Multimers

Abstract

Multimers of effector domains have recognized utility in medical and non-medical applications. The present invention relates to tetramers of polypeptides and multimers, such as tetramers, octamers, dodecamers and hexadecamers, of epitopes or effector domains, such as antigen binding sites (e.g., antibody or TCR binding sites that specifically bind to an antigen or pMHC or to a variable domain thereof), or of peptides, such as incretins, insulin or hormone peptides.

Description

[Technical Field]

[0001] The present invention relates to tetramers of polypeptides and multimers, such as tetramers, octamers, dodecamers and hexadecamers, of epitopes or effector domains, such as antigen binding sites (e.g., antibody or TCR binding sites that specifically bind to an antigen or pMHC or to a variable domain thereof), or of peptides, such as incretins, insulin or hormone peptides. [Background technology]

[0002] Multimers of effector domains have recognized utility in medical and non-medical applications to enhance biological or binding activity, for example, to provide higher avidity of antigen binding (e.g., in the case of effector domains that are antibody or TCR binding domains), to combine to increase the activity and presence of effector domains, or to provide bispecific or multispecific targeting or interaction with target ligands in vivo or in vitro.

[0003] Multimerization domains that induce self-assembly of protein monomers into multimers are known in the art. Examples include domains found in transcription factors such as p53, p63, and p73, as well as domains found in ion channels such as TRP cation channels. The transcription factor p53 can be divided into various functional domains, such as an N-terminal transactivation domain, a proline-rich domain, a DNA-binding domain, a tetramerization domain, and a C-terminal regulatory region. The tetramerization domain of human p53 extends from residues 325 to 356 and has a four-helix bundle fold (Jeffrey et al., Science (New York, NY) 1995, 267(5203):1498-1502). The TRPM tetramerization domain is a short, inverted coiled-coil tetramerization domain found in transient receptor potential cation channel subfamily M member proteins 1 to 8. It is held together by extensive core packing and interchain polarity interactions (Fujiwara et al., Journal of Molecular Biology 2008, 383(4):854-870). Transient receptor potential (TRP) channels constitute a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Another example is the potassium channel BTB domain. This domain is found at the N-terminus of voltage-gated potassium channel proteins and represents a cytoplasmic tetramerization domain (T1) involved in the assembly of α subunits into functional tetrameric channels (Bixby et al., Nature Structural Biology 1999, 6(1):38-43). This domain is also found in non-potassium channel proteins, such as KCTD1 (potassium channel tetramerization domain-containing protein 1; Ding et al., DNA and Cell Biology 2008, 27(5):257-265).

[0004] Multimeric antibody fragments have been generated using a variety of multimerization techniques, including biotin, dHLX, ZIP and BAD domains, and p53 (Thie et al., Nature Boitech., 2009:26, 314-321). Biotin is a bacterial protein that is efficient to produce and elicits an immune response in humans.

[0005] Human p53 (UniProtKB-P04637(P53_HUMAN)) functions as a tumor suppressor in many tumor types, inducing growth arrest or apoptosis depending on the physiological context and cell type. It is involved in cell cycle regulation as a transactivator, acting to negatively regulate cell division by controlling a set of genes required for this process. Human p53 is found in elevated amounts in a variety of transformed cells. It is frequently mutated or inactivated in approximately 60% of cancers. Human p53 deficiency is seen in Barrett's metaplasia, a condition in which the normal stratified squamous epithelium of the lower esophagus is replaced by metaplastic columnar epithelium. This condition develops as a complication in approximately 10% of patients with chronic gastroesophageal reflux disease and predisposes to the development of esophageal adenocarcinoma.

[0006] Nine isoforms of p53 occur naturally and are expressed in a wide range of normal tissues, but in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues but is undetectable in the brain, lung, prostate, muscle, fetal brain, spinal cord, and fetal liver. Isoform 3 is expressed in most normal tissues but is undetectable in the lung, spleen, testis, fetal brain, spinal cord, and fetal liver. Isoform 7 is expressed in most normal tissues but is undetectable in the prostate, uterus, skeletal muscle, and breast. Isoform 8 is detected only in the colon, bone marrow, testis, fetal brain, and intestine. Isoform 9 is expressed in most normal tissues but is undetectable in the brain, heart, lung, fetal liver, salivary gland, breast, or intestine. Summary of the Invention

[0007] The present invention provides a polypeptide comprising an antibody Fc region, the Fc region comprising an antibody CH2 and an antibody CH3, and a self-associating multimerization domain (SAM), wherein CH2 comprises an antibody hinge sequence and lacks a core hinge region. Advantageously, the Fc does not directly pair with another Fc, which is useful for generating multimers by multimerization using the SAM domain. For example, the benefit may be to support desired multimer formation and / or to increase the purity of the multimers formed by such multimerization.

[0008] The present invention also provides a multimer of multiple antibody Fc regions, wherein each Fc is composed of a respective polypeptide and is not paired with another Fc region, and optionally, the multimer is for medical use.

[0009] The present invention also provides: In the first configuration A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., a protein domain or peptide), wherein the multimer is multimerized by first, second, third and fourth self-associating tetramerization domains (TDs) associated together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of the protein domain or peptide.

[0010] In the second configuration An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of a TCR binding site, insulin peptide, incretin peptide or peptide hormone, or a plurality of said tetramers or octamers. An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of an antibody binding site or antibody variable domain (e.g., a single variable domain), or a plurality of said tetramers or octamers. In examples, the tetramer or octamer is soluble in an aqueous solution (e.g., aqueous eukaryotic cell culture medium). In examples, the tetramer or octamer is expressible in eukaryotic cells. Examples are provided below.

[0011] In the third configuration The following (a) a TCR V domain or TCR binding site, wherein the tetramer or octamer is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (b) an antibody single variable domain, wherein the tetramer or octamer is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (c) a TCR V domain or TCR binding site, wherein the tetramer or octamer can be expressed intracellularly and / or extracellularly by HEK293 cells; or (d) a tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., a tetramer or octamer) of an antibody variable domain (e.g., an antibody single variable domain), wherein the tetramer or octamer can be expressed intracellularly and / or extracellularly by HEK293 cells.

[0012] In the fourth configuration Multimeric, tetrameric, octameric, dodecamer, hexadecamer or 20-mer (eg, tetramer or octamer) engineered polypeptides or monomers of the invention.

[0013] In the fifth configuration An engineered (and optionally isolated) engineered polypeptide (P1) comprising (in the N-terminal to C-terminal direction): (a) TCR V1-TCR C1-antibody CH1 (e.g., IgG CH1)-optional linker-TD; (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) TCR V1-TCR C1-antibody CH1 (e.g., IgG CH1)-optional linker-TD, where V1 is Vδ and C1 is Cδ; or (b) TCR V1-antibody CH1 (e.g., IgG CH1)-optional linker-TD, (i) V1 is Vα, (i) V1 is Vβ, (iii) V is V, or (iv) TCR V1-antibody CH1 (e.g., IgG CH1)-optional linker-TD, where V1 is Vδ; or (c) antibody V1-antibody CH1 (e.g., IgG CH1)-optional linker-TD; (i) V1 is VH, or (ii) antibody V1-antibody CH1 (e.g., IgG CH1)-optional linker-TD, where V1 is VL (e.g., Vλ or Vκ); or (d) antibody V1—any antibody CH1 (e.g., IgG CH1)-antibody Fc (e.g., IgG Fc)-any linker-TD; (i) V1 is VH, or (ii) antibody V1--any antibody CH1 (e.g., IgG CH1)-antibody Fc (e.g., IgG Fc)-any linker-TD, wherein V1 is VL (e.g., Vλ or Vκ); or (e) antibody V1-antibody CL (e.g., Cλ or Cκ)-optional linker-TD; (i) V1 is VH, or (ii) antibody V1-antibody CL (e.g., Cλ or Cκ)-optional linker-TD, where V1 is VL (e.g., Vλ or Vκ); or (f) TCR V1-TCR C1-optional linker-TD, (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) An engineered (and optionally isolated) engineered polypeptide (P1) comprising TCR V1-TCR C1-optional linker-TD, where V1 is Vδ and C1 is Cδ.

[0014] In the sixth configuration A nucleic acid encoding an engineered polypeptide or monomer of the invention, optionally wherein the nucleic acid is comprised by an expression vector for expressing the polypeptide.

[0015] In the seventh configuration Use of a nucleic acid or vector of the invention in a method for producing a protein multimer to produce a multimer that is expressed and / or secreted intracellularly, the method comprising expressing and / or secreting the multimer from a eukaryotic cell containing the nucleic acid or vector.

[0016] In the eighth configuration, (a) TCR V domain multimers, wherein the method comprises soluble and / or intracellular expression of a TCR V-TD (e.g., NHR2 TD or TCR V-p53 TD) fusion protein expressed in a eukaryotic cell, and optionally, the method comprises isolating a plurality of said multimers; (a) antibody V domain multimers, wherein the method comprises soluble and / or intracellular expression of an antibody V (e.g., single variable domain)-TD (e.g., V-NHR2 TD or V-p53 TD) fusion protein expressed in a eukaryotic cell, and optionally, the method comprises isolating a plurality of said multimers; (c) an incretin peptide (e.g., GLP-1, GIP, or insulin) multimer, wherein the method comprises soluble and / or intracellular expression of an incretin peptide-TD (e.g., incretin peptide-NHR2 TD or incretin peptide-p53 TD) fusion protein expressed in a eukaryotic cell, such as a HEK293T cell, and optionally, the method comprises isolating a plurality of said multimers; or (d) A method for producing peptide hormone multimers, the method comprising soluble and / or intracellular expression of a peptide hormone-TD (e.g., peptide hormone-NHR2 TD or peptide hormone-p53 TD) fusion protein expressed in a eukaryotic cell such as a HEK293T cell, and optionally isolating a plurality of said multimers.

[0017] In the ninth configuration Use of a nucleic acid or vector of the present invention in a method for producing a protein multimer to produce glycosylated multimers in a eukaryotic cell containing the nucleic acid or vector.

[0018] In the tenth configuration Use of a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog thereof) in a method for producing a tetramer of a polypeptide to produce a higher yield of the tetramer relative to a monomer and / or dimer of the polypeptide.

[0019] In the 11th configuration 1. Use of an engineered polypeptide in a method for producing a tetramer of a polypeptide comprising multiple copies of a protein domain or peptide to produce a higher yield of tetramer relative to a monomer and / or dimer of the polypeptide, wherein the engineered polypeptide comprises one or more copies of the protein domain or peptide and further comprises a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD or a homolog or ortholog).

[0020] In the 12th configuration Use of a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog thereof) in a method for producing a tetramer of a polypeptide to produce multiple tetramers that are not a mixture with monomers, dimers, or trimers.

[0021] In the 13th configuration A host eukaryotic cell comprising a nucleic acid or vector for intracellular and / or secretory expression of a multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., tetramer, octamer), engineered polypeptide or monomer of the invention.

[0022] In the 14th configuration 1. Use of an engineered polypeptide in a method for producing tetramers of a polypeptide comprising multiple copies of a protein domain or peptide to produce multiple tetramers that are not a mixture with monomers, dimers, or trimers, wherein the engineered polypeptide comprises one or more copies of the protein domain or peptide and further comprises a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog).

[0023] In the 15th configuration A multivalent heterodimeric soluble T cell receptor capable of binding to a pMHC complex comprising: (i) a TCR extracellular domain; (ii) an immunoglobulin constant domain; and (iii) a multivalent heterodimeric soluble T cell receptor comprising the NHR2 multimerization domain of ETO.

[0024] In the 16th configuration A polymeric immunoglobulin (i) an immunoglobulin variable domain; (ii) a multimeric immunoglobulin comprising the NHR2 multimerization domain of ETO.

[0025] In the 17th configuration 1. A method for assembling soluble multimeric polypeptides, comprising: (a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO; (b) assembling multiple copies of said monomers, thereby obtaining a multimeric soluble polypeptide.

[0026] In the 18th configuration, A mixture comprising (i) a cell line encoding a polypeptide of the present invention (e.g., a eukaryotic mammalian cell line, such as a HEK293, CHO, or Cos cell line), and (ii) a tetramer of the present invention.

[0027] In the 19th configuration 1. A method for improving the yield of tetramers of protein effector domains (e.g., antibody variable domains or binding sites), the method comprising expressing a tetramer of a polypeptide from a cell line (e.g., a mammalian cell, CHO, HEK293 or Cos cell line), wherein the polypeptide is a polypeptide of the invention and comprises one or more effector domains, and optionally isolating the expressed tetramer.

[0028] In the 20th configuration (N-terminal to C-terminal or C-terminal to N-terminal) (i) an immunoglobulin superfamily domain; (ii) an optional linker; and (iii) a self-associating multimerization domain (SAM) (optionally a self-associating tetramerization domain (TD)), a polypeptide comprising:

[0029] The present invention also provides pharmaceutical compositions, cosmetics, foods, beverages, cleaning products, and detergents comprising the multimers, tetramers, octamers, dodecamers, hexadecamers, or 20-mers (e.g., tetramers or octamers) of the present invention.

[0030] A multimer herein is, for example, a dimer, trimer, tetramer, octamer, dodecamer, hexameric, or 20-mer.

[0031] As shown in Example 22, dodecameric and hexadecameric multimers surprisingly exhibit very high functional affinities for antigen binding due to the increased avidity effect. These functional affinities toward 8-12 binding sites (compare Tables 15 and 16) or 8-16 binding sites are more than additive, and increased synergy is seen as a result of enhanced avidity. Thus, in one embodiment, multimers that are 12-valent toward the antigen (i.e., dodecameric as described herein) are preferred, and in another embodiment, multimers that are 16-valent toward the antigen (i.e., hexadecamers as described herein) are preferred. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 is a schematic diagram depicting the stepwise self-assembly of tetravalent heterodimeric soluble TCR protein complexes via monomers and homodimers, assisted by the NHR2 tetramerization domain. [Figure 2] FIG. 1 is a schematic diagram showing the stepwise self-assembly of an octavalent heterodimeric soluble TCR protein complex via monomer 2 and homodimer 2, assisted by the NHR2 tetramerization domain and immunoglobulin hinge domain. [Figure 3]FIG. 1 is a schematic diagram of the domain arrangement of the α and β chains used in the expression and assembly of the ts-NY-ESO-1 TCR. [Figure 4] FIG. 1 is a schematic diagram of the domain arrangement of the α and β chains used in the expression and assembly of the os-NY-ESO-1 TCR. [Figure 5] Amino acid sequences of the α and β chains of the ts-NY-ESO-1 TCR protein complex. The amino acid sequences of the alternative domains are underlined. [Figure 6] Amino acid sequences of the α and β chains of the os-NY-ESO-1 TCR protein complex. The amino acid sequences of alternative domains are underlined. [Figure 7] FIG. 1 is a schematic diagram of the domain arrangement of the α and β chains used in the expression and assembly of the ts-NY-ESO-1 TCR-IL2 fusion protein complex. [Figure 8] FIG. 1 is a schematic diagram of the domain arrangement of the α and β chains used in the expression and assembly of the os-NY-ESO-1 TCR-IL2 fusion protein complex. [Figure 9]

[0033] Figure 1 shows the amino acid sequences of the α and β chains of the ts-NY-ESO-1 TCR-IL2 fusion protein complex. The amino acid sequences of the alternative domains are underlined. [Figure 10] Amino acid sequences of the α and β chains of the os-NY-ESO-1 TCR-IL2 fusion protein complex. The amino acid sequences of the alternative domains are underlined. [Figure 11A] FIG. 1 is a schematic diagram depicting the stepwise self-assembly of tetravalent single-domain antibody (dAb) complexes via monomers and homodimers assisted by the NHR2 tetramerization domain. [Figure 11B] FIG. 1 is a schematic diagram of a domain arrangement for assembling a tetravalent dAb, including a linker and NHR2 domain. [Figure 12A] FIG. 1 is a schematic diagram depicting the stepwise self-assembly of tetravalent Fab complexes via monomers and homodimers assisted by the NHR2 tetramerization domain. [Figure 12B]FIG. 1 is a schematic diagram of the domain arrangement for assembling a tetravalent Fab, including the linker and NHR2 domains in the heavy chain and the variable and constant domains of the light chain. [Figure 13A] FIG. 1 is a schematic diagram depicting the stepwise self-assembly of an octavalent Fab complex via monomers and homodimers assisted by an antibody hinge region linked to an NHR2 tetramerization domain and a CH1 domain. [Figure 13B] Schematic diagram of the domain arrangement for assembling an octavalent Fab, including the hinge, linker and NHR2 domains in the heavy chain and the variable and constant domains in the light chain. [Figure 14] Schematic diagram of Quad (quadruple mutant) 16 and Quad 17. [Figure 15] The monomer sequences and organization of (A) Quad16 and (B) Quad17 are shown. [Figure 16] Analysis of secreted proteins using anti-Ig Western blot is shown. (A) PAGE gel under SDS-denaturing conditions - 16 = Quad16; 17 = Quad17; (B) PAGE gel under native (i.e., non-denaturing) conditions - 16 = Quad16; 17 = Quad17. [Figure 17] Western blot prepared from a denaturing SDS-PAGE gel probed with anti-human IgG HRP detection antibody. (A) Protein samples for Quads 3 and 4 were prepared from whole cell extracts and loaded in lanes 1 and 2, respectively. The expected Mw for Quads 3 and 4 is 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quads 12 and 13 were prepared from whole cell extracts and loaded in lanes 1 and 2, respectively. The expected Mw for Quads 12 and 13 is 47.8 and 48.1 kDa, respectively. [Figure 18]Western blot prepared from a denaturing SDS-PAGE gel probed with anti-human IgG HRP detection antibody. (A) Protein samples from Quads 3 and 4 were prepared by concentrating cell supernatants and loaded in lanes 1 and 2, respectively. The expected Mw for Quads 3 and 4 is 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quads 12 and 13 were prepared by concentrating cell supernatants and loaded in lanes 1 and 2, respectively. The expected Mw for Quads 12 and 13 is 47.8 and 48.1 kDa, respectively. [Figure 19] Western blots prepared from denaturing SDS-PAGE gels probed with anti-HIS HRP detection antibody. (A) Protein samples from Quads 14, 15, 18, and 19 were prepared from whole-cell extracts and loaded in lanes 1 and 4, respectively. The expected Mw for Quads 14, 15, 18, and 19 are 22.0, 22.3, 37.4, and 37.7 kDa, respectively. (B) Protein samples from Quads 23, 24, 26, and 27 were prepared from whole-cell extracts and loaded in lanes 1–4, respectively. The expected Mw for Quads 23, 24, 26, and 27 are 32.1, 32.4, 33.7, and 34.0 kDa, respectively. (C) Protein samples from Quads 34 and 38 were prepared from whole-cell extracts and loaded in lanes 1–2, respectively. The expected Mw for Quads 34 and 38 are 25.5 and 25.4 kDa, respectively. (D) Protein samples from Quads 40, 42, 44, and 46 were prepared from whole cell extracts and loaded in lanes 1–4, respectively. The expected Mw for Quads 40, 42, 44, and 46 are 25.4, 37.6, 25.5, and 38.0 kDa, respectively. Lane U contains concentrated serum prepared from untransfected HEK293T cells (negative control), and lane C is a His-tagged protein used as a positive control for the anti-His HRP detection antibody. The serum anti-His background band is highlighted with a black arrow, which can be consistently detected in all blots. [Figure 20](A) Western blot prepared from a denaturing SDS-PAGE gel and probed with anti-human IgG HRP detection antibody. Protein samples from Quads 14 and 15 prepared from whole cell extracts were loaded in lanes 1 and 2, respectively. The expected Mw for Quads 14 and 15 is 22.0 and 22.3 kDa, respectively. (B) Western blot prepared from a native PAGE gel and probed with anti-human IgG HRP detection antibody. Lanes 1 and 2 contain protein samples of Quads 14 and 15 prepared from whole cell extracts. [Figure 21] It is a Quad polypeptide fused to a leader and tag sequence. If a linker is present, it is a G4S (only one G4S). * indicates a TCR constant domain with an introduced cysteine ​​residue that allows S-S bond formation between the TCR alpha and beta chains. A human IgG1 hinge was used. All C regions are human. The TCR V domain is specific for NY-ESO-1. GFP = green fluorescent protein. [Figure 22] Schematic representation of the multimeric structure of Quad formats A-AC. The description and monomeric building blocks from which tetravalent Quad molecules are assembled are shown in Table 8. [Figure 23] SDS-PAGE analysis of monospecific tetravalent dAb Quad57 protein purified from culture supernatant. Quad protein migrated according to the expected MW, as indicated by the arrow, without visible impurities. [Figure 24] SDS-PAGE analysis of bispecific tetravalent dAb Quad54 protein purified from culture supernatant. (A) Quad protein migrates according to the expected MW as indicated by the arrows without visible impurities. (B) Direct binding ELISA using Quad54 protein serially diluted in a fixed concentration of recombinant TNFα protein coated on a plate. Quad54 binds to TNFα protein in a dose-dependent manner. [Figure 25]SDS-PAGE analysis of the monospecific tetravalent scFv Quad. Quad51 and 63 proteins were purified from culture supernatant and analyzed by SDS-PAGE. (A) Quad proteins migrated according to their expected MW, as indicated by the arrows, without visible impurities. (B) Direct binding ELISA using Quad51 and 63 proteins serially diluted with a fixed concentration of recombinant TNFα protein coated on a plate. Both Quad51 and 63 bind to TNFα protein in a dose-dependent manner with similar binding strength. (C) SDS-PAGE analysis of the W51ScFv monovalent anti-TNFα control protein. (D) Western blot analysis of TNFα-mediated caspase-3 signaling in the presence of Quad51, Humira (Hum), and W51ScFv. Culture medium (CM) alone or culture medium containing actinomycin D (AD) was used as a negative control. The detection antibodies used for each blot are indicated next to each blot. Western blot detected with anti-tubulin represents an internal loading control. (E) SDS-PAGE analysis of Quad53 Tet protein purified from culture supernatant. Quad protein migrated according to the expected MW, as indicated by the arrow. (F) Direct binding ELISA using serially diluted Quad53 Tet protein coated onto a plate containing a fixed concentration of recombinant CD20 protein. Quad53 Tet binds to CD20 protein in a dose-dependent manner. [Figure 26]SDS-PAGE analysis of the monospecific octavalent scFv Quad53 protein purified from culture supernatant. (A) The Quad protein migrates according to the expected MW, as indicated by the arrow, without visible impurities. (B) Direct binding ELISA using serially diluted Quad53 Oct protein coated onto a plate containing a fixed concentration of recombinant CD20 protein. Quad53 Oct binds to CD20 protein in a dose-dependent manner. (C) Monovalent, tetravalent, and octavalent versions of Quad53 analyzed by SDS-PAGE. (D) Direct binding ELISA comparing the binding strength of monovalent, tetravalent, and octavalent versions of Quad53 to recombinant CD20. Increasing binding strength is observed with increasing Quad53 valency. [Figure 27] SDS-PAGE analysis of bispecific tetravalent SCfV Quad55 protein purified from culture supernatant. (A) Quad protein migrates according to the expected MW as indicated by the arrows without visible impurities. (B) Direct binding ELISA using Quad55 protein serially diluted with a fixed concentration of recombinant TNFα protein coated on a plate. Quad55 binds to TNFα protein in a dose-dependent manner. [Figure 28] SDS-PAGE analysis of bispecific tetravalent Quad. Bispecific scFv x dAb Quad56 protein was purified from culture supernatant and analyzed by SDS-PAGE. (A) Quad protein migrated according to the expected MW as indicated by the arrows without visible impurities. (B) Direct binding ELISA using serially diluted Quad56 protein coated onto a plate containing a fixed concentration of recombinant TNFα protein. Quad56 binds to TNFα protein in a dose-dependent manner. (C) Direct binding ELISA comparing the binding strength of three different bispecific tetravalent Quad formats (Quad54: dAb x dAb, Quad55: scFv x scFv, and Quad56: ScFv x dAb) to recombinant CD20. All three bispecific tetravalent Quad formats bind to CD20 with similar binding strength and in a dose-dependent manner. [Figure 29] SDS-PAGE analysis of monospecific tetravalent monomeric Ig scFv Quad64 version 1 protein purified from culture supernatant. "Monomeric Ig" refers to a multimer of the polypeptide of the present invention, including Fc, where CH2 contains a hinge sequence but lacks the core hinge region, which advantageously prevents the Fc regions from multimerizing together; instead, multimerization is mediated by the SAM (e.g., TD) domains of the polypeptides in the multimer. (A) Quad protein migrates according to the expected MW, as indicated by the arrow, without visible impurities. (B) Direct binding ELISA using Quad64 protein serially diluted with a fixed concentration of recombinant CD20 protein coated on a plate. Quad64 binds to CD20 protein in a dose-dependent manner. [Figure 30] SDS-PAGE analysis of the monospecific tetravalent monomeric Ig scFv Quad65 version 2 protein purified from culture supernatant. (A) The Quad protein migrates according to the expected MW as indicated by the arrows without visible impurities. (B) Direct binding ELISA using serially diluted Quad65 protein coated onto a plate containing a fixed concentration of recombinant CD20 protein. Quad65 binds to CD20 protein in a dose-dependent manner. [Figure 31] (A&B) Schematic representation of Quad68 and Quad69. The specificity of the dAbs for PD-L1 and 4-1BB is indicated by the arrows. (C&D) SDS-PAGE analysis of Quad68 and Quad69 proteins purified from culture supernatants. The Quad proteins migrated according to the expected MW, as indicated by the arrows, without visible impurities. [Figure 32] Alignment of p53 tetramerization domains (TDs) from different species. Sequence variations from the human TD are highlighted in bold and underlined. [Figure 33]Schematic representation of the molecular (A) and structural (B & C) configurations of the tetravalent and octavalent anti-TNFα dAb monomeric Ig Quad. Purified Quad proteins were analyzed by SDS-PAGE (D). The tetravalent and octavalent Quad proteins migrated according to the expected molecular weights, as shown, without any visible impurities. The core hinge region has been deleted in these formats, indicated as * or CH2' in the figure. The Q92 chain contains a C-terminally located His tag, not shown in the figure. [Figure 34] SDS-PAGE analysis of octavalent bispecific anti-PDL1 / 4-1BB dAb monomeric Ig Quad (A) and octavalent monospecific anti-PDL1 dAb monomeric Ig Quad (B) proteins purified from culture supernatants. The Quad proteins migrated according to the expected molecular weight, as indicated by the arrows, without visible impurities. [Figure 35] SDS-PAGE analysis of Avelumab Fab monomer Ig Quad (A) and Humira Fab monomer Ig Quad (B) proteins purified from culture supernatant. The Quad proteins migrated according to the expected molecular weight, as indicated by the arrows, without visible impurities. Humira (adalimumab) Fab monomer Ig Quad was further analyzed by SEC, revealing that the fully assembled tetrameric protein eluted as a single peak at the expected molecular weight (315.8 kDa), with no visible dimeric or monomeric forms. [Figure 36] Schematic representation of the structural arrangement of aflibercept monomeric Ig Quad (Q96). * indicates that the hinge region lacks the core hinge region. Analysis of the Q96 Quad protein by SDS-PAGE revealed a single protein band at the expected molecular weight (B). The binding profile of Q96 to VEGF-A was analyzed by ELISA, revealing dose-dependent binding. [Figure 37]A schematic diagram of the molecular configuration of the monovalent, tetravalent, and octavalent anti-TNFα dAb Quads (A). All constructs contain a C-terminally located His tag, not shown. Purified Quad proteins were analyzed by SDS-PAGE, revealing single protein bands at the expected molecular weights for the monovalent, tetravalent, and octavalent versions (lanes 1–3, respectively) (B). TNFα binding assays using the Q88 monovalent, tetravalent, and octavalent Quad proteins (C) in addition to the Q92 and Q92+Q93 (D) Quad proteins were performed by ELISA, demonstrating dose-dependent binding. The TNFα neutralization capacity of each Quad protein was analyzed using a WEHI cell-based bioassay (E and F, respectively). Enhanced TNFα neutralization capacity can be seen in Quads with increasing numbers of anti-TNFα dAb binding domains. [Figure 38] Schematic representation of the molecular (A) and structural (B) sequences of the dodecavalent and hexahexavalent anti-TNFα dAb multimers (alternatively referred to as multimeric Quads). The light chain constant region is represented by CL, which is composed of either a kappa or lambda constant region. The Q142 construct contains a C-terminally located His tag, not shown. Purified Quad proteins were analyzed by SDS-PAGE (C). Dodecavalent anti-TNFα dAb Quads with either lambda (lane 1) or kappa (lane 2) constant regions migrated on the SDS-PAGE gel according to the expected molecular weight. Dodecavalent anti-TNFα dAb Quads with either lambda (lane 3) or kappa (lane 4) constant regions also migrated on the SDS-PAGE gel according to the expected molecular weight. Figure 10. TNFα neutralization potency of dodecavalent and hexahexavalent anti-TNFα dAb Quad proteins bearing either kappa (D) or lambda (E) light chain constant regions as analyzed using the WEHI cell-based bioassay. Enhanced potency is seen with increasing anti-TNFα dAb binding domains. [Figure 39]Schematic structures of dodecavalent trispecific (A) and hexavalent tetraspecific (B) multimers. For each format, the schematic structure of the monomeric building blocks is also shown. Regions within these molecules containing optional flexible linkers are indicated by arrows. CL domains can be either kappa or lambda C regions. The dodecavalent trispecific Quad contains three different dAbs, labeled 1 through 3, which can bind to three different antigens on three different cells, three antigens on the same cell, or three different epitopes on the same antigen. This format represents a 4+4+4 trispecific dodecavalent Quad. The dodecavalent trispecific Quad contains four different dAbs, labeled 1 through 4, which can bind to four different antigens on four different cells, four antigens on the same cell, or four different epitopes on the same antigen. This format represents a 4+4+4+4 tetraspecific dodecavalent Quad. [Figure 40] Schematic structure of the tetravalent, non-Ig-like Humira Fab-TD Quad (A). The schematic structure of the monomeric building blocks is also shown. The region within this molecule containing the optional flexible linker is indicated by an arrow (e.g., each linker is a G4S linker, as described herein). * indicates the absence of the core hinge region (i.e., the lower hinge sequence is present, and optionally, the upper hinge sequence is also present). Purified Humira Fab-TD protein was analyzed by SDS-PAGE, revealing a single protein band at the expected molecular weight (B). TNFα binding assays using Humira Fab-TD and Humira Fab monovalent control proteins were performed by ELISA, revealing dose-dependent binding (C). The TNFα neutralization ability of each of the Quad proteins was analyzed using a WEHI cell-based bioassay (D). Enhanced TNFα neutralization ability can be seen with Humira Fab-TD compared to the Humira Fab monovalent control. [Figure 41]Schematic structures of the non-Ig-like Quad (A) or Ig-like Quad (B) of an octavalent Fab. For each format, the schematic structure of the monomeric building blocks is also shown. Regions within these molecules containing optional flexible linkers are indicated by arrows (e.g., each linker is a G4S linker, as described herein). [Figure 42] FIG. 23 is a schematic diagram of the monomer building blocks of formats A-AC outlined in FIG. 22.

[0033] All polypeptide diagrams and amino acid sequences herein are written from N-terminus to C-terminus. All nucleotide sequences herein are written from 5' to 3'. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention relates to tetramers of polypeptides, and multimers, such as tetramers, octamers, dodecamers, and hexadecamers or 20-mers (e.g., tetramers and octamers), of epitopes or effector domains (such as antigen-binding sites (e.g., antibody or TCR binding sites that specifically bind to an antigen or pMHC or to a variable domain thereof)), or of peptides, such as incretins, insulin, or hormone peptides. In embodiments, the multimers of the invention can be usefully produced in eukaryotic systems and secreted from eukaryotic cells in a soluble form, which is useful for various industrial applications, for example, the production of pharmaceuticals, diagnostic agents, imaging agents, detergents, and the like. Higher order multimers, such as tetramers or octamers, of effector domains or peptides are useful for enhancing antigen or pMHC binding activity. This may be useful for producing effective pharmaceuticals or for increasing the sensitivity of diagnostic reagents comprising multimers, such as tetramers or octamers. An additional or alternative advantage is that the multimers of the invention have an improved in vivo half-life when administered to a human or animal subject, e.g., to treat or prevent a disease or condition in the subject. Usefully, the present invention can also provide multispecific (e.g., bispecific or trispecific) multivalent binding proteins. Specificity can be related to the specificity of antigen or pMHC binding. By using a single engineered polypeptide comprising a binding domain or peptide, the present invention, in certain embodiments, usefully provides a means for producing multivalent (e.g., bispecific) proteins with high purity. The use of a single species of engineered polypeptide monomer avoids the problem of mixed products seen when two or more different polypeptide species are used to produce multivalent (e.g., bispecific) or multivalent proteins.

[0035] The present invention provides the following items, aspects, paragraphs, and concepts (which are not intended to represent "claims"; claims are presented at the end of this disclosure after the examples and tables). Any item herein can be combined with any aspect or concept herein. Any aspect herein can be combined with any concept herein.

[0036] Aspects: 1. A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., a protein domain) or peptide, wherein the multimer is multimerized by first, second, third and fourth self-associating tetramerization domains (TDs) associated together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of the protein domain or peptide.

