Multimers, tetramers and octamers

Abstract

To provide higher avidity of antigen binding or enhance biological or binding activity, such as for providing bi- or multi-specific targeting or interaction with target ligands in vivo or in vitro, in multimers of effector domains that have recognized utility in medical and non-medical applications for combining and multiplying the activity and presence of effector domains.SOLUTION: The invention discloses multimers such as tetramers of polypeptides and tetramers and octamers of effector domains, such as antigen binding sites (e.g., antibody or TCR binding sites that specifically bind to antigen or pMHC, or variable domains thereof) or peptides such as incretin, insulin or hormone peptides.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to tetramers and multimers, such as tetramers and octamers, of polypeptides, such as effector domains (such as antigen binding sites (e.g., antibodies or TCR binding sites that specifically bind to an antigen or pMHC, or variable domains thereof)) or 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 combine and multiply the activities and presence of effector domains, e.g., to enhance biological or binding activity, such as to provide higher avidity of antigen binding (e.g., for effector domains that are antibody or TCR binding domains) or to provide bi- or multi-specific targeting or interaction with target ligands in vivo or in vitro.

[0003] Multimerization domains that cause 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. Transcription factor p53 can be divided into different functional domains: N-terminal transactivation domain, proline-rich domain, DNA binding domain, tetramerization domain and C-terminal regulatory region. The tetramerization domain of human p53 extends from residues 325-356 and has a 4-helical bundle fold (Jeffrey et al., Science (New York, NY) 1995, 267(5203):1498-1502). The TRPM tetramerization domain is a short antiparallel coiled-coil tetramerization domain of member proteins 1-8 of the transient receptor potential cation channel subfamily M. It is held together by extensive core packing and interchain polar 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 BTB domain of potassium channels. This domain can be found at the N-terminus of voltage-gated potassium channel proteins and represents a cytoplasmic tetramerization domain (T1) involved in the assembly of alpha subunits into functional tetrameric channels (Bixby et al., Nature Structural Biology 1999, 6(1):38-43). This domain can also be 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 produced using various multimerization techniques, including biotin, dHLX, ZIP and BAD domains as well as p53 (Thie et al., Nature Boitech., 2009:26, 314-321). Biotin, which is efficient to produce, is a bacterial protein that induces an immune response in humans.

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

[0006] Nine isoforms of p53 naturally exist and are expressed in a wide range of normal tissues in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues, but is not detected in the brain, lung, prostate, muscle, fetal brain, spinal cord, and fetal liver. Isoform 3 is expressed in most normal tissues, but is not detected in the lung, spleen, testis, fetal brain, spinal cord, and fetal liver. Isoform 7 is expressed in most normal tissues, but is not detected in the prostate, uterus, skeletal muscle, and breast. Isoform 8 is only detected in the colon, bone marrow, testis, fetal brain, and intestine. Isoform 9 is expressed in most normal tissues, but is not detected in the brain, heart, lung, fetal liver, salivary gland, breast, or intestine. Summary of the Invention

[0007] The present invention provides the following:

[0008] 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) that associate together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of said protein domain or peptide.

[0009] In the second configuration An isolated tetramer or octamers of a TCR binding site, an insulin peptide, an incretin peptide or a peptide hormone, or a plurality of said tetramers or octamers. An isolated tetramer or octamer of an antibody binding site or antibody variable domain (e.g., a single variable domain), or a plurality of said tetramers or octamers. In one example, the tetramer or octamer is soluble in an aqueous solution (e.g., aqueous eukaryotic cell culture medium). In one example, the tetramer or octamer is expressible in a eukaryotic cell. Examples are provided below.

[0010] In the third configuration (a) a tetramer or octamer of a TCR V domain or TCR binding site that is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (b) a tetramer or octamer of an antibody single variable domain that is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (c) a tetramer or octamer of a TCR V domain or a TCR binding site, which is expressible intracellularly and / or extracellularly by HEK293 cells; or (d) A tetramer or octamer of an antibody variable domain (e.g., an antibody single variable domain), which can be expressed intracellularly and / or extracellularly by HEK293 cells.

[0011] In the fourth configuration Multimeric, tetrameric or octameric engineered polypeptides or monomers of the invention.

[0012] In the fifth configuration (From N-terminus to C-terminus) (a) TCR V1-TCR C1-antibody CH1 (e.g., IgG CH1)-optionally a 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) V1 is Vδ and C1 is Cδ; or (b) TCR V1-antibody CH1 (e.g., IgG CH1)-optionally linker-TD; (i) V1 is Vα, (ii) V1 is Vβ, (iii) V1 is Vγ, or (iv) V1 is Vδ, or (c) antibody V1-antibody CH1 (e.g., IgG CH1)-optional linker-TD; (i) V1 is VH, or (ii) V1 is a VL (e.g., Vλ or Vκ); or (d) antibody V1 - optional antibody CH1 (e.g., IgG CH1) - antibody Fc (e.g., IgG Fc) - optional linker - TD; (i) V1 is VH, or (ii) V1 is a 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) V1 is a 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) V1 is Vδ and C1 is Cδ; An engineered (and optionally isolated) engineered polypeptide (P1).

[0013] In the sixth configuration A nucleic acid encoding an engineered polypeptide or monomer of the invention, optionally comprised by an expression vector for expressing said polypeptide.

[0014] In the seventh configuration Use of a nucleic acid or vector of the present invention in a method for producing a protein multimer to produce a multimer that is expressed and / or secreted within a cell, the method comprising expressing the multimer in a eukaryotic cell containing the nucleic acid or vector and / or secreting the multimer from the eukaryotic cell.

[0015] In the eighth configuration (a) a method of producing a TCR V domain multimer comprising 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 isolating a plurality of said multimers; (b) a method for producing an antibody V domain multimer comprising 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 isolating a plurality of said multimers; (c) a method for producing an incretin peptide (e.g., GLP-1, GIP or insulin) multimer, comprising 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 isolating a plurality of said multimers; or (d) A method for producing a peptide hormone multimer, 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.

[0016] In the ninth configuration Use of a nucleic acid or vector of the invention in a method for producing a protein multimer to produce a glycosylated multimer in a eukaryotic cell containing said nucleic acid or vector.

[0017] 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 the tetramer in higher yield than monomeric and / or dimeric polypeptides.

[0018] In the eleventh 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 tetramers in higher yield than monomeric and / or dimeric polypeptides, wherein the engineered polypeptide comprises one or more copies of said 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).

[0019] In the 12th configuration 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 multiple tetramers that are not mixed with monomers, dimers, or trimers.

[0020] In the thirteenth configuration A eukaryotic host cell comprising a nucleic acid or vector for the intracellular and / or secretory expression of the multimers, tetramers, octamers, engineered polypeptides or monomers of the invention.

[0021] In the fourteenth configuration

[0022] 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 in a mixture with monomers, dimers or trimers, wherein the engineered polypeptide comprises one or more copies of said 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 fifteenth configuration (i) a TCR extracellular domain, (ii) an immunoglobulin constant domain, and (iii) NHR2 multimerization domain of ETO A multivalent heterodimeric soluble T cell receptor capable of binding to the pMHC complex, comprising:

[0024] In the 16th configuration (i) an immunoglobulin variable domain, and (ii) the NHR2 multimerization domain of ETO A polymeric immunoglobulin comprising:

[0025] In the seventeenth configuration 1. A method for assembling a soluble multimeric polypeptide comprising the steps of: (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. A method comprising:

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

[0027] In the 19th configuration A method for increasing 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 a polypeptide of the invention and comprising one or more effector domains, and the method optionally comprising isolating the expressed tetramer.

[0028] The present invention also provides pharmaceutical compositions, cosmetics, foodstuffs, beverages, cleaning products, detergents comprising the multimers, tetramers or octamers of the present invention. [Brief description of the drawings]

[0029] [Figure 1] Schematic representation of the stepwise self-assembly of tetravalent heterodimeric soluble TCR protein complexes via monomers and homodimers, assisted by the NHR2 tetramerization domain. [Diagram 2] Schematic representation of the stepwise self-assembly of an octavalent heterodimeric soluble TCR protein complex via 2 monomers and 2 homodimers, assisted by the NHR2 tetramerization domain and an immunoglobulin hinge domain. [Diagram 3] FIG. 1 is a schematic diagram of the a and β chain domain configurations used to express and assemble the ts-NY-ESO-1 TCR. [Figure 4] FIG. 1 is a schematic diagram of the a- and β-chain domain configuration used to express and assemble the os-NY-ESO-1 TCR. [Diagram 5] Amino acid sequences of the a and β chains of the ts-NY-ESO-1 TCR protein complex. Alternating domain amino acid sequences are underlined. [Figure 6] Amino acid sequences of the a- and β-chains of the os-NY-ESO-1 TCR protein complex. Alternating domain amino acid sequences are underlined. [Figure 7] FIG. 1 is a schematic diagram of the a- and β-chain domain configuration used to express and assemble the ts-NY-ESO-1 TCR-IL2 fusion protein complex. [Figure 8] FIG. 1 is a schematic diagram of the a- and β-chain domain configuration used to express and assemble the os-NY-ESO-1 TCR-IL2 fusion protein complex. [Figure 9] Amino acid sequences of the a and β chains of the ts-NY-ESO-1 TCR-IL2 fusion protein complex. Alternating domain amino acid sequences are underlined. [Figure 10] Amino acid sequences of the a and β chains of the os-NY-ESO-1 TCR-IL2 fusion protein complex. Alternating domain amino acid sequences are underlined. [Figure 11-1] FIG. 1 is a schematic illustrating the stepwise self-assembly of tetravalent single domain antibody (dAb) complexes via monomers and homodimers, assisted by the NHR2 tetramerization domain. [Figure 11-2] Schematic diagram of domain configuration for assembly of tetravalent dAbs, including linker and NHR2 domains. [Figure 12-1] Schematic representation of the stepwise self-assembly of tetravalent Fab complexes via monomers and homodimers assisted by the NHR2 tetramerization domain. [Figure 12-2] Schematic diagram of the domain arrangement for assembly of a tetravalent Fab, including the linker and NHR2 domains in the heavy chain, and the light chain variable and constant domains. [Figure 13-1] Schematic representation of 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 13-2] Schematic diagram of the domain arrangement for assembly of an octavalent Fab, including the hinge, linker and NHR2 domains in the heavy chain, and the light chain variable and constant domains. [Figure 14] This is a diagram of the Quad 16 and Quad 17. [Figure 15-1] The monomer sequences and organization of (A) Quad 16 and (B) Quad 17 are shown. [Figure 15-2] Same as above [Figure 15-3] Same as above [Figure 15-4] Same as above [Figure 16] Analysis of secreted proteins using anti-Ig Western blot is shown: (A) PAGE gel under SDS denaturing conditions-16=Quad16; 17=Quad17; and (B) PAGE gel under native (i.e., non-denaturing) conditions-16=Quad16; 17=Quad17. [Figure 17] Western blots prepared from denaturing SDS-PAGE gels probed with anti-human IgG HRP detection antibody. (A) Protein samples from Quad 3 and Quad 4 were prepared from whole cell extracts and run in lanes 1 and 2, respectively. The expected Mw for Quad 3 and Quad 4 are 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quad 12 and Quad 13 were prepared from whole cell extracts and run in lanes 1 and 2, respectively. The expected Mw for Quad 12 and Quad 13 are 47.8 and 48.1 kDa, respectively. [Figure 18]Western blots prepared from denaturing SDS-PAGE gels probed with anti-human IgG HRP detection antibody. (A) Protein samples from Quad 3 and Quad 4 were prepared by concentrating cell supernatants and run in lanes 1 and 2, respectively. The expected Mw for Quad 3 and Quad 4 are 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quad 12 and Quad 13 were prepared by concentrating cell supernatants and run in lanes 1 and 2, respectively. The expected Mw for Quad 12 and Quad 13 are 47.8 and 48.1 kDa, respectively. [Figure 19-1]Western blots prepared from denaturing SDS-PAGE gels probed with anti-HIS HRP detection antibody. (A) Protein samples from Quad 14, Quad 15, Quad 18 and Quad 19 were prepared from whole cell extracts and run in lanes 1-4, respectively. The predicted Mw for Quad 14, Quad 15, Quad 18 and Quad 19 are 22.0, 22.3, 37.4 and 37.7 kDa, respectively. (B) Protein samples from Quad 23, Quad 24, Quad 26 and Quad 27 were prepared from whole cell extracts and run in lanes 1-4, respectively. The predicted Mw for Quad 23, Quad 24, Quad 26 and Quad 27 are 32.1, 32.4, 33.7 and 34.0 kDa, respectively. (C) Protein samples from Quad 34, and Quad 38 were prepared from whole cell extracts and run in lanes 1-2, respectively. The expected Mw for Quad 34, and Quad 38 is 25.5 and 25.4 kDa, respectively. (D) Protein samples from Quad 40, Quad 42, Quad 44, and Quad 46 were prepared from whole cell extracts and run in lanes 1-4, respectively. The expected Mw for Quad 40, Quad 42, Quad 44, and Quad 46 is 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 19-2] Same as above [Figure 20](A) Western blot prepared from a denaturing SDS-PAGE gel and probed with anti-human IgG HRP detection antibody. Protein samples from Quad 14 and Quad 15 prepared from whole cell extracts were run in lanes 1 and 2, respectively. The expected Mw for Quad 14 and Quad 15 is 22.0 and 22.3 kDa, respectively. (B) Western blot prepared from a native PAGE gel probed with anti-human IgG HRP detection antibody. Lanes 1 and 2 contain protein samples from Quad 14 and Quad 15 prepared from whole cell extracts. [Figure 21-1] Quad polypeptides fused to leader and tag sequences. When present, the linker is G4S (one G4S only). * indicates TCR constant domain with introduced cysteine ​​residue allowing formation of S-S bond between alpha and beta chains of TCR. Human IgG1 hinge was used. All C regions are human. TCR V domains are specific for NY-ESO-1. GFP=Green Fluorescent Protein. [Figure 21-2] Same as above [Figure 21-3] Id. 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 PREFERRED EMBODIMENTS

[0030] Detailed Description The present invention relates to multimers, such as tetramers and octamers of effector domains of polypeptides, such as antigen binding sites (e.g., antibodies or TCR binding sites that specifically bind antigens or pMHC, or variable domains thereof) or peptides, such as incretins, insulin or hormone peptides. In embodiments, the multimers of the invention are useful for production in eukaryotic systems and can be secreted from eukaryotic cells in a soluble form useful for various industrial applications, such as the production of pharmaceuticals, diagnostics, imaging agents, detergents, etc. Higher order multimers, such as tetramers or octamers of effector domains or peptides, are useful for increasing antigen or pMHC binding avidity. This may be useful for the production of effective drugs or for increasing the sensitivity of diagnostic reagents that include the multimers, tetramers or octamers. An additional or alternative advantage is the increased half-life in vivo when the multimers of the invention are administered to a human or animal subject, for example to treat or prevent a disease or condition in the subject. Usefully, the present invention can also provide multispecific (e.g., bi- or trispecific) multivalent binding proteins. The specificity can be with respect to antigen or pMHC binding specificity. By using a single engineered polypeptide comprising a binding domain or peptide, the present invention provides a means to produce multivalent (e.g., bispecific) proteins, usefully in certain instances with high purity. The use of a single type of engineered polypeptide monomer avoids the problem of mixed products seen when two or more different polypeptide species are used to produce multi-(e.g., bi)specific or multivalent proteins.

[0031] The present invention provides the following provisions, aspects and concepts. Any provision herein can be combined with any aspect or concept herein. Any aspect herein can be combined with any concept herein.

[0032] 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, said multimer being multimerized by first, second, third and fourth self-associating tetramerization domains (TDs) that associate together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of said protein domain or peptide.

[0033] In one example, each TD is any one of proteins 1-119 listed in Table 2. In one example, each TD is a p53 TD or a homolog or ortholog thereof. In one example, each TD is an NHR2 TD or a homolog or ortholog thereof. In one example, each TD is a p63 TD or a homolog or ortholog thereof. In one example, each TD is a p73 TD or a homolog or ortholog thereof. In one example, each TD is not an NHR2 TD. In one example, each TD is not a p53 TD. In one example, each TD is not a p63 TD. In one example, each TD is not a p73 TD. In one example, each TD is not a p53, 63 or 73 TD. In one example, each TD is not an NHR2, p53, 63 or 73 TD.

[0034] By "associated together" is meant that the TD of 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 is soluble in an 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., human NHR TD, p53 TD, p63 TD and p73 TD) or their orthologues or homologues.

[0035] In one example, the TD is not p53 TD (or a homolog or orthologue thereof), e.g., not human p53 TD (or a homolog or orthologue thereof). In one example, the TD is NHR2 TD or a homolog or orthologue thereof, but not p53 TD or a homolog or orthologue thereof. In one example, the TD is human NHR2 TD or a homolog or orthologue thereof, but not human p53 TD or a homolog or orthologue thereof. In one example, the TD is human NHR2. In one example, 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 one example, the domain or peptide is not naturally constituted by a polypeptide that also includes the NHR2 TD.

[0036] In one example, all of the domains of the polypeptide are human.

[0037] The engineered polypeptide may comprise one or more copies of said domain or peptide at the N-terminus of said copy of said TD. Additionally or alternatively, the engineered polypeptide may comprise one or more copies of said domain or peptide at the C-terminus of said copy of said TD. In one example, the engineered polypeptide comprises a first said domain or peptide and a TD, wherein the first domain or peptide is separated from the TD 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, and no further said domain or peptide is present between the first domain or peptide and the TD.

