Immunoglobulin single variable domains targeting t cell receptors

Abstract

The present technology provides immunoglobulin single variable domains (ISVD) that bind to both the constant domain of human T cell receptor (TCR) on T cells and the constant domain of non-human primate TCR on T cells. The technology of the invention also relates to polypeptides comprising ISVDs according to the technology of the invention 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 of producing these ISVDs or polypeptides. In addition, the present technology relates to methods for treatment using ISVDs or polypeptides of the present technology.

Description

[0001] manual 1. Technical Field

[0002] The present technology provides immunoglobulin single variable domains (ISVDs) that bind 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. The present technology also relates to multispecific polypeptides, which comprise 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 ISVD or polypeptides, and vectors, hosts, and methods for producing the ISVD or polypeptides. In addition, the present technology relates to methods for treatment using an ISVD or polypeptide according to the present technology. 2. Background technology

[0003] Cancer takes a huge toll worldwide. Today, it is the second leading cause of death worldwide, after heart disease and stroke. Cancer is one of 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 increase further in the coming decades. Population growth, aging and changes in lifestyle have been identified as contributing factors to the growing cancer burden. In 2013, the WHO predicted that by 2030, cancer will surpass ischemic heart disease and become the most common cause of death worldwide (Source: WHO Cancer).

[0004] In 2008, the total economic impact of premature cancer deaths and disabilities worldwide was about $900 billion, accounting for 1.5% of the world's GDP at the time. As cancer becomes increasingly common, the total economic impact is sure to have increased significantly. Available treatment options for solid tumors typically include a combination of surgical resection, chemotherapy, and radiotherapy. Little progress has been made in 40 years of clinical experience, especially in the late stages of cancer. People are eagerly looking forward to new therapies to combat cancer.

[0005] Antibody therapy is now an important part of the means for doctors to fight diseases and especially cancer. Monoclonal antibodies have been established as a key treatment method for a series of diseases for several years. Most of the antibody therapies approved at the same time rely on monospecific monoclonal antibodies (mAbs). Until today, most targets of mAbs require exciting methods or antagonistic methods. Although targeting cell surface antigens themselves can mediate anti-tumor activity by inducing apoptosis, most mAb-based activities for hematological malignancies rely on Fc-mediated effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0006] Recently, immunotherapy has become a rapidly growing area of ​​cancer research. Immunotherapy is the directing of the body's immune surveillance system, and in particular T cells, to cancer cells.

[0007] Cytotoxic T cells (CTL) are T lymphocytes that kill cancer cells, cells infected (especially infected with viruses), or otherwise damaged cells. T lymphocytes (also referred to as T cells) express T cell receptors (TCRs) and CD3 receptors on the cell surface. αβTCR-CD3 complexes (or "TCR complexes") are composed of six different type I single-pass transmembrane proteins: TCRα and TCRβ chains that form TCR heterodimers responsible for ligand recognition, and CD3γ, CD3δ, CD3ε and ζ chains with non-covalent associations of cytoplasmic sequence motifs, which are tyrosine phosphorylated after receptor activation and recruit a large number of signal transduction components (Call et al. 2004, Molecular Immunology [Molecular Immunology] 40: 1295-1305).

[0008] The α and β chains of the heterodimeric T cell receptor (TCR) are composed of constant domains and variable domains. T cells are activated after TCR recognizes cognate peptides presented by self-MHC molecules, and signal transduction is initiated by the tyrosine phosphorylated CD3 complex, leading to T cell proliferation and differentiation.

[0009] Different from inducing specific T cell response (depending on the expression of MHC molecules and the presence, production, transport and display of specific peptide antigens by cancer cells), recent developments have attempted to combine the advantages of immunotherapy with antibody therapy by mobilizing all T cells of patients in a polyclonal manner via recombinant antibody-based technology. Bispecific antibodies have been engineered, and they have a tumor recognition portion on one arm (target binding arm), while the other arm (effector binding arm) of the molecule (so-called T cell engager (TCE), which usually targets CD3) has specificity for T cell antigens. These bispecific antibodies are multi-targeted molecules that enhance the immune response of patients to malignant cells. T cells and tumor cells are co-engaged by bispecific antibodies to cause the formation of cytolytic synapses between T cells and tumor cells, which induces T cell activation and causes tumor cell killing.