[0037] In examples, each TD is a TD of any one of proteins 1-119 listed in Table 2. In examples, each TD is a p53 TD or a homolog or ortholog thereof. In examples, each TD is an NHR2 TD or a homolog or ortholog thereof. In examples, each TD is a p63 TD or a homolog or ortholog thereof. In examples, each TD is a p73 TD or a homolog or ortholog thereof. In examples, each TD is not an NHR2 TD. In examples, each TD is not a p53 TD. In examples, each TD is not a p63 TD. In examples, each TD is not a p73 TD. In examples, each TD is not a p53, 63, or 73 TD. In examples, each TD is not an NHR2, p53, 63, or 73 TD.

[0038] By "associating together," the TD in embodiment 1 multimerizes the first, second, third, and fourth copies of the engineered polypeptide to provide a multimeric protein, e.g., a multimer that can be expressed intracellularly in a eukaryotic or mammalian cell (e.g., HEK293 cell) and / or secreted extracellularly from a eukaryotic or mammalian cell (e.g., HEK293 cell), and / or that is soluble in aqueous medium (e.g., eukaryotic or mammalian cell (e.g., HEK293 cell) culture medium). Examples are the NHR TD, p53 TD, p63 TD, and p73 TD (e.g., NHR TD, p53 TD, p63 TD, and p73 TD), or orthologs or homologs thereof.

[0039] In examples, the TD is not p53 TD (or a homolog or ortholog thereof), for example, it is not human p53 TD (or a homolog or ortholog thereof). In examples, the TD is NHR2 TD or a homolog or ortholog thereof, but excluding p53 TD or a homolog or ortholog thereof. In examples, the TD is human NHR2 TD or a homolog or ortholog thereof, but excluding human p53 TD or a homolog or ortholog thereof. In examples, the TD is human NHR2. In examples, the amino acid sequence of the TD is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of human NHR2. In examples, the domain or peptide is not naturally encompassed by a polypeptide that also includes NHR2 TD.

[0040] In an example, all of the domains of the polypeptide are human.

[0041] The engineered polypeptide may comprise one or more copies of the domain or peptide N-terminal to the copy of the TD. Additionally or alternatively, the engineered polypeptide may comprise one or more copies of the domain or peptide C-terminal to the copy of the TD. In examples, the engineered polypeptide comprises a first of the domains or peptides and a TD, wherein the first domain or peptide is spaced apart by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive amino acids from the TD, and there are no further domains or peptides between the first domain or peptide and the TD.

[0042] In examples, a multimer (e.g., a tetramer of the engineered polypeptide) comprises four (but not more than four) TDs (e.g., identical TDs) and 4, 8, 12, or 16 (but not more than said 4, 8, 12, or 16, respectively) copies of the domain or peptide. In examples, each TD and each of the domains or peptides is human.

[0043] In examples, a multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer (e.g., a tetramer or octamer) comprises first, second, third, and fourth identical copies of an engineered polypeptide, where the polypeptide comprises a TD and one (but not more than one), two (but not more than two), or more copies of the protein domain or peptide. For example, a tetramer of an epitope or effector domain has four identical copies of a polypeptide comprising a TD, where each polypeptide has one such epitope or effector domain. For example, an octamer of an epitope or effector domain has four identical copies of a polypeptide comprising a TD, where each polypeptide has two such epitopes or effector domains. For example, a dodecamer of an epitope or effector domain has four identical copies of a polypeptide comprising a TD, where each polypeptide has three such epitopes or effector domains. For example, a hexadecameric epitope or effector domain has four identical copies of a polypeptide containing a TD, each polypeptide having four such epitopes or effector domains. For example, a 20-mer epitope or effector domain has four identical copies of a polypeptide containing a TD, each polypeptide having five such epitopes or effector domains. Generally, for example, an X-mer epitope or effector domain has four identical copies of a polypeptide containing a TD, each polypeptide having X / 4 such epitopes or effector domains, where X=any multiple of 4, e.g., 4, 8, 12, 16, 20, 24, 28, or 32.

[0044] In some embodiments, by requiring only one type of engineered polypeptide to form a multimer of the invention, e.g., a tetramer or octamer, the invention advantageously provides a format that can be easily isolated as a pure (or highly pure, i.e., >90, 95, 96, 97, 98, or 99% pure) format, as well as methods for producing such formats in pure (or highly pure) form. Purity is indicated by the multimer of the invention not being mixed in a composition with any other multimers or polypeptide monomers, or by the multimer of the invention comprising more than 90, 95, 96, 97, 98, or 99% of species in the composition that contain the multimer of the invention and other multimers and / or polypeptide monomers, including the engineered polypeptide. Thus, admixture of different types of polypeptides in these embodiments is avoided or minimized. Thus, this also advantageously provides a plurality of multimers (e.g., a plurality of tetramers or octamers or dodecamers or hexamers) comprising only one (and not more than one) type of engineered polypeptide, wherein the multimer is monospecific (but multivalent) for antigen binding, or bispecific or multivalent for antigen binding. Thus, the present invention provides a plurality of multimers (e.g., a plurality of tetramers or octamers or dodecamers or hexamers) wherein each polypeptide is at least tetravalent for antigen binding, and is (i) bispecific (i.e., capable of specifically binding to two different antigens), or (ii) monospecific and at least tetravalent for antigen binding. When antigen binding is referred to herein, this may alternatively be pMHC binding if the domain is a TCR V domain. Advantageously, the plurality is in pure form (i.e., not mixed with a multimer comprising two or more types of polypeptide monomers (e.g., a tetramer or octamers or dodecamers or hexamers) comprising multiple types of polypeptide monomers). In examples, the multimer comprises at least two different types of antigen binding sites. In examples, the multimer is bispecific, trispecific, or tetraspecific.In examples, the multimer has a valency of 4, 6, 8, 10, or 12, preferably 4 or 8, antigen binding sites or pMHC binding sites.

[0045] In examples, the peptide MHC (pMHC) is a class I or class II pMHC.

[0046] As used herein, the term "specifically binds," e.g., with respect to a domain, antibody, or binding site, means a domain, antibody, or binding site that recognizes a particular antigen (or pMHC) with a binding affinity of 1 mM or less as determined by SPR.

[0047] Target binding capacity, specificity and affinity (KD (also called Kd), K オフ and / or K オン) can be determined by any routine method in the art, for example, by surface plasmon resonance (SPR). As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant of a particular binding site / ligand, receptor / ligand, or antibody / antigen interaction. In one embodiment, surface plasmon resonance (SPR) is performed at 25°C. In another embodiment, SPR is performed at 37°C. In one embodiment, SPR is performed at a physiological pH, such as about pH 7, or at pH 7.6 (e.g., using pH 7.6 buffered saline (also known as HBS-EP)). In one embodiment, SPR is performed at a physiological salt level, for example, 150 mM NaCl. In one embodiment, SPR is performed at a detergent level of 0.05% or less, for example, in the presence of 0.05% P20 (polysorbate 20; e.g., Tween-20™) and 3 mM EDTA. In one example, SPR is performed at 25°C or 37°C in a buffer solution of pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25°C or 37°C. HBS-EP is available from Teknova Inc. (California, catalog number H8022). In an example, affinity (e.g., of a VH / VL binding site) is determined using SPR by using any standard SPR instrument, such as a Biacore™ or ProteOn XPR36™ (Bio-Rad®). Binding data can be fitted to a 1:1 model using standard techniques, such as using the model inherent in the ProteOn XPR36™ analysis software.

[0048] In examples, the multimers, tetramers, or octamers, or dodecamers, or hexamers, or 20-mers of the invention are isolated multimers, tetramers, or octamers, or dodecamers, or hexamers, or 20-mers. In examples, the multimers, tetramers, or octamers of the invention are comprised of copies of the engineered polypeptide. Optionally, the multimers, tetramers, or octamers, or dodecamers, or hexamers, or 20-mers of the invention comprise 4 or 8 or 12 or 16 or 20 copies of the engineered polypeptide, respectively, but not more than 4 or 8 or 12 or 16 or 20 copies.

[0049] By "engineered" it is meant that the polypeptide does not occur in nature, for example a protein domain or peptide not naturally encompassed by a polypeptide that also comprises the TD.

[0050] Each protein domain or peptide may be a biologically active domain or peptide (e.g., biologically active in humans or animals), such as a domain that specifically binds to an antigen or peptide-MHC (pMHC), or the domain is constituted by an antigen or pMHC binding site. Alternatively, the domain or peptide is a carbohydrate, glucose, or glucoregulator, such as an incretin or insulin peptide. In another embodiment, the domain or peptide is an inhibitor of an enzyme, or an inhibitor of a biological function or pathway in humans or animals. In another embodiment, the domain or peptide is an iron regulator. Thus, in examples, each protein domain or peptide is selected from an antigen or pMHC binding domain or peptide, a hormone, a carbohydrate, a glucose or glucoregulator, an iron regulator, and an enzyme inhibitor. 2. The multimer of any one of the preceding embodiments, wherein the multimer is a tetramer, octamer, 12-mer, 16-mer, or 20-mer of said domains or peptides (e.g., a tetramer, octamer, 12-mer, or 16-mer). 3. The multimer of any one of aspects 1 or 2, comprising a tetramer, octamer, 12-mer, 16-mer or 20-mer (e.g., a tetramer, octamer, 12-mer or 16-mer) of an immunoglobulin superfamily binding site (antibody or TCR binding site, such as an scFv or scTCR).

[0051] The immunoglobulin superfamily (IgSF) is a large protein superfamily of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on shared structural features with immunoglobulins (also known as antibodies), all of which possess domains known as immunoglobulin domains or folds. IgSF members include cell surface antigen receptors, coreceptors and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. They are commonly associated with immune system roles.

[0052] A T cell receptor (TCR) domain can be Vα (e.g., paired with Vβ), Vβ (e.g., paired with Vα), Vγ (e.g., paired with Vδ), or Vδ (e.g., paired with Vγ). 4. The multimer of embodiment 3, wherein the binding site comprises a first variable domain paired with a second variable domain.

[0053] In a first example, the first and second variable domains are constituted by engineered polypeptides, in another example, the first domain is constituted by an engineered polypeptide and the second domain is constituted by a further polypeptide different from the engineered polypeptide (and optionally comprising or lacking a TD).

[0054] In another embodiment, the domain is a constant region domain. In another embodiment, the domain is an FcAb. In another embodiment, the domain is a non-Ig antigen-binding site or is constituted by a non-Ig antigen-binding site, such as an affibody.

[0055] Antigen-binding sites and effector domains In examples, the or each antigen-binding site (or effector domain) may be an antibody variable domain (e.g., VL or VH, antibody single variable domain (domain antibody or dAb), camelid VHH antibody single variable domain, shark immunoglobulin single variable domain (NAV), Nanobody™ or camelized VH single variable domain); a T-cell receptor binding domain; an immunoglobulin superfamily domain; an agnathan variable lymphocyte receptor (J Immunol; 2010 Aug 1; 185(3):1367-74; "Alternative adaptive immunity in jawless vertebrates"; Herrin BR & Cooper M D.); fibronectin domains (e.g., Adnectin™); scFv; (scFv)2; sc-diabody; scFab; antigen binding sites derived from scaffolds selected from centrin and CTLA-4 (Evibody™); lipocalin domains; protein A such as the Z domain of protein A (e.g., Affibody™ or SpA); A domains (e.g., Avimer™ or Maxibody™); heat shock proteins (such as epitope binding domains derived from GroEI and GroES); transferrin domains (e.g., Transbody); ankyrin repeat proteins (e.g., DARPin™); peptide aptamers; C-type lectin domains (e.g., Tetranectin™): human gamma-crystallin or human ubiquitin (affilin); PDZ domains; scorpion toxins; and Kunitz-type domains of human protease inhibitors.

[0056] A further source of antigen-binding sites are the antibody variable domains and VH / VL pairs disclosed in WO2007 / 024715 at page 40, line 23 to page 43, line 23. This particular disclosure is incorporated herein by reference as if expressly set forth herein to provide the basis for epitope-binding moieties for use in the present invention and which may be included in the claims herein.

[0057] A "domain" is a folded protein structure that has a tertiary structure independent of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be added, deleted, or transferred to other proteins without losing the function of the rest of the protein and / or domain. A "single antibody variable domain" is a folded polypeptide domain that contains sequences characteristic of antibody variable domains. Thus, this includes complete antibody variable domains and modified variable domains, such as those in which one or more loops are replaced by sequences not characteristic of antibody variable domains, or by antibody variable domains that are truncated or contain N- or C-terminal extensions, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.

[0058] The phrase "immunoglobulin single variable domain" or "antibody single variable domain" refers to an antibody variable domain (VH, VHH, VL) that specifically binds to an antigen or epitope independently of a different V region or domain. Immunoglobulin single variable domains can exist in formats (e.g., homo- or heteromultimers) with other, different variable regions or variable domains, where other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds to the antigen independently of additional variable domains). A "domain antibody" or "dAb," as the term is used herein, is equivalent to an "immunoglobulin single variable domain" capable of binding to an antigen. Immunoglobulin single variable domains can be human antibody variable domains, but also include single antibody variable domains from other species, such as rodents (e.g., as disclosed in WO 00 / 29004), nurse shark, and camelid VHH immunoglobulin single variable domains. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species such as camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy-chain antibodies that naturally lack light chains. Such VHH domains can be humanized according to standard techniques available in the art, and such domains are still considered "domain antibodies" according to the present invention. As used herein, "VH" includes camelid VHH domains. NAVs are another type of immunoglobulin single variable domain identified in cartilaginous fishes, including nurse sharks. These domains are also known as novel antigen receptor variable regions (commonly abbreviated as V(NAR) or NARV). For further details, see Mol. Immunol. 44, 656-665 (2006) and US 2005 / 0043519A. CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. The extracellular domain has an Ig-fold-like variable domain. Loops corresponding to the CDRs of an antibody can be replaced with heterologous sequences to confer different binding properties.CTLA-4 molecules engineered with different binding specificities are also known as evibodies. For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, bilins, retinoids, and lipids. They have a rigid beta-sheet secondary structure with multiple loops at the open end of a conical structure that can be engineered to bind different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1, and US2007 / 0224633. Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. The domains are composed of three-helical bundles of approximately 58 amino acids. The library was generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818 A1. Avimers™ are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide-bond structure. Diversity is generated by shuffling the natural mutations exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include trans-bodies.For details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins™) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. They can be designed to bind to different target antigens by randomizing the residues of the first α-helix and the β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007), and US 2004 / 0132028 A1. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins™ are composed of a backbone of the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US2008 / 0139791, WO2005 / 056764, and US6818418B1. Peptide aptamers are combinatorial recognition molecules consisting of a certain scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins 25-50 amino acids long containing 3-4 cysteine ​​bridges; examples of microproteins include KalataBI, conotoxins, and knottins.Microproteins have loops that can be designed to contain up to 25 amino acids without affecting the overall folding of the microprotein. For details on engineered knottin domains, see WO2008 / 098796. Other epitope-binding moieties and domains have been used as scaffolds to engineer different target antigen-binding properties, including human gamma-crystallin and human ubiquitin (affilin), Kunitz-type domains of human protease inhibitors, the PDZ-domain of the Ras-binding protein AF-6, scorpion toxin (charybdotoxin), and C-type lectin domains (tetranectin), as discussed in Chapter 7—Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006).

[0059] In examples, the or each antigen-binding site (or effector domain) comprises a non-Ig scaffold, e.g., selected from the group consisting of affibodies, affilins, anticalins, atrimers, avimers, bicycle peptides, Cys knots, DARpins, fibronectin type III, Fyomers, Kunitz domains, OBodies, aptamers, adnectins, armadillo repeat domains, beta-heparin mimetics, and lipocalins. 5. The multimer of any one of aspects 1-4, wherein each polypeptide comprises a first and a second copy of said protein domain or peptide, and wherein the polypeptide comprises (in the N-terminal to C-terminal direction) (i) said first copy-TD- said second copy, (ii) TD- and the first and second copies, or (iii) said first and second copies-TD. 6. A multimer according to any one of aspects 1 to 5, wherein the TD is an NHR2 TD and the domain or peptide is not an NHR2 domain or peptide, or wherein the TD is a p53 TD and the domain or peptide is not a p53 domain or peptide. 7. The multimer of any one of aspects 1-6, wherein the engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, and the second type of protein domain or peptide is different from the first protein domain or peptide.

[0060] For example, the polypeptide comprises, N-terminally, (i) P1-TD-P2, or (ii) TD-P1-P2, where P1 = a copy of a first type of domain or peptide (i.e., a type of domain or peptide of the multimer of embodiment 1) and P2 = a copy of said second type of domain or peptide. 8. The multimer of any one of aspects 1 to 7, wherein the domain is an immunoglobulin superfamily domain. 9. The multimer of any one of aspects 1 to 8, wherein the domain or peptide is an antibody variable or constant domain (e.g., an antibody single variable domain), a TCR variable or constant domain, an incretin, an insulin peptide, or a hormone peptide. 10. The multimer of any one of aspects 1-9, wherein the multimer comprises first, second, third and fourth identical copies of said engineered polypeptide, wherein the polypeptide comprises one (but not more than one), two (but not more than two), or more copies of the TD and said protein domain or peptide. 11. The multimer of any one of aspects 1-10, wherein the engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD. 12. The multimer of embodiment 11, wherein the polypeptide comprises (from N-terminal to C-terminal direction): (i) V1-optional linker-NHR2 TD; (ii) V1-optional linker-NHR2 TD-optional linker-V2; or (iii) V1-optional linker-V2-optional linker-NHR2 TD, wherein V1 and V2 are TCR variable domains and are the same or different, or wherein V1 and V2 are antibody variable domains and are the same or different. 13. The multimer of embodiment 12, wherein V1 and V2 are antibody single variable domains. 14. The multimer of embodiment 11, wherein each engineered polypeptide comprises (from N-terminal to C-terminal direction): V1-optional linker-NHR2 TD, wherein V1 is an antibody or TCR variable domain, each engineered polypeptide is paired with a respective second engineered polypeptide, and a polypeptide comprising V2, V2 being an antibody or TCR variable domain that pairs with V1 to form an antigen or pMHC binding site, respectively, and optionally one polypeptide comprises an antibody Fc or comprises an antibody CH1, and the other polypeptide comprises an antibody CL paired with CH1. 15. The multimer of any one of aspects 1-14, wherein the TD comprises (i) an amino acid sequence identical to, or at least 80% identical to, SEQ ID NO: 10 or 126, or (ii) an amino acid sequence identical to, or at least 80% identical to, SEQ ID NO: 120 or 123. 16. The multimer is selected from the group consisting of ReoPro™, abciximab, Rituxan™, rituximab, Zenapax™, daclizumab, Simulect™, basiliximab, Synagis™, palivizumab, Remicade™, infliximab, Herceptin™, Mylotarg™, gemtuzumab, Campath™, alemtuzumab, Zevalin™, ibritumomab, Humira™, and others. Mark), Adalimumab, Xolair™, Omalizumab, Bexxar™, Tositumomab, Raptiva™, Efalizumab, Erbitux™, Cetuximab, Avastin™, Bevacizumab, Tysabri™, Natalizumab, Actemra™, Tocilizumab, Vectibix™, Panitumumab, Lucentis™, Ranibizumab, Soliris™, Eculizumab, Cimzi a™, certolizumab, Simponi™, golimumab, Ilaris™, canakinumab, Stelara™, ustekinumab, Arzerra™, ofatumumab, Prolia™, denosumab, Numax™, motavizumab, ABThrax™, raxibacumab, Benlysta™, belimumab, Yervoy™, iplizumab, Adcetris™, brentuximab, Ved 16. The multimer of any one of aspects 1-15, comprising a tetramer, octamer, 12-mer, 16-mer, or 20-mer (e.g., a tetramer, octamer, 12-mer, or 16-mer, or a tetramer or octamer) of an antigen-binding site of an antibody selected from the group consisting of otin™, Perjeta™, pertuzumab, Kadcyla™, ado-trastuzumab, Keytruda™, Opdivo™, Gazyva™, and obinutuzumab. Optionally, the binding site of the polypeptides of the multimer comprises the VH of the antibody binding site and the CH1 of the antibody (i.e., from N-terminal to C-terminal direction, VH-CH1 and SAM). In embodiments, the polypeptide may be paired with an additional polypeptide comprising (from N-terminal to C-terminal direction, e.g., VL-CL, where CL is the CL of the antibody).

[0061] For example, the protein domain of the engineered polypeptide is the V domain (VH or VL) of the antibody binding site of an antibody selected from the group, wherein the multimer comprises an additional V domain (VL or VH, respectively) that pairs with the V domain of the engineered polypeptide to form the antigen binding site of the selected antibody. Advantageously, therefore, the present invention provides tetramers, octamers, 12-mers, 16-mers or 20-mers (e.g., tetramers, octamers, 12-mers or 16-mers, or tetramers or octamers) of the selected binding site, which may beneficially have improved affinity, avidity and / or efficacy for binding to its cognate antigen, or for treating or preventing a disease or condition in a human or animal to which the multimer is administered to bind to the cognate antigen in vivo.

[0062] For example, a multimer, tetramer, octamer, 12-mer, 16-mer, or 20-mer (e.g., a tetramer, octamer, 12-mer, or 16-mer, or a tetramer or octamer) comprises four (or the X / 4, as described above) copies of the antigen-binding site of an antibody, where the antibody is adalimumab, sarilumab, dupilumab, bevacizumab (e.g., AVASTIN™), cetuximab (e.g., ERBITUX™), tocilizumab (e.g., ACTEMRA™), or trastuzumab (e.g., HERCEPTIN™). Alternatively, the antibody is an anti-CD38 antibody, an anti-TNFα antibody, an anti-TNFR antibody, an anti-IL-4Ra antibody, an anti-IL-6R antibody, an anti-IL-6 antibody, an anti-VEGF antibody, an anti-EGFR antibody, a PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PCSK9 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD138 antibody, an anti-IL-1 antibody. Alternatively, the antibody is selected from the antibodies disclosed on page 40, line 23 to page 43, line 23 of WO2007 / 024715, the disclosures of which are incorporated herein by reference.

[0063] The binding site herein can be, for example, a ligand (e.g., a cytokine or growth factor, e.g., VEGF or EGFR) binding site of a receptor (e.g., KDR or Flt). The binding site herein can be, for example, the binding site of Eyelea™, Avastin™, or Lucentis™, for example, for ophthalmic or oncological medical use in humans or animals. When the ligand or antigen is VEGF, the multimer, tetramer, or octamer can be used to treat or prevent cancer or ocular conditions (e.g., wet or dry AMD or diabetic retinopathy), or as an inhibitor of angiogenesis in human or animal subjects. 17. An isolated tetramer, octamer, dodecamer, 16mer or 20mer of a TCR binding site, insulin peptide, incretin peptide or peptide hormone, or a plurality of such tetramers, octamers, dodecamers, 16mers or 20mers.

[0064] Several important peptide hormones are secreted by the pituitary gland. The anterior pituitary gland secretes three hormones: prolactin, which acts on the mammary glands; adrenocorticotropic hormone (ACTH), which acts on the adrenal cortex to regulate glucocorticoid secretion; and growth hormone, which acts on bone, muscle, and liver. The posterior pituitary gland secretes vasopressin and antidiuretic hormone, also known as oxytocin. However, peptide hormones are produced by a variety of organs and tissues, including the heart (atrial natriuretic peptide (ANP) or atrial natriuretic factor (ANF)), pancreas (glucagon, insulin, somatostatin), gastrointestinal tract (cholecystokinin, gastrin), and adipose tissue (leptin). In examples, the peptide hormones of the present invention are selected from prolactin, ACTH, growth hormone (somatotropin), vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin (e.g., human prolactin, ACTH, growth hormone, vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin).

[0065] In examples, the incretin is a GLP-1, GIP, or exendin-4 peptide.

[0066] The present invention provides, in embodiments, the following engineered multimers: An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of an incretin. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of insulin peptide. An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of GLP-1 (glucagon-like peptide-1 (GLP-1)) peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of GIP (glucose-dependent insulinotropic polypeptide) peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of an exendin (eg, exendin-4) peptide. An isolated tetramer, octamer, dodecamer, hexadecamer or 20amer of a peptide hormone. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of prolactin or a prolactin peptide. An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of ACTH or an ACTH peptide. An isolated tetrameric, octameric, dodecamer, hexameric or 20-meric peptide of growth hormone or growth hormone peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of vasopressin or a vasopressin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of oxytocin or an oxytocin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of glucagon or a glucagon peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of insulin or an insulin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of somatostatin or a somatostatin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of cholecystokinin or a cholecystokinin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of gastrin or a gastrin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of leptin or a leptin peptide. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of an antibody binding site (eg, scFv or Fab). An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of a TCR binding site (e.g., scTCR). An isolated tetramer, octamer, dodecamer, hexadecamer or 20mer of a TCR Vα / Vβ binding site. An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of a TCR Vγ / Vδ binding site. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of an antibody single variable domain binding site. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of an FcAb binding site.

[0067] In any of these tetramers, octamers, dodecamers, 16-mers, or 20-mer examples, the domain or peptide is human. In any of these tetramers, octamers, dodecamers, 16-mers, or 20-mer examples, the tetramers, octamers, dodecamers, 16-mers, or 20-mers comprise an NHR2 TD (e.g., human NHR2). In any of these tetramers, octamers, dodecamers, 16-mers, or 20-mer examples, the tetramers, octamers, dodecamers, 16-mers, or 20-mers comprise a p53 TD (e.g., human p53 TD). In any of these tetramers, octamers, dodecamers, 16-mers, or 20-mer examples, the tetramers, octamers, dodecamers, 16-mers, or 20-mers comprise a p63 TD (e.g., human p63 TD). In any of these tetramers, octamers, dodecamers, hexamers, or 20-mer examples, the tetramer, octamer, dodecamer, hexamer, or 20-mer comprises a p73 TD (e.g., human p73 TD). In any of these tetramers, octamers, dodecamers, hexamers, or 20-mer examples, the tetramer, octamer, dodecamer, hexamer, or 20-mer comprises a tetramer of a TD (e.g., human NHR2 TD), thereby forming a multimer of four or eight domains or peptides.

[0068] In examples, the plurality is pure and not a mixture with multimers of the binding site or peptide, eg, where the multimers comprise more than one type of polypeptide monomer. 18. A multimer, tetramer, octamer, dodecamer, hexadecamer or 20amer, (a) soluble in aqueous solutions (e.g., aqueous eukaryotic cell growth media or buffers); (b) capable of being secreted by eukaryotic cells; and / or (c) The multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer according to any one of embodiments 1 to 17, which is an expression product of a eukaryotic cell.

[0069] In examples, multimers, tetramers, octamers, dodecamers, hexadecamers or 20-mers can be stably secreted from HEK293T (or other eukaryotic, mammalian, CHO or Cos) cells, as shown by a single band on a PAGE gel using a sample of supernatant from such cells at the molecular weight expected for the multimer, tetramer, octamers, dodecamers, hexadecamers or 20-mer detected using Western blot. 19. The following (a) a TCR V domain or TCR binding site, wherein the tetramer, octamer, dodecamer, hexadecamer, or 20-mer is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (b) an antibody single variable domain, wherein the tetramer, octamer, dodecamer, hexameric or 20-mer is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (c) a TCR V domain or TCR binding site, wherein a tetramer, octamer, dodecamer, hexameric or 20-mer can be expressed intracellularly and / or extracellularly by HEK293 cells; or (d) A tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., a tetramer or octamer) of an antibody variable domain (e.g., an antibody single variable domain), wherein the tetramer, octamer, dodecamer, hexadecamer or 20-mer can be expressed intracellularly and / or extracellularly by HEK293 cells.

[0070] An example of the medium is SFMII growth medium supplemented with L-glutamine (e.g., complete SFMII growth medium supplemented with 4 mM L-glutamine). In an example, the medium is serum-free HEK293 cell culture medium. In an example, the medium is serum-free CHO cell culture medium.

[0071] For example, the cells herein are human cells, for example, HEK293 cells (such as HEK293T cells). 20. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of aspects 1 to 19, wherein the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is bispecific for antigen or pMHC binding. 21. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of aspects 1 to 20, wherein the domains are identical. 22. The multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer of any one of embodiments 1-21, wherein the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer comprises eukaryotic glycosylation.

[0072] For example, the glycosylation is glycosylation of a CHO cell. For example, the glycosylation is glycosylation of a HEK (e.g., HEK293, such as HEK293T) cell. For example, the glycosylation is glycosylation of a Cos cell. For example, the glycosylation is glycosylation of a Picchia cell. For example, the glycosylation is glycosylation of a Saccharomyces cell. 23. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to aspect 22, wherein the cell is a HEK293 cell. 24. A plurality of multimers, tetramers, octamers, dodecamers, hexadecamers, or 20-mers according to any one of embodiments 1 to 23. 25. A pharmaceutical composition comprising the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer according to any one of aspects 1 to 24, and a pharmaceutically acceptable carrier, diluent, or excipient. 26. A cosmetic, food, beverage, cleaning product, or detergent comprising the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20amer according to any one of aspects 1 to 24. 27. The engineered (and optionally isolated) multimeric, tetrameric, octameric, dodecamer, hexadecameric or 20-meric engineered polypeptide or monomer (optionally isolated) according to any one of aspects 1 to 26.

[0073] A monomer is a polypeptide engineered as disclosed herein, comprising the protein domain or peptide, and further comprising a TD.

[0074] Optionally, the engineered polypeptide comprises (from N- to C-terminus) a variable domain (V1)-a constant domain (C) (eg, CH1 or Fc)-an optional linker-TD. 28. An engineered (and optionally isolated) engineered polypeptide (P1) comprising (in the N-terminal to C-terminal direction): (a) TCR V1-TCR C1-antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ))-optional linker-TD, (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) TCR V1-TCR C1-antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ))-optional linker-TD, wherein V1 is Vδ and C1 is Cδ; or (b) TCR V1-antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ))-optional linker-TD, (i) V1 is Vα, (i) V1 is Vβ, (iii) V is V, or (iv) TCR V1-antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ))-optional linker-TD, wherein V1 is Vδ; or (c) antibody V1-antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ))-optional linker-TD, (i) V1 is VH, or (ii) antibody V1-antibody C (e.g., CH, CH1 (e.g., IgG CH1), or CL (e.g., Cλ or Cκ))-optional linker-TD, wherein V1 is VL (e.g., Vλ or Vκ); or (d) antibody V1 - any antibody C (e.g., CH, CH1 (e.g., IgG CH1) or CL (e.g., Cλ or Cκ)) - antibody Fc (e.g., IgG Fc) - any linker - TD, (i) V1 is VH, or (ii) antibody V1-any antibody C (e.g., CH, CH1 (e.g., IgG CH1), or CL (e.g., Cλ or Cκ))-antibody Fc (e.g., IgG Fc)-any linker-TD, wherein V1 is VL (e.g., Vλ or Vκ); or (e) antibody V1-antibody CL (e.g., Cλ or Cκ)-optional linker-TD; (i) V1 is VH, or (ii) antibody V1-antibody CL (e.g., Cλ or Cκ)-optional linker-TD, where V1 is VL (e.g., Vλ or Vκ); or (f) TCR V1-TCR C1-optional linker-TD, (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) An engineered (and optionally isolated) engineered polypeptide (P1) comprising TCR V1-TCR C1-optional linker-TD, where V1 is Vδ and C1 is Cδ.

[0075] In (a) or (b), in examples, the TCR V is comprised of a single-chain TCR binding site (scTCR) that specifically binds to pMHC, and the binding site comprises TCR V-linker-TCRV. In examples, the engineered polypeptide comprises (from N- to C-terminus): (i) V1-linker-V-anyC-anylinker-TD, or (ii) Va-linker-V1-anyC-anylinker-TD, where Va is a TCR V domain and C is an antibody C domain (e.g., CH1 or CL) or TCR C.

[0076] Preferably, antibody C is a CH1 (eg, an IgG CH1).

[0077] In examples, the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer has a size of 155 kDa or less, for example, the protein domain is an antibody variable domain comprising a CDR3 of at least 16, 17, 18, 19, 20, 21 or 22 amino acids, such as a camelid CDR3 or a bovine CDR3.

