EA049999B1 - METHODS OF MODULATING THE IMMUNE RESPONSE - Google Patents

Abstract

The invention provides methods for modulating the immune response in an individual. The invention provides methods of treatment. The invention provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The invention relates to methods comprising administering a multimeric polypeptide (synTac) to an individual undergoing treatment with an immune checkpoint inhibitor.

Description

Cross reference

This application claims priority to U.S. Provisional Patent Application No. 62/471,832, filed March 15, 2017, and U.S. Provisional Patent Application No. 62/521,009, filed June 16, 2017, which applications are incorporated herein by reference in their entirety.

Introduction

The adaptive immune response involves the interaction of the T cell receptor (TCR), present on the surface of T cells, with a small peptide antigen noncovalently presented on the surface of the antigen-presenting cell (APC) by the major histocompatibility complex (MHC; also referred to in humans as the human leukocyte antigen (HLA) complex). This interaction represents the targeting mechanism of the immune system and is a necessary molecular interaction for T cell modulation (activation or inhibition) and effector function. Following epitope-specific targeting of target cells, T cells are activated through the interaction of costimulatory proteins found on the APC with T cell costimulatory protein analogues. Both signals - epitope binding to the TCR and interaction of APC costimulatory proteins with T cell costimulatory proteins - are necessary to direct T cell specificity and activation or inhibition. The TCR is specific for a given epitope; However, the costimulatory protein is not epitope specific and is instead typically expressed on all T cells or large subsets of T cells.

The essence of the invention

The invention provides methods for modulating an immune response in an individual. The invention provides methods for treatment. The invention provides methods comprising administering a multimeric polypeptide (synTac) and an immune checkpoint inhibitor to an individual. The invention relates to methods comprising administering a multimeric polypeptide (synTac) to an individual who is being treated with an immune checkpoint inhibitor.

Brief description of graphic materials

Fig. 1A-1D schematically depict various embodiments of a T cell modulating multimeric polypeptide. In these embodiments, disulfide bonds are formed between MHC polypeptides (e.g., HLA) present in individual polypeptides.

Fig. 2A-2Q show the amino acid sequences of wild-type human IL-2 (Fig. 2A); and the amino acid sequences of variant IL-2 polypeptides (Fig. 2B-2Q).

Figures 3A-3C show the amino acid sequences of the IL-2 receptor alpha chain (Figure 3A), beta chain (Figure 3B), and gamma chain (Figure 3C).

Fig. 4A-4C show the amino acid sequences of immunoglobulin Fc polypeptides.

Figures 5A-5C show the amino acid sequences of the human leukocyte antigen (HLA) class I heavy chain polypeptides. Signal sequences are underlined.

Fig. 6 shows a multiple amino acid sequence alignment of beta-2-microglobulin (P2M) precursors (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 95), Pan troglodytes (NP_001009066.1; SEQ ID NO: 195), Macaca mulatta (NP_001040602.1; SEQ ID NO: 96), Bos taurus (NP776318.1; SEQ ID NO: 97), and Mus musculus (NP_033865.2; SEQ ID NO: 98). Amino acids 1-20 represent the signal peptide.

Fig. 7A-7B show the production of IL-2/synTac (Cue-IL-2-a and Cue-IL-2-b) of the present invention after transient transfection. Fig. 7A shows the crude yield, Fig. 7B shows the purified product.

Fig. 8A-8B illustrate the production of IL-2/synTac according to the present invention, in which the IL-2 polypeptide is present in the light chain (a polypeptide chain with a light chain (e.g., P2M) of an MHC class I molecule) or in the heavy chain (a polypeptide chain with a heavy chain of an MHC class I molecule).

Fig. 9 shows the expression level of IL-2/syn-Tac in which IL-2 is wild type (wt) or contains different combinations of F42A, D20K, Q126A, E15A, Y45A and H16A.

Fig. 10 shows the expression of IL-2/synTac according to the present disclosure, wherein IL-2 is present in one copy (1X), two copies (2X), or three copies (3X) in synTac.

Fig. 11 shows in vitro stimulation of antigen-specific CD8+ T cells and non-specific CD8' T cells with IL-2/synTac of the present invention, wherein the IL-2 variant containing the F42A and H16A substitutions is present in synTac in two copies.

Figure 12 shows IL-2/synTac binding to specific (lymphocytic choriomeningitis virus; LCMV) or non-specific (OT1; ovalbumin-recognizing) CD8+ T cells.

Figure 13 shows IL-2/synTac-mediated signaling in antigen-specific (LCMV) or non-specific (BL6) CD8+ T cells.

Fig. 14A-14F show the percentage of phosphosignal transducer and activator of transcription 5 (pSTAT5)-positive cells after stimulation of CD8+ antigen-specific (LCMV) or non-specific (BL6) cells with IL-2/synTac of the present invention at various concentrations of IL-2/synTac.

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Fig. 15 shows the in vivo activity of IL-2/synTac of the present invention. The left panel shows the fold change in the number of antigen-specific CD8+ T cells after administration of phosphate-buffered saline (PBS), recombinant IL-2 (rIL-2), or IL-2/synTac of the present invention. The right panel depicts antigen-specific and non-antigen-specific responses after administration of PBS, rIL-2, or IL-2/synTac of the present invention.

Fig. 16A-16B show the effects of increasing the dose (Fig. 16A) and route of administration (Fig. 16B).

Figures 17A-17B show the effect of IL-2 copy number on in vivo anti-tumor efficacy.

Fig. 18 shows the serum half-life of IL-2/synTac of the present invention following intraperitoneal administration of IL-2/synTac at 10 mg/kg.

Fig. 19 shows the stability of IL-2/synTac of the present disclosure 2 hours after intraperitoneal administration of IL-2/synTac at 10 mg/kg.

Fig. 20 shows size exclusion chromatography data of IL-2/synTac of the present invention after incubation of IL-2/synTac at 4°C or 37°C for 5 days.

Fig. 21 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention with a leader peptide, wherein the IL-2/synTac heavy chain comprises an IgG1 Fc with an N297A substitution.

Fig. 22 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention without the leader peptide, wherein the IL-2/synTac heavy chain comprises an IgG1 Fc with an N297A substitution.

Fig. 23A-23B shows the nucleotide sequence (Fig. 23A) encoding the IL-2/synTac heavy chain depicted in Fig. 21; and a key (Fig. 23B) to the sequence.

Fig. 24 shows the amino acid sequence of the IL-2/synTac heavy chain with the leader peptide, where the IL-2/synTac heavy chain contains IgG1 Fc with L234A and L235A substitutions.

Fig. 25 shows the amino acid sequence of the IL-2/synTac heavy chain without the leader peptide, where the IL-2/synTac heavy chain contains IgG1 Fc with L234A and L235A substitutions.

Fig. 26A-26B shows the nucleotide sequence (Fig. 26A) encoding the IL-2/synTac heavy chain depicted in Fig. 24; and a key (Fig. 26B) to the sequence.

Fig. 27 shows the amino acid sequence of the IL-2/synTac heavy chain with the leader peptide, where the IL-2/synTac heavy chain contains IgG1 Fc with L234F, L235E, and P331S substitutions.

Fig. 28 shows the amino acid sequence of the IL-2/synTac heavy chain without the leader peptide, where the IL-2/synTac heavy chain contains IgG1 Fc with L234F, L235E, and P331S substitutions.

Fig. 29A-29B shows the nucleotide sequence (Fig. 29A) encoding the IL-2/synTac heavy chain depicted in Fig. 27; and a key (Fig. 29B) to the sequence.

Fig. 30 shows the amino acid sequence of the IL-2/synTac light chain with a leader peptide, where the IL-2/synTac light chain contains the E7 epitope of human papillomavirus (HPV).

Fig. 31 shows the amino acid sequence of the IL-2/synTac light chain without the leader peptide, where the IL-2/synTac light chain contains the HPV E7 epitope.

Fig. 32 shows the nucleotide sequence encoding the IL-2/synTac light chain depicted in Fig. 30.

Figures 33A-33D show the amino acid sequences of wild-type human IgG1 Fc (Figure 33A), IgG1 Fc with L234F, L235E, and P331S substitutions (Figure 33B), IgG1 Fc with N297A substitution (Figure 33C), and IgG1 Fc with L234A and L235A substitutions (Figure 33D).

Figures 34A-34C show the amino acid sequence of the P2 microglobulin polypeptide (R12C) (Figure 34A), the IL-2 polypeptide variant (H16A; F42A) (Figure 34B), and the MHC-H class I chain A0201 (Y84A; A236C) (Figure 34C).

Fig. 35 shows the synergistic effect of IL-2/synTac and anti-PD1 antibody on tumor volume reduction.

Fig. 36A-36PP shows the amino acid sequence of 4-1BBL (Fig. 36A) and examples of 4-1BBL polypeptide variants (Fig. 36B-36IIII).

Fig. 37 shows the amino acid sequence of 4-1BB.

Fig. 38A-38B illustrate interferon-gamma (IFN-γ) secretion by target cells exposed to a synTac polypeptide for 3 days (Fig. 38A) or 5 days (Fig. 8B) in accordance with an embodiment of the present disclosure.

Fig. 39A-39B illustrate interleukin-2 (IL-2) secretion by target cells exposed to a synTac polypeptide for 3 days (Fig. 39A) or 5 days (Fig. 9B) in accordance with an embodiment of the present disclosure.

Fig. 40A-40B illustrate interleukin-6 (IL-6) secretion by target cells exposed to a synTac polypeptide for 3 days (Fig. 40A) or 5 days (Fig. 40B) in accordance with an embodiment of the present disclosure.

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Fig. 41A-41B illustrate the secretion of tumor necrosis factor-alpha (TNFa) by target cells contacted with a synTac polypeptide for 3 days (Fig. 41A) or 5 days (Fig. 41B) in accordance with an embodiment of the present disclosure.

Fig. 42A-42B illustrate interleukin-10 (IL-10) secretion by target cells exposed to a synTac polypeptide for 3 days (Fig. 42A) or 5 days (Fig. 42B) in accordance with an embodiment of the present disclosure.

Fig. 43A-43B illustrate the secretion of interleukin-17A (IL-17A) by target cells contacted with a synTac polypeptide for 3 days (Fig. 43A) or 5 days (Fig. 43B) in accordance with an embodiment of the present disclosure.

Fig. 44A-44B illustrate interleukin-4 (IL-4) secretion by target cells exposed to a synTac polypeptide for 3 days (Fig. 44A) or 5 days (Fig. 44B) in accordance with an embodiment of the present disclosure.

Fig. 45 shows the proliferation of target cells contacted with a synTac polypeptide in accordance with an embodiment of the present invention.

Fig. 46 shows the viability of target cells contacted with a synTac polypeptide in accordance with an embodiment of the present invention.

Fig. 47 shows the expression levels of various synTac polypeptides produced in CHO cells.

Fig. 48 shows the in vivo effect of the synTac polypeptide of the present invention on tumor volume.

Fig. 49 shows the effect of co-administration of different doses of 4-1BBL/synTac and anti-PD1 antibody on tumor mass and the percentage of granzyme B ⁺ tumor-infiltrating lymphocytes (TILs).

Fig. 50A-50B show the amino acid sequences of PD-L1 polypeptides.

Fig. 51 shows the amino acid sequence of the CD80 polypeptide.

Fig. 52 shows the amino acid sequence of the ICOS-L polypeptide.

Fig. 53 shows the amino acid sequence of the OX40L polypeptide.

Fig. 54 shows the amino acid sequence of the PD-L2 polypeptide.

Fig. 55 shows the amino acid sequence of the CD86 (B7-2) polypeptide.

Fig. 56 shows the amino acid sequence of the Fas ligand polypeptide (FAS-L).

Definitions

The terms polynucleotide and nucleic acid, as used interchangeably herein, refer to a polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

The terms peptide, polypeptide and protein are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids and polypeptides with modified peptide backbones.

A polynucleotide or polypeptide has a certain percentage of sequence identity with another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids are the same and they appear in the same relative positions when the two sequences are compared. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403–10.

The term conservative amino acid substitution refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, the group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids having amide-containing side chains consists of asparagine and glutamine; the group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains consists of lysine, arginine, and histidine; the group of amino acids having acidic side chains consists of aspartate; and the group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Typical groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

As used herein, the term "binding" (e.g., in relation to the binding of a T cell modulating multimeric polypeptide to a polypeptide (e.g., a T cell receptor)

- 3 049999 cells) on a T cell) refers to the non-covalent interaction between them. Binding interactions are typically characterized by a dissociation constant (KD) 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. Affinity refers to the strength of binding, with increased binding affinity correlating with a lower _KD .

As used herein, the term immunological synapse or immune synapse generally refers to a natural interface between two interacting immune cells of the adaptive immune response, including, for example, the interface between an antigen-presenting cell (APC) or target cell and an effector cell, such as a lymphocyte, effector T cell, natural killer cell, and the like. The immunological synapse between an APC and a T cell is typically initiated by the interaction of a T cell antigen recognition receptor and major histocompatibility complex molecules, such as described in Bromley et al., Annu Rev Immunol. 2001;19:375-96; the contents of which are hereby incorporated by reference in their entirety.

T cell includes all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Treg), and NK-T cells.

A costimulatory polypeptide (also referred to herein as an immunomodulatory polypeptide) as the term is used herein includes a polypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.) that specifically binds a recognized costimulatory polypeptide (also referred to herein as a recognized immunomodulatory polypeptide) on a T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, binding of a TCR/CD3 complex to a major histocompatibility complex (MHC) polypeptide loaded with the peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The costimulatory ligand may 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, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4, HVEM, an agonist, or an antibody that binds a toll-like receptor to a ligand that specifically binds to B7-H3. A costimulatory ligand also includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

The modulating domain (MOD) of the T cell modulating multimeric polypeptide includes a costimulatory polypeptide, for example an IL-2 polypeptide, such as a variant IL-2 polypeptide.

In the context of this document, heterologous means a nucleotide or polypeptide that is not found in a naturally occurring nucleic acid or naturally occurring protein, respectively.

As used herein, the term recombinant 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 distinct 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 produce a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained within 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 one insert. Recombinant expression vectors are typically generated for the purpose of expressing and/or propagating the insert(s) or for constructing other recombinant nucleotide sequences. Inserts may or may not be operably linked to a promoter sequence and may or may not be operably linked to regulatory DNA sequences.

The terms antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. In addition, the term encompasses Fab', Fv, F(ab') ₂ -, and other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. As used herein, a monoclonal antibody

- 4 049999 is an antibody produced by a group of identical cells that are produced in a single cell by repeated cellular replication. In other words, a clone of cells produces only one type of antibody. While a monoclonal antibody can be produced using hybridoma technology, other production methods known to those skilled in the art (e.g., antibodies obtained from antibody phage display libraries) can also be used. The antibody can be monovalent or divalent. The antibody can be an immunoglobulin (Ig) monomer, which is a Y-shaped molecule consisting of four polypeptide chains: two heavy chains and two light chains, linked by disulfide bonds.

As used herein, the term "humanized antibody" refers to an antibody comprising portions of antibodies of different origins, wherein at least one portion comprises amino acid sequences of human origin. For example, a humanized antibody may comprise portions derived from a non-human immunoglobulin with the desired specificity, such as a mouse, and derived from human immunoglobulin sequences (e.g., a chimeric immunoglobulin), chemically linked together using conventional techniques (e.g., synthetically) or produced as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding portions of the chimeric antibody protein can be expressed to produce a contiguous polypeptide chain). Another example of a humanized antibody is an antibody comprising one or more antibody chains that comprise a CDR derived from an antibody of non-human origin and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed within the term humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1. See also Ladner et al., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786; and Bird, R. E. et al., Science, 242: 423–426 (1988) for single-chain antibodies.

For example, humanized antibodies can be produced using synthetic and/or recombinant nucleic acids to produce genes (e.g., DNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences targeting humanized variable regions can be engineered using PCR mutagenesis techniques to alter DNA sequences encoding the human or humanized chain, e.g., a pre-humanized variable region template DNA (see Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471–2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297–302 (1991)). Variants can also be readily generated using these or other suitable methods. For example, cloned variable regions can be mutagenized and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see Krebber et al., U.S. Patent No. 5,514,548; Hoogenboom et al., WO 93/06213, published April 1, 1993)).

Antibody fragments are portions of an intact antibody, such as the antigen-binding region or the variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057–1062 (1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site and a residual Fc fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen-binding active sites and is still capable of cross-linking with antigen.

Fv is the smallest antibody fragment that contains a complete antigen recognition site and an antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight noncovalent association. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the V _H -V _L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv, containing only the three CDRs specific for antigen) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH ₁ ) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of several residues at the carboxyl terminus of the CH ₁ domain of the heavy chain, including one or more cysteines from the hinge

- 5 049999 region of the antibody. In this document, Fab'-SH is the designation for a Fab' fragment in which the cysteine residues of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other methods for chemically linking antibody fragments are also known.

The light chains of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further divided into types, such as IgG2a and IgG2b.

Single chain Fv, or sFv, or scFv antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term diabodies refers to small antibody fragments with two antigen-binding sites, the fragments of which comprise a heavy-chain variable domain (V _H ) linked to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L ). By using a linker that is too short to allow the association of the two domains in one chain, the domains are forced to associate with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described in more detail, for example, in European Patent 404,097; WO 93/11161 and in Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

In the context of this document, the term affinity refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as the dissociation constant (K _D ). The 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 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater or more greater than the affinity of the antibody for unrelated amino acid sequences. The affinity of an antibody for a target protein can, for example, be 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 femtomole (fM), or more. As used herein, the term avidity refers to the resistance of a complex of two or more agents to dissociation upon dilution. As used herein, the terms immunoreactive and preferentially bind are used interchangeably with respect to antibodies and/or antigen-binding fragments.

The term binding refers to the direct association of two molecules due to, for example, covalent, electrostatic, hydrophobic, ionic, and/or hydrogen bonding interactions, including such interactions as salt and water bridges. The term specific binding refers to binding with an affinity of at least 10 ^-7 M or more, such as 5 x 10 ^-7 M, 10 ^-8 M, 5 x 10 ^-8 M, and more. The term non-specific binding refers to binding with an affinity of less than about 10 ^-7 M, such as binding with an affinity of 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, etc.

As used herein, the term CDR or complementarity determining region is intended to refer to non-contiguous antigen-binding active sites that are located within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, Sequences of proteins of immunological interest (1991) (also referred to herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared to one another. However, the use of any of the definitions to refer to CDRs of an antibody or grafted antibody or variants thereof is encompassed within the scope of the term as defined and used herein. The amino acid residues that comprise the CDRs, as defined in the references cited above, are presented in the table below for comparison. The CDRs listed in Table 2 have been defined according to Kabat 1991.

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CDR Definitions

Kabat ¹ Chothia ² MacCallum ³ Vh CDR-1 31-35 26-32 30-35 VhCDR-2 50-65 53-55 47-58 Vh CDR-3 95-102 96-101 93-101 Vl CDR-1 24-34 26-32 30-36 Vl CDR-2 50-56 50-52 46-55 Vl CDR-3 89-97 91-96 89-96

¹ Residue numbering according to the nomenclature of Kabat et al. above ² Residue numbering according to the nomenclature of Chothia et al. above ³ Residue numbering according to the nomenclature of MacCallum et al. above

As used herein, the terms CDR-L1, CDR-L2 and CDR-L3 refer to the first, second and third CDRs of the variable region of the light chain, respectively. As used herein, the terms CDR-H1, CDR-H2 and CDR-H3 refer to the first, second and third CDRs of the variable region of the heavy chain, respectively. As used herein, the terms CDR1, CDR-2 and CDR-3 refer to the first, second and third CDRs of the variable region of either chain, respectively.

As used herein, the term framework, when used in relation to the variable region of an antibody, is intended to refer to all amino acid residues outside the CDR regions within the variable region of the antibody. The framework of a variable region is typically a discontinuous amino acid sequence of approximately 100-120 amino acids in length, but is intended to refer only to those amino acids that are outside the CDRs. As used herein, the term framework region is intended to refer to each domain of the framework that is separated by a CDR.

The terms treatment, cure and the like are used herein to denote the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of total or partial prevention of a disease or its symptoms and/or may be therapeutic in terms of partial or total cure of a disease and/or an adverse event associated with the disease. As used herein, the term treatment encompasses any treatment of a disease or symptom in a mammal, and includes: (a) preventing the onset of a disease or symptom in a subject that may be predisposed to developing the disease or symptom but in whom the disease or symptom has not yet been diagnosed; (b) inhibiting the disease or symptom, i.e. stopping its development; or (c) alleviating the disease, i.e. causing the disease to regress. The therapeutic agent may be administered before, during or after the onset of a disease or injury. Treatment of an ongoing disease, in which the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably carried out before complete loss of function in the affected tissues. It is desirable that the therapy be administered to the subject 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 which diagnosis, treatment, or therapy is sought. Mammals include, for example, humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), etc.

Before further describing the present invention, it should be understood that the invention is not limited to the specific embodiments described, as they may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not limiting, since the scope of the present invention is limited only by the appended claims.

When a range of values is given, it is to be understood that the invention covers each intermediate value, to the accuracy of the tenth decimal place of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of the range, as well as any other stated or intermediate value in such stated range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the invention, subject to any specifically excluded limit in the stated range. If a stated range includes one or both limits, ranges excluding one or both of these included limits are also included in the invention.

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 invention pertains. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention,

- 7 049999 of the present invention, preferred methods and materials will now be described. All publications mentioned herein are herein incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which such publications are cited.

It should be noted that the use of the singular in this document and in the appended claims includes reference to the plural unless the context clearly dictates otherwise. Thus, for example, a reference to a variant of an IL-2 polypeptide includes a plurality of such polypeptides, and a reference to an HLA class I heavy chain polypeptide includes reference to one or more HLA class I heavy chain polypeptides and equivalents thereof known to those skilled in the art, etc. It should also be noted that the claims may be amended to exclude any optional elements. Accordingly, this statement should serve as a preliminary basis for the use of such inclusive terminology as exclusively, only, etc. in connection with the indication of elements of the claims, or the use of a negative feature.

It should be understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be presented in combination as a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be presented separately or in any suitable subcombination. All combinations of embodiments that are within the scope of the invention are expressly included in the invention and are described herein in the same way as if each and every combination were individually and expressly described. In addition, all subcombinations of the various embodiments and their elements are also expressly included in the invention and are described herein in the same way as if each and every subcombination were individually and expressly described.

The publications discussed herein are presented solely with respect to their contents prior to the filing date of this application. Nothing herein should be interpreted as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Moreover, the dates of the publications presented may differ from the actual publication dates, which requires independent confirmation.

Detailed description of the essence of the invention

The present invention relates to methods of treatment comprising administering to an individual in need thereof a multimeric polypeptide that modulates T cells (synTac multimeric polypeptide) and at least one additional therapeutic substance. In some embodiments, the at least one additional therapeutic substance is an immune checkpoint inhibitor. In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The present invention relates to methods comprising administering a multimeric polypeptide (synTac) to an individual who is being treated with an immune checkpoint inhibitor.

The T cell modulating multimeric polypeptide is also referred to herein as a synTac polypeptide or a synTac multimeric polypeptide or simply synTac. The synTac polypeptide comprises a modulatory domain. In some cases, the modulatory domain comprises a wild-type amino acid sequence, such as an amino acid sequence found in a naturally occurring modulatory polypeptide. In some cases, the modulatory domain is a variant modulatory domain, wherein the variant modulatory domain exhibits reduced binding affinity for the immunomodulatory polypeptide as compared to the affinity of the wild-type modulatory domain for the immunomodulatory polypeptide. The synTac polypeptide can modulate the activity of a target T cell. A synTac polypeptide comprising a variant modulatory domain provides increased target cell specificity.

In some cases, the method of treatment of the present invention comprises administering to an individual in need thereof synTac and an immune checkpoint inhibitor. In some cases, synTac and the immune checkpoint inhibitor provide a synergistic effect compared to the effect of synTac when administered alone (in monotherapy) or the immune checkpoint inhibitor when administered alone (in monotherapy).

The combination of synTac and an immune checkpoint inhibitor is, in some cases, more effective than the additive effects of synTac administered as monotherapy or the immune checkpoint inhibitor administered as monotherapy. For example, in some cases, the synergistic effect of synTac and an immune checkpoint inhibitor allows for lower doses of synTac or the immune checkpoint inhibitor to be used and/or for synTac or the immune checkpoint inhibitor to be administered less frequently to an individual in need thereof. The ability to use lower doses of the therapeutic agents (synTac or immune checkpoint inhibitor) and/or to administer such agents less frequently may reduce toxicity or other adverse effects.