[0010] While most bispecific antibodies that activate T cells target the CD3 complex on T cells, some bispecific binders that target the constant domain of the αβ T cell receptor have been described in WO 2016 / 180969A1. However, one of the main problems with these bispecific antibodies that activate T cells is that almost no cross-reactivity with the cynomolgus monkey T cell receptor is observed.

[0011] Various formats of bispecific antibody constructs have been proposed. For example, the bispecific antibody format may 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).

[0012] However, the disadvantages of such bispecific antibody formats include high molecular weight and high viscosity at high concentrations, making, for example, subcutaneous administration challenging, and that each binding unit requires the interaction of two variable domains for specific and high affinity binding, which has an impact on polypeptide stability and production efficiency. Such bispecific antibody formats may also potentially lead to CMC issues associated with low production efficiency and low titer and / or light chain mispairing or heavy chain mispairing.

[0013] Currently, only one bispecific antibody, blinatumomab (BiTE molecule recognizing CD19 and CD3), is currently available on the market for clinical treatment of cancer. Although this T-cell engaging format was approved by the FDA for second-line treatment in December 2014, many obstacles had to be overcome. The first clinical trial of blinatumomab was stopped prematurely due to neurological adverse events, cytokine release syndrome (CRS) and infections on the one hand, and due to the lack of objective clinical response or robust signs of biological activity on the other hand. CRS is the most important adverse event reported in first-line T-cell engaging therapies.

[0014] In order to minimize the risk of adverse events and systemic side effects (such as cytokine storm), extreme care must be taken in choosing the T cell antigen arm. The latter must bind the TCR complex in a monovalent manner and must not trigger T cell signaling in the absence of targeted cancer cells. Only specific binding of the two arms of the bispecific antibody to its target (tumor and T cell antigen) can trigger the formation of cytolytic synapses and subsequent killing of tumor cells.

[0015] Non-human primates (such as cynomolgus monkeys or rhesus monkeys) are generally considered to be the most suitable animal species for preclinical studies (including efficacy and toxicity studies). In order to enable the evaluation of the toxicity of bispecific T cell engaging antibodies in non-human primates, good species cross-reactivity of the antibodies to human and non-human primate TCRs is desirable.

[0016] Thus, there remains a need for additional multispecific T cell engaging formats, and in particular multispecific T cell engaging formats that target T cell receptors other than CD3.

[0017] In addition, it is necessary to bind both target cells and T cells with sufficient affinity to induce antibody constructs of cytotoxic responses. At the same time, such constructs should not induce cytotoxic responses to non-target cells (i.e., cells that do not express the target antigen or only express the target antigen at a low level). Thus, a balance can be achieved between efficacy and safety. It is further desirable that such constructs can be efficiently produced, for example, in a microbial host. In addition, when used in treatment, the construct should not exert undesirable side effects or only exert minimal undesirable side effects, such as side effects caused by cytotoxic activity to non-target cells. 3. Summary of the invention

[0018] The inventors have found novel T cell engagers (TCE) immunoglobulin single variable domains (ISVDs) that can specifically target and bind to TCR. These ISVDs (also referred to as TCEs) that bind to TCR can be connected to parts that can bind to cell-specific targets. Pathologies (such as cancer) caused by abnormal cells can be targeted by the immune system through the combination of TCE ISVDs and T cells, and these abnormal cells present the specific targets on their cell surfaces. Therefore, T cells are directed to tumor cells expressing the specific targets, and through this combination of T cells and TCEs, T cell activation is induced. Once T cells are activated, effective target cell killing is triggered.

[0019] The inventors have found that constructs comprising TCR-targeting ISVDs and cell-specific targets according to the technology of the present invention simultaneously result in efficient T cell-mediated in vitro killing of cells expressing the target. In addition, such constructs only show limited activity against cells that do not express the target or express low levels of the target. This suggests the possibility of inducing highly specific T cell-mediated cytotoxic responses against specific target cells while exhibiting favorable safety characteristics.