[0078] In an example, the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer comprises a TCR binding site and an antibody binding site. For example, each polypeptide comprises a TCR V (e.g., comprised of an scTCR that specifically binds to pMHC) and an antibody V (e.g., comprised of an scFv, or paired with a second V domain comprised of the second polypeptide to form a V / V paired binding site that specifically binds to an antigen). In an example, the pMHC comprises a RAS peptide. In an example, the antigen is selected from the group consisting of PD-1, PD-L1, or any other antigen disclosed herein. For example, the antigen is PD-1, and the pMHC comprises a RAS peptide. 29. The engineered polypeptide P1 is paired with a further polypeptide (P2), wherein P2 is (in the N-terminal to C-terminal direction): (g) TCR V2-TCR C2-antibody CL (e.g., Cλ or Cκ), wherein P1 is as described in (a) of embodiment 28; (i) If P1 conforms to (a)(ii), then V2 is Vα and C2 is Cα; (ii) If P1 conforms to (a)(i), then V2 is Vβ and C2 is Cβ; (iii) if P1 conforms to (a)(iv), then V2 is Vγ and C2 is Cγ; or (iv) TCR V2-TCR C2-antibody CL (e.g., Cλ or Cκ), where V2 is Vδ and C2 is Cδ, if P1 is according to (a)(iii); or (h) a TCR V2-antibody CL (e.g., Cλ or Cκ), wherein P1 is according to (b) as described in embodiment 28; (i) If P1 obeys (b)(ii), then V2 is Vα; (ii) If P1 conforms to (b)(i), then V2 is Vβ; (iii) if P1 conforms to (b)(iv), then V2 is Vγ; or (iv) TCR V2-antibody CL (e.g., Cλ or Cκ), where V2 is Vδ if P1 is according to (b)(iii); or (i) antibody V2-CL (e.g., Cλ or Cκ), wherein P1 is according to (c) of embodiment 28; (i) If P1 complies with (c)(ii), then V2 is VH; (ii) antibody V2-CL (e.g., Cλ or Cκ), where P1 is according to (c)(i), and V2 is a VL (e.g., Vλ or Vκ); or (j) antibody V2—any CL (e.g., Cλ or Cκ), wherein P1 is according to (d) as described in embodiment 28; (i) If P1 complies with (d)(ii), then V2 is VH; (ii) when P1 is according to (d)(i), V2 is a VL (e.g., Vλ or Vκ), antibody V2 - any CL (e.g., Cλ or Cκ); or (k) antibody V2-CH1 (e.g., IgG CH1), wherein P1 is according to (e) of embodiment 28; (i) If P1 complies with (e)(ii), then V2 is VH; (ii) antibody V2-CH1 (e.g., IgG CH1), where P1 is according to (e)(i), and V2 is a VL (e.g., Vλ or Vκ); or (l) TCR V2-TCR C2, wherein P1 is according to (f) as described in embodiment 28; (i) If P1 conforms to (f)(ii), then V2 is Vα and C2 is Cα; (ii) If P1 conforms to (f)(i), then V2 is Vβ and C2 is Cβ; (iii) If P1 conforms to (f)(iii), then V2 is Vγ and C2 is Cγ; or (iv) The polypeptide of embodiment 28, comprising a TCR V2-TCR C2, wherein when P1 is according to (f)(iv), then V2 is Vδ and C2 is Cδ.

[0079] Optionally, V1 and V2 form a paired variable domain binding site capable of specifically binding to an antigen or pMHC. In examples, V1 and V2 are selected from the group consisting of ReoPro™, abciximab, Rituxan™, rituximab, Zenapax™, daclizumab, Simulect™, basiliximab, Synagis™, palivizumab, Remicade™, infliximab, Herceptin™, Mylotarg™, gemtuzumab, Campath™, alemtuzumab, Zev alin™, ibritumomab, Humira™, adalimumab, Xolair™, omalizumab, Bexxar™, tositumomab, Raptiva™, efalizumab, Erbitux™, cetuximab, Avastin™, bevacizumab, Tysabri™, natalizumab, Actemra™, tocilizumab, Vectibix™, panitumumab , Lucentis™, Ranibizumab, Soliris™, Eculizumab, Cimzia™, Certolizumab, Simponi™, Golimumab, Ilaris™, Canakinumab, Stelara™, Ustekinumab, Arzerra™, Ofatumumab, Prolia™, Denosumab, Numax™, Motavizumab, ABThrax™, Raxyvac The variable domain of an antibody selected from the group consisting of imabu, Benlysta™, belimumab, Yervoy™, iplizumab, Adcetris™, brentuximab, Vedotin™, Perjeta™, pertuzumab, Kadcyla™, ado-trastuzumab, Keytruda™, Opdivo™, Gazyva™, and obinutuzumab. Optionally, the binding site of the multimeric polypeptide comprises the VH of the antibody binding site and the CH1 of the antibody (i.e., from N- to C-terminus, VH-CH1 and SAM). In one embodiment, the polypeptide can be paired with an additional polypeptide comprising (from N- to C-terminus, for example, VL-CL, where CL is the CL of the antibody).

[0080] In one embodiment, the antibody is Avastin.

[0081] In one embodiment, the antibody is Actemra.

[0082] In one embodiment, the antibody is Erbitux.

[0083] In one embodiment, the antibody is Lucentis.

[0084] In one embodiment, the antibody is sarilumab.

[0085] In one embodiment, the antibody is dupilumab.

[0086] In one embodiment, the antibody is alirocumab.

[0087] In one embodiment, the antibody is bococizumab.

[0088] In one embodiment, the antibody is evolocumab.

[0089] In one embodiment, the antibody is pembrolizumab.

[0090] In one embodiment, the antibody is nivolumab.

[0091] In one embodiment, the antibody is ipilimumab.

[0092] In one embodiment, the antibody is Remicade.

[0093] In one embodiment, the antibody is golimumab.

[0094] In one embodiment, the antibody is ofatumumab.

[0095] In one embodiment, the antibody is Benlysta.

[0096] In one embodiment, the antibody is Campath.

[0097] In one embodiment, the antibody is rituximab.

[0098] In one embodiment, the antibody is Herceptin.

[0099] In one embodiment, the antibody is durvalumab.

[0100] In one embodiment, the antibody is daratumumab.

[0101] In examples, any binding domain (e.g., dAb or scFv or Fab) or V1 herein may be selected from the group consisting of ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, Aggrecan, AGR2, AICDA, AWI, AIG1, AKAP1, AKAP2, AIYIH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), B7.1, B7.2, BAD, BAFF, BAG1, BA I1, BCL2, BCL6, BDNF, BLNK, BLRl (MDR15), BlyS, BMPl, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRIA, BMPRIB, BMPR2, BPAG1 (plectin), BRCA1, CI9orfl O(IL27w), C3, C4A, C5, C5R1, CANT1, CASP1, CASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCL11(eotaxin), CCL13(MCP-4), CCL15(MIP-id), CCL16(HCC -4), CCL17(TARC), CCL18(PARC), CCL19(MIP-3b), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MIP-2), SLC, exodus-2, CCL22(MDC / STC-1), CCL23(MP IF-1), CCL24 (MPIF-2I eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-la), CCL4 (MIP-lb), CCL5 (RANTES), CCL7 (M CP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-1RB / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13) , CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI1), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), CD164, CD19,CD1C、CD20、CD200、CD-22、CD24、CD28、CD3、CD37、CD38、CD3E、CD3G、CD3Z、CD4、CD40、CD40L、CD44、CD45RB、CD52、CD69、CD72、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CDH1(E-カドヘリン)、CDH10、CDH12、CDH13、CDH18、CDH19、CDH20、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKN1A(p2IWapl / Cipl)、CDKN1B(p27Kipl)、CDKNIC、CDKN2A(pl6INK4a)、CDKN2B、CDKN2C、CDKN3、CEBPB、CER1、CHGA、CHGB、Chitinase、CHST10、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7(クローディン-7)、CLN3、CLU(クラステリン)、CMKLR1、CMKOR1(RDC1)、CNR1、COL18A1、COL1A1、COL4A3、COL6A1、CR2、CRP、CSFl(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTLA4、CTNNBl(b-カテニン)、CTSB(カテプシンB)、CX3CL1(SCYDi)、CX3CR1(V28)、CXCL1(GROl)、CXCLIO(IP-10)、CXCL11(l-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GR02)、CXCL3(GR03)、CXCL5(ENA-78ILIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR ISTRL33 I Bonzo)、CYB5、CYC1、CYSLTR1、DAB2IP、DES、DKFZp451J0118、DNCL1、DPP4、E2F1、ECGF1、EDG1、EFNAI、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、EN01、EN02、EN03、EPHB4、EPO、ERBB2(Her-2)、EREG、ERK8、ESR1、ESR2、F3(TF)、FADD、FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF 20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR3, FIGF(VEGFD), FILl(EPSILON), FILl(ZET) A), FU12584, FU25530, FLRTl (fibronectin), FLTl, FOS, FOSLl (FRA-I), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-65T, GATA3, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRFl, GPR2 (CCRIO), GPR31, GPR44, GPR81 (FKSG80), GRCCIO (CIO), GRP, GSN (gelsolin), GSTPl, HAVCR2, HDAC4, EDAC5, HDAC7A, HDAC9, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, ICEBERG, ICOSL, 1D2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN gamma, TFNW1, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL10, IL10RA, IL10RB, IL11, IL1 1RA, IL-12, IL12A, IL12B, IL12RB1, IL12RB2, 1L13, IL13RA1, IL13RA2, 1L14, 1L15, IL15RA, IL16, 1L17, IL17B, IL17C, IL17R, 1L18, IL18BP, IL18 R1, IL18RAP, 1L19, ILIA, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2 IL1RN, 1L2, 1L20, IL20RA, IL21R, 1L22, 1L22R,1L22RA2, 1L23, 1L24, 1L25, 1L26, 1L27, 1L28A, 1L28B, 1L29, IL2RA, IL2RB. IL2RG.1L3.1L30.IL3RA.1L4.IL4R.1L5.IL5RA.1L6 0) 1L7 TL7R 1L8 IL8RA IL8RB IL8RB 1L9 IL9R ILK INHA INHBA INSL 3 INSL4, IRAQ, IRAQ2, ITGA1, ITGA2, 1TGA3, ITGA6(a6インテリン), ITGAV, ITG B3, ITGB4(b4, JAK1, JAK3, JUN, K6HF, KAI1, KDR, MTLG, KLF5( GCBoxBP)、KLF6、KLK10、KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK6 KLK9, KRT1, KRT19(ラン19), KRT2A, KRTHB6 (Green Protein Layer II, LAMA5, LEP(レプチン), Lingo-p75, Lingo-Troy, LPS, LTA(TNF-b), LTB, LTB4R(GPR16), LTB4R 2. LTBR, ​​MACMARCKS, MAG, Omgp, MAP2K7(c-Jun), MDK, MIB1, ミドカイン, MIF IP-2, MK167(Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (MK167(Ki-ifi), MTSS1 MUC1(MYC), MYD88, NCK2, NFKB1, NFKB2, NGFB(NGF), NGFR, Ng R-Lingo, NgR-Nogo66(Nogo), NgR-p75, NgR-Troy, NME1(NM23A), NOX5, NPPB NROB1, NROB2, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, N R2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A 3. NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZ1, OPRD1, P2RX7, PAP ARTI, PATE, PAWR, PCA3, PCNA, PDGFA, PDGFB, PECAM1, PF4(CXCL4), PGF, PGR.Phosphacan, PIAS2, PIK3CG, PLAU (uPA), PLG, PLXDC1, PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, RAC2 (p2IRac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobulin 2), SCGB2A2 (mammaglobulin 1), SCYE1 (endothelial monocyte-activating cytokines), SDF2, SERPINA1, SERPINIA3, SERPINB5 (maspin), SERPINE1 (PAT-i), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPPl, SPRRIB (Spri), ST6GAL1, STABl, STAT6, STEAP, STEAP2, TB4R2, TBX21, TCPIO, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2 , TGFBR3, TH1L, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-i), T]MP3, tissue factor, TLRIO, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-a (also referred to herein as TNF alpha or TNFα), TNFAIP2 (B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSFIO (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase IA),TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, Versican, VHLC5, VLA-4, XCL1, (lymphectin), XCL2 (SCM-lb), XCR1 (GPR5 / CCXCR1), YY1, and ZFPM2 (by itself, as a single variable domain, or when paired with V2).

[0102] For example, in any configuration of the invention, a multimer, octamer, dodecamer, hexadecamer, or 20-mer may specifically bind to a first and a second epitope or antigen (e.g., in the case of an octamer, dodecamer, hexadecamer, or 20-mer), optionally specifically bind to a first, second, and third epitope or antigen (e.g., in the case of a dodecamer, hexadecamer, or 20-mer), or optionally specifically bind to a first, second, third, and fourth epitope or antigen. and optionally binds (e.g., in the case of a 16-mer or 20-mer) or specifically binds (e.g., in the case of a 20-mer) to a first, second, third, fourth, and fifth epitope or antigen, each of which may be selected from the group consisting of EpCAM and CD3; CD19 and CD3; VEGF and VEGFR2; VEGF and EGFR; CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and CD138; CD 38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20; CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTN02; CSPGs and RGMA; IGF1 and IGF2; IGF1 and / or 2 and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAM8; IL-13 and CL25; IL-13 and IL -l beta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-beta; IL-1 alpha and IL-1 beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; RGM A and RGM B; Te38 and TNF alpha; TNF alpha and IL-12; TNF alpha and IL-12p40; TNF alpha and IL-13; TNF alpha and IL-15; TNF alpha and IL-17; TNF alpha and IL-18; TNF alpha and IL-1 beta;Selected from the group consisting of TNFalpha and IL-23; TNFalpha and MIF; TNFalpha and PEG2; TNFalpha and PGE4; TNFalpha and VEGF; and VEGFR and EGFR; TNFalpha and RANK ligand; TNFalpha and Blys; TNFalpha and GP130; TNFalpha and CD-22; and TNFalpha and CTLA-4;

[0103] For example, the first epitope or antigen is selected from the group consisting of CD3, CD16, CD32, CD64, and CD89, and the second epitope or antigen is selected from the group consisting of EGFR, VEGF, IGF-1R, Her2, c-Met (also known as HGF), HER3, CEA, CD33, CD79a, CD19, PSA, EpCAM, CD66, CD30, HAS, PSMA, GD2, ANG2, IL-4, IL-13, VEGFR2, and VEGFR3.

[0104] In examples, any binding domain (e.g., dAb or scFv or Fab) or V1 herein can specifically bind to an antigen selected from the group consisting of human IL-1A, IL-1β, IL-1RN, IL-6, BLys, APRIL, activin A, TNF alpha, a BMP, BMP2, BMP7, BMP9, BMP10, GDF8, GDF11, RANKL, TRAIL, VEGFA, VEGFB or PGF (by itself as a single variable domain or when paired with V2), and optionally, the multimer comprises a cytokine amino acid sequence (e.g., at the C-terminus of the TD) such as IL-2 or an IL2-peptide, and the multimer, octamer, dodecamer, hexadecamer or 20-mer is for treating or preventing cancer in a human subject. In examples, the effector or protein domain is capable of binding to such an antigen, and optionally the multimer comprises a cytokine amino acid sequence such as IL-2 or an IL2-peptide (e.g., the C-terminus of the TD), and the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is for treating or preventing cancer in a human subject. 30. A multimer (e.g. a dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer) of P1 as defined in embodiment 28, or of P1 paired with P2 as defined in embodiment 29, or a plurality of such multimers, wherein the multimer is as defined in any one of embodiments 1 to 24.

[0105] Preferably, the multimer is a tetramer of the engineered polypeptide and / or effector domain, hi one example, the tetramers are not a mixture of polypeptide monomers, dimers, or trimers.

[0106] In one example, a multimer, such as a tetramer, can specifically bind to two different pMHCs. 31. A nucleic acid encoding an engineered polypeptide or monomer according to any one of aspects 27 to 29, optionally wherein the nucleic acid is comprised by an expression vector for expressing the polypeptide.

[0107] In an example, the nucleic acid is DNA, optionally operably linked to or including a promoter for expression of the polypeptide or monomer. In another example, the nucleic acid is RNA (e.g., mRNA). 32. A eukaryotic host cell comprising a nucleic acid or vector according to aspect 31 for the intracellular and / or secretory expression of a multimeric, tetrameric, octameric, dodecamer, hexadecameric or 20-meric engineered polypeptide or monomer according to any one of aspects 1 to 24. 33. Use of a nucleic acid or vector according to embodiment 31 in a method for producing a protein multimer to produce a multimer that is expressed and / or secreted intracellularly, the method comprising expressing and / or secreting the multimer from a eukaryotic cell containing the nucleic acid or vector. 34. Use of a nucleic acid or vector according to aspect 31 in a method for producing a protein multimer, for producing a glycosylated multimer in a eukaryotic cell comprising the nucleic acid or vector.

[0108] Glycosylation of mammals of the present invention is useful for producing pharmaceuticals comprising or consisting of the multimers, tetramers, octamers, dodecamers, hexadecamers, or 20-mers of the present invention for the treatment or prevention of mammalian, e.g., human, diseases or conditions. Accordingly, the present invention provides such methods of use, as well as the multimers, tetramers, octamers, dodecamers, hexadecamers, or 20-mers of the present invention for this purpose. Similarly, intracellular and / or secreted expression in one or more mammalian host cells (or cell lines thereof) of the present invention is useful for producing such pharmaceuticals. Particularly useful is expression in HEK293, CHO, or Cos cells, which are commonly used for pharmaceutical production.

[0109] In one embodiment, the invention includes a detergent or personal healthcare product comprising a multimer, tetramer, octamer, dodecamer, hexadecamer, or 20amer of the invention. In one embodiment, the invention includes a food or beverage comprising a multimer, tetramer, octamer, dodecamer, hexadecamer, or 20amer of the invention.

[0110] In examples, the multimers, monomers, dimers, trimers, tetramers, octamers, dodecamers, hexadecamers or 20-mers, polypeptides, compositions, mixtures, uses or methods of the invention are for or used in industrial or domestic applications, such as for or in the agriculture, petroleum or oil industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dental industry, energy industry, consumer goods industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp and paper industry, textile industry, clothing industry, leather or suede or animal skin industry, tobacco industry or steel industry. 35. A mixture comprising: (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of aspects 27 to 29; and (ii) a multimer, monomer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer as defined in any one of aspects 1 to 24. 36. The mixture of aspect 35, wherein the cell line is in a medium comprising a secreted product of the cells, and the secreted product comprises the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20amer. 37. A multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of aspects 1 to 24 for medical use. 38. A method for producing: (a) TCR V domain multimers (methods for producing TCR V domain multimers include soluble and / or intracellular expression of TCR V-NHR2 TD or TCR V-p53 TD fusion proteins expressed in eukaryotic cells, which methods optionally include isolating a plurality of said multimers); (a) antibody V domain multimers (methods for producing antibody V domain multimers include soluble and / or intracellular expression of antibody V (e.g., single variable domain)-NHR2-TD or V-p53 TD fusion proteins expressed in eukaryotic cells, which methods optionally include isolating a plurality of said multimers); (c) incretin peptide (e.g., GLP-1, GIP, or insulin) multimers (methods for producing incretin peptide multimers include soluble and / or intracellular expression of incretin peptide-NHR2 TD or incretin peptide-p53 TD fusion proteins expressed in eukaryotic cells, such as HEK293T cells, which methods optionally include isolating a plurality of said multimers); or (a) A method for producing a peptide hormone multimer (the method for producing a peptide hormone multimer comprises soluble and / or intracellular expression of a peptide hormone-NHR2 TD or peptide hormone-p53 TD fusion protein expressed in a eukaryotic cell, such as a HEK293T cell, and optionally, isolating a plurality of said multimers). 39. Provided is the use of a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog thereof) in a method for producing a tetramer of a polypeptide to produce a higher yield of the tetramer relative to a monomer and / or dimer of the polypeptide. 40. Use of an engineered polypeptide in a method for producing a tetramer of a polypeptide comprising multiple copies of a protein domain or peptide to produce a higher yield of the tetramer relative to a monomer and / or dimer of the polypeptide, wherein the engineered polypeptide comprises one or more copies of the protein domain or peptide and further comprises a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD or a homolog or ortholog). 41. Use of a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog thereof) in a method for producing a tetramer of a polypeptide to produce multiple tetramers that are not a mixture with monomers, dimers, or trimers. 42. Use of an engineered polypeptide in a method for producing tetramers of a polypeptide comprising multiple copies of a protein domain or peptide to produce multiple tetramers that are not a mixture with monomers, dimers, or trimers, wherein the engineered polypeptide comprises one or more copies of the protein domain or peptide and further comprises a self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD, or p73 TD, or a homolog or ortholog). 43. The use according to any one of aspects 39-42, wherein the yield of tetramer is at least 10, 20, 30, 40, or 50 times the yield of monomer and / or dimer. 44. The use of any one of aspects 39-43, wherein the ratio of tetramers produced by this method to monomers and / or dimers produced is at least 90:10 (e.g., at least 95:5, or 98:2, or 99:1). 45. The use according to any one of aspects 39 to 44, wherein each monomer has a size of less than or equal to 40, 35, 30, 25, or 20 kDa. 46. ​​The use according to any one of aspects 39 to 45, wherein each tetramer has a size of less than or equal to 200, 160, 155, or 150 kDa. 47. The use according to any one of aspects 39 to 46, wherein the method comprises expressing the tetramer from a eukaryotic cell line. 48. A multivalent heterodimeric soluble T cell receptor capable of binding to a pMHC complex, comprising: (i) a TCR extracellular domain; (ii) an immunoglobulin constant domain; and (iii) a multivalent heterodimeric soluble T cell receptor comprising the NHR2 multimerization domain of ETO. 49. Polymeric immunoglobulins (i) an immunoglobulin variable domain; (ii) a multimeric immunoglobulin comprising the NHR2 multimerization domain of ETO. 50. A method for assembling a soluble multimeric polypeptide, comprising: (a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO; (b) assembling multiple copies of said monomers, thereby obtaining a multimeric soluble polypeptide.

[0111] The present invention provides (i) a monomer as shown in Figure 1; (ii) homodimers, as shown in Figure 1; (iii) homotetramers as shown in Figure 1; (iv) Monomers as shown in Figure 2 2 , (v) Homodimers as shown in Figure 2 2 , (vi) Homotetramers as shown in Figure 2 2 , (vii) a monomer as shown in Figure 11a; (viii) homodimers as shown in Figure 11a; (ix) homotetramers as shown in Figure 11a; (x) a monomer as shown in Figure 12a; (xi) homodimers as shown in Figure 12a; (xii) homotetramers as shown in Figure 12a; (xiii) Monomers as shown in Figure 13a 2 , (xiv) Homodimers as shown in Figure 13a 2 , (xv) Homotetramer as shown in Figure 13a 2 ,or (xvi) A multimeric protein comprising any one of (i) to (xv) (e.g., any one of Quads 3, 4, 12, 13, 14, 15, 16, and 17) or a multimer of any protein shown in Figure 21 (excluding the leader or tag); (xvii) a multimer of (xvi), or (xviii) Further provided is a pharmaceutical composition comprising any one of (i) to (xvii) and a pharmaceutically acceptable carrier, diluent, or excipient.

[0112] The present invention also provides (i) tetravalent or octavalent antibody V molecules; (ii) tetravalent or octavalent antibody Fab molecules; (iii) tetravalent or octavalent antibody dAb molecules; (iv) tetravalent or octavalent antibody scFv molecules; (v) a tetravalent or octavalent antibody TCR V molecule, or (vi) a tetravalent or octavalent antibody scFv molecule; This molecule is (a) soluble in aqueous solution (e.g., a solution or cell culture medium disclosed herein); and / or (b) can be expressed intracellularly and / or extracellularly by HEK293 cells;

[0113] The present invention provides claimed multimers (e.g., tetramers) of NHR2 or p53 (or another TD disclosed herein) fused at their N- and / or C-termini to an amino acid sequence (e.g., a peptide, protein domain, or protein) that is not an NHR2 sequence. For example, the sequence is selected from a TCR (e.g., TCRα, TCRβ, Cα, or Cβ), a cytokine (e.g., an interleukin, e.g., IL-2, IL-12, IL-12, and IFN), an antibody fragment (e.g., scFv, dAb, or Fab), and an antibody domain (e.g., a V or C domain, e.g., a VH, VL, Vκ, Vλ, CH, CH1, CH2, CH3, hinge, Cκ, or Cλ domain). Optionally, the multimer comprises: a) soluble in aqueous solution (e.g., a solution disclosed herein or cell culture medium); and / or b) A molecule that can be expressed intracellularly and / or extracellularly by HEK293 cells.

[0114] The present invention provides: (i) Use of NHR2 or p53 (or another TD disclosed herein) for the production of a polypeptide for soluble expression of multimers of the polypeptide from cells, such as eukaryotic cells, such as mammalian, HEK293, CHO or Cos cells. (ii) Use of NHR2 or p53 (or another TD disclosed herein) for the production of a polypeptide for intracellular expression of a multimer of the polypeptide in a cell, such as a eukaryotic cell, such as a mammalian, HEK293, CHO or Cos cell. (iii) A cell comprising an intracellular expression product, the product comprising a multimer of a polypeptide comprising NHR2 or p53 (or another TD disclosed herein) fused at its N-terminus and / or C-terminus to an amino acid sequence (e.g., a peptide, protein domain, or protein) that is not an NHR2 sequence. (iv) Use of NHR2 as a promiscuous tetramerization domain for tetramerizing peptides, protein domains, polypeptides or proteins in the production of intracellularly and / or solubly expressed multimers from host cells.

[0115] Optionally, the amino acid is the amino acid sequence of a human peptide, protein domain, or protein, such as a TCR (e.g., TCRα, TCRβ, Cα, or Cβ), a cytokine (e.g., an interleukin, such as IL-2, IL-12, IL-12, and IFN), an antibody fragment (e.g., an scFv, dAb, or Fab), or an antibody domain (e.g., a V or C domain, such as a VH, VL, Vκ, Vλ, CH, CH1, CH2, CH3, hinge, Cκ, or Cλ domain).

[0116] Optionally, the or each polypeptide comprises a polypeptide selected from the group consisting of Quads 1-46 (i.e., a polypeptide as shown in Figure 21, but excluding any leader or tag sequences). Optionally, the invention provides multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, or octamers, preferably tetramers or octamers) of such polypeptides selected from the group consisting of Quads 1-46 (i.e., 2, 3, 4, 5, 6, 7, or 8 copies of such polypeptides), e.g., for medical or diagnostic uses, e.g., to treat or prevent a disease or condition in a human or animal (e.g., a human).

[0117] Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequence) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-50. Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequence) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-115. Optionally, the invention provides multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers or octamers, preferably tetramers or octamers) of such polypeptides, e.g., for medical or diagnostic uses, e.g., for treating or preventing a disease or condition in humans or animals (e.g., humans).

[0118] In examples, the TD is a TD composed of any one of SEQ ID NOs: 1-9. In examples, the TD is a TD comprising SEQ ID NO: 10 or 126. In examples, the TD is encoded by SEQ ID NO: 124 or 125. In examples, the amino acid sequence of each TD is SEQ ID NO: 10 or 126, or is at least 80, 85, 90, 95, 96m, 97, 98 or 99% identical to SEQ ID NO: 10 or 126.

[0119] In examples, the TD is a TD comprising SEQ ID NO: 120 or 123. In examples, the TD is encoded by SEQ ID NO: 116 or 119. In examples, the amino acid sequence of each TD is SEQ ID NO: 120 or 123, or is at least 80, 85, 90, 95, 96m, 97, 98 or 99% identical to SEQ ID NO: 120 or 123.

[0120] Optionally, the domain or peptide comprised by the engineered polypeptide or monomer comprises an amino acid selected from SEQ ID NOs: 51-82.

[0121] High purity tetramer As exemplified herein, the present invention in one configuration is based on the surprising realization that a tetramerization domain (TD), e.g., the p53 tetramerization domain (p53 TD), can be used to preferentially produce tetramers of effector domains over the production of lower-order structures such as dimers and monomers. This is particularly useful for secreting tetramers, which is a desirable yield from mammalian expression cell lines such as CHO, HEK293, and Cos cell lines. The present invention is also particularly useful for producing tetramers of 200, 160, 155, or 150 kDa or less in size.

[0122] Thus, the present invention provides the following concepts: concept 1. Use of a tetramerization domain (TD) (e.g., p53 tetramerization domain (p53 TD) or NHR2 TD), or a homolog or ortholog thereof, in a method for producing a tetramer of a polypeptide to produce a higher yield of the tetramer relative to a monomer and / or dimer of the polypeptide.

[0123] Monomers and dimers contain one or two copies of the TD, a homolog or ortholog, respectively.

[0124] In examples, the TD, ortholog or homolog is a human domain.

[0125] As used herein, TD refers to a human TD or a homolog, e.g., a TD selected from any of the p53 TD sequences disclosed in UniProt (www.uniprot.org), e.g., a p53 TD disclosed in Table 13. In an example, the homolog is a p53 TD from a non-human animal species, e.g., a mouse, rat, horse, cat, or dog p53 TD. See Figure 32, which shows a high level of conservation between p53 TDs from different species and supports the use of a non-human p53 TD as a substitute for human p53 TD. In an example, the homolog is a p53 TD from a non-human mammalian species. In an example, the homolog is identical to human p53 TD except for up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change.

[0126] In examples, the yield of tetramer is higher than the yield of monomer; in examples, the yield of tetramer is higher than the yield of dimer; in examples, the yield of tetramer is higher than the yield of trimer; in examples, the yield of tetramer is higher than the yield of monomer and dimer; in examples, the yield of tetramer is higher than the yield of monomer and trimer; in examples, the yield of tetramer is higher than the yield of monomer, dimer, and trimer.

[0127] For example, the TD is a TD of p53 isoform 1. In examples, the TD comprises or consists of an amino acid sequence identical to positions 325-356 (or 319-360, or 321-359) of human p53 (e.g., isoform 1). Optionally, the TD, ortholog, or homolog comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, 126, 11, or 12. For example, the sequence is identical to the selected sequence. Optionally, the TD, ortholog, or homolog comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 120, 121, 122, or 123. For example, the sequence is identical to the selected sequence. 2. The use according to concept 1, wherein the first, second, third and fourth copies of the same TD or its homologue or ortholog are used. 3. The use according to any one of concepts 1 to 2, wherein the TD is an NHR2, p53, p63 or p73 tetramerization domain. For example, each TD is a p53 TD. In examples, the TD is an ortholog or homolog of a p53 TD, for example, a human p53 TD. 4. The use according to any one of concepts 1 to 3, wherein the yield of tetramer is at least 10 times the yield of monomer and / or dimer.

[0128] Optionally, the yield is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the yield of monomer and / or dimer. Optionally, the ratio of tetramer to monomer and / or dimer produced is at least 90:10, for example, at least 95:5, or 96:4, or 97:3, or 98:2, or 99:1. Optionally, only tetramer is produced.

[0129] In one embodiment, each domain comprised by each monomer, dimer, or tetramer is a human domain, and optionally, the monomer, dimer, or tetramer does not include a non-human amino acid sequence or linker. 5. The use of any one of concepts 1-4, wherein the ratio of tetramer produced by the method to monomer and / or dimer produced is at least 90:10 (i.e., 9 times the amount of monomer, 9 times the amount of dimer, or 9 times the amount of combined monomer and dimer). 6. The use of concept 4 or 5, wherein the yield or ratio is determinable or determined by obtaining a sample of the product of the tetramer production process, using a protein separation technique on the sample to resolve the tetramer, monomer, and dimer, and comparing the amount of tetramer to the amount of monomer and dimer.

[0130] The amount of tetramer, monomer, dimer and trimer can be determined, for example, using Western blot analysis of a gel as described herein, e.g., a native gel, i.e., a gel not under denaturing conditions, such as in the absence of SDS. 7. Yield or ratio is (a) obtaining a sample of the product of the tetramer production process; (b) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to resolve the sample into a band corresponding to the tetramer and a band corresponding to the monomer and / or the dimer; (c) determining the termination or ratio of the tetramer band compared to the monomer and / or dimer bands, e.g., by comparing relative band intensities and / or band sizes. The use of concepts 4 or 5, wherein the termination or ratio is determinable or determined by: 8. The yield or ratio is (d) obtaining a sample of the product of the tetramer production process; (e) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to resolve the sample into bands corresponding to the tetramer, e.g., performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions, e.g., where the gel is under non-denaturing conditions (e.g., in the absence of sodium dodecyl sulfate (SDS)), to resolve the sample into bands corresponding to the tetramer; (f) determining the absence of a band corresponding to the monomer and / or the absence of a band corresponding to the dimer. 9. (g) obtaining a second sample of the product of the tetramer production process; (h) performing polyacrylamide gel electrophoresis (PAGE) under reducing conditions to resolve the second sample into bands corresponding to the monomer and / or the dimer, e.g., performing polyacrylamide gel electrophoresis (PAGE) under reducing conditions where the gel is under non-denaturing conditions (e.g., in the absence of sodium dodecyl sulfate (SDS)), to resolve the second sample into bands corresponding to the monomer and / or the dimer; 9. The use of concept 7 or 8, comprising: (i) comparing the gel produced by step (h) with the gel of step (b) or (e) to determine the location of monomer and / or dimer bands in the gel of step (b) or where such bands are expected to be in the gel of step (e). 10. The use of any one of concepts 1 to 9, wherein each monomer has a size of 40 kDa or less.

[0131] For example, the monomer has a size of 35, 30, 25, 24, 23, 22, 21 or 20 kDa or less. 11. The use of any one of concepts 1 to 10, wherein each monomer has a size of 150 kDa or less.

[0132] For example, the monomer has a size of less than or equal to 80, 90, 100, 110, 120, 130 or 140 kDa. 12. The use of any one of concepts 1-11, wherein the method comprises expressing the tetramer from a mammalian cell line, such as a HEK293, CHO, or Cos cell line.

[0133] For example, the cell line is a HEK293 (e.g., HEK293T) cell line. Alternatively, the cell line is a yeast (e.g., Saccharomyces or Pichia, e.g., P pastoris) or bacterial cell line. 13. The use of any one of concepts 1-12, wherein the method comprises secreting the tetramer from a mammalian cell line, such as a HEK293, CHO, or Cos cell line.

[0134] Thus, in an advantageous example, the use of tetramers is for expression from mammalian cell lines (e.g., HEK293, CHO or Cos cell lines) or eukaryotic cell lines, which is useful for large-scale production of products of the invention, such as tetramers.