- 8,049,999 effects that can be associated with the administration of a therapeutic agent in monotherapy, without reducing the effectiveness of the therapeutic agent in the treatment. In addition, the synergistic effect of synTac and an immune checkpoint inhibitor may result in an enhanced clinical benefit compared to the clinical benefit obtained with synTac monotherapy or immune checkpoint inhibitor monotherapy. Examples of clinical benefit include, for example, a decrease in tumor mass in an individual; a decrease in the number of cancer cells in an individual; an increased survival time in an individual; an increased time of remission; and the like. Finally, the synergistic effect of synTac and an immune checkpoint inhibitor may reduce the adverse or undesirable side effects associated with synTac monotherapy or immune checkpoint inhibitor monotherapy.

Immune checkpoint inhibitors

Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is a checkpoint inhibitory molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

In some cases, the immune checkpoint inhibitor is an antibody specific to an immune checkpoint. In some cases, the immune checkpoint antibody is a monoclonal antibody. In some cases, the immune checkpoint antibody is humanized or deimmunized such that the antibody does not substantially elicit an immune response in a human. In some cases, the antibody is a humanized monoclonal antibody. In some cases, the immune checkpoint antibody is a deimmunized monoclonal antibody. In some cases, the immune checkpoint antibody is a fully human monoclonal antibody. In some cases, the immune checkpoint antibody inhibits the binding of an immune checkpoint polypeptide to an immune checkpoint polypeptide ligand. In some cases, the immune checkpoint antibody inhibits the binding of an immune checkpoint polypeptide to an immune checkpoint polypeptide receptor.

Antibodies, such as monoclonal antibodies, that are specific for immune checkpoints and that act as immune checkpoint inhibitors are known in the art. See, for example, Wurz et al. (2016) Ther. Adv. Med. Oncol. 8:4; and Naidoo et al. (2015) Ann. Oncol. 26:2375.

Suitable immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI-6469 (MedImmune/AZ), CP870,893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035 and combinations thereof.

Suitable antibodies to LAG3 include, for example, BMS-986016 and LAG525. Suitable antibodies to GITR include, for example, TRX518, MK-4166, INCAGN01876 and MK-1248. Suitable antibodies to OX40 include, for example, MEDI0562, INCAGN01949, GSK2831781, GSK-3174998, MOXR-0916, PF04518600 and LAG525. Suitable antibodies to VISTA are provided, for example, in WO 2015/097536.

A suitable dosage of the anti-immune control antibody is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, including from about 50 mg/kg to about 1200 mg/kg per day. Other typical dosages of such agents include approximately 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg and 2300 mg/kg per day. An effective dose of the antibody may be administered as two, three, four, five, six or more divided doses administered separately at appropriate intervals throughout the day.

Antibodies to PD-1

In some cases, the immune checkpoint inhibitor is an antibody to PD-1.

- 9 049999

Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224. In some cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001. Suitable anti-PD1 antibodies are described in U.S. Patent Publication No. 2017/0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18.

In some cases, the PD1 antibody is pembrolizumab. The amino acid sequence of the heavy chain of pembrolizumab is:

OVOLVOSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGOGLEWMGGI

NPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLOFDDTAVYYCARRDYRFDMGFDY WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGL YSLS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ Ш NO:51).

The amino acid sequence of the heavy chain variable region (VH) is underlined.

The amino acid sequence of the light chain of pembrolizumab is:

EIVLTOSPATLLSPGERATLSCRASKGVSTSGYSYLHWYOOKPGOAPRLLIYLAS YLESGVPARF SSGSGSGTDFTLTIS SLEPEDF AVYYCQHSRDLPLTFGGGTKVEIKRT VAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:52).

The amino acid sequence of the light chain variable region (VL) is underlined.

In some cases, the anti-PD-1 antibody contains the VH and VL regions of pembrolizumab. In some cases, the anti-PD-1 antibody contains the CDRs of the heavy chain and light chain of pembrolizumab.

In some cases, the anti-PD-1 antibody is nivolumab (also known as MDX1106 or BMS-936558; see, e.g., Topalian et al. (2012) N. Eng. J. Med. 366:2443–2454; and U.S. Patent No. 8,008,449). The amino acid sequence of the nivolumab heavy chain is:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIW

YDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:53).

The amino acid sequence of the light chain of nivolumab is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA TGIPARFSGSGSGTDFTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:54).

In some cases, the anti-PD-1 antibody contains the CDRs of the heavy chain and light chain of nivolumab. Anti-CTLA4 antibodies

In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol. 31:616–22.

In some cases, the anti-CTLA-4 antibody is ipilimumab. The amino acid sequence of the ipilimumab heavy chain is:

- 10 049999

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVTFISY DGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:55). The amino acid sequence of the VH region is underlined.

The amino acid sequence of the ipilimumab light chain is:

EIVLTOSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGOAPRLLIYGAFSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOOYGSSPWTFGOGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:56). The amino acid sequence of the VL region is underlined.

In some cases, the anti-CTLA4 antibody contains the VH and VL regions of ipilimumab. In some cases, the anti-CTLA4 antibody contains the CDRs of the heavy chain and light chain of ipilimumab.

Antibodies to PD-L1

In some cases, the immune checkpoint inhibitor is an anti-PD-L1 monoclonal antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some cases, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO 2011/066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149.

In some cases, the PD-L1 antibody is atezolizumab. The amino acid sequence of the heavy chain of atezolizumab is:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPY GGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57).

The amino acid sequence of the light chain of atezolizumab is:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:58).

In some embodiments, the anti-PD-L1 antibody comprises the CDRs of the heavy chain and light chain of atezolizumab.

In some cases, the anti-PDL1 antibody is KN035, a fully humanized single-domain anti-PD-L1 antibody fused to a human IgG1 Fc polypeptide. Zhang et al. (2017) Cell Discov. 3:17004; and WO 2017/020801. The single-domain portion of the KN035 antibody may comprise the amino acid sequence:

OVOLQESGGGLVOPGGSLRLSCAASGKMSSRRCMAWFROAPGKERERVAKLLTTSGST

YLADSVKGRFTISONNAKSTVYLOMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFO

YWGQGTQVTVS (SEQ ID NO:216), where the underlined amino acids represent CDR1, CDR2 and CDR3.

T cell modulating multimeric polypeptides (synTac)

Multimeric (e.g., heterodimeric, heterotrimeric) polypeptides suitable for use in the method of the present invention are described below. The multimeric polypeptides are

- 11 049999 are T cell modulating polypeptides and are also referred to herein as T cell modulating multimeric polypeptides or synTac (from immunological synapse for T cell activation).

A T cell modulating multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a first major histocompatibility complex (MHC) polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory (MOD) domains, wherein the one or more immunomodulatory domains are: A) at the C-terminus of the first polypeptide; B) at the N-terminus of the second polypeptide; C) at the C-terminus of the second polypeptide; or D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide. In some cases, the T cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an IgFc polypeptide. In some cases, the T cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) an immunomodulatory domain; iii) a second MHC polypeptide; and ii) an IgFc polypeptide. In some cases, the T cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory (MOD) domain. In some cases, the T cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an immunomodulatory domain. In some cases, the T cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, from N-terminal to C-terminal direction: i) an immunomodulatory domain; and ii) a second MHC polypeptide. In some cases, the T-cell multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide.

In some cases, the multimeric polypeptide contains a non-Ig scaffold. For example, in some cases, the non-Ig scaffold is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

In some cases, the first MHC polypeptide is a β2microglobulin polypeptide (β2M); and the second MHC polypeptide is a class I MHC heavy chain polypeptide. A suitable β2-M polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the P2M polypeptide depicted in Fig. 6. In some cases, the class I MHC heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in one of Figs. 5A-5C. In some cases, the first MHC polypeptide is an MHC class II alpha chain polypeptide; and the second MHC polypeptide is an MHC class II beta chain polypeptide.

The epitope present in the multimeric polypeptide may be a T-cell epitope.

In some cases, the multimeric polypeptide comprises an IgFc polypeptide. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the IgFc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in Fig. 4A-4C.

The first polypeptide and the second polypeptide of the multimeric polypeptide may be non-covalently linked. The first polypeptide and the second polypeptide of the multimeric polypeptide may be covalently linked. The first polypeptide and the second polypeptide of the multimeric polypeptide may be covalently linked, wherein the covalent linkage is via a disulfide bond. In some cases,

- 12 049999 the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to produce a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to produce a second Cys residue, and wherein the disulfide bond is between the first and second Cys residues.

The multimeric polypeptide may include a linker between one or more of: an epitope and a first MHC polypeptide; two copies of an immunomodulatory (MOD) polypeptide; an immunomodulatory polypeptide and a second MHC polypeptide; and a second MHC polypeptide and an Ig Fc polypeptide.

Immunomodulatory polypeptides suitable for inclusion in a T cell multimeric polypeptide include, but are not limited to, 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, IL-2 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide.

A multimeric polypeptide may include 2 or more immunomodulatory polypeptides. A multimeric polypeptide may include 2 immunomodulatory polypeptides. In some cases, the 2 immunomodulatory polypeptides are in tandem. A multimeric polypeptide may include 3 immunomodulatory polypeptides. In some cases, the 3 immunomodulatory polypeptides are in tandem.

The multimeric polypeptide may comprise a third polypeptide, wherein the third polypeptide comprises an immunomodulatory polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the immunomodulatory polypeptide of the first polypeptide or the second polypeptide. In some cases, the third polypeptide is covalently linked to the first polypeptide.

Examples of suitable multimeric polypeptides are described in WO 2017/151940; WO 2017/201210; and PCT/US2017/067663. The disclosures of WO 2017/151940, WO 2017/201210, and PCT/US2017/067663 are incorporated herein by reference.

MHC polypeptides

As noted above, the multimeric polypeptide of the present invention includes MHC polypeptides. For the purposes of the present invention, the term major histocompatibility complex (MHC) polypeptides includes MHC polypeptides of various species, including human MHC polypeptides (also referred to as human leukocyte antigens (HLA)), rodent MHC polypeptides (e.g., mouse, rat, etc.), and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., horses, bovines, sheep, goats, etc.), etc.). The term MHC polypeptide is intended to include MHC class I polypeptides (e.g., β-2-microglobulin and MHC class I heavy chain) and MHC class II polypeptides (e.g., MHC class II α-polypeptide and MHC class II β-polypeptide).

As noted above, in some embodiments of the multimeric polypeptide of the present invention, the first and second MHC polypeptides are MHC class I polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class I β2microglobulin (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain). In other cases, the first and second MHC polypeptides are MHC class II polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide.

In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is a human MHC polypeptide, wherein human MHC polypeptides are also referred to as human leukocyte antigen (HLA) polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is an HLA class I polypeptide, such as a β2-microglobulin polypeptide or an HLA class I heavy chain polypeptide. HLA class I heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides and HLA-G heavy chain polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is an HLA class II polypeptide, such as an HLA class II α chain or an HLA class II β chain. Class II MHC polypeptides include class II MHC α and β DP polypeptides, α and β DM polypeptides, α and β DOA polypeptides, α and β DOB polypeptides, α and β DQ polypeptides, and α and β DR polypeptides.

In some cases, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in one of Figs. 5A-5C.

- 13 049999

HLA-A

As an example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:59). HLA-A (Y84A; A236C)

In some cases, the MHC class I heavy chain polypeptide contains Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain (Y84A; A236C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRG YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAA QTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSD HEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP WITH

GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 50), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant containing the R12C substitution.

HLA-A (Y84C; A139C)

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence (Y84C; A139C):

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 196), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-A11 (HLA-A11)

As one non-limiting example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence of the human HLA-A A11 heavy chain (also referred to as HLA-A11):

- 14 049999

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 197).

Such MHC class I heavy chain may be seen in Asian populations, including populations of people of Asian descent.

HLA-A A11 (Y84A; A236C)

As one non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-A A11 allele that comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A A11 heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGAYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 198), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant containing the R12C substitution.

HLA-B

As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of a human HLA-B heavy chain:

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY

WDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY

AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE

NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV

ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 199).

HLA-B (Y84A; A236C)

As one non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-B polypeptide that comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-B heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY WDRNTQIYKAQAQTDRESLRNLRGAYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 200), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant containing the R12C substitution.

HLA-B (Y84C; A139C)

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%,

- 15 049999 at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity with the following amino acid sequence of the human HLA-B heavy chain (Y84C; A139C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY WDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTCAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:201), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139. HLA-C

As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of a human HLA-C heavy chain:

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQNYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 202). HLA-C (Y84A; A236C)

As one non-limiting example, in some cases the MHC class I heavy chain polypeptide is an HLA-C polypeptide that contains the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain (Y84A; A236C) amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQNYKRQAQADRVSLRNLRGAYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:203), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant containing the R12C substitution.

HLA-C (Y84C; A139C)

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain amino acid sequence (Y84C; A139C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTCAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:204), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

In some cases, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%,

- 16 049999 at least 99% or 100% identity of the amino acid sequence with the amino acid sequence depicted in one of Figs. 3A-3C.

As an example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25365 of the human HLA-A heavy chain polypeptide amino acid sequence depicted in Fig. 3A.

As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide can comprise an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-362 of the human HLA-B heavy chain polypeptide amino acid sequence depicted in Fig. 3B.

As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-362 of the amino acid sequence of the human HLA-C heavy chain polypeptide depicted in Fig. 3C.

As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQ

EGPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLR

GYQQFAYDGRDYIALNEDLKTWTAADTAALITRRKWEQAGDAEYYRAYLEGECVEWL

RRYLELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQ

DMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRW (SEQ ID NO:60).

The b2-microglobulin (b2M) polypeptide of the multimeric polypeptide may be a human b2M polypeptide, a non-human primate b2M polypeptide, a mouse b2M polypeptide, and the like. In some cases, the b2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the b2M amino acid sequence depicted in Fig. 6. In some cases, the b2M polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 21 through 119 of the b2M amino acid sequence depicted in Fig. 6.

In some cases, the MHC polypeptide comprises a single amino acid substitution relative to a reference MHC polypeptide (wherein the reference MHC polypeptide may be a wild-type MHC polypeptide), wherein the single amino acid substitution replaces the amino acid with a cysteine (Cys) residue. Such cysteine residues, when present in the MHC polypeptide of the first polypeptide of the multimeric polypeptide of the present invention, can form a disulfide bond with a cysteine residue present in the second polypeptide chain of the multimeric polypeptide of the present invention.

In some cases, the first MHC polypeptide in the first polypeptide of the multimeric polypeptide and/or the second MHC polypeptide in the second polypeptide of the multimeric polypeptide comprises an amino acid substitution for replacing an amino acid with cysteine, wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a cysteine in the second MHC polypeptide, wherein the cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide, or wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide.

For example, in some cases, one of the following pairs of residues in HLA β2 microglobulin and HLA class I heavy chain is replaced by cysteines (where the residue numbers correspond to those of the mature polypeptide): 1) residue β2M 12, HLA class I heavy chain residue 236; 2) residue β2M 12, HLA class I heavy chain residue 237; 3) residue β2M 8, HLA class I heavy chain residue 234; 4) residue

- 17 049999 β2M 10, HLA class I heavy chain residue 235; 5) β2M 24, HLA class I heavy chain residue 236; 6) b2M 28, HLA class I heavy chain residue 232; 7) b2M 98, HLA class I heavy chain residue 192; 8) b2M 99, HLA class I heavy chain residue 234; 9) b2M 3, HLA class I heavy chain residue 120; 10) b2M 31, HLA class I heavy chain residue 96; 11) b2M 53, HLA class I heavy chain residue 35; 12) b2M 60, HLA class I heavy chain residue 96; 13) b2M 60, HLA class I heavy chain residue 122; 14) B2M residue 63, HLA class I heavy chain residue 27; 15) B2M residue Arg3, HLA class I heavy chain residue Gly120; 16) B2M residue His31, HLA class I heavy chain residue Gln96; 17) B2M residue Asp53, HLA class I heavy chain residue Arg35; 18) B2M residue Trp60, HLA class I heavy chain residue Gln96; 19) B2M residue Trp60, HLA class I heavy chain residue Asp122; 20) B2M residue Tyr63, HLA class I heavy chain residue Tyr27; 21) B2M residue Lys6, HLA class I heavy chain residue Glu232; 22) B2M residue Gln8, HLA class I heavy chain residue Arg234; 23) B2M residue Tyr10, HLA class I heavy chain residue Pro235; 24) B2M residue Ser11, HLA class I heavy chain residue Gln242; 25) B2M residue Asn24, HLA class I heavy chain residue Ala236; 26) B2M residue Ser28, HLA class I heavy chain residue Glu232; 27) B2M residue Asp98, HLA class I heavy chain residue His192; and 28) B2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC/HLA class I heavy chain refers to the mature MHC/HLA class I heavy chain without the signal peptide. For example, in the amino acid sequence depicted in Fig. 5A, which includes the signal peptide, Gly120 is Gly144; Gln96 is Gln120; etc. In some cases, the B2M polypeptide contains an R12C substitution and the HLA class I heavy chain contains an A236C substitution; in such cases, a disulfide bond is formed between Cys-12 of the B2M polypeptide and Cys-236 of the HLA class I heavy chain. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence (i.e., residue 260 of the amino acid sequence depicted in Fig. 5A) is substituted with Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence (i.e., residue 260 of the amino acid sequence depicted in Fig. 5B) is substituted with Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence (i.e., residue 260 of the amino acid sequence depicted in Fig. 5C) is substituted with Cys. In some cases, residue 32 (corresponding to Arg-12 of mature B2M) of the amino acid sequence shown in Fig. 6 is substituted with Cys.

In some cases, the B2M polypeptide contains the amino acid sequence: IQRTPKIQVY SRHPAENGKS NFLNCYVSGF

HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:61).

In some cases, the B2M polypeptide contains the amino acid sequence: IQRTPKIQVY SCHPAENGKS

NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF

TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE

GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ IDNO:59).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE

GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ Ш NO:62).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

- 18 049999

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:50).

In some cases, the β2Μ polypeptide contains the following amino acid sequence: IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE

HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48);

and the HLA class I heavy chain polypeptide of the multimeric polypeptide of the present invention comprises the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE

GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE

WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ

Ш NO:62), where the Cys residues, which are underlined and in bold, form a disulfide bond with each other in the multimeric polypeptide.

In some cases, the B2M polypeptide contains the amino acid sequence:

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW

SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:48).

Framework polypeptides

The T cell modulating multimeric polypeptide comprises an Fc polypeptide or other suitable scaffold polypeptide.

Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffold polypeptides. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptide, transferrin, Fc receptor polypeptide, elastin-like polypeptide (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215; for example, a pentapeptide repeating unit containing polypeptide (Val-Pro-Gly-X-Gly; SEQ ID NO: 212), where X is any amino acid other than proline), albumin-binding polypeptide, silk-like polypeptide (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), and silk-elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075) and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

Suitable scaffold polypeptides are, in some cases, half-life enhancing polypeptides. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a multimeric polypeptide compared to a control multimeric polypeptide lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide, compared to a control multimeric polypeptide that lacks the scaffold polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, 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. For example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide, compared to a control multimeric polypeptide that lacks the Fc polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, 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.

- 19 049999

Fc polypeptides

In some cases, the first and/or second polypeptide chain of the multimeric polypeptide comprises an Fc polypeptide. The Fc polypeptide of the multimeric polypeptide can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence of the Fc region depicted in Fig. 4A-C. In some cases, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide depicted in Fig. 4A. In some cases, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide depicted in Fig. 4A; and comprises an N77 substitution; for example, the Fc polypeptide comprises an N77A substitution. In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG2 Fc polypeptide depicted in Fig. 4A; for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide depicted in Fig. 4A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, with at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide depicted in Fig. 4A; for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide depicted in Fig. 4A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, with at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide depicted in Fig. 4B; for example, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in Fig. 4B. In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, with at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in Fig. 4C; for example, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in Fig. 4C.

In some cases, the Fc polypeptide present in the multimeric polypeptide comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc).

- 20 049999 comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for the substitution of N297 with an amino acid other than asparagine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33C (human IgG1 Fc comprising the substitution N297A). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for the substitution of L234 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for the substitution of L235 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33D (human IgG1 Fc comprising a L234A substitution and a L235A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for a substitution of P331 with an amino acid other than proline; in some cases, the substitution is P331S. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for substitutions at L234 and L235 with amino acids other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33A (human IgG1 Fc), except for substitutions at L234 and L235 with amino acids other than leucine and a substitution of P331 with an amino acid other than proline. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence depicted in Fig. 33B (human IgG1 Fc comprising substitutions L234F, L235E, and P331S). In some cases, the Fc polypeptide present in the multimeric polypeptide is an IgG1 Fc polypeptide that comprises substitutions L234A and L235A.

Linkers

The multimeric polypeptide may include linker peptides located, for example, between an epitope and an MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an IgFc polypeptide; between a first immunomodulatory polypeptide and a second immunomodulatory polypeptide; or between a second immunomodulatory polypeptide and a third immunomodulatory polypeptide.

For example, the multimeric polypeptide can include linker peptides located, for example, between an epitope and an MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an IgFc polypeptide; between a first IL-2 polypeptide variant and a second IL-2 polypeptide variant; or between a second IL-2 polypeptide variant and a third IL-2 polypeptide variant. As another example, the multimeric polypeptide can include linker peptides located, for example, between an epitope and an MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an IgFc polypeptide; between a first 4-1BBL polypeptide variant and a second 4-1BBL polypeptide variant; or between a second 4-1BBL polypeptide variant and a third 4-1BBL polypeptide variant.

Suitable linkers (also called spacers) can be easily selected and can be any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids. 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, or 25 amino acids in length.

Examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS) _n , (GSGGS) _n (SEQ ID NO: 210), and (GGGS) _n (SeQ ID NO: 211), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers can be used, Gly and Ser are both relatively unstructured, so they can serve as a neutral connection between the components. Glycine polymers can be used, glycine gaining significantly more phi-psi space than even alanine, and is significantly less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).

Examples of linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 65), GGSGG (SEQ ID NO: 66), GSGSG (SEQ ID NO: 67), GSGGG (SEQ ID NO: 68), GGGSG (SEQ ID NO: 69), GSSSG (SEQ ID NO: 70), and the like. Examples of linkers may include, for example, Gly (Ser ₄ ) n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the amino acid sequence (GSSSS) n (SEQ ID NO: 71), where n is 4. In some cases, the linker comprises the amino acid sequence (GSSSS) n (SEQ ID NO: 72), where n is 5.

- 21 049999

In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 205), wherein n is 1. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 206), wherein n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 207), wherein n is 3. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 208), wherein n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 209), wherein n is 5. In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 73).

In some cases, a linker polypeptide present in a first polypeptide of a multimeric polypeptide of the present invention includes a cysteine residue that can form a disulfide bond with a cysteine residue present in a second polypeptide of a multimeric polypeptide of the present invention. In some cases, for example, a suitable linker comprises the amino acid sequence GCGASGGGGSGGGGS (SEQ ID NO.74).

Epitopes

An epitope (a peptide presenting one or more epitopes) present in a multimeric polypeptide of the present invention may be from about 4 amino acids to about 25 amino acids in length, for example, an epitope may be from 4 amino acids (aa) to 10 aa, from 10 to 15 aa, from 15 to 20 aa, or from 20 to 25 aa in length. For example, an epitope present in a multimeric polypeptide of the present invention may be 4 amino acids (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 length. In some cases, an epitope present in a multimeric polypeptide of the present invention is from 5 amino acids to 10 amino acids in length, such as 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

An epitope present in a multimeric polypeptide of the present invention is specifically bound to a T cell, i.e., the epitope is specifically bound to an epitope-specific T cell. The epitope-specific T cell binds an epitope having a reference amino acid sequence, but does not substantially bind an epitope that differs from the reference amino acid sequence. For example, an epitope-specific T cell binds an epitope having a reference amino acid sequence and binds an epitope that differs from the reference amino acid sequence, if at all, with an affinity of less than 10-6 M, less than ^10-5 M, or less than ^10-4 M. An epitope-specific T cell can bind the 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.

