Immunoglobulin single variable domains targeting the T cell receptor

Abstract

The present technology provides immunoglobulin single variable domains (ISVDs) that bind to both the constant domain of a human T cell receptor (TCR) on a T cell and the constant domain of a non-human primate TCR on a T cell. It also relates to polypeptides comprising an ISVD of the present technology and at least one ISVD capable of binding to an antigen on a target cell. The present technology further provides nucleic acids encoding the ISVDs or polypeptides, as well as vectors, hosts, and methods for producing such ISVDs or polypeptides. Furthermore, the present technology relates to treatment methods utilizing the ISVDs or polypeptides of the present technology.

Description

[Technical Field]

[0001] The present technology provides immunoglobulin single variable domains (ISVDs) that bind to both the constant domain of a human T cell receptor (TCR) on a T cell and the constant domain of a non-human primate TCR on a T cell. It also relates to multispecific polypeptides comprising an ISVD according to the present technology and at least one ISVD capable of binding to an antigen on a target cell. The present technology further provides nucleic acids encoding the ISVDs or polypeptides, as well as vectors, hosts, and methods for producing such ISVDs or polypeptides. Furthermore, the present technology relates to treatment methods utilizing the ISVDs or polypeptides according to the present technology. [Background technology]

[0002] Cancer exacts an enormous human toll worldwide. It is currently the second leading cause of death globally, surpassed only by heart disease and heart attack. Cancer stands out among the leading causes of morbidity and mortality worldwide, with approximately 19.3 million new cases and 10 million cancer-related deaths in 2020. The number of new cases is expected to rise further over the next decades. Population growth, aging, and lifestyle changes have been cited as contributing factors to the increasing cancer burden. In 2013, the WHO estimated that by 2030, cancer would surpass ischemic heart disease as the most common cause of death worldwide (Source: WHO Cancer).

[0003] The total economic impact of premature death and disability from cancer worldwide was already $900 billion in 2008, accounting for 1.5% of world gross domestic product at that time. As cancer becomes increasingly common, the total economic impact is sure to have also increased significantly. Available treatment regimens for solid tumors typically include a combination of surgical resection, chemotherapy, and radiation therapy. In 40 years of clinical experience, little progress has been achieved, especially in advanced stages of cancer. New therapies to combat cancer are desperately needed.

[0004] Antibody therapy is now an important part of physicians' armamentarium for fighting disease, especially cancer. Monoclonal antibodies have been established as a key therapeutic approach for a range of diseases for several years. Most of the antibody therapies approved over that time rely on single-specific monoclonal antibodies (mAbs). To date, most mAb targets require either agonistic or antagonistic approaches. While targeting cell surface antigens themselves can mediate antitumor activity via apoptosis induction, most mAb-based activity against hematological malignancies relies on one of several Fc-mediated effector functions, such as complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0005] More recently, immunotherapy has emerged as a rapidly growing area of ​​cancer research, in which the body's immune surveillance system, specifically T cells, are directed against cancer cells.

[0006] Cytotoxic T cells (CTLs) are T lymphocytes that kill cancer cells, cells infected (especially with viruses), or otherwise damaged cells. T lymphocytes (also called T cells) express T cell receptors (TCRs) and CD3 receptors on their cell surface. The αβTCR-CD3 complex (or "TCR complex") is composed of six distinct type I single-spanning transmembrane proteins: the TCRα and TCRβ chains, which form a TCR heterodimer involved in ligand recognition, and the noncovalently associated CD3γ, CD3δ, CD3ε, and CD3ζ chains, which are tyrosine phosphorylated upon receptor activation and carry cytoplasmic sequence motifs that recruit multiple signaling components (Call et al. 2004, Molecular Immunology 40:1295-1305).

[0007] Both the α and β chains of the heterodimeric T cell receptor (TCR) consist of constant and variable domains. T cells are activated by TCR recognition of cognate peptides presented by self MHC molecules, followed by signal transduction initiated by the tyrosine-phosphorylated CD3 complex, leading to T cell proliferation and differentiation.

[0008] Rather than eliciting specific T cell responses that rely on the expression of MHC molecules by cancer cells and the presence, development, transport, and presentation of specific peptide antigens, more recent developments have attempted to combine the benefits of immunotherapy and antibody therapy by engaging all of a patient's T cells in a polyclonal manner through recombinant antibody-based technologies. Bispecific antibodies, so-called T cell engagers (TCEs), have been engineered to carry a tumor-recognition moiety on one arm (the target-binding arm) and specificity for a T cell antigen on the other arm (the effector-binding arm), often targeting CD3. These bispecific antibodies are multitargeting molecules that enhance a patient's immune response against malignant cells. Coengagement of T cells and tumor cells by bispecific antibodies leads to the formation of a cytolytic synapse between the T cells and tumor cells, thereby inducing T cell activation and tumor cell killing.

[0009] While the majority of bispecific antibodies for T cell activation target the CD3 complex on T cells, WO 2016 / 180969 A1 describes several bispecific binding agents that target the constant domain of the αβ T cell receptor. However, one major challenge with these bispecific antibodies for T cell activation is that little cross-reactivity with the cynomolgus monkey T cell receptor was observed.

[0010] Bispecific antibody constructs have been proposed in multiple formats, for example, bispecific antibody formats can involve chemical conjugation of two antibodies or fragments thereof (Brennan M. et al. 1985, Science 229(4708):81-83; Glennie MJ et al. 1987, J Immunol 139(7):2367-2375).

[0011] However, drawbacks of such bispecific antibody formats include high molecular weight and high viscosity at high concentrations, which make them difficult to address, for example, subcutaneous administration, and have implications for polypeptide stability and production efficiency, in that each binding unit requires the interaction of two variable domains for specific and high-affinity binding. Such bispecific antibody formats can also potentially result in poor production efficiency and low titers and / or CMC issues related to light chain mispairing or heavy chain mispairing.

[0012] Currently, only one bispecific antibody, blinatumomab (a BiTE molecule recognizing CD19 and CD3), is on the market for use in the clinic for the treatment of cancer. This T cell-engaging format was approved by the FDA in December 2014 for second-line treatment, but many hurdles had to be overcome. The first clinical trial of blinatumomab was prematurely terminated due to neurological adverse events, cytokine release syndrome (CRS), and infections on the one hand, and the lack of objective clinical responses or robust signs of biological activity on the other. CRS was the most significant adverse event reported with the first T cell-engaging therapy.

[0013] To minimize the risk of adverse events and systemic side effects, such as cytokine storm, utmost care must be taken when selecting the T cell antigen arm. The latter must bind to the TCR complex in a monovalent manner and must not trigger T cell signaling in the absence of target cancer cells. Only specific binding of both arms of a bispecific antibody to their targets (tumor and T cell antigen) can trigger the formation of a cytolytic synapse and subsequent tumor cell killing.

[0014] Non-human primates, such as cynomolgus and rhesus monkeys, are generally considered the most suitable animal species for preclinical studies, including efficacy and toxicity studies. To enable assessment of the toxicity of bispecific T cell-engaging antibodies in non-human primates, good species cross-reactivity of antibodies against human and non-human primate TCRs is recommended.

[0015] Thus, there remains a need for other multispecific T cell engaging formats, particularly multispecific T cell engaging formats that target T cell receptors other than CD3.

[0016] Additionally, there is a need for antibody constructs that bind with sufficient affinity to both target cells and T cells to induce a cytotoxic response. At the same time, such constructs should not induce a cytotoxic response in non-target cells, i.e., cells that do not express the target antigen or express it only at low levels. This allows for a good compromise between efficacy and safety. It is further desirable that such constructs can be efficiently produced, for example, in a microbial host. Furthermore, when used therapeutically, the constructs should have no or only minimal undesired side effects, such as those caused by cytotoxic activity in non-target cells. Summary of the Invention [Problem to be solved by the invention]

[0017] The present inventors have discovered novel T cell engager (TCE) immunoglobulin single variable domains (ISVDs) capable of specifically targeting and binding to TCRs. These TCR-binding ISVDs, also referred to as TCEs, can be linked to moieties capable of binding to cell-specific targets. Pathologies caused by abnormal cells, such as cancer, that display the specific target on their cell surface can be targeted by the immune system through binding of the TCE ISVD to T cells. T cells are then directed to tumor cells expressing the specific target, and T cell activation is induced through such binding of the T cells to the TCE. Once activated, effective target cell killing is triggered.

[0018] The present inventors have found that constructs containing an ISVD that simultaneously targets a TCR and a cell-specific target in accordance with this technology result in efficient T cell-mediated killing of target-expressing cells in vitro. Moreover, such constructs exhibited only limited activity against cells that do not express the target or express it at low levels. This suggests the possibility of inducing highly specific T cell-mediated cytotoxic responses against specific target cells while exhibiting a favorable safety profile.

[0019] In stability studies of earlier-developed TCR-binding ISVDs, the inventors observed a lack of chemical stability at some amino acid residues at certain positions in the ISVD. Specifically, they observed isomerization at aspartic acid (D) at position 61 and tryptophan (W) oxidation at position 99 (Kabat numbering). They hypothesized that trace levels of transition metals catalyzed downstream processing by polysorbates led to the observed tryptophan oxidation. They observed that several amino acid substitutions at selected positions in the TCR-binding ISVD could reduce or even completely prevent these deleterious effects.

[0020] Provided herein are TCR-binding ISVDs that have potent binding to TCRs of different species and improved chemical stability. [Means for solving the problem]

[0021] Thus, in a first aspect, the present technology relates to an ISVD that specifically targets the constant domain of a human and / or non-human primate T cell receptor present on a T cell, wherein the ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively): a. The amino acid sequence of CDR1 (according to Kabat) is INFYG (SEQ ID NO: 79), b. The amino acid sequence of CDR2 (according to Kabat) is HISIGDQTDYAX1SAKG (SEQ ID NO: 80), and c. The amino acid sequence of CDR3 (according to Kabat) is LSRIX2PYDY (SEQ ID NO: 81), where: - the amino acid residue X1 is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or the amino acid residue X2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I.

[0022] Another aspect of the present technology relates to an ISVD that specifically binds to a constant domain of a human and / or non-human primate T cell receptor (TCR) present on a T cell, wherein the ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively), a. The amino acid sequence of CDR1 (according to AbM) is GYVHKINFYG (SEQ ID NO: 82), b. The amino acid sequence of CDR2 (according to AbM) is HISIGDQTD (SEQ ID NO: 83), and c. The amino acid sequence of CDR3 (according to AbM) is LSRIX2PYDY (SEQ ID NO: 84), where: the amino acid residue at position 61 (according to Kabat) is chosen from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or the amino acid residue X2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I.

[0023] In one embodiment, X1 is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S. In one embodiment, X1 is selected from E or D, for example, X1 is E.

[0024] In another embodiment, position 61 (according to Kabat) is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S, for example, position 61 (according to Kabat) is E or D.

[0025] In one embodiment, position 61 (according to Kabat) is E.

[0026] In one embodiment, X2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I. In one embodiment, X2 is Y, A, Q, F, S, T, or H. In one embodiment, X2 is Y, Q, S, or T. In one embodiment, X2 is Y.

[0027] In one embodiment, the amino acid residue at position 103 (Kabat numbering) in the ISVD is selected from the group consisting of W, R, A, E, Y, L, H, I, Q, V, K, S, G, P, F, T, for example, the amino acid at position 103 (Kabat numbering) is W.

[0028] In another embodiment, the technology provides an ISVD in which X1 is E, X2 is Y, and the amino acid residue at position 103 (Kabat numbering) is W; or an ISVD in which the amino acid residue at position 61 (Kabat numbering) is E, X2 is Y, and the amino acid residue at position 103 (Kabat numbering) is W.

[0029] In a further embodiment, the ISVD is a heavy chain ISVD. In one embodiment, the ISVD is selected from a VHH, a humanized VHH, a camelized VH, a domain antibody, a single domain antibody, and a dAb. In one embodiment, the ISVD is selected from a VHH, a humanized VHH, and a camelized VH. In one embodiment, the ISVD has a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% to a sequence of SEQ ID NOs: 2-57, wherein the amino acid residues forming the CDR sequences are disregarded for purposes of determining the degree of sequence identity.

[0030] A further aspect of the technology relates to an ISVD, wherein the sequence of the ISVD is: X0VQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYAX1SAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIX2PYDYX3GQGTLVTVSS (SEQ ID NO: 85) where: a. X0 is selected from E and D; b. X1 is selected from the group consisting of E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S; c. X2 is selected from the group consisting of Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I; and d. X3 is selected from the group consisting of W, R, A, E, Y, L, H, I, Q, V, K, S, G, P, F, and T.

[0031] In one embodiment, X0 is D. In one embodiment, X1 is selected from D or E. In one embodiment, X1 is E. In one embodiment, X2 is selected from the group consisting of Y, T, S, and Q. In one embodiment, X2 is Y. In one embodiment, X3 is W.

[0032] In another aspect of the present technology, the ISVD of the present technology is part of a multispecific polypeptide that further comprises a moiety capable of binding to a specific cell surface target.

[0033] Thus, in some embodiments of the present technology, the present technology provides a polypeptide comprising a first ISVD and at least one further ISVD, wherein the first ISVD specifically binds to a constant domain of a human TCR and / or a non-human primate TCR present on a T cell, and the at least one further ISVD specifically binds to an antigen on a target cell, wherein the first ISVD is an ISVD according to the present technology.

[0034] In these embodiments, the amino acid sequence of the first ISVD can have at least 80% sequence identity to at least one of the amino acid sequences of SEQ ID NOs: 2-57, where the amino acid residues forming the CDR sequences are disregarded for purposes of determining the degree of sequence identity. In one embodiment, the first ISVD is selected from the group of amino acid sequences consisting of SEQ ID NOs: 37, 42, 46, 50, and 52, e.g., SEQ ID NO: 37 or SEQ ID NO: 42.

[0035] In another embodiment, the polypeptide may further comprise a third ISVD that specifically binds to a second antigen on a target cell.

[0036] In some embodiments of the present technology, the polypeptide further comprises one or more other groups, residues, moieties, or binding units, optionally linked via one or more peptidic linkers, wherein the one or more other groups, residues, moieties, or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, moieties, or binding units. For example, the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.

[0037] Also provided are nucleic acid molecules encoding the ISVDs or polypeptides of the present technology, or vectors comprising the nucleic acids.

[0038] The present technology also relates to non-human hosts or host cells transformed or transfected with nucleic acids or vectors encoding the ISVDs or polypeptides of the present technology.

[0039] The present technology further relates to compositions comprising the ISVDs or polypeptides of the present technology, preferably the compositions are pharmaceutical compositions.

[0040] Further provided is a method for producing an ISVD or polypeptide disclosed herein, said method comprising: a. expressing a nucleic acid sequence encoding an ISVD or polypeptide in a suitable host cell or host organism or other suitable expression system, optionally followed by: b. isolating and / or purifying the ISVD or polypeptide; At least includes.

[0041] Moreover, the technology relates to compositions or polypeptides for use as pharmaceutical agents.

[0042] In one embodiment of the present technology, the polypeptide or composition is for use in the treatment of a proliferative disease, an inflammatory disease, an infectious disease, or an autoimmune disease, hi one embodiment of the present technology, said proliferative disease is cancer.

[0043] The present technology also provides methods of treatment comprising administering the composition or polypeptide to a subject in need thereof.

[0044] In one embodiment of the present technology, the method of treatment is for treating a proliferative disease, an inflammatory disease, an infectious disease, or an autoimmune disease, hi one embodiment, said proliferative disease is cancer.

[0045] The present technology also provides compositions or polypeptides for use in the preparation of a pharmaceutical agent, in one embodiment of the present technology, the pharmaceutical agent is used in the treatment of a proliferative disease, an inflammatory disease, an infectious disease, or an autoimmune disease.

