Multi-domain binding molecules

Abstract

To provide soluble multi-domain binding molecules comprising T cell receptors (TCR) having specificity for an antigen, an immunoglobulin Fc domain or an albumin-binding moiety; and an immune effector domain.SOLUTION: The present invention provides a multi-domain binding molecule comprising: a peptide-major histocompatibility complex (pMHC) binding moiety linked to a T cell engaging immune effector; and a half-life extending domain, comprising an immunoglobulin Fc or an albumin binding domain. The multi-domain binding molecule is advantageous because it displays improved half-life while retaining its function.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a T cell receptor (TCR), an immunoglobulin Fc domain, and a cellular endothelial cell line having specificity for an antigen. a soluble portion comprising an albumin-binding portion; and an immune effector domain. The present invention relates to multi-domain binding molecules of the present invention, which are advantageous because they exhibit improved half-life while retaining function. [Background technology]

[0002] 2. Background of the Invention Many protein-based therapeutics, including antibody fragments and fusion proteins, They are rapidly eliminated from the body after administration. Their short circulating half-life is typically attributed to their small size (which allows efficient clearance by renal filtration) and lack of protection from intracellular degradation. In this case, frequent dosing or long infusion times are necessary to maintain effective concentrations of the drug over long periods of time. To improve dosing, several strategies have been used to extend circulating half-life. These include the use of flexible hydrophilic molecules (e.g., hydrocarbons) These include increasing the hydrodynamic radius of the protein by attaching a saccharide or PEG (polyethylene glycol) and leveraging recycling via the fetal Fc receptor (FcRn) by attaching an antibody Fc domain or serum albumin (Konnteman, Curr Opin Biotechnol. 2011 Dec;22(6):868-76).

[0003] Strategies that exploit FcRn-mediated recycling are particularly attractive due to the lower risk of inducing immunogenicity in vivo and the long half-life extension that can be achieved. The half-life of a T-cell binding bispecific antibody in the TriTac® format has been reported to exceed 200 hours following attachment of an Fc domain (Lorenczewski et al., Blood 2017. 130(Suppl 1), 2815). Similarly, a bispecific antibody in the TriTac® format incorporating an albumin binding domain has been reported to have a half-life of more than 4 days (Wesche et al., Cancer Res 2018;78(13 Suppl):Abstract nr 3814). Fusion protein comprising a soluble T cell receptor fused to an anti-CD3 antibody fragment Proteins are a novel category of T cell-binding bispecific fusion proteins with in vivo half-lives of 6-8 hours (Sato et al., 2018 J Clin Onc 2018 36, no. 15_suppl 9521-9521; Middleton et al., J Clin Onc 2016 34, no. 15_suppl 3016-3016). Unlike traditional antibodies, T cell receptors are designed to recognize short peptides (peptide-HLA) derived from intracellular antigens and presented by human leukocyte antigens on the cell surface. Effective immune synapse formation between peptide-HLA complexes on antigen-presenting cells and T cells relies on a fixed interaction geometry (that is perturbed by increased intramembrane distances) (Choudhuri et al., 2005 Nature Jul 28;436(7050):578-82). Summary of the Invention [Problem to be solved by the invention]

[0004] Description of the Invention There is a need for T cell-binding bispecific proteins that have increased half-lives and are capable of mediating efficient immune synapse formation. [Means for solving the problem]

[0005] Contrary to expectations in the art, the present inventors have demonstrated that TCR-anti-CD3 fusion proteins bind to antibody Fc domains. It has been surprisingly found that fusion of the antibody to a domain or albumin binding moiety results in efficient immune synapse formation.

[0006] In the first aspect, i) peptide-major histocompatibility complex (pMHC) linked to a T cell-binding immune effector A binding moiety; ii) a half-life extending domain comprising an immunoglobulin Fc or albumin binding domain; A multi-domain binding molecule is provided comprising: [Brief description of the drawings]

[0007] [Figure 1] A) Design of TCR-anti-CD3-Fc fusion proteins; B) Exemplary sequences of TCR-anti-CD3-Fc fusion proteins of the invention [Diagram 2] Figure 2 shows that TCR-anti-CD3 fusions incorporating an Fc domain can mediate T cell activation in the presence of antigen-positive target cells. Data for three IgG1-Fc fusions are shown. [Diagram 3] A) shows that TCR-anti-CD3 fusions incorporating an albumin-binding peptide can mediate T cell activation in the presence of antigen-positive target cells. Three formats are shown where the albumin-binding peptide is attached to either C-α (F1), N-α (F2) or C-β (F3). B) shows that TCR-anti-CD3 fusions incorporating an albumin-binding nanobody can mediate T cell activation in the presence of antigen-positive target cells. Two formats are shown where the albumin-binding peptide is attached to either C-α (R) or C-β (Y). C) shows that TCR-anti-CD3 fusions incorporating an albumin-binding domain antibody (Albudab™) can mediate T cell activation in the presence of antigen-positive target cells. One format is shown where Albudab™ is attached to the C-α of the TCR-anti-CD3 fusion. [Figure 4]A) shows the PK profile of the TCR-anti-CD3-Albudab™ fusion in mouse serum. B) shows the theoretical PK profile of the TCR-anti-CD3-Albudab™ fusion in humans based on evaluation in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In the first aspect, i) peptide-major histocompatibility complex (pMHC) linked to a T cell-binding immune effector A binding moiety; ii) a half-life extending domain comprising an immunoglobulin Fc or albumin binding domain; A multi-domain binding molecule is provided comprising:

[0009] Preferably, the pMHC binding moiety is a T cell receptor (TCR) or a TCR and / or antibody. A TCR-like antibody is one that comprises a variable domain and at least one constant domain. In the present invention, the pMHC binding moiety comprises at least one immunoglobulin constant domain. Preferably, the constant domain may correspond to the constant domain of the TCR α or β chain (TRAC or TRBC, respectively). Alternatively, the TCR constant domain of the pMHC binding moiety may be replaced by the constant domain of an antibody light or heavy chain (CL, CH1, CH2, CH3 or CH4). The constant domain may be full length or may be truncated. The TCR constant domain may be truncated to remove the transmembrane domain. When the constant domain is truncated, preferably only the membrane-binding part is removed. With respect to the native constant domain, additional mutations may be introduced into the amino acid sequence of the constant domain. The constant region may contain residues (naturally occurring or introduced) that allow dimerization, for example by a disulfide bond between two cysteine ​​residues.