[0038] In one example, a multimer (e.g., a tetramer of said 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 said domain or peptide. In one example, each TD and each of said domains or peptides is human.

[0039] In one example, the multimer, tetramer or octamer comprises first, second, third and fourth identical copies of an engineered polypeptide comprising a TD and one (but not more than one), two (but not more than two) or more copies of said protein domain or peptide.

[0040] In some embodiments, by requiring only one engineered polypeptide to form the multimers, tetramers or octamers of the invention, the invention advantageously provides formats that can be readily isolated in a pure (or highly purified, i.e., greater than 90, 95, 96, 97, 98 or 99% pure) format, as well as methods for producing such formats in pure (or highly purified) form. Purity is indicated by the multimers of the invention being free of admixture in a composition with any other multimers or polypeptide monomers, or comprising greater than 90, 95, 96, 97, 98 or 99% of the species in a composition comprising the multimers of the invention and other multimers and / or polypeptide monomers, including engineered polypeptides. Thus, in these embodiments, admixture of different types of polypeptides is avoided or minimized. Thus, this also advantageously provides for multiple multimers (e.g., multiple tetramers or octamers) comprising only one (and not more than one) type of engineered polypeptide, which are monospecific (but multivalent) for antigen binding, or alternatively, which are bi- or multispecific for antigen binding. Thus, the invention provides a plurality of multimers (e.g. a plurality of tetramers or octamers, 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. Where antigen binding is described herein, this may alternatively be pMHC binding where the domain is a TCR V domain. Advantageously, the plurality is in pure form (i.e. not mixed with multimers comprising more than one type of polypeptide monomer, such as tetramers or octamers). In one example, the multimer comprises at least two different types of antigen binding sites. In one example, the multimer is bispecific, trispecific or tetraspecific. In one example, the multimer has a valency of antigen binding sites or pMHC binding sites of 4, 6, 8, 10 or 12, preferably 4 or 8.

[0041] In one example, the peptide MHC (pMHC) is a class I or class II pMHC.

[0042] For example, the term "specifically binds" as used herein with respect to a domain, antibody or binding site means that the domain, antibody or binding site recognizes a particular antigen (or pMHC) with a binding affinity of 1 mM or less as determined by SPR.

[0043] Target binding capacity, specificity and affinity (KD (also called Kd), K off and / or K. on) 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 physiological pH, for example, about pH 7 or pH 7.6 (e.g., using Hepes buffered saline (also referred to as HBS-EP) at pH 7.6). In one embodiment, SPR is performed at physiological salt levels, for example, 150 mM NaCl. In one embodiment, SPR is performed in the presence of a detergent level of 0.05% by volume or less, for example, 0.05% P20 (polysorbate 20; e.g., Tween-20™) and 3 mM EDTA. In one example, SPR is performed in pH 7.6 buffer, 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA at 25° C. or 37° C. The buffer may contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25° C. or 37° C. HBS-EP can be obtained from Teknova Inc (California; catalog number H8022). In one example, affinity (e.g., affinity of VH / VL binding sites) is determined using SPR using any standard SPR instrument such as Biacore™ or by using ProteOn XPR36™ (Bio-Rad®). Binding data can be fitted to a unique 1:1 model using standard techniques, for example, using the model unique to ProteOn XPR36™ analysis software.

[0044] In one example, the multimer, tetramer or octamer of the present invention is an isolated multimer, tetramer or octamer. In one example, the multimer, tetramer or octamer of the present invention is composed of copies of the engineered polypeptide. Optionally, the multimer, tetramer or octamer of the present invention comprises four or eight, but not more than four or eight copies of the engineered polypeptide, respectively.

[0045] "Engineered" means that the polypeptide does not occur in nature, for example a protein domain or peptide is not naturally constituted by a polypeptide that also comprises said TD.

[0046] Each of said protein domains or peptides may be a biologically active domain or peptide (e.g. biologically active in humans or animals), for example a domain that specifically binds to an antigen or peptide MHC (pMHC), or a domain that 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. Alternatively, the domain or peptide is an inhibitor or enzyme, or an inhibitor of a biological function or pathway in humans or animals. Alternatively, the domain or peptide is an iron regulator. Thus, in one example, each of the protein domains or peptides is selected from an antigen or pMHC binding domain or peptide; a hormone; a carbohydrate, glucose or glucoregulator; an iron regulator; and an enzyme inhibitor. 2. The multimer of any preceding embodiment, which is a tetramer or octamer of said domain or peptide. 3. The multimer of any one of embodiments 1 or 2, comprising a tetramer or octamer of an immunoglobulin superfamily binding site (e.g. an antibody or TCR binding site, such as scFv or scTCR).

[0047] The immunoglobulin superfamily (IgSF) is a large protein superfamily of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features they share with immunoglobulins (also known as antibodies), and they all have domains known as immunoglobulin domains or folds. Members of the IgSF 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 usually associated with a role in the immune system.

[0048] The T cell receptor (TCR) domain can be Vα (e.g., paired with Vβ), Vβ (e.g., paired with Vα), Vy (e.g., paired with Vδ) or Vδ (e.g., paired with Vy). 4. The multimer of embodiment 3, wherein said binding site comprises a first variable domain paired with a second variable domain.

[0049] In a first example, the first and second variable domains are constituted by an engineered polypeptide, 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 a TD or lacking a TD).

[0050] Alternatively, the domain is a constant region domain. Alternatively, the domain is an FcAb. Alternatively, the domain is a non-Ig antigen binding site or is constituted by a non-Ig antigen binding site, such as an affibody.

[0051] Antigen-binding sites and effector domains In one example, the or each antigen-binding site (or effector domain) may be an antibody variable domain (e.g. VL or VH, an antibody single variable domain (domain antibody or dAb), a camelid VHH antibody single variable domain, a shark immunoglobulin single variable domain (NAV), a Nanobody™ or a camelized VH single variable domain); a T-cell receptor binding domain; an immunoglobulin superfamily domain; a jawless 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.

[0052] Further sources of antigen binding sites are the antibody variable domains and VH / VL pairs disclosed in WO2007024715, 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 a basis for epitope binding moieties for use in the present invention and for possible inclusion in the claims of this application.

[0053] A "domain" is a folded protein structure that has a tertiary structure independent of the rest of the protein. In general, a domain is responsible for a distinct functional property of a protein and can often be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or the domain. A "single antibody variable domain" is a folded polypeptide domain that contains sequences characteristic of an antibody variable domain. Thus, single antibody variable domains include complete antibody variable domains and modified variable domains, e.g., those in which one or more loops are replaced by sequences that are not characteristic of antibody variable domains, or 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.

[0054] 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 independent 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 domains that are not required for antigen binding by a single immunoglobulin variable domain (i.e., the immunoglobulin single variable domain binds to an antigen independent of additional variable domains). A "domain antibody" or "dAb" as the term is used herein is the same as an "immunoglobulin single variable domain" that can bind 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., those disclosed in WO 00 / 29004), nurse sharks and camelid VHH immunoglobulin single variable domains. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species such as camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of 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. NAV is another type of immunoglobulin single variable domain identified in cartilaginous fish such as the nurse shark. 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 Patent Publication No. 20050043519. CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold.The loops corresponding to the CDRs of an antibody can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have 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 hydrophobic small molecules such as steroids, villins, retinoids and lipids. They have a rigid beta-sheet secondary structure with numer of 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), U.S. Patent No. 7,250,297 and U.S. Patent Publication No. 20070224633. Affibodies are scaffolds derived from Protein A of Staphylococcus aureus that can be engineered to bind antigens. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818. Avimer™ are multi-domain proteins derived from the A-domain scaffold family. Natural domains of approximately 35 amino acids adopt a defined disulfide bond structure. Diversity is generated by shuffling the natural variations exhibited by the family of A-domains. 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 further 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 essential membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two a-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and beta-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 Patent Publication No. 20040132028. Fibronectin is a scaffold that can be engineered to bind antigens. Adnectin™ consists of a backbone of the natural amino acid sequence of the tenth domain of the 15 repeat units of human fibronectin type III (FN3). The three loops at one end of the β-sandwich can be engineered to allow Adnectin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435- 444 (2005), US Patent Application Publication No. 20080139791, WO 2005056764 and US Patent No. 6,818,418. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), which contains a constrained variable peptide loop inserted into the active site.For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring small proteins of 25-50 amino acids in length that contain 3-4 cysteine ​​bridges, examples of small proteins include KalataBI and conotoxins and knottins. The small proteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall fold of the small protein. For further details of engineered knottin domains, see WO2008098796. Other epitope-binding moieties and domains include proteins that 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), which are reviewed in Chapter 7 of Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). 5. The multimer of any preceding embodiment, wherein each polypeptide comprises a first and a second copy of said protein domain or peptide, said polypeptide comprising (in the N-terminal to C-terminal direction): (i) said first copy-TD-said second copy; (ii) TD-and said first and second copies; or (iii) said first and second copies-TD. 6. The multimer of any preceding embodiment, wherein said TD is an NHR2 TD and said domain or peptide is not an NHR2 domain or peptide; or, wherein said TD is a p53 TD and said domain or peptide is not a p53 domain or peptide. 7. The multimer of any preceding embodiment, wherein said engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, said second type of protein domain or peptide being different from said first protein domain or peptide.

[0055] For example, the polypeptide comprises, from the N-terminus, (i) P1-TD-P2; or (ii) TD-P1-P2, where P1 is a copy of a domain or peptide of a first type (i.e., a domain or peptide type of the multimer of embodiment 1) and P2 is a copy of said second type of domain or peptide. 8. The multimer of any preceding embodiment, wherein said domain is an immunoglobulin superfamily domain. 9. The multimer of any preceding embodiment, wherein said 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 preceding embodiment, wherein said multimer comprises first, second, third and fourth identical copies of said engineered polypeptide, said polypeptide comprising a TD and one (but not more than one), two (but not more than two) or more copies of said protein domain or peptide. 11. The multimer of any preceding embodiment, wherein said engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD. 12. The multimer of embodiment 11, wherein the polypeptide comprises (in the 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 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 (in the N-terminal to C-terminal direction) V1-optionally a linker-NHR2 TD, wherein V1 is an antibody or TCR variable domain, and each engineered polypeptide pairs with a respective second engineered 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 wherein one polypeptide comprises an antibody Fc, or comprises an antibody CH1, and the other polypeptide comprises an antibody CL that pairs with CH1. 15. The multimer of any preceding embodiment, wherein said 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; Cambusa™; path(trademark); Alemtuzumab; Zevalin(trademark); Ibritumomab; Humira(trademark); Adalimumab; Xolair(trademark); Omalizumab; Bexxar(trademark); Tositumomab; Raptiva(trademark); Efalizumab; Erbitux(trademark); Cetuximab; Avastin(trademark); Bevacizumab; Tysabri(trademark); Natalizumab; Actemra(trademark); Actemra(trademark); tocilizumab; Vectibix(trademark); panitumumab; Lucentis(trademark); ranibizumab; Soliris(trademark); eculizumab; Cimzia(trademark); certolizumab; Simponi(trademark); golimumab, Ilaris(trademark); canakinumab; Stelara(trademark); ustekinumab; Arzerra(trademark); ofatumumab; Prolia(TM); denosumab; Numax(TM); motavizumab; ABThrax(TM); raxibacumab; Benlysta(TM); belimumab; Yervoy(TM); ipilimumab; Adcetris(TM); brentuximab; Vedotin(TM); Perjeta(TM); pertuzumab; Kadcyla(TM); ado-trastuzumab;The multimer of any preceding embodiment comprising a tetramer or octamer of the antigen binding site of an antibody selected from the group consisting of Keytruda™, Opdivo™, Gazyva™, and obinutuzumab;

[0056] For example, said protein domain of the engineered polypeptide is a V domain (VH or VL) of an antibody binding site of an antibody selected from said group, and the multimer comprises a further V domain (VL or VH, respectively) which pairs with the V domain of the engineered polypeptide to form the antigen binding site of the selected antibody. Thus, advantageously, the invention provides tetramers or octamers of the binding site of said selected antibody, 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 humans or animals to which the multimer is administered to bind to the cognate antigen in vivo.

[0057] For example, the multimer, tetramer or octamer contains four 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 (Herceptin™). Alternatively, the antibody is an anti-CD38 antibody, an anti-TNFa 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, an anti-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 in WO2007024715, page 40, line 23 to page 43, line 23, the disclosure of which is incorporated herein by reference.

[0058] The binding site in the present invention may 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). For example, the binding site in the present invention may be a binding site of Eyelea™, Avastin™ or Lucentis™, for example, for eye or tumor medical applications in humans or animals. When the ligand or antigen is VEGF, the mutlimers, tetramers or octamers may be for the treatment or prevention of a cancer or eye condition (e.g., wet or dry AMD or diabetic retinopathy) in a human or animal subject, or as an inhibitor of angiogenesis. 17. An isolated tetramer or octamers of a TCR binding site, an insulin peptide, an incretin peptide or a peptide hormone, or a plurality of said tetramers or octamers.

[0059] A number of important peptide hormones are secreted by the pituitary gland. The anterior pituitary 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 secretes antidiuretic hormone, also called vasopressin, and oxytocin. Peptide hormones are produced by many different organs and tissues, including the heart (atrial natriuretic peptide (ANP) or atrial natriuretic factor (ANF)) and pancreas (glucagon, insulin, and somatostatin), gastrointestinal tract (cholecystokinin, gastrin), and adipose tissue stores (leptin). In one example, the peptide hormone of the invention is selected from prolactin, ACTH, growth hormone (somatotropin), vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin (e.g., selected from human prolactin, ACTH, growth hormone, vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin).

[0060] In one example, the incretin is a GLP-1, GIP or exendin-4 peptide.

[0061] The present invention provides, in embodiments, the following engineered tetramers and octamers: Isolated tetramers or octamers of incretins. An isolated tetramer or octamer of insulin peptide. Isolated tetramer or octamer of GLP-1 (Glucagon-like peptide-1 (GLP-1) peptide. An isolated tetramer or octamer of GIP (glucose-dependent insulinotropic polypeptide) peptide. An isolated tetramer or octamer of an exendin (eg, exendin-4) peptide. Isolated tetramers or octamers of peptide hormones. An isolated tetramer or octamer of prolactin or a prolactin peptide. An isolated tetramer or octamer of ACTH or ACTH peptide. An isolated tetramer or octamer of growth hormone or a growth hormone peptide. An isolated tetramer or octamer of vasopressin or vasopressin peptides. An isolated tetramer or octamer of oxytocin or an oxytocin peptide. An isolated tetramer or octamer of glucagon or a glucagon peptide. An isolated tetramer or octamer of insulin or an insulin peptide. An isolated tetramer or octamer of somatostatin or a somatostatin peptide. An isolated tetramer or octamer of cholecystokinin or a cholecystokinin peptide. An isolated tetramer or octamer of gastrin or a gastrin peptide. An isolated tetramer or octamer of leptin or a leptin peptide. An isolated tetramer or octamer of an antibody binding site (e.g., scFv or Fab). An isolated tetramer or octamer of a TCR binding site (e.g., scTCR). Isolated tetramers or octamers of TCR Vα / Vβ binding sites. Isolated tetramers or octamers of the TCR Vy / Vδ binding site. An isolated tetramer or octamer of antibody single variable domain binding sites. Isolated tetramers or octamers of FcAb binding sites.

[0062] In one example of any of these tetramers or octamers, the domain or peptide is human. In one example of any of these tetramers or octamers, the tetramer or octamers comprises an NHR2 TD (e.g., human NHR2). In one example of any of these tetramers or octamers, the tetramer or octamers comprises a p53 TD (e.g., human p53 TD). In one example of any of these tetramers or octamers, the tetramer or octamers comprises a p63 TD (e.g., human p63 TD). In one example of any of these tetramers or octamers, the tetramer or octamers comprises a p73 TD (e.g., human p73 TD). In one example of any of these tetramers or octamers, the tetramer or octamers comprises a tetramer of a TD (e.g., human NHR2 TD), whereby the domain or peptide forms a multimer of four or eight domains or peptides.

[0063] In one example, the plurality is pure and not mixed with multimers of said binding sites or peptides, eg, which contain more than one type of polypeptide monomer. 18. (a) is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (b) capable of being secreted from a eukaryotic cell; and / or (c) is a eukaryotic expression product; The multimer, tetramer, or octamer of any preceding embodiment.

[0064] In one example, the multimer, tetramer or octamer can be secreted from HEK293T (or other eukaryotic, mammalian, CHO or Cos) cells in a stable form as indicated by a single band of the predicted molecular weight for said multimer, tetramer or octamer on a PAGE gel using a sample of supernatant from such cells and detected using Western blot. 19. (a) A tetramer or octamer of a TCR V domain or TCR binding site that is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (b) a tetramer or octamer of an antibody single variable domain that is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer); (c) a tetramer or octamer of a TCR V domain or a TCR binding site, which is expressible intracellularly and / or extracellularly by HEK293 cells; or (d) A tetramer or octamer of an antibody variable domain (e.g., an antibody single variable domain), which can be expressed intracellularly and / or extracellularly by HEK293 cells.

[0065] 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 one example, the medium is serum-free HEK293 cell culture medium. In one example, the medium is serum-free CHO cell culture medium.

[0066] For example, the cell in the present invention is a human cell, such as a HEK293 cell (such as a HEK293T cell). 20. The multimer, tetramer, or octamer of any preceding embodiment, wherein said tetramer or octamer is bispecific for antigen or pMHC binding. 21. The multimer, tetramer, or octamer of any preceding embodiment, wherein said domains are identical. 22. The multimer, tetramer, or octamer of any preceding embodiment, comprising eukaryotic glycosylation.