[0046] In one embodiment of the present technology, said proliferative disease is cancer. [Brief explanation of the drawings]

[0047] [Figure 1] 1 shows binding by a TCE ISVD according to the present technology to primary human T cells as determined by flow cytometry (FACS). [Figure 2] 1 shows binding by a TCE-CD123-ALB ISVD construct according to the present technology to primary human T cells as determined by flow cytometry (FACS). [Figure 3A-B] Figure 3 shows the dose-response curve of TCE-CD123-ALB in a flow cytometry-based human T cell-mediated MOLM-13 cell killing assay using an effector-to-target ratio of 10 to 1 in the presence of 30 μM HSA. Cells were obtained from two human donors. Results for donor 1 are shown in Figures 3A, 3C, and 3E, while results for donor 2 are shown in Figures 3B, 3D, and 3F. [Figure 3C-D] Figure 3 shows the dose-response curve of TCE-CD123-ALB in a flow cytometry-based human T cell-mediated MOLM-13 cell killing assay using an effector-to-target ratio of 10 to 1 in the presence of 30 μM HSA. Cells were obtained from two human donors. Results for donor 1 are shown in Figures 3A, 3C, and 3E, while results for donor 2 are shown in Figures 3B, 3D, and 3F. [Figure 3E-F] Figure 3 shows the dose-response curve of TCE-CD123-ALB in a flow cytometry-based human T cell-mediated MOLM-13 cell killing assay using an effector-to-target ratio of 10 to 1 in the presence of 30 μM HSA. Cells were obtained from two human donors. Results for donor 1 are shown in Figures 3A, 3C, and 3E, while results for donor 2 are shown in Figures 3B, 3D, and 3F. [Figure 4] 4A and 4B show dose-response curves for TCE-CD123-ALB in flow cytometry-based human (4A) and cynomolgus monkey (4B) T cell-mediated KG-1a cell killing assays using an effector-to-target ratio of 10 to 1 in the presence of 30 μM HSA. [Figure 5A-B]Dose-response curves of the TCE-GPC3-GPC3-ALB construct in impedance-based human (Figures 5A, 5B) and cynomolgus monkey (Figures 5C, 5D) T cell-mediated HuH-7 cell killing assays using an effector-to-target ratio of 15 to 1 in the presence of 30 μM HSA are shown. [Figure 5C-D] Dose-response curves of the TCE-GPC3-GPC3-ALB construct in impedance-based human (Figures 5A, 5B) and cynomolgus monkey (Figures 5C, 5D) T cell-mediated HuH-7 cell killing assays using an effector-to-target ratio of 15 to 1 in the presence of 30 μM HSA are shown. [Figure 6] The progress of relative tryptophan oxidation at 25° C., quantified as the sum of peaks with RRT<1.0 by reversed-phase chromatography, is shown for both ISVD constructs A022600424 and A022600462. [Figure 7] The progress of tryptophan oxidation, quantified as the sum of peaks with RRT<1.0 by reversed-phase chromatography, is shown after 1 day and 2 weeks under forced degradation conditions for both ISVD constructs A022600424 and A022600462. [Figure 8] 1 shows the results of peptide mapping of ISVD constructs A022600424 and A022600462. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present inventors have previously found that the introduction of certain amino acid mutations—and combinations thereof—into the CDRs of ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969) results in improved binding to the constant domains of human TCRs and / or non-human primate TCRs. However, the developed improved TCR-binding ISVD, designated T017000700 (SEQ ID NO: 1), has been shown to have several challenges in chemical stability studies related to isomerization and tryptophan oxidation.

[0049] Therefore, there remains a need for further improved TCR-binding ISVDs.

[0050] The inventors have now found that the introduction of certain amino acid mutations at specific positions within the sequence of the ISVD can eliminate the issues observed with T017000700, while retaining improved TCR binding relative to T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969).

[0051] Amino acid residues will be referred to interchangeably herein according to the standard three-letter or one-letter amino acid codes listed in Table B-1 below.

[0052] [Table 1]

[0053] When an amino acid residue is represented as "X" or "Xaa," this means that the amino acid residue is unspecified unless a more restrictive interpretation is required by the context. For example, when there is a description of the amino acid sequence of a CDR in which one (or more) amino acid residues are represented by "X," the description may further specifically indicate which amino acid residue is (can be) present at that specific position in the CDR.

[0054] 5.1 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD) defines an immunoglobulin molecule in which the antigen-binding site resides and is formed by a single immunoglobulin domain. This distinguishes it from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, dis-scFv), in which two immunoglobulin domains, specifically two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L) interact to form the antigen-binding site. In this case, V H and V L Both of these contribute their complementarity determining regions (CDRs) to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0055] In view of the above definitions, antigen-binding domains of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD, or IgE molecules known in the art) or Fab fragments, F(ab')2 fragments, Fv fragments, e.g., disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) would not normally be considered immunoglobulin single variable domains, since in these cases, binding to each epitope of an antigen will not normally occur with one (single) immunoglobulin domain, but with paired (associated) immunoglobulin domains, e.g., light and heavy chain variable domains, i.e., the V domains of immunoglobulin domains that together bind to the respective epitopes of the antigen. H -V L Because in pairs, it will happen.

[0056] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope on an antigen without pairing with other immunoglobulin variable domains. The binding site of an immunoglobulin single variable domain consists of a single V H , single V HH , or a single V L It is formed by domains.

[0057] Thus, a single variable domain may be any light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). Lsequence) or a suitable fragment thereof or a heavy chain variable domain sequence (e.g., V H Array or V HH sequence) or a suitable fragment thereof.

[0058] Immunoglobulin single variable domains (ISVDs) include, for example, camelized V H or humanized V HH Including V H , V HH Preferably, it is a heavy chain ISVD such as camelized V H or humanized V HH Including V HH The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0059] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or a suitable amino acid sequence for use as a single domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb), other single variable domain, or any suitable fragment of any one of them. In particular, the immunoglobulin single variable domain may be a NANOBODY® immunoglobulin single variable domain (e.g., a humanized V HH Or Camelization V H Including V HH ) or a suitable fragment thereof. NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV.

[0060] V HH , V HH Antibody fragments, and V HH Also known as antibodies, "V HH The "V domain" was originally described as the antigen-binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. 1993, Nature 363:446-448). HH The term "domain" refers to the heavy chain variable domain (referred to herein as "V" in a conventional four-chain antibody) present inH domain”) and the light chain variable domain (referred to herein as “V L The term was chosen to distinguish these variable domains from the V domains. HH For a further description, see the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302).

[0061] Typically, the generation of immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening naive or synthetic libraries, e.g., by phage display.

[0062] The generation of immunoglobulin sequences has been extensively described in various publications, among which WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001. In these methods, camelids are immunized with a target antigen to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind to the target antigen.

[0063] In these cases, the development of immunoglobulins requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production.

[0064] Immunization and / or screening of immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0065] The present technology may use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The present technology also includes fully human, humanized, or chimeric sequences. For example, the present technology includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs described by Ward et al. (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 and 1996). Moreover, the present technology also includes fusion immunoglobulin sequences, such as those that form multivalent and / or multispecific constructs (e.g., one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al. 2001, J. Biol. Chem. 276(10):7346-7350; see also, for example, WO 96 / 34103 and WO 99 / 23221), as well as immunoglobulin sequences containing tags or other functional moieties, such as toxins, labels, radioactive chemicals, etc., which can be derived from the immunoglobulin sequences of the present technology.

[0066] "Humanized V HH " is a naturally occurring V HH The amino acid sequence of the naturally occurring domain may be "humanized", i.e., may contain an amino acid sequence corresponding to the amino acid sequence of the naturally occurring domain. HH One or more amino acid residues in the amino acid sequence (particularly in the framework sequence) of the V H The humanized V domains are substituted with one or more amino acid residues present at the corresponding positions in the V domain (e.g., as shown above). This can be done in a manner known per se, which will be clear to those skilled in the art based on the further description herein and the prior art (e.g., WO 2008 / 020079). Again, such humanized V domains can be used to HHcan be obtained by any suitable method known per se, and it should be noted that the method is not strictly limited to polypeptides obtained using naturally occurring polypeptides containing a VHH domain as a starting material.

[0067] "Camelization V H " is a naturally occurring V H domains, but which have been "camelized," i.e., the naturally occurring V domains from traditional four-chain antibodies. H One or more amino acid residues in the amino acid sequence of the V domain of a heavy chain antibody HH This can be done in a manner known per se, which will be clear to the skilled person on the basis of the further disclosure herein and the prior art (e.g. WO 2008 / 020079). Such "camelizing" substitutions, as defined herein, are preferably substituted with one or more amino acid residues present in the corresponding positions of V. H -V L The amino acid residues are inserted at amino acid positions that form and / or are present at the interface and / or the so-called Camelidae hallmark residues (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 and 1996, supra). H V used as a starting material or starting point to generate or design H The sequence is preferably a V H sequence, more preferably human V H An array, e.g., V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, wherein the naturally occurring V H It should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0068] A preferred structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FR"), referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively, interposed between three complementarity-determining regions ("CDR"), referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0069] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079, the amino acid residues of immunoglobulin single variable domains are given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) and are also described in the article by Riechmann and Muyldermans 2000 (J. Immunol. Methods 240(1-2):185-195, see e.g., Figure 2 of this publication) for the sequence of V from camelids. HH applied to the domain, V H Can be numbered according to the common numbering scheme for domains. H Domain and V HH It should be noted that, as is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.H and V HH The total number of amino acid residues in the sequence is usually in the range of 110 to 120, often 112 to 115. However, it should be noted that for the purposes described herein, shorter and longer sequences may also be suitable.

[0070] In this application, unless otherwise indicated, CDR sequences were determined according to Kabat (Martin 2010, In: Kontermann and Duebel (eds.), Antibody Engineering Vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 1-30, CDR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 31-35, FR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 36-49, CDR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 50-65, FR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 66-94, CDR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 95-102, and FR4 of the immunoglobulin single variable domain comprises amino acid residues at positions 103-113.

[0071] The determination of CDR regions can also be carried out according to different methods. In the present application, the CDR sequences were also determined according to the AbM definition described in Martin 2010 (in: Kontermann and Duebel (Eds.) 2010, Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1 to 25, CDR1 contains amino acid residues at positions 26 to 35, FR2 contains amino acids at positions 36 to 49, CDR2 contains amino acid residues at positions 50 to 58, FR3 contains amino acid residues at positions 59 to 94, CDR3 contains amino acid residues at positions 95 to 102, and FR4 contains amino acid residues at positions 103 to 113.

[0072] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequence, and examples of suitable framework sequences will be clear to those skilled in the art, e.g., based on the standard handbooks and further disclosures and prior art cited herein.

[0073] The framework sequences are preferably immunoglobulin framework sequences or (suitable combinations of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of a light chain variable domain (e.g., V L sequence) and / or heavy chain variable domains (e.g., V H Array or V HH In a particularly preferred embodiment, the framework sequences are derived from V HH framework sequences derived from the V sequences (in which case the framework sequences may optionally be partially or fully humanized) or camelized conventional V sequences. H The sequence may be either (as defined herein) or (as defined herein).

[0074] Specifically, the framework sequences present in the ISVD sequences used in the present technology are those in which the ISVD sequence is a humanized V HH or Camelization V H Including V HH and / or one or more hallmark residues (as defined herein) such that: Some preferred, but non-limiting, examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0075] Again, as generally described herein for immunoglobulin sequences, it is possible to use any suitable fragment (or combination of fragments) of the above, for example a fragment containing one or more CDR sequences suitably flanked by and / or linked via one or more framework sequences (e.g., in the same order as these CDR and framework sequences may be present in the full-size immunoglobulin sequence from which the fragment is derived).

[0076] It should be noted, however, that the present technology is not limited with respect to the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor with respect to the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific, but non-limiting, aspects, the ISVD sequence may be, but is not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, particularly a partially or fully humanized VHV). HHimmunoglobulin sequences), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR-grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the above.

[0077] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (particularly an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0078] As described above, the ISVD may be an ISVD or a suitable fragment thereof. For a general description of ISVDs, see the further description below as well as the prior art cited herein. However, in this regard, the description and prior art do not contain any reference to so-called "V H 3 Class ISVD (i.e., DP-47, DP-51, DP-29, etc.) H It should be noted that in the present application, mainly ISVDs (ISVDs with high degrees of sequence homology to three classes of human germline sequences) have been described. However, in the broadest sense, any type of ISVD can generally be used, for example, so-called "V" ISVDs, as described in WO 2007 / 118670. HISVD (i.e., V such as DP-78) belonging to the "4th class" H It should be noted that four classes of ISVDs with high degrees of sequence homology to human germline sequences are also used.

[0079] In general, ISVD (particularly (partially) humanized V HH Sequence and camelization V H V including array HH A sequence) can be characterized by the presence of one or more "hallmark residues" (as defined herein) in one or more of the framework sequences (again as defined herein). Thus, in general, an ISVD can be defined by the (generic) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and one or more of the hallmark residues are as further defined herein.) An immunoglobulin sequence can be defined as an immunoglobulin sequence having the following structure:

[0080] Specifically, ISVD has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein.) It can be an immunoglobulin sequence having the following structure:

[0081] More specifically, the ISVD has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering system are selected from the hallmark residues listed in Table A-0 below.) It can be an immunoglobulin sequence having the following structure:

[0082] [Table 2]

[0083] [Table 3]

[0084] The present technology particularly uses an ISVD capable of binding to the constant domain of a TCR. In the context of the present technology, "binding to" a particular target molecule has its usual meaning in the art as understood in the context of an antibody and its respective antigen.

[0085] Therefore, the target molecule of the ISVD used in this technology is the constant domain of the TCR.

[0086] Binding to the TCR can be achieved, for example, by binding to the TCR alpha subunit and / or the TCR beta subunit. An example is a mammalian TCR. Human TCRs are preferred, but versions from other species, such as mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primate, such as cynomolgus monkey (also referred to herein as "cyno"), or camelid, such as llama or alpaca, are also applicable to the present technology.

[0087] The sequences of the TCR-α / β constant domains of human and cynomolgus origin are provided in Table A-1 (SEQ ID NOs: 106 and 108 are for the TCR α constant domains of human and cynomolgus origin, respectively, and SEQ ID NOs: 107 and 109 are for the TCR β constant domains of human and cynomolgus origin, respectively). The origin of each of these sequences, expressed as a UniProt or Genbank file identifier, is listed for each of the above sequences in Table A-1. The exact identity of the amino acid sequence originally derived from the rhesus source to that of the cynomolgus source was confirmed by in-house sequencing.

[0088] In one embodiment, the ISVD specifically binds to the constant domain of human T cell receptor alpha (TCR-α) (SEQ ID NO: 106) and / or the constant domain of human T cell receptor beta (TCR-β) (SEQ ID NO: 107) or a polymorphic variant or isoform thereof.

[0089] In one embodiment, the ISVD specifically binds to the constant domain of a non-human primate TCR. In one embodiment, the non-human primate TCR is a macaque or rhesus TCR. In one embodiment, the macaque or rhesus TCR comprises the constant domain of TCR-α of SEQ ID NO: 108 and / or the constant domain of TCR-β of SEQ ID NO: 109, or a polymorphic variant or isoform thereof.

[0090] Isoforms are alternative protein sequences that can arise from the same gene due to a single or combination of biological events, such as alternative promoter usage, alternative splicing, alternative initiation, ribosomal frameshifting (all known in the art).

[0091] [Table 4]

[0092] [Table 5]

[0093] [Table 6]

[0094] Thus, in a first aspect, the present technology relates to an ISVD that specifically binds to a constant domain of a human and / or non-human primate T cell receptor (TCR) present on a T cell, wherein the ISVD essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), a. The amino acid sequence of CDR1 (according to Kabat) is INFYG (SEQ ID NO: 79), b. The amino acid sequence of CDR2 (according to Kabat) is HISIGDQTDYAX1(SAKG (SEQ ID NO: 80) c. The amino acid sequence of CDR3 (according to Kabat) is LSRIX2PYDY (SEQ ID NO: 81), where: - the amino acid residue X1 is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or the amino acid residue X2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I.

[0095] An ISVD sequence may also be represented using the AbM definition for CDRs as follows: an ISVD that specifically binds to the constant domain of a human and / or non-human primate T cell receptor (TCR) present on a T cell, wherein the ISVD consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively); a. The amino acid sequence of CDR1 (according to AbM) is GYVHKINFYG (SEQ ID NO: 82), b. The amino acid sequence of CDR2 (according to AbM) is HISIGDQTD (SEQ ID NO: 83), and c. The amino acid sequence of CDR3 (according to AbM) is LSRIX2PYDY (SEQ ID NO: 84), where: the amino acid residue at position 61 (according to Kabat) is chosen from E, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or the amino acid residue X2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I.

[0096] The present inventors have found that the ISVDs having the CDRs according to the present technology have potent TCR binding ability and do not show isomerization or tryptophan oxidation at the relevant positions 61 and 99 (Kabat numbering), respectively, which means that the molecules are effective in engaging T cells and are also chemically stable.

[0097] In one embodiment of the present technology, the amino acid sequence of the ISVD that specifically binds to a human TCR may exhibit greater than 85%, for example, greater than 90%, greater than 95%, or greater than 99% sequence identity to any of SEQ ID NOs: 2 to 57, wherein the CDRs are as defined herein.