Suitable epitopes include, but are not limited to, epitopes present in a cancer-associated antigen. Cancer-associated antigens include, but are not limited to, alpha-folate receptor; carbonic anhydrase IX (CAIX); CD19; CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor; ganglioside antigen GD2; HER2/Neu; IL-13R-a2; kappa light chain; LeY; cell adhesion molecule L1; melanoma-associated antigen (MAGE); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); tumor-associated glycoprotein-72 (TAG-72); and vascular endothelial growth factor receptor-2 (VEGF-R2). See, e.g., Vigneron et al. (2013) Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int'l Article ID 948501. In some cases, the epitope is an epitope of the E7 antigen of human papillomavirus; see, e.g., Ramos et al. (2013) J. Immunother. 36:66.

In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO: 75). In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO: 76). In some cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO: 77). In some cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO: 78). See, e.g., Ressing et al. ((1995) J. Immunol. 154:5934) for additional relevant HPV epitopes.

Immunomodulatory polypeptides

Suitable immunomodulatory polypeptides include, but are not limited to, IL-2 polypeptide, 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the PD-L1 polypeptide depicted in Fig. 50A or Fig. 50B.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the CD80 polypeptide depicted in Fig. 51.

- 22 049999

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the ICOS-L polypeptide depicted in Fig. 51.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the OX40L polypeptide depicted in Fig. 53.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the PD-L2 polypeptide depicted in Fig. 54.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the CD86 polypeptide depicted in Fig. 55.

In some cases, the immunomodulatory polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the FAS-L polypeptide depicted in Fig. 56.

In some instances, an immunomodulatory polypeptide present in synTac exhibits reduced binding affinity for a co-immunomodulatory co-recognized polypeptide expressed on the surface of a T cell compared to the binding affinity of a wild-type immunomodulatory polypeptide for the same co-immunomodulatory co-recognized polypeptide. In some instances where synTac comprises an immunomodulatory polypeptide with reduced affinity, the synTac polypeptide exhibits reduced binding to a co-immunomodulatory co-recognized polypeptide expressed on the surface of a T cell. For example, in some cases, a synTac polypeptide that comprises an immunomodulatory polypeptide with reduced affinity binds a recognized co-immunomodulatory polypeptide with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, and at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a control synTac polypeptide comprising the wild-type an immunomodulatory polypeptide of the same type as the related immunomodulatory polypeptide.

Determination of binding affinity

The binding affinity between an immunomodulatory polypeptide and its co-immunomodulatory polypeptide recognition can be determined by biolayer interferometry (BLI) using purified immunomodulatory polypeptide and purified co-immunomodulatory polypeptide recognition. The binding affinity between synTac of the present invention and its co-immunomodulatory polypeptide recognition can also be determined by BLI using purified synTac and the co-immunomodulatory polypeptide recognition. BLI methods are well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014) J. Vis. Exp. 18:e51383. The specific and relative binding affinities described in this disclosure between an immunomodulatory polypeptide and its recognized immunomodulatory polypeptide or between synTac and its recognized immunomodulatory polypeptide can be determined using the following procedures.

To determine the binding affinity between synTac of the present invention and its recognized co-immunomodulatory polypeptide, a BLI assay can be performed using an Octet RED 96 (Pal ForteBio) or similar instrument as described below. To determine the binding affinity of a T cell modulating multimeric polypeptide (e.g., synTac of the present invention or a control T cell modulating multimeric polypeptide (wherein the control T cell modulating multimeric polypeptide comprises a wild-type immunomodulatory polypeptide)), the T cell modulating multimeric polypeptide is immobilized on an insoluble support (biosensor). The immobilized T cell modulating multimeric polypeptide is a target. The immobilization can be accomplished by immobilizing a capture antibody on an insoluble support, wherein the capture antibody immobilizes the T cell modulating multimeric polypeptide. For example, immobilization can be achieved by immobilizing antibodies to Fc (e.g., human Fc IgG) on an insoluble carrier, where

- 23 049999 immobilized antibodies to Fc bind to and immobilize a T cell modulating multimeric polypeptide (where the T cell modulating multimeric polypeptide comprises an Ig Fc polypeptide). The co-immunomodulatory polypeptide is applied at several different concentrations to the immobilized T cell modulating multimeric polypeptide and the instrument response is recorded. The assays are performed in a liquid medium containing 25 mM HEPES at pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin and 0.02% nonionic detergent Tween 20. Binding of the co-immunomodulatory polypeptide to the immobilized T cell modulating multimeric polypeptide is performed at 30°C. An anti-MHC class I monoclonal antibody can be used as a positive control for binding affinity. For example, anti-HLA class I monoclonal antibody W6/32 (American Type Culture Collection, no. HB-95; Parham et al. (1979) J. Immunol. 123:342) with a KD of 7 nM can be used. A standard curve can be generated using serial dilutions of the anti-MHC class I monoclonal antibody. The anti-MHC class I coimmunomodulatory polypeptide or mAb is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces: i) an immobilized polypeptide (target); and ii) an internal reference layer. Changing the number of molecules (analyte; eg, coimmunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a change in the interference pattern; This shift in the interference pattern can be measured in real time. The two kinetic terms that describe the affinity of the target-analyte interaction are the association constant (k _a ) and the dissociation constant (kd). The ratio of these two values (k _d/a ) yields the affinity constant KD.

As noted above, the determination of the binding affinity between an immunomodulatory polypeptide (e.g., IL-2 or an IL-2 variant) and its co-immunomodulatory polypeptide (e.g., IL-2R) recognized therein can also be determined using BLI. The assay is similar to that described above for the synTac multimeric polypeptide. The BLI assay can be performed using an Octet RED 96 (Pal ForteBio) or similar instrument as follows. A component of the synTac immunomodulatory polypeptide of the present invention (e.g., an IL-2 variant polypeptide of the present invention); and a control immunomodulatory polypeptide (wherein the control immunomodulatory polypeptide comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL2) are immobilized on an insoluble support (biosensor). The immunomodulatory polypeptide is a target. The immobilization can be accomplished by immobilizing the capture antibody on an insoluble support, wherein the capture antibody immobilizes the immunomodulatory polypeptide. For example, if the target is fused with an immunoaffinity label (e.g., FLAG, human IgG Fc), the immobilization can be accomplished by immobilization with the corresponding antibody on the immunoaffinity label (e.g., anti-human IgG Fc) on an insoluble support, wherein the immobilized antibodies bind to and immobilize the immunomodulatory polypeptide (wherein the immunomodulatory polypeptide includes an Ig Fc polypeptide). The co-immunomodulatory polypeptide (or polypeptides) is applied at several different concentrations to the immobilized immunomodulatory polypeptide, and the response of the device is recorded. Alternatively, the co-immunomodulatory polypeptide (or polypeptides) are immobilized in a biosensor (e.g., for an IL-2 receptor heterotrimer, as a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer) and the immunomodulatory polypeptide is applied at several different concentrations to the immobilized co-immunomodulatory polypeptide and the instrument response is recorded. Assays are performed in a liquid medium containing 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% nonionic detergent Tween 20. Binding of the co-immunomodulatory polypeptide to the immobilized polypeptide is performed at 30°C. An anti-MHC class I monoclonal antibody can be used as a positive control for binding affinity. For example, anti-HLA class I monoclonal antibody W6/32 (American Type Culture Collection, no. HB-95; Parham et al. (1979) J. Immunol. 123:342) with a KD of 7 nM can be used. A standard curve can be generated using serial dilutions of the anti-MHC class I monoclonal antibody. The anti-MHC class I coimmunomodulatory polypeptide or mAb is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces: i) an immobilized polypeptide (target); and ii) an internal reference layer. Changing the number of molecules (analyte; eg, coimmunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a change in the interference pattern; this shift in the interference pattern can be measured in real time. The two kinetic terms that describe the affinity of the target-analyte interaction are the association constant ( _ka ) and the dissociation constant ( _kd ). The ratio of these two values (kd/ _a ) yields the affinity constant Kd. Determining the binding affinity of a wild-type immunomodulatory polypeptide (e.g., IL-2) for its receptor (e.g., IL-2R), as well as a variant immunomodulatory polypeptide (e.g., an IL-2 variant as described herein) for its co-recognized co-immunomodulatory polypeptide (e.g., its receptor) (e.g., IL2R), thus allows one to determine the relative binding affinity of the variant co-immunomodulatory polypeptide (e.g., its receptor) (e.g., IL2R).

- 24 049999 modulating polypeptide compared to a wild-type co-immunomodulatory polypeptide for a recognized co-immunomodulatory polypeptide. That is, it can be determined whether the binding affinity of a variant immunomodulatory polypeptide for its receptor (its recognized immunomodulatory polypeptide) is reduced compared to the binding affinity of a wild-type immunomodulatory polypeptide for the same recognized immunomodulatory polypeptide, and if so, what is the percentage reduction in binding affinity of the wild-type co-immunomodulatory polypeptide.

The BLI assay is performed on a multi-well plate. To perform the assay, the plate loading scheme is defined, the assay steps are defined, and the biosensors are assigned in the Octet Data Acquisition software. The biosensor kit is hydrated. The hydrated biosensor kit and assay plate are equilibrated for 10 minutes on the Octet instrument. Once the data is acquired, the acquired data are loaded into the Octet data analysis software. The data are processed in the Processing window by specifying the reference subtraction method, Y-axis alignment, interstep correction, and Savitzky-Golay filtering. The data are analyzed in the Analysis window by specifying the steps for the analysis (association and dissociation), selection of the curve fitting model (1:1), fitting method (global), and window of interest (in seconds). The quality of the fit is assessed. The KD values for each data curve (analyte concentration) can be averaged if they are within a 3-fold range. KD error values should be within one order of magnitude of the affinity constant values; R ² values should be greater than 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

In some cases, the ratio of: i) the binding affinity of a control T cell modulating multimeric polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to the co-immunomodulatory polypeptide (e.g., IL-2R) to ii) the binding affinity of a T cell modulating multimeric polypeptide of the present invention comprising a wild-type immunomodulatory polypeptide variant (e.g., IL-2 variant) to the co-immunomodulatory polypeptide (e.g., IL-2R), as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 103:1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1. In some cases, the ratio of: i) the binding affinity of a control T cell modulating multimeric polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide) to the co-immunomodulatory polypeptide recognition to ii) the binding affinity of a T cell modulating multimeric polypeptide of the present invention comprising a wild-type immunomodulatory polypeptide variant to the co-immunomodulatory polypeptide recognition, as measured by BLI, is in the range of from 1.5:1 to 10 ⁶ :1, such as from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 10 ² :1, from 10 ² :1 to 103:1, from 103:1 to 10 ⁴ :1, from 10 ⁴ :1 to 10 ⁵ :1, or from 10 ⁵ :1 to 10 ⁶ :1.

In some cases, the ratio of: i) the binding affinity of a control immunomodulatory polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to a cognate coimmunomodulatory polypeptide (e.g., IL-2R) to ii) the binding affinity of an immunomodulatory polypeptide of the present invention comprising a variant wild-type immunomodulatory polypeptide (e.g., variant IL-2) to a cognate immunomodulatory polypeptide (e.g., IL-2R), as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 103:1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1. In some cases, the ratio of: i) the binding affinity of a control immunomodulatory polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide) to the co-immunomodulatory polypeptide to ii) the binding affinity of an immunomodulatory polypeptide of the present disclosure comprising a wild-type variant immunomodulatory polypeptide to the co-immunomodulatory polypeptide, as measured by BLI, is in the range of 1.5:1 to 10 ⁶ :1, such as 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 ² :1, 10 ² :1 to 103:1, 103:1 to 10 ⁴ :1, 10 ⁴ :1 to 10 ⁵ :1, or 10 ⁵ :1 to 10 ⁶ :1.

IL-2/synTac

In some cases, the multimeric polypeptide comprises wild-type (naturally occurring) IL-2 as a modulating domain. In some cases, the multimeric polypeptide comprises a variant IL-2 polypeptide as a modulating domain.

The T cell modulating multimeric polypeptide, which contains the IL-2 polypeptide as a modulation domain (MOD), is also called IL-2/synTac, IL-2/synTac polypeptide, or IL2/multimer polypeptide.

- 25 049999

In some cases, the IL-2/synTac polypeptide comprises a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises one copy of a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises two copies of a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises three copies of a wild-type IL-2 polypeptide. In some cases, the wild-type IL-2 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2A. The amino acid sequence of a wild-type human IL2 polypeptide may be as follows:

APTSSSTKKT

QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1).

In some cases, the synTac polypeptide comprises a variant IL-2 polypeptide. The variant IL-2 polypeptide present in the multimeric polypeptide exhibits reduced binding affinity for the IL2R compared to the binding affinity of wild-type IL-2 for the IL2R. The multimeric polypeptide that comprises the variant IL-2 polypeptide also exhibits reduced binding affinity for the IL2R compared to a control multimeric polypeptide comprising wild-type IL-2 for the IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Fig. 3A-3C).

In some cases, the IL-2/synTac polypeptide exhibits a reduced binding affinity for the IL2R compared to the binding affinity of an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A for the IL2R. For example, in some cases, the IL2/synTac polypeptide binds the IL2R with a binding affinity that is less than the binding affinity of a control synTac polypeptide comprising an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A for the IL2R, comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Figs. 3A-3C. For example, in some cases, the IL-2/synTac polypeptide binds the IL2R with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, and at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a control synTac polypeptide comprising an IL-2 polypeptide comprising the amino acid sequence depicted in FIG. 2A for IL2R (e.g., IL2R comprising alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in Fig. 3A-3C).

In some cases, the IL-2/synTac polypeptide has a binding affinity for the IL2R that is from 100 nM to about 100 μM. In some cases, the IL-2/synTac polypeptide has a binding affinity for the IL2R that is from about 100 nM to about 500 nM. For example, in some cases, the IL-2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Figs. 3A-3C) that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 to about 400 nM, from about 400 to about 450 nM, or from about 450 to about 500 nM. In some cases, the IL-2/synTac polypeptide has a binding affinity for the IL2R (e.g., the IL2R comprising the alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in Fig. 3A-3C) that is from about 500 nM to 1 μM. For example, in some cases, the IL-2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Figs. 3A-3C) that is from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 to about 800 nM, from about 800 to about 900 nM, or from about 900 nM to about 1 μM. In some cases, the IL2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in Fig. 3A-3C) that is from about 1 μM to 10 μM. For example, in some cases, the IL-2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Figs. 3A-3C) that is from about 1 μM to 2 μM, from about 2 μM to about 3 μM, from about 3 μM to about 4 μM, from about 4 μM to about 5 μM, from about 5 μM to about 6 μM, from about 6 μM to about 7 μM, from about 7 μM to about 8 μM, from

- 26 049999 about 8 μM to about 9 μM or from about 9 μM to about 10 μM. In some cases, the IL-2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Fig. 3A-3C) that is from about 10 μM to 100 μM. For example, in some cases, the IL-2/synTac polypeptide has a binding affinity for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptides comprising the amino acid sequences (mature form) depicted in Figs. 3A-3C) that is from about 10 μM to about 20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 40 μM, from about 40 μM to about 50 μM, from about 50 μM to about 60 μM, from about 60 μM to about 70 μM, from about 70 μM to about 80 μM, from about 80 μM to about 90 μM, or from about 90 μM to about 100 μM.

The IL2 polypeptide variant present in the IL-2/synTac polypeptide may have one amino acid substitution relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as depicted in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in the IL-2/synTac polypeptide has 2 to 10 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as depicted in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in the synTac polypeptide of the present invention has 2 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as depicted in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 3 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 4 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 5 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 6 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 7 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 8 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 9 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1). In some cases, the IL2 polypeptide variant present in a synTac polypeptide of the present invention has 10 amino acid substitutions relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide comprising the amino acid sequence depicted in Fig. 2A or as set forth in SEQ ID NO: 1).

In some cases, a multimeric polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from amino terminus (N-terminus) to carboxyl terminus (C-terminus): a) an epitope (e.g., a T-cell epitope); b) a first major histocompatibility complex (MHC) polypeptide; and c) an immunomodulatory polypeptide (e.g., an IL2 variant polypeptide of the present invention); and wherein the second polypeptide comprises, from N-terminus to C-terminus: a) a second MHC polypeptide; and b) an immunoglobulin (Ig) Fc polypeptide. In other cases, a multimeric polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus: a) an epitope (e.g., a T-cell epitope); and b) a first MHC polypeptide; and wherein the second polypeptide comprises, in order from N-terminus to C-terminus: a) an immunomodulatory polypeptide (e.g., a variant IL2 polypeptide of the present invention); b) a second MHC polypeptide; and c) an IgFc polypeptide. In some cases, the first and second MHC polypeptides are MHC class I polypeptides; for example, in some cases, the first MHC polypeptide is a B2-microglobulin (B2M) polypeptide.

- 27 049999 or β2M) MHC class I and the second MHC polypeptide is an MHC class I heavy chain (H chain); or the first MHC polypeptide is an MHC class I H chain and the second MHC polypeptide is an MHC class I β2M polypeptide). In other cases, the first and second MHC polypeptides are MHC class II polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide. In some cases, the multimeric polypeptide comprises two or more immunomodulatory polypeptides, wherein at least one of the immunomodulatory polypeptides is a variant of the IL2 immunomodulatory polypeptide of the present invention. When the multimeric polypeptide of the present invention comprises two or more immunomodulatory polypeptides, in some cases the two or more immunomodulatory polypeptides are present in the same polypeptide chain and may be in tandem. When the multimeric polypeptide of the present invention comprises two or more immunomodulatory polypeptides, in some cases the two or more immunomodulatory polypeptides are present in separate polypeptides. In some cases, the multimeric polypeptide of the present disclosure is a heterodimer. In some cases, the multimeric polypeptide of the present disclosure is a trimeric polypeptide.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory domain (e.g., an IL2 polypeptide variant of the present invention). In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an immunomodulatory domain (e.g., an IL2 polypeptide variant of the present invention). In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an immunomodulatory domain (e.g., an IL2 polypeptide variant of the present invention); and ii) a second MHC polypeptide. In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain (e.g., an IL2 polypeptide variant of the present invention); and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide. In some cases, when the multimeric polypeptide of the present invention comprises a non-Ig framework, the non-Ig framework is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

In some cases, the multimeric polypeptide of the present disclosure is monovalent. In some cases, the multimeric polypeptide of the present disclosure is multivalent. In some cases, the multivalent multimeric polypeptide of the present invention comprises an immunoglobulin Fc polypeptide on one of the first or second polypeptide. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present invention, the multimeric polypeptide can be a homodimer, wherein two molecules of the multimeric polypeptide are present in a homodimer, wherein the two molecules of the multimeric polypeptide can be linked to each other by a disulfide bond, such as through an Fc polypeptide present in the two molecules. As another example, the multimeric polypeptide of the present invention can comprise three, four or five molecules of the multimeric polypeptide, wherein the molecules of the multimeric polypeptide can be linked to each other by a disulfide bond, such as through an Fc polypeptide present in the molecules.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a β2M polypeptide; and iii) an IL2 polypeptide variant of the present invention; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a β2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an IL2 polypeptide variant of the present invention; ii) an MHC class I heavy chain; and iii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a B2M polypeptide; iii) a first variant of the IL2 polypeptide of the present invention; iv) a second variant of the IL2 polypeptide of the present invention; and v) a third variant of the IL2 polypeptide of the present invention; and b)

- 28 049999 a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, the first, second, and third variant IL2 polypeptides have the same amino acid sequence. In some cases, the first, second, and third variant IL2 polypeptides differ from each other in amino acid sequence. In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a first variant IL2 polypeptide of the present invention; ii) a second variant IL2 polypeptide of the present invention; and iii) a third variant IL2 polypeptide of the present invention; iv) an MHC class I heavy chain; and v) an Fc polypeptide. In some cases, the first, second, and third variant IL2 polypeptides have the same amino acid sequence. In some cases, the first, second, and third variant IL2 polypeptides differ from each other in amino acid sequence.

Replacement F42

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Val. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Leu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2B, wherein amino acid 42 is Ile. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 or synTac polypeptide variant comprising the same has a binding affinity for the IL2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from at

- 29 049999 measured 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacement Y45

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is an amino acid other than tyrosine, such as wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is Val. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is Leu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2F, wherein amino acid 45 is He. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 or synTac polypeptide variant comprising the same has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100

- 30,049,999 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacement Q126

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is an amino acid other than glutamine, such as wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Val. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Leu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2G, wherein amino acid 126 is Ile. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

F42 and H16 Replacements

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and wherein amino acid 16 is an amino acid other than histidine, such as wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Ala and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Ala and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Val and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Leu and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2H, wherein amino acid 42 is Ile and amino acid 16 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the multimeric polypeptide of the present invention comprises 2 copies of an IL-2 variant comprising the F42A and H16A substitutions, wherein the multimeric polypeptide comprises HLA class I and B2M heavy chain polypeptides, and wherein the 2 copies of IL-2 (F42A, H16A) are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, the IL-2 variant polypeptide or synTac comprising it has a binding affinity for IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure has a length of 133

- 32,049,999 amino acids. In some cases, the IL-2 polypeptide variant comprises the amino acid sequence shown in Fig. 34B (containing the H16A and F42A substitutions).

F42 and D20 Replacements

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and wherein amino acid 20 is an amino acid other than aspartic acid, such as wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 20 is Asn, Gln, Lys, Arg, or His. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Val and amino acid 20 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Leu and amino acid 20 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is He and amino acid 20 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Asn. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gln. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Lys. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid

- 33 049999 amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Arg. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is His. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, D20 and E15

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, such as wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His, or Glu; and wherein amino acid 15 is an amino acid other than glutamic acid, such as wherein amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, an IL-2 polypeptide variant of the present invention comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 15 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; and wherein amino acid 15 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid

- 34 049999 a sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In some cases, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2I, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, the IL-2 or synTac polypeptide variant comprising the same has a binding affinity for the IL2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM,

- 35 049999 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, D20 and H16

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala.

- 36 049999

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in a multimeric polypeptide of the present invention. In some cases, a multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, a multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising a b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, D20 and Q126

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, a variant IL-2 polypeptide present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, a variant polypeptide

- 37 049999

IL-2 present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; and wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is He, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide.

- 38 049999 in some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, D20 and Y45

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; and wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence of the

- 39 049999 depicted in Fig. 2M, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 45 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 45 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F4, D20, Y45 and H16

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is an amino acid other than phenylalanine, such as where amino acid

- 40 049999 amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; where amino acid 45 is an amino acid other than tyrosine, for example where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, such as wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Val. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid

- 41 049999 amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, D20, Y45 and Q126

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 126 is an amino acid other than glutamine, such as wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least

- 42 049999 at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 is Val. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 126 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 20, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention.

- 43 049999 n. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, for example, where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, for example, where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising a b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising it has a binding affinity for IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements F42, D20, Y45, H16 and Q126

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg, or His; wherein amino acid 45 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to

- 44 049999 amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as where the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain

- 45 049999 pi containing the β2M polypeptide. In some cases, the IL-2 or synTac polypeptide variant comprising the same has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids in length.

Replacements for F42, Q126 and H16

In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is an amino acid other than phenylalanine, such as wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu, or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu, or Ile; wherein amino acid 126 is Asn, Gln, Lys, Arg, or His; and wherein amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Val, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptide present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptide present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ile, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, a variant

- 46 049999 IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Arg, and amino acid 16 is Ala. In some cases, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In some cases, a single copy of the IL-2 polypeptide variant is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention comprises two copies of the IL-2 polypeptide variant, such as where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention comprises three copies of the IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2/synTac of the present invention comprises an HLA class I heavy chain and a b2M, the IL-2 polypeptide(s) is/are on a polypeptide chain comprising the b2M polypeptide. In some cases, the IL-2 polypeptide variant or synTac comprising the IL-2 polypeptide variant has a binding affinity for the IL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the IL-2 polypeptide variant is 133 amino acids long.

4-1BBL

In some cases, a synTac suitable for use in the method of the present invention comprises a 4-1BBL polypeptide as an immunomodulatory domain(s). Suitable immunomodulatory domains of 4-1BBL include a wild-type 4-1BBL immunomodulatory domain and a variant 4-1BBL immunomodulatory domain.

The amino acid sequence of wild-type human 4-1BBL is shown in Fig. 36A. The tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL comprises amino acids 81-254, amino acids 80-254, or amino acids 80-246 of the amino acid sequence shown in Fig. 36A. Thus, the amino acid sequence of wild-type human 4-1BBL THD may be, for example, one of SEQ ID NOs: 213-215, as follows:

- 47 049999

PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT

KELVVAKAGV YYVFFQLELR RWAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:213).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RWAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:214).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RWAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ Ш NO:215).