[0010] The inventors have unexpectedly discovered a multi-domain binding molecule comprising a pMHC-binding portion, an immune effector domain and an immunoglobulin Fc domain or an albumin-binding portion. They found that TCR-pMHC interactions remain functional, confirming the finding that TCR-pMHC interactions rely on a fixed binding geometry and that TCR triggering is sensitive to increased intermembrane distances. This is especially surprising in light of the knowledge in the field (Garboczi et al., Nature. 1996 Nov 14;384(6605):134-41; Choudhuri et al., 2005 Nature Jul 28;436(7050):578-82; Rudolph et al., Annu Rev Immunol. 2006;24:419-66). In fact, the kinetic dissociation model proposes that TCR triggering is the result of tethering of the TCR-CD3 complex within a region of close contact (where tyrosine phosphorylation is favored due to size-dependent exclusion of tyrosine phosphatases, such as CD45) (Choudhuri et al., 2005 Nature Jul 28;436(7050):578-82; Davis et al., Nat Immunol. 2006 Aug;7(8):803-9). Thus, the small size (approximately 100 Å) and fixed binding geometry of the TCR-pMHC complex are understood to be important for immune synapse formation and TCR triggering. Based on this knowledge, one skilled in the art will appreciate that the antigen-binding polypeptides of the invention are expected to result in poor immune synapse formation, perturbation of the TCR-pMHC binding geometry, and ultimately ineffective TCR triggering.

[0011] The pMHC binding moiety can be a TCR-like antibody. Preferably, the pMHC binding moiety comprises the variable domain of a TCR-like antibody. Antibodies do not naturally recognize pMHC; however, it is known that it is possible to engineer antibodies with specificity for pMHC. Such antibodies are called TCR-like or TCR mimetic antibodies (Chang et al., Expert Opin Biol Ther. 2016 Aug;16(8):979-87 and Dahan et al., Expert Rev Mol Med. 2012 Feb 24;14:e6). The TCR may be a heterodimeric α / β or γ / δ TCR polypeptide pair. Alternatively, the TCR may be a single chain α / β or γ / δ TCR polypeptide. The amino acid sequence of the TCR variable domain may be any of the naturally occurring and may correspond to those found in, or contain one or more mutations relative to the native TCR. Such mutations may be such as to increase the affinity of the TCR for a given antigen. Additionally or alternatively, mutations that improve stability and manufacturability may be incorporated. The TCR can bind to the MHC complexed with a peptide antigen. Preferably, the peptide antigen is any disease-associated antigen. Preferably, the peptide antigen is any tumor-associated antigen. The peptide antigen can be a peptide derived from GP100, NYESO, MAGEA4 or PRAME, as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319. TCRs may be used as described in WO2011001152, WO2017109496, WO2017175006, and WO2018234319. It may have an amino acid sequence such as:

[0012] The T cell-binding immune effector domain may be a CD3 effector domain. Cell-associated immune effectors are activated by T cells through interactions with CD3 and / or the TCR / CD3 complex. The CD3 effector may be an antibody scFv (or an antibody-like scaffold of similar size) that activates Anti-CD3 antibodies or antibody fragments, particularly anti-CD3 scFv or antibody-like scaffolds, are also included. Further immune effectors include, but are not limited to, cytokines (e.g., IL-2 and IFN- γ; superantigens and their variants; chemokines (e.g., IL-8, platelet factor 4, melanoma growth stimulating protein; antigens on immune cells (e.g., T cells or NK cells) (e.g., anti-CD28 or Antibodies that bind to complement activators, including, but not limited to, anti-CD16 or any molecule located in the immune synapse, and antibodies that bind to complement activators, including fragments, derivatives and variants thereof. stomach.

[0013] The half-life prolonging domain may be linked to the C- or N-terminus of the PMHC binding moiety or the C- or N-terminus of the T cell binding immune effector. The half-life extending domain may comprise an immunoglobulin Fc. The immunoglobulin Fc domain can be any antibody Fc region. The Fc region is the antibody tail region that interacts with cell surface Fc receptors and several proteins of the complement system. Fc regions typically consist of two polypeptides, which together contain two or three heavy chain constant domains (termed CH2, CH3, and CH4) and a hinge region. The Fc domain of the immunoglobulin subclasses IgG1, IgG2, and IgG4 binds to and undergoes FcRn-mediated recycling, providing a long circulating half-life (3-4 weeks). IgG The interaction of FcRn with the IgG1 and FcRn is localized to the Fc region, which covers part of the CH2 and CH3 domains. Immunoglobulin Fc domains suitable for use in the present invention include, but are not limited to, IgG1 or IgG4 Fc domains. Preferably, the Fc domain is derived from a human sequence. The Fc region also preferably contains KiH mutations to facilitate dimerization as well as a cytochrome P450 mutation to interact with the activating receptor. The molecule may have a mutation that prevents the interaction of the two (i.e., a functionally silent molecule). The immunoglobulin Fc domain may be combined with other domains (i.e., TCR or immunoglobulins). The immunoglobulin Fc may be fused to the C- or N-terminus of the antibody (effector). domain (i.e., TCR or immune effector) via a linker. Alternatively, no linker may be used. The linker sequence is typically flexible in that it is made up primarily of amino acids, such as glycine, alanine, and serine, that do not have bulky side chains that may limit flexibility. Alternatively, a linker with greater rigidity may be desirable. Usable or optimal lengths of linker sequences can be readily determined. In many cases, the linker sequence is about 12 amino acids or less in length, such as 10 amino acids or less in length or 2-10 amino acids in length. The linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Examples of suitable linkers that may be used in the multi-domain binding molecules of the present invention include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828). When fused to a TCR, an immunoglobulin Fc may be fused to either the alpha or beta chain, or both the alpha and beta chains, with or without a linker. Additionally, individual chains of the Fc may be fused to individual chains of the TCR.

