sequence (wherein italicized aas 1-24 form the signal peptide): MALWMRLLPL LALLALWGPD PAAA FVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN (SEQ ID NO:169).
  • 122. The MAPP or higher order MAPP (e.g., duplex MAPP) of aspect 119, wherein the T1D peptide epitope has the aa sequence: GAGSLQPLALEGSLQKRG (SEQ ID NO:170) (prolns 73-90), SLQPLALEGSLQKRG (SEQ ID NO:171) (prolns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:172) (prolns 76-90; K88S), QPLALEGSLQKRG (SEQ ID NO:173), or QPLALEGSLQSRG (SEQ ID NO:174).
  • 123. The MAPP or duplex MAPP of any preceding aspect, wherein the peptide epitope is from about 4 aas (aa) to about 25 aa (e.g., the epitope can have a length of from 4 aa to 10 aa, from about 6 aa to about 12 aa, from 10 aa to 15 aa, from 10 aa to 20 aa, from 15 aa to 20 aa, or from 20 aa to 25 aa).
  • 124. The MAPP or duplex MAPP of any preceding aspect wherein the peptide epitope is from 10 aa to 20 aa.
  • 125. The MAPP or duplex MAPP of any preceding aspect further comprising a disulfide bond stabilizing the presenting sequence or presenting complex, or assisting in positioning the peptide epitope in an epitope binding pocket/groove of a presenting sequence or presenting complex (e.g., a disulfide between a linker attaching the epitope to an MHC peptide sequence and an MHC peptide sequence such as a DRA peptide sequence set forth in Table 3, Table 4, or Table 5).
  • 126. A pharmaceutical composition comprising a MAPP or duplex MAPP of any of the preceding aspects.
  • 127. A method of treatment or prophylaxis of a T1D comprising administering to a patient/subject (e.g., a diabetic or prediabetic patient in need thereof) a pharmaceutical composition comprising an effective amount of one or more MAPPs or higher order MAPP complexes (e.g., duplex MAPPs) of any of aspects 1-125.
  • 128. The method of aspect 127, wherein the MAPP or duplex MAPP reduces hemoglobin A1C or blood sugar (e.g., glucose) levels in a diabetic or prediabetic patient/subject relative to the hemoglobin A1C or blood sugar levels prior to administration of the MAPP or duplex MAPP.
  • 129. The method of any of aspects 127-128, wherein the one or more MAPPs or duplex MAPPs are administered to a mammalian patient or subject.
  • 130. The method of any of aspects 127-129, wherein the subject is human.
  • 131. The method of any of aspects 127-129, wherein the subject is non-human (e.g., mammal, rodent, lagomorph, bovine, canine, feline, rodent, murine, caprine, simian, ovine, equine, porcine, etc.).
  • 132. One or more nucleic acids comprising one or more nucleic acid sequences encoding a MAPP or higher order MAPP complex (e.g., a duplex MAPP) according to any of aspects 1-125.
  • 133. The one or more nucleic acids of aspect 132 comprising a nucleic acid sequence encoding the framework polypeptide operably linked to a promoter and/or a nucleic acid sequence encoding the dimerization polypeptide operably linked to a promoter.
  • 134. One or more recombinant expression vectors comprising one or more nucleic acid molecules comprising sequences encoding a MAPP of any of aspects 1 to 125.
  • 135. A method of producing cells expressing a MAPP or higher order map (e.g., duplex MAPP), the method comprising introducing one or more nucleic acid molecules according to aspect 132 or 133, or one or more expression vectors according to aspect 134, 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.
  • 136. The method of aspect 135, wherein the cell is a cell of a mammalian cell line selected from HeLa cells, CHO cells, 293 cells, Vero cells, NIH 3T3 cells, Huh-7 cells, BHK cells, PC12 cells, COS cells, COS-7 cells, RAT1 cells, mouse L cells, human embryonic kidney (HEK) cells, and HLHepG2 cells.
  • 137. One or more cells transiently or stably expressing a MAPP or duplex MAPP prepared by the method of aspect 135 or 136.
  • 138. The cells of aspect 137, wherein the cells express from about 25 to about 350 (e.g., 20-50, 50-100, 100-200, 200-300, 300-350) mg/liter or more of the MAPP or duplex MAPP without a substantial reduction (e.g., less than a 5%, 10%, or 15% reduction) in viability relative to otherwise identical cells not expressing the MAPP or duplex MAPP.
  • 139. A method of selectively delivering one or more MOD polypeptides and/or variant MOD polypeptides to a cell, tissue, patient or subject having or suspected of having T1D, the method comprising administering to a patient/subject having or suspected of having T1D an effective amount of one or more MAPPs or higher order MAPPS (e.g., duplex MAPPs) of any of aspects 1-125.