Wild-type 4-1BBL binds to 4-1BB (CD137). The amino acid sequence of 4-1BB is shown in Fig. 37. The 4-1BBL polypeptide variant of the present invention binds to 4-1BB with reduced affinity compared to the binding of wild-type 4-1BBL to 4-1BB.

Variant 4-1BBL polypeptides include polypeptides having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a corresponding wild-type 4-1BBL polypeptide and includes variant 4-1BBL polypeptides that differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, or more than 15 amino acids, from a corresponding wild-type 4-1BBL polypeptide. In some cases, a variant 41BBL polypeptide differs in amino acid sequence from a wild-type 4-1BBL polypeptide by only one amino acid. In some cases, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 2 amino acids. In some cases, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 3 amino acids. In some cases, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 4 amino acids. In some cases, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 5 amino acids.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure exhibits a reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A for 4-1BB. For example, in some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is less than the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A to a 4-1BB polypeptide comprising the amino acid sequence depicted in Fig. 37. For example, in some cases, a 41BBL polypeptide variant present in a multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less in affinity binding of a 41BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence depicted in Fig. 37).

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure exhibits a reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 213 for 4-1BB. For example, in some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is less than the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 213 for a 4-1BB polypeptide comprising the amino acid sequence depicted in one of Figs. 37A-37C. For example, in some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 213 for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence depicted in Fig. 37).

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB that is from 100 nM to 100 μM. As another example, in some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence depicted in Fig. 37) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 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 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure exhibits increased production in a mammalian host cell compared to the production in the same mammalian host cell of a control multimeric polypeptide comprising a 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). For example, in some cases, a variant 4-1BBL polypeptide present in a multimeric polypeptide, when expressed in a mammalian host cell, is produced in an amount that is from 25% higher to about 50% higher, from about 50% higher to about 75% higher, from about 75% higher to about 2-fold higher, from about 2-fold higher to about 5-fold higher, from about 5-fold higher to about 10-fold higher, from about 10-fold higher to about 20-fold higher, from about 20-fold higher to about 30-fold higher, from about 30-fold higher to about 40-fold higher, from about 40-fold higher to about 50-fold higher, from about 50-fold higher to about 75-fold higher, from about 75-fold higher to about 100-fold higher, or more than 100-fold higher than the amount of a control a multimeric polypeptide comprising a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213) produced in the same mammalian host cell.

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide is produced in a mammalian host cell in an amount of from about 50 mg/L to about 75 mg/L, from about 75 mg/L to about 100 mg/L, from about 100 mg/L to about 150 mg/L, from about 150 mg/L to about 200 mg/L, from about 200 mg/L to about 250 mg/L, from about 250 mg/L to about 500 mg/L, or more than 500 mg/L. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide is produced in a mammalian host cell in an amount of from about 10 mg/L to about 15 mg/L, from about 15 mg/L to about 20 mg/L, from about 20 mg/L to about 25 mg/L, from about 25 mg/L to about 30 mg/L, from about 35 mg/L to about 40 mg/L, from about 40 mg/L to about 45 mg/L, or from about 45 mg/L to about 50 mg/L.

A 4-1BBL polypeptide variant present in a multimeric polypeptide may have one amino acid substitution relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as depicted in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 2 to 10 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as depicted in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 2 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 41BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 3 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence

- 49 049999 the amino acid sequence depicted in Fig. 36A or as depicted in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 4 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as depicted in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 5 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as depicted in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 6 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant of the present disclosure has 7 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 8 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 9 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 10 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213).

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has from 11 to 50 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence depicted in Fig. 36A or as set forth in SEQ ID NO: 213). For example, in some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 11 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence shown in Fig. 36A or as shown in SEQ ID NO: 213).

Suitable 4-1BBL polypeptide variants that may be included in the multimeric polypeptide of the present invention include those described above.

4-1BBL with K127 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (denoted by x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 47 is an amino acid other than lysine, such as wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at K48. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at K48. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at K48. In some cases, a 4-1BBL polypeptide variant present in

- 50 049999 multimeric polypeptide according to the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at K48.

Replacements K127 + M91

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at M91, wherein amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 91 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 11 is other than methionine, such as where amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 11 is Ala.

Replacements K127 + F92

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at F92, wherein amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 92 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 12 is other than phenylalanine, such as where amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 12 is Ala.

Replacements K127 + Q94

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at Q94, wherein amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 94 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 14 is other than glutamine, such as where amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 14 is Ala.

- 51 049999

Replacements K127 + L95

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at L95, wherein amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 95 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 15 is other than leucine, such as where amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 15 is Ala.

Replacements K127 + V96

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at V96, wherein amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 96 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 16 is other than valine, such as where amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 16 is Ala.

Replacements K127 + Q98

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at Q98, wherein amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 98 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 18 is other than glutamine, such as where amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 18 is Ala.

Replacements K127 + N99

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 52 049999 of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at N99, wherein amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 99 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 19 is other than asparagine, such as where amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 19 is Ala.

Replacements K127 + V100

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at V100, wherein amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 100 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 20 is other than valine, such as where amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 20 is Ala.

Replacements K127 + L101

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at L101, wherein amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 101 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 21 is other than leucine, such as where amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 21 is Ala.

Replacements K127 + L102

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 53 049999

98% or at least 99% relative to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (designated x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at L102, wherein amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala; and amino acid 102 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 22 is other than leucine, such as where amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 22 is Ala.

Replacement K127 + I103

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at I103, wherein amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 103 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 23 is other than isoleucine, such as where amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 23 is Ala.

Replacements K127 + D104

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at D104, wherein amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, amino acid 127 is Ala; and amino acid 104 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 24 is other than aspartic acid, such as where amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, amino acid 47 is Ala; and amino acid 24 is Ala.

Replacements K127 + G105

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (designated x) is an amino acid other than

- 54 049999 zine, for example, wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at G105, wherein amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala; and amino acid 105 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 25 is other than glycine, such as where amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 25 is Ala.

Replacements K127 + P106

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at P106, wherein amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 106 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 26 is other than proline, such as where amino acid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 26 is Ala.

Replacements K127 + L107

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at L107, wherein amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 107 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 27 is other than leucine, such as where amino acid 27 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 27 is Ala.

Replacements K127+ S108

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at S108, wherein amino acid 108

- 55 049999 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 108 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 28 is other than serine, such as where amino acid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 28 is Ala.

Replacements K127 + W109

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at W109, wherein amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 109 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 29 is other than tryptophan, such as where amino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 29 is Ala.

Replacements K127 + Y110

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at Y110, wherein amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 110 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 30 is other than tyrosine, such as where amino acid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 30 is Ala.

Replacements K127 + S111

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at S111, wherein amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 111 is

- 56 049999 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 31 is other than serine, such as where amino acid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 31 is Ala.

Replacements K127 + D112

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at D112, wherein amino acid 112 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, amino acid 127 is Ala; and amino acid 112 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 32 is other than aspartic acid, such as where amino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, amino acid 47 is Ala; and amino acid 32 is Ala.

Replacements K127 + P113

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at P113, wherein amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 113 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 33 is other than proline, such as where amino acid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 33 is Ala.

Replacements K127 + G114

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at G114, wherein amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 114 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence that is identical to

- 57 049999 an amino acid sequence identity of at least 90%, at least 95%, at least 98% or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, for example wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 34 is other than glycine, for example wherein amino acid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala; and amino acid 34 is Ala.

Replacements K127 + L115

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein: i) amino acid 127 (denoted by x) is an amino acid other than lysine, such as where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) an amino acid substitution at L115, wherein amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and amino acid 115 is Ala. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein: i) amino acid 47 is an amino acid other than lysine, such as where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and ii) amino acid 35 is other than leucine, such as where amino acid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; and amino acid 35 is Ala.

4-1BBL with replacement Q227

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 147 is other than glutamine, such as wherein amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q148. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q148. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q148. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q148.

4-1BBL with M91 replacement

In some instances, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36E, wherein amino acid 91 (designated x) is an amino acid other than methio

- 58 049999 nina, for example, where amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 11 is other than methionine, such as wherein amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at M12. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at M12. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at M12. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at M12.

4-1BBL with F92 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36F, wherein amino acid 92 (denoted by x) is an amino acid other than phenylalanine, such as wherein amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 12 is other than phenylalanine, such as wherein amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F13. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F13. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F13. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F13.

4-1BBL with Q94 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36G, wherein amino acid 94 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 14 is other than glutamine,

- 59 049999 example where amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q15. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q15. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q15. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q15.

4-1BBL with L95 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36H, wherein amino acid 95 (designated x) is an amino acid other than leucine, such as wherein amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 15 is other than leucine, such as wherein amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L16. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L16. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L16. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L16.

4-1BBL with V96 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36I, wherein amino acid 96 (denoted by x) is an amino acid other than valine, such as wherein amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 16 is other than valine, such as wherein amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in

- 60 049999

SEQ ID NO: 214, with an amino acid substitution at V17. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at V17. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at V17. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at V17.

4-1BBL with Q98 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36J, wherein amino acid 98 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 18 is other than glutamine, such as wherein amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q19. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q19. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q19. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q19.

4-1BBL with N99 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36K, wherein amino acid 99 (denoted by x) is an amino acid other than asparagine, such as wherein amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 19 is other than asparagine, such as wherein amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at N20. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or less amino acid sequence identity to the

- 61 049999 at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at N20. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at N20.

4-1BBL with V100 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36L, wherein amino acid 100 (denoted by x) is an amino acid other than valine, such as wherein amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 20 is other than valine, such as wherein amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V21. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V21. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V21. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V21.

4-1BBL with L101 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36M, wherein amino acid 101 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 21 is other than leucine, such as wherein amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L22. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L22. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L22. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L22.

4-1BBL with L102 replacement

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 62 049999 of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36N, wherein amino acid 102 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 22 is other than leucine, such as wherein amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L23. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L23. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L23. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L23.

4-1BBL with I103 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 360, wherein amino acid 103 (denoted by x) is an amino acid other than isoleucine, such as wherein amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 23 is other than isoleucine, such as wherein amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at I24. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at I24. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at I24. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at I24.

4-1BBL with replacement D104

In some instances, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36P, wherein amino acid 104 (denoted by x) is an amino acid other than aspartic acid, such as wherein amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some instances, a 4-1BBL polypeptide variant

1BBL present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 24 is different from aspartic acid, such as wherein amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D25. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D25. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D25. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D25.

4-1BBL with G105 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36Q, wherein amino acid 105 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 25 is other than glycine, such as wherein amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G26. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G26. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G26. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G26.

4-1BBL with replacement P106

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36R, wherein amino acid 106 (denoted by x) is an amino acid other than proline, such as wherein amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 26 is other than proline, such as wherein amino acid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

- 64 049999

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P27. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P27. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P27. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P27.

4-1BBL with L107 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36S, wherein amino acid 107 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 27 is other than leucine, such as wherein amino acid 27 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L28. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L28. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L28. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L28.

4-1BBL with S108 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36T, wherein amino acid 108 (denoted by x) is an amino acid other than serine, such as wherein amino acid 108 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 28 is other than serine, such as wherein amino acid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S29. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid

- 65 049999 the sequence presented in SEQ ID NO: 214, with an amino acid substitution at S29. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at S29. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at S29.

4-1BBL with W109 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36U, wherein amino acid 109 (denoted by x) is an amino acid other than tryptophan, such as wherein amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 29 is other than tryptophan, such as wherein amino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at W30. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at W30. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at W30. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at W30.

4-1BBL with Y110 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36V, wherein amino acid 110 (denoted by x) is an amino acid other than tyrosine, such as wherein amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 30 is other than tyrosine, such as wherein amino acid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Y31. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Y31. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Y31. In some cases, a 4-1BBL polypeptide variant present in

- 66 049999 multimeric polypeptide according to the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at Y31.

4-1BBL with S111 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36W, wherein amino acid 111 (denoted by x) is an amino acid other than serine, such as wherein amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 31 is other than serine, such as wherein amino acid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S32. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S32. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S32. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S32.

4-1BBL with replacement D112

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36X, wherein amino acid 112 (denoted by x) is an amino acid other than aspartic acid, such as wherein amino acid 112 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a 41BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 32 is other than aspartic acid, such as wherein amino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D33. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D33. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D33. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D33.

4-1BBL with replacement P113

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 67 049999

98% or at least 99% relative to the amino acid sequence depicted in Fig. 36Y, wherein amino acid 113 (denoted by x) is an amino acid other than proline, for example, wherein amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 33 is other than proline, such as wherein amino acid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P34. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P34. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P34. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P34.

4-1BBL with G114 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36Z, wherein amino acid 114 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 34 is other than glycine, such as wherein amino acid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G35. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G35. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G35. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G35.

4-1BBL with L115 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36AA, wherein amino acid 115 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90% amino acid sequence identity,

- 68 049999 at least 95%, at least 98% or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 213, wherein amino acid 35 is different from leucine, for example, wherein amino acid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L36. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L36. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L36. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L36.

4-1BBL with G117 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36BB, wherein amino acid 117 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 117 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 37 is other than glycine, such as wherein amino acid 37 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G38. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G38. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G38. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G38.

4-1BBL with V118 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36CC, wherein amino acid 118 (denoted by x) is an amino acid other than valine, such as wherein amino acid 118 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 38 is other than valine, such as wherein amino acid 38 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having identity

- 69 049999 an amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at V39. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at V39. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at V39. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 with an amino acid substitution at V39.

4-1BBL with S119 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36DD, wherein amino acid 119 (denoted by x) is an amino acid other than serine, such as wherein amino acid 119 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 39 is other than serine, such as wherein amino acid 39 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S40. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S40. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S40. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S40.

4-1BBL with L120 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36EE, wherein amino acid 120 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 120 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 40 is other than leucine, such as wherein amino acid 40 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L41. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L41. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure

- 70 049999 disclosure, comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at L41. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at L41.

4-1BBL with T121 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36FF, wherein amino acid 121 (denoted by x) is an amino acid other than threonine, such as wherein amino acid 121 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 41 is other than threonine, such as wherein amino acid 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T42. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T42. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T42. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T42.

4-1BBL with G122 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36GG, wherein amino acid 122 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 122 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 42 is other than glycine, such as wherein amino acid 42 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G43. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G43. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G43. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G43.

- 71 049999

4-1BBL with G123 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36HH, wherein amino acid 123 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 123 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 43 is other than glycine, such as wherein amino acid 43 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G44. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G44. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G44. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G44.

4-1BBL with L124 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36II, wherein amino acid 124 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 124 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 44 is other than leucine, such as wherein amino acid 44 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L45. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L45. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L45. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L45.

4-1BBL with S125 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36JJ, wherein amino acid 125 (designated x) is an amino acid other than serine,

- 72 049999 for example, where amino acid 125 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 45 is other than serine, such as wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S46. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S46. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S46. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S46.

4-1BBL with Y126 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36KK, wherein amino acid 126 (designated x) is an amino acid other than tyrosine, such as wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 46 is other than tyrosine, such as wherein amino acid 46 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Y47. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Y47. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Y47. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Y47.

4-1BBL with replacement E128

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36LL, wherein amino acid 128 (denoted by x) is an amino acid other than glutamic acid, such as wherein amino acid 128 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 41BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 48 differs from

- 73 049999 glutamic acid, for example, where amino acid 48 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E49. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E49. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E49. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E49.

4-1BBL with replacement D129

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36MM, wherein amino acid 129 (denoted by x) is an amino acid other than aspartic acid, such as wherein amino acid 129 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 49 is other than aspartic acid, such as wherein amino acid 49 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D50. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D50. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D50. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D50.

4-1BBL with T130 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36NN, wherein amino acid 130 (denoted by x) is an amino acid other than threonine, such as wherein amino acid 130 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 50 is other than threonine, such as wherein amino acid 50 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in

- 74 049999

SEQ ID NO: 214, with an amino acid substitution at T51. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at T51. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at T51. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at T51.

4-1BBL with replacement K131

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36OO, wherein amino acid 131 (denoted by x) is an amino acid other than lysine, such as wherein amino acid 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 51 is other than lysine, such as wherein amino acid 51 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at K52. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at K52. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at K52. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at K52.

4-1BBL with replacement E132

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36PP, wherein amino acid 132 (designated x) is an amino acid other than glutamic acid, such as wherein amino acid 132 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 41BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 52 is other than glutamic acid, such as wherein amino acid 52 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E53. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or less amino acid sequence identity to the

- 75 049999 at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at E53. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at E53.

4-1BBL with F144 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36QQ, wherein amino acid 144 (denoted by x) is an amino acid other than phenylalanine, such as wherein amino acid 144 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 64 is other than phenylalanine, such as wherein amino acid 64 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F65. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F65. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F65. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F65.

4-1BBL with F145 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36RR, wherein amino acid 145 (denoted by x) is an amino acid other than phenylalanine, such as wherein amino acid 145 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 65 is other than phenylalanine, such as wherein amino acid 65 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F66. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F66. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F66. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F66.

4-1BBL with replacement Q146

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 76 049999 of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36SS, wherein amino acid 146 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 66 is other than glutamine, such as wherein amino acid 66 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q67. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q67. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q67. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q67.

4-1BBL with L147 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36TT, wherein amino acid 147 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 147 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 67 is other than leucine, such as wherein amino acid 67 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L68. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L68. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L68. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L68.

4-1BBL with replacement E148

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36UU, wherein amino acid 148 (denoted by x) is an amino acid other than glutamic acid, such as wherein amino acid 148 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 4-1BBL polypeptide variant

- 77 049999

1BBL present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 68 is different from glutamic acid, such as wherein amino acid 68 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E69. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E69. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E69. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E69.

4-1BBL with L149 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36VV, wherein amino acid 149 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 149 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 69 is other than leucine, such as wherein amino acid 69 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L70. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L70. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L70. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L70.

4-1BBL with R150 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36WW, wherein amino acid 150 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 70 is other than arginine, such as wherein amino acid 70 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

- 78 049999

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R71. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R71. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R71. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R71.

4-1BBL with R151 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36XX, wherein amino acid 151 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 151 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 71 is other than arginine, such as wherein amino acid 71 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R72. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R72. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R72. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R72.

4-1BBL with V152 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36YY, wherein amino acid 152 (denoted by x) is an amino acid other than valine, such as wherein amino acid 152 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 72 is other than valine, such as wherein amino acid 72 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V73. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid

- 79 049999 the sequence presented in SEQ ID NO: 214, with an amino acid substitution at V73. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at V73. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at V73.

4-1BBL with V153 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36ZZ, wherein amino acid 153 (denoted by x) is an amino acid other than valine, such as wherein amino acid 153 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 73 is other than valine, such as wherein amino acid 73 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V74. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V74. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V74. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V74.

4-1BBL with G155 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36AAA, wherein amino acid 155 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 155 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 75 is other than glycine, such as wherein amino acid 75 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G76. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G76. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G76. In some cases, a 4-1BBL polypeptide variant present in

- 80 049999 multimeric polypeptide according to the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at G76.

4-1BBL with replacement E156

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36BBB, wherein amino acid 156 (denoted by x) is an amino acid other than glutamic acid, such as wherein amino acid 156 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 41BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 76 is other than glutamic acid, such as wherein amino acid 76 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E77. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E77. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E77. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E77.

4-1BBL with G157 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36CCC, wherein amino acid 157 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 157 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 77 is other than glycine, such as wherein amino acid 77 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G78. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G78. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G78. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G78.

4-1BBL with S158 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 81 049999

98% or at least 99% relative to the amino acid sequence depicted in Fig. 36DDD, wherein amino acid 158 (designated x) is an amino acid other than serine, for example, wherein amino acid 158 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 78 is other than serine, such as wherein amino acid 78 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S79. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S79. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S79. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S79.

4-1BBL with replacement D184

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36EEE, wherein amino acid 184 (designated x) is an amino acid other than aspartic acid, such as wherein amino acid 184 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 104 is other than aspartic acid, such as wherein amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D105. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at D105. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D105. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at D105.

4-1BBL with L185 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36FFF, wherein amino acid 185 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 185 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90% amino acid sequence identity,

- 82 049999 at least 95%, at least 98% or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 213, wherein amino acid 105 is different from leucine, for example, wherein amino acid 105 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L106. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L106. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L106. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L106.

4-1BBL with replacement P186

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36GGG, wherein amino acid 186 (denoted by x) is an amino acid other than proline, such as wherein amino acid 186 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 106 is other than proline, such as wherein amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P107. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at P107. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P107. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at P107.

4-1BBL with replacement P187

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36HHH, wherein amino acid 187 (denoted by x) is an amino acid other than proline, such as wherein amino acid 187 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 107 is other than proline, such as wherein amino acid 107 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having identity

- 83 049999 an amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at P108. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at P108. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at P108. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 with an amino acid substitution at P108.

4-1BBL with S189 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36III, wherein amino acid 189 (denoted by x) is an amino acid other than serine, such as wherein amino acid 189 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 109 is other than serine, such as wherein amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S110. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S110. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S110. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S110.

4-1BBL with S190 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36JJJ, wherein amino acid 190 (denoted by x) is an amino acid other than serine, such as wherein amino acid 190 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 110 is other than serine, such as wherein amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S111. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S111. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure

- 84 049999 disclosure, comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at S111. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at S111.

4-1BBL with replacement E191

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36KKK, wherein amino acid 191 (denoted by x) is an amino acid other than glutamic acid, such as wherein amino acid 191 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 111 is other than glutamic acid, such as wherein amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E112. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E112. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E112. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E112.

4-1BBL with R193 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36LLL, wherein amino acid 193 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 193 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 113 is different from arginine, such as wherein amino acid 113 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R114. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R114. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R114. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R114.

- 85 049999

4-1BBL with replacement N194

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36MMM, wherein amino acid 194 (designated x) is an amino acid other than asparagine, such as wherein amino acid 194 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 114 is other than asparagine, such as wherein amino acid 114 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at N115. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at N115. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at N115. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at N115.

4-1BBL with S195 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36NNN, wherein amino acid 195 (denoted by x) is an amino acid other than serine, such as wherein amino acid 195 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 115 is other than serine, such as wherein amino acid 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S116. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at S116. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S116. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at S116.

4-1BBL with F197 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36OOOO, wherein amino acid 197 (designated x) is an amino acid other than phe

- 86 049999 nilalanine, for example, where amino acid 197 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 117 is other than phenylalanine, such as wherein amino acid 117 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F118. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at F118. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F118. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at F118.

4-1BBL with Q210 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36PPP, wherein amino acid 210 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 210 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 130 is other than glutamine, such as wherein amino acid 130 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q131. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q131. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q131. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q131.

4-1BBL with R211 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36QQQ, wherein amino acid 211 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 211 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 131 is other than arginine,

- 87 049999 example, where amino acid 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R132. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R132. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R132. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R132.

4-1BBL with L212 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36RRR, wherein amino acid 212 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 212 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 132 is other than leucine, such as wherein amino acid 132 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L133. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L133. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L133. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L133.

4-1BBL with G213 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36SSS, wherein amino acid 213 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 213 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 133 is other than glycine, such as wherein amino acid 133 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in

- 88 049999

SEQ ID NO: 214, with an amino acid substitution at G134. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 214, with an amino acid substitution at G134. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at G134. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 with an amino acid substitution at G134.

4-1BBL with V214 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36TTT, wherein amino acid 214 (denoted by x) is an amino acid other than valine, such as wherein amino acid 214 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 134 is other than valine, such as wherein amino acid 134 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V135. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V135. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V135. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V135.

4-1BBL with replacement H215

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36UUU, wherein amino acid 215 (denoted by x) is an amino acid other than histidine, such as wherein amino acid 215 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 135 is other than histidine, such as wherein amino acid 135 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 with an amino acid substitution at H136. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or less amino acid sequence identity to the

- 89 049999 at least 99% relative to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at H136. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at H136.

4-1BBL with L216 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36VVV, wherein amino acid 216 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 216 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 136 is other than leucine, such as wherein amino acid 136 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L137. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L137. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L137. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at L137.

4-1BBL with replacement H217

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36WWW, wherein amino acid 217 (denoted by x) is an amino acid other than histidine, such as wherein amino acid 217 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 137 is other than histidine, such as wherein amino acid 137 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at H138. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at H138. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at H138. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at H138.

4-1BBL with T218 replacement

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 90 049999 of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in Fig. 36XXX, wherein amino acid 218 (designated x) is an amino acid other than threonine, such as wherein amino acid 218 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 138 is other than threonine, such as wherein amino acid 138 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T139. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T139. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T139. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T139.