[0014] Preferably, the Fc region may be derived from an IgG1 or IgG4 subclass. The two chains may comprise the CH2 and CH3 constant domains and all or part of the hinge region. The hinge region may substantially or partially correspond to the hinge region of IgG1, IgG2, IgG3 or IgG4. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region. Preferably, the hinge region contains at least one disulfide bond linking the two chains. The Fc region may contain mutations with respect to the WT Fc sequence. Mutations include substitutions, insertions and deletions. The mutations may be engineered to introduce desired therapeutic properties. For example, a KiH mutation may be engineered into the CH3 domain to facilitate heterodimerization. In this case, one chain is engineered to contain a bulky protruding residue (i.e., knob), e.g., Y, and the other chain is engineered to contain a complementary pocket (i.e., hole). Suitable locations for KiH mutations are known in the art. Additionally or alternatively, mutations may be introduced that abolish or reduce binding to Fcy receptors and / or increase binding to FcRn, and / or that prevent Fab arm exchange or remove protease sites. Additionally or alternatively, mutations may be made for manufacturing reasons, e.g., to remove or replace amino acids that may be subject to post-translational modifications (e.g., glycosylation).

[0015] Examples include: IgG4 (underlined residues indicate mutations relative to the wild-type sequence) "knob" YGPPCPPCPAFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK "hole" YGPPCPPCPAFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0016] IgG (underlined residues indicate mutations relative to the wild-type sequence) "knob" VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK "hole" VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0017] The half-life extending domain may comprise an albumin-binding domain. As is known in the art, albumin has a long circulating half-life of 19 days due in part to its size (above the renal threshold) and due to its specific interactions and recycling via FcRn. Attachment to albumin is a well-known strategy to improve the circulating half-life of therapeutic molecules in vivo. Albumin may be attached non-covalently by the use of specific albumin binding domains or covalently by conjugation or direct genetic fusion. Examples of therapeutic molecules in which attachment to albumin has been utilized to improve half-life can be found in Sleep et al., Biochim Biophys Acta. 2013 Dec;1830(12):5526-34.

[0018] The albumin-binding domain may be any moiety capable of binding to albumin, including any known albumin-binding moiety. Albumin-binding domains may be selected from endogenous or exogenous ligands, small organic molecules, fatty acids, peptides and proteins that specifically bind to albumin. Examples of suitable albumin-binding domains include short peptides, such as those described in Dennis et al., J Biol Chem. 2002 Sep 20;277(38):35035-43 (e.g., peptide QRLMEDICLPRWGCLWEDDF); proteins engineered to bind to albumin, such as those described in J Biol Chem. 2002 Sep 20;277(38):35035-43 (e.g., peptide QRLMEDICLPRWGCLWEDDF); and proteins based on albumin binding domains found in nature, such as the streptococcal protein G protein (Stork et al., Eng Des Sel. 2007 Nov;20(11):569-76), e.g. Albumod®, marketed by Affibody. Preferably, the albumin is human serum albumin (HSA). The affinity of the agonist for human albumin may be in the picomolar to micromolar range. Considering the extremely high concentration of albumin in human serum (35-50 mg / ml, approximately 0.6 mM), it is calculated that substantially all of the albumin binding domain binds to albumin in vivo.

[0019] The albumin-binding moiety may be linked to the C- or N-terminus of the other domain (i.e., TCR or immune effector). or immune effector) via a linker. Linkers are generally flexible in that they are made primarily of amino acids, such as glycine, alanine, and serine, that do not have bulky side chains that may limit their flexibility. Alternatively, linkers with greater rigidity may be desirable. Usable or optimal lengths of linker sequences can be readily determined. In many cases, the linker sequence is about 12 amino acids or less in length, such as 10 amino acids or less in length, or 2-10 amino acids in length. Linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Examples of suitable linkers that may be used in the multi-domain binding molecules of the invention include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP and GGEGGGSEGGGS (as described in WO2010 / 133828). When fused to a TCR, the albumin-binding moiety may be fused to either the alpha or beta chain, or to both the alpha and beta chains, with or without a linker.

[0020] A multi-domain binding molecule according to the first aspect may be used as a medicament. In a further aspect there is provided a pharmaceutical composition comprising a multi-domain binding molecule according to the first aspect. In another further aspect, there is provided a nucleic acid encoding a multi-domain binding molecule according to the first aspect. An expression vector comprising the nucleic acid of this aspect is provided. Additionally, there is provided a host cell comprising the nucleic acid or vector of this aspect, in which the nucleic acid encoding the multi-domain binding molecule is present as a single open reading frame or as two separate open reading frames encoding the α and β chains, respectively. Also in a further aspect there is provided a method of producing a multi-domain binding molecule according to the first aspect comprising maintaining a host cell as described above under optimal conditions for expression of the nucleic acid and isolating the multi-domain antigen binding polypeptide. In a still further aspect there is provided a method of treatment comprising administering to a patient in need thereof a multi-domain binding molecule according to the first aspect.

[0021] Phenotypically silent variants of any of the molecules disclosed herein are also within the scope of the present invention. As used herein, the term "phenotypically silent variant" is understood to refer to a variant that incorporates, in addition to those mentioned above, one or more additional amino acid changes (including substitutions, insertions and deletions) and that has a phenotype similar to the corresponding molecule without said changes. In the context of this application, phenotype includes binding affinity (KD and / or binding half-life) and specificity. Preferably, the phenotype of the soluble multi-domain binding molecule is , binding affinity and specificity, as well as immunostimulatory potency and purification yield. A phenotypically silent variant may have one or more conservative substitutions and / or one or more The above tolerated substitutions may be included. Tolerated substitutions refer to substitutions that are not within the definition of conservative substitutions below, but are silent in expression. Those skilled in the art know that various amino acids have similar properties and are therefore "conservative". One or more such amino acids of a protein, polypeptide or peptide can often be substituted with one or more other such amino acids without destroying the desired activity of the protein, polypeptide or peptide.