[0067] 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 or octamer of embodiment 22, wherein said cell is a HEK293 cell. 24. A multiple of the multimer, tetramer, or octamer of any preceding embodiment. 25. A pharmaceutical composition comprising the multimer, tetramer, or octamer of any preceding embodiment and a pharma- ceutically acceptable carrier, diluent, or excipient. 26. A cosmetic, food product, beverage, cleaning product, or detergent comprising the multimer, tetramer, or octamer of any one of embodiments 1 to 24. 27. The engineered (and optionally isolated) polypeptide or (optionally isolated) monomer of the multimer, tetramer, or octamer of any preceding embodiment.

[0068] The monomer is an engineered polypeptide as disclosed herein that comprises said protein domain or peptide and further comprises a TD.

[0069] Optionally, the engineered polypeptide comprises (in the N-terminal to C-terminal direction) a variable domain (V1)-a constant domain (C) (eg, CH1 or Fc)-an optional linker-TD. 28.(From N-terminus to C-terminus): (a) TCR V1-TCR C1-Antibody C (e.g. CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ))-optionally a linker-TD; (i) V1 is Vα and C1 is Cα; (ii) V1 is Vβ and C1 is Vβ; (iii) V1 is Vy and C1 is Cy; or (iv) V1 is Vδ and C1 is C8; or (b) TCR V1-Antibody C (e.g. CH, CH1 (such as IgG CH1) or CL (such as Cλ or Cκ))-optionally a linker-TD; (i) V1 is Vα, (ii) V1 is Vβ, (iii) V1 is Vy, or (iv) V1 is Vδ, or (c) antibody V1 - antibody C (e.g. CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ)) - an optional linker - TD; (i) V1 is VH, or (ii) V1 is a VL (e.g., Vλ or Vκ); or (d) antibody V1 - optional antibody C (e.g. CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ)) - antibody Fc (e.g. IgG Fc) - optional linker - TD; (i) V1 is VH, or (ii) V1 is a VL (e.g., Vλ or Vκ); or (e) antibody V1 - antibody CL (e.g. Ck or Cκ) - optional linker - TD; (i) V1 is VH, or (ii) V1 is a 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) V1 is Vδ and C1 is Cδ; An engineered (and optionally isolated) engineered polypeptide (P1).

[0070] In (a) or (b), in one example, the TCR V is constituted by a single chain TCR binding site (scTCR) that specifically binds to pMHC, the binding site comprising TCR V-linker-TCRV. In one example, the engineered polypeptide comprises (from N-terminal to C-terminal direction) (i) V1-linker-V-optional C-optional linker-TD, or (ii) Vα-linker-V1-optional C-optional linker-TD, where Vα is a TCR V domain and C is an antibody C domain (e.g., CH1 or CL) or TCR C.

[0071] Preferably, antibody C is a CH1 (eg, an IgG CH1).

[0072] In one example, the multimer, tetramer or octamer has a size of 155 kDa or less, for example, said 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.

[0073] In one example, the multimer, tetramer, or octamer comprises a TCR binding site and an antibody binding site. For example, each polypeptide comprises a TCR V (e.g., comprised by a scTCR that specifically binds to the pMHC) and an antibody V (e.g., comprised by a scFv, or paired with a second V domain comprised by said second polypeptide to form a V / V paired binding site that specifically binds to an antigen). In one example, the pMHC comprises a RAS peptide. In one 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 comprises (in the N-terminal to C-terminal direction): (g) TCR V2-TCR C2-antibody CL (e.g. Cλ or Cκ), wherein P1 is according to (a) as described in embodiment 28; and (i) when P1 is according to (a)(ii), V2 is Vα and C2 is Cα; (ii) when P1 is according to (a)(i), V2 is Vβ and C2 is Cβ; (iii) when P1 is according to (a)(iv), V2 is Vγ and C2 is Cγ; or (iv) when P1 is according to (a)(iii), V2 is Vδ and C2 is Cδ; or (h) a TCR V2-antibody CL (e.g., Ck or Cκ), wherein P1 is according to (b) as described in embodiment 28; and (i) if P1 is according to (b)(ii), then V2 is Vα; (ii) when P1 is according to (b)(i), V2 is Vβ; (iii) when P1 is according to (b)(iv), V2 is Vγ; or (iv) if P1 is according to (b)(iiii), then V2 is Vδ; or (i) antibody V2-CL (e.g. Ck or Cκ), wherein P1 is according to (c) as described in embodiment 28; and (i) when P1 is according to (c)(ii), then V2 is VH; or (ii) when P1 is according to (c)(i), V2 is VL (e.g., Vλ or VK); or (j) antibody V2 - with an optionally present CL (e.g. Ck or Cκ) and P1 is according to (d) as described in embodiment 28; and (i) when P1 is according to (d)(ii), then V2 is VH; or (ii) when P1 is according to (d)(i), V2 is a VL (e.g., Vλ or Vκ); or (k) antibody V2-CH1 (e.g., IgG CH1), wherein P1 is according to (e) as described in embodiment 28; and (i) when P1 is according to (e)(ii), then V2 is VH; or (ii) when P1 is according to (e)(i), 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; and (i) when P1 is according to (f)(ii), V2 is Vα and C2 is Cα; (ii) when P1 is according to (f)(i), V2 is Vβ and C2 is Cβ; (iii) when P1 is according to (f)(iii), V2 is Vγ and C2 is Cγ; or (iv) if P1 is according to (f)(iv), then V2 is Vδ and C2 is Cδ; The polypeptide of embodiment 28.

[0074] In some cases, V1 and V2 form a paired variable domain binding site capable of specifically binding to an antigen or pMHC. In one example, V1 and V2 are selected from the group consisting of, for example, ReoPro™; Abciximab; Rituxan™; Rituximab; Zenapax™; Daclizumab; Simulect™; Basiliximab; Synagis™; Palivizumab; Remicade™; Infliximab; Herceptin™; Mylotarg™; Gemtuzumab; Campus™; Alemtuzumab. ;Zevalin(trademark);Ibritumomab;Humira(trademark);Adalimumab;Xolair(trademark);Omalizumab;Bexar(trademark);Tositumomab;Raptiva(trademark);Efalizumab;Erbitux(trademark);Cetuximab;Avastin(trademark);Bevacizumab;Tysabri(trademark);Natalizumab;Actemra(trademark);Tocilizumab;Vectibix(trademark);Pa The variable domain of an antibody selected from the group consisting of nitumumab; Lucentis(TM); ranibizumab; Soliris(TM); eculizumab; Cimzia(TM); certolizumab; Simponi(TM); golimumab, Ilaris(TM); canakinumab; Stelara(TM); ustekinumab; Arzera(TM); ofatumumab; Prolia(TM); denosumab; Numax(TM); motavizumab; Abthrax(TM); raxibacumab; Benlysta(TM); belimumab; Yervoy(TM); ipilimumab; Adcetris(TM); brentuximab; Vedotin(TM); Perjeta(TM); pertuzumab; Kadcyla(TM); ado-trastuzumab; Keytruda(TM), Opdivo(TM), Gazyva(TM) and obinutuzumab.

[0075] In one embodiment, the antibody is Avastin.

[0076] In one embodiment, the antibody is Actemra.

[0077] In one embodiment, the antibody is Erbitux.

[0078] In one embodiment, the antibody is Lucentis.

[0079] In one embodiment, the antibody is sarilumab.

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

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

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

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

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

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

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

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

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

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

[0090] In one embodiment, the antibody is a Cambus.

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

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

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

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

[0095] In one example, V1 (by itself when it is a single variable domain, or when paired with V2) is 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-a-glycoprotein);B7.1;B7.2;BAD;BAFF;BAG1;BAI1;BCL2;BCL6;BDNF;BLNK;BLRl (MDR15);BlyS;BM Pl;BMP2;BMP3B (GDFIO);BMP4;BMP6;BM P8;BMPRIA;BMPRIB;BM PR2;BPAG1(plectin);BRCA1;CI9orflO(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1;CCBP2(D6 / JAB61);CC L1(1-309);CCL11(eotaxin);CCL13(MCP-4);CCL15(MIP-id);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(M IP-3b);CCL2(MCP-1);MCAF;CCL20(MIP-3a);CCL21(MIP-2);SLC;Exodus-2;CCL22(MDC / STC-1);CCL23(M PIF-1);CCL24(MPIF-2 I eotaxin-2);CCL25(TECK);CCL26(eotaxin-3);CCL27(CTACK / ILC);CCL28;CCL3(MIP-la);CCL4(M IP-lb);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1;CCNA2;CCND1;CCNE1;CCNE2;CCR1(CKR1 / HM145);CCR2(mcp-1RB / RA);CCR3(CKR3 / CMKBR3);CCR4;CCR5(CM KBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6);CCR7(CKR7 / EBI1);CCR8(CM KBR8 / 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;キチナーゼ;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7(クローディン-7);CLN3;CLU(クラスタリン);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-78 I LIX);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);FGF20;FGF21;FGF22;FGF23;FGF3(int-2);FGF4(HST);FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR3;FIGF(VEGFD);FILl(epsilon);FILl(ZETA);FU1 2584;FU25530;FLRTl(fibronectin);FLTl;FOS;FOSLl(FRA-I);FY(DARC);GABRP(GABAa);GAGEB1;GAGEC1;GALNAC4S-65T;GATA3;GDF5;GFI1;GGT1;GM-CSF;GNAS1;GNRHl;GPR2(CCRIO);GPR31;GPR44;GPR81(FKSG80);GRCCIO(CIO);GRP;GSN(gelsolin);GSTPl;HAVCR2;HDAC4;EDAC5;HDAC 7A;HDAC9;HGF;HIF1A;HIP1;histamine and histamine receptor;HLA-A;HLA-DRA;HM74;HMOX1;HUMCYT2A;ICEBERG;ICOSL;1D2;IFN-a;IFNA1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFN gamma;TFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-1;IL10;IL10RA;IL10RB;IL11;IL11RA;I L-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;1L4;IL4R;1L5;IL5RA;1L6;IL6R;IL6ST (glycoprotein 130);1L7;TL7R;1L8;IL8RA;IL8RB;I L8RB;1L9;IL9R;ILK;INHA;INHBA;INSL3;INSL4;IRAKI;IRAK2;ITGA1;ITGA2;1TGA3;ITGA6(a6 integrin);ITGAV;ITGB3;ITGB4(b4 integrin);JAG1;JAK1;JAK3;J UN;K6HF;KAI1;KDR;MTLG;KLF5 (GC box BP);KLF6;KLK10;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19 (keratin 19);KRT2A;KRTHB6 (hair-specific type II keratin);LAMA5;LEP (leptin);Lingo-p75;Lingo-Troy;LPS;LTA (TNF-b);LTB;LTB4R (GPR16);LTB4R2;LTBR;MACMARCKS;MAG or Omgp;MAP2K7 (c-Jun);MDK;M IB1;Midkine;M IF;M IP-2;MK167 (Ki-67);MMP2;M MP9;MS4A1;MSMB;MT3 (metallothionectin-ifi);MTSS 1;MUC 1 (mucin);MYC;MYD88;NCK2;Neurocan;NFKB 1;NFKB2;NGFB (NGF);NGFR;NgR-Lingo;NgR-Nogo66 (Nogo);NgR-p75;NgR-Troy;NM E1(NM23A);NOX5;NPPB;NROB1;NROB2;NR1D1;NR1D2;NR1H2;NR1H3;NR1H4;NR1I2;NR1I3;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;N R4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZ1;OPRD1;P2RX7;PAP;PARTI;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(mammaglobin 2);SCGB2A2(mammaglobin 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;TGFA;TGFB1;TGFB1I1;TGFB2;TGFB3;TGFBI;TGFBR1;T GFBR2;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;TNFRSF1 1A;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSFIO(TRAIL);TNFSF1 1(TRANCE);TNFSF12(AP03L);TNFSF13(April);TNFSF13B;TNFSF14(HVEM-L);TNFSF1 5(VEGI);TNFSF1 8;TNFSF4(0X40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSF8(CD30 ligand);TNFSF9(4-lBB ligand);TOLLIP;Toll-like receptor;TOP2A(topoisomerase lia);TP53;TPM 1;TPM2;TRADD;TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREM 1;TREM2;TRPC6;TSLP;TWEAK;VEGF;VEGFB;VEGFC;versican;VHL C5;VLA-4;XCL1(lymphotactin);XCL2(SCM-lb);XCR1(GPR5; / CCXCR1); YY1; and ZFPM2.

[0096] For example, in any of the configurations of the invention, the multimer, tetramer or octamer specifically binds to a first and second epitope or antigen, the first and second epitopes or antigens being, respectively, EpCAM and CD3; CD19 and CD3; VEGF and VEGFR2; VEGF and EGFR; CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and CD138; CD38 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; CSPG and RGM A;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-1 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; TNF alpha and IL-23; TNF alpha and M IF; TNF alpha and PEG2; TNF alpha and PGE4; TNF alpha and VEGF; and VEGFR and EGFR; TNF alpha and RANK ligand; TNF alpha and Blys; TNF alpha and GP130; TNF alpha and CD-22; and TNF alpha and CTLA-4.

[0097] 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.

[0098] In one example, V1 (by itself if it is a single variable domain or when paired with V2) 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, BMP, BMP2, BMP7, BMP9, BMP10, GDF8, GDF11, RANKL, TRAIL, VEGFA, VEGFB or PGF, optionally the multimer comprises the amino acid sequence (e.g., C-terminus to TD) of a cytokine, such as IL-2 or an IL2-peptide, and the multimer, tetramer or octamer is for treating or preventing cancer in a human subject. In one example, the effector or protein domain is capable of binding to such an antigen, and optionally the multimer comprises an amino acid sequence (e.g., C-terminus to TD) of a cytokine, such as IL-2 or an IL2-peptide, and the multimer, tetramer or octamer is for treating or preventing cancer in a human subject. 30. A multimer (such as a dimer, trimer, tetramer or octamer) of P1 as defined in embodiment 28, or P1 paired with P2 as defined in embodiment 29, or a plurality of said multimers, optionally wherein said multimer is according to any one of embodiments 1 to 24.

[0099] Preferably, the multimer is a tetramer of the engineered polypeptide and / or effector domain, in one example, the tetramers are not mixed with monomers, dimers or trimers of the polypeptide.

[0100] In one example, a multimer, such as a tetramer, is capable of specifically binding to two different pMHC. 31. A nucleic acid encoding an engineered polypeptide or monomer of any one of aspects 27-29, optionally wherein said nucleic acid is comprised by an expression vector for expressing said polypeptide.

[0101] In one example, the nucleic acid is DNA, optionally operably linked to or including a promoter for expression of the polypeptide or monomer, hi another example, the nucleic acid is RNA (e.g., mRNA). 32. A eukaryotic host cell comprising a nucleic acid or vector of embodiment 31 for the intracellular and / or secretory expression of a multimer, tetramer, octamer, engineered polypeptide or monomer of any one of embodiments 1 to 24. 33. Use of the 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 within a cell, said method comprising expressing said multimer in a eukaryotic cell comprising said nucleic acid or vector and / or secreting said multimer from said eukaryotic cell. 34. Use of a nucleic acid or vector according to embodiment 31 in a method for producing a protein multimer for producing a glycosylated multimer in a eukaryotic cell comprising said nucleic acid or vector.

[0102] The glycosylation of the mammal of the invention is useful for producing a medicament comprising or consisting of the multimer, tetramer or octamer of the invention for the medical treatment or prevention of a disease or condition in a mammal, e.g., a human. The invention therefore provides such a method of use as well as the multimer, tetramer or octamer of the invention for this purpose. Similarly, intracellular and / or secretory expression in one or more host cells (or cell lines thereof) that are mammalian according to the invention is useful for producing such a medicament. Such expression in HEK293, CHO or Cos cells is particularly useful, since these cells are often used for the production of pharmaceuticals.

[0103] In one embodiment, the invention includes a cleaning product or personal health care product comprising a multimer, tetramer or octamer of the invention.In one embodiment, the invention includes a foodstuff or beverage comprising a multimer, tetramer or octamer of the invention.