[0020] In stability studies with an earlier developed TCR-binding ISVD, the inventors observed that some amino acid residues at certain positions in the ISVD lacked chemical stability. Specifically, they observed that isomerization occurred at aspartic acid (D) at position 61, and tryptophan (W) oxidation occurred at position 99 (Kabat numbering). It is assumed that trace levels of transition metals are catalyzed by polysorbates during downstream processing, which leads to the observed tryptophan oxidation. The inventors made several amino acid substitutions at selected positions of the TCR-binding ISVD and observed that these deleterious effects could be attenuated or even completely prevented.

[0021] Provided herein are TCR-binding ISVDs that have potent binding to TCRs of different species and improved chemical stability.

[0022] 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 T cells, wherein the ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively), and wherein:

[0023] a. The amino acid sequence of CDR1 (according to Kabat) is INFYG (SEQ ID NO: 79);

[0024] b. The amino acid sequence of CDR2 (according to Kabat) is HISIGDQTDYAX1SAKG (SEQ ID NO: 80); and

[0025] c. The amino acid sequence of CDR3 (according to Kabat) is LSRIX2PYDY (SEQ ID NO: 81);

[0026] in

[0027] - the amino acid residue 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; and / or

[0028] - the amino acid residue 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.

[0029] Another aspect of the present technology relates to 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 comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein

[0030] a. The amino acid sequence of CDR1 (according to AbM) is GYVHKINFYG (SEQ ID NO: 82);

[0031] b. The amino acid sequence of CDR2 (according to AbM) is HISIGDQTD (SEQ ID NO: 83); and

[0032] c. The amino acid sequence of CDR3 (according to AbM) is LSRIX2PYDY (SEQ ID NO: 84);

[0033] And among them

[0034] - the amino acid residue at position 61 (according to Kabat) is selected from the group consisting of E, D, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S; and / or

[0035] - the amino acid residue 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.

[0036] 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 an embodiment, X1 is selected from E or D, such as X1 is E.

[0037] 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, such as position 61 (according to Kabat) is E or D.

[0038] In one embodiment, position 61 (according to Kabat) is E.

[0039] 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.

[0040] 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, such as the amino acid at position 103 (Kabat numbering) is W.

[0041] In another embodiment, the present technology provides an ISVD wherein X1 is E, X2 is Y, and the amino acid residue at position 103 (Kabat numbering) is W; or an ISVD wherein 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.

[0042] In further embodiments, the ISVD is a heavy chain ISVD. In one embodiment, the ISVD is selected from VHH, humanized VHH, camelized VH, domain antibodies, single domain antibodies and dAbs. In one embodiment, the ISVD is selected from VHH, humanized VHH and 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% with the sequence of SEQ ID NO: 2-57, wherein for the purpose of determining the degree of sequence identity, the amino acid residues forming the CDR sequence are not taken into account.

[0043] Another aspect of the present technology relates to ISVD, wherein the sequence of ISVD is

[0044] X0VQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKER EKVAHISIGDQTDYAX1SAKGRFTISRDESKNTVYLQMNSLRPEDTAAYY CRALSRIX2PYDYX3GQGTLVTVSS(SEQ ID NO:85),

[0045] in

[0046] a.X0 is selected from E and D.

[0047] 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;

[0048] 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

[0049] 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.

[0050] 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.

[0051] In another aspect of the present technology, an ISVD according to the present technology is part of a multispecific polypeptide that further comprises a portion capable of binding to a specific cell surface target.

[0052] In some embodiments of the present technology, the present technology thus provides a polypeptide comprising a first ISVD and at least one additional ISVD, wherein the first ISVD specifically binds to a constant domain of a human and / or non-human primate TCR present on a T cell, and the at least one additional ISVD specifically binds to an antigen on a target cell, wherein the first ISVD is an ISVD according to the present technology.

[0053] In these embodiments, the amino acid sequence of the first ISVD may have at least 80% sequence identity with at least one of the amino acid sequences of SEQ ID NOs: 2-57, wherein the amino acid residues forming the CDR sequences are not taken into account for the purpose 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, such as SEQ ID NO: 37 or SEQ ID NO: 42.

[0054] In another embodiment, the polypeptide may further comprise a third ISVD that specifically binds to a second antigen on the target cell.

[0055] 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 peptide 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).

[0056] Also provided are nucleic acid molecules encoding the ISVD or polypeptide of the present technology or vectors comprising the nucleic acid.