[0022] Thus, the amino acids glycine, alanine, valine, leucine and isoleucine (aliphatic The amino acids that make up the cyclic chain can often be substituted for one another. Among the substitutions, glycine and alanine were substituted for each other (because they have relatively short side chains). It is preferred to substitute valine, leucine and isoleucine for one another (because they have larger aliphatic side chains which are hydrophobic). Other amino acids include: phenylalanine, tyrosine and tryptophan (aromatic Examples of amino acids that may be used include: lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present invention may be made with naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group of alanine may be replaced with an ethyl group and / or minor changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0023] Substitutions of this nature are sometimes referred to as "conservative" or "semi-conservative" amino acid substitutions. Thus, the present invention includes any of the above amino acid sequences, but having one or more conservative substitutions and / or one or more tolerated substitutions in said sequence, This extends to the use of molecules whose amino acid sequence of the TCR has at least 90% identity to the TCR sequences disclosed herein, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. "Identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, identity also means the degree of sequence relatedness, as appropriate, between polypeptide or polynucleotide sequences, as determined by the match of sequence alignment. Although there are several methods for measuring the identity between two polypeptide or polynucleotide sequences, the commonly used methods for determining identity are computer programs. Suitable computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic acid Research, 12, 387 (1984), BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0024] Amino acid sequences can be compared using a program such as the CLUSTAL program. This program compares amino acid sequences and, if necessary, inserts spaces into either sequence to find the best alignment. The best alignment is evaluated based on amino acid identity or similarity (identity + amino acid type conservation) can be calculated. Programs such as BLASTx can be used to The method aligns the longest possible sequence and assigns a value to its fit. Thus, it is possible to obtain a comparison that finds several similar regions, each with a different score. Both types of identity analysis are contemplated in the present invention. The percent identity of two amino acid sequences or two nucleic acid sequences can be determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced into the first sequence for best alignment). The amino acid residues or nucleotides at corresponding positions are compared. The "best alignment" is the alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (see, for example, "best alignment"). i.e., % identity = number of identical positions / total number of positions × 100).

[0025] The determination of the percent identity between two sequences can be accomplished using a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTn and BLASTp programs of Altschul et al. (1990), J. Mol. Biol. 215:403-410. The determination of the percent identity between two nucleotide sequences can be performed using the BLASTn program. The determination of the percent identity between two protein sequences can be performed using the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997), Nucleic acid Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches that detect distant relatedness between molecules (supra). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTn and BLASTp) can be used. See http: / / www.ncbi.nlm.nih.gov. Default general parameters can include, for example, Word Size=3, Expect Threshold=10. Parameters can be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.Other sequence analysis algorithms known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994), Comput. Appl. Biosci., 10:3-5; and FASTA, described in Pearson and Lipman (1988), Proc. Natl. Acad. Sci. 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. For purposes of assessing percent identity in this disclosure, BLASTp with default parameters is used as the comparison method. In addition, where the percent identity given is a non-integer value for an amino acid, the resulting value is rounded down to the nearest integer (i.e., a sequence of 25 amino acids with 90% sequence identity would be "22.5" but would be "22"). Thus, in the example provided, a sequence in which 22 of the 25 amino acids are matched is within 90% sequence identity.

[0026] As will be appreciated by those skilled in the art, the sequences provided at the C-terminus and / or N-terminus may affect the function of the molecule. 1, 2, 3, 4, 5, or 6 or more without substantially affecting the functional properties (e.g., the TCR portion) of the The residues provided at the C-terminus and / or N-terminus can be shortened or extended. The amino acid sequence may be shortened or extended by 1, 2, 3, 4 or 5 residues, and all such variants are encompassed by the present invention. Mutations (conservative and tolerated substitutions, insertions and Modifications (including deletions) may be introduced into the sequence. Such methods include, but are not limited to, polymerase chain reaction (PCR) based methods, restriction enzyme based cloning or ligation independent cloning (LIC) procedures. These methods are detailed in many standard molecular biology textbooks. For further details regarding polymerase chain reaction (PCR) and restriction enzyme based cloning, see Sambrook and Russell (2001), Molecular Cloning - A Laboratory Manual (3rd ed.) CSHL Press. Further information regarding ligation independent cloning (LIC) procedures can be found in Rashtchian (1995), Curr Opin Biotechnol 6(1):30-6. The TCR sequences provided by the present invention may be obtained from solid phase synthesis or any other suitable method known in the art.

[0027] The molecules of the invention may have an ideal safety profile for use as therapeutic agents. An ideal safety profile means that the molecules of the invention, in addition to having demonstrated good specificity, have passed further preclinical safety tests. Examples of such tests include low cytokine release in the presence of whole blood (and thus a feasible in vivo Whole blood assay confirms that the risk of developing cytokine release syndrome is low. and alloreactivity tests that confirm that the recipient is unlikely to recognize alternative HLA types. can be done. The molecules of the invention may be capable of high-yield purification. The yield may be determined based on the amount of material retained during the purification process (i.e., the amount of solubilized material obtained before refolding). (amount of correctly folded material obtained at the end of the purification process relative to the amount of protein) and / or yields are the exact formula obtained at the end of the purification process relative to the original culture volume. The yield may be determined based on the amount of material deposited. High yield means a yield of 1% or more, more preferably 5% or more, or higher. High yield means a yield of 1 mg / ml or more, more preferably 3 mg / ml or more, 5 mg / ml or more, or higher.

[0028] Binding affinity (equilibrium constant K D Methods for determining the binding affinity (which is inversely proportional to T) and the binding half-life (which is denoted as T) are known to those of skill in the art. In a preferred embodiment, the binding affinity and binding half-life are determined by This is measured using surface plasmon resonance (SPR) or Bio-Layer Interferometry (BLI) (e.g., using a BIAcore or Octet instrument, respectively). A doubling of affinity is called a K D It is understood that T1 / 2 is ln2 / dissociation rate (k off ) Therefore, if T1 / 2 doubles, k off becomes 1 / 2. K of TCR D value and k off Values ​​are usually measured for a soluble form of the TCR, i.e. a form truncated to remove cytoplasmic and transmembrane domain residues. To account for variability between individual measurements, especially for interactions with dissociation times greater than 20 hours, the binding affinity and / or binding half-life of a given TCR is measured using the same assay protocol for several hours, 3 hours or more, and the results averaged. To compare binding data between two samples (i.e. two different TCRs and / or two preparations of the same TCR), measurements are preferably measured using the same assay conditions (e.g. temperature). The measurement methods described for TCRs may also be applied to the multi-domain antigen-binding polypeptides described herein.

[0029] Certain preferred multi-domain binding molecules of the invention are capable of generating highly potent T cell responses in vitro against antigen positive cells, in particular cells presenting low levels of antigens typical of cancer cells (i.e. 5-100, e.g. 50 antigens / cell (Bossi et al. (2013) Oncoimmunol. 1;2(11):e26840; Purbhoo et al. (2006), J Immunol 176(12):7308-7316)). Such TCRs may be suitable for incorporation into the multi-domain antigen binding polypeptides described herein. The T cell response measured may be release of T cell activation markers (e.g. interferon gamma or granzyme B) or target cell killing or other measures of T cell activation (e.g. T cell proliferation). Preferably, a highly potent response will be in the nM to pM range, preferably EC50 or less than 500 nM, most preferably EC50 or less than 1 nM or 500 pM. 50 It has value.