[0104] In one example, the multimer, monomer, dimer, trimer, tetramer, octamer, polypeptide, composition, mixture, use or method of the present invention is for or used in a method for industrial or domestic use.For example, it is for or used in the agriculture, oil or petroleum 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, health care industry, dental industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry or steel industry. 35. A mixture comprising (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of embodiments 27-29, and (ii) a multimer, tetramer or octamer as defined in any one of embodiments 1-24. 36. The mixture of embodiment 35, wherein the cell line is in a medium comprising a secretion product of the cell, and the secretion product comprises the multimer, tetramer or octamer. 37. A multimer, tetramer or octamer of any one of embodiments 1 to 24 for medical use. 38. (a) A method for producing a TCR V domain multimer comprising soluble and / or intracellular expression of a TCR V-NHR2 TD or TCR V-p53 TD fusion protein expressed in a eukaryotic cell, and optionally isolating a plurality of said multimers; (b) a method of producing an antibody V domain multimer comprising soluble and / or intracellular expression of an antibody V (e.g. single variable domain)-NHR2 TD or V-p53 TD fusion protein expressed in a eukaryotic cell, and optionally isolating a plurality of said multimers; (c) a method of producing an incretin peptide (e.g., GLP-1, GIP or insulin) multimer comprising soluble and / or intracellular expression of an incretin peptide-NHR2 TD or incretin peptide-p53 TD fusion protein expressed in a eukaryotic cell, such as a HEK293T cell, and optionally isolating a plurality of said multimers; or (d) A method for producing a peptide hormone multimer comprising 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. 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 the tetramer in higher yield than monomeric and / or dimeric polypeptides. 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 tetramers in higher yield than monomeric and / or dimeric polypeptides, wherein said engineered polypeptide comprises one or more copies of said 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 mixed 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 mixed with monomers, dimers or trimers, wherein said engineered polypeptide comprises one or more copies of said 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 of any one of aspects 39-42, wherein said yield of tetramer is at least 10, 20, 30, 40, or 50 times higher than said yield of monomer and / or dimer. 44. The use of any one of aspects 39 to 43, wherein the ratio of tetramers produced in the 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 of any one of aspects 39-44, wherein each monomer has a size of less than 40, 35, 30, 25 or 20 kDa. 46. ​​The use of any one of aspects 39 to 45, wherein each tetramer has a size of less than 200, 160, 155 or 150 kDa. 47. The use of any one of aspects 39 to 46, wherein the method comprises expressing the tetramer from a eukaryotic cell line. 48.(i) TCR extracellular domain, (ii) an immunoglobulin constant domain, and (iii) NHR2 multimerization domain of ETO A multivalent heterodimeric soluble T cell receptor capable of binding to the pMHC complex, comprising: 49.(i) an immunoglobulin variable domain, and (ii) the NHR2 multimerization domain of ETO A polymeric immunoglobulin comprising: 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. A method comprising:

[0105] The present invention further provides: (i) The monomer shown in Figure 1 ; (ii) the homodimer shown in Figure 1 ; (iii) the homotetramer shown in Figure 1 ; (iv) Monomers shown in FIG. 2 ; (v) The homodimer shown in Figure 2 2 ; (vi) The homotetramer shown in Figure 2 2 ; (vii) the monomer shown in Figure 11a; (viii) homodimer shown in Figure 11a; (ix) homotetramer shown in Figure 11a; (x) the monomer shown in Figure 12a; (xi) homodimer shown in Figure 12a; (xii) homotetramer as shown in Figure 12a; (xiii) Monomers shown in Figure 13a 2 ; (xiv) The homodimer shown in Figure 13a 2 ; (xv) Homotetramer as shown in FIG. 13a 2 ;or (xvi) A multimeric protein comprising any one of (i)-(xv) (e.g., any one of Quad 3, Quad 4, Quad 12, Quad 13, Quad 14, Quad 15, Quad 16, and Quad 17), or a multimer of any protein shown in FIG. 21 (excluding any leader or tag). (xvii) a multimer of a plurality of (xvi); or (xviii) A pharmaceutical composition comprising any one of (i) to (xvii) and a pharma- ceutically acceptable carrier, diluent or excipient.

[0106] 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) Tetravalent or octavalent antibody scFv molecules wherein the molecule comprises (a) is soluble in an 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;

[0107] The invention provides claimed multimers (e.g., tetramers) of 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. For example, the sequence is selected from a TCR (e.g., TCRa, TCRβ, Ca or Oβ), 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 Cx domain). Optionally, the multimer comprises: (i) is soluble in an aqueous solution (e.g., a solution or cell culture medium disclosed herein); and / or (ii) expressible intracellularly and / or extracellularly by HEK293 cells; It is a molecule.

[0108] The invention provides: (i) The 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 a cell, e.g., a eukaryotic cell, e.g., a mammalian, HEK293, CHO or Cos cell. (ii) The 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, e.g., a eukaryotic cell, e.g., 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) The use of NHR2 as a promiscuous tetramerization domain to tetramerize peptides, protein domains, polypeptides or proteins in the production of multimers that are expressed intracellularly and / or soluble from a host cell.

[0109] In some cases, the amino acid is the amino acid sequence of a human peptide, protein domain or protein, such as a TCR (e.g., TCRa, TCRβ, Ca 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).

[0110] 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, e.g. for medical or diagnostic uses, e.g. for medical uses to treat or prevent a disease or condition in a human or animal (e.g. a human).

[0111] Optionally, the or each polypeptide comprises a polypeptide encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-50 (excluding any leader or tag sequence). Optionally, the or each polypeptide comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-115 (excluding any leader or tag sequence). Optionally, the invention provides multimers of such polypeptides (e.g. dimers, trimers, tetramers, pentamers, hexamers, heptamers or octamers, preferably tetramers or octamers), for example for medical or diagnostic uses, e.g. for medical uses to treat or prevent a disease or condition in a human or animal (e.g. human).

[0112] In one example, the TD is a TD consisting of any one of SEQ ID NOs: 1-9. In one example, the TD is a TD comprising SEQ ID NO: 10 or 126. In one example, the TD is encoded by SEQ ID NO: 124 or 125. In one example, the amino acid sequence of each TD is SEQ ID NO: 10 or 126, or is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10 or 126.

[0113] In one example, the TD is a TD comprising SEQ ID NO: 120 or 123. In one example, the TD is encoded by SEQ ID NO: 116 or 119. In one example, the amino acid sequence of each TD is SEQ ID NO: 120 or 123, or is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 120 or 123.

[0114] Optionally, the domain or peptide comprised by the engineered polypeptide or monomer comprises an amino acid selected from SEQ ID NOs: 51-82.

[0115] Highly pure tetramer As exemplified herein, the present invention is based on the surprising realization that, in one configuration, a tetramerization domain (TD), such as the p53 tetramerization domain (p53 TD), can be used to produce tetramers of effector domains preferentially over the production of lower order structures such as dimers and monomers. This is particularly useful since secretion of tetramers is the desired yield from mammalian expression cell lines such as CHO, HEK293 and Cos cell lines. The present invention is also particularly useful for the production of tetramers of sizes up to 200, 160, 155 or 150 kDa.

[0116] Thus, the present invention provides the following concept: 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 the tetramer in higher yield than monomeric and / or dimeric polypeptides.

[0117] Monomers and dimers contain one or two copies of TD, homologues or orthologs, respectively.

[0118] In one example, the TD, ortholog or homolog is a human domain.

[0119] In one example, the yield of tetramer is higher than the yield of monomer; in one example, the yield of tetramer is higher than the yield of dimer; in one example, the yield of tetramer is higher than the yield of trimer; in one example, the yield of tetramer is higher than the yield of monomer and dimer; in one example, the yield of tetramer is higher than the yield of monomer and trimer; in one example, the yield of tetramer is higher than the yield of monomer, dimer and trimer.

[0120] For example, the TD is a TD of p53 isoform 1. In one example, 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 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 said selected sequence. Optionally, the TD, ortholog, or homolog comprises or consists of an amino acid sequence 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 said selected sequence. 2. Use of concept 1, where the first, second, third and fourth copies of the same TD or its homologue or ortholog are used. 3. The use of any preceding concept, wherein said TD is a tetramerization domain of NHR2, p53, p63 or p73. For example, the TD is a p53 TD. In one example, the TD is an ortholog or homolog of a p53 TD, such as a human p53 TD. 4. Use of any preceding concept, wherein the yield of said tetramer is at least 10 times said yield of monomer and / or dimer.

[0121] Optionally, the yield is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x the yield of monomer and / or dimer. Optionally, the ratio of tetramer:monomer and / or dimer produced is at least 90:10, e.g., at least 95:5; or 96:4; or 97:3; or 98:2; or 99:1. Optionally, only tetramer is produced.

[0122] 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. Use of any preceding concept, wherein the ratio of tetramer produced in said 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 a combination of 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 separate tetramers, monomers and dimers, and comparing the amount of tetramers to the amount of monomers and dimers.

[0123] The amount of tetramers, monomers, dimers and trimers can be determined, for example, using Western blot analysis of gels described herein, e.g., native gels, i.e., gels that are not under denaturing conditions, such as in the absence of SDS. 7. The yield or ratio is (a) obtaining a sample of the product of said tetramer production process; (b) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to separate the sample into a band corresponding to the tetramer and a band corresponding to the monomer and / or the dimer; and (c) determining the yield or ratio of the tetramer band 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, determinable or determined by. 8. The yield or ratio is (d) obtaining a sample of the product of said tetramer production process; (e) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to separate the sample into bands corresponding to the tetramer, e.g., the gel is under non-denaturing conditions (e.g., in the absence of sodium dodecylsuphate (SDS)); (f) determining the absence of a band corresponding to the monomer and / or a band corresponding to the dimer; The use of concepts 4 or 5, determinable or determined by. 9. (g) obtaining a second sample of the product of said tetramer production process; (h) performing polyacrylamide gel electrophoresis (PAGE) under reducing conditions to separate the second sample into bands corresponding to the monomer and / or the dimer, e.g., the gel is under non-denaturing conditions (e.g., in the absence of sodium dodecyl sulfate (SDS)); and (i) comparing the gel produced by step (h) with the gels of steps (b) or (e) to determine the location of the monomer and / or dimer bands in the gel of step (b), or determining that such a gel is expected to be the gel of step (e). Use of concepts 7 or 8, including: 10. Use of any preceding concept, wherein each monomer has a size of 40 kDa or less.

[0124] For example, the monomer has a size of less than or equal to 35, 30, 25, 24, 23, 22, 21 or 20 kDa. 11. Use of any preceding concept, wherein each tetramer has a size of 150 kDa or less.

[0125] For example, the tetramer has a size of less than 80, 90, 100, 110, 120, 130 or 140 kDa. 12. Use of any preceding concept, wherein the method comprises expressing the tetramer from a mammalian cell line, such as a HEK293, CHO or Cos cell line.

[0126] 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. Use of any of the preceding concepts, wherein the method comprises secreting the tetramer from a mammalian cell line, such as a HEK293, CHO or Cos cell line.

[0127] Thus, advantageously, in one example, the use or tetramer is for expression from a mammalian cell line (e.g., HEK293, CHO or Cos cell line) or a eukaryotic cell line, which is useful for large-scale production of the products of the invention, such as tetramers.

[0128] 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. Use of any of the preceding concepts, wherein each polypeptide or monomer comprises said TD, a homologue or ortholog, and one or more protein effector domains (such as one or more antibody domains, e.g., one or more antibody domains forming an antigen binding site). 15. The polypeptide, (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 Use of concept 14, including one or more of the following: 16. Use of any of the preceding concepts, wherein each polypeptide or monomer comprises said TD, a homologue or ortholog, and one or more incretin, insulin, GLP-1 or exendin-4 domains. 17. Use of any of the preceding concepts, wherein each polypeptide or monomer comprises said TD, a homologue or ortholog, 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 Use of concept 17, provided by 19. Use of concept 18, wherein each binding site is provided by an antibody single variable domain. 20. The use of any one of concepts 14 to 18, wherein the TD, homolog or ortholog is directly fused to the further domain. 21. Use of concept 20, wherein each monomer or polypeptide comprises the TD, homologue or ortholog fused directly or via a peptide linker to the C-terminus of the 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 that connects (i) to (ii) (e.g., connects the C-terminus of (ii) to the N-terminus of (i)). Including, Optionally, each tetramer has a size of 150 or 200 kDa or less. Tetramer.

[0129] For example, tetramer has a size of 80, 90, 100, 110, 120, 130 or 140 kDa or less. In one example, any multimer, dimer, trimer, tetramer or octamer in the present invention has a size of at least 60 or 80 kDa, which may be useful, for example, to increase half-life in human or animal subjects administered the multimer, dimer, trimer, tetramer or octamer (e.g., to treat or prevent disease or condition in the subject). The size of these ranges may be larger than the size of renal filtration.

[0130] Alternatively, the invention provides a monomer, dimer, tetramer or octamer 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 The tetramer of concept 22, comprising one or more of: 24. The tetramer of concept 22 or 23, wherein each polypeptide comprises said TD, a 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 A tetramer of concept 25 provided by. 27. The tetramer of 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, homolog or ortholog is directly fused to the effector domain. 29. A tetramer according to any one of concepts 22 to 27, wherein each polypeptide comprises the TD, homologue or ortholog fused directly or via a peptide linker to the C-terminus of the effector domain.

[0131] In one embodiment, each polypeptide comprises only two (i.e. only a first and a second, but not a third) effector domains or only two dAbs, VHHs, scFvs, scTCRs, Fabs or antigen binding sites. 30. A pharmaceutical composition comprising any one of the tetramers of Concepts 22-29 and a pharma- ceutically acceptable carrier, diluent or excipient.

[0132] Optionally, the composition is contained in a sterile medical container or device, such as a syringe, vial, inhaler or injection device. 31. A cosmetic, food, beverage, cleaning product, or detergent comprising any one of the tetramers of concepts 22 to 29. 32. A mixture comprising a cell line (e.g., a mammalian cell line, such as a HEK293, CHO or Cos cell line) encoding a polypeptide as described in any preceding concept, and a tetramer as defined in any preceding concept.

[0133] Optionally, the mixture is contained in a sterile container. 33. The mixture of concept 32, wherein the cell line is in a medium containing a secretion product of the cell, the secretion product comprising the tetramer. 34. The mixture of concept 33, wherein the secretion 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 secretion product contains 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 secretion product comprises the tetramer in a ratio of tetramer:monomer and / or dimer of at least 90:10. 37. A method for enhancing the yield of a tetramer of a protein effector domain (e.g., an antibody variable domain or binding site), comprising expressing a tetramer of a polypeptide from a cell line (e.g., a mammalian cell, a CHO, HEK293 or Cos cell line), wherein the polypeptide is as defined in any of the preceding concepts and comprises one or more effector domains, and wherein the method optionally comprises isolating the expressed tetramer.

[0134] The homologues, orthologs or equivalents have a tetramerization function.

[0135] Homolog: A gene, nucleotide or protein sequence related to a second gene, nucleotide or protein sequence by originating from a common ancestral DNA or protein sequence. The term homologue may be applied to the relationship between genes separated by the event of replication or between genes separated by the event of gene duplication.

[0136] Orthologs: Orthologs are gene, nucleotide or protein sequences in different species that have evolved from a common ancestral gene, nucleotide or protein sequence by speciation. Orthologs usually retain the same function during evolution.

[0137] In one example, the TD, ortholog or homolog is a TD of any one of proteins 1-119 listed in Table 2. In one 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 the use of the p53 tetramerization domain (p53-TD) or a homolog or ortholog thereof, all aspects of the invention herein can be read as relating to the use or inclusion of a TD of any one of proteins 1-119 listed in Table 2 or a homolog or ortholog thereof in a polypeptide, monomer, dimer, trimer or tetramer. The TD can be a TD of NHR2 (e.g., human NHR2) or an ortholog or homolog thereof. The TD can be a TD of p63 (e.g., human p63) or an ortholog or homolog thereof. The TD can be a TD of p73 (e.g., human p73) or an ortholog or homolog thereof. This can have one or more advantages as follows: - secretion of the tetramer from a mammalian or other eukaryotic cell, e.g., a mammalian cell as disclosed herein, such as CHO, HEK293 or Cos; - Enhancement of the yield of secreted tetramers versus monomers; - Enhanced yield of secreted tetramers versus dimers; - Enhancement of the yield of secreted tetramers versus trimers; - Enhanced yield of secreted tetramers versus monomers and dimers combined; - Enhanced yield of secreted tetramers versus monomers, dimers and trimers combined; - increasing the affinity or avidity of antigen binding in tetramers that contain antigen-binding sites; - Enhanced production and / or expression of tetramers of size less than or equal to 200 or 160 or 150 kDa. In one embodiment, each polypeptide or monomer is selected from the group consisting of ReoPro(TM); abciximab; Rituxan(TM); rituximab; Zenapax(TM); daclizumab; Simulect(TM); basiliximab; Synagis(TM); Palivizumab; Remicade(TM); infliximab; Herceptin(TM); trastuzumab; Mylotarg(TM); gemtuzumab; Campath(TM); alemtuzumab; Zevalin(TM); ibritumomab; Humira(TM); adalimumab; Xolair(TM); omalizumab; Bexar(TM); tositumomab; Raptiva(TM); efalizumab; Erbitux(TM); cetuximab; Avastin(TM); bevacizumab; Tysabri(TM); natalizumab; Actemra(TM); tocilizumab; Vectimab The antibody comprises one or more VH, VL or VH / VL binding sites of an antibody selected from: VIX(TM); panitumumab; Lucentis(TM); ranibizumab; Soliris(TM); eculizumab; Cimzia(TM); certolizumab; Simponi(TM); golimumab, Ilaris(TM); canakinumab; Stelara(TM); ustekinumab; Arzera(TM); ofatumumab; Prolia(TM); denosumab; Numax(TM); motavizumab; Abthrax(TM); raxibacumab; Benlysta(TM); belimumab; Yervoy(TM); ipilimumab; Adcetris(TM); brentuximab; Vedotin(TM); Perjeta(TM); pertuzumab; Kadcyla(TM); ado-trastuzumab; Gazyva(TM) and obinutuzumab. Alternatively, (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.

[0138] In one example, the tetramer comprises four copies of an 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 an 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.

[0139] In one example, the tetramer includes four copies of an antigen binding site for Avastin. In one example, the tetramer includes four copies of an antigen binding site for Humira. In one example, the tetramer includes four copies of an antigen binding site for Erbitux. In one example, the tetramer includes four copies of an antigen binding site for Actemra™. In one example, the tetramer includes four copies of an antigen binding site for Sarilumab. In one example, the tetramer includes four copies of an antigen binding site for Dupilumab. In one example, the tetramer includes four copies of an antigen binding site for Alirocumab or Evolocumab. In one example, the tetramer includes four copies of an antigen binding site for In one example, the tetramer includes four copies of an antigen binding site for Remicade. In one example, the tetramer includes four copies of an antigen binding site for Lucentis. In one example, the tetramer includes four copies of an 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, e.g., where the binding site is that of Avastin, Lucentis, or Eylea). Such tetramers are useful for administration to humans to treat or prevent angiogenesis.