[0057] The present technology also relates to non-human hosts or host cells transformed or transfected with nucleic acids or vectors encoding ISVDs or polypeptides according to the present technology.

[0058] In addition, the present technology relates to a composition comprising an ISVD or a polypeptide of the present technology, preferably, the composition is a pharmaceutical composition.

[0059] Further provided is a method for producing an ISVD or polypeptide as disclosed herein, the method comprising at least the following steps:

[0060] a. expressing the nucleic acid sequence encoding the ISVD or polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally followed by:

[0061] b. Isolating and / or purifying the ISVD or polypeptide.

[0062] Furthermore, the present technology relates to compositions or polypeptides for use as medicaments.

[0063] In one embodiment of the present invention, the polypeptide or composition is used in the treatment of a proliferative disease, an inflammatory disease, an infectious disease or an autoimmune disease. In one embodiment of the present invention, the proliferative disease is cancer.

[0064] The technology of the present invention also provides a treatment method, which includes the step of administering the composition or polypeptide to a subject in need.

[0065] In one embodiment of the present technology, the method of treatment is used to treat a proliferative disease, an inflammatory disease, an infectious disease, or an autoimmune disease. In one embodiment, the proliferative disease is cancer.

[0066] The present invention also provides a composition or polypeptide for use in preparing a medicament. In one embodiment of the present invention, the medicament is used to treat a proliferative disease, an inflammatory disease, an infectious disease or an autoimmune disease.

[0067] In one embodiment of the present technology, the proliferative disease is cancer. 4. Description of the drawings

[0068] Figure 1 Binding of TCE ISVD according to the present technology to primary human T cells as determined in flow cytometry (FACS) is shown.

[0069] Figure 2 Binding of TCE-CD123-ALB ISVD constructs according to the present technology to primary human T cells as determined in flow cytometry (FACS) is shown.

[0070] FIG3 shows the dose-response curve of the TCE-CD123-ALB construct 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 2 human donors. Results for donor 1 are shown in Figure 3A , Figure 3C and Figure 3E , while the results for donor 2 are shown in Figure 3B , Figure 3D and Figure 3F middle.

[0071] Figure 4 shows the dose-response curves of the TCE-CD123-ALB construct in a flow cytometry-based human (4A) and cynomolgus monkey (4B) T cell-mediated KG-1a cell killing assay in the presence of 30 μM HSA using an effector to target ratio of 10 to 1.

[0072] Figure 5 shows that in the presence of 30 μM HSA, using an effector to target ratio of 15 to 1, in the impedance-based human ( Figure 5A , Figure 5B ) and cynomolgus monkeys ( Figure 5C , Figure 5D ) Dose response curve of TCE-GPC3-GPC3-ALB construct in T cell-mediated HuH-7 cell killing assay.

[0073] Figure 6 Shown is the evolution of relative tryptophan oxidation at 25°C quantified as the sum of peaks with RRT < 1.0 in reverse phase chromatography for both ISVD constructs A022600424 and A022600462.

[0074] Figure 7 Shown is the evolution of tryptophan oxidation quantified as the sum of peaks with RRT<1.0 in reverse phase chromatography for both ISVD constructs A022600424 and A022600462 after 1 day and after 2 weeks under forced degradation conditions.

[0075] Figure 8 Results of peptide mapping of ISVD constructs A022600424 and A022600462 are shown. 5. Specific implementation methods

[0076] The inventors found in previous studies that the introduction of certain amino acid mutations and combinations thereof in the CDRs of ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969) resulted in improved binding to the constant domains of human TCRs and / or non-human primate TCRs. However, the developed improved TCR-binding ISVD, called T017000700 (SEQ ID NO: 1), showed some problems related to isomerization and tryptophan oxidation in chemical stability studies.

[0077] Therefore, there is still a need for further improved TCR-binding ISVDs.

[0078] The inventors have now discovered that introducing certain amino acid mutations at specific positions within the sequence of ISVD can remove the problems observed with T017000700, while retaining the improved TCR binding for T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969).

[0079] Amino acid residues will be referred to herein interchangeably according to the standard three-letter or one-letter amino acid code, as described in Table B-1 below.