[0098] In a further embodiment, the amino acid sequence of the ISVD that specifically binds to a human TCR may exhibit greater than 85%, preferably at least 90%, more preferably at least 95% sequence identity. Preferably, the sequence comprises SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:52, e.g., SEQ ID NO:37 or SEQ ID NO:42.

[0099] In another embodiment of the present technology, the amino acid sequence of an ISVD that specifically binds to a human TCR may exhibit greater than 85%, for example, greater than 90%, greater than 95%, or greater than 99% sequence identity to any of SEQ ID NOs: 2 to 57, in which case the amino acid residues that form the CDR sequences are disregarded for purposes of determining the degree of sequence identity.

[0100] In a further embodiment, the amino acid sequence of the ISVD that specifically binds to a human TCR may exhibit greater than 85%, preferably at least 90%, more preferably at least 95% sequence identity, where the amino acid residues that form the CDR sequences are disregarded for purposes of determining the degree of sequence identity. Preferably, the sequence comprises SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:52, e.g., SEQ ID NO:37 or SEQ ID NO:42.

[0101] When an ISVD exhibits greater than 85%, e.g., greater than 90%, greater than 95%, or greater than 99% sequence identity to any of SEQ ID NOs: 2-57, the ISVD preferably exhibits at least half the binding affinity, and more preferably at least the same binding affinity, to a human TCR compared to one of the ISVDs specified in Table A-4, where the binding affinity is measured using the same method, e.g., surface plasmon resonance (SPR).

[0102] Additionally, when an ISVD exhibits greater than 85%, e.g., greater than 90%, greater than 95%, or greater than 99% sequence identity to any of SEQ ID NOs: 2-57, the ISVD preferably exhibits at least half the potency of T cell-mediated target cell killing, and more preferably at least the same potency of T cell-mediated target cell killing, as compared to one of the ISVDs specified in Table A-4, where T cell-mediated target cell killing is measured using the same method, e.g., a flow cytometry-based T cell-mediated cell killing assay or an impedance-based T cell-mediated killing assay.

[0103] The percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%, where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered to be the difference of a single amino acid residue (a single position).

[0104] Generally, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation methods outlined above, the amino acid sequence having the greatest number of amino acid residues will be considered the "first" amino acid sequence, and the other amino acid sequence will be considered the "second" amino acid sequence.

[0105] As used herein, an "amino acid difference" means a deletion, insertion or substitution, preferably a substitution, of a single amino acid residue compared to a reference sequence.

[0106] In one embodiment of the present technology, the amino acid substitution is a conservative substitution, preferably in which one amino acid in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly, (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln, (c) polar positively charged residues: His, Arg, and Lys, (d) large aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys, and (e) aromatic residues: Phe, Tyr, and Trp. (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0107] In another embodiment of the present technology, conservative substitutions are as follows: Ala to Gly or Ser, Arg to Ly, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, He to Leu or Val, Leu to He or Val, Lys to Arg, Gln, or Glu, Met to Leu, Tyr, or He, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, He, or Leu.

[0108] In one embodiment of the present technology, the entire amino acid sequence of the ISVD is: X0VQLVESGGGVVVQPGGSLRSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYAX1SAKGRFTISRDESKNTVYLQMNSLRPEDTAAYCRALSRIX2PYDYX3GQGTLVTVSS, wherein a. X0 is selected from E and D; b. X1 is selected from the group consisting of E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S; c. X2 is selected from the group consisting of Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I; and d. X3 is selected from the group consisting of W, R, A, E, Y, L, H, I, Q, V, K, S, G, P, F, and T.

[0109] In one embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is any of Y, T, S, or Q, and / or X3 is W.

[0110] In another embodiment of the present technology, X0 is E, and / or X1 is E, and / or X2 is any of Y, T, S, or Q, and / or X3 is W.

[0111] In one embodiment of the present technology, X0 is E, and / or X1 is E, and / or X2 is T, and / or X3 is W.

[0112] In further embodiments of the present technology, X0 is D, and / or X1 is E, and / or X2 is T, and / or X3 is W.

[0113] In another embodiment of the present technology, X0 is E, and / or X1 is E, and / or X2 is S, and / or X3 is W.

[0114] In one embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is S, and / or X3 is W.

[0115] In one embodiment of the present technology, X0 is E, and / or X1 is E, and / or X2 is Q, and / or X3 is W.

[0116] In another embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is Q, and / or X3 is W.

[0117] In further embodiments of the present technology, X0 is E, and / or X1 is E, and / or X2 is Y, and / or X3 is W.

[0118] In one embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is Y, and / or X3 is W.

[0119] The inventors found that substituting the amino acid at the first position (Kabat numbering) of the sequence from E to D was able to prevent pyroglutamate formation while leaving the efficacy of the ISVD unaffected.

[0120] In another embodiment of the present technology, the ISVD of the present technology has the amino acid sequence of any one of SEQ ID NOs: 37, 42, 46, 50, and 52.

[0121] In further embodiments of the present technology, the ISVD has the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 42.

[0122] The inventors observed that ISVDs according to the present technology having an E at position 61 (Kabat numbering) and a Y, A, S, or H at position 99 have similar or higher melting temperatures (Tm), higher aggregation onset temperatures (Tagg), and reduced oligomeric fractions when compared to reference ISVD T017000700. ISVDs having an E at position 61 (Kabat numbering) and a Q or T at position 99 did not show any significant decrease in Tm and maintained or had higher Tag and similar or reduced oligomeric fractions (Δ% Oligo).

[0123] Thus, in one embodiment, an ISVD of the present technology or an ISVD exhibiting greater than 90%, for example greater than 95% or greater than 99% sequence identity to any of SEQ ID NOs: 2 to 57 has a melting temperature (Tm) of at least 71°C, at least 72°C, preferably at least 72.5°C, more preferably at least 73°C.

[0124] In another embodiment, the ISVD of the present technology or an ISVD exhibiting greater than 90%, for example greater than 95% or greater than 99% sequence identity to any of SEQ ID NOs: 2 to 57, has an aggregation onset temperature (Tagg) of at least 67°C, at least 68°C, at least 69°C, at least 71°C, preferably at least 72°C, more preferably at least 73°C.

[0125] In yet further embodiments, an ISVD of the present technology or an ISVD exhibiting greater than 90%, for example greater than 95% or greater than 99% sequence identity to any of SEQ ID NOs: 2-57 has an oligomerization fraction (Δ% oligo) of less than 0.5%, preferably less than 0.4%, more preferably less than 0.3%, even more preferably less than 0.2%, or even less than 0.1%.

[0126] A list of the occurring ISVDs can be found in Table A-4. Additionally, the CDR sequence combinations of the occurring ISVDs can be found in Table A-5.

[0127] In one embodiment of the present technology, the TCE ISVD has CDR1, CDR2, and CDR3 sequences selected from the CDR1, CDR2, and CDR3 sequences presented in Table A-5.

[0128] In another embodiment of the present technology, the TCE ISVD has CDR1, CDR2, and CDR3 sequences selected from the combination of CDR sequences presented in the same row of Table A-5.

[0129] 5.2 Multispecific Polypeptides The present inventors have found that the introduction of certain amino acid mutations—and combinations thereof—into the CDRs of ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969) results in improved binding to the constant domains of human TCRs and / or non-human primate TCRs. Additional amino acid mutations at specific positions within the sequence of the ISVD could further improve the chemical stability of the ISVD by minimizing and even preventing isomerization and tryptophan oxidation.

[0130] Therefore, in one aspect of the present technology, there is provided a polypeptide comprising a first ISVD capable of specifically binding to a constant domain of a human and / or non-human primate T cell receptor (TCR) present on a T cell, and a second ISVD capable of specifically binding to a first antigen on a target cell, wherein the first antigen is different from the TCR, the target cell is different from the T cell, the first and second ISVDs essentially consist of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), and the first ISVD is an ISVD related to the present technology.

[0131] In this multispecific polypeptide, the CDR regions are as defined herein (see Section 5.1). The inventors have found that polypeptides comprising an ISVD with CDRs according to the present technology have potent TCR binding ability and do not exhibit isomerization or tryptophan oxidation at the relevant positions 61 and 99 (Kabat numbering), respectively.

[0132] In one embodiment, the amino acid sequence of the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity to at least one of the amino acid sequences of any of SEQ ID NOs: 2 to 57, wherein the sequence of the CDR region is as defined herein, and preferably the sequence of the first ISVD comprises SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, or SEQ ID NO: 52.

[0133] In one embodiment, the amino acid sequence of the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity to at least one of the amino acid sequences of any of SEQ ID NOs: 2 to 57, where the amino acid residues forming the CDR sequences are disregarded for purposes of determining the degree of sequence identity, and preferably the sequence of the first ISVD comprises SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, or SEQ ID NO: 52.

[0134] In another embodiment, the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 32, 33, and / or 35-57, wherein the sequence of the CDR region is as defined herein, and preferably the sequence comprises SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, or SEQ ID NO: 52.

[0135] In another embodiment, the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 32, 33, and / or 35-57, where for purposes of determining the degree of sequence identity, the amino acid residues forming the CDR sequences are disregarded, and preferably the sequence comprises SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, or SEQ ID NO: 52.

[0136] In a further embodiment, the first ISVD comprises or consists of SEQ ID NO:37 or SEQ ID NO:42.

[0137] In yet another embodiment, the first ISVD comprises or consists of SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:52.

[0138] As shown in the Examples further provided herein, substitution of specific amino acid residues at positions 61 and 99 in T017000700 (SEQ ID NO: 1) did not affect the affinity of the ISVD for the constant domains of human and / or non-human primate TCRs, but the resulting variants had better chemical stability compared to the previously developed T017000700 (SEQ ID NO: 1). T017000978 (SEQ ID NO: 37) and T017000991 (SEQ ID NO: 42) were found to be particularly potent and stable. Additionally, T017000995 (SEQ ID NO: 46), T017000999 (SEQ ID NO: 50), and T017001001 (SEQ ID NO: 52) showed lower affinity for TCRs but maintained high cell-killing potency, suggesting better biodistribution and potentially more target-specific activity.

[0139] In one embodiment of the present technology, the polypeptide is at least bispecific, but can also be, for example, trispecific, tetraspecific, pentavalent, etc. Moreover, the polypeptide is at least bivalent, but can also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0140] The terms "bispecific," "trispecific," "tetraspecific," "pentaspecific," etc. are all included within the term "multispecific" and refer to binding to different numbers of target molecules, such as 2, 3, 4, 5, etc., respectively.

[0141] The terms "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," etc. are all included in the term "multivalent" and indicate the presence of 2, 3, 4, 5, 6, etc. binding units / building blocks, e.g., ISVDs, respectively.

[0142] For example, the polypeptide may be bispecific, bivalent, e.g., a polypeptide comprising or consisting of two ISVDs, where one ISVD specifically binds to the constant domain of a human and / or non-human primate TCR on a T cell, and one ISVD specifically binds to a cell surface-specific target antigen, and the TCR and target antigen are preferably of human origin.

[0143] The polypeptides may also be bispecific trivalent, e.g., comprising or consisting of three ISVDs, where two ISVDs specifically bind to the same cell surface-specific target antigen and one ISVD specifically binds to the constant domain of a human and / or non-human primate TCR on a T cell.

[0144] In another example, the polypeptide can be trispecific trivalent, e.g., a polypeptide comprising or consisting of three ISVDs, where one ISVD specifically binds to the constant domain of a human and / or non-human primate TCR on a T cell, and one ISVD specifically binds to a first antigen on a target cell, and one ISVD specifically binds to a second antigen on the same target cell.

[0145] In yet another example, the trispecific trivalent polypeptide comprises one ISVD that specifically binds to a constant domain of a human and / or non-human primate TCR on a T cell and one ISVD that specifically binds to a first antigen on a target cell, next to one ISVD that specifically binds to human serum albumin.

[0146] Further examples of multispecific, multivalent polypeptides will be apparent to those skilled in the art based on the disclosure herein.

[0147] Such a polypeptide can simultaneously be biparatopic, for example, if the two ISVDs bind to two different epitopes of the target antigen.

[0148] The term "biparatopic" refers to binding to two different parts (eg, epitopes) of the same target molecule.

[0149] The components of the multispecific multivalent polypeptides described herein, preferably the ISVDs, may be linked to each other by one or more suitable linkers, eg, peptidic linkers.

[0150] The use of linkers to connect two or more (poly)peptides is well known in the art.

[0151] One frequently used class of peptidic linkers is known as the "Gly-Ser" or "GS" linker, which consists essentially of glycine (G) and serine (S) residues and usually contains one or more repeats of a peptide motif, such as the GGGGS (SEQ ID NO: 86) motif (e.g., a repeat of the formula (Gly-Gly-Gly-Gly-Ser) n (where n can be 1, 2, 3, 4, 5, 6, 7, or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 87), the 15GS linker (n=3), and the 35GS linker (n=7). See, for example, Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10):1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27(10):325-330). In the polypeptides disclosed herein, the use of 5GS and 9GS linkers to link components of the polypeptide to each other is preferred. Preferably, a linker of less than 10 amino acids is used to link a first ISVD capable of specifically binding to a TCR to a second ISVD capable of specifically binding to a cell surface-specific target antigen.

[0152] Examples of suitable linkers are given in Table A-2 below.

[0153] [Table 7]

[0154] Thus, in one embodiment, the polypeptide comprises a first ISVD capable of specifically binding to a constant domain of a human and / or non-human primate TCR of the present technology and a second ISVD capable of specifically binding to a cell surface-specific target antigen, which are linked by a 5GS and / or 9GS linker.

[0155] In another embodiment of the present technology, the polypeptide comprises a first ISVD capable of specifically binding to a constant domain of a human and / or non-human primate TCR of the present technology and a second ISVD capable of specifically binding to a cell surface-specific target antigen, which are linked by a 9GS linker.

[0156] The present inventors have surprisingly found that such a configuration can increase the efficiency of the polypeptide to elicit a T cell-mediated cytotoxic response.

[0157] It will be appreciated that the ISVD that binds to the TCR and the ISVD that binds to the first antigen on the target cell can be positioned in either order in the multispecific, multivalent polypeptide of the present technology (as also demonstrated in the Examples section).

[0158] Thus, in one embodiment of the present technology, the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: a first ISVD that specifically binds to a TCR, a second ISVD that specifically binds to a cell surface-specific target antigen, and any binding unit that provides a polypeptide with increased half-life as defined herein. A first ISVD that specifically binds to a TCR, a second ISVD that specifically binds to CD33, and a third ISVD that specifically binds to CD123, and any binding unit that provides a polypeptide with increased half-life as defined herein. In one embodiment, the ISVDs are linked by a 9GS linker. The binding unit that provides a polypeptide with increased half-life is preferably an ISVD that preferentially binds to serum albumin.

[0159] Such configurations of the polypeptide can provide strong efficacy for the treatment of cancer.

[0160] Again, this does not exclude that other binding units / building blocks, such as other ISVDs that bind to other antigens on target cells or that bind to other targets, may be present in the polypeptide. Moreover, it does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be interposed. By way of example, as further described below (see in particular Section 5.4 "(In vivo) Half-Life Extension") below), the polypeptide may further comprise another ISVD that specifically binds to human serum albumin, which may even be located, for example, between the "first ISVD" and the "second ISVD."

[0161] The second ISVD of the polypeptide of the present technology specifically binds to an antigen on target cells, preferably on cancer cells. "Target cells" as referred to herein are cells that display specific antigens on their surface. In one embodiment, "target cells" are cancer cells.

[0162] The membrane (also called the plasma membrane or phospholipid bilayer), which is the outer boundary of a cell, surrounds the cell's cytoplasm. In other words, the membrane is the cell's surface. This membrane serves to separate and protect the cell from its surrounding environment and is made mostly of a phospholipid bilayer. Various protein molecules, such as channels, pumps, and cell receptors, are embedded within this membrane. Because the membrane is fluid, protein molecules can move within the membrane. As used herein, the term "antigen on a target cell" refers to a molecule displayed on the surface of a cell. In most cases, this molecule will be located in or on the plasma membrane of the cell so that at least a portion of the molecule remains accessible from the outside of the cell in its tertiary form. A non-limiting example of a cell surface molecule located in the plasma membrane is a transmembrane protein that contains hydrophilic and hydrophobic regions in its tertiary conformation. Here, at least one hydrophobic region allows the cell surface molecule to be embedded or inserted into the hydrophobic plasma membrane of the cell, while the hydrophilic regions extend on either side of the plasma membrane into the cytoplasm and extracellular space, respectively.

[0163] The antigen can be any target on a cell, e.g., a tumor antigen. In one embodiment, the antigen is specific to the target cell, e.g., a cancer cell, e.g., a tumor antigen or tumor-associated antigen (TAA) on the cancer cell.