[0135] For example, the cell line is a HEK293 (e.g., HEK293T) cell line. Alternatively, the cell line is a yeast (e.g., Saccharomyces or Pichia, e.g., P pastoris) or bacterial cell line. 14. The use according to any one of concepts 1 to 13, wherein each polypeptide or monomer comprises the TD, a homologue or an ortholog, and one or more protein effector domains, such as one or more antibody domains, e.g., one or more antibody domains that form an antigen-binding site. 15. A polypeptide comprising: (i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding to an antigen; (ii) an scFv capable of binding to antigen or an scTCR capable of binding to pMHC; (iii) a Fab capable of binding to the antigen, or (iv) a TCR variable domain or a pMHC binding site. 16. The use according to any one of concepts 1 to 15, wherein each polypeptide or monomer comprises the TD, homologue or ortholog, and one or more incretin, insulin, GLP-1 or exendin-4 domains. 17. The use according to any one of concepts 1 to 16, wherein each polypeptide or monomer comprises the TD, a homologue or an orthologue, and a first and a second antigen binding site. 18. Each binding site is (i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding to an antigen; (ii) an scFv capable of binding to antigen or an scTCR capable of binding to pMHC; (iii) a Fab capable of binding to the antigen, or (iv) a TCR variable domain or a pMHC binding site. 19. The use according to concept 18, wherein each binding site is provided by an antibody single variable domain. 20. The use according to any one of concepts 14 to 18, wherein the TD, homologue or ortholog is directly fused to said further domain. 21. The use according to concept 20, wherein each monomer or polypeptide comprises a TD, homologue or orthologue fused directly or via a peptide linker to the C-terminus of said further domain. 22. A tetramer of polypeptides, each polypeptide comprising: (i) a tetramerization domain (TD) (e.g., p53 TD or NHR2 TD) or a homolog or ortholog thereof; (ii) one or more protein effector domains; and (iii) optionally, a linker connecting (i) to (ii) (e.g., connecting the C-terminus of (ii) to the N-terminus of (i)); Optionally, each tetramer has a size of 150 or 200 kDa or less.

[0136] For example, the monomer has a size of 80, 90, 100, 110, 120, 130 or 140 kDa or less. In some examples, any multimer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer has a size of at least 60 or 80 kDa, which may be useful, for example, to extend the half-life in human or animal subjects administered in the form of multimer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., to treat or prevent a disease or condition in the subject). The size of these ranges may exceed the renal filtration size.

[0137] Alternatively, the invention provides a monomer, dimer, octamer, dodecamer, hexadecamer, or 20mer instead of a tetramer. 23. Each polypeptide is (i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding to an antigen; (ii) an scFv capable of binding to antigen or an scTCR capable of binding to pMHC; (iii) a Fab capable of binding to the antigen, or (iv) a TCR variable domain or a pMHC binding site; 24. The tetramer of concept 22 or 23, wherein each polypeptide comprises the TD, homolog or ortholog, and one or more incretin, insulin, GLP-1 or exendin-4 domains. 25. The tetramer of concept 22 or 23, wherein each polypeptide comprises the TD, a homolog or ortholog, and a first and a second antigen binding site. 26. Each binding site is (i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding to an antigen; (ii) an scFv capable of binding to antigen or an scTCR capable of binding to pMHC; (iii) a Fab capable of binding to the antigen, or (iv) a TCR variable domain or a pMHC binding site, the tetramer of concept 25 provided by 27. The tetramer according to concept 26, wherein each binding site is provided by an antibody single variable domain. 28. The tetramer of any one of concepts 22 to 27, wherein the TD, homologue or ortholog is fused directly to the effector domain. 29. The tetramer of any one of concepts 22 to 27, wherein each polypeptide comprises a TD, homolog, or ortholog fused to the C-terminus of the effector domain, either directly or via a peptide linker.

[0138] In embodiments, each polypeptide comprises only two (i.e. only the first and second, but not the third) effector domains, or only two dAb, VHH, scFv, scTCR, Fab or antigen binding sites. 30. A pharmaceutical composition comprising the tetramer of any one of concepts 22-29 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0139] Optionally, the composition is comprised in a sterile medical container or device, such as a syringe, vial, inhaler, or infusion device. 31. A cosmetic, food, beverage, cleaning product, or detergent comprising the tetramer of any one of concepts 22-29. 32. A mixture comprising a cell line (e.g., a mammalian cell line, e.g., a HEK293, CHO, or Cos cell line) encoding a polypeptide according to any one of concepts 1 to 31 and a tetramer as defined in any one of concepts 1 to 31.

[0140] Optionally, the mixture is constituted in a sterile container. 33. The mixture of concept 32, wherein the cell line is in a medium containing a secreted product of the cell, and the secreted product comprises the tetramer. 34. The mixture according to concept 33, wherein the secreted product does not contain monomers and / or dimers as defined in any one of concepts 1 to 31. 35. The mixture of concept 33, wherein the secreted product comprises the tetramer in an amount at least 10 times the amount of the monomer and / or dimer. 36. The mixture of concept 33, wherein the secreted product comprises the tetramer in a ratio of tetramer:monomer and / or dimer of at least 90:10. 37. A method for improving the yield of a tetramer of a protein effector domain (e.g., an antibody variable domain or binding site), the method comprising expressing a tetramer of a polypeptide from a cell line (e.g., a mammalian cell, a CHO, HEK293 or Cos cell line), the polypeptide being as defined in any one of concepts 1-36 and comprising one or more effector domains, and optionally isolating the expressed tetramer.

[0141] Homologues, orthologs or equivalents have multimerization or tetramerization function.

[0142] Homolog: A gene, nucleotide, or protein sequence related to a second gene, nucleotide, or protein sequence by descent from a common ancestral DNA or protein sequence. The term homolog can apply to the relationship between genes separated by an event or the relationship between genes separated by the event of gene duplication.

[0143] Orthologs: Orthologs are gene, nucleotide, or protein sequences in different species that have evolved from a common ancestral gene, nucleotide, or protein sequence through speciation. Orthologs usually retain the same function during evolution.

[0144] In an example, the TD, ortholog, or homolog is a TD of any one of proteins 1-119 listed in Table 2. In an example, the ortholog or homolog is an ortholog or homolog of a TD of any one of proteins 1-119 listed in Table 2. In one embodiment, instead of using a p53 tetramerization domain (p53-TD) or a homolog or ortholog thereof, all aspects of the invention herein can instead be read to relate to the use or inclusion of a polypeptide, monomer, dimer, trimer, or tetramer of a TD of proteins 1-119 listed in Table 2, or a homolog or ortholog thereof. The TD can be an NHR2 (e.g., human NHR2) TD or an ortholog or homolog thereof. The TD can be a p63 (e.g., human p63) TD or an ortholog or homolog thereof. The TD can be a p73 (e.g., human p73) TD or an ortholog or homolog thereof. This has one or more of the following advantages: - secretion of the tetramer from mammalian or other eukaryotic cells, e.g., mammalian cells disclosed herein, such as CHO, HEK293 or Cos; - improved yield of secreted tetramers versus monomers, -Improved yield of secreted tetramers versus dimers, -Improved yield of secreted tetramers versus trimers, - improved yield of secreted tetramers versus combined monomers and dimers; -Improved yield of secreted tetramers versus combined dimers and trimers, - improved antigen binding affinity or avidity in tetramers containing antigen-binding sites; - Improved production and / or expression of tetramers, wherein the size of the tetramers is 200 or less, or 160 or less, or 150 kDa or less. In examples, each polypeptide or monomer may be selected from the group consisting of ReoPro™, abciximab, Rituxan™, rituximab, Zenapax™, daclizumab, Simulect™, basiliximab, Synagis™, palivizumab, Remicade™, infliximab, Herceptin™, Mylotarg™, gemtuzumab, Campath™, Alembic, and others. Tuzumab, Zevalin™, Ibritumomab, Humira™, Adalimumab, Xolair™, Omalizumab, Bexxar™, Tositumomab, Raptiva™, Efalizumab, Erbitux™, Cetuximab, Avastin™, Bevacizumab, Tysabri™, Natalizumab, Actemra™, Tocilizumab, Vectibix™ trademark), panitumumab, Lucentis™, ranibizumab, Soliris™, eculizumab, Cimzia™, certolizumab, Simponi™, golimumab, Ilaris™, canakinumab, Stelara™, ustekinumab, Arzerra™, ofatumumab, Prolia™, denosumab, Numax™, motavizumab, ABThr The antibody comprises a VH, VL, or VH / VL binding site of an antibody selected from the group consisting of: ax™, raxibacumab, Benlysta™, belimumab, Yervoy™, iplizumab, Adcetris™, brentuximab, Vedotin™, Perjeta™, pertuzumab, Kadcyla™, ado-trastuzumab, Gazyva™, and obinutuzumab. In another method, (e.g., for treating or preventing cancer in a human), each polypeptide or monomer comprises one or more VH, VL, or VH / VL binding sites of an antibody selected from ipilimumab (or YERVOY™), tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™), pidilizumab, BMS-936559, durvalumab, and atezolizumab.Optionally, the binding site of the multimeric polypeptide comprises the VH of the antibody binding site and the CH1 of the antibody (i.e., from N- to C-terminus, VH-CH1 and SAM). In one embodiment, the polypeptide can be paired with an additional polypeptide comprising (from N- to C-terminus, e.g., VL-CL, where CL is the CL of the antibody).

[0145] In an example, the tetramer comprises four copies of the antigen-binding site of a first antibody selected from the group consisting of ipilimumab (or YERVOY™), tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™), pidilizumab, BMS-936559, durvalumab, and atezolizumab, and optionally four copies of the antigen-binding site of a second antibody selected from the group, wherein the first and second antibodies are different. For example, the first antibody is ipilimumab (or YERVOY™) and, optionally, the second antibody is nivolumab (or OPDIVO™) or pembrolizumab (or KEYTRUDA™). This is useful for treating or preventing cancer in humans.

[0146] In an example, the tetramer comprises four copies of the antigen binding site for Avastin. In an example, the tetramer comprises four copies of the antigen binding site for Humira. In an example, the tetramer comprises four copies of the antigen binding site for Erbitux. In an example, the tetramer comprises four copies of the antigen binding site for Actemra™. In an example, the tetramer comprises four copies of the antigen binding site for sarilumab. In an example, the tetramer comprises four copies of the antigen binding site for dupilumab. In an example, the tetramer comprises four copies of the antigen binding site for alirocumab or evolocumab. In an example, the tetramer comprises four copies of the antigen binding site for Remicade. In an example, the tetramer comprises four copies of the antigen binding site for Remicade. In an example, the tetramer comprises four copies of the antigen binding site for Lucentis. In an example, the tetramer comprises four copies of the antigen binding site for Eylea™. Such tetramers are useful for administration to humans to treat or prevent cancer. Such tetramers are useful for administration to humans to treat or prevent ocular conditions (e.g., wet AMD or diabetic retinopathy, for example, when the binding site is an Avastin, Lucentis, or Eylea site). Such tetramers are useful for administration to humans to treat or prevent angiogenesis.

[0147] In an example, the tetramer comprises four copies of insulin. In an example, the tetramer comprises four copies of GLP-1. In an example, the tetramer comprises four copies of GIP. In an example, the tetramer comprises four copies of exendin-4. In an example, the tetramer comprises four copies of insulin and four copies of GLP-1. In an example, the tetramer comprises four copies of insulin and four copies of GIP. In an example, the tetramer comprises four copies of insulin and four copies of exendin-4. In an example, the tetramer comprises four copies of GLP-1 and four copies of exendin-4. Such tetramers are useful for administration to humans to treat or prevent diabetes (e.g., type II diabetes) or obesity.

[0148] Exemplary Antigens The polypeptide, multimer, or each binding site herein (e.g., a dAb or scFv binding site) can bind to one or more antigens or epitopes. In examples, the (or each) antigen herein is selected from the following list: In examples, the (or each) epitope herein is an epitope of an antigen selected from the following list:

[0149] Activin type II receptor, activin type IIB receptor, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM1A, ADAM1B, ADAM2, ADAM20, ADAM21, ADAM22, ADAM23, ADAM24P, ADAM28, ADAM29, ADAM30, ADAM32, ADAM33, ADAM3A, ADAM3B, ADAM5, ADAM6, ADAM7, ADAM8, ADAM9, ADORA2A, AKT, ALK, alpha-4 integrin, alpha-synuclein, anthrax protective antigen, BACE1, BCMA, beta-amyloid, BRAF, BTLA, BTNL2, CCR4, CCR5, CD126, CD151, C D16, CD160, CD19, CD20, CD22, CD226, CD244, CD27, CD274 (PDL1), CD276, CD28, CD3, CD30, CD300A, CD300C, CD300E, CD300LB, CD300LF, CD33, CD38, CD3, CD3 epsilon, CD3 delta, CD3 gamma, CD40, CD40L, CD47, CD48, CD5, CD52, CD59, CD6, CD70, CD72, CD73, CD80, CD81, CD84, CD86, CD96, CDK4 / 6, CEA, CEACAM1, CEACAM3, CGRP receptor, CLEC12A, CLEC1B, CLEC4A, CLEC5A, CLEC7A, Clostridium difficile toxin, cMET, complement factor C5, complement factor D, CSF1R, CSF2RA, CTAG1B, CTLA4, CXCL12, CXCR2, CXCR4, DR4, DR5, EDA, EDA2R, EGFR, EGFRvIII, EMR1, ENTPD1, ​​EpCAM, factor IX, factor X, factor VII , FAP, FAS, FCAR, FCER1G, FCER2, TFR2, 4-1BB, FCGR2A, FCGR2B, FCGR3A, FCGR3B, FCRL1, FCRL3, FCRL4, FCRL6, FGRF1 / 2 / 3, FLT3, GAL, GEM, GITR, GITRL, GM-CSF, GM-CSF receptor, GP IIb IIIa, gpNMB, TIM3, HDAC1, HER-2, HER3, HFE, HHLA2, histone H1 modulator, HLA-C, HLA-G, HMGB1, HMMR, HVEM, ICAM-1,ICOS, ICOSL, IDO1, IFNG, IL-1 IL-12, IL-13, IL-2, IL-22R, IL-23, IL-23a, IL-24, IL-2R, IL-34, I L-8, IL10, IL11, IL13, IL17A, IL17D, IL22, IL2RA, IL36G, IL4, IL4a, IL5 IL6 .Environment E. E. E. .G INHBA, INHBB, I and Aquarius, INF- a-2a / 2b, INF-b-1a / 1b, ITGA2B, ITGB3, KIR, KIR2DL1, KIR2DL2, KIR2DL3 KIR2DL4、KIR2DL5A、KIR3DL1、KIR3DL3、KIR3DS1、KIT、KLRC1、KLRC2、KLR F1, KLRG1, KLRK1, KRAS, LAG3, LAIR1, LAIR2, LFA-1, LIGHT, LILRA1, LILRA 2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LIL RB5, LILRP1, LILRP2, LTA, LTBR, ​​LY9, MadCam, MAGE-C1, MAGE-C2, MARCO EK-1 / 2, MIA3, MIC, MICA, MICB, MMP9, MS4A1, MS4A2, mTOR, MUC1, MUCIN-1 Nav1.7, Nav1.8, NCR1, NCR2, NCR3, NGF, NGFR, NT5E, NY-ESO-1, OX40, OX4 0L, p53, PARP, PCSK9, PD-1, PDCD1LG2, PDCD6, PDGF scavenging PECAM1, PI3K Like PILRA, PPP1R1B, PSMA, PTPN6, PVR, PVRL2, PVRL3, RANKL Link SIGLEC10, SIGLEC12, SIGLEC15, SIGLEC5, SIGLEC6, SIGLEC7, SIG LEC9, SIRPA, SIRPB1, SLAMF1, SLAMF6, SLAMF7, SLAMF8, SNCA, SOD1, STAT3 、STING、SURVIVIN、TARM1、Tau、TDP43、TfR1、TGF-b、TGM2、TIGIT、TIM-3、T LR-4, TLR03, TMEM30a, TMIGD2, TNFa, TNFRSF10A, TNFRSF10B, TNFRSF10C.TNFRSF10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF4, TNFRSF6B, TNFRSF8, TNFRSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, transmembrane glycoprotein NMB modulator, TREML1, TREML2, TSLP, VEGF, VEGF-2R, VEGF1, VEGFA, VEGFL, VEGFR, viral envelope glycoprotein, viral protein hemagglutinin, VISTA, VSIG4, VSTM1, VTCN1, and WEE-1. In examples, the antigen herein is PCSK9, e.g., human PCSK9, and optionally, the multimer has 4, 8, 12, or 16 copies of an anti-PCSK9 binding site (e.g., a dAb).

[0150] An exemplary antigen is a toxin, such as a snake venom toxin, e.g., a multimer of the invention is administered to a human or animal subject (e.g., systemically or by IV injection), and the antigen-binding site comprised by the multimer specifically binds to the toxin of interest. Preferably, each binding site or domain of the multimer is a dAb (e.g., a Nanobody™). For example, the toxin antigen binding site for each snake venom of the multimer of the invention is a C33 single domain VH as disclosed in Figure 4 of Richard et al., PLoS One. 2013 Jul 22;8(7):e69495. doi:10.1371 / journal.pone.0069495; "In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody," ... For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C15 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C7 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of the multimer of the invention is a C13 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety by the present invention for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH, as disclosed in said Figure 4.For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C19 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C34 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C31 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C20 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C2 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen binding site of each snake venom of a multimer of the invention is a C29 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4.For example, the toxin antigen-binding site of each snake venom of the multimer of the present invention is a C42 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. For example, the toxin antigen-binding site of each snake venom of the multimer of the present invention is a C43 single domain VH as disclosed in Figure 4 of Richard et al., the amino acids of which are incorporated herein in their entirety for possible use in the present invention as a binding site or domain or dAb or Nanobody or VHH or VH as disclosed in Figure 4. Examples of snake venom toxins are 3FTx, dendrotoxin, or PLA2 toxin. Optionally, the toxin is, for example, an alpha neurotoxin from a cobra.

[0151] Another example of a toxin is a circulating toxin, for example, when a multimer of the present invention is administered to a human or animal subject (e.g., systemically or by IV injection), the antigen-binding site formed by the multimer specifically binds to the toxin in the subject's blood. These examples are useful for sequestering the toxin, reducing the toxic effects of the toxin on the subject, or promoting the excretion or metabolism of the toxin.

[0152] In examples, the antigens are viral antigens, each of which is a capsid protein or a carbohydrate (e.g., a sugar). In examples, the multimers of the invention bind to a virus or viral antigen, e.g., a virus selected from Table 19, where the virus comprises a surface antigen bound by the multimer, or the multimers of the invention bind to a cell or viral antigen, e.g., selected from the antigens disclosed in Table 20. Binding to a virus can, for example, reduce or inhibit attachment of the virus to a host cell or infection of a cell by the virus. For example, the invention provides a method of treating or preventing (e.g., reducing the risk of) a viral or cellular infection in a human, animal, or plant subject (e.g., a human subject), comprising administering a multimer of the invention to the subject, wherein the multimer binds to a surface antigen of the virus, thereby inhibiting attachment of the virus to a host cell, inhibiting infection of the host cell by the virus, and / or sequestering the virus in the subject (e.g., to mark the bound virus for clearance from the subject's systemic circulation or tissues). In another method, the present invention provides a method for treating or preventing (e.g., reducing the risk of) bacterial or archaeal cell infection in a human, animal, or plant subject (e.g., a human subject), the method comprising administering a multimer of the present invention to the subject, wherein the multimer binds to a surface antigen of the cell, thereby inhibiting infection of the subject by the cell and / or sequestering the cell in the subject (e.g., marking the bound cells for clearance from the subject's systemic circulation or tissues). In another method, for example, the present invention provides a method for treating or preventing (e.g., reducing the risk of) cancer in a human or animal subject (e.g., a human subject), the method comprising administering a multimer of the present invention to the subject, the multimer binds to a surface antigen of a tumor cell, thereby sequestering the subject's cells (e.g., marking the bound cells for clearance from the systemic circulation or tissues of the subject) or marking the cells for targeting by the immune system or another therapy administered to the subject (e.g., immune checkpoint therapy or CAR-T therapy).

[0153] In an example, the antigen is selected from CXCR2, CXCR4, GM-CSF, ICAM-1, IFN-g, IL-1, IL-10, IL-12, IL-1R1, IL-1R2, IL-1Ra, IL-1, IL-4, IL-6, IL-8, MIF, TGF-, TNF-, TNFR1, TNFR2, and VCAM-1. Targeting one or more of these antigens can be useful for treating or preventing sepsis in a subject. Thus, in an example, the multimer of the present invention comprises one or more antigen binding sites (e.g., each provided by a dAb), and the multimer is for use in a method for treating or preventing sepsis in a human or animal subject, wherein the multimer is administered to the subject (e.g., systemically or intravenously). Optionally, the multimer is monospecific, bispecific, trispecific, or tetraspecific with respect to antigen binding. For example, the multimers are bispecific, trispecific, or tetraspecific for an antigen selected from CXCR2, CXCR4, GM-CSF, ICAM-1, IFN-γ, IL-1, IL-10, IL-12, IL-1R1, IL-1R2, IL-1Ra, IL-1, IL-4, IL-6, IL-8, MIF, TGF-, TNF-, TNFR1, TNFR2, and VCAM-1. Pharmaceutical compositions comprising such multimers and a pharmaceutically acceptable diluent, carrier, or excipient are also provided. Methods of treating or preventing sepsis in a human or animal subject are also provided, including, for example, administering the multimers to the subject, e.g., systemically or intravenously.

[0154] Diseases and Conditions Polypeptide monomers or multimers (e.g., dimers, trimers, tetramers, or octamers) of the invention can be used in methods for administration to a human or animal subject to treat or prevent a disease or condition in that subject.

[0155] Optionally, the disease or condition is selected from the following: (a) a neurodegenerative disease or condition; (b) a brain disease or condition; (c) a CNS disease or condition; (d) memory loss or impairment; (e) heart or cardiovascular disease or condition, e.g., heart attack, stroke, atrial fibrillation; (f) liver disease or condition; (g) kidney disease or condition, e.g., chronic kidney disease (CKD); (h) pancreatic disease or condition; (i) a pulmonary disease or condition, e.g., cystic fibrosis or COPD; (j) gastrointestinal disease or condition; (k) throat or oral cavity disease or condition; (l) eye disease or condition; (m) diseases or conditions of the genitals, such as diseases or conditions of the vagina, labia, penis, or scrotum; (n) Sexually transmitted diseases or conditions, such as gonorrhea, HIV infection, syphilis, or chlamydial infection; (o) ear disease or condition; (p) skin diseases or conditions; (q) heart disease or condition; (r) nasal diseases or conditions; (s) a hematological disease or condition, such as anemia, e.g., anemia of chronic disease or cancer; (t) viral infection, (u) Pathogenic bacterial infection; (v) cancer, (w) an autoimmune disease or condition, e.g., SLE; (x) an inflammatory disease or condition, such as rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease or IBD; (y) autism; (z) ADHD, (aa) bipolar disorder, (bb) ALS [amyotrophic lateral sclerosis], (cc) osteoarthritis, (dd) congenital or developmental disability or condition; (ee) miscarriage, (ff) blood coagulation status, (gg) bronchitis, (hh) Dry or wet AMD, (ii) angiogenesis (e.g., of tumors or of the eye); (jj) common cold, (kk) epilepsy, (ll) fibrosis, e.g., liver or lung fibrosis, (mm) fungal diseases or conditions, e.g., thrush; (nn) A metabolic disease or condition, such as obesity, anorexia, diabetes, type I or type II diabetes. (oo) ulcers, e.g., gastric or skin ulcers, (pp) dry skin, (qq) Sjögren's syndrome, (rr) Cytokine storm, (ss) deafness, hearing loss or hearing impairment; (tt) Hypo- or hyper-metabolism (i.e., slower or faster than average for the subject's weight, sex, and age). (uu) Fertility disorders, e.g., infertility or subfertility; (vv) Jaundice; (lol) Skin rash, (xx) Kawasaki disease, (yy) Lyme disease, (zz) allergies, e.g., nut, pollen, dust mite, cat or dog fur or dander allergies; (aaa) malaria, typhoid, tuberculosis or cholera, (bbb) depression, (ccc) Mental retardation, (ddd)microcephaly, (eee) malnutrition, (fff) conjunctivitis, (ggg) pneumonia, (hhh) Pulmonary embolism, (iii) pulmonary hypertension, (jjj) bone disorders, (kkk) Sepsis or septic shock, (lll) sinusitis, (mmm) stress (e.g., occupational stress), (nnn) thalassemia, anemia, von Willebrand disease, or hemophilia, (ooo) Shingles or cold sores, (ppp) Menstruation, (qqq) Decrease in sperm count.

[0156] Neurodegenerative or CNS Nervous System Diseases or Conditions to Treat or Prevent In certain embodiments, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer's disease, reproductive psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinsonism, Huntington's chorea, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinsonism.

[0157] In an example, when the method of the present invention is performed on a human or animal subject to treat a CNS or neurodegenerative disease or condition, the method causes downregulation of the subject's Treg cells, thereby promoting the entry of systemic monocyte-derived macrophages and / or Treg cells across the choroid plexus into the subject's brain, thereby treating, preventing, or reducing the progression of the disease or condition (e.g., Alzheimer's disease). In one embodiment, the method causes an increase in IFN-gamma in the subject's CNS system (e.g., brain and / or CSF). In an example, the method restores nerve fibers and / or reduces the progression of nerve fiber damage. In an example, the method restores nerve myelin and / or reduces the progression of nerve myelin damage. In examples, the methods of the present invention treat or prevent a disease or condition disclosed in WO2015 / 136541 and / or the methods can be used in conjunction with any of the methods disclosed in WO2015 / 136541 (the disclosure of this document is incorporated herein by reference in its entirety to provide a disclosure of such methods, diseases, conditions and potential therapeutic agents, e.g., agents such as immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed herein, that can be administered to a subject to effect treatment and / or prevention of CNS and neurodegenerative diseases and conditions).

[0158] Cancer treatment or prevention Cancers that can be treated include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors.Cancers can include non-solid tumors (e.g., blood tumors such as leukemia and lymphoma) or solid tumors.The types of cancers that can be treated by the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0159] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include acute leukemia (acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroblastic leukemia), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0160] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, lung cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, sebaceous gland carcinoma, papillary car ... papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, sebaceous gland carcinoma, papillary carcinoma, medullary carcin These include tumors such as cystoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0161] Autoimmune diseases for treatment or prevention Acute disseminated encephalomyelitis (ADEM) Acute necrotizing hemorrhagic leukoencephalitis Addison's disease Agammaglobulinemia Alopecia areata Amyloidosis ·Ankylosing spondylitis ·Anti-GBM / anti-TBM nephritis Antiphospholipid syndrome (APS) ·Autoimmune angioedema ·Autoimmune aplastic anemia Autoimmune autonomic dysfunction ·Autoimmune hepatitis ·Autoimmune hyperlipidemia ·Autoimmune immunodeficiency ·Autoimmune inner ear disease (AIED) Autoimmune myocarditis Autoimmune oophoritis Autoimmune pancreatitis ·Autoimmune retinopathy ·Autoimmune thrombocytopenic purpura (ATP) ·Autoimmune thyroid disease ·Autoimmune urticaria Axonal and neuronal neuropathies Baro's disease Behçet's disease Bullous pemphigoid Cardiomyopathy Castleman's disease Celiac disease Chagas disease Chronic fatigue syndrome Chronic inflammatory demyelinating polyneuropathy (CIDP) Chronic recurrent multifocal osteomyelitis (CRMO) Churg-Strauss syndrome Cicatricial pemphigoid / benign mucous membrane pemphigoid Crohn's disease Cogan's syndrome ·Cold agglutinin disease Congenital heart block Coxsackie's myocarditis ·CREST disease Essential mixed cryoglobulinemia Demyelinating neuropathy ·Dermatitis herpetiformis ·Dermatomyositis Devic's disease (neuromyelitis optica) Discoid lupus erythematosus Dressler syndrome ·Endometriosis Eosinophilic esophagitis Eosinophilic fasciitis ·Erythema nodosum Experimental allergic encephalomyelitis Evans syndrome ·Fibromyalgia Fibrotic alveolitis ·Giant cell arteritis (temporal arteritis) Giant cell myocarditis Glomerulonephritis Goodpasture's syndrome ·Granulomatosis with polyangiitis (GPA) (formerly Wegener's granulomatosis) Graves' disease Guillain-Barré syndrome Hashimoto's encephalitis Hashimoto's thyroiditis ·Hemolytic anemia Henoch-Schönlein purpura ·Pregnancy herpes Hypogammaglobulinemia Idiopathic thrombocytopenic purpura (ITP) IgA nephropathy IgG4-related sclerosing disease Immunomodulatory lipoproteins Inclusion body myositis ·Interstitial cystitis Juvenile arthritis Juvenile diabetes (type 1 diabetes) Juvenile myositis Kawasaki syndrome Lambert-Eaton syndrome ·Leukocytoclastic vasculitis ·lichen planus ·Lichen sclerosus ·Lyny conjunctivitis Linear IgA disease (LAD) Lupus (SLE) Lyme disease, chronic Meniere's disease Microscopic polyangiitis ·Mixed connective tissue disease (MCTD) Mooren's ulcer Mukka-Habermann's disease Multiple sclerosis ·Myasthenia gravis Myositis Narcolepsy Neuromyelitis optica (Devic's disease) ·Neutropenia Ocular cicatricial pemphigoid ·Optic neuritis Relapsing rheumatism PANDAS (pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection) Paraneoplastic cerebellar degeneration Paroxysmal nocturnal hemoglobinuria (PNH) Parry-Romberg syndrome Neuralgic amyotrophy Pars planitis (peripheral uveitis) ·Pemphigus Peripheral neuropathy Perivenous encephalitis Pernicious anemia POEMS syndrome Polyarteritis nodosa Polyglandular autoimmune syndrome types I, II, and III Polymyalgia rheumatica Polymyositis Post-myocardial infarction syndrome Postpericardiotomy syndrome Progesterone dermatitis Primary biliary cirrhosis Primary sclerosing cholangitis ·psoriasis Psoriatic arthritis Idiopathic pulmonary fibrosis Pyoderma gangrenosum ·Erythroblastic aplasia Raynaud's phenomenon Reactive arthritis Reflex sympathetic dystrophy Reiter's syndrome Relapsing polychondritis Restless legs syndrome Retroperitoneal fibrosis Rheumatic fever Rheumatoid arthritis ·sarcoidosis Schmidt syndrome ·Scleritis Scleroderma Sjögren's syndrome Semen-testicular autoimmunity Stiff-person syndrome Subacute bacterial endocarditis (SBE) Susac syndrome ·Sympathetic ophthalmia ·Takayasu arteritis ·Temporal arteritis / giant cell arteritis Thrombocytopenic purpura (TTP) Tolosa-Hunt syndrome Transverse myelitis ·Type 1 diabetes ·Ulcerative colitis ·Undifferentiated connective tissue disease (UCTD) Uveitis ·Vasculitis ·Blistering dermatosis ·Vitiligo Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA))

[0162] Inflammatory diseases for treatment or prevention Alzheimer's disease ·Ankylosing spondylitis Arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) ·asthma Atherosclerosis Crohn's disease Colitis ·Dermatitis ·Diverticulitis ·Fibromyalgia ·hepatitis ·Irritable bowel syndrome (IBS) Systemic lupus erythematosus (SLE) ·Nephritis Parkinson's disease ·Ulcerative colitis.

[0163] Multivalent soluble TCR This composition relates to a multivalent soluble TCR protein. In one aspect, the present invention relates to tetravalent and octavalent soluble TCR analogs. The TCR protein of the present invention is capable of self-assembly from monomers and is entirely of human origin. The protein is a multimer comprising the ETO NHR2 multimerization domain. The present invention also relates to methods for constructing multimeric soluble TCRs and methods for using such proteins.

[0164] Attempts to utilize alternative soluble TCR formats arise because therapeutic molecules have lagged significantly behind numerous antibody formats. This is primarily due to TCRs being heterodimeric transmembrane proteins that have inherent solubility challenges once the extracellular TCR α / β chains dissociate from the transmembrane and cytoplasmic domains. Second, the inherent low affinity and avidity of these molecules for their cognate ligands at the target site has significantly hindered their development as therapeutic molecules.

[0165] To overcome these drawbacks, this aspect of the invention provides a TCR protein that is both multivalent and soluble. Multivalency enhances the avidity of the TCR to its cognate pMHC, while solubility allows the TCR to be used outside of a transmembrane environment. Thus, in a first aspect, there is provided a multivalent heterodimeric soluble T cell receptor capable of binding to a pMHC complex, comprising: (i) a TCR extracellular domain; and (ii) an immunoglobulin constant domain; (iii) a multivalent heterodimeric soluble T cell receptor comprising: (iii) an NHR2 multimerization domain of ETO;

[0166] The use of Ig constant domains provides stability and solubility to the TCR extracellular domain, and multimerization via the NHR2 domain provides multivalency and improved avidity. Advantageously, all of the domains are of human origin or correspond to human protein sequences.

[0167] The use of Ig constant domains to stabilize and soluble TCRs avoids the use of non-native disulfide bonds, and therefore advantageously the TCRs of the present invention do not contain non-native disulfide bonds.