4-1BBL with replacement E219

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36YYY, wherein amino acid 219 (denoted by x) is an amino acid other than glutamic acid, such as wherein amino acid 219 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 139 is other than glutamic acid, such as wherein amino acid 139 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E140. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at E140. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E140. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at E140.

4-1BBL with R221 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36ZZZ, wherein amino acid 221 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 221 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant, when

- 91 049999 present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence presented in SEQ ID NO: 213, wherein amino acid 141 is other than arginine, such as wherein amino acid 141 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R142. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R142. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R142. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R142.

4-1BBL with R223 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36AAAA, wherein amino acid 223 (denoted by x) is an amino acid other than arginine, such as wherein amino acid 223 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 143 is other than arginine, such as wherein amino acid 143 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R144. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at R144. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R144. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at R144.

4-1BBL with replacement H224

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36BBBB, wherein amino acid 224 (denoted by x) is an amino acid other than histidine, such as wherein amino acid 224 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 144 is other than histidine, such as wherein amino acid 144 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

- 92 049999

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at H145. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at H145. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at H145. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at H145.

4-1BBL with W226 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36CCCC, wherein amino acid 226 (denoted by x) is an amino acid other than tryptophan, such as wherein amino acid 226 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 146 is other than tryptophan, such as wherein amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at W147. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at W147. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at W147. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at W147.

4-1BBL with L228 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36DDDD, wherein amino acid 228 (denoted by x) is an amino acid other than leucine, such as wherein amino acid 228 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 148 is other than leucine, such as wherein amino acid 148 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at L149. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid

- 93 049999 the sequence presented in SEQ ID NO: 214, with an amino acid substitution at L149. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at L149. In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at L149.

4-1BBL with replacement T229

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36EEEE, wherein amino acid 229 (designated x) is an amino acid other than threonine, such as wherein amino acid 229 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 149 is other than threonine, such as wherein amino acid 149 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T150. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T150. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T150. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T150.

4-1BBL with Q230 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36FFFF, wherein amino acid 230 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 230 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 150 is other than glutamine, such as wherein amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q151. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at Q151. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at Q151. In some cases, a 4-1BBL polypeptide variant present in a

- 94 049999 multimeric polypeptide according to the present disclosure comprises the amino acid sequence presented in SEQ ID NO: 215, with an amino acid substitution at Q151.

4-1BBL with G231 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36GGGG, wherein amino acid 231 (denoted by x) is an amino acid other than glycine, such as wherein amino acid 231 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 151 is other than glycine, such as wherein amino acid 151 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G152. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at G152. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G152. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at G152.

4-1BBL with replacement T233

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36HHHH, wherein amino acid 233 (denoted by x) is an amino acid other than threonine, such as wherein amino acid 233 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 153 is other than threonine, such as wherein amino acid 153 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T154. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at T154. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T154. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at T154.

4-1BBL with V234 replacement

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 95 049999

98% or at least 99% relative to the amino acid sequence depicted in Fig. 36IIII, wherein amino acid 234 (designated x) is an amino acid other than valine, for example, wherein amino acid 234 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 154 is other than valine, such as wherein amino acid 154 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V155. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214, with an amino acid substitution at V155. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V155. In some cases, a 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215, with an amino acid substitution at V155.

Representative multimeric polypeptides containing the immunomodulatory polypeptide 4-1BBL

Exemplary multimeric polypeptides that are suitable for use in the method of the present invention are described below.

K127

In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a B2M polypeptide; and iii) a variant 4-1BBL polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (designated x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; or a variant 4-1BBL polypeptide of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at K47, such as wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Arg, His, Asp, or Glu; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a 4-1BBL polypeptide variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (denoted by x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; or a variant 4-1BBL polypeptide of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at K47, such as wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Arg, His, Asp, or Glu; ii) an MHC class I heavy chain; and iii) an Fc polypeptide. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a B2M polypeptide; iii) a first variant 4-1BBL polypeptide of the present invention; iv) a second variant of the 4-1BBL polypeptide of the present invention; and v) a third variant of the 4-1BBL polypeptide of the present invention; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, each of the first, second and

- 96 049999 of the third variant 4-1BBL polypeptides comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (designated x) is an amino acid other than lysine, for example, wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; for example, each of the first, second, and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at K47, for example, wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, BMetyl, Asn, Gln, Arg, His, Asp, or Glu. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a first variant 4-1BBL polypeptide of the present invention; ii) a second variant 4-1BBL polypeptide of the present invention; and iii) a third variant 4-1BBL polypeptide of the present invention; iv) an MHC class I heavy chain; and v) an Fc polypeptide. In some cases, each of the first, second, and third variant 41BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (designated x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; for example, each of the first, second, and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at K47, for example, wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Metyl, Asn, Gln, Arg, His, Asp, or Glu. In some cases, a variant 4-1BBL polypeptide comprises a K127 substitution and an M91 substitution (based on the numbering depicted in Fig. 36A). In some cases, a variant 4-1BBL polypeptide comprises a K127 substitution and an F92 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a Q94 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L95 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a V96 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a Q98 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 41BBL polypeptide variant comprises a K127 substitution and a N99 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a V100 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and an L101 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and an L102 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and an I103 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a D104 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a G105 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a P106 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L107 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a S108 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a W109 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a Y110 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a S111 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a D112 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a P113 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a G114 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L115 substitution (based on the numbering depicted in Fig. 36A).

In some cases, the multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b)

- 97 049999 a second polypeptide comprising, in N-terminal to C-terminal direction: i) a first 4-1BBL polypeptide variant of the present invention; ii) a linker; iii) a second 4-1BBL polypeptide variant of the present invention; iv) a linker; v) a third 4-1BBL polypeptide variant of the present invention; vi) an MHC class I heavy chain; and vii) an Fc polypeptide. In some cases, each of the first, second, and third 4-1BBL polypeptide variants comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence depicted in Fig. 36B, wherein amino acid 127 (designated x) is an amino acid other than lysine, such as wherein amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; for example, each of the first, second, and third variant 4-1BBL polypeptides comprises an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at K47, for example, wherein amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases, the linker comprises the sequence (GSSSS)n (SEQ ID NO: 131), wherein n is 1, 2, 3, 4, or 5. In some embodiments, n is 4. In some embodiments, n is 5. In some cases, the 4-1BBL polypeptide variant comprises a K127 substitution and an M91 substitution (based on the numbering depicted in Fig. 36A). In some cases, the 4-1BBL polypeptide variant comprises a K127 substitution and an F92 substitution (based on the numbering depicted in Fig. 36A). In some cases, the 4-1BBL polypeptide variant comprises a K127 substitution and a Q94 substitution (based on the numbering depicted in Fig. 36A). In some cases, the 4-1BBL polypeptide variant comprises a K127 substitution and a L95 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a V96 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 41BBL polypeptide variant comprises a K127 substitution and a Q98 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a N99 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a V100 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L101 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L102 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a I103 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a D104 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a G105 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a P106 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L107 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a S108 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a W109 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a Y110 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a S111 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a D112 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a P113 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a G114 substitution (based on the numbering depicted in Fig. 36A). In some cases, a 4-1BBL polypeptide variant comprises a K127 substitution and a L115 substitution (based on the numbering depicted in Fig. 36A).

Q227

In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a B2M polypeptide; and iii) a variant 4-1BBL polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or a 4-1BBL polypeptide variant of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at Q147, such as wherein amino acid 147 is Gly, Ala, Val, Leu, Ile,

- 98 049999

Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys Arg, His, Asp, or Glu; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a variant of a 4-1BBL polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (designated x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or a variant 4-1BBL polypeptide of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at Q147, such as wherein amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys Arg, His, Asp, or Glu; ii) an MHC class I heavy chain; and iii) an Fc polypeptide. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a B2M polypeptide; iii) a first variant 4-1BBL polypeptide of the present invention; iv) a second variant 4-1BBL polypeptide of the present invention; and v) a third variant 4-1BBL polypeptide of the present invention; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, each of the first, second, and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (designated x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or each of the first, second and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at Q147, such as wherein amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a first variant 4-1BBL polypeptide of the present invention; ii) a second variant 4-1BBL polypeptide of the invention; and iii) a third variant 4-1BBL polypeptide of the invention; iv) an MHC class I heavy chain; and v) an Fc polypeptide. In some cases, each of the first, second, and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or each of the first, second and third variant 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, with an amino acid substitution at Q147, such as wherein amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, a multimeric polypeptide of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a first variant 4-1BBL polypeptide of the present invention; ii) a linker; iii) a second embodiment of a 4-1BBL polypeptide of the invention; iv) a linker; v) a third embodiment of a 4-1BBL polypeptide of the invention; vi) an MHC class I heavy chain; and vii) an Fc polypeptide. In some cases, each of the first, second, and third embodiment 4-1BBL polypeptides comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Fig. 36D, wherein amino acid 227 (denoted by x) is an amino acid other than glutamine, such as wherein amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or each of the first, second and third variant 4-1BBL polypeptides contains

- 99 049999 an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98% or at least 99% to the amino acid sequence presented in SEQ ID NO: 213, with an amino acid substitution at Q147, for example, wherein amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the linker comprises the sequence (GSSSS)n (SEQ ID NO: 131), wherein n is 1, 2, 3, 4, or 5. In some cases, the linker comprises the sequence (GSSSS)n (SEQ ID NO: 131), wherein n is 4. In some cases, the linker comprises the sequence (GSSSS)n (SEQ ID NO: 131), wherein n is 5. In some cases, the linker comprises the sequence (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the sequence (GGGGS)n, wherein n is 2. In some cases, the linker comprises the sequence (GGGGS)n, wherein n is 3. In some cases, the linker comprises the sequence (GGGGS)n, wherein n is 4. In some cases, the linker comprises the sequence (GGGGS)n, where n is 5.

Multiple immunomodulatory domains

As noted above, in some cases, the multimeric polypeptide comprises two or more immunomodulatory polypeptides. In some cases, at least one of the two or more immunomodulatory polypeptides is a variant of the immunomodulatory polypeptide. For example, in the case of IL-2/synTac, in some cases, at least one of the two or more immunomodulatory polypeptides is a variant of the IL-2 polypeptide. As another example, in the case of 4-1BBL/synTac, in some cases, at least one of the two or more immunomodulatory polypeptides is a variant of the 4-1BBL polypeptide.

In some cases, the multimeric polypeptide comprises two or more copies of the IL-2 polypeptide variant of the present invention. In some cases, the two or more IL-2 polypeptide variants are on the same polypeptide chain of the multimeric polypeptide. In some cases, the two or more IL-2 polypeptide variants are on separate polypeptide chains of the multimeric polypeptide.

In some cases, the multimeric polypeptide comprises a first immunomodulatory polypeptide and at least a second immunomodulatory polypeptide, wherein the first immunomodulatory polypeptide is a variant of the IL-2 polypeptide of the present invention, and the second immunomodulatory polypeptide is not an IL-2 polypeptide. For example, in some cases, the second immunomodulatory polypeptide is a member of the tumor necrosis factor (TNF) superfamily; for example, FasL polypeptide, 4-1BBL polypeptide, CD40 polypeptide, OX40L polypeptide, CD30L polypeptide, CD70 polypeptide, etc. In some cases, the second immunomodulatory polypeptide of the multimeric polypeptide is a T cell costimulator polypeptide and is a member of the immunoglobulin (Ig) superfamily; for example, CD7 polypeptide, CD86 polypeptide, ICAM polypeptide, etc. In some cases, the second immunomodulatory polypeptide is 4-1BBL, OX40L, ICOSL, ICAM, PD-L1, CD86, FasL and PD-L2. Suitable immunomodulatory polypeptides of the multimeric polypeptide of the present invention include, for example, CD7, CD30L, CD40, CD70, CD83, HLAG, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4 or HVEM.

Additional T cell modulating domains (MOD) that may be included in a multimeric polypeptide of the present invention include naturally occurring or synthetic human gene products (protein), affinity reagents (e.g., antibody, antibody fragment, single chain Fvs, aptamers, nanobody) targeting a human gene product, including but not limited to all secreted proteins resulting from classical and non-classical (e.g., FGF2, IL1, S100A4) secretion mechanisms, and ecto-domains of all cell surface proteins anchored via natural genetically encoded protein segments (monotopic or polytopic membrane or post-translational modifications such as GPI linkages). Any natural or synthetic affinity reagent (e.g., antibody, antibody fragment, single-chain Fv, aptamer, nanobody, lectin, etc.) that targets a cell surface glycan or other post-translational modification (e.g., sulfated sites). Examples include, but are not limited to, members of the TNF/TNFR family (OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70) or affinity reagents directed to members of the TNF/TNFR family; members of the immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains, ICOS, ICOS ligand, HHLA2, butyrophilins, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR), killer cell immunoglobulin-like receptors (KIR)), members of the lectin superfamily, selectins, cytokine/chemokine receptors and cytokine/chemokine receptors, growth factors and growth factor receptors, molecules adhesion (integrins, fibronectins, cadherins) or ecto-domains of polytopic integral membrane proteins or affinity reagents directed to the immunoglobulin superfamily and the listed gene products. As well as active homologues/orthologues of such gene products, including but not limited to viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences

- 100 049999 sequences, eukaryotic pathogens (e.g. Schistosoma, Plasmodium, Babesia, Eimeria, Theileria, Toxoplasma), Entamoeba, Leishmania and Trypanosoma) and mammalian coding regions extracted. In addition, the MOD may contain a small molecule drug targeting a human gene product.

Additional polypeptides

The polypeptide chain of the multimeric 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 may be included at the N-terminus of the polypeptide chain of the multimeric polypeptide, at the C-terminus of the polypeptide chain of the multimeric polypeptide, or within the polypeptide chain of the multimeric polypeptide.

Epitope tags

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 79); FLAG (e.g., DYKDDDDK (SEQ ID NO: 80); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 81), and the like.

Affine domains

Affinity domains include peptide sequences that can interact with a binding partner, such as one immobilized on a solid support, such a partner being useful for identification and purification. DNA sequences encoding multiple consecutive single-stranded amino acids, such as histidine, when condensed with the expressed protein can be used to perform a one-step purification of a recombinant protein by high-affinity binding to a resin column such as nickel-sepharose. Examples of affinity domains include His5 (HHHHH) (SEQ ID NO: 82), HisX6 (HHHHHH) (SEQ ID NO: 83), Cmyc (EQKLISEEDL) (SEQ ID NO: 81), Flag (DYKDDDDK) (SEQ ID NO: 80), StrepTag (WSHPQFEK) (SEQ ID NO: 84), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 79), glutathione-S transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 85), Phe-His-HisThr (SEQ ID NO: 86), chitin binding domain, S peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 87), metal binding domains, such as zinc binding domains or calcium binding domains, such as those derived from calcium binding proteins, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, centrin, large calpain subunits, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K and calretinin, inteins, biotin, streptavidin, MyoD, hazel zipper sequences and maltose binding protein.

Examples of IL-2/multimeric polypeptides

The following are non-limiting embodiments of an IL2/synTac multimeric polypeptide suitable for use in the treatment method of the present invention.

In some cases, the IL-2/synTac multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence depicted in Fig. 34A; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an IL-2 variant polypeptide of the present invention; ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. In some cases, the IL-2 variant polypeptide comprises an H16A and F42A substitution. In some cases, the IgG1 Fc polypeptide comprises the N297A substitution. In some cases, the IgG1 Fc polypeptide comprises the L234A substitution and the L235A substitution. In some cases, the IgG1 Fc polypeptide comprises the L234F substitution and the L235E substitution. In some cases, the IgG1 Fc polypeptide comprises the L234F substitution, the L235E substitution, and the P331S substitution. In some cases, the second polypeptide comprises two copies of a variant IL-2 polypeptide. In some cases, the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide. In some cases, the second polypeptide comprises a peptide linker between one or more of (a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide; (b) a variant IL-2 polypeptide and an MHC heavy chain polypeptide; and (c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 207), (GGGGS) ₄ (SEQ ID NO: 208), and AAAGG (SEQ ID NO: 73). In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33B. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33C. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33D.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a B2-microglobulin polypeptide comprising the amino acid sequence depicted in Fig. 34A; and b) a second polypeptide comprising, in

- 101 049999 in a row from the N-terminus to the C-terminus: i) an IL-2 variant polypeptide comprising the amino acid sequence depicted in Fig. 34B; ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. In some cases, the IgG1 Fc polypeptide comprises an N297A substitution. In some cases, the IgG1 Fc polypeptide comprises an L234A substitution and an L235A substitution. In some cases, the IgG1 Fc polypeptide comprises a L234F substitution and an L235E substitution. In some cases, the IgG1 Fc polypeptide comprises a L234F substitution, a L235E substitution, and a P331S substitution. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33B. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33C. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33D. In some cases, the second polypeptide comprises two copies of an IL-2 variant polypeptide. In some cases, the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide. In some cases, the second polypeptide comprises a peptide linker between one or more of: (a) a first copy of an IL-2 variant polypeptide and a second copy of an IL2 variant polypeptide; (b) an IL-2 variant polypeptide and an MHC heavy chain polypeptide; and c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 207), (GGGGS) ₄ (SEQ ID NO: 208), and AAAGG (SEQ ID NO: 73).

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77); ii) a B2 microglobulin polypeptide comprising the amino acid sequence depicted in Fig. 34A; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an IL-2 variant polypeptide comprising the amino acid sequence depicted in Fig. 34B; ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising the amino acid sequence depicted in Fig. 33A, 33B, 33C, or 33D. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33B. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33C. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33D. In some cases, the second polypeptide comprises two copies of the IL-2 variant polypeptide. In some cases, the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide. In some cases, the second polypeptide comprises a peptide linker between one or more of: (a) a first copy of the IL-2 variant polypeptide and a second copy of the IL-2 variant polypeptide; (b) a IL-2 variant polypeptide and an MHC heavy chain polypeptide; and (c) between an MHC heavy chain polypeptide and the IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO:207), (GGGGS) ₄ (SEQ ID NO:208), and AAAGG (SEQ ID NO:73). In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33B. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33C. In some cases, the IgG1 Fc polypeptide comprises the amino acid sequence shown in Fig. 33D.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising the amino acid sequence depicted in Fig. 31; and b) a second polypeptide comprising the amino acid equivalent depicted in Fig. 22.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising the amino acid sequence depicted in Fig. 31; and b) a second polypeptide comprising the amino acid equivalent depicted in Fig. 25.

In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising the amino acid sequence depicted in Fig. 31; and ab) a second polypeptide comprising the amino acid equivalent depicted in Fig. 28.

Compositions, doses and routes of administration

In carrying out the method of treatment of the present invention, synTac can be formulated in a composition containing a pharmaceutically acceptable excipient, and the immune checkpoint inhibitor can be formulated in a composition containing a pharmaceutically acceptable excipient. For simplicity, the term active ingredient is used below to refer to synTac or the immune checkpoint inhibitor. In general, synTac and the immune checkpoint inhibitor are present in separate compositions.

The composition may contain a pharmaceutically acceptable excipient, a wide variety of which are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients have been described in detail in various publications including, for example, Remington: The Science and Practice of Pharmacy, ^19th Ed. (1995), or the latest edition, Mack Publishing Co; A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition,

- 102 049999

Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., ^3rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutical composition may comprise synTac or an immune checkpoint inhibitor and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition will be suitable for administration to a subject, such as being sterile. For example, in some cases, the pharmaceutical composition will be suitable for administration to a human, such as when the composition is sterile and does not contain detectable pyrogens and/or other toxins.

The compositions may include other components such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. of pharmaceutical grade. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate them to physiological conditions, such as pH adjusting agents and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochlorides, sulfates, solvates (e.g., mixed ionic salts, water, organic substances), hydrates (e.g., water), and the like.

For example, the compositions may include an aqueous solution, a powder form, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition may be formulated according to the various routes of administration described below.

When the active substance (synTac or immune checkpoint inhibitor) is administered by injection (e.g., subcutaneously, intraperitoneally, intramuscularly and/or intravenously) directly into tissue, the formulation can be provided as a ready-to-use dosage form or as a non-aqueous form (e.g., a reconstitutable, shelf-stable powder) or as an aqueous form, such as a liquid, consisting of pharmaceutically acceptable carriers and excipients. The compositions can also be provided with the ability to increase the serum half-life of the active substance after administration. For example, the protein can be presented as a liposomal composition, prepared as a colloid, or according to other conventional methods to increase the serum half-life. A variety of methods are available for the preparation of liposomes, as described, for example, in Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9467, U.S. Patents 4,235,871, 4,501,728, and 4,837,028. The formulations may also be provided in controlled release or slow release forms.

Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, antioxidants, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickeners, stabilizers and preservatives. For example, the pharmaceutical composition in question may be present in a container, for example, a sterile container such as a syringe. The compositions may be presented in sealed single-dose or multi-dose containers such as ampoules and vials and may be stored under freeze-drying conditions (lyophilized conditions), requiring only the addition of a sterile liquid vehicle, such as water for injection, immediately before use. Injectable solutions and suspensions for immediate administration may be prepared from sterile powders, granules and tablets.

The concentration of the active substance (synTac or immune checkpoint inhibitor) in the preparation can vary widely (e.g., from less than about 0.1%, typically 2% or at least about 2%, to 20% to 50% or more by weight) and is typically selected based primarily on fluid volumes, viscosity, and patient-related factors, in accordance with the particular route of administration selected and the needs of the patient.

The present disclosure provides a container containing an active substance (synTac or an immune checkpoint inhibitor), such as a container containing a liquid composition containing the active substance. The container may be, for example, a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, the container and the composition are sterile.

The present disclosure relates to compositions, including pharmaceutical compositions, comprising an active substance (synTac or an immune checkpoint inhibitor). The composition may comprise: a) an active substance (synTac or an immune checkpoint inhibitor); and b) an excipient, as described above. In some embodiments, the excipient is a pharmaceutically acceptable excipient.

Compositions containing a nucleic acid or a recombinant expression vector

In some cases, synTac is administered as a multimeric polypeptide per se. In other cases, instead of administering synTac as a multimeric polypeptide per se, one or more nucleic acids comprising nucleotide sequences encoding synTac are administered. The nucleic acids may be present in a pharmaceutical composition. The pharmaceutical composition may comprise one or more recombinant expression vectors comprising one or more nucleic acids. A wide variety of pharmaceutical

- 103 049999 pharmaceutically acceptable excipients and there is no need to discuss them in detail here. Pharmaceutically acceptable excipients have been described in detail in various publications including, for example, A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., ^3rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutical composition may comprise a nucleic acid or recombinant expression vector of the present invention in an amount of from about 0.001% to about 90% (w/w). In the description of the formulations below, the term nucleic acid or recombinant expression vector will be understood to include a nucleic acid or recombinant expression vector containing nucleotide sequences encoding synTac.

The nucleic acid or recombinant expression vector may be mixed, encapsulated, conjugated or otherwise linked to other compounds or mixtures of compounds; such compounds may include, for example, liposomes or receptor molecules. The nucleic acid or recombinant expression vector may be combined in a formulation with one or more components that promote uptake, distribution and/or absorption.

The nucleic acid or recombinant expression vector composition may be formulated in any of a variety of possible dosage forms, such as, but not limited to, tablets, capsules, gelcaps, liquid syrups, soft gels, suppositories, and enemas. The nucleic acid or recombinant expression vector composition may also be formulated as a suspension in an aqueous, non-aqueous, or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

The composition comprising the nucleic acid or recombinant expression vector may be a liposomal composition. As used herein, the term liposome means a vesicle consisting of amphiphilic lipids arranged in a spherical or bilayered bilayer. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed of lipophilic material and an aqueous interior that contains the delivery composition. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form a stable complex. It is believed that pH-sensitive or negatively charged liposomes entrap DNA rather than form complexes with it. Both cationic and non-cationic liposomes can be used to deliver the subject nucleic acid or recombinant expression vector.

Liposomes also include sterically stabilized liposomes, a term which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in an increased circulation duration compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their use are further described in US Pat. No. 6,287,860, incorporated herein by reference in its entirety.

The compositions and formulations of the present invention may also include surfactants. The use of surfactants in drug products, formulations, and emulsions is well known in the art. Surfactants and their use are further described in US Pat. No. 6,287,860.