[0164] The term "tumor antigen" as used herein can be understood as an antigen presented on tumor cells. Such antigens can be presented on the cell surface, with the extracellular portion often combined with the transmembrane and cytoplasmic portions of the molecule. Sometimes, such antigens can be presented only by tumor cells and not at all by normal or healthy cells. Tumor antigens can be expressed exclusively on tumor cells or can exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. However, this will not generally be the case. More commonly, they are antigens presented by tumor cells and normal cells and are called "tumor-associated antigens (TAA)." Such tumor-associated antigens can be overexpressed on tumor cells compared to normal cells, or they are more accessible for antibody binding in tumor cells due to the more compact structure of tumor tissue compared to normal tissue. TAAs are preferably antigens that are expressed on cells of a specific tumor but preferably not expressed on normal cells. In many cases, TAAs are antigens that are normally expressed in cells only at a particular time in an organism's development (e.g., during fetal development) and are inappropriately expressed in the organism at this point in development, or are antigens that are not expressed in normal tissues or cells of the organ that currently expresses the antigen.

[0165] In one embodiment, the first antigen on the target cell is a tumor-associated antigen (TAA).

[0166] In one embodiment, the first antigen on the target cell is more abundant on cancer cells than on normal cells. The antigen on the target cell is preferably a tumor-associated antigen (TAA).

[0167] In one embodiment, the first antigen on the target cell is a tumor antigen or a tumor-specific antigen (TSA).

[0168] In one embodiment, the multispecific, multivalent polypeptide of the present technology comprises a second ISVD that specifically binds to CD123 or glypican-3.

[0169] In a further embodiment, the polypeptide of the present technology further comprises a third ISVD that specifically binds to a second antigen on a target cell.

[0170] The target cells bound by the polypeptides of the present technology are particularly mammalian cells, preferably primate cells, and even more preferably human cells. The target cells are preferably hyperproliferative cells, such as cancer cells.

[0171] In another embodiment of the present technology, the multispecific, multivalent polypeptide exhibits reduced binding by pre-existing antibodies in human serum. To this end, in one embodiment of the present technology, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD (preferably at the C-terminus of the ISVD of the polypeptide), but preferably of each ISVD.

[0172] In another embodiment of the present technology, the polypeptide exhibits an extension of 1 to 5 amino acids (preferably naturally occurring), e.g., a single alanine (A) extension, at the C-terminus of the C-terminal ISVD, which is typically VTVSS (SEQ ID NO: 88).

[0173] In another embodiment of the present technology, the polypeptide presents a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) of at least one ISVD.

[0174] In another embodiment of the present technology, the ISVDs present a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) of at least one ISVD. In such embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 89), VQVSS (SEQ ID NO: 90), VTVKS (SEQ ID NO: 91), VTVQS (SEQ ID NO: 92), VKVKS (SEQ ID NO: 93), VKVQS (SEQ ID NO: 94), VQVKS (SEQ ID NO: 95), or VQVQS (SEQ ID NO: 96), so that after addition of a single alanine, the C-terminus of the polypeptide exhibits, for example, the sequence VTVSSA (SEQ ID NO: 97), VKVSSA (SEQ ID NO: 98), VQVSSA (SEQ ID NO: 99), VTVKSA (SEQ ID NO: 100), VTVQSA (SEQ ID NO: 101), VKVKSA (SEQ ID NO: 102), VKVQSA (SEQ ID NO: 103), VQVKSA (SEQ ID NO: 104), or VQVQSA (SEQ ID NO: 105), preferably VTVSSA.

[0175] In another embodiment of the present technology, the polypeptide exhibits at least a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) of the C-terminal ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) of at least one ISVD, and an extension of 1 to 5 amino acids (preferably naturally occurring), such as a single alanine (A), at the C-terminus of the C-terminal ISVD (so that the C-terminus of the polypeptide consists, for example, of the sequence VTVSSA, VKVSSA, or VQVSSA, preferably VTVSSA). For further information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0176] As will be apparent from the foregoing and further description herein, the ISVDs of the present technology can be used as "building blocks" to form polypeptides of the present technology, e.g., by suitably combining them with other groups, residues, moieties, or binding units to form compounds or constructs described herein that combine one or more desired properties or biological functions in a single molecule (e.g., but not limited to, the bivalent / trivalent / tetravalent / multivalent and bi / tri / quadruplex / multispecific polypeptides of the present technology described herein). Polypeptides having multiple ISVDs are also referred to herein as "constructs" or "ISVD formats."

[0177] 5.3 Specificity The terms "specificity," "specifically binds," or "specific binding" refer to the number of different target molecules, e.g., antigens, from the same organism to which a particular binding unit, e.g., an ISVD, can bind with sufficiently high affinity (see below). "Specificity," "specifically binds," or "specific binding" are used interchangeably herein with "selectivity," "selectively binds," or "selective binding." A binding unit, e.g., an ISVD, preferably binds specifically to its designated target.

[0178] The specificity / selectivity of a binding unit can be determined based on affinity, which describes the strength or stability of a molecular interaction. Affinity is usually expressed in moles / liter (or M) as K D or dissociation constant. Affinity can also be expressed as the association constant K A It can also be expressed as 1 / K D is equal to (moles / liter) -1 (or M -1 ) units.

[0179] Affinity is a measure of the binding strength between a moiety and a binding site on a target molecule, K D The lower the value, the stronger the binding strength between the target molecule and the targeting moiety.

[0180] Typically, the binding units (e.g., ISVDs) used in this technology are 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, more preferably 10 -8 ~10 -12 Dissociation constant in moles / liter (K D ) (i.e., 10 5 ~10 12 liters / mole or more, preferably 10 7 ~10 12 liters / mole or more, even more preferably 10 8 ~10 12 Association constant (K) in liters / mole A ) to bind to its target.

[0181] 10 -4 Any K greater than mol / liter D The value (or 10 4 Any K less than liter / mol A values), are generally considered to indicate nonspecific binding.

[0182] The K of a biological interaction that is considered specific, e.g., the binding of an immunoglobulin sequence to an antigen D is typically 10 -5 moles / liter (10,000 nM or 10 μM) to 10 -12 in the range of 0.001 nM or 1 pM or less.

[0183] Therefore, specific / selective binding can be measured using the same measurement method, e.g., SPR. -5 ~10 -12 moles / liter or less K D Binds to TCR at a value of 10 -4 moles / liter K D It may refer to a binding unit (or a polypeptide comprising same) that binds to the relevant target at a given value.

[0184] Thus, the ISVD preferably exhibits at least half the binding affinity, and more preferably at least the same binding affinity, to a human TCR compared to an ISVD consisting of the amino acids of SEQ ID NO: 1, provided that the binding affinity is measured using the same method, e.g., SPR.

[0185] Specific binding to a particular target from a particular species does not exclude that the binding unit can also specifically bind to an analogous target from a different species, for example, specific binding to a human TCR does not exclude that the binding unit (or a polypeptide comprising it) can also specifically bind to a TCR from a cynomolgus monkey.

[0186] When an ISVD is said to exhibit "improved cross-reactivity with respect to binding to human and non-human primate TCRs" compared to another ISVD, it refers to the ISVD exhibiting improved avidity (e.g., K D or k off This means that the ratio of the ISVDs (represented by ) is lower than the aforementioned ratios calculated for other ISVDs in the same assay.

[0187] Good cross-reactivity with respect to binding to human and non-human primate TCRs allows for assessment of toxicity of multispecific T cell engaging polypeptides in preclinical studies conducted in human primates.

[0188] Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as different variants thereof known per se in the art, as well as other techniques mentioned herein.

[0189] The dissociation constant may be an actual or apparent dissociation constant, as will be apparent to those skilled in the art. Methods for determining dissociation constants will be apparent to those skilled in the art, and include, for example, the techniques listed below. -4 moles / liter or 10 -3 moles / liter (e.g., 10 -2 It will also be apparent that dissociation constants above 1000 kJ / mol / liter may be unmeasurable. Optionally, as will also be apparent to those skilled in the art, the dissociation constant (actual or apparent) may be determined by the relationship [K D =1 / K A ] to calculate the (actual or apparent) association constant (K A ) can be calculated based on

[0190] The affinity of a molecular interaction between two molecules can be measured via various techniques known per se, for example, the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology 13:1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting alterations in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions, thereby determining k on , k off Measurement, and therefore K D (or K A) values ​​are obtained. This can be done, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991, Biotechniques 11:620-627), Johnson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnson et al. (1991, Anal. Biochem. 198:268-277).

[0191] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the term "biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and an immobilized protein layer (signal beam) on a biosensor tip. Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0192] Alternatively, affinity can be measured by kinetic exclusion assay (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method that measures the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrated solution of antibody / antigen complexes is passed through a column containing beads pre-coated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved by a fluorescently labeled protein that binds to the antibody (or antigen).

[0193] The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0194] In one embodiment, the ISVD of the present technology is at least about 10 for binding to human TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25°. 3 M -1 s -1 , at least about 10 4 M -1 s -1 , and at least about 10 5 M -1 s -1 an on-rate constant (k on )

[0195] In one embodiment, the ISVD of the present technology exhibits a binding affinity of at least about 10 for binding to a non-human primate TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C. 3 M -1 s -1 , at least about 104 M -1 s -1 , and at least about 10 5 M -1 s -1 k selected from the group consisting of on It has.

[0196] In one embodiment, the ISVD of the present technology is at most about 10 for binding to human TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C. -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , and at most about 10 -4 s -1 k selected from the group consisting of off It has.

[0197] In one embodiment, the ISVD of the present technology is at most about 10 for binding to a non-human primate TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C. -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , and at most about 10- -4 s -1 k selected from the group consisting of off It has.

[0198] ISVDs with Y, F, H, K, L, or R at position 99 have particularly advantageous kappa sequences compared to ISVDs with different amino acids at position 99. off Thus, in one embodiment, the ISVD has Y, F, H, K, L, or R at position 99. In one embodiment, the ISVD has Y, F, H, or R at position 99.

[0199] ISVDs having W, A, E, F, H, I, K, L, Q, R, S, T, V, or Y at position 103 (Kabat numbering) showed particularly favorable koff compared to ISVDs having another amino acid at position 103. Thus, in one embodiment, an ISVD has W, A, E, F, H, I, K, L, Q, R, S, T, V, or Y at position 103. In one embodiment, an ISVD has W, A, E, F, H, I, K, L, Q, S, T, or V at position 103.

[0200] ISVDs with A, E, F, H, I, K, L, N, P, Q, R, S, T, V, or Y at position 61 (Kabat numbering) have kappa cleavage properties that are as good as a reference TCE ISVD with D at position 61. off Since isomerization at D61 was observed in the reference ISVD, there was a need to obtain ISVDs with at least similar off-rates that have different amino acids at position 61. Thus, in one embodiment, the ISVD has A, E, F, H, I, K, L, N, P, Q, R, S, T, V, or Y at position 61, and preferably the ISVD has E at position 61.

[0201] In one embodiment, the ISVD of the present technology is at most about 10 for binding to human TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C. -6 M, at most about 10 -7 M, at most about 10 -8 , and at most about 10 -9 M D )

[0202] In one embodiment, the ISVD of the present technology is at most about 10 for binding to a non-human primate TCR, preferably as measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C. -5 M, at most about 10 -6 M, at most about 10 -7 M, and at most about 10 -8 K selected from the group consisting of D It has.

[0203] In some embodiments, the TCR-binding ISVDs of the present technology have the same or a lower off-rate constant (k off In some embodiments, the ISVDs of the present technology bind to human TCRs with the same or lower k compared to the ISVD of SEQ ID NO: 1. off In some embodiments, the TCR-binding ISVDs of the present technology bind to non-human primate TCRs with the same or a lower off-rate constant (k off ) and binds to human TCR.

[0204] In some embodiments, the ISVD of the present technology has the same or a lower k compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969). off binds to non-human primate TCRs at the off-rate (k off ) can be measured by any method known to one of skill in the art. In one embodiment, the off-rate (k off ) is measured by surface plasmon resonance (SPR), preferably at 25° C., preferably performed on a ProteOn XPR36 instrument.

[0205] The inventors have demonstrated that the affinities (K D ) according to the present technology have also been found to have similar or higher potency than the reference ISVD when tested in cell killing assays.

[0206] Surprisingly, the inventors have found that the affinity (K ) of the reference ISVD T017000700 (SEQ ID NO: 1) for binding to human TCR is 40-fold less than that of the reference ISVD T017000700 (SEQ ID NO: 1). DWe have found a subset of ISVDs of this technology that are substituted with an E at position 61 and a Q, S, or T at position 99 (Kabat numbering), yet maintain high potency in cell killing assays. These specific ISVDs, which have an E at position 61 and a Q, S, or T at position 99, present interesting opportunities for the creation of biotherapeutics with better biodistribution.

[0207] In one embodiment, the ISVD of the present technology has improved cross-reactivity for binding to human and non-human primate TCRs compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969). Thus, in certain embodiments, the ISVD of the present technology has improved k for binding to human TCRs. off k for binding to non-human primate TCRs within a 5-fold range of off It has.

[0208] In another embodiment, the ISVD of the present technology has the same or a lower K as compared to SEQ ID NO: 1, preferably as measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. D In another embodiment, the ISVD of the present technology binds to human TCR at the same or a lower K as compared to SEQ ID NO: 1, preferably as measured by surface plasmon resonance (SPR) preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. D In another embodiment, the ISVD of the present technology binds to a non-human primate TCR at the same or a lower K as compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), preferably as measured by surface plasmon resonance (SPR) preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. DIn another embodiment, the ISVD of the present technology binds to human TCR at the same or a lower K as compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), preferably as measured by surface plasmon resonance (SPR) preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. D binds to non-human primate TCRs.

[0209] In one embodiment, the ISVD of the present technology is preferably measured by SPR, preferably performed on a ProteOn XPR36 instrument at 25° C., and is at most about 10 for binding to human TCR. -6 M, at most about 10 -7 M, at most about 10 -8 M, at most about 10 -8 M, and at most about 10 -9 M D ), and (ii) has at most about 10 for binding to a non-human primate TCR -5 M, at most about 10 -6 M, at most about 10 -7 M, and at most about 10 -8 K selected from the group consisting of M D It has.

[0210] In one embodiment, the ISVDs of the present technology exhibit improved cross-reactivity, i.e., avidity (e.g., K) with respect to binding to human and non-human primate TCRs. D or k off The ratio of K is lower than that for ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969). D or k off is preferably determined by SPR, preferably performed at 25°C, preferably on a ProteOn XPR36 instrument.

[0211] For example, the ISVD of this technology is DPreferably, the antibody exhibits a lower human-cynomolgus monkey cross-reactivity difference compared to the human-cynomolgus monkey cross-reactivity difference in ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969) based on the above. D is determined by SPR, preferably performed at 25°C, preferably on a ProteOn XPR36 instrument.

[0212] 5.4 (in vivo) half-life extension The polypeptides of the present technology may further comprise one or more other groups, residues, moieties, or binding units, optionally linked via one or more peptidic linkers, as previously disclosed, where the one or more other groups, residues, moieties, or binding units provide the polypeptide with increased half-life (in vivo) compared to a corresponding polypeptide without the one or more other groups, residues, moieties, or binding units. Increased in vivo half-life means, for example, that the polypeptide exhibits increased half-life in a mammal, such as a human subject, after administration. Half-life can be expressed, for example, as t1 / 2 beta.

[0213] The type of group, residue, moiety, or binding unit is generally not limited and may, for example, be selected from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

[0214] More specifically, the one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life can be selected from the group consisting of binding units capable of binding to serum albumins such as human serum albumin or serum immunoglobulins such as IgG, preferably binding units capable of binding to human serum albumin. The binding unit is preferably an ISVD.

[0215] For example, WO 04 / 041865 describes an ISVD that binds to serum albumin (specifically human serum albumin) that can be linked to other proteins (e.g., one or more other ISVDs that bind to a desired target) to increase the half-life of the protein.

[0216] International application WO 06 / 122787 describes several ISVDs for (human) serum albumin, including the ISVD designated Alb-1 (SEQ ID NO: 52 in WO 06 / 122787) and its humanized variants such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787), which again can be used to extend the half-life of therapeutic proteins and polypeptides and other therapeutic entities or moieties.

[0217] Moreover, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0218] In one embodiment of the present technology, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23, preferably Alb-8 or Alb-23 or a variant thereof, as set forth on pages 7 to 9 of WO 2012 / 175400, and an albumin binder described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, or WO 2018 / 134234.