[0140] In one example, the tetramer comprises four copies of insulin. In one example, the tetramer comprises four copies of GLP-1. In one example, the tetramer comprises four copies of GIP. In one example, the tetramer comprises four copies of exendin-4. In one example, the tetramer comprises four copies of insulin and four copies of GLP-1. In one example, the tetramer comprises four copies of insulin and four copies of GIP. In one example, the tetramer comprises four copies of insulin and four copies of exendin-4. In one 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.

[0141] Diseases and Conditions The monomers or multimers (e.g., dimers, trimers, tetramers or octamers) of the invention can be administered to a human or animal subject and used in methods for treating or preventing a disease or condition in the subject.

[0142] 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) cardiac or cardiovascular disease or condition, such as heart attack, stroke or 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) any disease or condition of the throat or oral cavity; (l) eye disease or condition; (m) A disease or condition of the genitals, such as a disease or condition of the vagina, labia, penis, or scrotum; (n) A sexually transmitted disease or condition, such as gonorrhea, HIV infection, syphilis or chlamydial infection; (o) Ear disease or condition; (p) skin disease or condition; (q) heart disease or condition; (r) Nasal disease or condition (s) a blood disease or condition, e.g., anemia, e.g., anemia of chronic disease or cancer; (t) viral infections; (u) Pathogenic bacterial infections; (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 defect or condition; (ee) miscarriage; (ff) blood coagulation status; (gg) bronchitis; (hh) dry or wet AMD; (ii) angiogenesis (e.g., of tumors or in the eye); (jj) common cold; (kk) epilepsy; (ll) fibrosis, e.g., liver or pulmonary fibrosis; (mm) Fungal diseases or conditions, e.g., thrush; (nn) A metabolic disease or condition, e.g., 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 impairment; (tt) slow or fast metabolism (i.e., slower or faster than average for the subject's weight, sex and age) (uu) pregnancy disorders, e.g., infertility or low fertility; (vv) Jaundice; (ww) skin rash; (xx) Kawasaki disease; (yy) Lyme disease; (zz) allergies, e.g. to nuts, grass, pollen, dust mites, cat or dog fur or dander; (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., industrial stress); (nnn) Thalassemia, anemia, von Willebrand's disease, or hemophilia; (ooo) Shingles or herpes labialis; (ppp) Menstruation; (qqq) Low sperm count.

[0143] Neurodegenerative or CNS Diseases or Conditions for Treatment or Prevention In one example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer's disease, geriatric psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinsonism, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinsonism.

[0144] In one 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 the downregulation of Treg cells in the subject, thereby promoting systemic monocyte-derived macrophages and / or Treg cells to cross the choroid plexus and enter 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., in the brain and / or CSF). In one example, the method restores nerve fibers and / or reduces the progression of nerve fiber damage. In one example, the method restores nerve myelin and / or reduces the progression of nerve myelin damage. In one example, the methods of the invention treat or prevent a disease or condition disclosed in WO2015136541 and / or the methods can be used in conjunction with any of the methods disclosed in WO2015136541 (the disclosure of which is incorporated herein by reference in its entirety, e.g., to provide disclosure of potential therapeutic agents, such as immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein, that can be administered to a subject to effect treatment and / or prevention of such methods, diseases, conditions, and CNS and neurodegenerative diseases and conditions).

[0145] Cancer for treatment or prevention Cancers that can be treated include vascularized tumors as well as non-vascularized or substantially non-vascularized tumors.Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or can include solid tumors.Types of cancers that can be treated using the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0146] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemias such as acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyeiocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0147] A solid tumor is an abnormal mass of tissue that does not usually contain cysts or liquid compartments. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). 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 tumors, 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, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors, including gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme) These include astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0148] To treat or prevent autoimmune diseases 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 neuropathic neuropathies Barrow'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 ostomyelitis (CRMO) Churg-Strauss syndrome Cicatricial pemphigoid / benign mucous membrane pemphigoid Crohn's disease Cogan's syndrome ·Cold agglutinin disease Congenital heart block Coxsackie myocarditis Crest Disease Essential mixed cryoglobulinemia Demyelinating neuropathy ·Dermatitis herpetiformis ·Dermatomyositis Devic's disease (neuromyelitis optica) ·Discoid lupus Dressler Syndrome ·Endometriosis ·Eosinophilic esophagitis ·Eosinophilic fasciitis ·Erythema nodosum Experimental allergic encephalomyelitis Evans syndrome ·Fibromyalgia Fibrinous alveolitis ·Giant cell arteritis (temporal arteritis) Giant cell myocarditis Glomerulonephritis Goodpasture's syndrome Granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis) Graves' disease Guillain-Barre syndrome Hashimoto's encephalitis Hashimoto's thyroiditis ·Hemolytic anemia Henoch-Schönlein purpura Herpes of pregnancy 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 ·Ligny conjunctivitis Linear immunoglobulin A disease (LAD) ·Lupus (SLE) Lyme disease, chronic Meniere's disease Microscopic polyangiitis ·Mixed connective tissue disease (MCTD) Mooren's ulcer Mucha-Habermann's disease Multiple sclerosis ·Myasthenia gravis Myositis Narcolepsy Neuromyelitis optica (Devik) ·Neutropenia Ocular cicatricial pemphigoid ·Optic neuritis Relapsing rheumatism · PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection) Paraneoplastic cerebellar degeneration Paroxysmal nocturnal hemoglobinuria (PNH) Parry-Romberg syndrome Parsonnage-Turner syndrome Pars planitis (peripheral uveitis) ·Pemphigus Peripheral neuropathy Perivenous encephalomyelitis Pernicious anemia POEMS syndrome Polyarteritis nodosa Autoimmune polyglandular syndromes types I, II, and III Rheumatic polymyalgia Polymyositis Post-myocardial infarction syndrome Post-pericardiotomy syndrome Progesterone dermatitis Primary biliary cholangitis 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 Leg Syndrome Retroperitoneal fibrosis Rheumatic fever Rheumatoid arthritis ·sarcoidosis Schmidt syndrome ·Scleritis Scleroderma Sjögren's syndrome Sperm and 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 skin disease ·Vitiligo Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA))

[0149] Inflammatory diseases for treatment or prevention Alzheimer's ·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

[0150] Multivalent soluble TCR This composition relates to multivalent soluble TCR proteins. In one aspect, the present invention relates to tetravalent and octavalent soluble TCR analogs. The TCR proteins of the present invention can self-assemble from monomers and are of fully human origin. The proteins are multimers that contain ETO NHR2 multimerization domains. The present invention also relates to methods of constructing multimeric soluble TCRs and methods of using such proteins.

[0151] Attempts to exploit alternative soluble TCR formats as therapeutic molecules have lagged far behind the plethora of antibody formats. This is in large part due to the inherent solubility issues of the heterodimeric transmembrane proteins of TCRs once the extracellular TCR α / β chains are separated from the transmembrane and cytoplasmic domains. Secondly, the inherent low affinity and avidity of these molecules for their cognate ligands at the target site has greatly hindered their development as therapeutic molecules.

[0152] To overcome these drawbacks, the present configuration of the invention provides a TCR protein that is both multivalent and soluble. Multivalency increases the avidity of the TCR for cognate pMHC, and solubility allows the use of the TCR outside of the transmembrane environment. Thus, in a first aspect: (i) TCR extracellular domain; (ii) an immunoglobulin constant domain, and (iii) NHR2 multimerization domain of ETO A multivalent heterodimeric soluble T cell receptor capable of binding to a pMHC complex is provided, comprising:

[0153] The use of Ig constant domains provided the TCR extracellular domain with stability and solubility, and multimerization via the NHR2 domain provided multivalency and increased avidity. Advantageously, all domains are of human origin or correspond to human protein sequences.

[0154] The use of Ig constant domains to stabilize and make the TCR soluble avoids the use of non-native disulfide bonds.Thus, advantageously, the TCRs of the present invention do not contain non-native disulfide bonds.

[0155] In one embodiment, the complex comprises a heavy chain and a light chain, and each light chain comprises a TCR Vα domain and an immunoglobulin C α domains, and each heavy chain contains a TCR Vβ domain and an immunoglobulin CH1 domain.

[0156] In one embodiment, each light chain additionally comprises a TCR Ca domain and each heavy chain additionally comprises a TCR Vβ domain.

[0157] In embodiments, the TCR and immunoglobulin domains may be separated by a flexible linker.

[0158] The NHR2 multimerization domain is advantageously attached to the C-terminus of an immunoglobulin domain, such that each heavy and light chain dimer is attached to one multimerization domain, and the heavy-light chain dimers associate into multivalent oligomers.

[0159] 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.

[0160] In embodiments, the TCR protein may further comprise an immunoglobulin hinge domain. The hinge domain allows the dimerization of heavy-light chain dimers, which allows further multimerization of the TCR protein. For example, the multimerization domain that forms a tetramer can use an immunoglobulin hinge domain to form multimers up to octamer. Similarly, the multimerization domain that dimerizes can form a tetramer in the presence of a hinge domain.

[0161] In an embodiment, the TCR proteins of the present invention are tetravalent.

[0162] In an embodiment, the TCR proteins of the present invention are octavalent.

[0163] The present invention provides a soluble TCR that stably assembles in a tetravalent heterodimeric format using the nervy homology region 2 (NHR2) domain found in ETO family proteins in humans (Liu et al. 2006). The NHR2 domain has been found to naturally form homotetramers formed from the pairing of two NHR2 homodimers. NHR2 operably linked to the extracellular TCRa chain or TCRβ chain preferentially forms tetravalent heterodimeric soluble TCR protein molecules that sequentially self-assemble from monomers and then homodimers (Figure 1).

[0164] TCR proteins that assemble into octamers can be made using the NHR2 domain by using an immunoglobulin hinge domain.

[0165] In a further embodiment, the TCR proteins of the present invention may be linked to biologically active polypeptides / effector molecules. Examples of such polypeptides may include immunologically active moieties such as cytokines, binding proteins such as antibodies or targeting polypeptides, etc.

[0166] 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 the use of these novel highly sensitive multivalent soluble TCR protein molecules. Applications for use include, but are not limited to, therapeutics, diagnostics, and drug discovery.

[0167] In a further aspect, the invention provides methods for constructing multivalent immunoglobulin molecules in an efficient manner without the use of non-human construct components.

[0168] therefore, (i) an immunoglobulin variable domain, and (ii) the NHR2 multimerization domain of ETO The present invention provides a multimeric immunoglobulin comprising:

[0169] 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 the immunoglobulin variable domain.

[0170] The ETO NHR2 domain is more efficient in producing immunoglobulin multimers than p53 and similar multimerization domains, allowing for the production of multimeric immunoglobulin molecules without the use of non-human components in the construction.

[0171] Also provided is a method of producing a multimeric immunoglobulin comprising expressing immunoglobulin variable domains in fusion with the NHR2 domain of ETO and assembling the variable domains into multimers.

[0172] Preferably, the immunoglobulin variable domain is attached to one or more immunoglobulin constant domains.

[0173] Advantageously, the immunoglobulin domain is an antibody domain. For example, the variable domain can be a VH and a VL antibody domain. For example, the constant domain is an antibody CH1 domain.

[0174] In one embodiment, the multimeric immunoglobulin molecules according to 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 coat protein, other immunoglobulin molecules are produced without coat protein, so that they can assemble on the phage surface as a result of NHR2 multimerization.

[0175] This configuration of the invention detailed above relates to nucleic acid sequences and methods for producing novel multivalent, e.g. tetravalent and octavalent, soluble proteins. In one embodiment, in particular, 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 the whole or part of the extracellular TCR α / β chains. The extracellular TCR α / β chains are linked to immunoglobulin CH1 and CL (either Cκ or CX) domains. This linkage allows the stable formation of the heterodimeric TCR α / β. The unique feature in the context of soluble tetravalent TCRs is the C H 1 C-terminus or C L The NHR2 homotetramer domain of the ETO family of proteins operably linked to the C-terminus of the ...

[0176] TCR extracellular domain The TCR extracellular domain is composed of a variable region and a constant region. These domains are present in T cell receptors in the same way that they are present in antibodies and other immunoglobulin domains. The repertoire of TCRs has a wide 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 (or diversity, D) regions that code for the complementarity determining region 3 (CDR3) of the a 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 of 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.

[0177] Immunoglobulin variable domains Antibody variable domains are known in the art and are available from a 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.

[0178] 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. (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).

[0179] One particularly advantageous approach is 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. Bioi., 222: 581; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et al. (1992) J Bioi. Chem., 267). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Refinement of phage display approaches are also known, as described, for example, in WO96 / 06213 and W092 / 01047 (Medical Research Council et al.) and W097 / 08320 (Morphosys), which are incorporated herein by reference.

[0180] Such techniques can be adapted for the production of multimeric immunoglobulins by fusion of the NHR2 multimerization domain to antibody variable domains.

[0181] Immunoglobulin constant domains The immunoglobulin constant domain referred to herein is preferably an antibody constant domain.Constant domains vary in sequence between antibody subtypes, and preferably, constant domains are IgG constant domains.Preferably, constant domains are CH1 constant domains.Antibody constant domains are well known in the art and can be obtained from many sources and databases, such as IMGT and Kabat databases.

[0182] 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.

[0183] 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 from other molecules in the multimer complex. Suitable linkers include, for example, glycine repeats, glycine-alanine repeats, Gly(4)Ser linkers, or flexible polypeptide linkers as described in Reddy Chichili et al., 2012 Protein Science 22:153-167.

[0184] Immunoglobulin hinge domain The Ig hinge domain, preferably the antibody hinge domain in the present invention, is a domain that connects antibody constant regions in natural antibodies. This domain therefore provides the natural dimerization of molecules that contain antibody constant domains. It is present in whole antibodies, such as IgG, as well as in F(ab)2 antibody fragments, for example. This region contains two natural interchain disulfide bonds that connect and hold together the two CH1 constant domains.

[0185] In one embodiment, the multimerization domain may be attached to the Ig constant domain or hinge domain. When the hinge domain is present, the multimerization domain forms a TRC octamer, which comprises four dimers of TCR variable-Ig constant domains joined at the hinge region. When the hinge region is absent, 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.

[0186] bioactive molecules One or more biologically active or effector molecules (EM) can be attached to the multimers of the invention, such as multimeric TCR proteins. Such molecules can be, for example, antibodies, such as anti-CD3 antibodies or antibody fragments, particularly antibodies that can aid in immune recognition and function of the TCR.

[0187] In some embodiments, the bioactive molecule can be a bioactive molecule such as a cytotoxic drug, a toxin, or a cytokine, as described in more detail below. Examples of bioactive 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, labels such as radioactive and fluorescent labels, etc. For examples of bioactive molecules that can be conjugated to TCRs, see US Patent Publication No. 20110071919.

[0188] In other embodiments, the biologically active molecule is, for example, selected from the group consisting of: 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 may be, for example, HSA or a 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 a cell matrix protein.

[0189] In one embodiment, the bioactive 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 specificity 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 that has increased avidity for target compared to a monovalent antibody fragment.

[0190] The biologically active molecule may further be an effector group, such as an antibody Fc region.

[0191] The attachment to the N-terminus or C-terminus may be performed before or after the assembly of the TCR molecule or engineered polypeptide into a multimer. Thus, TCR fusions with Ig constant domains may be produced (by synthesis or by expression of nucleic acid) where the N-terminus or C-terminus bioactive molecule is already present. However, in certain embodiments, the addition to the N-terminus or C-terminus is performed after the TCR fusion is produced. For example, fluorenylmethyloxycarbonyl chloride can be used to introduce an Fmoc protecting group at the N-terminus of the TCR fusion. Fmoc binds serum albumin such as HSA with high affinity, and Fmoc-Trp or FMOC-Lys binds with increased affinity. The peptide can be synthesized with the Fmoc protecting group attached and then linked to the scaffold through cysteine. An alternative is a palmitoyl moiety that also binds to HSA, which has been used, for example, in liraglutide to extend the half-life of this GLP-1 analogue.

[0192] 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, for example antibody Fc fragments.

[0193] NHR2 domain AML1 / ETO is a fusion protein resulting from t(8;21) 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). The nerve homology domain 2 of ETO is involved in many of the 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.

[0194] In one embodiment of the invention, the protein assembled in a soluble multivalent format is a TCR composed in part or in whole of the extracellular domains of the TCR a and β chains. H 1 and C L It is stabilized by domains and can be organized in the following configurations: 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

[0195] In one embodiment of the invention, the extracellular TCR domains are linked to immunoglobulin CH1 and CL domains via an optional peptide linker (L) that 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

[0196] In another embodiment of the invention, a tetramerization domain (TD), such as the NHR2 homotetramer domain, is linked to the C-terminus of either an immunoglobulin CH1 or CL domain, which is linked to the extracellular TCR a and β chains. The NHR2 domain may optionally be linked to the CH1 or CL domain via a peptide linker. The resulting tetravalent heterodimeric TCR protein may be organized in the following configuration ((L) is an optional 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

[0197] The sensitivity of soluble TCRs to their cognate pMHC can be enhanced by increasing the avidity effect. This is achieved by increasing the number of antigen binding sites, which is facilitated by the tetramerization domain. This also increases the molecular weight of the protein molecule compared to monovalent soluble TCRs, thus prolonging serum retention in circulation. The increased serum half-life also enhances the likelihood that these molecules will interact with their cognate target antigens.