[0168] In one embodiment, the complex comprises a heavy chain and a light chain, each light chain comprising a TCR Vα domain and an immunoglobulin C α Each heavy chain contains a TCR Vβ domain and an immunoglobulin C H Contains 1 domain.

[0169] In one embodiment, each light chain further comprises a TCR Cα domain and each heavy chain further comprises a TCR Cβ domain.

[0170] In embodiments, the TCR and immunoglobulin domains may be separated by a flexible linker.

[0171] The NHR2 multimerization domain is advantageously attached to the C-terminus of the immunoglobulin domain, such that each heavy and light chain dimer binds to one multimerization domain, resulting in the association of heavy-light chain dimers into multivalent oligomers.

[0172] In embodiments, the multimerization domain and the immunoglobulin domain are separated by a flexible linker, which in certain embodiments allows the multimerization domain to multimerize without interference from the immunoglobulin domain.

[0173] In embodiments, the TCR protein may further comprise an immunoglobulin hinge domain. The hinge domain allows the heavy chain-light chain dimer to dimerize, thereby allowing further multimerization of the TCR protein. For example, a multimerization domain that forms a polypeptide tetramer can use an immunoglobulin hinge domain to form multimers up to a polypeptide octamer. Similarly, a dimerization multimerization domain can form a tetramer in the presence of a hinge domain.

[0174] In an embodiment, the TCR proteins of the present invention are tetravalent.

[0175] In an embodiment, the TCR proteins of the present invention are octavalent.

[0176] The present invention provides a soluble TCR that stably assembles in a tetravalent heterodimeric format using the neurohomology region 2 (NHR2) domain found in human ETO family proteins (Liu et al. 2006). The NHR2 domain is naturally found to form homotetramers formed from the pairing of two NHR2 homodimers. When operably bound to the extracellular TCRα or TCRβ chain, NHR2 preferentially self-assembles into tetravalent heterodimeric soluble TCR protein molecules from monomers and subsequent homodimers (Figure 1).

[0177] TCR proteins that assemble into octamers can be created using the NHR2 domain by using an immunoglobulin hinge domain.

[0178] In a further embodiment, the TCR proteins of the present invention may be linked to a biologically active polypeptide / effector molecule, such as an immunologically active moiety such as a cytokine, a binding protein such as an antibody or a targeting polypeptide, etc.

[0179] The present invention further relates to methods for producing tetravalent and octavalent heterodimeric soluble TCRs, DNA vectors encoding the proteins used to transfect host cells of interest, and uses of these novel, highly sensitive, multivalent soluble TCR protein molecules for applications including, but not limited to, therapy, diagnostics, and drug discovery.

[0180] In a further aspect, the present invention provides methods for constructing polyvalent immunoglobulin molecules in an efficient manner without the use of non-human construct components.

[0181] Therefore, the following (i) an immunoglobulin variable domain; (ii) the NHR2 multimerization domain of ETO.

[0182] The immunoglobulin variable domain is preferably an antibody variable domain, which is fused to an ETO NHR2 multimerization domain, which provides a means for forming a tetramer of immunoglobulin variable domains.

[0183] The ETO NHR2 domain is more efficient than p53 and similar multimerization domains in producing immunoglobulin multimers, allowing for the production of multimeric immunoglobulin molecules without the use of non-human components in the construct.

[0184] Also provided is a method for producing a multimeric immunoglobulin comprising expressing immunoglobulin variable domains fused to the NHR2 domain of ETO and allowing the variable domains to assemble into multimers.

[0185] Preferably, the immunoglobulin variable domain is attached to one or more immunoglobulin constant domains.

[0186] Advantageously, the immunoglobulin domain is an antibody domain. For example, the variable domain is V H and V L It can be an antibody domain, for example, the constant domain is an antibody CH1 domain.

[0187] In one embodiment, the multimeric immunoglobulin molecules of the present invention, both TCR and non-TCR immunoglobulins, are produced for screening by phage display or another display technology. Thus, for example, multivalent immunoglobulins are produced as fusions with phage coat proteins. For each immunoglobulin produced fused to a coat protein, other immunoglobulin molecules are produced without the coat protein and can assemble on the phage surface as a result of NHR2 multimerization.

[0188] The compositions of the present invention detailed above relate to nucleic acid sequences and methods for producing novel multivalent, e.g., tetravalent and octavalent, soluble proteins. In one embodiment, the soluble proteins are TCRs assembled in a tetravalent heterodimeric format capable of binding to four pMHC with high sensitivity, affinity, and specificity. Soluble tetravalent heterodimeric TCRs are unique protein molecules composed of either all or part of the extracellular TCR α / β chains. The extracellular TCR α / β chains are linked to immunoglobulin C. H 1 and C L (either Cκ or Cλ) domains. This linkage allows the stable formation of heterodimeric TCRα / β. In the context of soluble tetravalent TCRs, a unique feature is the C H 1 C-terminus or C L The NHR2 domain is a homotetramerization domain of the ETO family of proteins operably linked to the C-terminus of the NHR2 domain. Binding of the NHR2 domain to heterodimeric α / β TCRs in this manner results in intracellular self-assembly into a tetravalent format that is then secreted into the supernatant as a soluble protein. TCR extracellular domain

[0189] The TCR extracellular domain is composed of a variable region and a constant region. These domains exist in T cell receptors in the same way as they exist in antibodies and other immunoglobulin domains. The TCR repertoire contains extensive diversity created by the same gene rearrangement mechanism used in antibody heavy and light chain genes (Tonegawa, S. (1988) Biosci. Rep. 8:3-26). Most of the diversity is generated at the junction of the variable (V) and joining (J) (or diversity D) regions, which encode the complementarity-determining regions 3 (CDR3) of the α and β chains (Davis and Bjorkman (1988) Nature 334:395-402). Databases of TCR genes are available, such as the IMGT LIGM database, and methods for cloning TCRs are known in the art, see, e.g., Bentley and Mariuzza (1996) Ann. Rev. Immunol. 14:563-590; Moysey et al., Anal Biochem. 2004 Mar 15;326(2):284-6; Walchli, et al. (2011) A Practical Approach to T-Cell Receptor Cloning and Expression. PLoS ONE 6(11):e27930.

[0190] Immunoglobulin variable domains Antibody variable domains are known in the art and are available from a wide variety of sources. Databases of antibody variable domain sequences exist, such as IMGT and Kabat, and variable domains can be produced by cloning and expression of natural sequences or by synthesis of artificial nucleic acids according to established techniques.

[0191] Methods for constructing bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al. (1990) Nature, 348:552; Kang et al. (1991) Proc. Natl. Acad. Sci. USA, 88:4363; Clackson et al. (1991) Nature, 352:624; Lowman et al. (1991) Biochemistry, 30:10832; Burton et al. (1991) Proc. Natl. Acad. Sci USA, 88:10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19:4133; Chang et al. (1991) J Immunol., 147:3610; Breitling et al. al. (1991) Gene, 104:147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins and Winter (1992) J Immunol., 22:867; Marks et al., 1992, J Bioi. Chem., 267:16007; Lerner et al. (1992) Science, 258:1313, incorporated herein by reference).

[0192] One particularly advantageous approach has been the use of scFv phage libraries (Huston et al., 1988, Proc. Natl. Acad. Sci USA, 85:5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci USA, 87:1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991) Nature, 352:624; Marks et al. (1991) J Mol. Biol., 222:581; Chiswell et al. (1992) Trends Biotech., 10:80; Marks et al. (1992) J Biol. Chem., 267). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Modifications of phage display approaches are also known, as described, for example, in WO96 / 06213 and WO92 / 01047 (Medical Research Council et al.) and WO97 / 08320 (Morphosys), which are incorporated herein by reference.

[0193] Such techniques can be adapted to produce multimeric immunoglobulins by fusing the NHR2 multimerization domain to antibody variable domains.

[0194] Immunoglobulin constant domains The immunoglobulin constant domain referred to herein is preferably an antibody constant domain. The sequence of the constant domain varies between antibody subtypes, and preferably the constant domain is an IgG constant domain. Preferably, the constant domain is a CH1 constant domain. Antibody constant domains are well known in the art and are available from many sources and databases, including the IMGT and Kabat databases.

[0195] The fusion of antibody constant domains to immunoglobulin variable domains is also known in the art, for example in the construction of engineered Fab antibody fragments.

[0196] Linker A flexible linker can be used to connect the TCR variable domain (Ig constant domain) to the NHR2 multimerization domain. This allows the TCR domain and the multimerization domain to function without steric hindrance from each other or other molecules in the multimeric complex. Suitable linkers include, for example, glycine repeats, glycine-alanine repeats, Gly(4)Ser linkers, or flexible polypeptide linkers such as those described in Reddy Chichili et al., 2012 Protein Science 22:153-167.

[0197] immunoglobulin hinge domain Ig hinge domain, preferably antibody hinge domain herein, is the domain that connects the antibody constant region of natural antibody.Therefore, this domain provides the natural dimerization of molecules that contain antibody constant domains.It is present in, for example, F(ab)2 antibody fragments, as well as whole antibodies such as IgG.This region forms two natural interchain disulfide bonds that connect two CH1 constant domains.

[0198] In one embodiment, the multimerization domain can be linked to an Ig constant domain or hinge domain. When the hinge domain is present, the multimerization domain forms a TRC octamer, which contains four dimers of TCR variable Ig constant domains linked by the hinge region. In the absence of the hinge region, the multimerization domain leads to the formation of a tetramer. Preferably, the multimerization domain is attached to the C-terminus of the constant domain or hinge region.

[0199] biologically active molecules One or more biologically active molecules or effector molecules (EMs) can be attached to the multimers of the invention, e.g., multimeric TCR proteins. Such molecules can be, for example, antibodies, particularly antibodies that can support immune recognition and function of the TCR, such as anti-CD3 antibodies or antibody fragments.

[0200] In some embodiments, the biologically active molecule can be a biologically active molecule such as a cytotoxic drug, a toxin, or a cytokine, as described in more detail below. Examples of biologically active molecules include chemokines such as MIP-1b, cytokines such as IL-2, growth factors such as GM-CSF or G-CSF, toxins such as ricin, cytotoxic agents such as doxorubicin or taxanes, radioactive and fluorescent labels, etc. For examples of biologically active molecules capable of binding to TCRs, see US2011 / 0071919.

[0201] In other embodiments, the biologically active molecule is selected from the group consisting of, for example, a group capable of binding to a molecule that extends the half-life of a polypeptide ligand in vivo, and a molecule that extends the half-life of a polypeptide ligand in vivo. Such a molecule can be, for example, an HSA or cell matrix protein, and the group capable of binding to a molecule that extends the half-life of a TCR molecule in vivo is an antibody or antibody fragment specific for HSA or the cell matrix.

[0202] In one embodiment, the biologically active molecule is a binding molecule, such as an antibody fragment.Two, three, four, five or more antibody fragments can be linked together using a suitable linker.The specificities of any two or more of these antibody fragments can be the same or different, and if they are the same, a multivalent binding structure is formed, which has increased binding activity to the target compared with a monovalent antibody fragment.

[0203] Additionally, the biologically active molecule may be an effector group, such as an antibody Fc region.

[0204] Attachment to the N- or C-terminus can occur before or after the TCR molecule or engineered polypeptide assembles into a multimer. Thus, TCR fusions with Ig constant domains can be generated (synthetically or by nucleic acid expression) with the N- or C-terminal biologically active molecule already in place. However, in certain embodiments, the N- or C-terminal addition occurs after the TCR fusion is generated. For example, fluorenylmethyloxycarbonyl chloride can be used to introduce an Fmoc protecting group at the N-terminus of the TCR fusion. Fmoc binds with high affinity to serum albumin, including HSA, while Fmoc-Trp or FMOC-Lys also bind with high affinity. The peptide can be synthesized with the Fmoc protecting group remaining and then conjugated to the scaffold via cysteine. Another method is the palmitoyl moiety, which also binds to HSA and is used, for example, in liraglutide to extend the half-life of this GLP-1 analog.

[0205] Alternatively, the TCR fusion can be modified at the N-terminus, for example, with the amine and sulfhydryl-reactive linker Ne-maleimidocaproyloxy)succinimide ester (EMCS), through which the TCR can be linked to other polypeptides, such as antibody Fc fragments.

[0206] NHR2 domain AML1 / ETO is a fusion protein resulting from the t(8:21) mutation found in M2 subtype acute myeloid leukemia (AML). AML1 / ETO contains the N-terminal 177 amino acids of RUNX1 fused in-frame with most of ETO (575 aa). Neurohomology domain 2 of ETO is involved in many biological activities associated with AML1 / ETO, including oligomerization and protein-protein interactions. This domain has been characterized in detail in Liu et al. (2006). See Genbank accession number NG_023272.2.

[0207] In one embodiment of the invention, the protein assembled in a soluble multivalent format is a TCR composed of either part or all of the extracellular domains of the TCR α and β chains. The α and β chains of the TCR are encoded by immunoglobulin C. H 1 and C L It is stabilized by domains and can be arranged in the following configuration: 1.Vα-C L and VβC H 1 2.Vα-C H 1 and Vβ-C L 3.VαCα-C L and VβCβ-C H 1 4.VαCαC H 1 and VβCβC L

[0208] In one embodiment of the present invention, the extracellular TCR domain is linked to immunoglobulin C via an optional peptide linker (L). H 1 and C L Linked to the domain, it promotes protein flexibility and facilitates optimal protein folding. 1.Vα-(L)-C L and Vβ-(L)-C H 1 2.Vα-(L)-C H 1 and Vβ-(L)-C L 3.VαCα-(L)-C L and VβCβ-(L)-C H 1 4.VαCα-(L)-C H 1 and VβCβ-(L)-C L

[0209] In another embodiment of the present invention, a tetramerization domain (TD), such as an NHR2 homotetramerization domain, is linked to the extracellular TCR α and β chains. H 1 or C LThe NHR2 domain is optionally linked to the C-terminus of either domain via a peptide linker. H 1 or C L The resulting tetravalent heterodimeric TCR protein can be arranged in the following configuration, where (L) is any peptide linker: 1.Vα-(L)-C L and Vβ-(L)-C H 1-(L)-TD 2.Vα-(L)-C H 1-(L)-TD and Vβ-(L)-C L 3.VαCα-(L)-C L and VβCβ-(L)-C H 1-(L)-TD 4.VαCα-(L)-C H 1-(L)-TD and VβCβ-(L)-C L 5.Vα-(L)-C L -(L)-TD and Vβ-(L)-C H 1 6.Vα-(L)-C H 1 and Vβ-(L)-C L -(L)-TD 7.VαCα-(L)-C L -(L)-TD and VβCβ-(L)-C H 1 8.VαCα-(L)-C H 1 and VβCβ-(L)-C L -(L)-TD

[0210] The sensitivity of soluble TCRs to their cognate pMHC can be improved by increasing their avidity. This is achieved by increasing the number of antigen-binding sites, facilitated by the tetramerization domain. This in turn increases the molecular weight of the protein molecule compared to monovalent soluble TCRs, thus prolonging their serum retention in the circulation. Increasing serum half-life also increases the likelihood that these molecules will interact with their cognate target antigen.

[0211] Tetravalent heterodimeric soluble TCR protein molecules can simultaneously bind one, two, three, or four pMHCs displayed on a single cell, or can simultaneously bind one, two, three, or four different cells displaying the cognate pMHC.

[0212] The TCR α and β chain sequences used in the present invention can be derived from known TCRs specific for a particular pMHC, or can be identified de novo by screening using techniques known in the art, such as phage display. Furthermore, TCR sequences are not limited to α and β chains in the present invention, but can also incorporate TCR δ and γ or ε chains and sequence variations thereof, cloned directly from human T cells or identified by directed evolution using recombinant DNA technology.

[0213] In another embodiment of the present invention, the tetravalent heterodimeric soluble TCR protein molecules are preferentially produced in mammalian cells for optimal production of soluble, stable, and correctly folded protein molecules.

[0214] Multimeric (e.g., tetrameric or octameric) or multivalent TCRs of the present invention can be expressed in cells, such as mammalian cells, using any suitable vector system. The pTT5 expression vector is an example of an expression system used to express multivalent soluble TCRs. The pTT5 expression system allows for high-level transient production of recombinant proteins in suspension-adapted HEK293 EBNA cells (Zhang et al. 2009). It contains an origin of replication (oriP) recognized by the viral protein Epstein-Barr nuclear antigen 1 (EBNA-1), which, together with host cell replication factors, mediates episomal replication of DNA plasmids, allowing for enhanced expression of recombinant proteins. Other suitable vector systems for mammalian cell expression known in the art and commercially available can be used in the present invention.

[0215] Tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced by transient expression of genes from expression vectors.

[0216] In another embodiment, tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced from engineered stable cell lines. The cell lines can be engineered to produce protein molecules using genome engineering techniques known in the art, in which the gene encoding the protein molecule is integrated into the genome of the host cell as either a single copy or multiple copies. The site of DNA integration can be a defined location within the host genome or can be randomly integrated to maximize expression of the desired protein molecule. Genome engineering techniques can include, but are not limited to, homologous recombination, transposon-mediated gene transfer such as the PiggyBac transposon system, site-specific recombinases including recombinase-mediated cassette exchange, endonuclease-mediated gene targeting such as CRISPR / Cas9, TALEN, zinc finger nucleases, meganucleases, and virus-mediated gene transfer such as lentivirus.

[0217] In yet another aspect of the invention, tetravalent heterodimeric soluble TCR protein molecules or other multimers are produced by overexpression in the cytoplasm of E. coli as inclusion bodies and, after purification by affinity chromatography, refolded in vitro to produce functional protein molecules capable of properly binding their cognate pMHC or antigen.

[0218] In another aspect of the present invention, expression of the tetravalent heterodimeric soluble TCR protein molecules or other multimers is not limited to mammalian or bacterial cells, but can also be expressed and produced in lower eukaryotic cells such as insect cells, plant cells, and yeast cells.

[0219] In another embodiment of the invention, heterodimeric soluble TCR molecules or other multimers are produced as octavalent protein complexes, e.g., with up to eight binding sites for their cognate pMHC (Figure 2). The multiple antigen binding sites allow the molecule to bind up to eight pMHC displayed on a single cell or to bind pMHC displayed on up to eight different cells, thereby creating a highly sensitive soluble TCR.

[0220] The heterodimeric soluble TCR portion of the molecule is C, which links either the α or β chain of the TCR itself. H 1 or C L The IgC domain is converted into a bivalent molecule by fusing an immunoglobulin hinge domain to the C-terminus of either domain. The hinge domain allows the connection of the two heavy chains, giving a structure similar to that of IgG. A tetramerization domain such as NHR2 is linked to the C-terminus of the hinge domain via an optional peptide linker. By linking the immunoglobulin hinge to the C-terminus and N-terminus of the IgCH1 or CL domain and the NHR2 domain, respectively, a monomer can be formed. 2 In this conformation, the assembly of two NHR2 monomers, called the hinge-like structure, is possible. We predict that structural constraints will likely prevent the two NHR2 domains from forming a homodimer by antiparallel binding unless a long, flexible linker is provided between the hinge and the NHR2 domain. H 1 or C L Tetramerization and hinge domain binding to heterodimeric soluble TCRs via domain immunoglobulins results in homotetramerization of NHR2 2 This allows for the stepwise self-assembly of octavalent soluble TCRs formed through the NHR2 monomer. 2 and homodimers 2This is achieved via an intermediate protein complex (Figure 2). The resulting octavalent heterodimeric soluble TCR protein molecule has excellent sensitivity to its cognate pMHC, thus offering the unique advantage of identifying unknown antigens or pMHCs without the need to affinity mature the TCR for its pMHC ligand, far beyond the affinity found in nature. This will be particularly useful for identifying pMHCs recognized by uncharacterized tumor-specific T cells and T cells involved in other diseases, such as autoimmune disorders. Many different configurations of octavalent heterodimeric soluble TCR protein molecules can be generated. Some examples are shown below. 1.Vα-(L)-C L and Vβ-(L)-C H 1-hinge-(L)-TD 2.Vα-(L)-C H 1-hinge-(L)-TD and Vβ-(L)-C L 3.Vα-Cα-(L)-C L and Vβ-Cβ-(L)-C H 1-hinge-(L)-TD 4.VαCα-(L)-C H 1-hinge-(L)-TD and VβCβ-(L)-C L 5.Vα-(L)-C L -(L)-TD and Vβ-(L)-C H 1-hinge 6.Vα-(L)-C H 1-hinge and Vβ-(L)-C L -(L)-TD 7.Vα-Cα-(L)-C L -(L)-TD and Vβ-Cβ(L)-C H 1-hinge 8.Vα-Cα-(L)-C H 1-hinge and Vβ-Cβ-(L)-C L -(L)-TD

[0221] In another embodiment of the present invention, the self-assembling multivalent proteins, preferentially tetravalent and octavalent heterodimeric soluble TCRs, are fused or conjugated to biologically active drug / effector molecules, thereby enabling these molecules to be targeted to desired cell populations, such as cancer cells, and specifically exert their therapeutic effects. The tumor-targeting ability of monoclonal antibodies to induce biologically active molecules, such as cytotoxic drugs, effector molecules such as cytotoxic drugs, toxins, or cytokines, is well established (Perez et al. 2014; Young et al. 2014). In a similar manner, the multivalent soluble TCR molecules outlined in this invention can also be fused to effector proteins and polypeptides or conjugated to cytotoxic agents. Examples of effector protein molecules suitable for use as fusion proteins with the multivalent protein complexes outlined in the present invention include, but are not limited to, IFNα, IFNβ, IFNγ, IL-2, IL-11, IL-13, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor [TNF]α, IL-7, IL-10, IL-12, IL-15, IL-21, CD40L, and TRAIL, costimulatory ligands B7.1 or B7.2, chemokines DC-CK1, SDF-1, fractalkine, lyphotactin, IP-10, Mig, MCAF, MIP-1α, MIP-1 / 3, IL-8, NAP-2, PF-4, and RANTES or active fragments thereof. Examples of toxic agents suitable for use as fusion proteins or conjugated to multivalent protein complexes according to the present invention include, but are not limited to, toxins such as diphtheria toxin, ricin, Pseudomonas exotoxin, etc., and cytotoxic drugs such as auristatins, maytansines, calicheamicins, anthracyclines, duocarmycins, pyrrolobenzodiazepines, etc. The cytotoxic drugs can be conjugated by selected linkers that are either non-cleavable or cleavable by proteases or are acid-labile.

[0222] To eliminate heterogeneity and improve conjugate stability, cytotoxic drugs can be site-specifically conjugated. This can be achieved by engineering specific cysteine ​​residues or by using enzymatic conjugation via glycotransferases and transglutaminases (Panowski et al. 2014).

[0223] In another aspect of the invention, the multivalent protein complex is covalently linked to a detectable molecule such as a fluorescent, radioactive, or electron transfer agent.

[0224] In another embodiment of the invention, an effector molecule (EM) is fused to a multivalent protein complex via the C-terminus of a tetramerization domain, such as NHR2, via an optional peptide linker. Fusion via the NHR2 domain can be configured to generate multivalent protein complexes in many different configurations. Examples of some protein configurations that can be generated using tetravalent heterodimeric soluble TCRs are provided below. 1.Vα-(L)-C L and Vβ-(L)-C H 1-(L)-TD-(L)-EM 2.Vα-(L)-C H 1-(L)-TD-EM and Vβ-(L)-C L 3.Vα-Cα-(L)-C L and Vβ-Cβ-(L)-C H 1-(L)-TD-(L)-EM 4.Vα-Cα-(L)-C H 1-(L)-TD-(L)-EM and Vβ-Cβ-(L)-C L 5.Vα-(L)-C L -(L)-TD-(L)-EM and Vβ-(L)-C H 1 6.Vα-(L)-C H 1 and Vβ-(L)-C L -(L)-TD-(L)-EM 7.Vα-Cα-(L)-C L -(L)-TD-(L)-EM and Vβ-Cβ-(L)-CH 1 8.Vα-Cα-(L)-C H 1 and Vβ-Cβ-(L)-C L -(L)-TD-(L)-EM

[0225] In another embodiment of the invention, an effector molecule (EM) is fused to the multivalent protein complex at the C-terminus of either the immunoglobulin CH1 or CL1 domain via an optional peptide linker. Fusion of the EM via the immunoglobulin domain can be configured to generate multivalent protein complexes in many different configurations. Examples of some protein configurations that can be generated using tetravalent heterodimeric soluble TCRs are shown below. 9.Vα-(L)-C L -(L)-EM and Vβ-(L)-C H 1-(L)-TD 10.Vα-(L)-C H 1-(L)-TD and Vβ-(L)-C L -(L)-EM 11.Vα-Cα-(L)-C L -(L)-EM and Vαβ-Cβ-(L)-C H 1-(L)-TD 12.Vα-Cα-(L)-C H 1-(L)-TD and Vβ-Cβ-(L)-C L -(L)-EM 13.Vα-(L)-C L -(L)-TD and Vβ-(L)-C H 1-(L)-EM 14.Vα-(L)-C H 1-(L)-EM and Vβ-(L)-C L -(L)-TD 15.Vα-Cα-(L)-C L -(L)-TD and Vβ-Cβ-(L)-C H 1--(L)-EM 16.Vα-Cα-(L)-C H 1-(L)-EM and Vβ-Cβ-(L)-C L -(L)-TD

[0226] In another embodiment of the present invention, an effector molecule (EM) is fused to the multivalent protein complex at the C-terminus of either the immunoglobulin CH1 or CL1 domain and at the C-terminus of the tetramerization domain (e.g., NHR2) via an optional peptide linker. This approach allows for the fusion of two effector molecules per TCR heterodimer complex. Fusion of the EM via the immunoglobulin domain and the tetramerization domain can be arranged to generate multivalent protein complexes in many different configurations. Examples of some protein configurations that can be generated using tetravalent heterodimeric soluble TCRs are shown below. 17.Vα-(L)-C L -(L)-EM and Vβ-(L)-C H 1-(L)-TD-(L)-EM 18.Vα-(L)-C H 1-(L)-TD-(L)-EM and Vβ-(L)-C L -(L)-EM 19.Vα-Cα-(L)-C L -(L)-EM and VβCβ-(L)-C H 1-(L)-TD-(L)-EM 20.Vα-Cα-(L)-C H 1-(L)-TD-(L)-EM and Vβ-Cβ-(L)-C L -(L)-EM 21.Vα-(L)-C L -(L)-TD-(L)-EM and Vβ-(L)-C H 1-(L)-EM 22.Vα-(L)-C H 1-(L)-EM and Vβ-(L)-C L -(L-)TD-(L)-EM 23.Vα-Cα-(L)-C L -(L)-TD-(L)-EM and Vβ-Cβ-(L)-C H 1-(L)-EM 24.Vα-(L)-C H 1-(L)-EM and Vβ-Cβ-(L)-C L -(L)-TD-(L)-EM

[0227] In another embodiment of the invention, the multivalent protein complex is fused to a protein tag to facilitate purification. Purification tags are known in the art and include, but are not limited to, the following tags: His, GST, TEV, MBP, Strep, FLAG.

[0228] Non-TCR multimers The present invention provides a unique method for assembling proteins in a soluble, multivalent format with the potential to bind multiple interaction domains or antigens. Proteins can be monomers, homodimers, heterodimers, or oligomers that have the potential to directly or indirectly participate preferentially in the immune system or modulate immune responses. Examples include, but are not limited to, TCRs, peptide MHC class I and class II, their antibody or antigen-binding portions, and binding proteins with alternative non-antibody protein scaffolds.

[0229] In another embodiment of the invention, the interacting domain or antigen can be any cell surface expressed or secreted protein, a peptide associated with MHC class I or II, or any protein associated with a pathogen, including viral and bacterial proteins.

[0230] The non-TCR multimer may be a multimer of antibodies or antibody fragments, for example, dAbs of Fabs. Examples of dAbs and Fabs according to the present invention include:

[0231] Examples of multivalent dAbs 25.VH-(L)-NHR2 26.VL(λ or κ)-(L)-NHR2 27.VH-(L)-NHR2-(L)-EM 28.VL(λ or κ)-(L)-NHR2-(L)-EM 29.VH-CH1-(L)-NHR2 30. VL(λ or κ)-CL-(L)-NHR2 31.VH-CH1-(L)-NHR2-(L)-EM 32.VL(λ or κ)-CL-(L)-NHR2-(L)-EM

[0232] Examples of multivalent Fabs 33. VH-CH1-(L)-NHR2 and VL(λ or κ)-CL 34. VL(λ or κ)-CL-(L)-NHR2 and VH-CH1 35. VH-CH1-hinge-(L)-NHR2 and VL(λ or κ)-CL 36. VL(λ or κ)-CL-hinge-(L)-NHR2 and VH-CH1 37. VH-CH1-(L)-NHR2-(L)-EM and VL(λ or κ)-CL 38. VL(λ or κ)-CL-(L)-NHR2-(L)-EM and VH-CH1 39. VH-CH1-hinge-(L)-NHR2-(L)-EM and VL(λ or κ)-CL 40. VL(λ or κ)-CL-hinge-(L)-NHR2-(L)-EM and VH-CH1 41. VH-CH1-(L)-NHR2 and VL(λ or κ)-CL-(L)-EM 42. VL(λ or κ)-CL-(L)-NHR2 and VH-CH1-(L)-EM 43. VH-CH1-hinge-(L)-NHR2 and VL(λ or κ)-CL-(L)-EM 44. VL(λ or κ)-CL-hinge-(L)-NHR2 and VH-CH1-(L)-EM

[0233] In the above example, (L) represents an optional peptide linker, while EM represents a biologically active agent or effector molecule such as a toxin, drug, cytokine, etc., including binding molecules such as antibodies, Fabs, and ScFvs.

[0234] The variable light chain can be either Vλ or Vκ.

[0235] In one aspect of the present invention, the assembled tetramerized protein molecule in one example can be a human pMHC for use in drug discovery using an animal drug discovery platform (e.g., rat, rabbit, chicken). In such a situation, the tetramerization domain is preferentially expressed and produced from a gene derived from the animal species for which it is intended. An example of such a drug discovery application would be the use of tetramerized human pMHC as an antigen for immunization, for example, in rats. When a rat is immunized with pMHC, the immune response is specifically directed against the human pMHC and not against the tetramerization domain of the protein complex.

[0236] Multivalent antibodies can be generated, for example, using single domain antibody sequences fused to an NHR2 multimerization domain.

[0237] In a related aspect of the present invention, the tetravalent protein can be a peptide used as a probe for molecular imaging of tumor antigens. The multivalent binding of such probes would have unique advantages over monovalent molecular probes, as it would enhance affinity, avidity, and retention time in vivo, which in turn would enhance in vivo tumor targeting.

[0238] The multimerization domain is the NHR2 domain described above.Preferably, the polypeptide is stabilized and / or soluble by using an Ig constant domain fused to the polypeptide, so that the fusion provides a tetramer of the polypeptide.An Ig hinge domain can be used to provide an octamer.

[0239] Use of multimers The multimeric TCR proteins according to the invention are useful in any application for which a soluble TCR protein is indicated. Particular advantages of the TCR proteins of the invention include increased avidity for a selected target and / or the ability to bind to multiple targets.

[0240] Therefore, in one embodiment, the multivalent heterodimeric soluble TCR protein molecules of the present invention can be used to selectively inhibit immune responses, such as the suppression of autoimmune responses. The multivalent, e.g., tetravalent, nature of these soluble protein molecules confers exquisite sensitivity and binding affinity for competing with antigen-specific interactions between T cells and antigen-presenting cells. This type of neutralizing effect can be therapeutically beneficial for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, Graves' disease, vasculitis, and type 1 diabetes.

[0241] Similarly, tetravalent heterodimeric soluble TCR protein molecules can be used to selectively suppress T cell recognition of specific transplantation antigens and self-antigens bound to target molecules, thus preventing tissue transplant rejection by inhibiting cell-cell interactions.

[0242] In another embodiment of the present invention, the tetravalent heterodimeric soluble TCR protein molecules can be used in clinical studies such as toxicology, infectious disease studies, neurological studies, behavioral and cognitive studies, reproduction, genetics and xenotransplantation studies.

[0243] Tetravalent heterodimeric soluble TCR protein molecules with improved sensitivity for cognate pMHC can be used for diagnostic purposes using biological samples obtained directly from human patients. The increased sensitivity of tetravalent heterodimeric soluble TCRs enables the detection of potentially disease-associated peptides displayed on MHC, which are naturally found expressed at low densities. These molecules can also be used for patient stratification to enroll patients in relevant clinical trials.

[0244] In another aspect of the present invention, the octavalent heterodimeric soluble TCR protein molecules may be used in pharmaceutical formulations for the treatment of various diseases.

[0245] In another aspect related to the present invention, the octavalent heterodimeric soluble TCR protein molecule may be used as a probe for tumor molecular imaging or may be prepared as a therapeutic protein.

[0246] Optionally, the polypeptide (first polypeptide) comprises or consists of a polypeptide disclosed in Table 8. In examples, the invention provides multimers (e.g., dimers, trimers, or tetramers, preferably tetramers) of such polypeptides. In examples, in Table 8, the multimerization domain (SAM) is a p53 domain (e.g., a human p53 domain). In examples, in Table 8, the multimerization domain (SAM) is an ortholog or homolog of a p53 domain (e.g., a human p53 domain).