[0219] In another embodiment, the polypeptide comprises a serum albumin binding moiety selected from Table A-3.

[0220] [Table 8]

[0221] [Table 9]

[0222] When such an ISVD that binds human serum albumin is at the C-terminal position, it can exhibit a C-terminal alanine (A) or glycine (G) stretch, preferably A. In some embodiments of the present technology, the ISVD that binds human serum albumin is at a position other than the C-terminal position (i.e., it is not the C-terminal ISVD of the polypeptide).

[0223] 5.5 Nucleic acid molecules Also provided are nucleic acid molecules encoding the ISVDs or polypeptides disclosed herein.

[0224] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, e.g., for the expression and / or production of polypeptides. Suitable hosts or host cells for production purposes will be apparent to those skilled in the art. They can, for example, be any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. (Non-human) hosts or host cells comprising a nucleic acid encoding a polypeptide are also encompassed by the present technology.

[0225] The nucleic acid can be, for example, DNA, RNA, or a hybrid thereof, and can also contain (e.g., chemically) modified nucleotides such as PNA. It can be single-stranded or double-stranded, and is preferably in the form of double-stranded DNA. For example, the nucleotide sequence can be genomic DNA or cDNA.

[0226] Nucleic acids can be prepared or obtained in a manner known per se and / or isolated from a suitable natural source. Nucleic acid sequences encoding naturally occurring (poly)peptides can be subjected to, for example, site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. It will also be clear to those skilled in the art that, to prepare nucleic acids, several nucleotide sequences, for example at least one nucleotide sequence encoding a targeting moiety and, for example, nucleic acids encoding one or more linkers, can also be linked together in a suitable manner.

[0227] Techniques for generating nucleic acids will be apparent to those skilled in the art. Examples may include, but are not limited to, automated DNA synthesis, site-directed mutagenesis, combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof), introducing mutations that result in expression of truncated expression products, introducing one or more restriction sites (e.g., creating cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes), and / or introducing mutations using a PCR reaction with one or more "mismatched" primers.

[0228] 5.6 Vectors Also provided are vectors comprising nucleic acid molecules encoding the ISVDs or polypeptides disclosed herein.

[0229] As used herein, a "vector" is a vehicle suitable for transporting genetic material into a cell. Vectors include naked nucleic acids such as plasmids or mRNA, or nucleic acids embedded within larger structures such as liposomes or viral vectors.

[0230] A vector generally comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. The vector is preferably an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g., when the vector is introduced into a (human) cell. In DNA-based vectors, this usually includes the presence of elements for transcription (e.g., promoter and polyA signal) and elements for translation (e.g., Kozak sequence).

[0231] Preferably, in a vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. By way of example, a promoter is considered to be "operably linked" to a coding sequence if it is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (in which case the coding sequence should be understood to be "under the control of" the promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They will also usually be essentially contiguous, although this may not be required.

[0232] Preferably, any regulatory elements of the vector are capable of providing their intended biological function in the intended host cell or host organism.

[0233] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, that is, for example, the promoter should be capable of initiating or otherwise controlling / regulating the transcription and / or expression of a nucleotide sequence, such as a coding sequence, to which it is operably linked.

[0234] 5.7 Composition The present technology also provides compositions comprising at least one ISVD or polypeptide disclosed herein, at least one nucleic acid molecule encoding an ISVD or polypeptide disclosed herein, or at least one vector comprising such a nucleic acid molecule. The compositions may be pharmaceutical compositions. The compositions may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient, and / or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and / or compounds.

[0235] 5.8 Host organisms The present technology also relates to host cells or host organisms comprising an ISVD or polypeptide disclosed herein, a nucleic acid encoding an ISVD or polypeptide disclosed herein, and / or a vector comprising a nucleic acid molecule encoding an ISVD or polypeptide disclosed herein.

[0236] Suitable host cells or host organisms will be apparent to those skilled in the art and may be, for example, any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli, or Pichia pastoris. The most preferred host is Pichia pastoris.

[0237] 5.9 Methods and Uses of ISVDs and Polypeptides The present technology also provides methods for producing the ISVDs or polypeptides disclosed herein. The methods may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the ISVD or polypeptide, expressing the ISVD or polypeptide in the host, optionally followed by one or more isolation and / or purification steps. Specifically, the methods include: a) expressing a nucleic acid sequence encoding an ISVD or polypeptide according to the present technology in a suitable host cell or (non-human) host organism or in another suitable expression system, optionally followed by b) isolating and / or purifying the polypeptide; may include:

[0238] Suitable host cells or host organisms for production purposes will be apparent to those skilled in the art. For example, they may be any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli, or Pichia pastoris. The most preferred host is Pichia pastoris.

[0239] Typically, the multispecific, multivalent polypeptides of the present technology combine high-affinity antigen recognition on target cells with T cell activation, resulting in activation independent of the T cell's natural specificity. The first ISVD of the polypeptide of the present technology has high affinity for / specifically binds to an effector cell, preferably a TCR of said effector cell, even more preferably a constant domain of the TCR.

[0240] The effector cell is a cell comprising a TCR complex, preferably an immune cell, such as a T cell, preferably a CD4+ T helper cell (also known as a CD4 cell, T helper cell, or T4 cell), more preferably a cytotoxic T cell (also known as a TC cell, CTL, or CD8+ T cell) or a natural killer T cell (NKT cell). In some embodiments, the cell is present in vivo. In some embodiments, the cell is present in vitro. The effector cells of the present technology particularly relate to mammalian cells, preferably primate cells, and even more preferably human cells.

[0241] As used herein, "T cell activation" refers to one or more cellular responses of T cells, e.g., cytotoxic T cells, selected from the following: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis. Proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis. The multispecific multivalent polypeptides of the present technology are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art and described herein, e.g., as described in WO 99 / 54440 or Schlereth et al. 2005 (Cancer Immunol. Immunother. 20:1-12), or as illustrated in the Examples or below.

[0242] In one embodiment, the present technology relates to a multispecific, multivalent polypeptide described herein, wherein the polypeptide induces T cell activation. Preferably, the polypeptide of the present technology induces T cell activation only when the second and / or additional ISVDs are bound to an antigen on a target cell.

[0243] In one embodiment, the technology relates to a multispecific, multivalent polypeptide as described herein, wherein said T cell activation depends on presenting said polypeptide bound to said first antigen on a target cell to a T cell.

[0244] T cell activation by the polypeptides of the present technology can be monitored by upregulation of CD69, CD25, and various cell adhesion molecules, de novo expression and / or release of cytokines (e.g., IFN-γ, TNF-α, IL-6, IL-2, IL-4, and IL-10), upregulation of granzyme and perforin expression, and / or cell proliferation, membrane blebbing, activation of caspase 3 and / or 7, nuclear DNA fragmentation, and / or cleavage of the caspase substrate poly(ADP-ribose) polymerase. Preferably, redirected lysis of target cells by multispecific, multivalent polypeptides is independent of T cell receptor specificity, the presence of MHC class I and / or β2-microglobulin, and / or the presence of any costimulatory stimuli.

[0245] In one embodiment, the technology relates to the multispecific, multivalent polypeptides described herein, wherein said T cell activation is independent of MHC recognition.

[0246] The multispecific, multivalent polypeptides of this technology are capable of inhibiting previously unstimulated peripheral polyclonal CD8 + and CD4 +The present invention demonstrates in vitro redirected lysis by positive T cells. Target cell redirection lysis via T cell recruitment by the polypeptides of the present technology involves cytolytic synapse formation and delivery of perforin and granzymes. Cell lysis by T cells is described, for example, in Atkinson and Bleackley 1995 (Crit. Rev. Immunol 15(3-4):359-384). Preferably, the engaged T cells are capable of sequential target cell lysis and are not affected by immune escape mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation (see, for example, WO 2007 / 042261). In vitro redirected lysis is observed at low picomolar concentrations, suggesting that very few polypeptides of the present technology need to bind to target cells to trigger T cells. Therefore, the present technology relates to potent polypeptides. Preferably, the multispecific, multivalent polypeptides of the present technology mediate killing of target cells, e.g., cancer cells, e.g., T cell stimulation with pore formation and delivery of pro-apoptotic components of cytotoxic T cell granules.

[0247] In one embodiment, the technology relates to the multispecific multivalent polypeptides described herein, wherein the T cell activation elicits one or more cellular responses of the T cells, the cellular responses being selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, expression of activation markers, and redirected target cell lysis.

[0248] As used herein, the term "potency" refers to a measure of the biological activity of an agent, such as a polypeptide or ISVD. The potency of an agent can be determined by any suitable method known in the art, such as, for example, those described in the Experimental Section. Cell culture-based potency assays are often the preferred format for determining biological activity because they measure a physiological response elicited by the agent and can generate results in a relatively short time. Various types of cell-based assays based on the mechanism of action of the product can be used, including, but not limited to, proliferation assays, cytotoxicity assays, cell killing assays, reporter gene assays, cell surface receptor binding assays, assays measuring the induction / inhibition of functionally essential proteins or other signaling molecules (e.g., phosphorylated proteins, enzymes, cytokines, cAMP, etc.), the Ramos B cell depletion model, and T cell-mediated tumor cell killing assays (as shown, for example, in the Examples Section), all of which are well known in the art.

[0249] In one embodiment, the multispecific, multivalent polypeptides of the present technology showed improved potency and efficacy in both human and cynomolgus T cell-mediated killing assays compared to the same format in which the first ISVD was replaced by T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969).

[0250] "Efficacy" (of a polypeptide of the present technology) is a measure of its maximum effective strength at saturating polypeptide concentrations. Efficacy represents the maximum response achievable by a polypeptide of the present technology. It refers to the ability of a polypeptide to produce a desired (therapeutic) effect.

[0251] Thus, in one embodiment, the present technology relates to a multispecific, multivalent polypeptide as described herein, wherein said T cell activation causes greater than about 10%, such as 20%, 30%, or 40%, or even greater than 50%, such as greater than 60%, such as 70%, 80%, or even greater than 90%, such as 100%, inhibition of said target cell activity, e.g., slowing or minimizing spread of target cells, inhibiting or slowing growth and / or proliferation of target cells, and / or killing target cells (e.g., causing regression of the disorder and / or symptoms). In particular embodiments, T cell activation causes greater than about 10%, such as 20%, 30%, or 40%, or even greater than 50%, such as greater than 60%, lysis of target cells.

[0252] In one embodiment, the multispecific, multivalent polypeptides described herein have at most about 10 -9 M, at most about 10 -10 M, and at most about 10 -11 M causes lysis of target cells by human T cells at an EC50 value selected from the group consisting of M. By way of example, the EC50 is determined in a flow cytometry-based assay as described in Examples 8 or 9 or an impedance-based cytotoxicity assay as described in Example 10.

[0253] In one embodiment, the multispecific, multivalent polypeptides described herein have at most about 10 -9 M, and at most about 10 -10 M, and at most about 10 -11 M causes lysis of target cells by non-human primate T cells at an EC50 value selected from the group consisting of M. For example, the EC50 is determined in a flow cytometry-based assay as described in Example 8 or 9, or an impedance-based cytotoxicity assay as described in Example 10.

[0254] Multispecific, multivalent polypeptides comprising a TCR-binding ISVD according to the present technology having an E at position 61 and a Y or S at position 99 have shown particularly high potency in flow cytometry-based T cell-mediated killing assays. Thus, in one embodiment, the multispecific, multivalent polypeptide comprises a TCR-binding ISVD having an E at position 61 and a Y or S at position 99. In one embodiment, the multispecific, multivalent polypeptide has a TCR-binding ISVD of at most about 5.10 -10 M, at most 10 -10 M, say at most about 5.10 -11 In one embodiment, the multispecific, multivalent polypeptide comprises a TCR-binding ISVD having an E at position 61 and a Y, Q, T, or S at position 99, and an EC50 value of at most about 5.10 M, wherein the EC50 value is determined in a flow cytometry-based T cell-mediated killing assay or an impedance-based T cell-mediated killing assay. -10 M, at most about 10 -10 M, say at most 5.10 -11 M causes lysis of target cells by human T cells at an EC50 value (determined by a flow cytometry-based T cell-mediated killing assay or an impedance-based T cell-mediated killing assay).

[0255] In one embodiment, the multispecific, multivalent polypeptide described herein is capable of activating human and / or non-human primate T cells to lyse target cells with an improved (lower) EC50 value compared to the same polypeptide in which the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969).

[0256] In one embodiment, the multispecific, multivalent polypeptide of the present technology causes lysis of target cells by human T cells with an improved (lower) EC50 value than that of the same polypeptide in which the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), as determined in a T cell-mediated killing assay. By way of example, the EC50 is determined as specified in the Examples section.

[0257] In one embodiment, the multispecific, multivalent polypeptide of the present technology causes lysis of target cells by non-human primate T cells with an improved (lower) EC50 value than that of the same polypeptide in which the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), as determined in a T cell-mediated killing assay. By way of example, the EC50 is determined as specified in the Examples section.

[0258] In one embodiment, the multispecific multivalent polypeptide described herein comprising a first ISVD of the present technology exhibits improved binding to the constant domains of human TCRs and / or non-human primate TCRs compared to the same polypeptide wherein the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), i.e., a polypeptide comprising as the first ISVD an ISVD having the CDR sequences of T0170056G05.

[0259] The binding properties of the ISVD of this technology are discussed in more detail below (Section 5.3 "Specificity").

[0260] In some embodiments, the multispecific, multivalent polypeptides of the present technology have an affinity for binding to a human TCR of at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s-1 , and at least about 10 5 M -1 s -1 an on-rate constant (k on )

[0261] In some embodiments, the multispecific, multivalent polypeptides described herein have an affinity for at least about 10 for binding to a non-human primate TCR. 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 k selected from the group consisting of on It has.

[0262] In some embodiments, the multispecific, multivalent polypeptides of the present technology have an affinity for binding to a human TCR of at most about 10 -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , and at most about 10 -4 s -1 an off-rate constant (k off )

[0263] In some embodiments, the multispecific, multivalent polypeptides described herein have a binding affinity of at most about 10 for binding to a non-human primate TCR. -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , and at most about 10 -4 s -1 k selected from the group consisting of off It has.

[0264] In some embodiments, the multispecific, multivalent polypeptides of the present technology have an affinity for binding to a human TCR of at most about 10 -5 M, at most about 10 -6 M, at most about 10 -7 M, at most about 10 -8 M, and at most about 10 -9 M D )

[0265] In some embodiments, the multispecific, multivalent polypeptides of the present technology have at most about 10 binding affinity to non-human primate TCRs. -5 M, at most about 10 -6 M, at most about 10 -7 M, and at most about 10 -8 K selected from the group consisting of M D It has.

[0266] In one embodiment, k on , k off , or K D is measured by surface plasmon resonance (SPR). For example, k on , k off , or K D is determined as specified in the Examples section.

[0267] In another embodiment, k on , k off , or K D Measured by biolayer interferometry (BLI).

[0268] In one embodiment, the multispecific, multivalent polypeptide of the present technology has an improved (lower) K than that of the same polypeptide where the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969). D It binds to human TCR.

[0269] In one embodiment, the multispecific, multivalent polypeptide of the present technology has an improved (lower) K than that of the same polypeptide where the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969). D binds to non-human primate TCRs.

[0270] The described polypeptides, nucleic acid molecules or vectors, or compositions comprising the ISVD or polypeptides, nucleic acid molecules or vectors - preferably the polypeptides or compositions comprising same - are useful as pharmaceutical agents.

[0271] Thus, the present technology provides a described polypeptide, nucleic acid molecule, or vector, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector, for use as a pharmaceutical agent.

[0272] Also provided is a polypeptide, nucleic acid molecule, or vector described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector, for use in preventing, treating, or ameliorating a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease.

[0273] Additionally provided is a composition comprising a polypeptide, nucleic acid molecule, or vector, or an ISVD or a polypeptide, nucleic acid molecule, or vector, described herein, for use in treating cancer.

[0274] Also provided are methods for preventing, treating, or ameliorating disease, said methods comprising administering to a subject in need thereof a pharmaceutically active amount of a polypeptide, nucleic acid molecule, or vector described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector.

[0275] Further provided is a method for preventing, treating, or ameliorating a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide, nucleic acid molecule, or vector described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector.

[0276] Additionally provided are methods for treating cancer, the methods comprising administering to a subject in need thereof a pharmaceutically active amount of a polypeptide, nucleic acid molecule, or vector described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector.

[0277] Further provided is the use of a polypeptide, nucleic acid molecule, or vector, or a composition comprising a polypeptide, nucleic acid molecule, or vector, described herein, in the preparation of a pharmaceutical agent.