[0198] A tetravalent heterodimeric soluble TCR protein molecule can simultaneously bind one, two, three or four pMHC presented on a single cell or simultaneously bind to one, two, three or four different cells presenting its cognate pMHC.

[0199] The TCR α and β chain sequences used in the present invention may be from known TCRs specific for a particular pMHC or may be identified de novo by screening using techniques known in the art such as phage display. Furthermore, the TCR sequences are not limited to α and β chains in the present invention but may also incorporate TCR8 and γ or ε chains and sequence variations thereof that have been cloned directly from human T cells or identified by directed evolution using recombinant DNA techniques.

[0200] 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.

[0201] Multimeric (e.g., tetrameric or octameric) or multivalent TCRs according to the 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 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 mediates episomal replication of DNA plasmids together with host cell replication factors to allow 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.

[0202] Tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced by transient expression of genes from expression vectors.

[0203] 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 genomic engineering techniques known in the art that integrate the gene encoding the protein molecule into the genome of the host cell as a single copy or multiple copies. The site of DNA integration can be a defined location in the host genome or can be randomly integrated to obtain maximum expression of the desired protein molecule. Genomic engineering techniques can include, but are not limited to, homologous recombination, transposon-mediated gene transfer such as the PiggyBac transposon system, site-specific recombinases such as 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.

[0204] In yet another embodiment 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 refolded in vitro after purification by affinity chromatography to produce functional protein molecules capable of properly binding to their cognate pMHC or antigen.

[0205] In another embodiment of the 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.

[0206] In another embodiment of the invention, heterodimeric soluble TCR molecules or other multimers are produced as octavalent protein complexes, for example 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 presented on a single cell or to pMHC presented on up to eight different cells, thus generating highly sensitive soluble TCRs.

[0207] The heterodimeric soluble TCR portion of the molecule is made bivalent by fusing an immunoglobulin hinge domain to the C-terminus of either the CH1 or CL domain, which is itself linked to either the TCR a or β chain. The hinge domain allows the connection of the two heavy chains, giving them a structure similar to IgG. A tetramerization domain such as NHR2 is linked to the C-terminus of the hinge domain, optionally via a peptide linker. Monomeric soluble TCRs can be formed by attaching an immunoglobulin hinge to the C-terminus and N-terminus of the Ig CH1 or CL domain and the NHR2 domain, respectively. 2This allows the assembly of two NHR2 monomers, termed NHR2 homotetramers. In this configuration, unless a long flexible linker is provided between the hinge and NHR2 domains, it is predicted that the two NHR2 domains will most likely not form homodimers by antiparallel association due to structural constraints. Linkage of the tetramerization and hinge domains to heterodimeric soluble TCRs via immunoglobulin CH1 or CL domains results in the formation of NHR2 homotetramers. 2 This allows for stepwise self-assembly of octavalent soluble TCRs formed through the synthesis of NHR2 monomers. 2 and homodimers 2 This is done through an intermediate protein complex of the octavalent heterodimeric soluble TCR protein molecule (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 to its pMHC ligands far beyond the affinity found in nature. In particular, it is useful for identifying pMHCs recognized by uncharacterized tumor-specific T cells and T cells involved in other diseases such as autoimmune diseases. Many different configurations of octavalent heterodimeric soluble TCR protein molecules can be produced. 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 H1-link Vβ-(L)-C L -(L)-TD 7.Vα-Cα-(L)-C L -(L)-TD and Vβ-Cβ(L)-C H 1-ヒンジ 8.Vα-Cα-(L)-C H 1-link Vβ-Cβ-(L)-C L -(L)-TD

[0208] In another embodiment of the present invention, the self-assembled multivalent proteins, preferentially tetravalent and octavalent heterodimeric soluble TCRs, are fused or conjugated to bioactive agents / effector molecules, thereby allowing these molecules to be guided to a desired cell population, such as cancer cells, and specifically exert their therapeutic effects. The tumor targeting ability of monoclonal antibodies to guide effector molecules, such as cytotoxic drugs, toxins, or cytokines, is well established (Perez et al. 2014; Young et al. 2014). Similarly, 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, IFNa, IFNβ, IFNy, 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-la, 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 the multivalent protein complexes described in the present invention include, but are not limited to, toxins such as diphtheria toxin, ricin, Pseudomonas exotoxin, and cytotoxic drugs such as auristatins, maytansines, calicheamicins, anthracyclines, duocarmycins, pyrrolobenzodiazepines, etc. The cytotoxic drugs can be conjugated by a selection linker, which is non-cleavable or cleavable by a protease, or is acid labile.

[0209] To remove heterogeneity and improve the stability of the conjugate, cytotoxic drugs can be conjugated in a site-specific manner, which can be achieved by engineering specific cysteine ​​residues or using enzymatic conjugation through glycosyltransferases and transglutaminases (Panowski et al. 2014).

[0210] In another embodiment of the invention, the multivalent protein complex is covalently linked to a molecule that allows for detection, such as a fluorescent, radioactive or electron transfer agent.

[0211] In another embodiment of the invention, the effector molecule (EM) is fused to the multivalent protein complex via the C-terminus of a tetramerization domain such as NHR2, optionally via a peptide linker. Fusion via the NHR2 domain can be arranged to produce multivalent protein complexes in many different configurations. Some examples of protein configurations that can be produced using tetravalent heterodimeric soluble TCRs are shown below: 1. Vα-(L)-C L and Vβ-(L)-C H 1-(L)-TD-(L)-EM 2. Vα-(L)-C H 1-(L)-TD-(L)-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)-C H 1 8. Vα-Cα-(L)-C H 1 and Vβ-Cβ-(L)-C L -(L)-TD-(L)-EM

[0212] 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, optionally via a peptide linker. Fusion of the EM via the immunoglobulin domains can be arranged to produce multivalent protein complexes in a number of different configurations. Some examples of protein configurations that can be produced 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

[0213] 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, optionally via a peptide linker, and also at the C-terminus of the tetramerization domain (e.g., NHR2). This approach allows the fusion of two effector molecules to be fused with the TCR heterodimer complex. The fusion of the EM via the immunoglobulin domain and the tetramerization domain can be arranged to produce multivalent protein complexes in many different configurations. Some examples of protein configurations that can be produced 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

[0214] In another embodiment of the invention, the multivalent protein complex is fused to a protein tag that facilitates purification. Purification tags are known in the art and include, but are not limited to, the following tags: His, GST, TEV, MBP, Strep, FLAG.

[0215] Non-TCR multimers The present invention provides a unique method for assembling proteins into soluble, multivalent formats with multiple interacting domains or the ability to bind antigens. Proteins can be monomers, homodimers, heterodimers or oligomers that preferentially participate directly or indirectly in the immune system or have the ability to modulate immune responses. Examples include, but are not limited to, TCRs, peptide MHC class I and class II, antibodies or antigen-binding portions thereof, and binding proteins with alternative non-antibody protein scaffolds.

[0216] 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, such as viral and bacterial proteins.

[0217] The non-TCR multimer may be a multimer of antibodies or antibody fragments, such as a dAb of a Fab. Examples of dAbs and Fabs according to the invention include:

[0218] Examples of multivalent DAbs 25.VH-(L)-NHR2 26. VL(lambda or kappa)-(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

[0219] Example of a multivalent Fab 33. VH-CH1-(L)-NHR2 and VL(lambda or kappa)-CL 34. VL(lambda or kappa)-CL-(L)-NHR2 and VH-CH1 35. VH-CH1-hinge-(L)-NHR2 and VL(lambda or kappa)-CL 36. VL(lambda or kappa)-CL-hinge-(L)-NHR2 and VH-CH1 37. VH-CH1-(L)-NHR2-(L)-EM and VL(lambda or kappa)-CL 38. VL(lambda or kappa)-CL-(L)-NHR2-(L)-EM and VH-CH1 39. VH-CH1-hinge-(L)-NHR2-(L)-EM and VL(lambda or kappa)-CL 40. VL(lambda or kappa)-CL-hinge-(L)-NHR2-(L)-EM and VH-CH1 41. VH-CH1-(L)-NHR2 and VL(lambda or kappa)-CL-(L)-EM 42. VL(lambda or kappa)-CL-(L)-NHR2 and VH-CH1-(L)-EM 43. VH-CH1-hinge-(L)-NHR2 and VL(lambda or kappa)-CL-(L)-EM 44. VL(lambda or kappa)-CL-hinge-(L)-NHR2 and VH-CH1-(L)-EM

[0220] In the above example, (L) represents an optional peptide linker, and EM represents a bioactive agent or effector molecule such as a toxin, drug or cytokine, including binding molecules such as antibodies, Fabs and ScFvs.

[0221] The variable light chain can be either Vλ or Vκ.

[0222] In one aspect of the present invention, the assembled tetramerized protein molecule can be, in one example, human pMHC for drug discovery applications using animal drug discovery platforms (e.g., mouse, rat, rabbit, chicken). In such a context, the tetramerization domain is preferentially expressed and produced from a gene originating from the intended animal species. One 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 to the human pMHC rather than the tetramerization domain of the protein complex.

[0223] Multivalent antibodies can be produced, for example, using single domain antibody sequences fused to an NHR2 multimerization domain.

[0224] In a related embodiment 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 has unique advantages over monovalent molecular probes, since they have enhanced affinity, avidity and retention time in vivo, which enhances tumor targeting in vivo.

[0225] The multimerization domain is the NHR2 domain described above.Preferably, the polypeptide is stabilized and / or made soluble by using an Ig constant domain fused to the polypeptide, the fusion providing a tetramer of the polypeptide.An Ig hinge domain can be used to provide an octamer.

[0226] Use of multimers The multimeric TCR proteins according to the invention are useful in any application in which soluble TCR proteins are indicated. Particular advantages of the TCR proteins of the invention include increased avidity for a selected target and / or the ability to bind multiple targets.

[0227] Thus, in one embodiment, the multivalent heterodimeric soluble TCR protein molecules of the present invention can be used to selectively inhibit immune responses, for example, to suppress autoimmune responses.The multivalent, e.g., tetravalent, nature of these soluble protein molecules gives them an exquisite sensitivity and binding affinity that competes with the antigen-specific interaction between T cells and antigen-presenting cells.This kind of neutralizing effect can be therapeutically beneficial in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, Graves' disease, vasculitis and type 1 diabetes.

[0228] Similarly, tetravalent heterodimeric soluble TCR protein molecules can be used to selectively suppress T cell recognition of specific transplantation antigens and self-antigens that bind to target molecules, thus preventing tissue transplant rejection by inhibiting cell-cell interactions.

[0229] 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.

[0230] Tetravalent heterodimeric soluble TCR protein molecules with enhanced sensitivity to cognate pMHC can be used for diagnostic purposes using biological samples obtained directly from human patients. The enhanced sensitivity of tetravalent heterodimeric soluble TCR allows the detection of potential disease-related peptides presented on MHC that are known to be naturally expressed at low density. These molecules can also be used for patient stratification to enroll patients in relevant clinical trials.

[0231] In another aspect of the invention, the octavalent heterodimeric soluble TCR protein molecules may be used in pharmaceutical preparations for the treatment of various diseases.

[0232] In another embodiment related to the present invention, the octavalent heterodimeric soluble TCR protein molecules can be used as probes for molecular imaging of tumors or prepared as therapeutic proteins. EXAMPLES

[0233] Example 1: Generation of tetravalent and octavalent soluble heterodimeric NY-ESO-1 TCR molecules This example demonstrates how to generate tetravalent and octavalent soluble heterodimeric TCR molecules, designated ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR, respectively. These formats overcome issues related to solubility at the target site and avidity for the cognate ligand.

[0234] To exemplify ts-NY-ESO-1 and os-NY-ESO-1 TCRs as stable and soluble molecules, a TCR αβ variable sequence with high affinity for NY-ESO-1 together with an immunoglobulin constant domain and an NHR2 tetramerization domain is used in this example.

[0235] SLLMWITQC-HLA-A used in this example * The high affinity NY-ESO TCR αβ chains specific for 0201 (comprising TCR Vα-Ca and Vβ-Cp, respectively) are reported in WO 2005 / 113595, including the signal peptide sequence (MGWSCIILFLVATATGVHS). To aid in protein purification, a histidine tag was incorporated at the C-terminus of the NHR2 domain.

[0236] DNA constructs encoding the components of the ts-NY-ESO-1 and os-NY-ESO-1 TCRs were synthetically constructed as a two-vector system to allow for soluble expression and functional assembly in mammalian cells. Schematic depictions of the two assembled TCR chains (a and β chains) for which DNA sequences were synthesized for cloning into expression vectors are shown in Figures 3 and 4, and their amino acid sequences are shown in Figures 5 and 6.

[0237] 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 mediates episomal replication of DNA plasmids together with host cell replication factors to allow enhanced expression of recombinant proteins. Therefore, we choose the pTT5 expression vector to clone the components for the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR molecules.

[0238] The synthesized DNA fragments containing the TCR αβ chains are digested with restriction enzymes (FastDigest, Fermantas) at the restriction sites (RS) and the DNA is separated on a 1% agarose gel. The DNA fragments of the correct size are excised and the DNA is purified using a gel extraction kit from Qiagen. The pTT5 vector is also digested with the same restriction enzymes and the linearized plasmid DNA is purified from the excised agarose gel. The digested TCR αβ chains are cloned into the digested pTT5 vector to obtain four expression vectors (pTT5-ts-NY-ESO-1-TCRa, pTT5-ts-ESO-1-TCRβ, pTT5-os-NY-ESO-1-TCRa and pTT5-os-ESO-1-TCRβ).

[0239] Expression of tetravalent and octavalent soluble NY-ESO TCRs

[0240] Functional expression of ts-NY-ESO-1 and os-NY-ESO-1 TCRs is performed in suspension-adapted HEK293 EBNA cells. HEK293-EBNA cells are cultured in serum-free Dulbecco's Modified Eagle Medium (DMEM, high glucose (4.5 g / L) with 2 mM L-glutamine) at 37 °C, 5% CO2 and 95% humidity.

[0241] For each transfection, select HEK293-EBNA cells (3 × 10) from approximately 60% confluent cells. 7Cells) are freshly seeded in 250 mL Erlenmeyer shaker flasks (Corning). Transfections are performed using FreeStyle MAX cationic lipid base reagent (Life Technologies) according to the supplier's guidelines. For expression of ts-NY-ESO-1 TCR, 37.5 μg of total plasmid DNA (18.75 μg of plasmid DNA for each of the vectors pTT5-ts-NY-ESO-1-TCRa and pTT5-ts-ESO-1-TCRβ or different amounts of the two expression plasmids) are used per transfection. Similarly, for expression of os-NY-ESO-1 TCR, 18.75 μg of plasmid DNA for each of the vectors pTT5-os-NY-ESO-1-TCRa and pTT5-os-ESO-1-TCRβ are used for transfection. After transfection, cells were harvested in fresh medium and cultured for 4-8 days in an orbital shaker at 110 rpm, 5% CO2, 37°C. Smaller scale transfections are performed similarly in 6-well or 24-well plates.

[0242] Expressed soluble eTCR 2 -Analysis of BiTE The ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR protein molecules secreted into the supernatant are analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) either directly or after protein purification. Protein samples and standards are prepared under both reducing and non-reducing conditions. SDS-PAGE was performed using cast minigels for protein electrophoresis on a Mini-PROTEAN Tetra cell electrophoresis system (Bio-Rad). Coomassie blue dye was used to stain the proteins in the SDS-PAGE gels.

[0243] Purification of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR protein molecules Soluble ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from cell supernatants are purified in two steps. In the first step, immobilized metal affinity chromatography (IMAC) is used with nickel-loaded nitrilotriacetic acid (NTA) agarose resin (HisPur Ni-NTA Superflow agarose-Thermo fisher). The binding and washing buffer consists of Tris-buffered saline (TBS) at pH 7.2 containing a low concentration of imidazole (10-25 mM). Elution and recovery of His-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from the IMAC column is achieved by washing with a high concentration of imidazole (>200 mM). Eluted protein fractions are analyzed by SDS-PAGE and fractions containing the protein of interest are pooled. The pooled protein fractions are either used directly in binding assays or further purified in a second step involving size exclusion chromatography (SEC): Superdex 200 increase prepacked columns (Gelifesciences) are used to separate out monomers, oligomers and any aggregated forms of the target protein.

[0244] Surface plasmon resonance Specific binding and affinity analysis of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR to their pMHC is performed using BIAcore. Briefly, purified histidine-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR proteins were bound to Ni in nitrilotriacetic acid (NTA). 2+ Captured on the sensor surface via chelation. Different concentrations of analyte solutions containing NY-ESO pep(SLLMwiTQv)-MHC(ProImmune) were injected and the binding signal was monitored.

[0245] Example 2: Generation of tetravalent and octavalent soluble NY-ESO TCR-IL2 fusion molecules DNA encoding the domains required to express the ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 protein complexes is synthesized as described above and cloned into the expression vector pTT5. Schematic depictions of the domains within the TCR αβ chain for ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 are shown in Figures 7 and 8, and the amino acid sequences are shown in Figures 9 and 10.

[0246] Expression, purification and characterization of ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 are carried out as described above.

[0247] Example 3: High yield of tetramer secretion Briefly, using conventional genetic engineering techniques, we generated a HEK293-T cell line that encodes Quad 16 (FIGS. 14 and 15) and another HEK293-T cell line that encodes Quad 17 (FIGS. 14 and 15).

[0248] Protein expression occurred and proteins were secreted from the cell lines. Samples of the medium in which the cells were incubated were subjected to PAGE under denaturing conditions (SDS-PAGE) or native conditions (without SDS), the former being further subjected to reducing conditions (using mercaptoethanol) whereas the latter was not.