In one embodiment, various penetration enhancers are included for efficient delivery of nucleic acids. In addition to facilitating diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the penetration of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their use are further described in U.S. Patent No. 6,287,860, incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gelcaps, sachets, tablets or minitablets. Thickening agents, flavoring agents, diluents, emulsifiers, dispersing agents or binders may be desirable. Suitable oral preparations include those in which the antisense nucleic acid is administered in combination with one or more penetration enhancers, surfactants and chelators. Suitable surfactants include, but are not limited to, fatty acids

- 104 049999 and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts and fatty acids and their use are further described in U.S. Patent No. 6,287,860. Also suitable are combinations of penetration enhancers, such as fatty acids/salts in combination with bile acids/salts. An example of a suitable combination is sodium lauric acid, capric acid and UDCA. Other penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and polyoxyethylene 20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanolamine, N.N-dimethylacetamide. KH-dimethylformamide. 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol and AZONE™.

Treatment methods

The present disclosure provides a method of treatment comprising administering synTac and an immune checkpoint inhibitor. In some cases, the method comprises administering to an individual in need thereof: a) a first composition comprising synTac; and b) a second composition comprising an immune checkpoint inhibitor. In some cases, the method of treatment of the present invention comprises administering to an individual in need thereof: a) a first composition comprising one or more recombinant expression vectors comprising nucleotide sequences encoding synTac; and b) a second composition comprising an immune checkpoint inhibitor. In some cases, the method of treatment of the present invention comprises administering to an individual in need thereof: a) a first composition comprising one or more mRNA molecules comprising nucleotide sequences encoding a multimeric polypeptide; and b) a second composition comprising an immune checkpoint inhibitor. In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint polypeptide.

Thus, for example, a method of treatment according to the present invention may comprise co-administering synTac (e.g., synTac 4-1BBL, synTac IL-2, etc., as described above) and an immune checkpoint specific antibody. By co-administering is meant that both synTac (e.g., synTac 4-1BBL, synTac IL-2, etc., as described above) and an immune checkpoint specific antibody are administered to the individual, although not necessarily at the same time, to achieve a therapeutic effect that results from the use of both synTac and the immune checkpoint inhibitor. The administration of synTac (e.g., synTac 4-1BBL, synTac IL-2, etc.) and the immune checkpoint specific antibody can be substantially simultaneous, such that synTac (e.g., synTac 4-1BBL, IL-2 synTac, etc.) can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) relative to the administration of the immune checkpoint specific antibody. In some cases, synTac (e.g., synTac 4-1BBL, synTac IL-2, etc.) is administered to an individual who is being treated with an immune checkpoint specific antibody. Administration of synTac (e.g. synTac 4-1BBL, synTac IL-2, etc.) and immune checkpoint specific antibody may occur at different times and/or frequencies.

Thus, for example, a method of treatment according to the present invention may comprise co-administration of synTac 4-1BBL and an immune checkpoint specific antibody. By co-administration is meant that synTac 4-1BBL and an immune checkpoint specific antibody are administered to an individual, although not necessarily at the same time, to achieve a therapeutic effect that results from the use of both synTac and an immune checkpoint inhibitor. The administration of synTac 4-1BBL and the immune checkpoint-specific antibody may be substantially simultaneous, such as synTac 4-1BBL may be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) relative to the administration of the immune checkpoint-specific antibody. In some cases, synTac 4-1BBL is administered to an individual who is being treated with an immune checkpoint-specific antibody. The administration of synTac 4-1BBL and the immune checkpoint-specific antibody may occur at different times and/or with different frequencies.

As another example, a method of treatment according to the present invention may comprise co-administering synTac (e.g., synTac IL-2, as described above) and an immune checkpoint specific antibody. By co-administering is meant that synTac IL-2 and the immune checkpoint specific antibody are administered to the individual, although not necessarily at the same time, to achieve a therapeutic effect that results from the use of both synTac and the immune checkpoint inhibitor. The administration of synTac IL-2 and the immune checkpoint specific antibody may be substantially simultaneous, e.g., synTac IL-2 may be administered to the individual within about 1 minute to about 24 hours (e.g.,

- 105 049999 for about 1 minute, for about 5 minutes, for about 15 minutes, for about 30 minutes, for about 1 hour, for about 4 hours, for about 8 hours, for about 12 hours, or for about 24 hours) relative to the administration of an immune checkpoint-specific antibody. In some cases, synTac IL-2 is administered to an individual who is being treated with an immune checkpoint-specific antibody. The administration of synTac IL-2 and the immune checkpoint-specific antibody may occur at different times and/or with different frequencies.

The present disclosure provides a method of treatment comprising administering synTac and an immune checkpoint inhibitor. The method of treatment of the present invention can modulate the activity of a target T cell. In some cases, such as when the target T cell is a CD8+ T cell, the multimeric polypeptide comprises MHC class I polypeptides (e.g., β2 microglobulin and MHC class I heavy chain). In some cases, such as when the target T cell is a CD4+ T cell, the multimeric polypeptide comprises MHC class II polypeptides (e.g., MHC class II α chain; MHC class II β chain).

When the multimeric polypeptide comprises an immunomodulatory polypeptide that is an activating polypeptide, the method of the present invention activates an epitope-specific T cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present in a cancer cell, and contact of the epitope-specific T cell with the multimeric polypeptide increases the cytotoxic activity of the T cell with respect to the cancer cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present in a cancer cell, and the method of the present invention increases the number of epitope-specific T cells.

In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the cytotoxic activity of the T cell towards the virus-infected cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the number of epitope-specific T cells.

When the multimeric polypeptide of the present invention includes an immunomodulatory polypeptide that is an inhibitory polypeptide, the method of the present invention inhibits an epitope-specific T cell. In some cases, the epitope-specific T cell is a self-reactive T cell that is specific for an epitope present in an autoantigen, and the method of the present invention reduces the number of self-reactive T cells.

In some cases, the immunomodulatory polypeptide is an activating polypeptide and the multimeric polypeptide activates an epitope-specific T cell. In some cases, the epitope is a cancer-associated epitope and the multimeric polypeptide enhances the activity of a T cell specific for the cancer-associated epitope.

In some cases, the method of treatment of the present invention treats cancer in an individual having cancer. Thus, the present disclosure provides a method of treating cancer in an individual, the method comprising administering to the individual: a) a multimeric polypeptide of the present invention or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) comprising nucleotide sequences encoding the multimeric polypeptide, wherein the multimeric polypeptide comprises a T-cell epitope that is a cancer epitope, and wherein the multimeric polypeptide comprises a stimulatory immunomodulatory polypeptide; and b) an immune checkpoint inhibitor. In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce the number of cancer cells in the individual. For example, in some cases, effective amounts of a multimeric polypeptide and an immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce the number of cancer cells in the individual by at least 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 the number of cancer cells in the individual before administration of the multimeric polypeptide and the immune checkpoint inhibitor or in the absence of administration of the multimeric polypeptide and the immune checkpoint inhibitor. In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce the number of cancer cells in the individual to undetectable levels. In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses,

- 106 049999 to an individual in need thereof, the tumor mass in the individual is reduced. For example, in some cases, effective amounts of a multimeric polypeptide and an immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce tumor mass in the individual by at least 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 the tumor mass in the individual before administration of the multimeric polypeptide and the immune checkpoint inhibitor or in the absence of administration of the multimeric polypeptide and the immune checkpoint inhibitor. In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, increase the survival time of the individual. For example, in some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, increase the survival time of the individual by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years compared to the expected survival time of the individual in the absence of administration of the multimeric polypeptide and the immune checkpoint inhibitor.

In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the cytotoxic activity of the T cell towards the virus-infected cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the number of epitope-specific T cells.

As noted above, in some instances, when performing a method of treating a subject, the multimeric polypeptide is administered to an individual in need thereof as the polypeptide itself. In other instances, when performing a method of treating a subject, one or more nucleic acids comprising nucleotide sequences encoding the multimeric polypeptide are administered to an individual in need thereof. Thus, in other instances, one or more nucleic acids encoding synTac are administered to an individual in need thereof.

Doses - synTac

Appropriate doses of synTac can be determined by the attending physician or other qualified healthcare professional based on a variety of clinical parameters. As is well known in the medical field, doses for any given patient will depend on many factors including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the time and route of administration, the patient's general health, and other concomitantly administered medications. The multimeric polypeptide (synTac) can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, such as from 0.1 mg/kg body weight to 10 mg/kg body weight, such as from 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below and above this exemplary range are contemplated, particularly taking into account the factors noted above. If the regimen is a continuous infusion, doses may also range from 1 mcg to 10 mg per kilogram of body weight per minute. The multimeric polypeptide can be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, such as from about 1 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, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of the multimeric polypeptide is from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition frequency for dosing based on the measured residence time and concentrations of the administered agent in body fluids or tissues. Following successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent relapse of the disease state, in which case the multimeric polypeptide is administered in maintenance doses ranging from 0.01 mcg to 100 g per kg body weight, from 0.1 mcg to 10 g per kg body weight, from 1 mcg to 1 g per kg body weight, from 10 mcg to 100 mg per kg body weight, from 100 mcg to 10 mg per kg body weight, or from 100 mcg to 1 mg per kg body weight.

It will be apparent to those skilled in the art that dosage levels may vary as a function of the particular multimeric polypeptide, the severity of the symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of the compound.

- 107 049999 by various means.

In some embodiments, multiple doses of the multimeric polypeptide (or nucleic acid or recombinant expression vector encoding them) are administered. The frequency of administration of the multimeric polypeptide may vary depending on any of a variety of factors, such as the severity of the symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once per month, twice per month, three times per month, every other week (qow), 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 per day (qid), or three times per day (tid).

The duration of administration of the multimeric polypeptide, such as the period of time during which the multimeric polypeptide is administered, may vary depending on any of a variety of factors, such as the patient's response, etc.

For example, the multimeric polypeptide can be administered for a period of time of from one day to one week, from two weeks to four weeks, from one month to two months, from two months to four months, from four months to 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.

Appropriate doses of synTac can be determined by the attending physician or other qualified healthcare professional based on a variety of clinical parameters. As is well known in the medical field, doses for any given patient will depend on many factors including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the time and route of administration, the patient's general health, and other concomitantly administered medications. The multimeric polypeptide (synTac) can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, such as from 0.1 mg/kg body weight to 10 mg/kg body weight, such as from 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below and above this exemplary range are contemplated, particularly taking into account the factors noted above. If the regimen is a continuous infusion, doses may also range from 1 mcg to 10 mg per kilogram of body weight per minute. The multimeric polypeptide can be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, such as from about 1 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, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of the multimeric polypeptide is from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition frequency for dosing based on the measured residence time and concentrations of the administered agent in body fluids or tissues. Following successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent relapse of the disease state, in which case the multimeric polypeptide is administered in maintenance doses ranging from 0.01 mcg to 100 g per kg body weight, from 0.1 mcg to 10 g per kg body weight, from 1 mcg to 1 g per kg body weight, from 10 mcg to 100 mg per kg body weight, from 100 mcg to 10 mg per kg body weight, or from 100 mcg to 1 mg per kg body weight.

It will be apparent to those skilled in the art that dosage levels may vary as a function of the particular multimeric polypeptide, the severity of the symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of the compound by a variety of means.

In some embodiments, multiple doses of the multimeric polypeptide (or nucleic acid or recombinant expression vector encoding them) are administered. The frequency of administration of the multimeric polypeptide may vary depending on any of a variety of factors, such as the severity of the symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once per month, twice per month, three times per month, every other week (qow), 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 per day (qid), or three times per day (tid).

The duration of administration of the multimeric polypeptide, such as the period of time during which the multimeric polypeptide is administered, may vary depending on any of a variety of factors, such as the patient's response, etc. For example, the multimeric polypeptide may be administered for a period of time of from one day to one week, from two weeks to four weeks, from one month to two months, from two months to four months, from four months to 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.

- 108 049999

Doses - Immune Checkpoint Inhibitor

Suitable doses of the immune checkpoint inhibitor can be determined by the attending physician or other qualified medical personnel based on a variety of clinical parameters. As is well known in the medical field, doses for any given patient will depend on many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the time and route of administration, the patient's general health, and other concomitantly administered medications. The immune checkpoint inhibitor can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, such as from 0.1 mg/kg body weight to 10 mg/kg body weight, such as from 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below and above this exemplary range are contemplated, particularly taking into account the factors noted above. If the regimen is a continuous infusion, doses may also range from 1 mcg to 10 mg per kilogram of body weight per minute. The immune checkpoint inhibitor can be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, such as from about 1 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, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of the immune checkpoint inhibitor is from 0.01 mcg to 100 g per kg body weight, from 0.1 mcg to 10 g per kg body weight, from 1 mcg to 1 g per kg body weight, from 10 mcg to 100 mg per kg body weight, from 100 mcg to 10 mg per kg body weight, or from 100 mcg to 1 mg per kg body weight. Those skilled in the art can readily estimate the frequency of repetition for dosing based on the measured residence time and concentrations of the administered agent in body fluids or tissues. Following successful treatment, it may be desirable to administer maintenance therapy to the patient to prevent relapse of the disease state, in which case the immune checkpoint inhibitor is administered at maintenance doses ranging from 0.01 mcg to 100 g per kg body weight, from 0.1 mcg to 10 g per kg body weight, from 1 mcg to 1 g per kg body weight, from 10 mcg to 100 mg per kg body weight, from 100 mcg to 10 mg per kg body weight, or from 100 mcg to 1 mg per kg body weight.

It will be apparent to those skilled in the art that dosage levels may vary as a function of the particular immune checkpoint inhibitor, the severity of the symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of the compound by a variety of means.

In some embodiments, multiple doses of the immune checkpoint inhibitor are administered. The frequency of administration of the immune checkpoint inhibitor may vary depending on any of a variety of factors, such as the severity of symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once per month, twice per month, three times per month, every other week (qow), 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 per day (qid), or three times per day (tid).

The duration of administration of the immune checkpoint inhibitor, such as the period of time over which the immune checkpoint inhibitor is administered, may vary depending on any of a variety of factors, such as the patient's response, etc. For example, the immune checkpoint inhibitor may be administered for a period of time of 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.

The following are non-limiting examples.

Pembrolizumab may be administered to an individual in need thereof in an amount of 2 mg/kg every 3 weeks. Pembrolizumab may be administered to an individual in need thereof in an amount of 200 mg every 3 weeks. In some cases, the method of the present invention involves reducing the amount of anti-PDT that must be administered to achieve a clinical effect. For example, in some cases, the amount of pembrolizumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of pembrolizumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Nivolumab may be administered to an individual in need thereof at 3 mg/kg every 2 weeks. Nivolumab may be administered to an individual in need thereof at 240 mg every 2 weeks. In some instances, the method of the present invention reduces the amount of anti-PDl that must be administered to achieve a clinical effect. For example, in some

- 109 049999 In some cases, the amount of nivolumab required to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of nivolumab required to achieve a clinical effect in the absence of treatment with synTac.

Atezolizumab may be administered to an individual in need thereof in an amount of 1200 mg every 3 weeks. In some cases, the method of the present invention comprises reducing the amount of αnmi-PD1 that must be administered to achieve a clinical effect. For example, in some cases, the amount of atezolizumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of atezolizumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Ipilimumab may be administered to an individual in need thereof at 3 mg/kg every 3 weeks. Ipilimumab may be administered to an individual in need thereof at 10 mg/kg every 3 weeks. Ipilimumab may be administered to an individual in need thereof at 10 mg/kg every 12 weeks. In some cases, the method of the present invention involves reducing the amount of anti-PDT that must be administered to achieve a clinical effect. For example, in some cases, the amount of ipilimumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of ipilimumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Routes of administration

The active substance (multimeric polypeptide; immune checkpoint inhibitor) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration. In some cases, synTac is administered by the first route of administration; and the immune checkpoint inhibitor is administered by a second route of administration that is different from the first route of administration. In some cases, synTac and the immune checkpoint inhibitor are administered by the same routes of administration.

Common and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral and other enteral and parenteral routes. Routes of administration may be combined or adjusted as needed depending on the multimeric polypeptide, immune checkpoint inhibitor and/or desired effect.

In some cases, the multimeric polypeptide is administered intravenously; and the immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered intramuscularly; and the immune checkpoint inhibitor is administered intramuscularly. In some cases, the multimeric polypeptide is administered locally; and the immune checkpoint inhibitor is administered locally. In some cases, the multimeric polypeptide is administered intratumorally; and the immune checkpoint inhibitor is administered intratumorally. In some cases, the multimeric polypeptide is administered peritumorally; and the immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intracranially; and the immune checkpoint inhibitor is administered intracranially. In some cases, the multimeric polypeptide is administered subcutaneously; and the immune checkpoint inhibitor is administered subcutaneously.

In some cases, the multimeric polypeptide is administered intravenously; and the immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intramuscularly; and the immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered systemically; and the immune checkpoint inhibitor is administered locally. In some cases, the multimeric polypeptide is administered intratumorally; and the immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered systemically; and the immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intravenously; and the immune checkpoint inhibitor is administered intracranially. In some cases, the multimeric polypeptide is administered subcutaneously; and the immune checkpoint inhibitor is administered intravenously.

The multimeric polypeptide and the immune checkpoint inhibitor may be administered to the host using any available conventional methods and routes suitable for the delivery of conventional drugs, including systemic or local routes. In general, routes of administration contemplated for use in the method of the present invention include, but are not limited to, enteral, parenteral and inhalation routes.

Parenteral routes of administration other than inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intratumoral, intratumoral, peritumoral, and intravenous, i.e., any route of administration other than via the alimentary canal. Parenteral administration

- 110 049999 may be administered for systemic or local delivery of the active substance (synTac or immune checkpoint inhibitor). If systemic delivery is desired, administration may include intravenous delivery.

Subjects suitable for treatment

Subjects suitable for treatment by the method of the present invention include individuals who have cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but who have not shown a response to the treatment, and individuals who have been treated for cancer and who initially showed a response to the treatment but subsequently became refractory to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have an infection (e.g., an infection with a pathogen such as a bacterium, virus, protozoa, etc.), including individuals who have been diagnosed with an infection and individuals who have been treated for an infection but who have not shown a response to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have a bacterial infection, including individuals who have been diagnosed with a bacterial infection, and individuals who have received treatment for a bacterial infection, but who have not shown a response to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have a viral infection, including individuals who have been diagnosed with a viral infection, and individuals who have received treatment for a viral infection, but who have not shown a response to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have an autoimmune disease, including individuals who have been diagnosed with an autoimmune disease, and individuals who have received treatment for an autoimmune disease, but who have not shown a response to the treatment.

In some cases, the method of the present invention includes administering synTac to an individual who is being treated with an immune checkpoint inhibitor. In some cases, the method of the present invention includes administering synTac to an individual who is being treated with an anti-PD1 antibody. For example, in some cases, the method of the present invention includes administering synTac to an individual who is being treated with pembrolizumab. As another example, in some cases, the method of the present invention includes administering synTac to an individual who is being treated with nivolumab. In some cases, the method of the present invention includes administering synTac to an individual who is being treated with an anti-PD-L1 antibody. For example, in some cases, the method of the present invention includes administering synTac to an individual who is being treated with atezolizumab. In some cases, the method of the present invention includes administering synTac to an individual who is being treated with an anti-CTLA4 antibody. For example, in some cases, the method of the present invention includes administering synTac to an individual who is being treated with ipilimumab. As another example, in some cases, the method of the present invention includes administering synTac to an individual who is being treated with tremelimumab.

In some cases, for example, when the epitope is an HPV epitope, a subject suitable for treatment by the method of the present invention is an individual who has been diagnosed with an HPV-associated cancer or an HPV-mediated cancer. HPV-associated and HPV-mediated cancers include, for example, head and neck cancer; cervical cancer; and genitourinary cancer.

Methods for selectively delivering a costimulatory polypeptide together with an immune checkpoint inhibitor to an individual

The present disclosure thus provides a method of delivering a costimulatory polypeptide, such as IL-2, or a reduced affinity variant of a naturally occurring costimulatory polypeptide, such as an IL-2 variant disclosed herein, to a selected T cell or selected T cell population, such as in a manner that it targets a TCR specific for a given epitope, together with co-administration of a checkpoint inhibitor, to provide a patient with the therapeutic effect of both selective delivery of the costimulatory polypeptide and the checkpoint inhibitor. The present disclosure provides a method of delivering a costimulatory polypeptide, such as IL-2, or a reduced affinity variant of a naturally occurring costimulatory polypeptide, such as an IL-2 variant disclosed herein, selectively to a target T cell bearing a TCR specific for an epitope present in a multimeric polypeptide of the present invention, together with co-administration of a checkpoint inhibitor, to provide a patient with the therapeutic effect of both the selective delivery of the costimulatory polypeptide and the checkpoint inhibitor. The method comprises contacting a population of T cells with a multimeric polypeptide of the present invention. The population of T cells may be a mixed population that comprises: i) a target T cell; and ii) non-target T cells that are not epitope-specific (e.g., T cells specific for epitopes other than the epitope to which the epitope-specific T cell binds). An epitope-specific T cell is specific for an epitope-presenting peptide present in a multi-

- 111 049999 meric polypeptide, and binds to an HLA peptide complex or an MHC peptide complex provided by the multimeric polypeptide. Contacting a population of T cells with the multimeric polypeptide delivers a costimulatory polypeptide (e.g., IL-2 or a reduced affinity variant of IL-2) present in the multimeric polypeptide selectively to T cells that are specific for the epitope presented in the multimeric polypeptide. The checkpoint inhibitor is co-administered with the multimeric polypeptide (either together or at a different time before and/or after administration of the multimeric polypeptide) to provide the patient with the therapeutic effect of selective delivery of both the costimulatory polypeptide and the checkpoint inhibitor.

Accordingly, the present disclosure provides a method of delivering to a patient (i) a checkpoint inhibitor as described above and (ii) a costimulatory polypeptide such as IL-2 or a reduced affinity variant of a naturally occurring costimulatory polypeptide such as an IL-2 variant disclosed herein, or a combination of both, selectively to a target T cell, the method comprising contacting a mixed population of T cells with a multimeric polypeptide of the present invention. The mixed population of T cells includes target T cells and non-target T cells. The target T cell is specific for an epitope present in the multimeric polypeptide. Contacting the mixed population of T cells with the multimeric polypeptide of the present invention delivers the costimulatory polypeptide present in the multimeric polypeptide to the target T cell. Thus, co-administration of a checkpoint inhibitor with a multimeric polypeptide (either together or at another time before and/or after administration of the multimeric polypeptide) provides the patient with the therapeutic effect of selective delivery of both the costimulatory polypeptide and the checkpoint inhibitor.

For example, a multimeric polypeptide of the present invention is contacted with a population of T cells comprising: i) target T cells that are specific for an epitope present in the multimeric polypeptide; and ii) non-target T cells, such as T cells that are specific for a second epitope that is not an epitope present in the multimeric polypeptide. Contacting the population results in selective delivery of a costimulatory polypeptide (e.g., a naturally occurring costimulatory polypeptide (e.g., a naturally occurring IL-2) or a reduced affinity variant of a naturally occurring costimulatory polypeptide (e.g., an IL-2 variant as disclosed herein)) present in the multimeric polypeptide to the target cell. Thus, for example, 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 non-target T cells bind the multimeric polypeptide, and as a result, the costimulatory polypeptide (e.g., IL-2 or an IL-2 variant) present in the multimeric polypeptides is substantially not delivered to the non-target T cells. Thus, co-administration of a checkpoint inhibitor with a multimeric polypeptide (either together or at another time before and/or after administration of the multimeric polypeptide) provides the patient with the therapeutic effect of selectively delivering both the costimulatory polypeptide and the checkpoint inhibitor.

In some cases, the T cell population is an in vitro population. In some cases, the T cell population is an in vitro population, and the biological response (e.g., activation and/or expansion of T cells and/or phenotypic differentiation) of the target T cell population to the multimeric polypeptide of the present invention is elicited in the context of an in vitro culture. For example, a mixed T cell population can be obtained from an individual and can be contacted with the multimeric polypeptide in vitro. Such contacting can include single or multiple exposures of the T cell populations to specific doses or exposure regimens. In some cases, said contacting results in selective binding/activation and/or expansion of target T cells in the T cell population and results in the formation of a population of activated and/or expanding target T cells. As an example, the mixed T cell population can be peripheral blood mononuclear cells (PBMCs). For example, patient PBMCs can be obtained by standard blood collection methods and enrichment of the PBMCs prior to exposure to 0.1-1000 nM of a multimeric polypeptide of the present invention under standard lymphocyte culture conditions. At time points before, during, and after exposure to a mixed T cell population at a defined dose and schedule, the abundance of target T cells in the in vitro culture can be monitored using specific peptide-MHC multimers and/or phenotypic markers and/or functional activity (e.g., cytokine ELISpot assay). In some cases, after achieving an optimal number and/or phenotype of antigen-specific cells in vitro, all or a portion of the population of activated and/or expanding target T cells is administered to an individual (the individual from whom the mixed T cell population was taken) who has already been administered a checkpoint inhibitor prior to administration of the target T cells and/or who will be administered a checkpoint inhibitor after administration of the target T cells.