[0247] Optionally, the invention provides a polypeptide (e.g., the polypeptide or the first polypeptide), the polypeptide comprising (in the N-terminal to C-terminal direction*): A. dAbs and Self-Associating Multimerization Domains (SAMs) B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, CH2, CH3 and SAM; G. A first scFv, CH2, CH3 and SAM; H.VH, CH1, CH2, CH3 and SAM, I.VL, CL, CH2, CH3 and SAM, J.dAb, SAM, CH2 and CH3, K.scFv, SAM, CH2 and CH3, L.VH, CH1, SAM, CH2 and CH3 M.VL, CL, SAM, CH2 and CH3, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. a first dAb, CH2, CH3, SAM and a second dAb; W. a first dAb, a CH2, a CH3, a SAM and a first scFv; X. a first dAb, a second dAb, a CH2, a CH3 and a SAM; Y. a first dAb, a second dAb, a CH2, a CH3, a SAM and a third dAb; Z. a first dAb, a second dAb, a CH2, a CH3, a SAM and a first scFv, or AA. 1st dAb, 2nd dAb, CH2, CH3, SAM, 3rd dAb and 4th dAb.

[0248] *Alternatively, components are written in the C-terminal to N-terminal direction.

[0249] In one embodiment, a polypeptide H, L, O, or Q is associated with a second polypeptide, and the second polypeptide comprises (from N-terminal to C-terminal) a VL and a CL, which is associated with a CH1 of the first polypeptide.

[0250] In one embodiment, polypeptide I, M, P, or R is associated with a second polypeptide, wherein the second polypeptide comprises (from N-terminal to C-terminal) a VH and a CH1, which is associated with a CL of the first polypeptide.

[0251] In an example, the polypeptide is encoded by a nucleotide sequence disclosed in Table 9. In an example, the polypeptide comprises or consists of an amino acid sequence disclosed in Table 10.

[0252] In examples, (i) the polypeptide comprises (in the N-terminal to C-terminal direction): A. dAbs and Self-Associating Multimerization Domains (SAMs) B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, CH2, CH3 and SAM; G. A first scFv, CH2, CH3 and SAM; H.VH, CH1, CH2, CH3 and SAM, I.VL, CL, CH2, CH3 and SAM, J.dAb, SAM, CH2 and CH3, K.scFv, SAM, CH2 and CH3, L.VH, CH1, SAM, CH2 and CH3 M.VL, CL, SAM, CH2 and CH3, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. a first dAb, CH2, CH3, SAM and a second dAb; W. a first dAb, a CH2, a CH3, a SAM and a first scFv; X. a first dAb, a second dAb, a CH2, a CH3 and a SAM; Y. a first dAb, a second dAb, a CH2, a CH3, a SAM and a third dAb; Z. a first dAb, a second dAb, a CH2, a CH3, a SAM and a first scFv, or AA. comprising a first dAb, a second dAb, a CH2, a CH3, a SAM, a third dAb and a fourth dAb; or (ii) the polypeptide is (in the C-terminal to N-terminal direction): A. dAbs and Self-Associating Multimerization Domains (SAMs) B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, CH2, CH3 and SAM; G. A first scFv, CH2, CH3 and SAM; H.VH, CH1, CH2, CH3 and SAM, I.VL, CL, CH2, CH3 and SAM, J.dAb, SAM, CH2 and CH3, K.scFv, SAM, CH2 and CH3, L.VH, CH1, SAM, CH2 and CH3 M.VL, CL, SAM, CH2 and CH3, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. a first dAb, CH2, CH3, SAM and a second dAb; W. a first dAb, a CH2, a CH3, a SAM and a first scFv; X. a first dAb, a second dAb, a CH2, a CH3 and a SAM; Y. a first dAb, a second dAb, a CH2, a CH3, a SAM and a third dAb; Z. a first dAb, a second dAb, a CH2, a CH3, a SAM and a first scFv, or AA. Includes a first dAb, a second dAb, CH2, CH3, SAM, a third dAb and a fourth dAb.

[0253] Optionally, the SAM is a tetramerization domain, eg, p53 TD.

[0254] In examples, the first, second, third (if present) and fourth (if present) dAbs have the same antigen binding specificity. In examples, the first, second, third (if present) and fourth (if present) dAbs have the same different antigen binding specificities.

[0255] In examples, the first and second scFvs have the same antigen binding specificity. In examples, the first and second scFvs have the same different antigen binding specificity.

[0256] In examples, the first dAb, the second dAb, and the first scFv have the same antigen binding specificity. In examples, the first dAb, the second dAb, and the first scFv have the same different antigen binding specificity.

[0257] Where a dAb is provided in the polypeptide herein, alternatively any different type of antigen binding domain may be provided instead, such as an scFv or Fab or a non-Ig binding domain (e.g. an affibody, avimer or fibronectin domain).

[0258] Where an scFv is provided in the polypeptide herein, alternatively any different type of antigen binding domain may be provided instead, such as a dAb or Fab or a non-Ig binding domain (e.g., an affibody, avimer or fibronectin domain).

[0259] Where a Fab is provided in the polypeptide herein, alternatively any different type of antigen binding domain may be provided instead, such as an scFv or a dAb or a non-Ig binding domain (e.g., an affibody, avimer or fibronectin domain).

[0260] Each antigen can be any antigen disclosed herein.

[0261] In an example, CH1 (if present), CH2, and CH3 are human Ig CH1, CH2, CH3, e.g., IgG1 CH1, CH2, and CH3. In an example, CH2 comprises a CH2 domain, CH3 comprises a CH3 domain, and CH2 comprises a hinge amino acid sequence. In this example, CH2 comprises (from the N-terminus to the C-terminus) a hinge amino acid sequence and a CH2 domain. In examples, the hinge amino acid sequence is (i) a complete hinge, (ii) a hinge amino acid sequence that is not functional for dimerizing the polypeptide with another such polypeptide, (iii) a hinge amino acid sequence that lacks a hinge core comprising the amino acid motif CXXC (and optionally also lacks an upper hinge amino acid sequence), or (iv) an upper hinge fused to a lower hinge but lacking a core hinge comprising the amino acid motif CXXC, or (v) a lower hinge but lacking a hinge core comprising the amino acid motif CXXC (and optionally also lacking an upper hinge amino acid sequence). Examples of upper, core, and lower hinge sequences are disclosed in Table 12. In examples, CH2 lacks a functional hinge region, i.e., the hinge region is non-functional for dimerizing the polypeptide with another such polypeptide. In examples, CH2 lacks a hinge region. In examples, CH2 lacks a complete hinge region sequence. In the example, CH2 lacks the core hinge region sequence.

[0262] In an example, CH2 includes (from the N-terminus to the C-terminus) any upper hinge region, lower hinge region, and CH2 domain, and CH2 (and the polypeptide) lacks a core hinge region that is functional for dimerizing the polypeptide with another such polypeptide.

[0263] In examples, CH2 includes (from N- to C-terminus) an optional upper hinge region, a lower hinge region, and a CH2 domain, and the CH2 (and polypeptide) lacks the core hinge region amino acid sequence CXXC, where X is any amino acid (optionally, each amino acid X is selected from P, R, and S).

[0264] In examples, CH2 comprises (from the N-terminus to the C-terminus) an amino acid and CH2 domain selected from SEQ ID NOs: 163-178, and the CH2 (and polypeptide) lacks a core hinge region amino acid sequence CXXC, where X is any amino acid (optionally, each amino acid X is selected from P, R, and S).

[0265] In one embodiment, the amino acid sequence of the core hinge region is selected from SEQ ID NOs: 180 to 182. In an embodiment, CH2 (polypeptide) lacks the amino acid sequence of SEQ ID NOs: 183 to 187.

[0266] In embodiments, the CH2 domain is a human IgG1 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence CPPC (SEQ ID NO: 180) of the core hinge region. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) of the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) of the core hinge region.

[0267] In embodiments, the CH2 domain is a human IgG2 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence ERKCCVE (SEQ ID NO: 184) of the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) of the core hinge region.

[0268] In embodiments, the CH2 domain is a human IgG3 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively, the CH2 (and polypeptide) lacks the upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181).

[0269] In embodiments, the CH2 domain is a human IgG4 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence ESKYGPP (SEQ ID NO: 187) of the upper hinge region and the amino acid sequence CPSC (SEQ ID NO: 182) of the core hinge region.

[0270] In examples, CH2 of the polypeptides herein lacks the amino acid sequence of the core hinge (and optionally also the upper hinge). In examples, CH2 of the polypeptides herein lacks the CXXC amino acid sequence of the core hinge, where X is any amino acid, preferably P, R, or S, most preferably P. In examples, CH2 comprises the APELLGGPSV amino acid sequence or the PAPELLGGPSV amino acid sequence. In examples, CH2 comprises the APPVAGPSV amino acid sequence or the PAPPVAGPSV amino acid sequence. In examples, CH2 comprises the APEFLGGPSV amino acid sequence or the PAPEFLGGPSV amino acid sequence.

[0271] In examples, the CH2 and CH3 of the polypeptides herein are human IgG1 CH2 and CH3 domains, and the CH2 lacks the amino acid sequence of the core hinge (and optionally also the upper hinge), e.g., the CH2 lacks the CPPC sequence. In examples, the CH2 comprises the amino acid sequence APELLGGPSV, or the amino acid sequence EPKSCDKTHT[P]APELLGGPSV, where the bracketed P is optional.

[0272] In examples, the CH2 and CH3 of the polypeptides herein are human IgG2 CH2 and CH3 domains, and the CH2 lacks the amino acid sequence of the core hinge (and optionally also the upper hinge), e.g., the CH2 lacks the CPPC sequence. In examples, the CH2 comprises the APPVAGPSV amino acid sequence, or the ERKCCVE[P]APPVAGPSV amino acid sequence, where the bracketed P is optional.

[0273] In examples, the CH2 and CH3 of the polypeptides herein are human IgG3 CH2 and CH3 domains, and the CH2 lacks the amino acid sequence of the core hinge (and optionally also the upper hinge), e.g., the CH2 lacks the CPRC sequence. In examples, the CH2 comprises the amino acid sequence APELLGGPSV, or the amino acid sequence ELKTPLGDTTHT[P]APELLGGPSV, where the bracketed P is optional. In examples, the CH2 comprises the amino acid sequence EPKSCDTPPP[P]APELLGGPSV, where the bracketed P is optional.

[0274] In examples, the CH2 and CH3 of the polypeptides herein are human IgG4 CH2 and CH3 domains, and the CH2 lacks the amino acid sequence of the core hinge (and optionally also the upper hinge), e.g., the CH2 lacks the CPSC sequence. In examples, the CH2 comprises the APEFLGGPSV amino acid sequence, or the ESKYGPP[P]APEFLGGPSV amino acid sequence, where the bracketed P is optional.

[0275] Where a polypeptide comprises a V-CH1, a CH2 may also be present, optionally lacking the core hinge region (or at least a sequence selected from CXXC and SEQ ID NOs: 180-182 as disclosed herein), and optionally lacking the upper and / or lower hinge regions to prevent F(ab')2 formation.

[0276] Aspects: By way of example, the present invention provides the following aspects, some of which are illustrated herein. 1. (N-terminal to C-terminal or C-terminal to N-terminal) (a) an immunoglobulin superfamily domain; (b) an optional linker; and (c) a self-associating multimerization domain (SAM) (optionally a self-associating tetramerization domain (TD)), a polypeptide comprising: In examples, each linker comprises (or up to comprises or consists of) 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acids. In another approach, the domain in (a) is a non-Ig domain or comprises a non-Ig scaffold.

[0277] In another method herein, instead of using a TD or a copy of a TD, in one embodiment, any other self-associating multimerization domain (SAM) can be used. In examples, the SAM (e.g., TD) is a human, dog, cat, horse, monkey (e.g., cynomolgus monkey), rodent (e.g., mouse or rat), rabbit, bird (e.g., chicken) or fish SAM (e.g., or TD).

[0278] Optionally, the domain of (a) is capable of specifically binding to an antigen selected from PD-L1, PD-1, 4-1BB, CTLA-4, 4-1BB, CD28, TNFalpha, IL17 (e.g., IL17A), CD38, VEGF-A, EGFR, IL-6, IL-4, IL-6R, IL-4R (e.g., IL-4Ra), OX40, OX40L, TIM-3, CD20, GITR, VISTA, ICOS, death receptor 5 (DR5), LAG-3, CD40, CD40L, CD27, HVEM, KRAS, hemagglutinin, transferrin receptor 1, amyloid beta, BACE1, Tau, TDP43, SOD1, alpha synuclein, and CD3.

[0279] In an example, the antigen is a peptide-MHC.

[0280] In some embodiments (e.g., some embodiments of aspect 16 or 17 below), the polypeptide comprises at least two binding moieties, e.g., two dAbs, two scFvs, or a dAb and an scFv. In examples, the binding moieties bind to the same antigen (e.g., an antigen disclosed herein or in the immediately preceding paragraph herein). In examples, the moieties bind to different antigens (e.g., an antigen disclosed herein or in the immediately preceding paragraph herein). For example, in aspect 16B, D, E, or F, the variable domains or scFvs can specifically bind to the same or different antigens selected from TNFalpha, CD38, IL17a, CD20, PD-1, PD-L1, CTLA-4, and 4-1BB. For example, one of the moieties binds to TNF-alpha and the other binds to IL17a; one of the moieties binds to PD-1 and the other binds to 4-1BB; or one of the moieties binds to PD-L1 and the other binds to 4-1BB; one of the moieties binds to PD-1 and the other binds to CTLA-4; or one of the moieties binds to PD-L1 and the other binds to CTLA-4. 2. The polypeptide of embodiment 1, wherein the (a) domain is an antibody variable domain.

[0281] For example, the variable domain herein is a VH (e.g., composed of an scFv or Fab polypeptide chain). In another example, it is a VHH (e.g., composed of an scFv or Fab polypeptide chain). In another example, it is a humanized VH, humanized VHH, or human VH (e.g., composed of an scFv or Fab polypeptide chain). In another example, it is a VL (e.g., composed of an scFv or Fab polypeptide chain). In another example, it is a Vκ. In another example, it is a Vλ.

[0282] In another example, the domain in (a) is a TCR variable domain (e.g., TCRα, TCRβ, TCRγ, or TCRδ).

[0283] An example of an immunoglobulin superfamily domain is an antibody single variable domain (dAb). 3. The polypeptide according to any one of aspects 1 to 2, wherein the variable domains of (a) are selected from antibody single variable domains, VH and VL, or the domains are constituted by scFv.

[0284] In examples, the single variable domain herein is a human or humanized dAb or nanobody, or is a camelid VHH domain.

[0285] In the example, domain (a) is composed of a single-chain TCR (scTCR). 4. The polypeptide of any one of aspects 1 to 3, wherein (a) is directly linked to (c), or (b) is directly linked to (a) and (c). 5. The polypeptide of any one of aspects 1 to 4, comprising (in N-terminal to C-terminal direction) a SAM, (d) an optional second linker, and (e) a second immunoglobulin superfamily domain. 6. The polypeptide of embodiment 5, wherein the second domain is selected from an antibody single variable domain, a VH and a VL, or wherein the domain is constituted by an scFv.

[0286] In examples, the single variable domain is a human or humanized dAb or nanobody, or is a camelid VHH domain. 7. The polypeptide of embodiment 5, wherein the second domain is an antibody single variable domain or an antibody constant domain. 8. The polypeptide of aspect 7, wherein the constant domain is constituted by an antibody Fc region. 9. The polypeptide of any one of aspects 5 to 8, wherein (c) is directly linked to (e), or (d) is directly linked to (c) and (e). 10. The polypeptide according to any one of aspects 1 to 9, wherein the TD is a p53, p63 or p73 TD or a homologue or orthologue thereof, or wherein the TD is an NHR2 TD or a homologue or orthologue thereof.

[0287] Optionally, the TD herein is a TD of a protein disclosed in Table 2. 11. The polypeptide of any one of aspects 1-10, wherein TD comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or 126. 12. The polypeptide of any one of aspects 1 to 11, comprising (f) an antibody variable domain, an antibody constant region, or an antibody Fc region between (a) and (c). 13. The polypeptide of aspect 12, wherein (f) (i) comprises an antibody CH1 constant domain, or (ii) an antibody Fc region (i.e., comprising CH2-CH3). 14. The polypeptide of embodiment 13, wherein the polypeptide is associated with a second polypeptide, and (iii) the second polypeptide comprises an antibody CL constant domain paired with the CH1 domain of (i), or (iv) the second polypeptide comprises an Fc region of a second antibody paired with the Fc region of (ii). 15. The polypeptide of aspect 12, wherein the variable domain of (f) is an antibody single variable domain. 16. A polypeptide (first polypeptide) comprising (in the N-terminal to C-terminal direction): A. A first antibody single variable domain (dAb), an optional linker, and the SAM; B. A first antibody single variable domain, an optional linker, the SAM and a second antibody single variable domain; C. A first scFv, an optional linker, and the SAM; D. A first scFv, an optional linker, the SAM and a second scFv; E. A first antibody single variable domain, an optional linker, the SAM and a first scFv; F. A first scFv, an optional linker, the SAM and a first antibody single variable domain; G. a first antibody variable domain, an optional first linker, a first antibody constant domain, an optional second linker, and the SAM; H. the SAM, an optional linker, and a first antibody single variable domain; I. the SAM, an optional linker and a first scFv; J. the SAM, an optional linker, a first antibody constant domain, a second optional linker and a first antibody variable domain; or K. The polypeptide of any one of embodiments 1 to 15, comprising or consisting of the SAM, an optional linker, a first antibody variable domain, a second optional linker, and a first antibody constant domain.

[0288] Optionally, each variable domain is a VH or VL (eg, Vκ or Vλ).

[0289] Optionally, each domain of the polypeptides herein is a human domain. Optionally, each domain of the polypeptides herein is a human or humanized domain. 17. (i) the polypeptide comprises (in the N-terminal to C-terminal direction): A. dAbs and Self-Associating Multimerization Domains (SAMs) B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, CH2, CH3 and SAM; G. A first scFv, CH2, CH3 and SAM; H.VH, CH1, CH2, CH3 and SAM, I.VL, CL, CH2, CH3 and SAM, J.dAb, SAM, CH2 and CH3, K.scFv, SAM, CH2 and CH3, L.VH, CH1, SAM, CH2 and CH3 M.VL, CL, SAM, CH2 and CH3, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. a first dAb, CH2, CH3, SAM and a second dAb; W. a first dAb, a CH2, a CH3, a SAM and a first scFv; X. a first dAb, a second dAb, a CH2, a CH3 and a SAM; Y. a first dAb, a second dAb, a CH2, a CH3, a SAM and a third dAb; Z. a first dAb, a second dAb, a CH2, a CH3, a SAM and a first scFv, or AA. comprising a first dAb, a second dAb, a CH2, a CH3, a SAM, a third dAb and a fourth dAb; or (ii) the polypeptide (in the C-terminal to N-terminal direction) A. dAbs and Self-Associating Multimerization Domains (SAMs) B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, CH2, CH3 and SAM; G. A first scFv, CH2, CH3 and SAM; H.VH, CH1, CH2, CH3 and SAM, I.VL, CL, CH2, CH3 and SAM, J.dAb, SAM, CH2 and CH3, K.scFv, SAM, CH2 and CH3, L.VH, CH1, SAM, CH2 and CH3 M.VL, CL, SAM, CH2 and CH3, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. a first dAb, CH2, CH3, SAM and a second dAb; W. a first dAb, a CH2, a CH3, a SAM and a first scFv; X. a first dAb, a second dAb, a CH2, a CH3 and a SAM; Y. a first dAb, a second dAb, a CH2, a CH3, a SAM and a third dAb; Z. a first dAb, a second dAb, a CH2, a CH3, a SAM and a first scFv, or AA. The polypeptide of any one of embodiments 1-15, comprising a first dAb, a second dAb, a CH2, a CH3, a SAM, a third dAb, and a fourth dAb.

[0290] Thus, in some embodiments, the polypeptide comprises an antibody Fc region, where the Fc comprises a CH2 and a CH3 domain. 18. The polypeptide of embodiment 16B, 16D, (i)17B, (i)17D, (i)17N, (i)17S, (i)17T, (i)17U, (i)17X, (i)17Y, (i)17Z, (i)17AA, (ii)17B, (ii)17D, (ii)17N, (ii)17S, (ii)17T, (ii)17U, (ii)17X, (ii)17Y, (ii)17Z or (ii)17AA, wherein the single variable domains (dAbs) are identical or the scFvs are identical. 19. The polypeptide of embodiment 16B, 16D, (i)17B, (i)17D, (i)17N, (i)17S, (i)17T, (i)17U, (i)17X, (i)17Y, (i)17Z, (i)17AA, (ii)17B, (ii)17D, (ii)17N, (ii)17S, (ii)17T, (ii)17U, (ii)17X, (ii)17Y, (ii)17Z or (ii)17AA, wherein the single variable domains are different or the scFvs are different. 20. (i) the first variable domain is a VH domain and the first constant domain is a CH1 domain, optionally, the polypeptide is associated with a second polypeptide, the second polypeptide comprising an antibody CL constant domain paired with the CH1 domain; (ii) the first variable domain is a VH domain and the first constant domain is a CL domain, optionally, the polypeptide is associated with a second polypeptide, the second polypeptide comprising an antibody CH1 constant domain paired with the CL domain; (iii) the first variable domain is a VL domain and the first constant domain is a CH1 domain, optionally, the polypeptide is associated with a second polypeptide; and the second polypeptide comprises an antibody CL constant domain paired with a CHI domain, or (iv) the first variable domain is a VL domain and the first constant domain is a CL domain, optionally wherein the polypeptide is associated with a second polypeptide, and the second polypeptide comprises an antibody CHI constant domain paired with a CL domain. 21. The polypeptide of any one of aspects 16 to 20, comprising an antibody Fc region or a further antibody single variable domain between (v) the first variable domain or scFv and (vi) the SAM, wherein the Fc comprises CH2 and CH3.

[0291] This further variable domain may be different to the first single variable domain or may have a target binding specificity which is different to that of the first single variable domain or scFv. 22. The polypeptide of any one of aspects 16 to 21, comprising an antibody Fc region or antibody single variable domain between (vii) the SAM and (viii) the most C-terminal variable domain, wherein Fc comprises CH2 and CH3. 23. The polypeptide of any one of aspects 16-22, comprising, in N-terminal to C-terminal direction, an antibody Fc region and an N-terminal most variable domain or scFv, wherein the Fc comprises CH2 and CH3. 24. The polypeptide of any one of aspects 16-23, comprising, in N-terminal to C-terminal direction, a SAM and an antibody Fc region, wherein Fc comprises CH2 and CH3. 25. The polypeptide according to any one of aspects 17 to 24, wherein CH2 lacks the amino acid sequence CXXC or an amino acid sequence selected from SEQ ID NOs: 180 to 182, and optionally lacks the amino acid sequence SEQ ID NOs: 183 to 187.

[0292] In examples, CH2 is a CH2' disclosed herein. Optionally, CH2 comprises (from N-terminal to C-terminal) an optional upper hinge region, a lower hinge region, and a CH2 domain, wherein the CH2 (and polypeptide) lacks a core hinge region that is functional for dimerizing the polypeptide with another such polypeptide. Optionally, CH2 comprises (from N-terminal to C-terminal) an optional upper hinge region, a lower hinge region, and a CH2 domain, wherein the CH2 (and polypeptide) lacks the core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally, each amino acid X is selected from P, R, and S). Optionally, CH2 comprises (from N-terminal to C-terminal) an amino acid and CH2 domain selected from SEQ ID NOs: 163-178, wherein the CH2 (and polypeptide) lacks the core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally, each amino acid X is selected from P, R, and S). In one embodiment, the amino acid sequence of the core hinge region is selected from SEQ ID NOs: 180-182. In embodiments, the CH2 (polypeptide) lacks the amino acid sequence of SEQ ID NOs: 183-187. In embodiments, the CH2 domain is a human IgG1 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence CPPC (SEQ ID NO: 180) in the core hinge region. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) in the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) in the core hinge region. In embodiments, the CH2 domain is a human IgG2 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3, respectively. Optionally, CH2 (and the polypeptide) lacks the amino acid sequence ERKCCVE (SEQ ID NO: 184) of the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) of the core hinge region.In embodiments, the CH2 domain is a human IgG3 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lack the upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively, the CH2 (and polypeptide) lack the upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181). In embodiments, the CH2 domain is a human IgG4 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3, respectively. Optionally, CH2 (and the polypeptide) lacks the amino acid sequence ESKYGPP (SEQ ID NO: 187) of the upper hinge region and the amino acid sequence CPSC (SEQ ID NO: 182) of the core hinge region. 26. The or each linker is (G4S) n 26. The polypeptide of any one of embodiments 1 to 25, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0293] In the example, n is 3. In the example, n is 4. In the example, n is 5. 27. The polypeptide of any one of aspects 1-26, wherein each domain and SAM is a human domain and SAM, respectively. 28. The polypeptide of any one of aspects 1 to 27, wherein each variable domain or scFv is capable of binding to an antigen.

[0294] In some instances, the binding is antagonistic to the antigen. In other instances, the binding is agonistic to the antigen. 29. The polypeptide of any one of aspects 1-28, wherein the polypeptide comprises binding specificities for two or more antigens, optionally 2, 3 or 4 different antigens.

[0295] For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (e.g., a dAb or scFv) and at least one anti-4-1BB binding domain. For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (e.g., a dAb or scFv) and at least one anti-PD-L1 binding domain. For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (e.g., a dAb or scFv) and at least one anti-PD-1 binding domain. For example, the polypeptide comprises at least one anti-TNF alpha binding domain (e.g., a dAb or scFv) and at least one anti-IL-17A binding domain.

[0296] For example, the polypeptide comprises a first antigen-binding domain (e.g., the VH, VL, VHH, dAb, scFv, or Fab variable region) at the N-terminus of the SAM and a second antigen-binding domain (e.g., the VH, VL, VHH, dAb, scFv, or Fab variable region) at the C-terminus of the SAM. In examples, the polypeptide comprises a third antigen-binding domain (e.g., the VH, VL, VHH, dAb, scFv, or Fab variable region) at the N-terminus of the SAM (e.g., also at the N-terminus of the first domain, or between the first domain and the SAM), and optionally, the polypeptide comprises a fourth antigen-binding domain (e.g., the VH, VL, VHH, dAb, scFv, or Fab variable region) at the C-terminus of the SAM (e.g., also at the C-terminus of the second domain, or between the second domain and the SAM).

[0297] In embodiments, the first domain is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3), and the second binding site is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In examples, the domains have the same antigen binding specificity. In examples, the domains have the same epitope binding specificity. In examples, the domains have different antigen binding specificities. In examples, the domains have different epitope binding specificities for the same antigen. In examples, the domains bind to TNF-alpha. In examples, the domains bind to CD20. In examples, the domains bind to PD-1. In examples, the domains bind to PD-L1. In examples, the domains bind to CTLA-4.

[0298] In embodiments, the first domain can specifically bind to 4-1BB, PD-1, or PD-L1, and the second binding site can specifically bind to CTLA-4. In embodiments, the second domain can specifically bind to 4-1BB, PD-1, or PD-L1, and the first binding site can specifically bind to CTLA-4. In examples, the first domain can specifically bind to 4-1BB, the second binding site can specifically bind to CTLA-4, the third binding site can specifically bind to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3), and the fourth binding site can specifically bind to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In an example, the first domain can specifically bind to PD-1, the second binding site can specifically bind to CTLA-4, the third binding site can specifically bind to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3), and the fourth binding site can specifically bind to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In examples, the first domain can specifically bind to PD-L1, the second binding site can specifically bind to CTLA-4, the third binding site can specifically bind to CTLA-4, PD-L1, CD3 or CD28, and the fourth binding site can specifically bind to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3).

[0299] In embodiments, the first domain is capable of specifically binding to TNF-alpha and the second binding site is capable of specifically binding to IL-17 (e.g., IL-17A). In embodiments, the second domain is capable of specifically binding to TNF-alpha and the first binding site is capable of specifically binding to IL-17 (e.g., IL-17A).

[0300] In examples, the polypeptide comprises a cytokine, e.g., IL-2, IL-15, or IL-21. In examples, the cytokine is a truncated cytokine, e.g., truncated IL-2, IL-15, or IL-21. In examples, the cytokine is C-terminal of the SAM (e.g., the C-terminal end of the most C-terminal of the antigen-binding domain). In examples, the cytokine is N-terminal of the SAM (e.g., the N-terminal end of the most N-terminal of the antigen-binding domain). In one embodiment of these examples, the first domain is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In an embodiment of these examples, the first domain is capable of specifically binding to 4-1BB, PD-1, PD-L1, or CTLA-4. In one embodiment of these examples, the second domain is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In one embodiment of these examples, the second domain is capable of specifically binding to 4-1BB, PD-1, PD-L1, or CTLA-4. In one embodiment of these examples, the third domain is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In one embodiment of these examples, the third domain is capable of specifically binding to 4-1BB, PD-1, PD-L1, or CTLA-4. In one embodiment of these examples, the fourth domain is capable of specifically binding to an immune checkpoint or T cell costimulatory antigen (e.g., selected from OX40, GITR, VISTA, CD40, CD28, LAG3, and TIM-3). In these example embodiments, the fourth domain is capable of specifically binding to 4-1BB, PD-1, PD-L1, or CTLA-4. 30. A multimer (optionally a tetramer) of a polypeptide according to any one of aspects 1 to 29.

[0301] In an example, the multimer is a polypeptide dimer.

[0302] In an example, the multimer is a polypeptide trimer.

[0303] In an example, the multimer is a polypeptide tetramer. 31. The polypeptide or multimer according to any one of aspects 1 to 30, which comprises eukaryotic glycosylation. 32. The polypeptide or multimer according to aspect 31, wherein the cell is a HEK293, CHO or Cos cell. 33. A polypeptide or multimer according to any one of aspects 1 to 32 for medical use. 34. A pharmaceutical composition comprising a polypeptide or multimer according to any one of aspects 1 to 33. 35. A nucleic acid encoding a polypeptide according to any one of aspects 1 to 29 and 31 to 33. 36. A eukaryotic cell or vector comprising the nucleic acid of embodiment 35. 37. A method for binding multiple copies of an antigen, the method comprising combining the copies with a multimer according to any one of aspects 30 to 33, wherein the copies are bound by polypeptides of the multimer, and optionally, the method comprises isolating the multimers bound to the antigen copies. 38. The method of embodiment 37, wherein the method is a diagnostic method for detecting the presence of a substance in a sample, the substance comprising an antigen, and the method comprises providing a sample (e.g., a body fluid, a food, a food ingredient, a beverage, a beverage ingredient, soil, or a forensic sample), mixing the sample with a multimer of any one of embodiments 30 to 33, and detecting binding of the multimer to the antigen in the sample.

[0304] In examples, the bodily fluid is a sample of blood, saliva, semen, or urine.

[0305] In examples, the method is for pregnancy testing or diagnosing a disease or condition in a subject from whom a sample has previously been obtained. 39. A method of treating or reducing the risk of a disease or condition in a human or animal subject, the method comprising administering to the subject a composition according to aspect 34, wherein a multimer comprised in the composition specifically binds to a target antigen in the subject, and said binding mediates the treatment or reduction of the risk. 40. The method of embodiment 39, wherein the antigen is an immune checkpoint or T cell co-stimulatory antigen (e.g., PD-L1, PD-1, or CTLA4), or the antigen is TNFalpha or IL-17A. 41. The method of embodiment 39, wherein the antigen mediates the disease or condition of the subject, and optionally, the binding antagonizes the antigen.

[0306] In another embodiment, the binding is antigen-driven. 42. A composition comprising a plurality of polypeptides according to any one of aspects 1 to 29 and 31 to 33, wherein at least 90% of the polypeptides are constituted by tetramers of the polypeptides.

[0307] Optionally, at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the polypeptide is made up of tetramers of the polypeptide. 43. The composition of aspect 42, wherein at least 98% of the polypeptide is composed of tetramers of the polypeptide. 44. The composition of aspect 42 or 43, wherein the remaining (i.e., the remainder of 100% of the polypeptides) polypeptides are selected from one or more of polypeptide monomers, dimers, and trimers. 45. A method for producing a composition comprising a plurality of polypeptides according to any one of aspects 1 to 29 and 31 to 33 (optionally a composition according to any one of aspects 42 to 44), the method comprising providing a eukaryotic host cell according to aspect 34, culturing the host cell, allowing expression and secretion from the cell of a tetramer of the polypeptides, and optionally isolating or purifying the tetramer. Any of these aspects may be combined with any of the other disclosures herein, for example, any of the sections or paragraphs.

[0308] FC-containing polypeptides and multimers The present invention also provides polypeptides and multimers that contain antibody Fc region.This is useful for utilizing FcRn recycling when administered to subjects such as humans or animals, which can contribute to desirable half-life in vivo.Fc region is also useful for providing Fc effector function.For example, IgG1 Fc can be useful when multimers are used to treat cancer, or when cell killing is desired, for example, by ADCC.

[0309] To this end, the present invention provides: Provided is a polypeptide comprising an antibody Fc region, the Fc region comprising an antibody CH2 and an antibody CH3, and a self-associating multimerization domain (SAM), wherein CH2 comprises an antibody hinge sequence and lacks a core hinge region.