[0249] The reduced gel showed a clear band at the expected monomer size (Figure 16). Surprisingly, the non-reduced, native gel showed no detectable bands at the monomer, dimer or trimer sizes, but instead a strong band at the tetramer size, indicating a very high yield of the tetramer was obtained, which was confirmed by SEC to be of high purity.

[0250] For Quad 16, the tetramer peak from SEC was run on SDS-PAGE and the resulting band was excised for mass spectrometry. Data were obtained with trypsin digestion and p53 was detected in 100% of the protein, conclusive evidence that secreted Quad 16 was multimerized.

[0251] Example 4: Intracellular protein expression of the extracellular portion of the TCR fused to NHR2 TP Expression vector All DNA fragments were synthesized and cloned into the expression vector pEF / myc / cyto (Invitrogen) by Twist Bioscience (California). The schematic diagram and sequence of the synthesized DNA fragments and Quad polypeptides are shown in FIG. 21 and in the sequence listing herein.

[0252] Preparation of DNA Lyophilized plasmid DNA synthesized by Twist Bioscience was resuspended in MQ water to a concentration of 50ng / μl. 50ng of DNA was transformed into 50μl of competent DH5a cells using conventional heat shock method. Cells were plated on LB agar plates containing 100μg / mL ampicillin and grown overnight at 37°C. Individual colonies were picked and grown overnight at 37°C at 220 rpm. DNA was purified from cells using QIAprep Spin Miniprep Kit according to the manufacturer's instructions (Qiagen).

[0253] Transfection into HEK293T cells Briefly, HEK293T cells were maintained in high glucose DMEM supplemented with 10% FBS and Pen / Strep. Cells were plated at 6 × 10 per well of a 6-well plate in 2 ml of medium. 5Cells were seeded and allowed to adhere overnight at 37°C, 5% CO2. 7.5 μl Lipofectamine 2000 was diluted in 150 μl OptiMem and incubated at room temperature for 5 min. Plasmid DNA (2.5 μg) was diluted in 150 μl OptiMem. The diluted DNA was combined with the diluted Lipofectamine 2000, mixed gently and incubated at room temperature for 20 min. 300 μl of the complex was added to one well of a 6-well plate. If the analysis required the medium to be serum-free, the medium was aspirated 6 hours after transfection and replaced with CD293 medium. Cells were incubated for 48 hours at 37°C, 5% CO2 before analysis.

[0254] Therefore, different formats of TCR-linked NHR2 tetramerization domain (TD) constructs (Quads) were transfected into HEK293T cells. Quad 3 and Quad 4, which resemble the tetravalent format of TCR (structures depicted diagrammatically in Figures 1 and 3), and Quad 12 and Quad 13, which resemble the octavalent format of TCR (structures depicted diagrammatically in Figures 2 and 4), were transfected for protein expression analysis. To confirm the proteins expressed in the cells, protein samples were prepared from the transfected HEK293T cells as follows. Briefly, the cells were washed once with 2 ml of PBS and then aspirated. 150 μl of trypsin-EDTA (0.05%) was added to each well and the cells were incubated at room temperature for about 1 minute. The plate was tapped to detach any strongly adherent cells. 850 μl of medium was added to each well to inactivate the trypsin. Cells were transferred to a 1.5 ml eppendorf and centrifuged at 1,000 rpm for 5 min. The supernatant was aspirated and the pellet was stored on ice. Cells were resuspended in 400 μl of cell lysis buffer (10 mM Tris, pH 7.5, 1% SDS) containing Protease Inhibitor Cocktail Set III (Calbiochem) and diluted 1 / 200. Samples were vortexed vigorously and incubated on ice for 20 min. Cells were sonicated using a Branson Ultrasonics Sonifier™ (Thermo Fisher Scientific). Amplitude was set at 30% and cells were sonicated for a total of 24 s (6 s on, 3 s off, 4 times). Total protein concentration was quantified using the Pierce BCA Protein Assay Kit™ according to the manufacturer's instructions. 100 μg was diluted with MQ water to a volume of 80 μl. 20 μl of 5x SDS loading buffer was then added to give a 1 mg / ml sample. Samples were incubated at 95° C. for 5 min prior to SDS-PAGE and Western blot analysis.

[0255] Protein samples were separated on SDS-PAGE under denaturing conditions. Typically, 25 μg of total cell lysate (25 μl) was run on the gel for Western blot analysis. 5 μl of PageRule Prestained 10-180 kDa Protein Ladder was run on the gel next to the protein sample. Gels were run in Tris-glycine buffer containing 0.1% SDS. A constant voltage of 150 volts was used and the gel was run for approximately 70 minutes until the dye front had migrated sufficiently.

[0256] SDS-PAGE (15% Bis-Tris) gels were prepared using the following separating and stacking gels:

[0257] Separating gel: 5ml 30% Bis-acrylamide 2.6ml 1.5M Tris (pH 8.8) 50μl 20% SDS 100μl 10% APS 10 μl TEMED ·2.2ml MQ water

[0258] Stacking gel: 0.75ml 30% Bis-acrylamide 1.25ml 1.5M Tris (pH 8.8) 25μl 20% SDS 50μl 10% APS 5 μl TEMED 2.9ml MQ water

[0259] Western blotting was performed for specific and sensitive detection of protein expression of TCR-NHR2 TD fusion proteins from Quad 3, Quad 4, Quad 12 and Quad 13. Proteins separated by SDS-PAGE were transferred to Amersham Hybond™ 0.45 μM PVDF membrane as follows. Briefly, Amersham Hybond 0.45 μM PVDF membrane was activated with MeOH for about 1 min and rinsed with transfer buffer (25 mM Tris, 190 mM glycine, 20% MeOH) before use. A stack of sponge, filter paper, gel, membrane, filter paper and sponge was prepared and placed in a cassette for transfer. Transfer was performed at 280 mA for 75 min on ice. The membrane was incubated in blocking buffer (TBST, 5% milk powder) for about 2 h. The membrane was washed briefly with TBST and then incubated with anti-human IgG HRP (Thermo, 31410) diluted 1 / 2500 in TBST, 1% milk powder overnight at 4° C. The membrane was washed extensively (3 washes with TBST, 15 min each) and then developed using Pierce ECL Western Blotting Substrate.

[0260] Using anti-human IgG detection antibodies to probe Western blots, specific protein bands of the expected molecular weights can be detected from samples prepared from Quad 3 (46.1 kDa), Quad 4 (46.4 kDa), Quad 12 (47.8 kDa) and Quad 13 (48.1 kDa) (Figures 17A and 17B). These data confirm the intracellular protein expression of the TCR-NHR2 TD fusion protein in HEK293T cells.

[0261] For all Quads analyzed, a clear single band could be detected, indicating that the fusion of TRVβ-TRVβ-IgG1-CH1 (+ / - IgG hinge domain) with the NHR2 TD is stable. These expression data also confirm that the tetravalent (Quad 3 and Quad 4) and octavalent (Quad 12 and Quad 13) molecules exemplified in this example can be assembled.

[0262] The difference between Quad 3 and Quad 4 is the presence of a small peptide linker (G4S) located between the IgG1 CH1 domain and the NHR2 TD. This is also true for Quad 12 and Quad 13, with Q13 containing a peptide linker between the IgG1 CH1 domain and the NHR2 TD. Expression data shows that the peptide linker does not result in protein expression. However, it may be desirable to include a peptide linker to help stabilize antigen binding and / or multimerization complexes in these TCR-NHR2 TD formats.

[0263] Example 5: Soluble protein expression of the extracellular portion of the TCR fused to NHR2 TP In Example 4, it was shown that TCR-NHR2 TD fusion proteins were expressed intracellularly in HEK293T cells. Here again, Quad 3, Quad 4, Quad 12 and Quad 13 were used to demonstrate soluble expression of these fusion proteins. Quad 3, Quad 4, Quad 12 and Quad 13 were transfected into HEK293T cells as described above, and soluble proteins from cell supernatants were concentrated. Briefly, 48 hours after transfection, media was collected and centrifuged at 2,000 rpm for 5 minutes to remove any cells or debris. Typically, 500 μl of media was concentrated to 100 μl using an Amicon™ Ultra 0.5 Centrifugal Unit with a MWCO of 10 kDa. 25 μl of 5× SDS loading buffer was added to the samples, followed by incubation at 95° C. for 5 minutes before gel / western blot analysis. Concentrated protein samples were separated on SDS-PAGE gels and transferred to Amersham Hybond 0.45 μM PVDF membranes. Western blotting and protein detection were performed using anti-human IgG HRP using the methods described above.

[0264] Protein samples concentrated and prepared from cell supernatants show specific protein bands at the expected molecular weights on Western blots corresponding to Quad 3 (46.1 kDa), Quad 4 (46.4 kDa), Quad 12 (47.8 kDa) and Quad 13 (48.1 kDa) (Figures 18A and 18B). Western blot expression data clearly demonstrate soluble expression of TCR-NHR2 TD fusion protein in HEK293T. These data are the first reported to demonstrate soluble expression of TCR-NHR2 TD fusion protein expressed in eukaryotic cells such as HEK293T cells.

[0265] The detection of soluble protein expression from both tetrameric (Quad 3 and Quad 4) and octameric (Quad 12 and Quad 13) TCR-NHR2 TD formats highlights the potential application of NHR2 TD in a wide range of contexts. The use of NHR2 TD fusion molecules can be used for the preparation of therapeutic molecules as well as protein molecules for use in diagnostics and imaging.

[0266] Example 6: Intracellular protein expression of antibody fragments fused to NHR2 TD To further illustrate the versatility of the NHR2 TD, several different antibody fragment formats fused to the NHR2 TD were constructed for testing expression in HEK293T cells.

[0267] Quad 14 and Quad 15 contain an antibody VH domain fused to NHR2 TD with or without a peptide linker located between the VH and NHR2 TD as depicted diagrammatically in Figure 11 and Figure 21. The VH domain in Quad 14 and Quad 15 is specific for GFP (Green Fluorescent Protein). Several other versions of this format were also constructed and tested with VHs specific for therapeutically useful drug targets. The sequences of the binding domains are listed in Table 4. Some of these include Quad 34 (specific for TNFa), Quad 38 (specific for VEGF), Quad 40 (specific for EGFR) and Quad 44 (specific for CD38).

[0268] Quad 38 and Quad 44 were further developed containing an additional binding arm with inclusion of a second VH domain specific for EGFR and CD138, respectively resulting in Quad 42 and Quad 46. Quad 42 and Quad 46 represent bispecific molecules capable of multimerizing via the NHR2 TD domain to form bispecific tetramers.

[0269] In another example, an effector molecule (human IL2) is linked to the C-terminus of Quad 14 and Quad 15 resulting in Quad 18 and Quad 19, thereby making the VH-NHR2-IL2 molecule tetravalent and bifunctional.

[0270] In another example, an antibody Fab fragment (VH-CH1) was linked to the NHR2 TD (Quad 23 and Quad 24) as depicted diagrammatically in Figure 12. Quad 23 and Quad 24 represent tetravalent Fab molecules when co-expressed or mixed and assembled in vitro with a second chain containing an immunoglobulin light chain (e.g., Quad 25).

[0271] In yet another example, a human IgG1 hinge domain is included in Quad 23 and Quad 24, designated Quad 26 and Quad 27, and depicted diagrammatically in Figure 13. Quad 26 and Quad 27 represent octavalent Fab molecules when co-expressed or mixed and assembled in vitro with a second chain containing an immunoglobulin light chain domain (e.g., Quad 25).

[0272] The following Quad vectors, all His-tagged, were transfected into HEK293T cells: Quad 14, Quad 15, Quad 18, Quad 19, Quad 23, Quad 24, Quad 26, Quad 27, Quad 34, Quad 38, Quad 40, Quad 42, Quad 44 and Quad 46. Protein samples were prepared from whole cell extracts as described above, separated on SDS-PAGE and transferred to Amersham Hybond 0.45μM PVDF membranes. Anti-His HRP (Sigma, A7058) diluted 1 / 2500 in TBST, 1% milk powder was used as a probe to detect specific protein expression.

[0273] Specific protein expression could be detected in whole cell extracts for all the different antibody-NHR2 TD fusion proteins tested using Quad 14, Quad 15, Quad 18, Quad 19, Quad 23, Quad 24, Quad 26, Quad 27, Quad 34, Quad 38, Quad 40, Quad 42, Quad 44 and Quad 46 (Figure 19A-D). Interestingly, Quad 18 and Quad 19, which contain an effector domain (IL2) fused to the C-terminus of the NHR2 TD domain in addition to the VH binding domain fused to the N-terminus of the NHR2 TD, showed good protein expression.

[0274] Expression of Quad 23 and Quad 24 polypeptides highlights the ability to use the NHR2 TD to form tetravalent antibody Fab molecules when co-expressed or mixed in vitro with a partner soluble Quad molecule (e.g., Quad 25). Similarly, expression of Quad 26 and Quad 27, which contain a human IgG1 hinge domain, highlights the ability to use the NHR2 TD to form octavalent antibody Fab molecules when co-expressed or mixed in vitro with a partner soluble second partner chain (e.g., Quad 25).

[0275] Quad 42 and Quad 46 bispecific molecules containing an additional VH domain fused to the C-terminus of the NHR2 TD domain also showed good protein expression. These data highlight the versatility of the NHR2 TD domain and its ability to be fused to different binding and effector molecules to develop a wide range of protein formats. The data also suggest that protein molecules can be fused to both the N-terminus and C-terminus of the NHR2 TD, allowing the development of bispecific, multivalent protein molecules.

[0276] Example 7: Multivalent assembly of antibody fragments fused to NHR2 TD The NHR2 TD is involved in the oligomerization of ETO into a tetrameric complex. As shown in Examples 4-6, the NHR2 TD domain can be used to fuse binding domains and effector molecules to the N-terminus or C-terminus or both N-terminus and C-terminus without affecting expression. The binding domains can be TCR variable and constant domains, antibodies and antibody fragments or effector molecules such as IL2. Although the NHR2 TD is part of an intracellularly expressed protein that is naturally expressed only intracellularly, proteins can also be expressed in a soluble format when fused to the NHR2 TD (Figure 18).

[0277] To demonstrate whether the NHR2 TD retains the ability to oligomerize when fused to a binding domain, Quad 14 and Quad 15 were expressed in HEK293T cells and protein samples were prepared from whole cell extracts as described above. Protein samples were resolved on PAGE gels under denaturing and non-denaturing (native) conditions. Native gels were prepared using the protocol described above without SDS. Proteins from the PAGE gels were transferred to Amersham Hybond 0.45 μM PVDF membranes. Specific protein expression was detected using anti-human IgG HRP detection antibody as a probe.

[0278] As expected, under denaturing conditions, the expression of VH-NHR2 TD from Quad 14 and Quad 15 can be seen as a monomer, and specific protein bands can be detected at the expected molecular weight (22 and 22.3 kDa) (Figure 20A). Interestingly, under non-denaturing, and therefore native, conditions, no monomer or dimer of VH-NHR2 TD from Quad 14 and Quad 15 can be detected (Figure 20B). Only a high molecular weight protein band, likely a tetramer of VH-NHR2 TD from Quad 14 and Quad 15, can be detected. The assembly of the tetramer appears to be efficient and pure, as judged by the protein intensity and the absence of any detectable monomers and dimers of Quad 14 and Quad 15.

[0279] Taken together, the data from Examples 4 to 7 provide conclusive evidence that the NHR2 TD is versatile and allows the fusion of a variety of protein binding domains and effector molecules. The NHR2 TD allows soluble expression of proteins from eukaryotic cells such as HEK293T cells, and results in the formation of highly stable and pure tetrameric molecules.