[0030] The TCR portion of the molecules of the invention may be an αβ heterodimer. The α-β heterodimeric TCR portion of the molecules of the invention typically comprises an α chain TRAC constant domain sequence and / or a β chain TRBC1 or TRBC2 constant domain sequence. The constant domains may be in soluble form (i.e., the transmembrane domains are also included in the cellular One or both constant domains may be a form that does not have a cytoplasmic domain. and / or TRBC1 / 2 sequences. also encompasses natural polymorphic variants such as N→K at position 4 of TRAC (Bragado et al., International Immunology. 1994 Feb;6(2):223-30). The α and β chain constant domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α and / or β chain constant domain sequences may have disulfide bonds introduced between residues of the respective constant domains, for example as described in WO 03 / 020763 and WO06000830. The α and β constant domains may be modified by substitution of a cysteine ​​residue at Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, which forms a disulfide bond between the α and β constant domains of the TCR. TRBC1 or TRBC2 may additionally comprise a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant domains present in the αβ heterodimer may have, at one or both C-termini, for example up to 15 or up to 10 or up to 8 or up to 7 amino acids deleted (truncated). The C-terminus of the α chain extracellular constant domain may have 8 amino acids deleted (truncated).

[0031] The TCR portion of the molecules of the invention may be in a single chain format. Single chain formats include Vα-L-Vβ, V β-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ type (where Vα and Vβ and Vβ are TCR α and β variable regions, respectively, and Cα and Cβ are TCR α and β constant regions, respectively. and L is a linker sequence). The length of the constant domain is not limited (Weidanz et al. (1998) J Immunol Methods. Dec 1;221(1-2):59-76; Epel et al. (2002), Cancer Immunol Immunother. Nov;51(10):565-73; WO 2004 / 033685; WO9918129). Single chain TCRs may have disulfide bonds introduced between residues of each constant domain as described in WO 2004 / 033685. Single chain TCRs are further described in WO2004 / 033685; WO98 / 39482; WO01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2): 59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10): 4759-4763; Schodin (1996) Mol Immunol 33(9): 819-829).

[0032] Therapeutic agents that may be combined with the molecules of the invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforin) or chemotherapeutic agents (e.g., cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxic agent may be present inside a liposome linked to a multi-domain antigen-binding polypeptide as described herein for slow release. This prevents damaging effects during transport in the human body and ensures that the toxic agent has its maximum effect after binding of the multi-domain antigen-binding polypeptide to the antigen-presenting cells of interest.

[0033] Other suitable therapeutic agents include, but are not limited to: Small molecule cytotoxic agents, i.e., those with a molecular weight of 700 or less that have the ability to kill mammalian cells. Compounds less than Daltons. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or transform under physiological conditions to release a cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolmide, topotecan, trimetrexate, larbourlate, auristatin E, vincristine, and doxorubicin; Peptide cytotoxins, i.e. proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNAases and RNAases; Radionuclides, i.e. unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, e.g. iodine-131, rhenium-186, indium-111 , yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents are used to facilitate the binding of these radionuclides to the high affinity TCR or its multimers. May be used; Immunostimulants, i.e. immune effector molecules that stimulate the immune response, such as cytokines (e.g. IL-2 and IFN-γ); ·Superantigens and their variants; TCR-HLA fusions, e.g., peptides that bind to common human pathogens, e.g., Epstein-Barr fusions with virus (EBV)-derived peptide-HLA complexes; · Chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g. anti-CD3, anti-CD28 or anti-CD16); Antibodies or fragments thereof that bind to molecules localized in the immune synapse ·Alternative protein scaffolds with antibody-like binding properties ·Complement activators; Heterologous protein domains, homologous protein domains, viral / bacterial proteins domain, viral / bacterial peptides.

[0034] Particularly preferred immune effectors are anti-CD3 antibodies, or functional fragments or variants of anti-CD3 antibodies. As used herein, the term "antibody" encompasses such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031, and 12F6. Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab') fragments, to name a few. 2 centre fragments, dsFv and scFv fragments, nanobodies TM (Available from Ablynx, Belgium) , constructs containing synthetic single-chain immunoglobulin variable heavy domains derived from camelid (e.g. camel or llama) antibodies) and domain antibodies (Domantis (Belgium); which contain affinity matured single-chain immunoglobulin variable heavy domains or immunoglobulin variable light domains) or Alternative protein scaffolds that exhibit antibody-like binding properties include Affibodies (Affibody (Sweden); including engineered Protein A scaffolds) or Anticalins (Anticalins) (Pieris (Germany); including engineered Anticalins). An immune effector is linked to the TCR portion of the multi-domain antigen-binding polypeptide. Here, preferably, the immune effector is an anti-CD3 antibody.

[0035] Linkage of the individual components of the multi-domain binding molecule can be via covalent or non-covalent attachment. Covalent attachment can be direct or indirect via a linker sequence. The linker sequence is usually flexible in that it is made up primarily of amino acids, such as glycine, alanine and serine, that do not have bulky side chains that may limit flexibility. Alternatively, a linker with greater rigidity may be desirable. Usable or optimal lengths of the linker sequence can be readily determined. In many cases, the linker sequence is about 12 amino acids or less in length, such as 10 amino acids or less or 2-10 amino acids in length. Examples of suitable linkers that can be used in the molecules of the invention include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP and GGEGGGSEGGGS (as described in WO2010 / 133828). In a further aspect, the invention provides a nucleic acid encoding a multi-domain binding molecule of the invention. In some embodiments, the nucleic acid is a cDNA. In some embodiments, the nucleic acid may be an mRNA. The nucleic acid may be a non-naturally occurring and / or purified and / or engineered nucleic acid. The nucleic acid sequence may be codon optimized according to the expression system to be utilized. As known to those skilled in the art, expression systems may include bacterial cells, such as E. coli, or yeast cells. The expression system may comprise cells, or mammalian cells, or insect cells, or may be a cell-free expression system.