VI. EXAMPLES

Example 1

Example 1 illustrates four MAPP heterodimer constructs that can multimerize to form higher order duplex complexes through interaction of their IgFc multimerization sequences (IgFc). The unduplexed MAPP heterodimers have the overall structure given in FIG. 14 as structures A, B, C, and D. Lines between the lines connecting elements represent optional linker peptide sequences. The peptide epitope is shown as a semicircle at the top of each structure between the al and 31 domains of the MHC Class II polypeptide sequences. FIG. 24 provides construct sequences 3877 and 3881 of the MAPP construct shown in FIG. 14 as structure A. FIG. 25 provides construct sequences 3883 and 3884 of the MAPP construct shown in FIG. 14 as structure B. FIG. 26 provides construct sequences 3885 and 3886 of the MAPP construct shown in FIG. 14 as structure C. FIG. 27 provides construct sequences 3888 and 3878 of the MAPP construct shown in FIG. 14 as structure D.

Accelerated stability testing in PBS pH7.4, 500 mM NaCl buffer demonstrates that the MAPP (duplex MAPPs) substantially retain their structure when tested at 37° C. or 42° C. Greater than 87.5% of the MAPPs remain as unaggregated duplex MAPP after 3 days at 37° C., and that greater than 85% of the MAPPs are present as unaggregated duplexes after 5 days at 37° C. Similarly, greater than 76% of the MAPPs are present as unaggregated duplex MAPPs after 3 days at 42° C., and that greater than 73% of the MAPPs are present as unaggregated duplexes after 5 days at 37° C. Reference data is provided in the table below. All percentages are relative to the amount of unaggregated duplex MAPPs present in the sample at the initiation of the test (taken as 100%).

Measurement of the melting points (Tm) in PBS pH7.4, 500 mM NaCl buffer demonstrates the HLA portion of the MAPPS may have a Tm greater than about 67° C., for example in the range of about 67° C. to about 69.5. Similarly, the Fc portions may have a Tm greater than about 78° C., for example in the range of about 78° C. to about 82° C.

Freeze thaw testing by freezing at −80° C. in PBS pH7.4, 500 mM NaCl buffer showed that the initial (“0” cycles) unaggregated duplex MAPP fraction (based on size exclusion chromatography) was greater than about 97.5% and ranged from about 97.5% to about 98.5%. After one freeze-thaw cycle (1×) the unaggregated fraction of material was greater than about 95.5% and ranged from about 95.5% to about 98%. After 3 freeze-thaw cycles (3×) the unaggregated fraction of material was greater than about 95.5% and ranged from about 95.5% to about 98%. The data are summarized in the Table that follows

	Construct
	3877-3881	3888-3878	3883-3884	3885-3886
Time	Day 3	Day 5	Day 3	Day 5	Day 3	Day 5	Day 3	Day 5
Temp. 37° C.	90.99%	89.59%	87.52%	86.59%	92.06%	92.34%	93.07%	91.93%
Temp. 42° C.	80.73%	76.19%	76.58%	73.11%	78.57%	74.78%	80.08%	74.58%
Tm pHLA	67.7° C.	68.2° C.	69.6° C.	69.1° C.
Tm Fc	81.6° C.	80° C.	76.4° C.	81.64° C.
Freeze -Thaw	0		0	97.6%	0	98.4%	0	98.3%
cycles	1x	97.8%	1x	95.7%	1x	97.9%	1x	97.7%
	3x	96.4%	3x	90.7%	3x	97.1%	3x	96%

Testing may be conducted by contacting the MAPPs with a population of PBMCs in vitro obtained from one or more mammalian individuals or by administration to a mammalian host in vivo. Where the MAPP comprises an inhibitory MOD (e.g., PD-L1 or FASL) the MAPP demonstrates a suppression or functional attenuation of antigen-specific CD4 T cells in vitro or in vivo. The suppression or functional attenuation may be manifested as, for example, either (i) a reduction in the number of proinsulin-specific T cells (e.g., demonstrated by proinsulin-tetramer staining) and/or (ii) a suppression of cytokine production (e.g., IL-2 and/or IFNγ) by proinsulin-specific CD4+ T Cells. In contrast, exposure of PBMC to MAPPs bearing an unrelated epitope does not demonstrate a suppression of antigen-specific CD4 T cells.