In some cases, the population of T cells is an in vitro population. For example, a mixed population of T cells is obtained from an individual and contacted with a multimeric polypeptide of the present invention in vitro. Such contacting, which may involve a single or

- 112 049999 repeated exposure of T cells to a given dose and/or exposure schedule in the context of in vitro cell culture can be used to determine whether a mixed population of T cells includes T cells specific for an epitope presented on a multimeric polypeptide. The presentation of T cells specific for an epitope of a multimeric polypeptide can be determined by analyzing a sample containing a mixed population of T cells, wherein the population of T cells includes T cells that are not specific for the epitope (non-target T cells) and may include T cells specific for the epitope (target T cells). Known assays can be used to detect activation and/or proliferation of target T cells, thereby providing an ex vivo assay that can determine whether a particular multimeric polypeptide (synTac) has an epitope that binds to T cells presented in an individual, and thus whether the multimeric polypeptide has potential use as a therapeutic composition for that individual. Suitable known assays for detecting activation and/or proliferation of target T cells include, for example, flow cytometric characterization of T cell phenotype and/or antigen specificity and/or proliferation. Such an assay for detecting the presence of epitope-specific T cells, such as a companion diagnostic, may further comprise additional assays (e.g., ELISpot assays for effector cytokines) and/or appropriate controls (e.g., antigen-specific and antigen-specific staining reagents for the multimeric peptide-HLA) to determine whether the multimeric polypeptide selectively binds/activates and/or promotes the growth of target T cells. Thus, for example, the present disclosure provides a method for detecting in a mixed population of T cells obtained from an individual the presentation 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 multimeric polypeptide of the present invention, wherein the multimeric polypeptide comprises the epitope of interest; and b) detecting activation and/or proliferation of T cells in response to said contacting, wherein activated and/or proliferated T cells indicate the presence of the target T cell. Alternatively and/or additionally, if activation and/or growth (proliferation) of the desired T cell population is obtained using the multimeric polypeptide, then all or part of the T cell population, including the activated/growing T cells, can be administered back to the individual (who was administered the checkpoint inhibitor prior to administration of the T cells and/or will be administered the checkpoint inhibitor after administration of the T cells) as a therapy.

In some cases, the T cell population is an in vivo population in an individual. In such cases, the method of the present invention for selectively delivering a costimulatory polypeptide (e.g., IL-2 or IL-2 with reduced affinity) to an epitope-specific T cell comprises administering a multimeric polypeptide to the individual.

The epitope-specific T cell to which the costimulatory polypeptide (e.g., IL-2 or IL-2 with reduced affinity) is selectively delivered is also referred to herein as the target T cell. In some cases, the target T cell is a regulatory T cell (Treg). In some cases, Treg inhibits or suppresses the activity of an autoreactive T cell.

In some cases, the target T cell is a cytotoxic T cell. For example, the target T cell may be a cytotoxic T cell specific for a cancer epitope c (e.g., an epitope presented by a cancer cell).

Examples of non-limiting aspects of the invention

The aspects, including embodiments, of the subject matter disclosed above may provide an advantage alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are set forth below, designated as Aspects 1-122, Aspects A-Z, Aspects AA-ZZ, and Aspects AAA-BBB. Upon reading this description, those skilled in the art will appreciate that each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide a basis for all such combinations of aspects, and is not limited to the combinations of aspects expressly set forth below:

Aspect 1. A variant IL-2 polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the variant IL-2 polypeptide has one or more amino acid substitutions relative to the sequence set forth in SEQ ID NO: 1, and wherein the variant IL-2 polypeptide exhibits reduced binding affinity for the IL-2 receptor (IL2R) comprising alpha, beta and gamma polypeptides having the amino acid sequences depicted in Figs. 3A-3C, compared to the binding affinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO: 1 for IL2R.

Aspect 2. A variant of the IL2 polypeptide according to aspect 1, wherein the variant comprises a substitution of one or more of E15, H16, D20, F42, Y45 and Q126.

Aspect 3. A variant of the IL2 polypeptide according to aspect 1 or aspect 2, wherein the variant immunomodule

- 113 049999 the binding polypeptide exhibits from less than 10% to less than 50% of the binding affinity exhibited by the IL2 amino acid sequence shown in SEQ ID NO: 1 for IL2R.

Aspect 4. A variant of the IL2 polypeptide according to any of aspects 1-3, wherein the variant comprises substitutions of F42 with Ala, Gly, Val, Ile or Leu.

Aspect 5. A variant of the IL2 polypeptide according to any of aspects 1-3, wherein the variant comprises substitutions F42 and D20.

Aspect 6. A variant of the IL2 polypeptide according to any of aspects 1-3, wherein the variant comprises substitutions F42 and HI6.

Aspect 7. A variant of the IL2 polypeptide according to any of aspects 1-3, wherein the variant comprises substitutions F42, D20 and Y45; or wherein the variant comprises substitutions F42, H16 and Q126.

Aspect 8. Multimeric polypeptide containing:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory domains, wherein the one or more immunomodulatory domains are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide, wherein at least one of the one or more immunomodulatory domains is a variant of the IL2 polypeptide according to any of aspects 1-7, and wherein the multimeric polypeptide exhibits a reduced binding affinity for the IL-2 receptor (IL2R) comprising alpha, beta and gamma polypeptides having the amino acid sequences depicted in Figs. 3A-3C, compared to the binding affinity of a control multimeric polypeptide comprising the IL2 amino acid sequence set forth in SEQ ID NO: 1 for the IL2R polypeptide.

Aspect 9. A multimeric polypeptide according to aspect 8, where:

a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope;

ii) a first MHC polypeptide; and iii) a variant IL2 polypeptide; and

b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an IgFc polypeptide.

Aspect 10. Multimeric polypeptide according to aspect 8, where:

a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and

b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) a variant of the IL2 polypeptide;

ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide.

Aspect 11. Multimeric polypeptide according to aspect 8, where:

a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and

b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) a variant IL2 polypeptide.

Aspect 12. Multimeric polypeptide according to aspect 8, where:

a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and

b) a second polypeptide comprising, in the direction from the N-terminus to the C-terminus:

i) a variant IL2 polypeptide; and ii) a second MHC polypeptide.

Aspect 13. Multimeric polypeptide according to aspect 8, where:

a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope;

ii) a first MHC polypeptide; and iii) a variant IL2 polypeptide; and

b) the second polypeptide comprises a second MHC polypeptide.

Aspect 14. The multimeric polypeptide of aspect 8, wherein the non-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide or a silk-like elastin-like polypeptide.

Aspect 15. A multimeric polypeptide according to any of aspects 8-14, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide.

- 114 049999

Aspect 16. The multimeric polypeptide of aspect 15, wherein the B2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in Fig. 6.

Aspect 17. The multimeric polypeptide of aspect 15, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B or HLA-C heavy chain.

Aspect 18. The multimeric polypeptide of aspect 15, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences shown in Figs. 5A-5C.

Aspect 19. A multimeric polypeptide according to any of aspects 8-14, wherein the first MHC polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect 20. A multimeric polypeptide according to any of aspects 8-19, wherein the epitope is a T-cell epitope.

Aspect 21. A multimeric polypeptide according to any of aspects 8-13 and 15-20, wherein the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide or an IgM Fc polypeptide.

Aspect 22. The multimeric polypeptide of aspect 21, wherein the IgFc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in Fig. 4A-4C.

Aspect 23. A multimeric polypeptide according to any of aspects 8-22, wherein the first polypeptide and the second polypeptide are non-covalently linked.

Aspect 24. A multimeric polypeptide according to any of aspects 8-22, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

Aspect 25. A multimeric polypeptide according to aspect 24, wherein the covalent bond is via a disulfide bond.

Aspect 26. The multimeric polypeptide of claim 25, wherein the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to produce a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to produce a second Cys residue, and wherein the disulfide bond is between the first and second Cys residues.

Aspect 27. A multimeric polypeptide according to any of aspects 8-26, comprising a linker located between the epitope and the first MHC polypeptide.

Aspect 28. A multimeric polypeptide according to any of aspects 8-26, comprising a linker located between the MHC polypeptide and the immunomodulatory polypeptide.

Aspect 29. A multimeric polypeptide according to any of aspects 8-28, comprising 2 variants of the IL2 polypeptide.

Aspect 30. A multimeric polypeptide according to any of aspects 8-28, comprising 3 variants of the IL2 polypeptide.

Aspect 31. A multimeric polypeptide according to aspect 29 or aspect 30, wherein 2 or 3 IL2 polypeptide variants are in tandem, and wherein the multimeric polypeptide comprises a linker between the IL2 polypeptide variants.

Aspect 32. A multimeric polypeptide according to any of aspects 8-28, wherein the IL2 variant comprises a substitution of one or more of E15, H16, D20, F42, Y45 and Q126.

Aspect 33. A multimeric polypeptide according to any of aspects 8-28, wherein the IL2 variant comprises a substitution of F42 with Ala, Gly, Val, Ile or Leu.

Aspect 34. The multimeric polypeptide of aspect 33, wherein the IL2 variant comprises the substitutions F42 and D20.

Aspect 35. The multimeric polypeptide of aspect 33, wherein the IL2 variant comprises the substitutions F42 and H16.

Aspect 36. The multimeric polypeptide of aspect 33, wherein the IL2 variant comprises substitutions F42, D20 and Y45; or wherein the IL-2 variant comprises substitutions F42, H16 and Q126.

Aspect 37. A nucleic acid containing a nucleotide sequence encoding a polypeptide,

i) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus:

a) epitope;

b) the first polypeptide of the major histocompatibility complex (MHC);

c) immunomodulatory polypeptide;

d) a proteolytically cleavable linker or ribosome skipping signal;

e) a second MHC polypeptide; and

f) immunoglobulin Fc polypeptide (Ig);

wherein the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide according to any of aspects 1-7; or ii) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus:

a) epitope;

- 115 049999

b) the first MHC polypeptide;

c) a proteolytically cleavable linker or ribosome skipping signal;

d) immunomodulatory polypeptide;

e) a second MHC polypeptide; and

f) an IgFc polypeptide, wherein the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide according to any of aspects 1-7.

Aspect 38. The nucleic acid of aspect 37, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide.

Aspect 39. The nucleic acid of aspect 38, wherein the B2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in Fig. 6.

Aspect 40. The nucleic acid of aspect 38, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B or HLA-C heavy chain.

Aspect 41. The nucleic acid of aspect 40, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences shown in Figs. 5A-5C.

Aspect 42. The nucleic acid of aspect 37, wherein the first MHC polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect 43. The nucleic acid of any of aspects 37-43, wherein the epitope is a T cell epitope.

Aspect 44. The nucleic acid of any of aspects 37-43, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide or an IgM Fc polypeptide.

Aspect 45. The nucleic acid of aspect 44, wherein the IgFc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in Fig. 4A-4C.

Aspect 46. The nucleic acid of any of aspects 37-45, wherein the variant IL2 immunomodulatory polypeptide comprises a substitution of one or more of E15, H16, D20, F42, Y45 and Q126.

Aspect 47. The nucleic acid of any one of aspects 37-46, wherein the multimeric polypeptide comprises a second immunomodulatory polypeptide selected from CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4 and HVEM.

Aspect 48. The nucleic acid of any of aspects 37-47, wherein the proteolytically cleavable linker or ribosome skipping signal comprises an amino acid sequence selected from:

a) LEVLFQGP (SEQ ID NO: 88);

b) ENLYTQS (SEQ ID NO: 90);

c) furin cleavage site;

d) LVPR (SEQ ID NO:89);

e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 91);

f) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 92);

g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 93); and h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 94).

Aspect 49. The nucleic acid of aspect 31, wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus:

a) the first leader peptide;

b) epitope;

c) the first MHC polypeptide;

d) immunomodulatory polypeptide;

e) a proteolytically cleavable linker or ribosome skipping signal;

f) the second leader peptide;

g) a second MHC polypeptide; and

h) immunoglobulin Fc polypeptide (Ig).

Aspect 50. The nucleic acid of aspect 49, wherein the first leader peptide and the second leader peptide are the leader peptide B2-M.

Aspect 51. A nucleic acid according to any of aspects 37-50, wherein the nucleotide sequence is operably linked to a transcriptional control element.

Aspect 52. The nucleic acid of aspect 51, wherein the transcriptional control element is pro

- 116 049999 motor, functional in the eukaryotic cell.

Aspect 53. The nucleic acid of any of aspects 37-52, wherein the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to produce a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to produce a second Cys residue, and wherein the first and second Cys residues provide a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

Aspect 54. A recombinant expression vector comprising a nucleic acid according to any of aspects 37-52.

Aspect 55. A recombinant expression vector according to aspect 54, wherein the vector is a viral vector or a non-viral vector.

Aspect 56. A host cell genetically modified with a recombinant expression vector according to aspect 48-55.

Aspect 57. The host cell according to aspect 56, wherein the host cell is in vitro.

Aspect 58. The host cell of aspect 57, wherein the host cell is genetically modified such that the cell does not produce endogenous MHC beta2-microglobulin polypeptide.

Aspect 59. Composition containing:

a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

i) epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain, wherein the immunomodulatory domain is a variant of the IL2 polypeptide according to any of aspects 1-7; and

b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) Fc Ig polypeptide.

Aspect 60. Composition containing:

a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; and

b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) an immunomodulatory domain, wherein the immunomodulatory domain is a variant of the IL2 polypeptide according to any of aspects 1-7;

ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide.

Aspect 61. The composition according to aspect 59 or aspect 60, wherein the first and/or second nucleic acid is present in a recombinant expression vector.

Aspect 62. A host cell genetically modified with the composition of any of aspects 5961.

Aspect 63. A method for producing a multimeric polypeptide according to any of aspects 8-36, the method comprising:

a) culturing the host cell of any of aspects 56-58 and 62 in vitro in a culture medium under conditions whereby the host cell synthesizes the multimeric polypeptide; and

b) isolating the multimeric polypeptide from the host cell and/or from the culture medium.

Aspect 64. The method of aspect 63, wherein the second polypeptide comprises an affinity label and wherein said isolating comprises contacting the multimeric polypeptide produced by the cell with a binding partner for the affinity label, wherein the binding partner is immobilized, thereby immobilizing the multimeric polypeptide.

Aspect 65. The method according to aspect 64, comprising eluting the immobilized multimeric polypeptide.

Aspect 66. A method for selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any of aspects 8-36, wherein said contacting selectively activates the epitope-specific T cell.

Aspect 67. The method according to aspect 66, wherein said contacting occurs in vitro.

Aspect 68. The method according to aspect 66, wherein said contacting occurs in vivo.

Aspect 69. The method of aspect 66, wherein the epitope is a cancer-associated epitope, and wherein said administration selectively increases the activity of a T cell specific for the cancer-associated epitope.

Aspect 70. A method of treating cancer in an individual, comprising administering to the individual an effective amount of:

- 117 049999

a) a multimeric polypeptide according to any of aspects 8-36; or

b) one or more recombinant expression vectors comprising nucleotide sequences encoding a multimeric polypeptide according to any of aspects 8-36; or

c) one or more mRNAs comprising nucleotide sequences encoding a multimeric polypeptide according to any of aspects 8-36, wherein the epitope is an epitope associated with cancer, and wherein said administration is effective for selectively activating an epitope-specific cancer T cell in an individual.

Aspect 71. The method according to aspect 70, wherein said administration is carried out subcutaneously.

Aspect 72. The method according to aspect 70, wherein said administration is carried out intravenously.

Aspect 73. The method according to aspect 70, wherein said administration is carried out intratumorally.

Aspect 74. The method according to aspect 70, wherein said administration is carried out systemically.

Aspect 75. The method according to aspect 70, wherein said administration is carried out distally relative to the treatment site.

Aspect 76. The method according to aspect 70, wherein said administration is carried out locally.

Aspect 77. The method according to aspect 70, wherein said administration is carried out at or near the treatment site.

Aspect 78. Composition containing:

a) a multimeric polypeptide according to any of aspects 8-36; and

b) a pharmaceutically acceptable excipient.

Aspect 79. Composition containing:

a) a nucleic acid according to any of aspects 37-53 or a recombinant expression vector according to aspect 54 or 55; and

b) a pharmaceutically acceptable excipient.

Aspect 80. A multimeric polypeptide containing:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) a β2 microglobulin polypeptide f2M) comprising the amino acid sequence depicted in Fig. 34A; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a variant of the IL-2 polypeptide according to any of aspects 1-7;

ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S (N77A, L14A, L15A, L14F, L15E, and P111S, respectively, based on the amino acid numbering shown in Fig. 33A).

Aspect 81. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises the substitution N297A (N77A based on the amino acid numbering shown in Fig. 33A).

Aspect 82. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises a L234A substitution and a L235A substitution (L14A and L15A based on the amino acid numbering shown in Fig. 33A).

Aspect 83. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises a L234F substitution and a L235E substitution (L14F and L15E based on the amino acid numbering shown in Fig. 33A).

Aspect 84. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises a L234F substitution, a L235E substitution and P331S (L14F, L15E and P111S substitutions based on the amino acid numbering shown in Fig. 33A).

Aspect 85. A multimeric polypeptide according to any of aspects 80-84, wherein the second polypeptide comprises two copies of a variant IL-2 polypeptide.

Aspect 86. The multimeric polypeptide of any of aspects 80-85, wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide.

Aspect 87. The multimeric polypeptide of any of aspects 80-86, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of the IL-2 polypeptide variant and a second copy of the IL-2 polypeptide variant;

b) a variant of the IL-2 polypeptide and an MHC heavy chain polypeptide; and

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 88. A multimeric polypeptide according to aspect 86 or aspect 87, wherein the peptide linker is selected from (GGGGS)3, (GGGGS)4 and AAAGG.

Aspect 89. A multimeric polypeptide containing:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) a B2-microglobulin polypeptide comprising the amino acid sequence depicted in Fig. 34A; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a variant IL-2 polypeptide comprising the amino acid sequence depicted in Fig. 34B;

- 118 049999 ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S (N77A, L14A, L15A, L14F, L15E and P111S, respectively, based on the amino acid numbering shown in Fig. 33A).

Aspect 90. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide comprises the substitution N297A (N77A based on the amino acid numbering shown in Fig. 33A).

Aspect 91. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide comprises a L234A substitution and a L235A substitution (L14A and L15A based on the amino acid numbering shown in Fig. 33A).

Aspect 92. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide comprises a L234F substitution and a L235E substitution (L14F and L15E based on the amino acid numbering shown in Fig. 33A).

Aspect 93. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide comprises a L234F substitution, a L235E substitution and a P331S substitution (L14F, L15E and P111S based on the amino acid numbering shown in Fig. 33A).

Aspect 94. The multimeric polypeptide of any of aspects 89-93, wherein the second polypeptide comprises two copies of the IL-2 polypeptide variant.

Aspect 95. The multimeric polypeptide of any of aspects 89-94, wherein the first polypeptide comprises a peptide linker between the epitope and the b2M polypeptide.

Aspect 96. The multimeric polypeptide of any of aspects 89-95, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of the IL-2 polypeptide variant and a second copy of the IL-2 polypeptide variant;

b) a variant of the IL-2 polypeptide and an MHC heavy chain polypeptide; and

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 97. A multimeric polypeptide according to aspect 95 or aspect 96, wherein the peptide linker is selected from (GGGGS)3, (GGGGSh and AAAGG.

Aspect 98. Multimeric polypeptide containing:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77);

ii) a B2-microglobulin polypeptide comprising the amino acid sequence depicted in Fig. 34A; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a variant IL-2 polypeptide comprising the amino acid sequence depicted in Fig. 34B;

ii) a major histocompatibility complex (MHC) heavy chain polypeptide comprising the amino acid sequence depicted in Fig. 34C; and iii) an IgG1 Fc polypeptide comprising the amino acid sequence depicted in Fig. 33A, 33B, 33C, or 33D.

Aspect 99. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence depicted in Fig. 33B.

Aspect 100. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence depicted in Fig. 33C.

Aspect 101. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence depicted in Fig. 33D.

Aspect 102. The multimeric polypeptide of any of aspects 98-101, wherein the second polypeptide comprises two copies of the IL-2 polypeptide variant.

Aspect 103. The multimeric polypeptide of any of aspects 98-102, wherein the first polypeptide comprises a peptide linker between the epitope and the b2M polypeptide.

Aspect 104. The multimeric polypeptide of any of aspects 98-103, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of the IL-2 polypeptide variant and a second copy of the IL-2 polypeptide variant;

b) a variant of the IL-2 polypeptide and an MHC heavy chain polypeptide; and

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 105. A multimeric polypeptide according to aspect 103 or aspect 104, wherein the peptide linker is selected from (GGGGS)3, (GGGGS^ and AAAGG.

Aspect 106. A multimeric polypeptide containing:

a) a first polypeptide comprising the amino acid sequence depicted in Fig. 31;

b) a second polypeptide comprising the amino acid sequence depicted in Fig. 22.

Aspect 107. A multimeric polypeptide containing:

a) a first polypeptide comprising the amino acid sequence shown in Fig.

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31;

b) a second polypeptide comprising the amino acid sequence depicted in Fig. 25.

Aspect 108. A multimeric polypeptide containing:

a) a first polypeptide comprising the amino acid sequence depicted in Fig. 31;

b) a second polypeptide comprising the amino acid sequence depicted in Fig. 28.

Aspect 109. A pharmaceutical composition containing:

a) a multimeric polypeptide according to any of aspects 80-108; and

b) a pharmaceutically acceptable excipient.

Aspect 110. One or more nucleic acids comprising nucleotide sequences encoding the first and/or second polypeptide of the multimeric polypeptide according to any of aspects 80-108.

Aspect 111. One or more nucleic acids according to aspect 110, wherein the nucleic acids are present in recombinant expression vectors.

Aspect 112. A method for selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any of aspects 80-108, wherein said contacting selectively activates the epitope-specific T cell.

Aspect 113. The method according to aspect 112, wherein said contacting occurs in vitro.

Aspect 114. The method according to aspect 112, wherein said contacting occurs in vivo.

Aspect 115. A method comprising administering to an individual an effective amount of:

a) a multimeric polypeptide according to any of aspects 80-108; or

b) one or more recombinant expression vectors comprising nucleotide sequences encoding a multimeric polypeptide according to any of aspects 80-108; or

c) one or more mRNAs comprising nucleotide sequences encoding a multimeric polypeptide according to any of aspects 80-108, wherein said administration induces a T cell response to the epitope in the individual.

Aspect 116. The method according to aspect 115, wherein said administration is carried out subcutaneously.

Aspect 117. The method according to aspect 115, wherein said administration is carried out intravenously.

Aspect 118. The method according to aspect 115, wherein said administration is carried out systemically.

Aspect 119. The method according to aspect 115, wherein said administration is carried out intramuscularly.

Aspect 120. The method according to aspect 115, wherein said administration is carried out distally relative to the treatment site.

Aspect 121. The method according to aspect 115, wherein said administration is carried out locally.

Aspect 122. The method according to aspect 115, wherein said administration is carried out at or near the treatment site.

Aspect A. A method for modulating an immune response in an individual in need thereof, the method comprising administering to the individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration modulates the immune response in an individual.

Aspect B. A method of treatment comprising administering to an individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are arranged:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

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C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats an individual.

Aspect C. A method of treating cancer in an individual, comprising administering to the individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats cancer in an individual.

Aspect D. A method of treatment comprising administering to an individual a multimeric polypeptide, wherein the individual is treated with an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope;

ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats an individual.

Aspect E. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) Fc Ig polypeptide.

Aspect F. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) immunomodulatory domain;

ii) a second MHC polypeptide; and iii) an immunoglobulin (Ig) Fc polypeptide.

Aspect G. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an IgFc polypeptide; and iii) an immunomodulatory domain.

Aspect H. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an immunomodulatory domain.

Aspect I. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

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a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) an immunomodulatory domain; and ii) a second MHC polypeptide.

Aspect J. The method according to any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) the second MHC polypeptide.

Aspect K. The method according to any of aspects A-D, wherein the non-^-scaffold multimeric polypeptide is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide or a silk-like elastin-like polypeptide.

Aspect L. The method according to any one of aspects A-K, wherein the first MHC polypeptide of the multimeric polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide.