[0278] Also provided is the use of a polypeptide, nucleic acid molecule, or vector, or a composition comprising a polypeptide, nucleic acid molecule, or vector described herein, in the preparation of a pharmaceutical agent for the prevention, treatment, or amelioration of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease.

[0279] Further provided is the use of a polypeptide, nucleic acid molecule or vector, or a composition comprising a polypeptide, nucleic acid molecule or vector, as described herein, in the preparation of a pharmaceutical composition, preferably for treating cancer.

[0280] A "subject" as referred to in the context of the present technology can be any animal, preferably a mammal. Among mammals, it is possible to distinguish between humans and non-human mammals. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., bovine, equine, ovine, caprine, or porcine animals), or animals commonly used for research purposes and / or antibody production (e.g., mice, rats, rabbits, cats, dogs, goats, ovine, equines, porcines, non-human primates such as cynomolgus monkeys, or camelids such as llamas or alpacas).

[0281] In the context of prophylactic and / or therapeutic purposes, the subject can be any animal, more particularly any mammal, but is preferably a human subject.

[0282] As used herein, the terms "treat," "treatment," and "treating," in the context of administering one or more therapies to a subject, refer to the reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., a disorder associated with cancer, and / or the amelioration of one or more symptoms thereof that occur as a result of the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In specific embodiments, the terms "treat," "treatment," and "treating," in the context of administering one or more therapies to a subject, refer to a reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, and refer to a reduction in cancer cells of at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to a control (e.g., a negative control such as phosphate buffered saline). In another embodiment, the terms "treat," "treatment," and "treating," in the context of administering one or more therapies to a subject, refer to reducing or ameliorating the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, with no change in cancer cell count, reduced length of hospital stay, reduced mortality rate, or increased survival of a cancer subject.

[0283] Substances (including polypeptides, nucleic acid molecules, and vectors) or compositions may be administered to a subject by any suitable route of administration, for example, enteral (e.g., oral or rectal) or parenteral (e.g., epicutaneous, sublingual, buccal, intranasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration. Parenteral administration, such as intramuscular, subcutaneous, or intradermal administration, is preferred. Subcutaneous administration is most preferred.

[0284] To provide the intended therapeutic result, an effective amount of the described polypeptide, nucleic acid molecule, or vector, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule, or vector, can be administered to a subject.

[0285] It can be administered one or more times. When administered two or more times, multiple administrations can be performed at suitable intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule, or vector.

[0286] [Table 10]

[0287] [Table 11]

[0288] [Table 12]

[0289] [Table 13]

[0290] [Table 14]

[0291] [Table 15]

[0292] [Table 16]

[0293] [Table 17]

[0294] [Table 18]

[0295] [Table 19]

[0296] [Table 20]

[0297] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art and will be apparent to those of ordinary skill in the art. See, e.g., Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual (2 nd Ed.) Vols.1-3, Cold Spring Harbor Laboratory Press), F. Ausubel et al. (1987, Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin (1985, Genes II, John Wiley & Sons, New York, NY), Old et al. (1981, Principles of Gene Manipulation: An Introduction to Genetic Engineering (2) nd Ed.)University of California Press,Berkeley,CA), Roitt et al.(2001,Immunology(6) th Ed.)Mosby / Elsevier,Edinburgh), Roitt et al.(2001,Roitt's Essential Immunology(10 thEd.) Blackwell Publishing, UK) and Janeway et al. (2005, Immunobiology (6 th See standard handbooks such as (Ed.) Garland Science Publishing / Churchill Livingstone, New York), as well as the general background art cited therein.

[0298] Unless otherwise indicated, all methods, steps, techniques, and operations not specifically described in detail can be and have been carried out in a manner known per se, as will be apparent to those skilled in the art. See, for example, again, the standard handbooks and the general background art cited herein and the further references cited therein, as well as the following reviews describing, for example, protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins: Presta (2006, Adv. Drug Deliv. Rev. 58(5-6):640-56), Levin and Weiss (2006, Mol. Biosyst. 2(1):49-57), Irving et al. (2001, J. Immunol. Methods 248(1-2):31-45), Schmitz et al. (2000, Placenta 21 Suppl. A:S106-12), Gonzales et al. (2005, Tumor Biol. 26(1):31-43).

[0299] The term "sequence" as used herein (e.g., terms such as "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "VHH sequence," or "protein sequence") should generally be understood to include both the related amino acid sequence as well as the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

[0300] "Amino acid sequence" is taken to mean a single amino acid or an unbranched sequence of two or more amino acids, depending on the context. A nucleotide sequence is taken to mean an unbranched sequence of three or more nucleotides.

[0301] The amino acids are L-amino acids commonly found in naturally occurring proteins and are listed in Table B-1. Amino acid sequences containing D-amino acids are not intended to be encompassed by this definition. Any amino acid sequence containing post-translationally modified amino acids may be written as the initially translated amino acid sequence using the symbols shown in Table B-1, along with positions of modifications such as hydroxylations and glycosylations, but such modifications shall not be explicitly shown in the amino acid sequence. Any peptide or protein capable of being represented as sequence modified linkages, bridges and end caps, non-peptidyl bonds, etc., is also encompassed by this definition.

[0302] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made up of a linear chain of two or more amino acids. The compound may have 10 or more amino acids, 25 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, or even 300 or more amino acids. Those of skill in the art will recognize that while polypeptides generally contain fewer amino acids than proteins, there is no art-recognized cutoff point for the number of amino acids that distinguishes a polypeptide from a protein; that polypeptides can be made by chemical synthesis or recombinant methods; and that proteins are generally made in vitro or in vivo by recombinant methods known in the art.

[0303] Unless the context clearly requires otherwise, throughout this specification and claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive, rather than an exclusive or exhaustive, sense, i.e., "including, but not limited to."

[0304] By way of example, when a nucleotide sequence, an amino acid sequence or a polypeptide is said to comprise or "consist essentially of" another nucleotide sequence, amino acid sequence or polypeptide, respectively, this can mean that the latter nucleotide sequence, amino acid sequence or polypeptide is incorporated into the first-mentioned nucleotide sequence, amino acid sequence or polypeptide, respectively, but more generally it means that the first-mentioned nucleotide sequence, amino acid sequence or polypeptide, respectively, comprises within its sequence a stretch of nucleotides or amino acid residues which has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, regardless of how the first-mentioned sequence was actually generated or obtained (which may for example be by any suitable method described herein). By way of non-limiting example, when a polypeptide of the present technology is said to comprise an immunoglobulin single variable domain, this can mean that said immunoglobulin single variable domain sequence is incorporated into the sequence of the polypeptide of the present technology, but more generally, it generally means that the polypeptide of the present technology contains within its sequence the sequence of an immunoglobulin single variable domain, regardless of how said polypeptide of the present technology was generated or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first-mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed as an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, the latter nucleotide sequence is in the same reading frame as the first-mentioned larger nucleic acid or nucleotide sequence).

[0305] When an amino acid sequence or polypeptide is said to "consist essentially of" an immunoglobulin single variable domain, it means that either the amino acid sequence or polypeptide is exactly identical to an immunoglobulin single variable domain, or corresponds to a polypeptide or amino acid sequence having a limited number of amino acid residues added to the amino terminus, the carboxy terminus, or both the amino terminus and the carboxy terminus of an immunoglobulin single variable domain, for example 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, for example 1, 2, 3, 4, 5, or 6 amino acid residues. When "consisting of" is used, it means that the amino acid sequence or polypeptide is exactly identical to an immunoglobulin single variable domain.

[0306] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the method described herein.

[0307] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to every element in the series. Many equivalents to the specific embodiments of the technology described herein will be known to those skilled in the art, or will be ascertainable using no more than routine experimentation. Such equivalents are intended to be encompassed by the technology.

[0308] The term "and / or" wherever used herein includes the meaning of "and", "or", and "all or any other combination of elements connected by said term".

[0309] As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range. [Example]

[0310] 6.1 Example 1: T Cell Engaging (TCE) ISVD Occurrence and Off-Rate Determination Variant occurrence Amino acids at positions 61, 99, and 103 (Kabat numbering) of ISVD T017000700 (SEQ ID NO: 1) were substituted to generate various variants that maintained function and overall generative potential but reduced or eliminated isomerization at position 61 and eliminated oxidation at sites 99 and / or 103 (Kabat numbering).

[0311] ISVD T017000700 and occurring variant ISVDs are shown above in Table A-4. All ISVDs were tagged with a FLAG-HIS tag (FLAG3-HIS6, SEQ ID NO: 77) for further experimental analysis.

[0312] Off-rate determination for human TCRαβ-zipper protein Binding of TCE ISVD variants to TCRαβ was assessed by determining the dissociation rate constant (kd) for recombinant human TCRαβ-zipper protein using an SPR-based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.) or an SPR-32 instrument (Bruker Daltonics SPR).

[0313] Setting up the ProteOn XPR36 Binding of crude extract monovalent TCE-FLAG3-HIS6 ISVD constructs to human TCRαβ-zipper protein (in-house produced huTCR(2XN9)-zipper) was probed by surface plasmon resonance (SPR) (Bio-Rad Laboratories, Inc., ProteOn XPR36). Targets were immobilized onto GLC sensor chips (short matrix, normal volume) using standard amine coupling chemistry. Crude extracts of ISVD constructs were injected at a 1:10 dilution, and binding parameters were assessed (in duplicate). Each ISVD was injected for 120 s, and dissociation was assessed for 600 s.

[0314] Data were double-referenced by subtracting the reference analyte lane and blank buffer injection. The off-rate constant (kd) for each interaction was calculated using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc., Version 3.1.0.6) applying a Langmuir 1:1 interaction model.

[0315] Setting up the SPR-32 Binding of crude extract monovalent TCE-FLAG3-HIS6 ISVD constructs to human TCRαβ-zipper protein (in-house produced huTCR(2XN9)-zipper) was probed by surface plasmon resonance (SPR) (Bruker Daltonics SPR, SPR-32). Targets were immobilized on high-capacity amine (HCA) sensor chips using standard amine coupling chemistry. Crude extracts of ISVD constructs were injected at a 1:10 dilution to assess binding parameters. Each ISVD was injected for 120 s, and dissociation was assessed for 600 s.

[0316] Data were double-referenced by subtracting a reference spot and a blank buffer injection. The off-rate constant (kd) for each interaction was calculated using the SPR-32 Analyser Software (Bruker Daltonics SPR, Version 3.2.0.19) applying a Langmuir 1:1 interaction model.

[0317] The results of the measurements can be seen in Tables 1-3.

[0318] [Table 21]

[0319] As can be seen from Table 1, binding of TCE ISVD variants with substitutions at position 61 (Kabat numbering) to the human TCRαβ-zipper protein is maintained at levels very similar to the reference (T017000700) for all tested constructs, i.e., constructs with amino acid residues A, E, F, H, I, K, L, N, P, Q, R, S, T, V, Y at position 61, as assessed by SPR-based off-rate determination.

[0320] [Table 22]

[0321] As can be seen in Table 2, binding of TCE ISVD variants with substitutions at position 99 (Kabat numbering) to the human TCRαβ-zipper protein was tested in constructs where residue 61 was fixed as glutamic acid (E). The variants with residues Y, A, D, E, F, G, H, I, K, L, P, Q, R, S, T, and V at position 99 maintained detectable levels of binding to the human TCRαβ-zipper protein but with approximately up to 100-fold faster off-rates when compared to the reference T017000700.

[0322] [Table 23]

[0323] As can be seen from Table 3, binding of TCE ISVD variants with substitutions at Kabat position 103 to the human TCRαβ-zipper protein, as assessed by SPR-based off-rate determination, is maintained at levels very similar to reference T017000700 for constructs with amino acid residues A, E, F, H, I, K, L, Q, R, S, T, V, Y at position 103, whereas no binding was observed for constructs with amino acid residues G or P at position 103.

[0324] Based on the results of the SPR analysis, it is concluded that it is possible to obtain a functional ISVD that binds to TCR when substituting one or more amino acids at positions 61, 99, and 103 (Kabat numbering) of the sequence of reference T017000700.

[0325] Several developmental ISVDs were selected and further analyzed in other experiments, which are discussed below.

[0326] 6.2 Example 2: Determination of Biophysical Properties (Tm, Tag, and Oligomerization) of TCE ISVD Variants Determination of melting temperature by thermal shift assay (TSA) Thermal shift assays (TSA) were performed in 96-well plates using a LightCycler 480II machine (Roche). One ISVD per row was analyzed at the following pH values: 4, 5, 6, 7, 8, and 9. Five µL of ISVD sample (0.8 mg / mL in PBS) per well was added to 5 µL of Sypro Orange (40x in MilliQ water, Invitrogen catalog no. S6551) and 10 µL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, and 115 mM NaCl, pH range 3.5-9). An applied temperature gradient (37 to 99°C at a rate of 0.03°C / s) induces unfolding of the ISVD, thereby exposing hydrophobic patches. Binding of Sypro Orange to these hydrophobic patches leads to an increase in fluorescence intensity (Ex / Em = 465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 serves as a measure of the melting temperature.

[0327] Determination of aggregation initiation temperature (Tagg assay) The temperature at which ISVD proteins initiate aggregation (= aggregation onset temperature = Tagg) was determined by dynamic light scattering (DLS) using a DynaPro plate reader (Wyatt). To this end, ISVDs bearing a 3xFLAG-HIS6 tag (SEQ ID NO: 77) were produced in E. coli, purified via IMAC followed by preparative SEC, filtered (0.22 μm), and used at a concentration of 1 mg / mL (D-PBS). After thawing, samples were filtered through a 0.1 μm membrane and centrifuged at 14,000 rpm for 5 min. While continuously recording the light scattering intensity, 30 μL samples (four replicates) were heated from 40 to 80°C at a constant rate of 0.25°C / min. The hydrodynamic radius derived from the measured intensity was plotted against temperature to determine the temperature at which the radius begins to increase (= Tagg°C).

[0328] Oligomerization assay Analytical size-exclusion chromatography (SE-HPLC) was used to investigate the oligomerization tendency of monovalent ISVDs under stress conditions (45°C for 1 week). To this end, ISVDs bearing a 3xFLAG-HIS6 tag were produced in E. coli, purified via IMAC followed by preparative SEC, filtered (0.22 μm), and used at a concentration of 1 mg / mL (DPBS). Two 100 μL aliquots were incubated: one sample at -20°C for 1 week and the other at 45°C for 1 week, and the SE-HPLC profiles were compared. Samples were clarified by centrifugation at 20,000 RCF for 5 min and subsequently analyzed on an Acquity UPLC BEH200 SEC system (mobile phase: 750 mM L-arginine·HCl + 10 mM phosphate pH 7.0, flow rate: 0.4 mL / min). The difference in relative pre-peak area between the stressed (+45°C) and unstressed (-20°C) samples was calculated and reported as Δ% Oligo (=% Oligo1W45°C-% OligoT0).

[0329] result Purified variants of TCE ISVD (see Table A-4) carrying selected amino acid substitutions at positions 61, 99, and 103 (Kabat numbering) were characterized by melting temperature (Tm), aggregation onset (Tagg), and oligomerization after 1 week at 45°C, as illustrated in Tables 4 and 5.

[0330] At position 61 of the TCE ISVD, the amino acid residue variants A, E, P, Q, R, S, and V were chosen because these are the most frequently occurring residues at this position in human VH genes.

[0331] At position 99 of the TCE ISVD, amino acid residue variants A, H, Q, S, T, and Y were selected. Y was selected because it was the residue that had the least effect on the off-rate of each ISVD variant for binding to TCRαβ (Table 2). Residues A, H, Q, S, and T were selected because each TCE ISVD exhibited a different range of off-rates for binding to TCRαβ.

[0332] Approximately 95% of human VH genes and naturally occurring ISVDs (in-house sequence analysis) have a tryptophan (W) residue at Kabat position 103. Other residues that can be found at position 103 in naturally occurring ISVDs are Y, R, and S. Therefore, we selected Y, R, and S as amino acid residue variants at position 103.

[0333] [Table 24]

[0334] [Table 25]

[0335] As can be seen from Table 4, T017000978, T017000992, T017000999, and T017001002 maintained or improved all three properties compared to the reference T017000700. For the ISVDs according to the present technology, a 0°C to 1°C increase in Tm was observed, a 1°C to 5°C increase in Tagg was observed, and a 0.2% to 0.0% or 0.1% decrease in oligomeric fraction was observed relative to the reference.