[0310] In one embodiment, the polypeptide comprises an epitope-binding site, e.g., an antibody VH single variable domain or antibody VH / VL pair, that binds to the epitope. Additionally or alternatively, the polypeptide comprises an epitope cognate to the antibody. This is useful, for example, when the multimer is contacted with a sample containing the antibody (e.g., for medical applications as disclosed herein), because the polypeptide can form multimers that bind copies of the antibody. In this way, for example, the multimer can be used in diagnostic or testing methods to determine the presence and / or amount (or relative amount) of the antibody in a sample. Because the multimer provides multiple copies of the epitope (at least one for each polypeptide comprised in the multimer), this is useful for binding many copies of an antibody that may be present in relatively small amounts in a sample, thereby improving the chances of detecting (or amplifying) a positive signal indicating the presence of the antibody. This can therefore increase the sensitivity of the assay, allowing relatively rare antibodies in a sample to be detected.

[0311] Optionally, CH2 lacks (i) a core hinge CXXC amino acid sequence, where X is any amino acid or each amino acid X is selected from P, R, and S, and / or (ii) an upper hinge amino acid sequence. CH2 lacks (i) a core hinge CXXC amino acid sequence, where X is any amino acid or each amino acid X is selected from P, R, and S, and the Fc does not directly pair with another Fc.

[0312] Optionally, the CXXC sequence is selected from SEQ ID NOs: 180-182, or CH2 lacks the amino acid sequence of SEQ ID NOs: 183-187.

[0313] Optionally, CH2 may be A. The amino acid sequence APELLGGPSV (SEQ ID NO: 163), or PAPELLGGPSV (SEQ ID NO: 164); B. The amino acid sequence APPVAGPSV (SEQ ID NO: 165), or PAPPVAGPSV (SEQ ID NO: 166); C. The amino acid sequence APEFLGGPSV (SEQ ID NO: 175), or PAPEFLGGPSV (SEQ ID NO: 176); D. The amino acid sequence EPKSCDKTHT[P]APELLGGPSV (SEQ ID NO: 167, or 168) (the P in parentheses is optional); E. The amino acid sequence ERKCCVE[P]APPVAGPSV (SEQ ID NO: 169, or 170) (the P in parentheses is optional); F. The amino acid sequence ELKTPLGDTTHT[P]APELLGGPSV (SEQ ID NO: 171, or 172) (the P in parentheses is optional); G. The amino acid sequence EPKSCDTPPP[P]APELLGGPSV (SEQ ID NO: 173, or 174) (the P in brackets is optional), or H. Contains the amino acid sequence ESKYGPP[P]APEFLGGPSV (SEQ ID NO: 177 or 178) (the P in parentheses is optional).

[0314] Optionally, CH2 comprises (from N-terminal to C-terminal) an optional upper hinge region, a lower hinge region, and a CH2 domain, wherein the CH2 (and polypeptide) lacks a core hinge region that is functional for dimerizing the polypeptide with another such polypeptide. Optionally, CH2 comprises (from N-terminal to C-terminal) an optional upper hinge region, a lower hinge region, and a CH2 domain, wherein the CH2 (and polypeptide) lacks the core hinge region amino acid sequence CXXC, where X is any amino acid (optionally, each amino acid X is selected from P, R, and S). Optionally, CH2 comprises (from N-terminal to C-terminal) an amino acid and CH2 domain selected from SEQ ID NOs: 163-178, wherein the CH2 (and polypeptide) lacks the core hinge region amino acid sequence CXXC, where X is any amino acid (optionally, each amino acid X is selected from P, R, and S). In one embodiment, the amino acid sequence of the core hinge region is selected from SEQ ID NOs: 180-182. In embodiments, the CH2 (polypeptide) lacks the amino acid sequence of SEQ ID NOs: 183-187. In embodiments, the CH2 domain is a human IgG1 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence CPPC (SEQ ID NO: 180) in the core hinge region. Optionally, the CH2 (and polypeptide) lacks the amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) in the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) in the core hinge region. In embodiments, the CH2 domain is a human IgG2 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3, respectively. Optionally, CH2 (and the polypeptide) lacks the amino acid sequence ERKCCVE (SEQ ID NO: 184) of the upper hinge region and the amino acid sequence CPPC (SEQ ID NO: 180) of the core hinge region.In embodiments, the CH2 domain is a human IgG3 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3, respectively. Optionally, the CH2 (and polypeptide) lack the upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively, the CH2 (and polypeptide) lack the upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and the core hinge region amino acid sequence CPRC (SEQ ID NO: 181). In embodiments, the CH2 domain is a human IgG4 CH2 domain, and the amino acid sequence of the core hinge region is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3, respectively. Optionally, CH2 (and the polypeptide) lacks the amino acid sequence ESKYGPP (SEQ ID NO: 187) of the upper hinge region and the amino acid sequence CPSC (SEQ ID NO: 182) of the core hinge region.

[0315] Optionally, the polypeptide comprises an antibody CH1-hinge sequence lacking the core region-CH2-CH3.

[0316] Optionally, CH2 and CH3 are A. Human IgG1 CH2 and CH3 domains, B. Human IgG2 CH2 and CH3 domains, C. Human IgG3 CH2 and CH3 domains, or D. Human IgG4 CH2 and CH3 domains.

[0317] Optionally, CH2 and CH3 are (a) a hinge sequence and core hinge region that is a human IgG1 CH2 and CH3 domain and a human IgG1 hinge sequence and hinge region; (b) a hinge sequence and core hinge region that is a human IgG2 CH2 and CH3 domain and a human IgG2 hinge sequence and hinge region; (c) a hinge sequence and core hinge region that is a human IgG3 CH2 and CH3 domain and a human IgG31 hinge sequence and hinge region; or (d) Human IgG4 CH2 and CH3 domains and a hinge sequence and core hinge region that is a human IgG4 hinge sequence and hinge region.

[0318] Optionally, A. The CH2 domain comprises a human IgG1 CH2 domain, and the core hinge i region amino acid sequence is SEQ ID NO: 180, and optionally, the CH2 lacks the upper hinge region amino acid sequence EPKSCDKTHT (SEQ ID NO: 183); or B. The CH2 domain comprises a human IgG2 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 180, and optionally, the CH2 lacks the upper hinge region amino acid sequence ERKCCVE (SEQ ID NO: 184); or C. The CH2 domain comprises a human IgG3 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 181, and optionally, the CH2 lacks the upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) or the upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186); or D. The CH2 domain is a human IgG4 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 182, and optionally, the CH2 lacks the upper hinge region amino acid sequence ESKYGPP (SEQ ID NO: 187).

[0319] Optionally, the polypeptide comprises (from N-terminus to C-terminus) said Fc region and said SAM, wherein the Fc region comprises (from N-terminus to C-terminus) a hinge sequence, a CH2 domain, and a CH3 domain.

[0320] Optionally, the polypeptide comprises one or more epitope-binding sites, e.g., an antibody variable domain capable of specifically binding to a first epitope, such as ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, Aggrecan, AGR2, AICDA, AWI, AIG1, AKAP1, AKAP2, AIYIH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), B7.1, B7.2, BAD, BAFF, BAG1, BAI1, BCL2, BCL6, BD NF, BLNK, BLRl (MDR15), BlyS, BMPl, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRIA, BMPRIB, BMPR2, BPAG1 (plectin), BRCA1, CI9orflO (IL27w ), C3, C4A, C5, C5R1, CANT1, CASP1, CASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCL11(eotaxin), CCL13(MCP-4), CCL15(MIP-id), CCL16(H CC-4), CCL17(TARC), CCL18(PARC), CCL19(MIP-3b), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MIP-2), SLC, exodus-2, CCL22(MDC / STC-1 ), CCL23 (MPIF-1), CCL24 (MPIF-2I eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-la), CCL4 (MIP-lb), CCL5(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-1RB / RA), CCR3(CKR3 / CMKBR 3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI1), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6),CCRL1(VSHK1)、CCRL2(L-CCR)、CD164、CD19、CD1C、CD20、CD200、CD-22、CD24、CD28、CD3、CD37、CD38、CD3E、CD3G、CD3Z、CD4、CD40、CD40L、CD44、CD45RB、CD52、CD69、CD72、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CDH1(E-カドヘリン)、CDH10、CDH12、CDH13、CDH18、CDH19、CDH20、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKN1A(p2IWapl / Cipl)、CDKN1B(p27Kipl)、CDKNIC、CDKN2A(pl6INK4a)、CDKN2B、CDKN2C、CDKN3、CEBPB、CER1、CHGA、CHGB、Chitinase、CHST10、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7(クローディン-7)、CLN3、CLU(クラステリン)、CMKLR1、CMKOR1(RDC1)、CNR1、COL18A1、COL1A1、COL4A3、COL6A1、CR2、CRP、CSFl(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTLA4、CTNNBl(b-カテニン)、CTSB(カテプシンB)、CX3CL1(SCYDi)、CX3CR1(V28)、CXCL1(GROl)、CXCLIO(IP-10)、CXCL11(l-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GR02)、CXCL3(GR03)、CXCL5(ENA-78ILIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR ISTRL33 I Bonzo)、CYB5、CYC1、CYSLTR1、DAB2IP、DES、DKFZp451J0118、DNCL1、DPP4、E2F1、ECGF1、EDG1、EFNAI、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、EN01、EN02、EN03、EPHB4、EPO、ERBB2(Her-2), EREG, ERK8, ESR1, ESR2, F3(TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF 14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR 3, FIGF (VEGFD), FILl (EPSILON), FILl (ZETA), FU12584, FU25530, FLRTl (fibronectin), FLTl, FOS, FOSLl (FRA-I), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-65T, GATA3, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRFl, GPR2 (CCRIO), GPR31, GPR44, GPR81 (FKSG80), GRCCIO (CIO), GRP, GSN (gelsolin (Gels olin), GSTPl, HAVCR2, HDAC4, EDAC5, HDAC7A, HDAC9, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, ICEBERG, ICOSL, 1D2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN gamma, TFNW1, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL10, IL10RA , IL10RB, IL11, IL11RA, IL-12, IL12A, IL12B, IL12RB1, IL12RB2, 1L13, IL13RA1, IL13RA2, 1L14, 1L15, IL15RA, IL16, 1L17, IL17B, IL17C, IL17R, 1L18, IL18BP, IL18R1, IL18RAP, 1L19, ILIA, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1,IL1RL2 IL1RN, 1L2, 1L20, IL20RA, IL21R, 1L22, 1L22R, 1L22RA2, 1L23, 1L24, 1L25 1L26, 1L27, 1L28A, 1L28B, 1L29, IL2RA, IL2RB, IL2RG, 1L3, 1L30, IL3RA L4, IL4R, 1L5, IL5RA, 1L6, IL6R, IL6ST (Flower Rate 130), 1L7, TL7R, 1L8, IL8RA IL8RB, IL8RB, 1L9, IL9R, ILK, INHA, INHBA, INSL3, INSL4, IRAQ, IRAQ2, I TGA1, ITGA2, 1TGA3, ITGA6(a6イシン), ITGAV, ITGB3, ITGB4(b4イテン) J. Chem AG1, JAK1, JAK3, JUN, K6HF, KAI1, KDR, MTLG, KLF5(GCBoxBP), KLF6, KLK10. KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK6、KLK9、KRT1、KRT19(ケラチン19), KRT2A, KRTHB6 (Large Enzyme II Linga), LAMA5, LEP (Lingo), Lingo-p75, Ling o-Troy, LPS, LTA(TNF-b), LTB, LTB4R(GPR16), LTB4R2, LTBR, ​​MACMARCKS, M.S AG: Omgp, MAP2K7(c-Jun), MDK, MIB1, Crab, MIF, MIP-2, MK167(Ki-67) MMP2, MMP9, MS4A1, MSMB, MT3 (Myster-ifi), MTSS1, MUC1 (MYC), MYC, MY D88, NCK2, NFKB1, NFKB2, NGFB(NGF), NGFR, NgR-Lingo, NgR-Nogo6 6(Nogo), NgR-p75, NgR-Troy, NME1(NM23A), NOX5, NPPB, NROB1, NROB2, NR1 D1, NR1D2, NR1H2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3 NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, N R6A1, NRP1, NRP2, NT5E, NTN4, ODZ1, OPRD1, P2RX7, PAP, PARTY, PATE, PAWR.PCA3, PCNA, PDGFA, PDGFB, PECAM1, PF4(CXCL4), PGF, PGR, phosphacan, PIAS2, PIK3CG, PLAU(uPA), PLG, PLXDC1, PPBP(CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL , PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2(COX-2), PTN, RAC2(p2IRac2), RARB, RGS1, RGS13, RGS3, RNF110(ZNF144), ROB02, S100A2, SCGB1D2(Lipofi Phospholipase B), SCGB2A1 (mammaglobulin 2), SCGB2A2 (mammaglobulin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPINA1, SERPINIA3, SERPINB5 (maspin), SERPINE1 (PAT-i), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPPl, SPRRIB (Spri), ST6GAL1, STABl, STAT6, STEAP, STEAP2, TB4R2, TBX21, TCPIO, TDGF1, TEK, TG FA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-i), T]MP3, tissue factor, TLRIO, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(F as), TNFRSF7, TNFRSF8, TNFRSF9, TNFSFIO (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor,TOP2A (topoisomerase lia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, versican, VHLC5, VLA-4, , XCL1 (lymphectin), XCL2 (SCM-1b), XCR1 (GPR5 / CCXCR1), YY1, and ZFPM2 (e.g., a human antigen). Optionally, the second epitope (as discussed below) is comprised of the same antigen as the first epitope (e.g., comprised of the same antigen molecule). In another example, the second antigen is comprised of the group. In an example, the first and second epitopes are comprised of different antigens selected from the group.

[0321] For example, the polypeptide has 1, 2, 3, 4, or 5 epitope-binding sites (optionally, the polypeptide comprises two or more binding sites (e.g., a single variable domain) that bind to different epitopes, or the polypeptide binding sites are identical). In one embodiment, the SAM is a TD (e.g., a p53 TD, such as human p53 TD) and the polypeptide has 2, 3, or 4 binding sites, such as 3 sites, or such as 4 sites. Preferably, the polypeptide has 3 binding sites. Preferably, the polypeptide has 4 binding sites. For example, the binding sites each bind to TNF-alpha (e.g., the binding sites are identical, e.g., are the same antibody single variable domain).

[0322] In an example, the multimer is an octavalent bispecific multimer comprising four copies of an anti-PD-L1 binding site (e.g., a dAb) and four copies of an anti-4-1BB binding site (e.g., a dAb).

[0323] In examples, the multimer is a tetravalent multimer comprising copies of an anti-PD-L1 binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-PD-L1 binding site (e.g., a dAb). In examples, the multimer is a dodecavalent multimer comprising copies of an anti-PD-L1 binding site (e.g., a dAb). In examples, the multimer is a hexavalent multimer comprising copies of an anti-PD-L1 binding site (e.g., a dAb). In examples, the anti-PD-L1 binding site comprises an avelumab or atezolizumab binding site that specifically binds to PD-L1. Preferably, in these examples, the SAM domain is a TD, for example, a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a dodecamer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide contains four copies of the binding site.

[0324] In examples, the multimer is a tetravalent multimer comprising copies of an anti-PD-1 binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-PD-1 binding site (e.g., a dAb). In examples, the multimer is a 12-valent multimer comprising copies of an anti-PD-1 binding site (e.g., a dAb). In examples, the multimer is a 16-valent multimer comprising copies of an anti-PD-1 binding site (e.g., a dAb). In examples, the anti-PD-1 binding site comprises a nivolumab or pembrolizumab binding site that specifically binds to PD-1. Preferably, in these examples, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a 12-mer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide contains four copies of the binding site.

[0325] In examples, the multimer is a tetravalent multimer comprising copies of an anti-DR5 (death receptor 5) binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-DR5 binding site (e.g., a dAb). In examples, the multimer is a dodecavalent multimer comprising copies of an anti-DR5 binding site (e.g., a dAb). In examples, the multimer is a hexavalent multimer comprising copies of an anti-DR5 binding site (e.g., a dAb). Preferably, in these examples, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a dodecamer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide comprises four copies of the binding site.

[0326] In some embodiments, the multimer is a tetravalent multimer containing copies of an anti-OX40 or OX40L binding site (e.g., a dAb). In some embodiments, the multimer is an octavalent multimer containing copies of an anti-OX40 or OX40L binding site (e.g., a dAb). In some embodiments, the multimer is a dodecavalent multimer containing copies of an anti-OX40 or OX40L binding site (e.g., a dAb). In some embodiments, the multimer is a hexavalent multimer containing copies of an anti-OX40 or OX40L binding site (e.g., a dAb). Preferably, in these embodiments, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide contains one copy of the binding site. In the case of an octamer, each polypeptide contains two copies of the binding site. In the case of a dodecamer, each polypeptide contains three copies of the binding site. In the case of a 16-mer, each polypeptide contains four copies of the binding site.

[0327] In examples, the multimer is a tetravalent multimer comprising copies of an anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-GITR binding site (e.g., a dAb). In examples, the multimer is a dodecavalent multimer comprising copies of an anti-GITR binding site (e.g., a dAb). In examples, the multimer is a hexavalent multimer comprising copies of an anti-GITR binding site (e.g., a dAb). Preferably, in these examples, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a dodecamer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide comprises four copies of the binding site.

[0328] In examples, the multimer is a tetravalent multimer comprising copies of an anti-antibody kappa light chain (KLC) binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-KLC binding site (e.g., a dAb). In examples, the multimer is a dodecavalent multimer comprising copies of an anti-KLC binding site (e.g., a dAb). In examples, the multimer is a hexavalent multimer comprising copies of an anti-KLC binding site (e.g., a dAb). Preferably, in these examples, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a dodecamer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide comprises four copies of the binding site.

[0329] In some embodiments, the multimer is a tetravalent multimer containing copies of an anti-VEGF binding site (e.g., a dAb). In some embodiments, the multimer is an octavalent multimer containing copies of an anti-VEGF binding site (e.g., a dAb). In some embodiments, the multimer is a dodecavalent multimer containing copies of an anti-VEGF binding site (e.g., a dAb). In some embodiments, the multimer is a hexavalent multimer containing copies of an anti-VEGF binding site (e.g., a dAb). In some embodiments, the anti-VEGF binding site comprises a VEGF receptor domain that specifically binds to VEGF (e.g., a VEGF binding site of human flt (e.g., flt-1) or KDR, e.g., Ig domain 2 from VEGFR1 or Ig domain 3 from VEGFR2). In some embodiments, the anti-VEGF binding site comprises an aflibercept, bevacizumab, or ranibizumab binding site that specifically binds to VEGF. Preferably, in these examples, the SAM domain is a TD, for example, a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide contains one copy of the binding site. In the case of an octamer, each polypeptide contains two copies of the binding site. In the case of a dodecamer, each polypeptide contains three copies of the binding site. In the case of a 16-mer, each polypeptide contains four copies of the binding site.

[0330] In examples, the multimer is a tetravalent multimer comprising copies of an anti-TNFα binding site (e.g., a dAb). In examples, the multimer is a tetravalent multimer comprising copies of an anti-TNFα binding site (e.g., a dAb). In examples, the multimer is an octavalent multimer comprising copies of an anti-TNF alpha binding site (e.g., a dAb). In examples, the multimer is a dodecavalent multimer comprising copies of an anti-TNF alpha binding site (e.g., a dAb). In examples, the multimer is a hexavalent multimer comprising copies of an anti-TNF alpha binding site (e.g., a dAb). Preferably, in these examples, the SAM domain is a TD, e.g., a p53 TD, such as human p53 TD. In the case of a tetramer, each polypeptide comprises one copy of the binding site. In the case of an octamer, each polypeptide comprises two copies of the binding site. In the case of a dodecamer, each polypeptide comprises three copies of the binding site. In the case of a 16-mer, each polypeptide contains four copies of the binding site.

[0331] Optionally, the variable domains are selected from antibody single variable domains, VH and VL, or the domains are comprised by an scFv. Optionally, the domains are comprised by an antibody VH / VL pair that binds to said first epitope. In examples, epitope binding herein is specific binding as defined herein.

[0332] Optionally, the polypeptide comprises (in the N-terminal to C-terminal direction): A. Variable domains, SAM and Fc regions, B. Fc region, SAM and variable domains, C. Variable domains, Fc region and SAM, D. SAM, variable domain and Fc region, or E.SAM, comprising an Fc region and a variable domain.

[0333] Optionally, the polypeptide comprises a second antibody variable domain at the N-terminus or C-terminus of the SAM, the second variable domain being capable of specifically binding to a second epitope, the first and second epitopes being the same or different.

[0334] Optionally, the SAM is a self-associating tetramerization domain (TD), and optionally the TD is a p53, p63, or p73 TD or a homolog or ortholog thereof, or the TD is an NHR2 TD or a homolog or ortholog thereof, or the TD comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10 or 126.

[0335] Optionally, (i) The polypeptide comprises (in the N-terminal to C-terminal direction): A. A first antibody single variable domain (dAb), an optional linker, and the SAM; B. A first antibody single variable domain, an optional linker, the SAM and a second antibody single variable domain; C. A first scFv, an optional linker, and the SAM; D. A first scFv, an optional linker, the SAM and a second scFv; E. A first antibody single variable domain, an optional linker, the SAM and a first scFv; F. A first scFv, an optional linker, the SAM and a first antibody single variable domain; G. a first antibody variable domain, an optional first linker, a first antibody constant domain, an optional second linker, and the SAM; H. the SAM, an optional linker, and a first antibody single variable domain; I. the SAM, an optional linker and a first scFv; J. the SAM, an optional linker, a first antibody constant domain, a second optional linker and a first antibody variable domain; or K. comprising the SAM, an optional linker, a first antibody variable domain, a second optional linker, and a first antibody constant domain; or (ii) the polypeptide comprises (in the N-terminal to C-terminal direction): A. dAb and SAM, B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, an Fc region and a SAM; G. First scFv, Fc region and SAM. H. VH, CH1, Fc region and SAM, I. VL, CL, Fc region and SAM, J. dAb, SAM and Fc region, K. scFv, SAM and Fc regions, L. VH, CH1, SAM and Fc regions; M. VL, CL, SAM and Fc regions, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. A first dAb, an Fc region, a SAM and a second dAb; W. A first dAb, an Fc region, a SAM and a first scFv; X. a first dAb, a second dAb, an Fc region and a SAM; Y. A first dAb, a second dAb, an Fc region, a SAM and a third dAb; Z. a first dAb, a second dAb, an Fc region, a SAM and a first scFv; or AA. comprises a first dAb, a second dAb, an Fc region, a SAM, a third dAb, and a fourth dAb; or (iii) the polypeptide comprises (in the C-terminal to N-terminal direction): A. dAb and SAM, B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, an Fc region and the SAM; G. A first scFv, an Fc region, and the SAM. H. VH, CH1, Fc region, and SAM; I. VL, CL, Fc region and SAM, J. dAb, SAM and Fc region, K. scFv, SAM and Fc regions, L. VH, CH1, SAM and Fc regions; M. VL, CL, SAM and Fc regions, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. A first dAb, an Fc region, a SAM and a second dAb; W. A first dAb, an Fc region, a SAM and a first scFv; X. a first dAb, a second dAb, an Fc region and a SAM; Y. A first dAb, a second dAb, an Fc region, a SAM and a third dAb; Z. a first dAb, a second dAb, an Fc region, a SAM and a first scFv; or AA. Includes a first dAb, a second dAb, an Fc region, a SAM, a third dAb and a fourth dAb.

[0336] Optionally, any single variable domain or dAb herein is a Nanobody™ or camelid VHH (eg a humanised camelid VHH).

[0337] In one embodiment, each polypeptide of the multimer is paired with a copy of an additional polypeptide, where the additional polypeptide comprises an antibody light chain constant region (e.g., Cκ or Cλ) paired with the Fc of the first polypeptide. In an example, the first polypeptide comprises an antibody VH domain, and the additional polypeptide comprises an antibody VL domain (e.g., Vκ or Vλ), where the VH and VL form an epitope-binding site. Thus, the multimer can be a Fab-like multimer, such as one containing multiple copies of the adalimumab (Humira) or avelumab (Bavencio) binding site, as illustrated in the examples below. All antibody domains in such a multimer can be, for example, human, and optionally, the SAM is a human domain. When the SAM is a TD (e.g., p53 TD), the multimer comprises a tetramer of VH / VL epitope-binding sites. The first polypeptide or the additional polypeptide can comprise a second epitope-binding site, for example, such that the multimer is octavalent. If the eight binding sites can bind to the same antigen, or if four VH / VL binding sites bind to a first antigen and the other four binding sites bind to a second antigen, where the antigens are different, the multimer can be octavalent and bispecific. If the first or additional polypeptides contain additional antigen-binding sites, the multimer can be 12-valent (and, for example, monospecific, bispecific, or trispecific for antigen binding). If the first or additional polypeptides contain additional antigen-binding sites, the multimer can be 16-valent (and, for example, monospecific, bispecific, trispecific, or tetraspecific for antigen binding).

[0338] The present invention further provides: A multimer (optionally a tetramer) of a polypeptide according to the invention, optionally wherein the multimer is for medical use. In an example, the medical use herein is the treatment or prevention of cancer, an autoimmune disease or condition, or any other disease or condition disclosed herein. A multimer of multiple antibody Fc regions, each Fc comprised of a respective polypeptide that is not paired with another Fc region, and optionally, the multimer is for therapeutic use. Optionally, each polypeptide comprises an epitope-binding domain or site as disclosed herein. A pharmaceutical composition comprising a polypeptide or multimer of the present invention. A nucleic acid encoding a polypeptide of the invention, optionally wherein the nucleic acid is comprised by a eukaryotic cell or vector. A method for binding multiple copies of an antigen, the method comprising combining the copies with a composition of the present invention, the copies being bound by a multimeric polypeptide, and optionally, isolating the multimers bound to the antigen copies. In an example, the multimers are contacted with a sample containing copies of the antigen, and the antigen copies are sequestered in the sample by binding to the multimers. For example, the multimers are administered to a human or animal patient (or the environment is exposed to the multimers), and the antigen copies are sequestered in humans (e.g., for the medical treatment), animals (e.g., for the medical treatment), or the environment. For example, the environment may be soil, water sources, waterways, or industrial fluids, for example, for environmental remediation, such as when the antigen is an environmental pollutant or contaminant. In an example, the method is for purifying a sample or isolating an antigen contained in the sample. A method of treating or reducing the risk of a disease or condition in a human or animal subject, the method comprising administering a composition of the invention to the subject, wherein a multimer comprised in the composition specifically binds to a target antigen in the subject, and wherein binding mediates the treatment or reduction of the risk of the disease or condition. A method for producing a composition comprising a plurality of polypeptides of the invention, wherein the SAM is a self-associating tetramerization domain (TD), the method comprising providing a eukaryotic host cell according to the invention, culturing the host cell, allowing expression and secretion of tetramers of the polypeptides from the cell, and optionally isolating or purifying the tetramers.