Claims

1. A multimeric antigen-presenting polypeptide (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, the dimerization sequence and counterpart dimerization sequence dimerizing through covalent and/or non-covalent interactions to form a MAPP heterodimer; and(iii) at least one presenting sequence and/or presenting complex;wherein (a) each presenting sequence comprises (i) a T1D peptide epitope, and (ii) MHC Class II α1, α2, β1, and β2 domain polypeptide sequences,(b) each presenting complex comprises a presenting complex 1st sequence and a presenting complex 2nd sequence, wherein the presenting complex 1st sequence or presenting complex 2nd sequence comprises the peptide epitope and at least one of the α1, α2, β1, and β2 polypeptide sequences, andthe presenting complex 1st sequence and presenting complex 2nd sequence together comprise a T1D peptide epitope and MHC Class II α1, α2, β1, and β2 domain polypeptide sequences,(c) one or both of the dimerization polypeptide and/or the framework polypeptide comprises a presenting sequence or a presenting complex 1st sequence, and(d) at least one framework polypeptide, dimerization peptide, presenting sequence, or presenting complex comprises one or more independently selected MOD and/or variant MOD polypeptide sequences; andwherein the framework polypeptide, dimerization polypeptide, presenting sequence, presenting complex 1st sequence and/or presenting complex 2nd sequence optionally comprise one or more independently selected linker sequences.

2. The MAPP of claim 1, wherein at least one presenting sequence or presenting complex comprises: (i) α1 and α2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to all or at least about 50 contiguous aas of a HLA DRA*01*01 or HLA DRA*01*02 polypeptide sequence; and(ii) β1 and β2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% 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 1, wherein at least one presenting sequence or presenting complex comprises: (i) α1 and α2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of an HLA DQ alpha 1 or DQ alpha 2 (DQA1 or DQA2) polypeptide sequence; and(ii) β1 and β2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of an HLA DQ beta 1 or DQ beta 2 (DQB1 or DQB2) polypeptide sequence.

4. The MAPP of claim 3, wherein at least one presenting sequence or presenting complex comprises either: (i) α1 and α2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of a DQA1*0501 polypeptide sequence, and β1 and β2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of a DQB1*0201 polypeptide sequence; or(ii) α1 and α2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of a DQA1*0301 polypeptide sequence, and β1 and β2 domain polypeptide sequences each having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to at least 60 contiguous aas of a DQB1*0302 polypeptide sequence.

5. The MAPP of claim 1, wherein the MAPP comprises at least one linker: (i) wherein the linker comprises an aa sequence selected from GSGGS (SEQ ID NO:124), GGGS (SEQ ID NO:125), GGSG (SEQ ID NO:126), GGSGG (SEQ ID NO:127), GSGSG (SEQ ID NO:128), GSGGG (SEQ ID NO:129), GGGSG (SEQ ID NO:130), GSSSG (SEQ ID NO:131), and GGGGS (SEQ ID NO:133), any of which may be present 1, 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:189), GCGGS (SEQ ID NO:190), GGCGS (SEQ ID NO:191), GGGCS (SEQ ID NO:192), and GGGGC (SEQ ID NO:193), with the remainder of the linker comprised of Gly and Ser residues.

6. The MAPP of claim 1, wherein the MAPP comprises a presenting sequence, and wherein the presenting sequence comprises, in the N-terminal to C-terminal direction: (i) the T1D peptide epitope, the β1, α1, α2, and β2 domain polypeptide sequences;(ii) the T1D peptide epitope, the β1, β2, α1, and α2 domain polypeptide sequences; or(iii) the T1D peptide epitope, the α1, α2, β1, and β2 domain polypeptide sequences,wherein the presenting sequence optionally comprises one or more wt. or variant MOD polypeptide sequences, andwherein said presenting sequence optionally comprises one or more independently selected linker sequences.