[0036] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one of the above nucleic acids. The present invention also provides recombinant host cells comprising one or more of the above constructs. As stated above, the nucleic acids encoding the specific binding molecules of the present invention constitute an aspect of the present invention, as do methods for producing specific binding molecules comprising expression from nucleic acids encoding the specific binding molecules. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under suitable conditions. After production by expression, the specific binding molecules may be isolated and / or purified using any suitable technique and then used as required. Systems for cloning and expressing polypeptides in various host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster liver cells, NSO mouse melanoma cells, and the like. A common suitable bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells, such as E. coli, is well established in the art. For an overview, see, for example, Pluckthun, Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for the production of specific binding molecules. For recent reviews, see, for example, Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0037] Suitable vectors can be selected or constructed containing appropriate regulatory sequences, promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as required. Vectors can be plasmids, viruses, such as phages or phagemids, as appropriate. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for the manipulation of nucleic acids, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and analysis of gene expression and proteins, are described in detail in Ausubel et al., eds., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons (1992). Thus, a further aspect of the invention provides a host cell containing a nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing the nucleic acid into a host cell. The introduction may use any available technique. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, and the like. Poration, liposome-mediated transfection and retroviruses or other viruses For bacterial cells, suitable techniques may include transduction using a vector (e.g., vaccinia or, for insect cells, baculovirus). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. Introduction may be followed by causing or allowing expression from the nucleic acid, for example by culturing the host cells under gene expression conditions.

[0038] The nucleic acids of the invention can be integrated into the host cell genome (e.g., chromosome. Integration can be achieved by standard This may be facilitated by the inclusion of sequences which facilitate recombination with the genome, according to conventional techniques. As is well known in the art, the molecules may be subject to post-translational modifications. Glycosylation is one such modification, which involves the attachment of oligosaccharide moieties to defined amino acids in the TCR chains. For example, asparagine residues or serine / threonine residues can be used in the well-known The glycosylation status of a particular protein depends on several factors, including the protein sequence, the protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation status (i.e., oligosaccharide type, covalent bonds, and total number of attachments) can affect the function of the protein. Thus, control of glycosylation is often desirable when producing recombinant proteins. Controlled glycosylation is used to improve antibody-based therapeutics (Jefferis et al. (2009) Nat Rev Drug Discov Mar;8(3):226-34.). For the TCR portion of the molecules of the invention, glycosylation may be controlled, for example, by using specific cell lines (including but not limited to mammalian cell lines, such as Chinese Hamster Ovary (CHO) cells or human embryonic liver (HEK) cells) or by chemical modification. Such modifications may be desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott (2005) Pharm Sci. Aug;94(8):1626-35).

[0039] For administration to a patient, the molecules of the invention (preferably with a detectable label or a therapeutic agent) are or a nucleic acid, expression vector or cell of the invention, It may be provided as part of a sterile pharmaceutical composition together with two or more pharma- ceutically acceptable carriers or excipients. This pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be provided in unit dosage form, typically in a hermetically sealed container, and may be provided as part of a kit. Such a kit may (but need not) include any other suitable components. The kit may contain a number of unit dosage forms. The pharmaceutical compositions can be administered by any suitable route, for example parenteral (subcutaneous, intramuscular, intrathecal or intravenous) suitable for administration by the enteral (including oral or rectal), inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. The dosage of the molecules of the invention can vary widely depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc. Suitable dosage ranges for the molecules of the invention are 25 ng / kg to 50 μg / kg. Or it may be 1 μg to 1 g. A physician will ultimately determine the appropriate dosage to be used. The multi-domain antigen-binding polypeptides, pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in a substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. may be provided.

[0040] The present invention also provides: a multi-domain antigen-binding polypeptide, nucleic acid, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in cancer or tumor therapy; use of a multi-domain antigen-binding polypeptide, a nucleic acid, a pharmaceutical composition or a cell of the invention in the manufacture of a medicament for the treatment of cancer or a tumor; a method for treating cancer or tumor in a patient comprising administering to the patient a multi-domain antigen-binding polypeptide, a nucleic acid, a pharmaceutical composition or a cell of the invention; An injectable formulation for administration to a human subject comprising a multi-domain antigen-binding polypeptide, a nucleic acid, a pharmaceutical composition or a cell of the invention.

[0041] The method of treatment may further comprise administering additional anti-neoplastic agents separately, in combination or sequentially. Examples of such agents are known in the art and may include immune activators and / or T cell modulating agents. Preferred features of each aspect of the invention are applicable to each of the other aspects, except that: The prior art documents referred to herein are presented to the fullest extent permitted by law. The entire area is incorporated.

[0042] Description of the drawings FIG. 1A) Design of TCR-anti-CD3-Fc fusion proteins; B) Exemplary sequences of TCR-anti-CD3-Fc fusion proteins of the invention. Figure 2 shows that TCR-anti-CD3 fusions incorporating an Fc domain bind to T cells in the presence of antigen-positive target cells. Figure 2 shows that IgG1-Fc fusions can mediate cell activation. Data for three IgG1-Fc fusions are shown. Figure 3A) shows that TCR-anti-CD3 fusions incorporating albumin-binding peptides bind to antigen-positive target cells. A) shows that TCR-anti-CD3 fusions incorporating albumin-binding nanobodies are able to mediate T cell activation in the presence of antigen-positive target cells. Three formats are shown, where the albumin-binding peptide is attached to either C-α (F1), N-α (F2) or C-β (F3). B) shows that TCR-anti-CD3 fusions incorporating albumin-binding nanobodies are able to mediate T cell activation in the presence of antigen-positive target cells. Two formats are shown, where the albumin-binding peptide is attached to either C-α (R) or C-β (Y). C) shows that albumin-binding domain antibodies (Albudab TM We show that TCR-anti-CD3 fusions incorporating Albudab ) can mediate T cell activation in the presence of antigen-positive target cells. TM One format in which is attached to C-α of a TCR-anti-CD3 fusion is shown. Figure 4A) shows the TCR-anti-CD3-Albudab antibody in mouse serum. TM B) PK characteristics of the fusion. Based on evaluation inTM The theoretical PK of the fusion in humans is shown. EXAMPLES

[0043] Working Example The following examples illustrate multi-domain binding molecules of the invention, which may also be referred to as TCR-anti-CD3-Fc fusion proteins or TCR-anti-CD3-albumin binding fusion proteins. Example 1 (Fc fusions) a) Design of TCR-anti-CD3-Fc fusion proteins In this example, a TCR-anti-CD3 fusion protein was used that comprises a high affinity TCR that binds to the HLA-A*02 restricted peptide of PRAME. Examples of such molecules are provided in WO2018234319. The human IgG1 Fc domain was fused to the C-terminus of TCR-anti-CD3 via a linker (see outline below). (See FIG. 1A for details). Two additional constructs were generated comprising functional variants of human IgG1 Fc known in the art. Variant 1 does not bind to Fcγ receptors (FcγRs) or the complement protein C1q and is therefore functionally silent. Variant 2 shows increased binding to FcRn, which may result in an extended in vivo half-life. In each case, the Fc domain comprises the known knobs-in-holes mutations Y86T and T22Y to facilitate heterodimerization. FIG. 1B shows the sequence of a TCR-anti-CD3-Fc fusion comprising a functionally silent human IgG1 Fc (variant 1).