[0339] paragraph: The present invention provides the following paragraphs: 1. (a) an antibody Fc region, the Fc region comprising an antibody CH2 domain and an antibody CH3 domain; (b) a self-associating multimerization domain (SAM), A polypeptide wherein CH2 lacks the core hinge CXXC amino acid sequence, where X is any amino acid. 2. The polypeptide of paragraph 1, wherein each amino acid X is selected from P, R, and S. 3. The polypeptide of paragraph 1 or 2, wherein CH2 lacks a complete upper hinge sequence. 4.CH2 is (a) the APELLGGPSV amino acid sequence, or the PAPELLGGPSV amino acid sequence; (b) the APPVAGPSV amino acid sequence, or the PAPPVAGPSV amino acid sequence; (c) The polypeptide of any one of paragraphs 1 to 3, comprising the amino acid sequence APEFLGGPSV or the amino acid sequence PAPEFLGGPSV. 5. CH2 and CH3 are (a) human IgG1 CH2 and CH3 domains; (b) human IgG2 CH2 and CH3 domains; (c) human IgG3 CH2 and CH3 domains, or (d) The polypeptide of any one of paragraphs 1 to 4, comprising human IgG4 CH2 and CH3 domains. 6. A polypeptide of paragraph 5(a) or (b), wherein CH2 lacks the CPPC sequence; or a polypeptide of paragraph 5(c), wherein CH2 lacks the CPRC sequence; or a polypeptide of paragraph 5(d), wherein CH2 lacks the CPSC sequence. 7.CH2 is (a) APELLGGPSV amino acid sequence; (b) the amino acid sequence EPKSCDKTHT[P]APELLGGPSV, where the P in parentheses is optional; (c) APPVAGPSV amino acid sequence; (d) ERKCCVE[P]APPVAGPSV amino acid sequence, wherein the P in parentheses is optional; (e) the amino acid sequence ELKTPLGDTTHT[P]APELLGGPSV, where the P in parentheses is optional; (f) the amino acid sequence EPKSCDTPPP[P]APELLGGPSV, where the P in parentheses is optional; (g) the APEFLGGPSV amino acid sequence, or (h) The polypeptide of any one of paragraphs 1 to 4, comprising the amino acid sequence ESKYGPP[P]APEFLGGPSV, wherein the bracketed P is optional. 8. The polypeptide of any one of paragraphs 1 to 7, wherein CH2 lacks a sequence selected from CXXC and SEQ ID NOs: 180-182 disclosed herein. 9. The polypeptide of any one of paragraphs 1 to 8, wherein the polypeptide comprises an antibody variable domain capable of specifically binding to a first epitope. 10. The polypeptide of paragraph 9, wherein the variable domains are selected from antibody single variable domains, VH and VL, or the domains are constituted by scFv. 11. The polypeptide of any one of paragraphs 1 to 10, wherein the Fc region is 3' to the SAM. 12. (a) the variable domain is 5' of the SAM and the Fc region is 3' of the SAM; (b) the variable domain is 3' of the SAM and the Fc region is 5' of the SAM; (c) the variable domain and the Fc region are 5' of the SAM; or (d) The polypeptide of paragraph 9, 10 or 11, wherein the variable domain and the Fc region are 3' of the SAM. 13. A polypeptide according to paragraph 12, comprising a second variable domain 5' or 3' of SAM, the second variable domain being capable of specifically binding to a second epitope, the first and second epitopes being the same or different. 14. The polypeptide of paragraph 13, wherein the epitopes are different epitopes of the same antigen or epitopes of different antigens. 15. The polypeptide of paragraph 13 or 14, wherein the second variable domain is selected from antibody single variable domains, VH and VL, or the domain is constituted by an scFv. 16. The polypeptide of paragraph 15, wherein the second variable domain is an antibody single variable domain or an antibody constant domain. 17. The polypeptide of any one of paragraphs 1 to 16, wherein the SAM is a self-associating tetramerization domain (TD). 18. A polypeptide according to paragraph 17, wherein the TD is a p53, p63 or p73 TD or a homologue or orthologue thereof, or wherein the TD is an NHR2 TD or a homologue or orthologue thereof. 19. The polypeptide of paragraph 17 or 18, wherein TD comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or 126. 20. The polypeptide of any one of paragraphs 1 to 19, comprising an antibody CH1 constant domain, and optionally CH1-CH2-CH3, wherein CH2 and CH3 are constituted by the Fc region. 21. A polypeptide (first polypeptide) comprising (in the N-terminal to C-terminal direction): A. A first antibody single variable domain (dAb), an optional linker, and the SAM; B. A first antibody single variable domain, an optional linker, the SAM and a second antibody single variable domain; C. A first scFv, an optional linker, and the SAM; D. A first scFv, an optional linker, the SAM and a second scFv; E. A first antibody single variable domain, an optional linker, the SAM and a first scFv; F. A first scFv, an optional linker, the SAM and a first antibody single variable domain; G. a first antibody variable domain, an optional first linker, a first antibody constant domain, an optional second linker, and the SAM; H. the SAM, an optional linker, and a first antibody single variable domain; I. the SAM, an optional linker and a first scFv; J. the SAM, an optional linker, a first antibody constant domain, a second optional linker and a first antibody variable domain; or K. The polypeptide of any one of paragraphs 1 to 20, comprising or consisting of the SAM, an optional linker, a first antibody variable domain, a second optional linker and a first antibody constant domain. twenty two. (i) the polypeptide (in the N-terminal to C-terminal direction) A. dAb and self-associating multimerization domain (SAM), B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, an Fc region and a SAM; G. First scFv, Fc region and SAM. H. VH, CH1, Fc region and SAM, I. VL, CL, Fc region and SAM, J. dAb, SAM and Fc region, K. scFv, SAM and Fc regions, L. VH, CH1, SAM and Fc regions; M. VL, CL, SAM and Fc regions, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. A first dAb, an Fc region, a SAM and a second dAb; W. A first dAb, an Fc region, a SAM and a first scFv; X. a first dAb, a second dAb, an Fc region and a SAM; Y. A first dAb, a second dAb, an Fc region, a SAM and a third dAb; Z. a first dAb, a second dAb, an Fc region, a SAM and a first scFv; or AA. comprises a first dAb, a second dAb, an Fc region, a SAM, a third dAb, and a fourth dAb; or (ii) the polypeptide (in the C-terminal to N-terminal direction) A. dAb and self-associating multimerization domain (SAM), B. A first dAb, a SAM and a second dAb, C. The first scFv and SAM, D. A first scFv, a SAM, and a second scFv; E. A first scFv, a SAM and a first dAb, F. A first dAb, an Fc region and a SAM; G. First scFv, Fc region and SAM. H. VH, CH1, Fc region, and SAM; I. VL, CL, Fc region and SAM, J. dAb, SAM and Fc region, K. scFv, SAM and Fc regions, L. VH, CH1, SAM and Fc regions; M. VL, CL, SAM and Fc regions, N. a first dAb, a second dAb and a SAM; O.VH, CH1 and SAM, P.V.L., C.L., and S.A.M. Q. VH, CH1, SAM and first dAb, R.VL, CL, SAM and the first dAb, S. a first dAb, a second dAb, a SAM and a third dAb; T. a first dAb, a second dAb, a SAM and a first scFv; U. a first dAb, a second dAb, a SAM, a third dAb and a fourth dAb; V. A first dAb, an Fc region, a SAM and a second dAb; W. A first dAb, an Fc region, a SAM and a first scFv; X. a first dAb, a second dAb, an Fc region and a SAM; Y. A first dAb, a second dAb, an Fc region, a SAM and a third dAb; Z. a first dAb, a second dAb, an Fc region, a SAM and a first scFv; or AA. The polypeptide of any one of paragraphs 1-20, comprising a first dAb, a second dAb, an Fc region, a SAM, a third dAb, and a fourth dAb. 23. The polypeptide of paragraph 21B, 21D, (i)22B, (i)22D, (i)22N, (i)22S, (i)22T, (i)22U, (i)22X, (i)22Y, (i)22Z, (i)22AA, (ii)22B, (ii)22D, (ii)22N, (ii)22S, (ii)22T, (ii)22U, (ii)22X, (ii)22Y, (ii)22Z or (ii)22AA, wherein the single variable domains (dAbs) are identical or the scFvs are identical. 24. The polypeptide of paragraph 21B, 21D, (i)22B, (i)22D, (i)22N, (i)22S, (i)22T, (i)22U, (i)22X, (i)22Y, (i)22Z, (i)22AA, (ii)22B, (ii)22D, (ii)22N, (ii)22S, (ii)22T, (ii)22U, (ii)22X, (ii)22Y, (ii)22Z or (ii)22AA, wherein the single variable domains are different or the scFvs are different. 25. (i) the first variable domain is a VH domain and the first constant domain is a CH1 domain, optionally, the polypeptide is associated with a second polypeptide, the second polypeptide comprising an antibody CL constant domain paired with the CH1 domain; (ii) the first variable domain is a VH domain and the first constant domain is a CL domain, optionally, the polypeptide is associated with a second polypeptide, the second polypeptide comprising an antibody CH1 constant domain paired with the CL domain; (iii) the first variable domain is a VL domain and the first constant domain is a CH1 domain, optionally, the polypeptide is associated with a second polypeptide; 22P, (i)22Q, (i)22R, (ii)22H, (ii)22I, (ii)22L, (ii)22M, (ii)22O, (ii)22P, (ii)22Q or (ii)22R, wherein (iv) the first variable domain is a VL domain and the first constant domain is a CL domain, optionally wherein the polypeptide is associated with a second polypeptide, and the second polypeptide comprises an antibody CH1 constant domain paired with a CL domain. 26. The polypeptide of any one of paragraphs 21 to 25, comprising an antibody Fc region or antibody single variable domain between (v) the first variable domain or scFv and (vi) the SAM, wherein the Fc comprises CH2 and CH3. 27. The polypeptide of any one of paragraphs 21 to 26, comprising an Fc region or antibody single variable domain between (i) the SAM and (ii) the most C-terminal variable domain. 28. A polypeptide according to any one of paragraphs 21 to 27, comprising, in N-terminal to C-terminal direction, an antibody Fc region and an N-terminal most variable domain or scFv. 29. A polypeptide according to any one of paragraphs 21 to 28, comprising, in N-terminal to C-terminal direction, a SAM and an antibody Fc region. 30. The or each linker is selected from the group consisting of: (G4S) n 30. The polypeptide of any one of paragraphs 21 to 29, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 31. The polypeptide of any one of paragraphs 1 to 30, wherein each domain and SAM is a human domain and SAM, respectively. 32. The polypeptide of any one of paragraphs 1 to 31, wherein the polypeptide comprises binding specificities for two or more antigens, optionally two, three, or four different antigens. 33. A multimer (optionally a tetramer) of a polypeptide according to any one of paragraphs 1 to 32. 34. A multimer of multiple antibody Fc regions, wherein each Fc is composed of its own polypeptide and is not paired with another Fc region. 35. The multimer of paragraph 34, wherein the multimer is a polypeptide tetramer comprising at least four Fc regions. 36. The multimer of paragraph 34 or 35, wherein each Fc region is identical. 37. The multimer of any one of paragraphs 1 to 36, wherein each Fc comprises a CH2 and a CH3, and each CH2 is a CH2 as described in any one of paragraphs 1 to 32. 38. A polypeptide or multimer according to any one of paragraphs 1 to 37, comprising eukaryotic glycosylation. 39. The polypeptide or multimer according to paragraph 38, wherein the cell is a HEK293, CHO or Cos cell. 40. A polypeptide or multimer according to any one of paragraphs 1 to 39 for medical use. 41. A pharmaceutical composition comprising a polypeptide or multimer according to any one of paragraphs 1 to 40. 42. A nucleic acid encoding a polypeptide according to any one of paragraphs 1-32 and 38-40. 43. A eukaryotic cell or vector comprising a nucleic acid according to paragraph 42. 44. A method of binding multiple copies of an antigen, the method comprising combining the copies with a multimer according to any one of paragraphs 33 to 40, wherein the copies are bound by polypeptides of the multimer, and optionally the method comprises isolating the multimers bound to the antigen copies. 45. The method of paragraph 37, wherein the method is a diagnostic method for detecting the presence of a substance in a sample, the substance comprising an antigen, and the method comprises providing a sample (e.g., a body fluid, a food, a food ingredient, a beverage, a beverage ingredient, soil or a forensic sample), mixing the sample with a multimer of any one of paragraphs 33 to 40, and detecting binding of the multimer to the antigen in the sample. 46. ​​A method of treating or reducing the risk of a disease or condition in a human or animal subject, the method comprising administering to the subject a composition according to paragraph 41, wherein a multimer comprised in the composition specifically binds to a target antigen in the subject, and said binding mediates the treatment or reduction of the risk. 47. The method of paragraph 46, wherein the antigen is an immune checkpoint (e.g., PD-L1, PD-1, or CTLA4), or the antigen is TNFalpha or IL-17A. 48. The method of paragraph 46, wherein the antigen mediates the disease or condition of interest, and optionally, the binding antagonizes the antigen. 49. A composition comprising a plurality of polypeptides according to any one of paragraphs 1 to 32 and 38 to 40, wherein at least 90% of the polypeptides are composed of tetramers of the polypeptides. 50. The composition of paragraph 49, wherein at least 98% of the polypeptide is composed of a tetramer of the polypeptide. 51. The composition of paragraph 49 or 50, wherein the remaining polypeptides are selected from one or more of polypeptide monomers, dimers, and trimers. 52. A method for producing a composition comprising a plurality of polypeptides according to any one of paragraphs 1 to 32 and 38 to 40 (optionally a composition according to any one of paragraphs 49 to 51), the method comprising providing a eukaryotic host cell according to paragraph 34, culturing the host cell, allowing expression and secretion from the cell of a tetramer of the polypeptides, and optionally isolating or purifying the tetramer.

[0340] concept: In certain embodiments, the present invention is useful for providing multimers for treating cancer in humans or animals. In this regard, it may be useful to use multimers to target tumors by binding to tumor-associated antigens and / or to bind to T cells to regulate their activity. For example, multimers may bind to antigens on T regulatory cells (Treg) and downregulate their activity. Additionally or alternatively, multimers may bind to T effector (Teff) cells to upregulate their activity. Providing an antibody Fc region in the multimeric polypeptide may be advantageous for providing Fc effector function and / or cytotoxicity for killing tumor cells. In one advantageous configuration, the present invention utilizes the ability to provide multiple identical antigen or epitope binding sites that can be used to bind to several copies of the same antigen or epitope on tumor cells, thereby providing an avidity effect in which multimers preferentially bind tumor cells over any non-target cells or normal cells because the surface of the former expresses more copies of the antigen than normal cells. In one configuration, the multimer also contains a binding site for an immune checkpoint modulator. In one example, the modulator is an immune checkpoint inhibitor, and the binding site antagonizes the inhibitor. This is useful, for example, when the inhibitor is expressed by Teff cells, to upregulate Teff activity near tumor cells targeted by the multimer (e.g., by binding a TAA to the tumor cells). In another example, the modulator is an immune checkpoint stimulator, and the binding site agonizes the inhibitor. This is useful, for example, when the inhibitor is expressed by Teff cells, to upregulate Teff activity near tumor cells targeted by the multimer (e.g., by binding a TAA to the tumor cells). Thus, upregulation of T cell activity can be stimulated near tumor cells, but not near non-target (e.g., normal or non-cancerous) cells. To this end, the present invention provides the following concepts. 1. A polypeptide comprising a self-associating multimerization domain (SAM), a first antigen-binding site, and a second antigen-binding site, wherein the first site specifically binds to a first antigen or epitope and the second binding site specifically binds to a second antigen or epitope, and each antigen or epitope is a tumor-associated antigen (TAA) or epitope, or an immune checkpoint modulator (e.g., inhibitor) antigen or epitope. 2. The polypeptide of concept 1, wherein the first antigen is a TAA and the second antigen is an immune checkpoint modulator (e.g., an inhibitor). 3. The polypeptide of concept 1, wherein the first antigen is a TAA and the second antigen is a TAA. 4. The polypeptide of concept 1, wherein the first antigen is an immune checkpoint inhibitor and the second antigen is an immune checkpoint modulator (e.g., inhibitor). 5. The polypeptide according to concept 3 or 4, wherein the binding sites are capable of specifically binding to the same epitope of the same antigen. 6. The polypeptide of concept 3 or 4, wherein the binding sites are capable of specifically binding to different epitopes of the same antigen. 7. The first antigen is 4-1BB, 4-1BBL, CD28, OX40, OX40L, ICOS, ICOSL, GITR, CD40, CD27, CD27L, CD40L, LIGHT, CD70, CD80, CD86, HER2, HER3, PSMA, WT1, MUC1, LMP2, EGFRvIII, MAGE A3, GD2, CEA, Melan a / MART1, Bcr-Abl, Survivin, PSA, hTERT, EphA2, PAP, EpCAM, ERG, PAX3, ALK, androgen receptor, cyclin B1, RhoC, GD3, PSCA, PAX5, LCK, VEGFR2, MAD CT-1, FAP, or MAD 7. The polypeptide of any one of concepts 1-6, wherein the polypeptide is selected from CT-2, PDGFR-beta, Fos-related antigen 1, NY-BR-1, ETV6-AML, RGS5, SART3, SSX2, XAGE-1, STn, PAP, and BCMA. 8. The polypeptide of any one of concepts 1-7, wherein the second antigen is selected from PDL1, PD1, CTLA4, BTLA, KIR, LAG3, TIM3, A2aR, HVEM, GAL9, VISTA, SIRPa, CD47, CD160, CD155, IDO, CEACAM1, 2B4, CD48, and TIGIT. 9. The polypeptide of any one of concepts 1-8, wherein the polypeptide comprises a third antigen-binding site capable of specifically binding to a third antigen or epitope. 10. The polypeptide of concept 9, wherein the third antigen is a TAA. 11. The polypeptide of concept 9, wherein the third antigen is an immune checkpoint modulator (e.g., inhibitor). 12. The polypeptide of any one of concepts 9-11, wherein the third antigen is the same as the first or second antigen. 13. The polypeptide of any one of concepts 9-12, wherein the third antigen is CD3 (e.g., CD3e) or CD28. 14. The polypeptide of any one of concepts 9-13, wherein the polypeptide comprises a fourth antibody binding site capable of specifically binding to a fourth antigen or epitope. 15. The polypeptide of concept 14, wherein the fourth antigen is a TAA. 16. The polypeptide of concept 14, wherein the fourth antigen is an immune checkpoint modulator (e.g., inhibitor). 17. The polypeptide of any one of concepts 1-16, wherein the polypeptide is according to any of the polypeptides disclosed herein.

[0341] In examples, the first dAb or first scFv of the polypeptide herein is the conceptual first antigen-binding site, and optionally, when a further dAb or scFv binding site is present, this is the conceptual second antigen-binding site.

[0342] In examples, the first dAb or first scFv of the polypeptide herein is the second antigen-binding site of these concepts, and optionally, when a further dAb or scFv binding site is present, this is the first antigen-binding site of the concepts. 18. A multimer of the polypeptide according to any one of concepts 1 to 17. 19. The multimer of concept 18 for administration to a human or animal subject for targeting immune checkpoint inhibitors and immune co-stimulatory molecules for the treatment of cancer. 20. A method of treating cancer in a human or animal subject, comprising administering to the subject the multimer of claim 18.

[0343] item: In terms of configuration, the present invention provides the following items: 1. A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., a protein domain or peptide), wherein the multimer is multimerized by first, second, third and fourth self-associating tetramerization domains (TDs) associated together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of the protein domain or peptide. 2. The multimer according to item 1, wherein the multimer is a tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., tetramer, octamer) of the domain or peptide. 3. The multimer according to item 1 or 2, comprising a tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., tetramer, octamer) of an immunoglobulin superfamily binding site. 4. The multimer according to item 3, wherein the binding site comprises a first variable domain paired with a second variable domain. 5. The multimer according to any one of items 1 to 4, wherein each polypeptide comprises a first and a second copy of the protein domain or peptide, and the polypeptide comprises (in the N-terminal to C-terminal direction) (i) the first of the copies - TD - the second of the copies, (ii) TD - and the first and second copies, or (iii) the first and second copies - TD. 6. The multimer according to any of items 1 to 5, wherein the TD is an NHR2 TD and the domain or peptide is not an NHR2 domain or peptide, or the TD is a p53 TD and the domain or peptide is not a p53 domain or peptide. 7. The multimer according to any one of items 1 to 6, wherein the engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, and the second type of protein domain or peptide is different from the first protein domain or peptide. 8. The multimer according to any one of items 1 to 7, wherein the domain is an immunoglobulin superfamily domain. 9. The multimer according to any one of items 1 to 8, wherein the domain or peptide is an antibody variable or constant domain, a TCR variable or constant domain, an incretin, an insulin peptide, or a hormone peptide. 10. The multimer according to any one of items 1 to 9, wherein the multimer comprises first, second, third and fourth identical copies of the engineered polypeptide, wherein the polypeptide comprises one (but not more than one), two (but not more than two), or more copies of the TD and the protein domain or peptide. 11. The multimer according to any one of items 1 to 10, wherein the engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD. 12. The multimer of item 11, wherein the polypeptide comprises (from N-terminus to C-terminus): (i) V1-optional linker-NHR2 TD; (ii) V1-optional linker-NHR2 TD-optional linker-V2; or (iii) V1-optional linker-V2-optional linker-NHR2 TD, wherein V1 and V2 are TCR variable domains and are the same or different, or wherein V1 and V2 are antibody variable domains and are the same or different. 13. The multimer according to item 12, wherein V1 and V2 are antibody single variable domains. 14. The multimer of item 11, wherein each engineered polypeptide comprises (from N-terminal to C-terminal direction) V1-optional linker-TD, wherein V1 is an antibody or TCR variable domain, each engineered polypeptide is paired with a respective second engineered polypeptide, and a polypeptide comprising V2, wherein V2 is an antibody or TCR variable domain that pairs with V1 to form an antigen or pMHC binding site, respectively, and optionally one polypeptide comprises an antibody Fc or comprises an antibody CH1, and the other polypeptide comprises an antibody CL paired with CH1. 15. The multimer according to any one of items 1 to 14, wherein the TD comprises (i) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 10 or 126, or (ii) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 120 or 123. 16. The multimer is selected from the group consisting of ReoPro™, abciximab, Rituxan™, rituximab, Zenapax™, daclizumab, Simulect™, basiliximab, Synagis™, palivizumab, Remicade™, infliximab, Herceptin™, Mylotarg™, gemtuzumab, Campath™, alemtuzumab, Zevalin™, ibritumomab, Hu mira™, adalimumab, Xolair™, omalizumab, Bexxar™, tositumomab, Raptiva™, efalizumab, Erbitux™, cetuximab, Avastin™, bevacizumab, Tysabri™, natalizumab, Actemra™, tocilizumab, Vectibix™, panitumumab, Lucentis™, ranibizumab, Soliris™, Eculizumab, Cimzia™, certolizumab, Simponi™, golimumab, Ilaris™, canakinumab, Stelara™, ustekinumab, Arzerra™, ofatumumab, Prolia™, denosumab, Numax™, motavizumab, ABThrax™, raxibacumab, Benlysta™, belimumab, Yervoy™, iplizumab, Adcetri 16. The multimer of any one of items 1 to 15, comprising a tetramer, octamer, dodecamer, hexadecamer, or 20-mer (e.g., a tetramer or octamer) of the antigen-binding site of an antibody selected from the group consisting of: ATZYME™, brentuximab, Vedotin™, Perjeta™, pertuzumab, Kadcyla™, ado-trastuzumab, Keytruda™, Opdivo™, Gazyva™, and obinutuzumab. 17. An isolated tetramer, octamer, dodecamer, hexameric or 20-mer of a TCR binding site, insulin peptide, incretin peptide or peptide hormone, or a plurality of said tetramers or octamers. 18. A multimer, tetramer, octamer, dodecamer, hexadecamer or 20amer, (a) soluble in aqueous solution; (b) capable of being secreted by eukaryotic cells; and / or (c) A multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer (e.g., a tetramer or octamer) according to any one of items 1 to 17, which is an expression product of a eukaryotic cell. 19. The following (a) a TCR V domain or TCR binding site, wherein the tetramer, octamer, dodecamer, hexameric or 20-mer is soluble in aqueous solution; (b) an antibody single variable domain, wherein the tetramer, octamer, dodecamer, hexameric or 20-mer is soluble in aqueous solution; (c) a TCR V domain or TCR binding site, wherein a tetramer, octamer, dodecamer, hexameric or 20-mer can be expressed intracellularly and / or extracellularly by HEK293 cells; or (d) A tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., a tetramer or octamer) of an antibody variable domain, wherein the tetramer, octamer, dodecamer, hexadecamer or 20-mer can be expressed intracellularly and / or extracellularly by HEK293 cells. 20. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of items 1 to 19, wherein the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is bispecific for antigen or pMHC binding. 21. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of items 1 to 20, wherein the domains are identical. 22. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to any one of items 1 to 21, wherein the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises eukaryotic glycosylation. 23. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer according to item 22, wherein the cell is a HEK293 cell. 24. A plurality of multimers, tetramers, octamers, dodecamers, hexadecamers or 20-mers (e.g., tetramers or octamers) according to any one of items 1 to 23. 25. A pharmaceutical composition comprising a multimer, tetramer, octamer, dodecamer, hexadecamer, or 20-mer (e.g., a tetramer or octamer) according to any one of items 1 to 24, and a pharmaceutically acceptable carrier, diluent, or excipient. 26. A cosmetic, food, beverage, cleaning product, or detergent comprising the multimer, tetramer, octamer, dodecamer, hexadecamer, or 20amer (e.g., tetramer or octamer) according to any one of items 1 to 24. 27. The engineered (and optionally isolated) multimeric, tetrameric, octameric, dodecamer, hexadecameric or 20-meric engineered polypeptide or monomer (optionally isolated) according to any one of items 1 to 26. 28. An engineered (and optionally isolated) polypeptide (P1) comprising (in the N-terminal to C-terminal direction): (a) TCR V1-TCR C1-antibody CH1-optional linker-TD, (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) TCR V1-TCR C1-antibody CH1-optional linker-TD, wherein V1 is Vδ and C1 is Cδ; or (b) TCR V1-antibody CH1-optional linker-TD, (i) V1 is Vα, (i) V1 is Vβ, (iii) V is V, or (iv) TCR V1-antibody CH1-optional linker-TD, wherein V1 is Vδ; or (c) antibody V1-antibody CH1-optional linker-TD, (i) V1 is VH, or (ii) antibody V1-antibody CH1-optional linker-TD, wherein V1 is VL; or (d) antibody V1-any antibody CH1-antibody Fc-any linker-TD; (i) V1 is VH, or (ii) antibody V1-optional antibody CH1-antibody Fc-optional linker-TD, wherein V1 is VL; or (E) Antibody V1-antibody CL-optional linker-TD, (i) V1 is VH, or (ii) antibody V1-antibody CL-optional linker-TD, wherein V1 is VL; or (f) TCR V1-TCR C1-optional linker-TD, (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Cβ; (iii) V1 is Vγ and C1 is Cγ; or (iv) An engineered (and optionally isolated) engineered polypeptide (P1) comprising TCR V1-TCR C1-optional linker-TD, where V1 is Vδ and C1 is Cδ. 29. The engineered polypeptide P1 is paired with a further polypeptide (P2), wherein P2 is (in the N-terminal to C-terminal direction): (g) TCR V2-TCR C2-antibody CL, wherein P1 is according to (a) described in item 28, (i) If P1 conforms to (a)(ii), then V2 is Vα and C2 is Cα; (ii) If P1 conforms to (a)(i), then V2 is Vβ and C2 is Cβ; (iii) if P1 conforms to (a)(iv), then V2 is Vγ and C2 is Cγ; or (iv) TCR V2-TCR C2-Antibody CL, where V2 is Vδ and C2 is Cδ, if P1 is according to (a)(iii); or (h) TCR V2-antibody CL, wherein P1 is according to (b) described in item 28, (i) If P1 obeys (b)(ii), then V2 is Vα; (ii) If P1 conforms to (b)(i), then V2 is Vβ; (iii) if P1 conforms to (b)(iv), then V2 is Vγ; or (iv) TCR V2-Antibody CL, where V2 is Vδ if P1 is according to (b)(iii); or (i) antibody V2-CL, wherein P1 is according to (c) described in item 28; (i) If P1 complies with (c)(ii), then V2 is VH; (ii) antibody V2-CL, where V2 is VL when P1 is according to (c)(i); or (j) antibody V2-any CL, wherein P1 is according to (d) described in item 28; (i) If P1 complies with (d)(ii), then V2 is VH; (ii) antibody V2-any CL, where V2 is a VL if P1 complies with (d)(i); or (k) antibody V2-CH1, wherein P1 is according to (e) of item 28; (i) If P1 complies with (e)(ii), then V2 is VH; (ii) antibody V2-CH1, wherein V2 is VL when P1 is according to (e)(i); or (l) TCR V2-TCR C2, wherein P1 is in accordance with (f) described in item 28; (i) If P1 conforms to (f)(ii), then V2 is Vα and C2 is Cα; (ii) If P1 conforms to (f)(i), then V2 is Vβ and C2 is Cβ; (iii) if P1 conforms to (f)(iv), then V2 is Vγ and C2 is Cγ; or (iv) The polypeptide of item 28, comprising TCR V2-TCR C2, wherein when P1 is according to (f)(iii), V2 is Vδ and C2 is Cδ. 30. A multimer or a plurality of multimers of P1 as defined in item 28 or P1 paired with P2 as defined in item 29, wherein the multimer is a multimer according to any one of items 1 to 24. 31. A nucleic acid encoding an engineered polypeptide or monomer according to any one of items 27 to 29, optionally wherein the nucleic acid is comprised by an expression vector for expressing the polypeptide. 32. A eukaryotic host cell comprising the nucleic acid or vector according to item 31 for the intracellular and / or secretory expression of a multimeric, tetrameric, octameric, dodecamer, hexadecamer or 20-mer (e.g., tetrameric, octameric) engineered polypeptide or monomer according to any one of items 1 to 24. 33. Use of the nucleic acid or vector according to item 31 in a method for producing a protein multimer to produce a multimer that is expressed and / or secreted in a cell, the method comprising expressing and / or secreting the multimer from a eukaryotic cell containing the nucleic acid or vector. 34. Use of the nucleic acid or vector according to item 31 in a method for producing a protein multimer to produce a glycosylated multimer in a eukaryotic cell containing the nucleic acid or vector. 35. A mixture comprising: (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of items 27 to 29; and (ii) a multimer, monomer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (e.g., a tetramer or octamer) as defined in any one of items 1 to 24. 36. The mixture of item 35, wherein the cell line is in a medium containing a secreted pr...

Claims

1. A polypeptide comprising: (a) an antibody Fc region, the Fc region comprising an antibody CH2 and an antibody CH3; (b) a self-associating multimerization domain (SAM), A polypeptide, wherein the polypeptide comprises an antibody hinge sequence lacking a core hinge region, and the SAM is a self-associating tetramerization domain (TD).

2. The hinge sequence is (i) a core hinge CXXC amino acid sequence, where X is any amino acid or where each amino acid X is selected from P, R and S; and / or (ii) A polypeptide according to claim 1, lacking an upper hinge amino acid sequence.

3. (a) the CXXC sequence is selected from SEQ ID NOs: 180-182; or (b) the polypeptide of claim 2(i), wherein the hinge sequence lacks the amino acid sequence of SEQ ID NO:183-187.

4. The hinge sequence is (a) the amino acid sequence APELLGGPSV (SEQ ID NO: 163), or PAPELLGGPSV (SEQ ID NO: 164); (b) the amino acid sequence APPVAGPSV (SEQ ID NO: 165), or PAPPVAGPSV (SEQ ID NO: 166); (c) the amino acid sequence APEFLGGPSV (SEQ ID NO: 175), or PAPEFLGGPSV (SEQ ID NO: 176); (d) the amino acid sequence EPKSCDKTHT[P]APELLGGPSV (SEQ ID NO: 167, or 168) (the P in brackets is optional); (e) the amino acid sequence ERKCCVE[P]APPVAGPSV (SEQ ID NO: 169, or 170) (the P in brackets is optional); (f) the amino acid sequence ELKTPLGDTTHT[P]APELLGGPSV (SEQ ID NO: 171, or 172) (the P in brackets is optional); (g) the amino acid sequence EPKSCDTPPP[P]APELLGGPSV (SEQ ID NO: 173, or 174) (the P in brackets is optional); or (h) The polypeptide according to any one of claims 1 to 3, comprising the amino acid sequence ESKYGPP[P]APEFLGGPSV (SEQ ID NO: 177 or 178) (the P in brackets is optional).

5. The CH2 and CH3 are (a) human IgG1 CH2 and CH3 domains; (b) human IgG2 CH2 and CH3 domains; (c) human IgG3 CH2 and CH3 domains, or (d) a polypeptide according to any one of claims 1 to 4, comprising human IgG4 CH2 and CH3 domains.

6. (a) the CH2 domain comprises a human IgG1 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 180, and optionally the hinge sequence lacks the upper hinge region amino acid sequence EPKSCDKTHT (SEQ ID NO: 183); (b) the CH2 domain comprises a human IgG2 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 180, and optionally the hinge sequence lacks the upper hinge region amino acid sequence ERKCCVE (SEQ ID NO: 184); (c) the CH2 domain comprises a human IgG3 CH2 domain, and the core hinge region amino acid sequence is SEQ ID NO: 181, and optionally the hinge sequence lacks the upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) or the upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186); or (d) the polypeptide of any one of claims 1 to 5, wherein the CH2 domain is a human IgG4 CH2 domain, the core hinge region amino acid sequence is SEQ ID NO: 182, and optionally the hinge sequence lacks the upper hinge region amino acid sequence ESKYGPP (SEQ ID NO: 187).

7. 7. The polypeptide of claim 1, wherein the polypeptide comprises (from N-terminal to C-terminal) the Fc region and the SAM, and the Fc region comprises (from N-terminal to C-terminal) the hinge sequence, CH2 domain and CH3 domain.

8. 8. The polypeptide of claim 1, wherein the polypeptide comprises one or more epitope binding sites and optionally comprises an antibody variable domain capable of specifically binding to a first epitope.

9. 9. The polypeptide of claim 8, wherein the variable domain is selected from an antibody single variable domain, VH and VL, or the domain is constituted by an scFv.

10. The polypeptide comprises (in the N-terminal to C-terminal direction): (a) the variable domain, the SAM and the Fc region, (b) the Fc region, the SAM, and the variable domain; (c) the variable domain, the Fc region, and the SAM; (d) the SAM, the variable domain, and the Fc region; or (e) a polypeptide according to claim 8 or 9, comprising the SAM, the Fc region and the variable domain.

11. 11. The polypeptide of claim 8, 9 or 10, comprising a second antibody variable domain at the N-terminus or C-terminus of the SAM, the second variable domain capable of specifically binding to a second epitope, the first and second epitopes being the same or different.

12. The polypeptide of any one of claims 1 to 11, wherein the TD is a p53, p63 or p73 TD; or the TD is an NHR2 TD; or the TD comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or 126.

13. (i) the polypeptide comprises (in the N-terminal to C-terminal direction): A. a first antibody single variable domain (dAb), an optional linker, and said SAM; B. A first antibody single variable domain, an optional linker, said SAM and a second antibody single variable domain; C. A first scFv, an optional linker and the SAM; D. A first scFv, an optional linker, the SAM and a second scFv; E. a first antibody single variable domain, an optional linker, said SAM and a first scFv; F. A first scFv, an optional linker, said SAM and a first antibody single variable domain; G. a first antibody variable domain, an optional first linker, a first antibody constant domain, a second optional linker, and said SAM; H. The SAM, an optional linker, and a first antibody single variable domain; I. The SAM, an optional linker, and a first scFv; J. The SAM, an optional linker, a first antibody constant domain, a second optional linker and a first antibody variable domain, or K. The SAM, an optional linker, a first antibody variable domain, a second optional linker, and a first antibody constant domain; or (ii) the polypeptide comprises (in the N-terminal to C-terminal direction): A. A dAb and said SAM, B. a first dAb, said SAM and a second dAb; C. a first scFv and the SAM; D. a first scFv, the SAM and a second scFv; E. a first scFv, the SAM and a first dAb; F. a first dAb, said Fc region and said SAM; G. a first scFv, the Fc region and the SAM; H. VH, CH1, the Fc region and the SAM, I. VL, CL, the Fc region and the SAM, J. A dAb, the SAM and the Fc region, K. an scFv, the SAM and the Fc region; L. VH, CH1, SAM and Fc regions; M. the VL, CL, the SAM and the Fc region; N. a first dAb, a second dAb and said SAM; O. VH, CH1 and the SAM, P. VL, CL and the SAM, Q. VH, CH1, said SAM and first dAb, R. VL, CL, the SAM and the first dAb, S. a first dAb, a second dAb, said SAM and a third dAb; T. a first dAb, a second dAb, said SAM and a first scFv; U. a first dAb, a second dAb, the SAM, a third dAb and a fourth dAb; V. a first dAb, said Fc region, said SAM and a second dAb; W. a first dAb, said Fc region, said SAM and a first scFv; X. a first dAb, a second dAb, the Fc region and the SAM; Y. a first dAb, a second dAb, the Fc region, the SAM and a third dAb; Z. a first dAb, a second dAb, the Fc region, the SAM and a first scFv, or AA. comprising a first dAb, a second dAb, the Fc region, the SAM, a third dAb and a fourth dAb; or (iii) the polypeptide comprises (in the C-terminal to N-terminal direction): A. A dAb and said SAM, B. a first dAb, said SAM and a second dAb; C. a first scFv and the SAM; D. a first scFv, the SAM and a second scFv; E. a first scFv, the SAM and a first dAb; F. a first dAb, said Fc region and said SAM; G. a first scFv, the Fc region and the SAM; H. VH, CH1, the Fc region, and the SAM; I. VL, CL, the Fc region and the SAM, J. A dAb, the SAM and the Fc region, K. an scFv, the SAM and the Fc region; L. the VH, CH1, SAM and Fc regions; M. the VL, CL, the SAM and the Fc region; N. a first dAb, a second dAb and said SAM; O. VH, CH1 and the SAM, P. VL, CL and the SAM, Q. VH, CH1, said SAM and first dAb, R. VL, CL, the SAM and the first dAb, S. a first dAb, a second dAb, said SAM and a third dAb; T. a first dAb, a second dAb, said SAM and a first scFv; U. a first dAb, a second dAb, the SAM, a third dAb and a fourth dAb; V. a first dAb, said Fc region, said SAM and a second dAb; W. a first dAb, said Fc region, said SAM and a first scFv; X. a first dAb, a second dAb, the Fc region and the SAM; Y. a first dAb, a second dAb, the Fc region, the SAM and a third dAb; Z. a first dAb, a second dAb, the Fc region, the SAM and a first scFv, or AA. The polypeptide of any one of claims 1 to 12, comprising a first dAb, a second dAb, the Fc region, the SAM, a third dAb and a fourth dAb.

14. A tetramer of the polypeptide of any one of claims 1 to 13, optionally wherein said tetramer is for medical use.

15. A pharmaceutical composition comprising a polypeptide or tetramer according to any one of claims 1 to 14.

16. A nucleic acid encoding a polypeptide according to any one of claims 1 to 14, optionally wherein said nucleic acid is constituted by a eukaryotic cell or vector.

17. 16. A method of antigen binding, the method comprising combining the antigen with the tetramer of claim 14 or the composition of claim 15, wherein the antigen is bound by a polypeptide of the tetramer, and optionally the method comprises isolating the tetramer bound to the antigen.

18. A method for producing a composition comprising a plurality of polypeptides according to any one of claims 1 to 14, the method comprising: providing a eukaryotic host cell according to claim 16; culturing the host cell; allowing expression and secretion from the cell of a tetramer of the polypeptides; and, optionally, isolating or purifying the tetramer.

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