7. The MAPP of claim 1, comprising at least one presenting complex, wherein the at least one presenting complex comprises a presenting complex 1st sequence and a presenting complex 2nd sequence, and wherein: (i) the presenting complex 1st sequence comprises the al domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the β1 domain polypeptide sequence;(ii) the presenting complex 1st sequence comprises the α2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the β2 domain polypeptide sequence;(iii) the presenting complex 1st sequence comprises the β1 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the al domain polypeptide sequence;(iv) the presenting complex 1st sequence comprises the β2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence; (v) the presenting complex 1st sequence comprises the al domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences;(vi) the presenting complex 1st sequence comprises the α2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences;(vii) the presenting complex 1st sequence comprises the α1 and/or α2 domain polypeptide sequences, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the β1 and β2 domain polypeptide sequences;(viii) the presenting complex 1st sequence comprises the β1 and/or β2 domain polypeptide sequences, and its associated presenting complex 2nd sequence comprises the T1D peptide epitope sequence and the α1 and α2 domain polypeptide sequences;(ix) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the al domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the β1 domain polypeptide sequence;(x) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the β2 domain polypeptide sequence;(xi) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the β1 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the al domain polypeptide sequence;(xii) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the β2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the α2 domain polypeptide sequence;(xiii) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the al domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the β1 and β2 domain polypeptide sequences;(xiv) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the α2 domain polypeptide sequence, and its associated presenting complex 2nd sequence comprises the β1 and β2 domain polypeptide sequences;(xv) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the al and/or α2 domain polypeptide sequences, and its associated presenting complex 2nd sequence comprises the β1 and β2 domain polypeptide sequences; or(xvi) the presenting complex 1st sequence comprises the T1D peptide epitope sequence and the 31 and/or β2 domain polypeptide sequences, and its associated presenting complex 2nd sequence comprises the α1 and α2 domain polypeptide sequences, and further wherein the at least one presenting complex optionally comprises one or more, or two or more, wt. or variant MODs.

8. The MAPP of any preceding claim, complexed to form a duplex or higher order MAPP comprising at least a first MAPP heterodimer and a second MAPP heterodimer of any of claims 1-7, 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; andwherein 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.

9. The duplex MAPP of claim 8, wherein, 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 wherein, 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, an Ig CH1 and Ig CL κ or λ constant region polypeptide pair, a knob-in-hole without disulfide (“KiH”) 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.

10. The duplex MAPP of claim 8, wherein the multimerization sequences comprise Ig Fc regions and the first and second dimerization sequences comprise independently selected Ig CH1, Ig CL K or leucine zipper, Fos or Jun domains.

11. The duplex MAPP of claim 8, 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.

12. The duplex MAPP of claim 8, comprising at least one wt. or variant MOD polypeptide sequence, wherein each MOD is selected independently from the group consisting of IL-2, FasL, PD-L1, TGF-β, CD80, CD86 and 4-1BBL polypeptide sequences.

13. The duplex MAPP of claim 12, comprising at least one wt. or variant PD-L1 polypeptide sequence.

14. The duplex MAPP of claim 13 wherein the peptide epitope is from about 10 aa to about 20 aa.

15. A method of treatment or prophylaxis of a T1D comprising administering to a patient/subject an effective amount of one or more MAPPs of any of claims 1-7 and/or one or more duplex MAPPs of any of claims 8-14.

16. One or more nucleic acid molecules comprising sequences encoding a MAPP of any one of claims 1-7.

17. A method of producing cells expressing a MAPP or a duplex MAPP, the method comprising introducing one or more nucleic acid molecules according to claim 16 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.

18. One or more cells transiently or stably expressing the MAPP or duplex MAPP prepared by the method of claim 17, 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.

19. A method of selectively delivering one or more MOD polypeptides and/or variant MOD polypeptides to a human patient having or suspected of having T1D, the method comprising administering to the patient an effective amount of one or more MAPPs of claims 1-7 and/or one or more duplex MAPPs of claims 8-14.