[0080] Table B-1: Common amino acids

[0081]

[0082]

[0083] When an amino acid residue is indicated as "X" or "Xaa", it means that the amino acid residue is unspecified, unless the context requires a more restrictive interpretation. For example, if the description provides the amino acid sequence of a CDR, in which one (or more) of the amino acid residues is indicated by "X", the description may further specify which (some) amino acid residues are (may be) present at that particular position of the CDR.

[0084] 5.1 Immunoglobulin Single Variable Domains

[0085] The term "immunoglobulin single variable domain" (ISVD) defines an immunoglobulin molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains (particularly two variable domains) interact to form an 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 an antigen binding site. In this case, V H and V L The complementarity determining regions (CDRs) of will contribute to the antigen binding site, that is, a total of 6 CDRs will participate in the formation of the antigen binding site.

[0086] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; as known in the art) or a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional 4-chain antibody (as known in the art) is generally not regarded as an immunoglobulin single variable domain, because in these cases, the binding to the corresponding epitope of the antigen usually does not occur through one (single) immunoglobulin domain, but rather through a pair of (related) immunoglobulin domains, such as a light chain and a heavy chain variable domain, i.e., through the V domains of the immunoglobulin domains that jointly bind to the epitope of the corresponding antigen. H -V L To what happened.

[0087] In contrast, an immunoglobulin single variable domain is able to specifically bind to an epitope of an antigen without the need to pair with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V H , Single V HH or a single V L Domain formation.

[0088] Thus, the single variable domain may be a light chain variable domain sequence (e.g., V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H Sequence or V HH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit essentially consisting 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).

[0089] An immunoglobulin single variable domain (ISVD) may for example be a heavy chain ISVD, such as V H 、V HH (Including camel-derived V H or humanized V HH ). Preferably, it is V HH , including camel-derived V H or humanized V HH The heavy chain ISVD may be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0090] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb); other single variable domains, or any suitable fragment of any of these.

[0091] In particular, an immunoglobulin single variable domain may be Immunoglobulin single variable domain (such as V HH , including humanized V HH or camel-derived V H ) or a suitable fragment thereof. and is a registered trademark of Ablynx NV.

[0092] “V HH domain", also called V HH s、V HH Antibody fragments and V HH Antibodies, originally described as “heavy chain antibodies” (i.e., “antibodies lacking light chains”; Hamers-Casterman et al. 1993, Nature 363:446-448), are antigen-binding immunoglobulin variable domains. The term “V HH The term "V domain" is used to describe the combination of these variable domains with the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "V H domain) and the light chain variable domain present in conventional 4-chain antibodies (referred to herein as "V L domain”). HH For more information, see the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74: 277-302).

[0093] Typically, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening natural or synthetic libraries (e.g., by phage display).

[0094] The generation of immunoglobulin sequences has been extensively described in various publications, of which WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 can be used as examples. In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. The VHH library obtained from the immunization is further screened for VHHs that bind to the target antigen.

[0095] In these cases, the generation of immunoglobulins requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0096] Peptide fragments of these antigens can be used for immunization and / or screening of immunoglobulin sequences.

[0097] The technology of the present invention can use immunoglobulin sequences from different sources, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The technology of the present invention also includes fully human, humanized or chimeric sequences. For example, the 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). In addition, the technology of the present invention also uses fused immunoglobulin sequences, such as to form multivalent and / or multispecific constructs (for antibodies containing one or more V HH Multivalent and multispecific polypeptides of the structural domain and their preparation, see also Conrath et al. 2001, J. Biol. Chem. [Journal of Biological Chemistry] 276 (10): 7346-7350, and see, for example, WO 96 / 34103 and WO 99 / 23221); and immunoglobulin sequences that can be derived from the immunoglobulin sequences of the present technology and contain tags or other functional parts (such as toxins, markers, radioactive chemicals, etc.).

[0098] “Humanized V HH " contains the corresponding naturally occurring V HH The amino acid sequence of the amino acid sequence of the structural domain of the human 4-chain antibody is similar to that of the human 4-chain antibody. H One or more amino acid residues at one or more corresponding positions of the V domain (e.g., as shown above) are substituted for the naturally occurring VHH One or more amino acid residues in the amino acid sequence of a sequence (particularly in the framework sequence) of the humanized V sequence can be modified in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g. WO 2008 / 020079). It should also be noted that these humanized V HH It can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material.