[0336] Surprisingly, as can be seen in Table 5, even though TCR binding of the ISVD variants at position 103 was not affected, substitutions at this position resulted in a 7.3°C to 15.1°C decrease in melting temperature and a 7°C to 20°C decrease in aggregation onset temperature when compared to the reference ISVD, suggesting that TCE W103 may be important for the biophysical stability of the ISVD.

[0337] Therefore, going forward, it was decided to keep position 103 a tryptophan while varying the amino acid residues at positions 61 and 99.

[0338] 6.3 Example 3: Affinity Determination of TCE ISVD Variants for Human and Cynomolgus TCRαβ Proteins Surface plasmon resonance (SPR) (Bio-Rad Laboratories, Inc., ProteOn XPR36) was used to probe the binding of purified monovalent TCE-FLAG3-HIS6 and TCE-HIS6 ISVD constructs to human TCRαβ-zipper protein (in-house produced huTCR(2XN9)-zipper) and cynomolgus monkey TCRαβ-zipper protein (in-house produced cyTCR(AEA41865)-zipper). Both targets were immobilized on a GLC sensor chip (short matrix, normal volume) using standard amine coupling chemistry. Six different concentrations of ISVD constructs were injected in multi-cycle kinetic (MCK) experiments. Each ISVD concentration was injected for 120 s, and dissociation was assessed for 600 s.

[0339] Data were double-referenced by subtracting the reference analyte lane and blank buffer injection. Affinity constants (k, k, and K) were calculated using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc., Version 3.1.0.6) applying a Langmuir 1:1 interaction model. D ) was calculated.

[0340] The results of affinity measurements of TCE ISVD amino acid variants to human and cynomolgus TCRαβ-zipper proteins are summarized below in Tables 6 and 7. T017000700 was used as reference.

[0341] [Table 26]

[0342] [Table 27]

[0343] As can be seen from Tables 6 and 7, the affinities of the TCE ISVD variants remain comparable to the reference TCE ISVD (K values ​​within 2.5-fold) for all constructs tested, with the exception of T017000995, T017000999, and T017001001.

[0344] Each ISVD has a K value 2-4 times higher for cynomolgus monkey TCRαβ protein than for human TCRαβ protein. D This indicates that the ISVD according to this technology has the potential to be developed and used for human therapeutic purposes.

[0345] 6.4 Example 4: Binding of Monovalent TCE ISVD to Purified Primary Human T Cells TCE ISVDs according to the present technology were tested for binding to human T cells by flow cytometry. The following ISVDs were tested: T017000700 (reference), T017000978, T017000995, T017000999, and T017001001. Negative controls were also included. Unstained cells (US), cells stained only with GaM-PE, and cells stained only with ANTI-FLAG® M2 antibody and GaM-PE (a-FLAG+GaM-PE) were included as negative controls.

[0346] Briefly, cells were harvested and transferred to V-bottom 96-well plates (5 × 1E4 cells / well in 50 μL) and incubated with serial dilutions of TCE ISVD in FACS buffer (D-PBS (Gibco, 14190) containing 2% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)) for 3.5 hours at 4°C.

[0347] Cells were then washed three times with FACS buffer, incubated with 1 μg / mL monoclonal ANTI-FLAG® M2 antibody (Sigma F1804) for 30 min at 4°C, washed again, and incubated with 1 / 100 diluted R-phycoerythrin-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgG, Fcγ fragment specific (Jackson Immunoresearch, 115-116-071, referred to as GaM-PE) for 30 min at 4°C.

[0348] To distinguish live from dead cells, cells were then resuspended in FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642 / 661) (Life Technologies-Molecular Probes, T3605). After staining, cells were analyzed using a MACSQuant® flow cytometer (Miltenyi) with FlowLogic software. First, a P1 population was selected based on FSC-SSC distribution, accounting for more than 80% of the total cell population. TO-PRO®-3 iodide-positive (dead) cells were excluded from this population (P1), and the mean fluorescence intensity (PE) value was calculated.

[0349] The results of the measurements can be seen in Figure 1. As expected, no staining of human T cells was observed in the control condition without TCE ISVD. Additionally, as can be seen in this figure, based on the measured mean fluorescence intensity, T017000978 has staining comparable to the reference T017000700. Variants T017000995, T017000999, and T017001001 have significantly lower staining, corresponding to the binding affinities determined with the zipper proteins shown in Table 6.

[0350] 6.5 Example 5: Generation of TCE-CD123-ALB construct TCE ISVD variants were formatted as multispecific ISVD constructs with a tumor-anchoring ISVD building block directed to CD123 and a human serum albumin-directed ISVD building block linked by a 9GS linker. The TCE variant building block was placed at either the N-terminal position (position 1) or the second position (position 2), followed by an anti-CD123 building block at position 2 or position 1, respectively. In all constructs, the anti-human serum albumin building block was placed at the C-terminal position 3. In addition to substitutions at positions 61 and 99 (Kabat numbering) in the TCE building block, when the TCE building block was placed at position 1 in the multispecific construct, the first residue of the TCE building block was either maintained as E or changed to D. The E1D mutation is commonly introduced to avoid pyroglutamate formation. The resulting constructs are listed in Table 8 below. The reference construct T017001017 contained the reference TCR binding ISVD T017000700.

[0351] [Table 28]

[0352] The following TCE ISVD building blocks were used in the constructs listed above: For T017001017 and T017001027, the reference TCE ISVD T017000700 (SEQ ID NO: 1) was used. For T017001018 and T017001028, T017000978 (sequence number 37) was used. T017001019, T017001024, and T017001029 used T017001001 (SEQ ID NO: 52). T017001024 additionally contained an E1D mutation in the TCE ISVD. T017001020, T017001025, and T017001030 used T017000999 (SEQ ID NO: 50). T017001025 additionally contained an E1D mutation in the TCE ISVD. T017001021, T017001026, and T017001031 used T017000995 (SEQ ID NO: 46). T017001026 additionally contained an E1D mutation in the TCE ISVD. For T017001022, T017000975 (SEQ ID NO: 34) was used, which is T017000700 with the E1D mutation. For T017001023, T017000991 (SEQ ID NO: 42) was used, which is T017000978 with the E1D mutation.

[0353] 6.6 Example 6: Affinity Determination of TCE-CD123-ALB Constructs to Human and Cynomolgus TCRαβ-Zipper Proteins Surface plasmon resonance (SPR) (Cytiva, Biacore 8K+) was used to probe the binding of purified TCE-CD123-ALB and CD123-TCE-ALB ISVD constructs to human TCRαβ-zipper protein (in-house produced huTCR(2XN9)-zipper) and cynomolgus monkey TCRαβ-zipper protein (in-house produced cyTCR(AEA41865)-zipper). Both targets were immobilized to a CM5 sensor chip using standard amine coupling chemistry. A total of 15 ISVD constructs (Table 8) were injected at 12 different concentrations (serial dilutions from 20 μM to 0.84 nM) in a multi-cycle kinetics experiment. Each ISVD concentration was injected for 180 s, and dissociation was assessed for 600 s.

[0354] Data were double-referenced by subtracting a reference flow cell (FC) and a blank buffer injection. Affinity constants (k, k, and K D ) was calculated applying the Langmuir 1:1 interaction model using Biacore Insight Evaluation Software (Cytiva, Version 3.0.12.15655). The results of affinity measurements of 15 multivalent ISVD constructs (Table 8) for human TCRαβ protein are summarized in Table 9, and for cynomolgus monkey TCRαβ protein are summarized in Table 10.

[0355] [Table 29]

[0356] [Table 30]

[0357] Tables 9 and 10 show that the TCE ISVD variants of the present technology retain their ability to bind to TCRs when used in constructs, demonstrating that the TCE ISVD variants of the present technology can be used in combination with targeting ISVDs, and are therefore suitable for development and use in target-specific therapeutic applications.

[0358] 6.7 Example 7: Determination of Binding by TCE-CD123-ALB ISVD Constructs to Purified Primary Human T Cells Dose-dependent binding of the TCE-CD123-ALB ISVD constructs of the present technology to primary human T cells was determined using flow cytometry. The following constructs were used: T017001017 (reference), T017001018, T017001019, T017001020, and T017001021. Unstained cells (US), cells stained only with GaM-PE, and cells stained only with ABH0074 and GaM-PE (ABH0074+GaM-PE) were included as negative controls.

[0359] Briefly, cells were harvested and transferred to a V-bottom 96-well plate (5 × 1E4 cells / well in 50 μL) and incubated with serial dilutions of TCE-CD123-ALB ISVD in FACS buffer (D-PBS (Gibco, 14190) containing 2% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)) for 2.5 hours at 4°C.

[0360] Next, the cells were washed three times with FACS buffer, incubated with 10 μg / mL anti-VHH mAb (prepared in-house) for 30 min at 4°C, washed again, and incubated with 1 / 100 diluted R-phycoerythrin-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgG, Fcγ fragment specific (Jackson Immunoresearch, 115-116-071) for 30 min at 4°C. Subsequently, to distinguish live from dead cells, the cells were resuspended in FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642 / 661) (Life Techn.-Molecular Probes, T3605). After staining, the cells were analyzed using a MACSQuant® flow cytometer (Miltenyi) with FlowLogic software. Initially, the P1 population, which accounted for more than 80% of the total cell population, was selected based on FSC-SSC distribution. TO-PRO®-3 iodide positive (dead) cells were excluded from this population (P1) and the mean fluorescence intensity PE value was calculated.

[0361] The results of the measurements are shown in Figure 2. As expected, no staining of human T cells was observed in the control condition without TCE ISVD. Additionally, as can be seen in this figure, T017001018 (containing TCE ISVD T017000978) and reference T017001017 (containing TCE ISVD T017000700) bind to human T cells with similar EC50s. Meanwhile, T017001019 (containing TCE ISVD T017000995), T017001020 (containing TCE ISVD T017000999), and T017001021 (containing TCE ISVD T017001001) have lower binding affinity to human T cells. This corresponds to the binding observed for the zipper proteins shown in Table 9.

[0362] 6.8 Example 8: Multispecific TCE-CD123-ALB Constructs Induce Human T Cell-Mediated Target Cell Killing Flow cytometry-based cytotoxicity assay The present ISVD constructs were further characterized for redirected T cell-mediated killing in a flow cytometry-based cytotoxicity assay using human primary T cells as effector cells and nonadherent target cells. Target cells were labeled with 4 μM PKH26 membrane dye using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions. Effector cells (2.5 × 10 cells / well) and PKH26-labeled target cells (2.5 × 10 cells / well) were co-incubated in a 96-well V-bottom plate (Greiner Bio-one, #651 180) in the target cell line's assay medium (target growth medium with 1% penicillin / streptomycin (Life Technologies, 15140) and 30 μM Alburex HSA (CSL Behring, 2160-679) at a 10:1 effector-to-target ratio. For analysis of concentration-dependent cell lysis, serial dilutions of ISVD constructs were added to cells in target assay medium and incubated at 37°C in a 5% CO2 atmosphere for 18 h. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)). Subsequently, cells were resuspended in 100 μL FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642 / 661) (ThermoFisher Scientific, T3605) to distinguish live from dead cells. Cells were analyzed using a MACSQuant X flow cytometer (Miltenyi Biotec). A total sample volume of 70 μL was ensured per sample. Gating was set on PKH26-positive cells, and TO-PRO®-3-positive cells were determined within this population. Percent specific lysis = (% TO-PRO-3+ without construct - % TO-PRO-3+ with construct) / (% TO-PRO-3+ without construct) x 100. Assays were run in the presence of excess HSA such that the ISVD was fully saturated with HSA as described above.

[0363] result To assess the functionality of TCE ISVD variants as T cell engagers, the TCE-CD123-ALB formats listed in Table 8 were evaluated in a flow cytometry-based T cell-mediated MOLM-13 cell killing assay using human primary T cells (2 donors) in combination with a CD123-expressing human MOLM-13 target cell line in the presence of 30 μM HSA, as described above. Graphical representations of the results are shown in Figures 3A-F. The calculated EC50 values ​​for target cell killing are shown in Table 11.

[0364] [Table 31]

[0365] As can be seen from Table 11 and Figure 3, all constructs with the TCE building block in the N-terminal position (position 1) mediated potent cell killing of target cells. In addition, mutation of the first position of the TCE ISVD from glutamic acid to aspartic acid did not significantly affect the killing potency of the constructs. Furthermore, having the TCE ISVD in the N-terminal position of the construct appears to result in increased potency when compared to placing the same TCE ISVD in the second position of the construct.

[0366] Surprisingly, ISVD constructs T017001019, T017001020, and T017001021, which contain TCE ISVDs T017001001, T01700999, and T017000995, respectively, have lower affinity for TCR zipper proteins and human T cells than the other constructs, yet are highly potent in terms of target cell killing. In fact, even though the observed affinity for human TCR zipper proteins and even human T cells was 40-fold lower than the reference (see Tables 6 and 9 and Figures 1 and 2), the potency of target cell killing was not significantly different.

[0367] It has been reported that lower affinity for T cell receptors compared with affinity for tumor-associated targets is important for distribution to tumor tissue rather than to T cell-rich secondary lymphoid tissues. Preferential targeting of tumor tissue is desirable to reduce T cell-mediated target clearance.

[0368] Therefore, these specific ISVD constructs with low affinity for the TCR and high potency of target cell killing may offer unique possibilities for generating T cell-engaging biotherapeutics that maintain high potency but can be administered at lower levels due to higher tumor tissue exposure, thus reducing the risk of toxicity.

[0369] In conclusion, the TCE ISVD variants according to the present technology are highly suitable for use in constructs for target-specific therapeutic applications in humans.

[0370] 6.9 Example 9: Multispecific TCE-CD123-ALB Construct Induces Human and Cynomolgus T Cell-Mediated Target Cell Killing of KG-1a Cells To assess the ability of the TCE-CD123-ALB ISVD constructs to kill tumor cells, cytotoxicity assays were performed using isolated human or cynomolgus monkey T cells as effector cells. ISVD constructs T017001017, T017001018, T017001019, T017001020, and T017001021 were tested. T017000968 (SEQ ID NO: 75) was included as a negative control. T017000968 contains the anti-TCR ISVD T017000975 and the albumin-binding ISVD, but does not contain the anti-CD123 ISVD.

[0371] Redirecting T cell-mediated killing of CD123 target cells by TCE-CD123-ALB constructs in a flow cytometry-based assay Human T cells were collected from buffy coat blood from healthy volunteers (blood bank Gent) using RosetteSep (StemCell Technologies, 15061) and subsequently enriched with Ficoll-Paque™ PLUS (GE Healthcare, 17-1440-03) according to the manufacturer's instructions. The quality and purity of purified human T cells were checked by flow cytometric assay using anti-CD3 (eBioscience, 12-0037-73), anti-CD8 (BD Biosciences, 555367), anti-CD4 (BD Biosciences, 345771), anti-CD45RO (BD Biosciences, 555493), anti-CD45RA (BD Biosciences, 550855), anti-CD19 (BD Biosciences, 555413), anti-CD25 (BD Biosciences, 557138), and anti-CD69 (BD Biosciences, 557050) fluorescently labeled antibodies. Cells were frozen in liquid nitrogen.

[0372] T cells from cynomolgus monkeys (Macaca fascicularis) were isolated in-house (Sanofi, Montpellier, France) from PBMCs (isolated via Ficoll density centrifugation) using a pan T cell isolation kit (Miltenyi, 130-091-993) according to the manufacturer's instructions. Cells were frozen in liquid nitrogen.

[0373] Human CD123-expressing KG1a cells were labeled with 4 μM PKH-26 membrane dye using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions and used as target cells. 2.5 × 1E5 effector cells (i.e., human or cynomolgus monkey primary T cells) and 2.5 × 1E4 target cells (i.e., PKH-labeled KG1a cells) were co-incubated in a 96-well V-bottom plate (10:1 effector-to-target ratio). To measure concentration-dependent cell lysis, serial dilutions of the TCE-CD123-ALB construct were added to the cells in assay medium containing 30 μM HSA (CSL Behring, Alburex 20 human serum albumin) and incubated for 18 hours at 37°C in a 5% CO2 atmosphere.

[0374] After incubation, cells were pelleted by centrifugation and resuspended in FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642 / 661) (Life Techn.-Molecular Probes, T3605) to distinguish live from dead cells. Cells were analyzed using a MACSQuant® flow cytometer (Miltenyi). Gating was set on PKH26-positive target cells, and the percentage of TO-PRO®-3 iodide-positive cells within this population was determined.

[0375] result The results can be seen in Table 12 below and in Figure 4.

[0376] [Table 32]

[0377] As can be seen from Table 12 and Figure 4, all TCE ISVD constructs were effective in killing KG-1a cells in both human and cynomolgus T cells. As expected, the negative control construct T017000689 did not show any cell killing.