Aspect M. The method according to aspect L, wherein the B2-microglobulin polypeptide of the multimeric polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in Fig. 6.

Aspect N. The method according to aspect L, wherein the MHC class I heavy chain polypeptide of the multimeric polypeptide is the heavy chain of HLA-A, HLA-B or HLA-C.

Aspect O. The method of aspect 11, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences shown in Figs. 5A-5C.

Aspect P. The method according to any one of aspects A-K, wherein the first MHC polypeptide of the multimeric polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect Q. The method according to any one of aspects A-P, wherein the epitope is a T-cell epitope.

Aspect R. The method according to any one of aspects A-J, wherein the multimeric polypeptide of the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide or an IgM Fc polypeptide.

Aspect S. The method of aspect R, wherein the IgFc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in Fig. 4A-4C.

Aspect T. The method according to any one of aspects A-S, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are non-covalently linked.

Aspect U. The method according to any one of aspects A-S, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are covalently linked.

Aspect V. The method of aspect U, wherein the covalent bond is via a disulfide bond.

Aspect W. The method according to aspect V, wherein the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution to obtain a first Cys residue and the second MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution to obtain a second Cys residue and wherein the disulfide bond is between the first and second Cys residues.

Aspect X. The method according to any one of aspects A-K, wherein the multimeric polypeptide comprises a linker between the epitope and the first MHC polypeptide, between the immunomodulatory polypeptide and the MHC polypeptide, or between the MHC polypeptide and the IgFc.

Aspect Y. The method according to any one of aspects A-K, wherein the immunomodulatory polypeptide of the multimeric polypeptide is selected from 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

Aspect Z. The method according to any of aspects A-Y, wherein the multimeric polypeptide comprises 2 or more immunomodulatory polypeptides.

Aspect AA. The method of aspect Z, wherein 2 or more immunomodulatory polypeptides are in tandem.

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Aspect BB. The method according to any of aspects A-Z and AA, wherein the immunomodulatory polypeptide is selected from 4-1BBL polypeptide, CD80 polypeptide, CD86 polypeptide, IL-2 polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

Aspect CC. The method according to any of aspects A-Z and AA-BB, wherein the immunomodulatory polypeptide is a variant of an immunomodulatory polypeptide having one or more amino acid substitutions relative to the natural form of the immunomodulatory polypeptide, and wherein the variant of the immunomodulatory polypeptide exhibits reduced binding affinity to the comodulatory polypeptide to which the natural form of the immunomodulatory polypeptide binds.

Aspect DD. The method according to any of aspects A-Z and AA-BB, wherein the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the variant IL-2 polypeptide has one or more amino acid substitutions relative to the sequence set forth in SEQ ID NO: 1, and wherein the variant IL-2 polypeptide exhibits reduced binding affinity for the IL-2 receptor (IL2R) comprising alpha, beta and gamma polypeptides having the amino acid sequences depicted in Fig. 3A-3C, compared to the binding affinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO: 1 for IL2R.

Aspect EE. The method of aspect DD, wherein the IL-2 polypeptide variant comprises a substitution of one or more of E15, H16, D20, F42, Y45 and Q126.

Aspect FF. The method of aspect EE, wherein the IL-2 polypeptide variant comprises:

a) replacement of F42 and D20;

b) replacement of F42 and H16;

c) replacements of F42, D20 and Y45; or

d) replacement of F42, H16 and Q126.

Aspect GG. The method according to any one of aspects A-Z and AA-FF, wherein the multimeric polypeptide comprises an Ig Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S.

Aspect HH. The method of aspect GG, wherein the IgFc polypeptide comprises:

a) replacement of N297A;

b) replacement of L234A and replacement of L235A;

c) replacement of L234F and replacement of L235E; or

d) L234F replacement, L235E replacement and P331S replacement.

Aspect II. The method according to any one of aspects A-Z and AA-HH, wherein the epitope of the multimeric polypeptide comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 77).

Aspect JJ. The method according to any one of aspects A-Z and AA-HH, wherein the β2microglobulin polypeptide of the multimeric polypeptide comprises the amino acid sequence depicted in Fig. 34A.

Aspect KK. The method according to any one of aspects A-Z and AA-JJ, wherein the heavy chain major histocompatibility complex (MHC) polypeptide of the multimeric polypeptide comprises the amino acid sequence depicted in Fig. 34C.

Aspect LL. The method according to any of aspects A-Z and AA-KK, wherein the immune checkpoint inhibitor is an antibody specific for the immune checkpoint inhibitor.

Aspect MM. The method of aspect LL, wherein the antibody is a monoclonal antibody.

Aspect NN. The method according to aspect KK or aspect LL, wherein the antibody comprises at least one framework region of a humanized light chain and/or heavy chain.

OO aspect. The method of aspect LL, wherein the antibody comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

Aspect PP. The method according to aspect LL, wherein the antibody is an Fv fragment, a nanobody or a Fab fragment.

Aspect QQ. The method of any one of aspects LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

Aspect RR. The method of any one of aspects LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for PD1.

Aspect SS. The method of aspect RR, wherein the antibody is pembrolizumab, nivolumab, pidilizumab or BMS-39886.

Aspect TT. The method of any one of the aspects of LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1.

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Aspect UU. The method of aspect TT, wherein the antibody is durvalumab, atezolizumab, KN035, or avelumab.

Aspect VV. The method of any one of aspects LL-PP, wherein the immune checkpoint inhibitor is an antibody specific to CTLA4.

Aspect WW. The method of aspect VV, wherein the antibody is ipilimumab or tremelimumab.

Aspect XX. The method according to any of aspects A-Z and AA-WW, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by the same route of administration.

Aspect YY. The method according to any one of aspects A-Z and AA-WW, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by different routes of administration.

Aspect ZZ. The method according to any of aspects A-Z and AA-YY, wherein the multimeric polypeptide is administered by an administration route selected from subcutaneous, intravenous, peritumoral and intramuscular.

Aspect AAA. The method according to any of aspects A-Z and AA-YY, wherein the immune checkpoint inhibitor is administered by an administration route selected from subcutaneous, intravenous, peritumoral and intramuscular.

Aspect BBB. A method according to any of the aspects A-Z, AA-ZZ and AAA, where the individual is a human.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, but they are not intended to limit the scope of what the inventors consider to be their invention, nor to indicate that the experiments reported below represent all or the only experiments performed. Care has been taken to maintain accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and variations should be allowed for. Unless otherwise indicated, parts refer to parts by weight, molecular weight refers to average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair(s); kb, kilobase pair(s); pL, picoliter(s); s or sec, second(s); min, minute(s); hr or h, hour(s); aa, amino acid(s); thousand(s) nucleotide pairs; b. o., base pair(s); nt, nucleotide(s); kiloDalton(s), kDa; i/m, intramuscular(s); i/p, intraperitoneal(s); s/c, subcutaneous(s), etc.

Example 1. IL-2/synTac production

The production of IL-2/synTac by transiently transfected mammalian cells was analyzed. As shown in Fig. 7A, the production levels (in mg/L culture medium) of two different IL2/synTac were greater than 90 mg/L 6-7 days after transient transfection of the cells.

IL-2/synTac produced by mammalian cells was purified and subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The results are shown in Fig. 7B. Sizes are given in kDa.

IL-2/synTac was generated in which the IL-2 polypeptide was in the light chain (i.e., a polypeptide containing an MHC class I light chain; e.g., β2M) or in the heavy chain (i.e., a polypeptide containing an MHC class I heavy chain). The expression levels and stability of IL-2/synTac were analyzed.

synTac was produced in mammalian cells. As shown in Fig. 8A, the level of production of IL2/synTac containing IL-2 in the heavy chain was approximately 25 times higher than that of IL-2/synTac containing IL-2 in the light chain.

IL-2/synTac produced by mammalian cells were subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions; and the gels were stained with Coomassie blue. As shown in Fig. 8B, IL-2/synTac containing IL-2 in the heavy chain was more stable than IL-2/synTac containing IL-2 in the light chain. Sizes are given in kDa.

The expression levels of IL-2/synTac containing the IL-2 variant were assessed. Figure 9 shows the expression level of IL-2/synTac in which IL-2 is wild-type (wt) or contains different combinations of F42A, D20K, Q126A, E15A, Y45A, and H16A. Expression levels are expressed as percentage change relative to synTac expression levels with wt IL-2.

The effect of IL-2 copy number in IL-2/synTac on expression levels was assessed. IL-2/synTac contained one copy (1X), two copies (2X), or three copies (3X) of synTac. Different IL-2/synTac were produced in mammalian cells and expression levels were analyzed. The data are shown in Fig. 10. IL-2/synTac with one or two copies of IL-2 showed similar expression levels, whereas IL-2/synTac with three copies of IL-2 showed lower expression levels. Expression levels are expressed as fold change relative to the expression level of IL-2/synTac with one copy of IL-2.

Example 2. IL-2/synTac activity in vitro

To achieve maximum targeting specificity via the T cell receptor, the affinity of the costimulatory polypeptide for its ligand should be lower than the affinity of MHC for the TCR. Affinity

- 124 049999 the affinity of the peptide/MHC to the TCR may be approximately 10 μM.

IL-2/synTac was generated containing two copies of an IL-2 variant containing the F42A and H16A substitutions. IL-2/synTac-induced costimulatory signaling was tested in antigen-specific CD8+ T cells and non-specific CD8+ T cells. Antigen-specific CD8+ T cells and non-specific CD8+ T cells bound to different concentrations of IL-2/synTac.

As shown in Fig. 11, IL-2/synTac induced costimulatory signaling in antigen-specific CD8+ T cells at a significantly lower concentration than in non-specific CD8+ T cells.

The selectivity of IL-2/synTac binding was tested. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. CD8+ T cells were incubated with several IL-2/synTac at different concentrations and allowed to bind for 20 min. Several IL-2/synTac contained IgG2a Fc. IL-2/synTac binding to CD8+ T cells was detected using phycoerythrin (PE)-labeled anti-IgG2a antibody. PE fluorescence was determined using flow cytometry to determine the percentage of cells bound to IL-2/synTac.

As shown in Fig. 12, IL-2/synTac binds in an antigen-specific manner to LCMV CD8+ T cells but does not exhibit significant binding to OT1 CD8+ T cells. Thus, IL2/synTac selectively binds to CD8+ T cells specific for an epitope present on IL2/synTac.

It was determined whether IL-2/synTac was able to selectively activate target T cells. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. The IL-2/synTac used included either a single amino acid substitution of F42A or substitutions of F42A and H16A. CD8' T cells were stimulated with IL-2/synTac at various concentrations for 20 min. Cells were then stained with PE-labeled phosphoSTAT5 antibody. PE fluorescence was determined by flow cytometry to determine the percentage of cells that were phospho-STAT5 positive, where (|)oc(|io-STAT5 is a marker of activation.

As shown in Fig. 13, IL-2/synTac induced CD8+ stimulation (as indicated by % phosphoSTAT5-positive cells) in antigen-specific (LCMV) CD8+ T cells at significantly lower concentrations than in non-specific (BL6) CD8 ⁺ T cells.

The specific activity of different IL-2/synTac was analyzed. IL-2/synTac containing one copy of IL-2, two copies of IL-2, or three copies of IL-2, where IL-2 contained different combinations of F42A, D20K, Q126A, E15A, H16A, and Y45A substitutions, were tested at different concentrations to assess stimulation of antigen-specific (LCMV) or non-specific (BL6) CD8+ cells. The percentage of phosphosignal transducer and activator of transcription 5 (pSTAT5)-positive was determined. The data are depicted in Fig. 14A-14F.

Example 3. IL-2/synTac activity in vivo

The in vivo activity of IL-2/synTac was tested. The in vivo fold change in antigen-specific CD8+ T cells was tested after administration of phosphate-buffered saline (PBS), recombinant IL-2 (rIL-2), or IL-2/synTac of the present invention. The data are shown in Fig. 15, left panel. The data show that IL-2/synTac is 10-fold more potent than rIL-2.

The specificity of IL-2/synTac in vivo was tested. Antigen-specific and non-antigen-specific responses after administration of PBS, rIL-2, or IL-2/synTac were assessed. Data are expressed as the percentage of lymph node cells that were antigen-specific or antigen-non-specific after administration of PBS, rIL-2, or IL-2/synTac. As shown in Fig. 15, right panel, IL-2/synTac induced an antigen-specific response (expressed as % of the maximal dilution of carboxyfluorescein succinimidyl ester (CFSE) as an index of T cell proliferation). In contrast, the response induced by rIL-2 was not antigen-specific.

A dose-response analysis was performed. IL-2/synTac (F42A, H16A) was administered intraperitoneally at concentrations of 4 mg/kg, 8 mg/kg, and 16 mg/kg. The results are shown in Fig. 16A. As shown in Fig. 16A, IL2/synTac administered at a dose of 4 mg/kg or 8 mg/kg gave similar results; IL-2/synTac administered at a dose of 16 mg/kg induced the most potent immunostimulatory activity.

The effect of the route of IL-2/synTac administration was tested. IL-2/synTac (F42A, H16A) was administered at a dose of 4 mg/kg either subcutaneously (SubQ) or intraperitoneally (IP). As shown in Fig. 16B, subcutaneous administration resulted in more potent immunostimulatory activity than IP administration.

The effect of IL-2 copy number on efficacy was determined. IL-2/synTac containing one copy of IL-2 (F42A, H16A) or two copies of IL-2 (F42A, H16A) were administered to mice bearing tumors expressing the HPV E7 epitope. The epitope included in these IL-2/synTac was the HPV E7 epitope. As shown in Fig. 17A and 17B, IL-2/synTac containing two copies of IL-2 (F42A, H16A) was more effective in reducing tumor size than IL-2/synTac containing only one copy of IL-2 (F42A, H16A).

Example 4. PK/PD and stability studies of IL-2/synTac

A pharmacokinetic (PK) analysis of IL-2/synTac was performed. IL-2/synTac (F42A, D20K, H16A) was administered intraperitoneally (IP) at a dose of 10 mg/kg. Serum samples were obtained at various time points after administration and the level of IL-2/synTac was measured in these serum samples. As shown in Fig. 18, the half-life of IL-2/synTac in serum was approximately 4 hours.

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IL-2/synTac was injected IP into C57BL/6 mice at 10 mg/kg and serum was collected two hours after injection. IL-2/synTac included a His ₆ tag. One hundred ng of injected protein or the equivalent of 40 μl serum was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-(His) ₆ or anti-β2M. The results shown in Fig. 19 indicate that IL-2/synTac remains stable and intact for at least 2 hours in vivo.

IL-2/synTac was stored at 4°C or 37°C for 5 days. 0.5 mg of each sample (at 10 mg/mL) was analyzed by size exclusion chromatography. As shown in Fig. 20, IL-2/synTac is stable and intact for at least 5 days at 4°C or 37°C.

Example 5. Effect of IL-2/synTac and anti-PD1 antibody on tumor volume

As shown in Fig. 35, administration of IL-2/synTac and anti-PD1 antibody to tumor-bearing mice reduced tumor volume.

Example 6. Production and characterization of synTac polypeptides with the 4-1BBL variant

synTac polypeptides have been synthesized and characterized. The following synTac polypeptides have been tested for activity against ovalbumin (OVA)-specific T cells:

1) Syn83/51. The light chain of Syn83/51 contains: a) the T-cell epitope OVA; b) amino acids 50-254 of the wild-type 4-1BBL polypeptide; and c) B2M; and the heavy chain of Syn83/51 contains: a) the heavy chain of MHC; and b) IgFc.

2) Syn239/345. The light chain of Syn239/345 contains: a) the T-cell epitope OVA; b) a trimer of amino acids 80-254 of wild-type 4-1BBL; and c) B2M; and the heavy chain of Syn239/345 contains: a) the heavy chain of MHC; and b) FcIgG2a.

3) Syn341/348. The Syn341/348 light chain comprises: a) the OVA T-cell epitope; b) the wild-type 4-1BBL trimer; and c) IgG2A Fc; and the Syn239/345 heavy chain comprises: a) the MHC heavy chain; and b) IgG2a Fc. In Syn341/348, the first unit of the 4-1BBL trimer contains amino acids 50-254 of wild-type 4-1BBL; the second and third units of the 4-1BBL trimer contain amino acids 80-254 of wild-type 4-1BBL.

4) Syn341/349. The light chain of Syn341/349 comprises: a T-cell epitope of OVA; b) a trimer of amino acids 80-254 of 4-1BBL, containing a K127A substitution in each unit of the trimer, with a GlySerSerSerSer linker between the first and second units and between the second and third units of the trimer; and c) B2M; and the heavy chain of Syn239/345 comprises: a) an MHC heavy chain; and b) an IgG2a Fc.

The resulting synTac heterodimers were cultured in vitro with ovalbumin-specific T cells for 3 days or 5 days at concentrations of 0; 1; 3.17; 10.01; 31.65, and 100 nM synTac. Controls included: a) medium only; b) phorbol 12-myristate 13-acetate (PMA) and ionophore A23187; and c) anti-CD3 antibody and anti-CD28 antibody.

After 3 days and after 5 days, the concentration of IFN-γ, IL-2, IL-6, TNF, IL-10, IL-17A and IL-4 in the culture medium were determined. In addition, the viability of OVA-specific T cells and the proliferation of OVA-specific T cells were determined.

The data are presented in Figs. 38-46.

As shown in Fig. 38-46, Syn 341/349 induces the production of IL-2 (a cellular fitness cytokine); induces the production of cytotoxic cytokines TNFa and IFN-γ; and induces proliferation and increases the viability of epitope-specific T cells.

Example 7. SynTac production in CHO cells

SynTac containing wild-type (wt) 4-1BBL or containing 4-1BBL with amino acid substitutions as shown in Fig. 47 were transiently expressed in CHO cells. The amount of synTac produced was determined. The amounts obtained are shown in Fig. 47.

Example 8. Effect of synTac 4-1BBL in vivo

SynTac containing the human papillomavirus (HPV) E7 antigenic peptide and the K127A 4-1BBL variant of the present invention (designated as CUE:4-1BBL (K127A) in Fig. 48) was administered at a dose of 5 mg/kg by intraperitoneal (IP) injection to mice bearing flank engrafted HPV TC-1 lung carcinoma. As a control, mice bearing the same tumor were administered phosphate-buffered saline (PBS). As shown in Fig. 48, the tumor volume in mice receiving CUE:4-1BBL (K127A) was reduced compared to mice receiving PBS.

Example 9. In vivo effects of co-administration of synTac 4-1BBL and an immune checkpoint inhibitor

As shown in Fig. 49, co-administration of synTac 4-1BBL of the present invention and an anti-PD1 antibody reduced tumor volume in a mouse tumor model and increased the percentage of tumor-infiltrating lymphocytes (TILs) that were granzyme B ^' .

Although the present invention has been described with reference to specific embodiments thereof, however, those skilled in the art should understand that various changes can be made and equivalents can be substituted without departing from the spirit and scope of the invention. In addition, numerous modifications can be made to adapt a particular situation, material, chemical composition, process, step or steps of the process to the purpose, spirit and

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Claims (19)

scope of the present invention. All such modifications are intended to be included within the scope of the appended claims. CLAUSE OF INVENTION 1. A method of treating cancer in an individual, comprising administering to the individual (i) an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody targeting PD-1, and (ii) a protein, wherein the protein comprises two heterodimers, wherein the two heterodimers have identical amino acid sequences, and wherein each heterodimer comprises: a) a first polypeptide comprising: i) a cancer-associated epitope, wherein the cancer-associated epitope is a peptide having a length of 8 to 14 amino acids; and ii) the polypeptide of β2-microglobulin f2M); and b) a second polypeptide comprising: i) two immunomodulatory polypeptides, wherein each of the two immunomodulatory polypeptides is a variant of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49; ii) a major histocompatibility complex (MHC) class I heavy chain polypeptide; and iii) an immunoglobulin (Ig) Fc polypeptide, wherein two heterodimers are linked by one or more disulfide bonds between the Ig Fc polypeptides, wherein the cancer-associated epitope is not an HPV epitope, and wherein the first polypeptide comprises a linker between the epitope and the B2M polypeptide, and wherein the second polypeptide comprises an independently selected peptide linker between one or more of: a) a variant of the IL-2 polypeptide and the MHC heavy chain polypeptide; b) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide; and c) two variants of the IL-2 polypeptide. 2. The method of treating cancer according to claim 1, wherein the cancer-associated epitope is an epitope present in a cancer-associated antigen selected from the group consisting of α-folate receptor; carbonic anhydrase IX (CAIX); CD19; CD20; CD22; CD30; cD33; CD44v7/8; carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor; ganglioside antigen GD2; Her2/neu; IL-13R-a2; kappa light chain; LeY; cell adhesion molecule L1; melanoma-associated antigen (MAGE); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); tumor-associated glycoprotein 72 (TAG-72); and vascular endothelial growth factor receptor 2 (VEGF-R2). 3. The method for treating cancer according to claim 1, wherein the B2M polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 21-119 of the amino acid sequence indicated in SEQ ID NO: 95. 4. The method of treating cancer according to claim 1, wherein the IgFc polypeptide comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 44-47. 5. The method for treating cancer according to claim 1, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence indicated in SEQ ID NO: 50. 6. A method for treating cancer according to claim 1, wherein: a) the first polypeptide of each heterodimer contains, in the direction from N-terminus to C-terminus: i) a peptide epitope, wherein the peptide epitope is 8-12 amino acids long; ii) a peptide linker; and iii) a b2M polypeptide, wherein the b2M polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence 21-119 of any of the amino acid sequences set forth in SEQ ID NO: 95-98; b) the second polypeptide of each heterodimer contains, in the direction from the N-terminus to the C-terminus: i) a first immunomodulatory polypeptide, wherein the first immunomodulatory polypeptide is a variant of an IL-2 polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 49; ii) a second immunomodulatory polypeptide, wherein the second immunomodulatory polypeptide is a variant of an IL-2 polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 49; iii) an MHC class I heavy chain polypeptide, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid - 127 049999 acidic sequence set forth in SEQ ID NO: 50; and iv) an IgFc polypeptide, and wherein the second polypeptide comprises a peptide linker independently selected from one or more of the following: a) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide; b) a second immunomodulatory polypeptide and an MHC class I heavy chain polypeptide; and c) MHC class I heavy chain polypeptide and Ig Fc polypeptide. 7. The method for treating cancer according to claim 6, wherein: i) the b2M polypeptide comprises a Cys at amino acid position 12 according to the amino acid numbering of SEQ ID NO: 48; and ii) the MHC class I polypeptide comprises a Cys at residue 236 according to the amino acid numbering of SEQ ID NO: 50, wherein each heterodimer comprises a disulfide bond linking the first polypeptide to the second polypeptide, and wherein the disulfide bond links the Cys at amino acid position 12 of the b2M polypeptide to the Cys at amino acid position 236 of the MHC heavy chain polypeptide, and wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO: 21. 8. The method for treating cancer according to claim 7, wherein i) the B2M polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48; ii) the first and second IL-2 polypeptide variants comprise the amino acid sequence set forth in SEQ ID NO: 49; iii) the MHC class I heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 50; and iv) the Ig Fc polypeptide comprises a L14A substitution and a L15A substitution according to the amino acid numbering set forth in SEQ ID NO: 44, and wherein the second polypeptide comprises a peptide linker between: a) the first and second immunomodulatory polypeptides; b) a second variant of an immunomodulatory polypeptide and an MHC class I heavy chain polypeptide; and c) MHC class I heavy chain polypeptide and Ig Fc polypeptide. 9. The method of claim 1, wherein the individual has become refractory to or has not responded to a prior cancer treatment. 10. The method of claim 8, wherein the individual has become refractory to or has not responded to a previous cancer treatment. 11. The method according to claim 6, wherein the protein is administered in an amount of 1 mg/kg to 10 mg/kg of body weight. 12. The method according to claim 7, wherein the protein is administered in an amount of 1 mg/kg to 10 mg/kg of body weight. 13. The method according to claim 8, wherein the protein is administered in an amount of 1 mg/kg to 10 mg/kg of body weight. 14. The method according to claim 1, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 51 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 52. 15. The method according to claim 1, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 53 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 54. 16. The method according to claim 6, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 51 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 52. 17. The method according to claim 6, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 53 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 54. 18. The method according to claim 8, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 51 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 52. 19. The method according to claim 8, wherein the immune checkpoint inhibitor comprises complementarity determining regions (CDRs) present in the variable region of the heavy chain of SEQ ID NO: 53 and comprises CDRs present in the variable region of the light chain of SEQ ID NO: 54. - 128 -