[0044] b) Expression and purification of TCR-anti-CD3-Fc fusion proteins Expression of Fc fusions was performed using a transient expression system based on suspension culture-adapted Chinese Hamster Ovary (CHO) cells (ExpiCHO Expression system, Thermo Fisher). Cells were transfected with mammalian expression plasmids containing the relevant TCR chains fused to various Ig Fc domains according to the manufacturer's instructions. After collection, cell culture supernatants were clarified by centrifugation at 4000-5000 × g for 30 min in a refrigerated centrifuge. Supernatants were filtered through a 0.22 μm filter and collected for further purification. For purification, the Fc fusions were first adjusted with buffer and then purified using mAbselect Sure prepacked columns (GE Healthcare or equivalent resin) according to the manufacturer's guidelines. Protein-containing fractions were pooled and further purified by size-exclusion chromatography using an appropriate column (GE Healthcare) in a physiologically relevant buffer. Fractions containing a particular protein were pooled and concentrated for downstream testing and storage.

[0045] c) Potent T cell activation by TCR-anti-CD3-Fc fusion protein TCR-anti-CD3-Fc fusion proteins potently redirect CD3+ T cells against antigen-presenting T2 cells Interferon-γ (IFN-γ) release was assessed as a marker for T cell activation. was used as a readout for. The assay was performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, T2 cells were used as target cells and pulsed with 5 nM PRAME peptide. Target cells were incubated in assay medium (10% heat-inactivated FBS and 1% penicillin-streptomycin) for 1 h at 37 °C for 2 h. 1 × 10 6Cells were prepared at a density of 100 / ml and plated at 50,000 cells / well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at 35,000 cells / well in a volume of 50 μl. TCR-anti-CD3-Fc fusion proteins were titrated to final concentrations of 10 nM to 0.0001 nM and added to the wells in a volume of 50 μl.

[0046] Plates were prepared according to the manufacturer's instructions. Wells containing target cells, effector cells and fusion proteins were brought to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Control wells were prepared omitting either the fusion protein, effector cells or target cells. Plates were incubated overnight (37° C. / 5% CO2). The following day, The plates were washed three times with wash buffer (1x PBS sachet, containing 0.05% Tween-20, made up in deionized water). Primary detection antibody was then added to each well in a volume of 50 μl. The plates were allowed to warm to room temperature. After incubation for 2 hours, the plates were washed again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP to each well and incubating at room temperature for 1 hour, and the washing step was repeated. Within 15 minutes before use, one drop (20 μl) of AEC chromogen was added to each 1 ml of AEC substrate, mixed, and 50 μl was added to each well. Spot development was monitored periodically and the plate was washed with tap water to terminate the color reaction. The plate was then dried at room temperature for at least 2 hours before counting the spots using a CTL analyzer with Immunospot software (Cellular Technology Limited). Data was prepared and analyzed using PRISM software. The results shown in Figure 2 demonstrate that the TCR-anti-CD3-Fc fusion proteins mediate efficient T cell activation in the presence of antigen-positive target cells. Ec50 values ​​were in the pM range (238 pM, 257 pM and 25 pM for fusions with IgG1-Fc, IgG1-Fc-variant 1 and IgG1-Fc-variant 2, respectively). Control experiments with antigen-negative target cells demonstrate that the functionally silent IgG1-variant 2 resulted in negligible background activity. Therefore, the functionally silent Fc domain was considered to be the most suitable for therapeutic applications.

[0047] Example 2 (Albumin Binding) a) Design of TCR-anti-CD3-albumin binding fusion protein In the first design, the TCR-anti-CD3 fusion protein contained a high affinity TCR that binds to the HLA-A*02 restricted peptide of gp100. The amino acid sequence of this molecule is disclosed in WO2011001152. Specifically, the TCR-anti-CD3 fusion comprises an alpha chain of SEQ ID NO: 45 of WO2011001152 (wherein, based on the numbering of SEQ ID NO: 45, amino acids 1-109 are replaced with SEQ ID NO: 8 of WO2011001152). and a β-chain of SEQ ID NO: 36 of WO2011001152 (wherein residues 259-370 correspond to SEQ ID NO: 27 of WO2011001152, and where the amino acids at positions 1 and 2 are A and I, respectively). The albumin-binding fragment had the amino acid sequence QRLMEDICLPRWGCLWEDDF. Sex peptides (as described in Dennis et al., J Biol Chem. 2002 Sep 20;277(38):35035-43) was attached to the TCR-anti-CD3 fusion via a linker. A suitable linker is GGGGS. Three variants were prepared in which the albumin binding peptide was fused to three different attachment sites: C-α (F1), N-α (F2) or C-β (F3).

[0048] In the second design, an albumin-binding nanobody was attached to the same TCR-anti-CD3 fusion as used in the first design. The synthetic nanobody was attached to the TCR-anti-CD3 fusion via a linker. A suitable linker is GGGGS. Two variants were prepared in which the albumin-binding nanobody was fused to two different attachment sites: C-α (R) or C-β (Y). In the third design, an albumin-binding domain antibody was attached to the C-terminus of the α chain of the TCR-anti-CD3 fusion. The antibody was Albudab TM The two domain antibodies belong to the Variants were used; DOM 7h-10-14 dAb and DOM 7h-11-15 dAb (provided in WO201010893 as SEQ ID NOs: 26 and 27, respectively). The antibodies were attached via a linker, either directly (i.e., without a linker). A suitable linker is GGGGS. The TCR-anti-CD3 fusion protein contained a high affinity TCR that binds to an HLA-A*01 restricted peptide of MAGEA3. The amino acid sequence of this molecule is provided in WO2013041865.