[0280] References ·Ali, SA et al., 1999. Transferrin Trojan Horses as a Rational Approach for the Biological Delivery of Therapeutic Peptide Domains. Journal of Biological Chemistry, 274(34), pp.24066-24073. Barbas et al. (1992) supra ·Bentley, GA & Mariuzza, RA, 1996. THE STRUCTURE OF THE T CELL ANTIGEN RECEPTOR. Annual Review of Immunology, 14(1), pp.563-590. ·Binz, H.K. et al., 2003. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. Journal of molecular biology, 332(2), pp.489-503. ·Bixby, K.A. et al., 1999. Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels. Nature structural biology, 6(1), pp.38-43. ·Borghouts, C., Kunz, C. & Groner, B., 2005. Peptide aptamers: recent developments for cancer therapy. Expert Opinion on Biological Therapy, 5(6), pp.783-797. ·Breitling, F. et al., 1991. A surface expression vector for antibody screening. Gene, 104(2), pp.147-53. ·Burton, D.R. et al., 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proceedings of the National Academy of Sciences of the United States of America, 88(22), pp.10134-7. ·Chang, C.N., Landolfi, N.F. & Queen, C., 1991. Expression of antibody Fab domains on bacteriophage surfaces. Potential use for antibody selection. Journal of immunology (Baltimore, Md.: 1950), 147(10), pp.3610-4. ·Chaudhary, V.K. et al., 1990. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins. Proceedings of the National Academy of Sciences, 87(3), pp.1066-1070. ·Chiswell, D.J. & McCafferty, J., 1992. Phage antibodies: will new "coliclonal" antibodies replace monoclonal antibodies? Trends in biotechnology, 10(3), pp.80-4. ·Clackson, T. et al., 1991. Making antibody fragments using phage display libraries. Nature, 352(6336), pp.624-8. ·Davis, M.M. & Bjorkman, P.J., 1988. T-cell antigen receptor genes and T-cell recognition. Nature, 334(6181), pp.395-402. ·Ding, X.-F. et al., 2008. Characterization and Expression of a Human KCTD1 Gene Containing the BTB Domain, Which Mediates Transcriptional Repression and Homomeric Interactions. DNA and Cell Biology, 27(5), pp.257-265. ·Fujiwara, Y. & Minor, D.L., 2008. X-ray Crystal Structure of a TRPM Assembly Domain Reveals an Antiparallel Four-stranded Coiled-coil. Journal of Molecular Biology, 383(4), pp.854-870. ·Hawkins, R.E. & Winter, G., 1992. Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool. European journal of immunology, 22(3), pp.867-70. ·Herrin, B.R. & Cooper, M.D., 2010. Alternative adaptive immunity in jawless vertebrates. Journal of immunology (Baltimore, Md.: 1950), 185(3), pp.1367-74. ·Hoogenboom, H.R. et al., 1991. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic acids research, 19(15), pp.4133-7. ·Hosse, R.J., Rothe, A. & Power, B.E., 2006. A new generation of protein display scaffolds for molecular recognition. Protein science: a publication of the Protein Society, 15(1), pp.14-27. ·Irving, R.A. et al., 2001. Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics. Journal of immunological methods, 248(1-2), pp.31-45. ·Jeffrey, P.D., Gorina, S. & Pavletich, N.P., 1995. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science (New York, N.Y.), 267(5203), pp.1498-502. ·Kang, A.S. et al., 1991. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proceedings of the National Academy of Sciences, 88(10), pp.4363-4366. ·Kohl, A. et al., 2003. Designed to be stable: Crystal structure of a consensus ankyrin repeat protein. Proceedings of the National Academy of Sciences, 100(4), pp.1700-1705. ·Lerner, R.A. et al., 1992. Antibodies without immunization. Science (New York, N.Y.), 258(5086), pp.1313-4. ·Liu, Y. et al., 2006. The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1 / ETO’s activity. Cancer Cell, 9(4), pp.249-260. ·Lowman, H.B. et al., 1991. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry, 30(45), pp.10832-8. ·Marks et al. (1991) supra ·Marks, J.D. et al., 1991. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. Journal of molecular biology, 222(3), pp.581-97. ·Marks, J.D. et al., 1992. Molecular evolution of proteins on filamentous phage. Mimicking the strategy of the immune system. The Journal of biological chemistry, 267(23), pp.16007-10. ·McCafferty et al. (1990) supra ·McCafferty, J. et al., 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348(6301), pp.552-4. ·Moysey, R., Vuidepot, A.-L. & Boulter, J.M., 2004. Amplification and one-step expression cloning of human T cell receptor genes. Analytical biochemistry, 326(2), pp.284-6. ·Panowski, S. et al., 2014. Site-specific antibody drug conjugates for cancer therapy. mAbs, 6(1), pp.34-45. ·Parker, M.H. et al., 2005. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two. Protein engineering, design & selection: PEDS, 18(9), pp.435-44. ·Perez, H.L. et al., 2014. Antibody-drug conjugates: Current status and future directions. Drug Discovery Today, 19(7), pp.869-881. ·Reddy Chichili, V.P., Kumar, V. & Sivaraman, J., 2013. Linkers in the structural biology of protein-protein interactions. Protein science: a publication of the Protein Society, 22(2), pp.153-67. ·Shao, C.-Y., Secombes, C.J. & Porter, A.J., 2007. Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library. Molecular immunology, 44(4), pp.656-65. ·Silverman, J. et al., 2005. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nature Biotechnology, 23(12), pp.1556-1561. ·Skerra, A., 2000. Lipocalins as a scaffold. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1482(1-2), pp.337-350. ·Thie, H. et al., 2009. Multimerization domains for antibody phage display and antibody production. New biotechnology, 26(6), pp.314-21. ·Tonegawa, S., 1988. Somatic generation of immune diversity. Bioscience reports, 8(1), pp.3-26. ·Walchli, S. et al., 2011. A Practical Approach to T-Cell Receptor Cloning and Expression M. M. Rodrigues, ed. PLoS ONE, 6(11), p.e27930. ·Wikman, M. et al., 2004. Selection and characterization of HER2 / neu-binding affibody ligands. Protein Engineering Design and Selection, 17(5), pp.455-462. ·Young, P.A., Morrison, S.L. & Timmerman, J.M., 2014. Antibody-cytokine fusion proteins for treatment of cancer: Engineering cytokines for improved efficacy and safety. Seminars in Oncology, 41(5), pp.623-636. ·Zahnd, C. et al., 2007. A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), pp.1015-28. ·Zhang, J. et al., 2009. Transient expression and purification of chimeric heavy chain antibodies. Protein Expression and Purification, 65(1), pp.77-82. ·Expert Opinion on Investigational Drugs 16(6) 909-917 (June 2007) ·Chapter 7 - Non- Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel)

[0281]

Table 1-1

[0282]

Table 1-2

[0283]

Table 2-1

[0284]

Table 2-2

[0285]

Table 2-3

[0286]

Table 2-4

[0287]

Table 2-5

[0288]

Table 2-6

[0289]

Table 2-7

[0290]

Table 2-8

[0291]

Table 3-1

[0292]

Table 3-2

[0293]

Table 3-3

[0294]

Table 3-4

[0295]

Table 3-5

[0296]

Table 3-6

[0297]

Table 3-7

[0298]

Table 3-8

[0299]

Table 3-9

[0300]

Table 3-10

[0301]

Table 3-11

[0302]

Table 3-12

[0303]

Table 4-1

[0304]

Table 4-2

[0305]

Table 4-3

[0306]

Table 5-1

[0307]

Table 5-2

[0308]

Table 5-3

[0309]

Table 5-4

[0310]

Table 6

[0311]

Table 7

Claims

1. A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., a protein domain) or peptide, said multimer being multimerized by first, second, third and fourth self-associating tetramerization domains (TDs) that associate together, each tetramerization domain being composed of a respective engineered polypeptide comprising one or more copies of said protein domain or peptide.

2. The multimer of claim 1 which is a tetramer or octamer of said domain or peptide.

3. 3. The multimer of claim 1 or 2, comprising a tetramer or an octamer of the immunoglobulin superfamily binding site.

4. 4. The multimer of claim 3, wherein the binding site comprises a first variable domain paired with a second variable domain.

5. 5. The multimer of any one of claims 1 to 4, wherein each polypeptide comprises a first and a second copy of said protein domain or peptide, said polypeptide comprising (in the N-terminal to C-terminal direction): (i) the first copy-TD-the second copy; (ii) TD-and the first and second copies; or (iii) the first and second copies-TD.

6. 6. The multimer of any one of claims 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. 7. The multimer of any one of claims 1 to 6, wherein the engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, wherein 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 claims 1 to 7, wherein the domain is an immunoglobulin superfamily domain.

9. The multimer of any one of claims 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. 10. The multimer of any one of claims 1 to 9, wherein the multimer comprises first, second, third and fourth identical copies of the engineered polypeptide, the polypeptide comprising a TD and one (but not more than one), two (but not more than two) or more copies of the protein domain or peptide.

11. The multimer of any one of claims 1 to 10, wherein the engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD.

12. 12. The multimer of claim 11, wherein the polypeptide comprises (in the 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 V1 and V2 are antibody variable domains and are the same or different.

13. 13. The multimer of claim 12, wherein V1 and V2 are antibody single variable domains.

14. 12. The multimer of claim 11, wherein each engineered polypeptide comprises (in the N-terminal to C-terminal direction) V1-optionally a linker-TD, where V1 is an antibody or TCR variable domain, and each engineered polypeptide pairs with a respective second engineered polypeptide comprising V2, where 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 that pairs with CH1.

15. 15. The multimer of any one of claims 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 multimers are selected from the group consisting of LeoPro™; Abciximab; Rituxan™; Rituximab; Zenapax™; Daclizumab; Simulect™; Basiliximab; Synagis™; Palivizumab; Remicade™; Infliximab; Herceptin™; Mylotarg™; Gemtuzumab; Campath™; Alemtuzumab; Zevalin™; Ibritum Mab; Humira (trademark); Adalimumab; Xolair (trademark); Omalizumab; Bexar (trademark); Tositumomab; Raptiva (trademark); Efalizumab; Erbitux (trademark); Cetuximab; Avastin (trademark); Bevacizumab; Tysabri (trademark); Natalizumab; Actemra (trademark); Tocilizumab; Vectibix (trademark); Panitumumab; Lucentis (trademark); Ranibizumab b; Soliris(tm); eculizumab; Cimzia(tm); certolizumab; Simponi(tm); golimumab, Ilaris(tm); canakinumab; Stelara(tm); ustekinumab; Arzera(tm); ofatumumab; Prolia(tm); denosumab; Numax(tm); motavizumab; Abslax(tm); raxibacumab; Benlysta(tm); belimumab; Yervoy (TM); ipilimumab; Adcetris™; brentuximab; Vedotin™; Perjeta™; pertuzumab; Kadcyla™; Ado-trastuzumab; KEYTRUDA™, OPDIVO™, GAZYVA™, and obinutuzumab.

17. An isolated tetramer or octamer of a TCR binding site, an insulin peptide, an incretin peptide or a peptide hormone, or a plurality of said tetramers or octamers.

18. (a) is soluble in aqueous solution; (b) capable of being secreted from a eukaryotic cell; and / or (c) is a eukaryotic expression product; A multimer, tetramer or octamer according to any one of claims 1 to 17.

19. (a) a tetramer or octamer of a TCR V domain or TCR binding site, which is soluble in aqueous solution; (b) a tetramer or octamer of an antibody single variable domain, which is soluble in aqueous solution; (c) a tetramer or octamer of a TCR V domain or TCR binding site, which is expressible intracellularly and / or extracellularly by HEK293 cells; or (d) A tetramer or octamer of antibody variable domains that can be expressed intracellularly and / or extracellularly by HEK293 cells.

20. 20. The multimer, tetramer or octamer of any one of claims 1 to 19, wherein the tetramer or octamer is bispecific for antigen or pMHC binding.

21. 21. The multimer, tetramer or octamer of any one of claims 1 to 20, wherein the domains are identical.

22. 22. The multimer, tetramer or octamer of any one of claims 1 to 21, comprising eukaryotic glycosylation.

23. 23. The multimer, tetramer or octamer of claim 22, wherein the cell is a HEK293 cell.

24. A plurality of the multimer, tetramer or octamer of any one of claims 1 to 23.

25. A pharmaceutical composition comprising the multimer, tetramer or octamer of any one of claims 1 to 24 and a pharma- ceutically acceptable carrier, diluent or excipient.

26. A cosmetic, food product, beverage, cleaning product, detergent comprising the multimer, tetramer or octamer according to any one of claims 1 to 24.

27. The engineered (and optionally isolated) polypeptide or (optionally isolated) monomer of a multimer, tetramer or octamer according to any one of claims 1 to 26.

28. (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) V1 is Vδ and C1 is Cδ; or (b) TCR V1-antibody CH1-optional linker-TD; (i) V1 is Vα, (ii) V1 is Vβ, (iii) V is Vγ; or (iv) V1 is Vδ, or (c) antibody V1-antibody CH1-optional linker-TD; (i) V1 is VH, or (ii) V is V; or (d) antibody V1-optional antibody CH1-antibody Fc-optional linker-TD; (i) V1 is VH, or (ii) V is V; or (e) antibody V1 - antibody CL - optional linker - TD; (i) V1 is VH, or (ii) V is V; 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) V1 is Vδ and C1 is Cδ; An engineered (and optionally isolated) polypeptide (P1).

29. The engineered polypeptide P1 is paired with a further polypeptide (P2), P2 comprising (in the N-terminal to C-terminal direction): (g) TCR V2-TCR C2-antibody CL, P1 being according to (a) of claim 28; and (i) when P1 is according to (a)(ii), V2 is Vα and C2 is Cα; (ii) when P1 is according to (a)(i), V2 is Vβ and C2 is Cβ; (iii) when P1 is according to (a)(iv), V2 is Vγ and C2 is Cγ; or (iv) when P1 is according to (a)(iii), V2 is Vδ and C2 is Cδ; or (h) TCR V2-antibody CL, P1 being according to (b) of claim 28; and (i) if P1 is according to (b)(ii), then V2 is Vα; (ii) when P1 is according to (b)(i), V2 is Vβ; (iii) when P1 is according to (b)(iv), V2 is Vγ; or (iv) if P1 is according to (b)(iii), then V2 is Vδ; or (i) antibody V2-CL, P1 being according to (c) as defined in claim 28, (i) when P1 is according to (c)(ii), V2 is VH; or (ii) when P1 is according to (c)(i), V2 is VL; or (j) antibody V2 - with an optional CL, P1 being according to (d) as defined in claim 28, and (i) when P1 is according to (d)(ii), V2 is VH; or (ii) when P1 is according to (d)(i), V2 is VL; or (k) antibody V2-CH1, wherein P1 is according to (e) of claim 28; and (i) when P1 is according to (e)(ii), V2 is VH; or (ii) when P1 is according to (e)(i), V2 is VL; or (l) TCR V2-TCR C2, P1 being according to (f) of claim 28; and (i) when P1 is according to (f)(ii), V2 is Vα and C2 is Cα; (ii) when P1 is according to (f)(i), V2 is Vβ and C2 is Cβ; (iii) when P1 is according to (f)(iii), V2 is Vγ and C2 is Cγ; or (iv) when P1 is according to (f)(iv), V2 is Vδ and C2 is Cδ; 29. The polypeptide of claim 28.

30. 27. A multimer of P1 as defined in claim 28, or P1 paired with P2 as defined in aspect 29, or a plurality of said multimers, optionally wherein said multimer is as defined in any one of claims 1 to 24.

31. 30. A nucleic acid encoding an engineered polypeptide or monomer according to any one of claims 27 to 29, optionally comprising an expression vector for expressing said polypeptide.

32. A eukaryotic host cell comprising a nucleic acid or vector according to claim 31 for the intracellular and / or secretory expression of a multimer, tetramer, octamer, engineered polypeptide or monomer according to any one of claims 1 to 24.

33. 32. Use of the nucleic acid or vector of claim 31 in a method for producing a protein multimer to produce a multimer that is expressed and / or secreted within a cell, said method comprising expressing said multimer in a eukaryotic cell containing said nucleic acid or vector and / or secreting said multimer from said eukaryotic cell.

34. 32. Use of the nucleic acid or vector according to claim 31 in a method for producing a protein multimer for producing a glycosylated multimer in a eukaryotic cell comprising said nucleic acid or vector.

35. 30. A mixture comprising: (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of claims 27 to 29; and (ii) a multimer, tetramer or octamer as defined in any one of claims 1 to 24.

36. 36. The mixture of claim 35, wherein the cell line is in a medium comprising a secretion product of the cell, the secretion product comprising the multimer, tetramer or octamer.

37. A multimer, tetramer or octamer according to any one of claims 1 to 24 for medical use.

38. (a) a method of producing a TCR V domain multimer comprising soluble and / or intracellular expression of a TCR V-NHR2 TD or TCR V-p53 TD fusion protein expressed in a eukaryotic cell, and optionally isolating a plurality of said multimers; (b) a method of producing an antibody V domain multimer comprising soluble and / or intracellular expression of an antibody V-NHR2 TD or V-p53 TD fusion protein expressed in a eukaryotic cell, and optionally isolating a plurality of said multimers; (c) a method of producing an incretin peptide multimer comprising soluble and / or intracellular expression of an incretin peptide-NHR2 TD or incretin peptide-p53 TD fusion protein expressed in a eukaryotic cell, such as a HEK293T cell, and optionally isolating a plurality of said multimers; or (d) A method of producing a peptide hormone multimer comprising 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 HEK293T cell, and optionally isolating a plurality of said multimers.

39. The use of a self-associating tetramerization domain (TD) in a method for producing tetramers of a polypeptide to produce tetramers in higher yield than monomeric and / or dimeric polypeptides.

40. 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 tetramers in higher yield than monomeric and / or dimeric polypeptides, wherein the engineered polypeptide comprises one or more copies of the protein domain or peptide and further comprises a self-associating tetramerization domain (TD).

41. The use of a self-associating tetramerization domain (TD) in a method for producing tetramers of a polypeptide to produce multiple tetramers that are not in a mixture with monomers, dimers or trimers.

42. 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 in 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).

43. 43. The use according to any one of claims 39 to 42, wherein the yield of tetramer is at least 10 times the yield of monomer and / or dimer.

44. 44. Use according to any one of claims 39 to 43, wherein the ratio of tetramer produced in the process to monomer and / or dimer produced is at least 90:

10.

45. The use according to any one of claims 39 to 44, wherein each monomer has a size of 40 kDa or less.

46. The use according to any one of claims 39 to 45, wherein each tetramer has a size of 150 kDa or less.

47. The use according to any one of claims 39 to 46, wherein the method comprises expressing the tetramer from a eukaryotic cell line.

48. (a) a TCR extracellular domain; (b) an immunoglobulin constant domain, and (c) NHR2 multimerization domain of ETO A multivalent heterodimeric soluble T cell receptor capable of binding to a pMHC complex, comprising:

49. (i) an immunoglobulin variable domain, and (ii) the NHR2 multimerization domain of ETO A polymeric immunoglobulin comprising:

50. 1. A method for assembling a soluble multimeric polypeptide comprising the steps of: (a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO; and (b) assembling multiple copies of said monomers, thereby obtaining a multimeric soluble polypeptide. A method comprising:

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