[0099] "Camel Source V H " contains the corresponding naturally occurring V H The amino acid sequence of the amino acid sequence of the heavy chain antibody V HH One or more amino acid residues occurring at one or more corresponding positions in the structural domain replace the naturally occurring V of a conventional four-chain antibody H This can be done in a manner known per se, as will be clear to the skilled person, for example based on the further description herein and the prior art (e.g. WO 2008 / 020079). Such "camelizing" substitutions are preferably inserted into the amino acid residues forming and / or present in the V H -V L The amino acid positions of the camelid interface and / or the so-called camelid hallmark residues as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 and 1996, supra). Preferably, the camelid V H The starting material or starting point V H The sequence is preferably from a mammalian V H Sequence, more preferably a human V H Sequence, such as V H 3 sequence. However, it should be noted that such camelization V H It can be obtained in any suitable manner known per se and is therefore not strictly limited to the use of compounds comprising naturally occurring V H A polypeptide having a structural domain is a polypeptide obtained as a starting material.

[0100] The preferred structure of the immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FR"), which are respectively 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"), and these framework regions are interrupted by three complementarity determining regions ("CDR"), which are respectively referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"), "Complementarity Determining Region 2" ("CDR2") and "Complementarity Determining Region 3" ("CDR3").

[0101] As further described on pages 58 and 59, paragraph q) of WO 08 / 020079, the amino acid residues of the immunoglobulin single variable domains may be selected according to the V given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH, Bethesda, Maryland, publication number 91). H The domains are numbered according to the common numbering of the domains, as described in Riechmann and Muyldermans 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, this publication Figure 2 ) applied to V from camelids HH It should be noted that, as is known in the art, H Domain and V HH As is well known in the art, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, 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 the number allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in a domain is typically in the range of 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0102] In this application, unless otherwise indicated, CDR sequences are determined according to Kabat (Martin 2010, In: Kontermann and Dübel (eds.), Antibody Engineering [antibody engineering] Volume 2, Springer Verlag [Springer Publishing House] Heidelberg Berlin [Heidelberg Berlin], Chapter 3, pages 33-51). According to this method, FR1 of an immunoglobulin single variable domain comprises amino acid residues at positions 1-30, CDR1 of an immunoglobulin single variable domain comprises amino acid residues at positions 31-35, FR2 of an immunoglobulin single variable domain comprises amino acids at positions 36-49, CDR2 of an immunoglobulin single variable domain comprises amino acid residues at positions 50-65, FR3 of an immunoglobulin single variable domain comprises amino acid residues at positions 66-94, CDR3 of an immunoglobulin single variable domain comprises amino acid residues at positions 95-102, and FR4 of an immunoglobulin single variable domain comprises amino acid residues at positions 103-113.

[0103] Determination in CDR district can also be carried out according to different methods.In the present application, CDR sequence is also determined according to AbM definition, as described in Martin 2010 (In:Kontermann and Dübel (editor) 2010, Antibody Engineering [antibody engineering], Volume 2, Springer Verlag [Springer Press] Heidelberg Berlin [Heidelberg Berlin], Chapter 3, Page 33-51).According to this method, FR1 is included in the amino acid residue at position 1-25, CDR1 is included in the amino acid residue at position 26-35, FR2 is included in the amino acid at position 36-49, CDR2 is included in the amino acid residue at position 50-58, FR3 is included in the amino acid residue at position 59-94, CDR3 is included in the amino acid residue at position 95-102, and FR4 is included in the amino acid residue at position 103-113.

[0104] In such immunoglobulin sequences, the framework sequence may be any suitable framework sequence and examples of suitable framework sequences will be clear to the skilled person, for example based on the standard handbooks and the further disclosure and prior art mentioned herein.

[0105] The framework sequence is preferably an immunoglobulin framework sequence or a (suitable combination of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequence can be derived from a light chain variable domain (e.g., V L-sequence) and / or from a heavy chain variable domain (e.g. V H -Sequence or V HH In a particularly preferred aspect, the framework sequence is derived from V HH -sequence (wherein the framework sequence may optionally have been partially or fully humanized) or a conventional V H Sequence (as defined herein).