[0049] b) Expression and purification of TCR-anti-CD3-albumin binding fusion proteins Using the same method as known in the art for TCR-anti-CD3 fusion proteins (e.g., WO2011001152, Example 2), the TCR-anti-CD3-albumin binding fusion protein was expressed as inclusion bodies in E. coli, then refolded and purified.

[0050] c) Potent T cell activation by TCR-anti-CD3-albumin binding fusion protein TCR-anti-CD3-albumin binding fusion protein was used to target CD3+ T cells against antigen-positive cancer cells. The ability of these cells to mediate potent redirection of interferon-γ (IFN-γ)-activated erythrocytes was evaluated. The output was used as a readout for T cell activation. The assay was performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, for fusions comprising albumin-binding peptides, melanoma Mel526 cells were used as target cells. Target cells were cultured in assay medium (RPMI 1640+150 in μM human serum albumin (HSA) and 1% penicillin-streptomycin-L-glutamine 1×10 6 Cells were prepared at a density of 100 / ml and plated at 50,000 cells / well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at 30,000 cells / well in a volume of 50 μl. TCR-anti-CD3-Fc fusion proteins were titrated to final concentrations of 10 nM to 0.0001 nM and added to the wells in a volume of 50 μl.

[0051] For fusions comprising Albudab®, myeloma EJM cells were used as target cells. Target cells were cultured at 1×10 in assay medium (RPMI 1640 + 45 μM HSA and 1% penicillin-streptomycin-L-glutamine). 6 Cells were prepared at a density of 100 / ml and plated at 50,000 cells / well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at 30,000 cells / well in a volume of 50 μl. TCR-anti-CD3-Fc fusion proteins were titrated to final concentrations of 10 nM to 0.0001 nM and added to the wells in a volume of 50 μl. Plates were prepared and developed as described in Example 1c. FIG. 3A-C shows that fusions of albumin-binding moieties with TCR-anti-CD3 fusion proteins are antigen-positive. These results demonstrate that the antibody mediates efficient T cell activation in the presence of target cells. Ec50 values ​​were in the pM range (137.4 / 178.0 / 137.1).

[0052] Example 3 (Extended Half-Life) a) PK evaluation of TCR-anti-CD3-albumin binding fusion protein The PK properties of the TCR-anti-CD3-AlbudAb fusion were examined in mouse serum. Mice were administered 0.1 mg / kg of fusion protein by intravenous bolus injection and serum samples were collected at regular intervals over 120 hours. PK evaluation was performed using an ELISA-based assay. Briefly, biotinylated pHLA complexes were attached to streptavidin-coated plates followed by serum samples. The detection step was performed using a primary goat antibody against anti-CD3 scFv and an HRP-conjugated anti-goat IgG activated for colorimetric detection with TMB at 450 nm. The results generated were used to confirm the presence and binding activity of the TCR-anti-CD3-Albudab fusion by serial dilution and analysis against a standard curve. Results were expressed as % activity. The results were used to generate plots of Cmax versus time.

[0053] The PK data obtained are shown in Figure 4A. Samples designated "TCR-Alb(10-14)" and "TCR-Alb(11-15)" refer to the TCR-anti-CD3 fusions described in Example 2a. "TCR-Alb(D)" refers to the non-albuminuria The control sample was fused to albumin-binding albumin. It is a TCR-anti-CD3 that does not contain Using the PK data in Figure 4A, a theoretical PK profile of the TCR-anti-CD3-AlbudAb fusion in humans was calculated. The predicted half-life of the fusion with Albudab was increased from 7 hours to 264 hours in vivo (Figure 4B).

Claims

1. i) peptide-major histocompatibility complex (pMHC) linked to a T cell-binding immune effector A binding moiety; ii) a half-life extending domain comprising an immunoglobulin Fc or albumin binding domain; A multi-domain binding molecule comprising:

2. The pMHC binding moiety may comprise a T cell receptor (TCR) or a TCR and / or an antibody variable domain and / or a small The multi-domain antibody of claim 1, which is a TCR-like antibody comprising at least one constant domain. In-binding molecules.

3. The multi-domain conjugate of claim 2, wherein the TCR is a heterodimeric α / β TCR polypeptide pair. sex molecule.

4. The multi-domain binding molecule of claim 2, wherein the TCR is a single chain α / β TCR polypeptide.

5. The T cell-binding immune effector domain is involved in the interaction with CD3 and / or the TCR / CD3 complex. The CD3 effector domain according to any one of claims 1 to 4, which activates T cells more effectively.

2. A multi-domain binding molecule as described in claim 1.

6. The complex of claim 5, wherein the CD3 effector domain comprises an antibody scFv or an antibody-like scaffold. Multidomain binding molecules.

7. The multi-domain binding molecule of any one of claims 1 to 6, wherein the half-life prolonging domain is an immunoglobulin Fc domain.

8. The multi-domain binding molecule of any one of claims 1 to 6, wherein the half-life prolonging domain comprises an albumin binding domain.

9. 9. The multi-domain binding molecule of any one of claims 1 to 8, wherein the half-life prolonging domain is linked to the C- or N-terminus of the pMHC binding moiety or the C- or N-terminus of the T cell binding immune effector.

10. 9. The multi-domain binding molecule of any one of claims 1 to 8, wherein the half-life prolonging domain is linked to the pMHC binding moiety or the T cell binding immune effector via a linker.

11. A multi-domain binding molecule according to any one of claims 1 to 10 for use as a medicament.

12. A pharmaceutical composition comprising a multi-domain binding molecule according to any one of claims 1 to 10.

13. A nucleic acid encoding a multi-domain binding molecule according to any one of claims 1 to 10.

14. An expression vector comprising the nucleic acid of claim 13.

15. A host cell comprising the nucleic acid of claim 13 or the vector of claim 14, wherein the nucleic acid encoding the multi-domain binding molecule is present as a single open reading frame or as two separate open reading frames encoding the alpha and beta chains, respectively.

16. A method for producing the multi-domain binding molecule of claim 1, comprising maintaining a host cell of claim 15 under conditions suitable for expression of the nucleic acid and isolating the multi-domain antigen binding polypeptide.

17. A method of treatment comprising administering to a patient in need thereof a multi-domain binding molecule according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 12.

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