[0378] Low-affinity TCE ISVDs T017001019, T017001020, and T017001021 were also highly potent in the KG1-a cell killing assay, further supporting that such TCE constructs with low-affinity anti-TCR building blocks have significant potential as T cell-engaging biotherapeutics, as described in Example 8.

[0379] Furthermore, in all TCE-CD123-ALB formats tested, the mediated cytotoxic activity observed in each of the formats using human T cells as effector cells was very similar to that mediated by cynomolgus monkey T cells.

[0380] Therefore, this cell killing assay further supports that the TCE ISVD variants of the present technology are highly suitable for use in constructs for targeted therapeutic applications in humans.

[0381] 6.10 Example 10: Functionality of TCE-GPC3-GPC3-ALB Fusions Generation of TCE-GPC3-GPC3-ALB fusions The TCE ISVD variants were formatted as multispecific ISVD constructs by linking two tumor-anchoring ISVD building blocks directed to glypican-3 (GPC3) and one directed to human serum albumin with 5GS and 9GS linkers. The first residue of the TCE building block was changed to D. The resulting constructs are listed in Table 13 below. The reference construct (A022600427) contained the reference TCR-binding ISVD T017000975 (T017000700 with an E1D mutation, SEQ ID NO: 34), two tumor-anchoring ISVD building blocks directed to glypican-3 (GPC3), and a human serum albumin-binding ISVD. The negative control (T017000698) did not contain any anti-GPC3 ISVD building blocks.

[0382] Impedance-based cytotoxicity assay The ISVD constructs were characterized for redirected T cell-mediated killing in an impedance-based cytotoxicity assay (e.g., as described in WO2018091606A1) using primary human or cynomolgus monkey effector T cells and adherent target cells. Impedance changes induced by target cell adhesion to the electrode surface were measured using an xCELLigence instrument (Roche). T cells are nonadherent and therefore do not affect impedance measurements. The xCELLigence® RTCA MP instrument quantifies the electrical impedance change and displays it as a dimensionless parameter called the cell index, which is directly proportional to the total area of ​​the tissue culture well covered by the cells. To each well of a 96E plate (ACEA Biosciences; 05 232 368 001), 50 μL of 120 μM Alburex HSA (CSL Behring, 2160-679) was added to give a final concentration of 30 μM in assay medium (target cell growth medium + 1% penicillin / streptomycin (Life Technologies Cat#15140)). Outer wells were not used and filled with 200 μL of medium or D-PBS. The 96E plate was placed in the xCELLigence® station (37°C incubator with 5% CO2) and a single measurement was performed in the absence of cells to measure the background impedance of the assay medium. Subsequently, 50 μL of target cells in assay medium (2 × 1E4 cells / well) were seeded onto the 96E plate, and 50 μL of serially diluted ISVD construct solution (4× concentration) in assay medium was added (final volume = 200 μL). After 30 min at room temperature, 50 μL of primary T5 cells in assay medium were added per well (3×1E5 cells / well) to achieve an effector-to-target ratio of 15:1. The plate was placed in the xCELLigence® station and impedance was measured every 15 min for 4 days. Data were analyzed at fixed time points (60 h).

[0383] The generated constructs are shown in Table 13 below.

[0384] [Table 33]

[0385] result Fusion constructs containing two ISVD building blocks directed against tumor-anchored GPC3 (glypican-3) and one directed against albumin, linked by a GS linker, were compared with the TCE ISVD variant T017000991 (SEQ ID NO: 42) and the reference T017000975 (SEQ ID NO: 34). As a negative control, a construct was generated without a GPC3-binding ISVD (T017000698, Table 13). As a positive control, a GPC3-binding bispecific antibody was generated. One arm of this antibody binds to GPC3 and the other to CD3. Graphical representations of the results are shown in Figures 5A-D. Calculated IC50 values ​​for target cell killing are shown in Table 14.

[0386] [Table 34]

[0387] The IC50 values ​​of the two constructs with the TCE building block fused to the GPC3-binding ISVD are comparable when using human or cynomolgus T cells. As expected, the construct without the GPC3-binding ISVD, i.e., the TCRαβ-binding ISVD fused only to the human serum albumin-binding ISVD, did not mediate any level of cell killing or mediated only a very low level of cell killing.

[0388] 6.11 Example 11: Chemical Stability Assessment of TCE ISVD To assess the chemical stability of the TCE ISVD of the present technology, forced degradation studies were performed using T017000991 (SEQ ID NO: 42) and compared with the reference T017000975 (SEQ ID NO: 34), both of which were equipped with a HIS6 tag (SEQ ID NO: 78).

[0389] The study consisted of forced oxidation with 10 mM HO and a 4-week incubation period at both 25°C and 40°C, mimicking accelerated and stressed conditions, respectively. After the stress test, the stability of T017000991 was analyzed by peptide mapping using tryptic digestion, peptide separation by reversed-phase chromatography, and mass spectrometry MS / MS detection. The molecule was screened for the following modifications: deamidation, isomerization / racemization, and methionine or tryptophan oxidation. To assess the value of sequence optimization of T017000991, the chemical stability of the ISVD with chemical modifications was compared to that of the reference T017000975.

[0390] result The results of the peptide mapping analysis under different temperature conditions are presented in Table 15 below.

[0391] [Table 35]

[0392] No significant changes were observed under forced oxidation conditions. Sequence coverage was approximately 85% for both molecules. 100% sequence coverage was obtained for both molecules in combination with AspN digestion. No other modifications were detected.

[0393] Two chemical liabilities were identified in T017000975: D61 is susceptible to isomerization and W99 is susceptible to oxidation (Kabat numbering). As described above, substitution of these two sensitive amino acids with T017000991 (D61E, W99Y) resulted in complete elimination of these two liabilities in the TCE ISVD of this technology under the conditions evaluated.

[0394] Therefore, the chemical stability of the TCE ISVD according to the present technology is improved relative to the reference TCE ISVD.

[0395] 6.12 Example 12: Chemical Stability of TCE ISVD as Part of a Format ISVD Construct To further demonstrate the improved stability of TCE ISVDs according to the present technology, the chemical stability of T017000991 was analyzed as part of a format ISVD construct.

[0396] A short-term storage study was performed. Two anti-GPC3 ISVDs, T017000991 and T017000975 (as a control), were formatted with an ISVD that binds to human serum albumin. The constructs are shown in Table 16 below.

[0397] [Table 36]

[0398] When the format molecules were stored as liquids containing the appropriate formulation buffer, tryptophan oxidation and aspartate isomerization were observed upon storage.

[0399] At that time, the tryptophan oxidation rate was found to be dependent on pH, temperature, ISVD concentration, transition metals, and polysorbate 20. Therefore, a stress test was performed to accelerate any potential oxidation events. Stress conditions consisted of incubation in plastic tubes at 40°C / 75% relative humidity (RH) ± 5% RH for 3 months, plus the addition of 100 ppm Fe(II) as an oxidant.

[0400] In this study, both ISVD constructs were formulated at 1 mg / mL in 25 mM histidine-HCl, 8% (w / v) sucrose, 0.01% (w / v) pH 6.5 buffer, and their stability at 25°C / 60% RH ± 5% RH was also monitored over a 3-month period.

[0401] The stability profile was monitored by reversed-phase chromatography following peaks with relative retention times (RRT) <1.0 as an indication of tryptophan oxidation, and by peptide mapping after tryptic digestion and reversed-phase LC-MS / MS.

[0402] result A graphical representation of the results can be seen in Figure 6, where the stability profile at 25°C is shown by following the peak with an RRT < 1.0 by reverse phase chromatography. A reduced oxidation rate of A022600462 is observed compared to A022600424, resulting in a 5-fold reduction in tryptophan oxidation.

[0403] A similar trend can be observed when evaluating the stability profile under stress conditions (Figure 7). The addition of Fe(II) to the formulation solution catalyzed the oxidation rate of the ISVD construct in the presence of polysorbate 20 and histidine. After 1 day of incubation at 40°C, the oxidation rate of A022600462 was nearly 4-fold lower compared to the control A022600424. The lower oxidation rate was still observed after 14 days of forced degradation conditions.

[0404] Peptide mapping was used to precisely determine where in the amino acid sequence various oxidation events occur. Temperature-stress stable samples were analyzed and compared for this purpose. After trypsin digestion, the resulting peptides covered approximately 85% of the sequence. Using Biopharma Finder software (BPF; version 3.2) on a mass spectrometer (MS), it was possible to identify the various peptides and elucidate their chemical modifications. The results indicated the presence of distinct sites of tryptophan oxidation in the selected formats, namely, on the TCE ISVD located at peptide number 13, and more precisely, on W99 (A022600424) and W103 (A022600424 and A022600462). The results are shown in Figure 8.

[0405] Peptide mapping analysis of samples from the storage stability study showed that the overall oxidation rate on A022600462 was significantly lower when compared to A0022600424 because tryptophan W99 had been removed from the original sequence. The oxidation rate on the second tryptophan appeared to remain stable, while tryptophan oxidation on other building blocks present in the format appeared unaffected by the introduced amino acid substitutions. As expected, removal of the D61 residue resulted in the elimination of isomerization liability in the final format.

[0406] In conclusion, the problems associated with tryptophan oxidation and isomerization in the ISVD were overcome by substituting amino acids at the relevant positions while maintaining the functionality of the ISVD.

[0407] 7 Industrial Applicability The ISVDs, polypeptides, nucleic acid molecules encoding them, vectors comprising such nucleic acids, and compositions described herein can be used, for example, to treat subjects suffering from cancer.

[0408] [Table 37]

[0409] Table 38

[0410] Table 39

[0411] Table 40

[0412] Table 41

[0413] Table 42

[0414] Table 43

[0415] Table 44

[0416] Table 45

[0417] Table 46

[0418] Table 47

[0419] Table 48

[0420] Table 49

[0421]

Table 50

[0422] Table 51

[0423] Table 52

[0424] Table 53

[0425] Table 54

[0426] Table 55

[0427] Table 56

Claims

1. An immunoglobulin single variable domain (ISVD) that specifically binds to the constant domain of human and / or non-human primate T cell receptors (TCRs) present on T cells, wherein the ISVD essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively). (A) (i) The amino acid sequence of CDR1 (according to Kabat) is INFYG (SEQ ID NO: 79), (ii) The amino acid sequence of CDR2 (according to Kabat) is HISIGDQTDYAX 1 It is SAKG (Sequence ID 80), and (iii) The amino acid sequence of CDR3 (according to Kabat) is LSRIX 2 PYDY (Sequence ID 81) Here, (I) Amino acid residue X 1 is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S, and optionally, amino acid residue X 1 is E or D, and optionally, amino acid residue X 1 is E and Amino acid residue X 2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I, or (II) Amino acid residue X 1 is selected from E, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or Amino acid residue X 2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I. or (B) (i) The amino acid sequence of CDR1 (following AbM) is GYVHKINFYG (SEQ ID NO: 82), (ii) The amino acid sequence of CDR2 (according to AbM) is HISIGDQTD (SEQ ID NO: 83), and (iii) The amino acid sequence of CDR3 (according to AbM) is LSRIX 2 PYDY (SEQ ID NO: 84), and Here, (I) The amino acid residue at position 61 (according to Kabat) is selected from E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S, and optionally the amino acid residue at position 61 (according to Kabat) is E or D, and optionally the amino acid residue at position 61 (according to Kabat) is E, and Amino acid residue X 2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I, or (II) The amino acid residue at position 61 (according to Kabat) is selected from E, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S, and / or Amino acid residue X 2 is selected from Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I. Immunoglobulin single variable domain (ISVD).

2. X 2 is Y, A, Q, F, S, T, or H, and arbitrarily X 2 is Y, Q, S, or T, and further optionally, X 2 The ISVD according to claim 1, wherein Y.

3. The ISVD according to claim 1, wherein the amino acid residue at position 103 in the ISVD (Kabat numbering) is selected from the group consisting of W, R, A, E, Y, L, H, I, Q, V, K, S, G, P, F, and T, and optionally the amino acid residue at position 103 in the ISVD (Kabat numbering) is W.

4. X 1 Alternatively, the amino acid residue at position 61 (Kabat numbering) is E, and X 2 The ISVD according to claim 1, wherein is Y and the amino acid residue at position 103 (Kabat numbering) is W.

5. The ISVD according to claim 1, wherein the amino acid residue at position 1 (Kabat numbering) is selected from E and D.

6. The ISVD is a heavy chain ISVD, and optionally, the heavy chain ISVD is V HH Humanization V HH Camelization V H The ISVD according to claim 1, selected from a domain antibody, a single-domain antibody, and a dAb.

7. The sequence identity of the ISVD is at least 85%, preferably at least 90%, and more preferably at least 95% to any sequence of sequence numbers 2 to 57, and for the purpose of determining the sequence identity, amino acid residues forming the CDR sequence are ignored, and optionally, the ISVD has at least 85%, preferably at least 90%, and more preferably at least 95% to sequences of sequence numbers 32, 33, and / or 35 to 57, preferably, the sequence is sequence number 37, sequence number 42, sequence number 46, sequence number 50, or sequence number 52, and optionally, (a) The ISVD includes or consists of Sequence ID No. 37 or Sequence ID No. 42, (b) The ISVD includes or consists of sequence number 46, sequence number 50, or sequence number 52, ISVD according to claim 1.

8. The following sequence: X 0 VELVESGGVVESLRLSCVASGYTHKIN$YGWYREINVAHISI X 1 SAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIX 2 PYDYX 3 GQGTLVTVSS (Sequence ID 85) (In the array, a. X 0 It is selected from E and D, b. X 1 It is selected from the group consisting of E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F, and S. c. X 2 is selected from the group consisting of Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L, and I, and d. X 3 It is selected from the group consisting of W, R, A, E, Y, L, H, I, Q, V, K, S, G, P, F, and T. Optionally, (i) X 0 D is, (ii) X 1 The group consisting of D or E is selected, and X is optionally selected. 1 E is, (iii)X 2 is selected from the group consisting of Y, T, S, and Q, and arbitrarily, X 2 Y is and / or (iv) X 3 (is W) An immunoglobulin single variable domain (ISVD) containing or consisting of a single variable domain.

9. A polypeptide comprising a first ISVD capable of specifically binding to the constant domain of human and / or non-human primate T cell receptors (TCRs) present on T cells, and a second ISVD capable of specifically binding to a first antigen on target cells, The first antigen is different from the TCR, the target cells are different from the T cells, the first and second ISVDs essentially consist of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), and the first ISVD is the ISVD according to any one of claims 1 to 6 or 8. Optionally, the amino acid sequence of the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, and more preferably at least 95% sequence identity with at least one of the amino acid sequences of SEQ ID NOs: 2 to 57, and for the purpose of determining the degree of sequence identity, amino acid residues forming the CDR sequence are ignored, and optionally, the first ISVD has at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 32, 33, and / or 35 to 57, and preferably, the sequence of the first ISVD includes SEQ ID NOs: 37, SEQ ID NOs: 42, SEQ ID NOs: 46, SEQ ID NOs: 50, or SEQ ID NOs:

52. Furthermore, optionally, (i) The first ISVD includes or consists of SEQ ID NO: 37 or SEQ ID NO: 42, (ii) The first ISVD includes or consists of sequence number 46, sequence number 50, or sequence number 52, Optionally, the polypeptide is (i) A third ISVD that specifically binds to a second antigen on the target cell, and / or (ii) one or more other groups, residues, moieties, or binding units optionally linked via one or more peptidic linkers, wherein the one or more other groups, residues, moieties, or binding units provide a polypeptide with an increased half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties, or binding units, and optionally the one or more other groups, residues, moieties, or binding units are ISVDs capable of binding to human serum albumin. A polypeptide that further includes this.

10. below: a. Expressing the nucleic acid sequence encoding the ISVD or polypeptide in a suitable host cell or host organism or other suitable expression system, and optionally subsequently... b. Isolating and / or purifying the ISVD or polypeptide, A method for producing ISVD according to any one of claims 1 to 8, including the following:

11. A nucleic acid encoding ISVD according to any one of claims 1 to 8.

12. A vector comprising the nucleic acid described in claim 11.

13. A non-human host or host cell transformed or transfected with the nucleic acid described in claim 11.

14. A composition comprising ISVD according to any one of claims 1 to 8, wherein the composition is optionally a pharmaceutical composition.

15. The polypeptide according to claim 9, for use as a pharmaceutical agent, optionally for use in the prevention, treatment, or recovery of a disease selected from the group consisting of proliferative disorders, inflammatory disorders, infectious disorders, and autoimmune disorders, wherein the disease is optionally cancer.

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