[0106] In particular, the framework sequences present in the ISVD sequences used in the present technology may contain one or more Hallmark residues (as defined herein) such that the ISVD sequence is V HH , including humanized V HH or camel-derived V H Some preferred but non-limiting examples of (appropriate combinations of) such framework sequences will become clear from the further disclosure herein.

[0107] Likewise, as generally described herein for immunoglobulin sequences, suitable fragments (or combinations of fragments) of any of the foregoing may also be used, such as fragments containing one or more CDR sequences suitably flanked by and / or connected via one or more framework sequences (e.g., in the same order as these CDR and framework sequences might appear in the full-size immunoglobulin sequence from which the fragment is derived).

[0108] However, it should be noted that the present technology is not limited as to the source of the ISVD sequence (or the nucleotide sequence for expressing the ISVD sequence), as 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 is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, in particular a partially or fully humanized V HH 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, and similar techniques well known to the skilled artisan for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing.

[0109] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may, for example, be 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 (in particular 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.

[0110] As mentioned above, the ISVD may be an ISVD or a suitable fragment thereof. For a general description of ISVDs, reference is made to the further description below and to the prior art cited herein. However, in this regard, it should be noted that the present specification and the prior art mainly describe so-called "ISVDs". H Type 3" ISVD (i.e. with V H However, it should be noted that the present technology in its broadest sense can generally use any type of ISVD and, for example, also use ISVDs belonging to the so-called "V H 4" ISVD (i.e. with V H Human germline sequences of Class 4 such as DP-78 have a high degree of sequence homology to ISVD), as described, for example, in WO 2007 / 118670.

[0111] Typically, ISVD (especially V HH Sequences, including (partially) humanized V HH Sequence and camelization V H Sequences) may be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more framework sequences (also as further described herein). Thus, in general, an ISVD may be defined as an immunoglobulin sequence having the following (general) structure:

[0112] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0113] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, wherein the one or more characteristic residues are as further defined herein.

[0114] In particular, an ISVD may be an immunoglobulin sequence having the following (general) structure:

[0115] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0116] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, wherein framework sequences are as further defined herein.

[0117] More specifically, an ISVD may be an immunoglobulin sequence having the following (general) structure:

[0118] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0119] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein 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 are selected from the hallmark residues mentioned in the following Table A-0.

[0120] Table A-0: Hallmark residues in ISVD

[0121]

[0122]

[0123] The present technology particularly uses ISVDs that can bind to the constant domain of a TCR.In the context of the present technology, "binding to" a target molecule has the usual meaning as understood in the art in the context of antibodies and their corresponding antigens.

[0124] Therefore, the target molecule of the ISVD used in the present technology is the constant domain of TCR.

[0125] The binding to TCR can be achieved, for example, by binding to TCR alpha subunits and / or TCR beta subunits. An example is a mammalian TCR. However, human TCRs are preferred, and versions from other species are also suitable for the technology of the present invention, such as TCRs from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates such as cynomolgus monkeys (also referred to herein as "cynomolgus monkeys (cyno)"), or camelids (such as llamas or alpacas).

[0126] The sequences of the TCR-α / β constant domains of human and cynomolgus monkey origin are provided in Table A-1 (SEQ ID NOs: 106 and 108 for the constant domains of TCRα from human and cynomolgus monkey origin, respectively; SEQ ID NOs: 107 and 109 for the constant domains of TCRβ from human and cynomolgus monkey origin, respectively). For each of the aforementioned sequences, the source of each of these sequences represented by UniProt or Genbank file identifiers is listed in Table A-1. In-house sequencing confirmed that the amino acid sequence originally derived from the rhesus monkey source was identical to the amino acid sequence from the cynomolgus monkey source.

[0127] 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 polymorphic variants or isoforms thereof.

[0128] 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 monkey TCR. In one embodiment, the macaque or rhesus monkey 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.

[0129] Isoforms are alternative protein sequences that can be produced from the same gene by a single biological event or by a combination of biological events, such as alternative promoter usage, alternative splicing, alternative initiation, and ribosomal frameshifting, all of which are known in the art.

[0130] Table A-1 Amino acid sequences associated with TCR ("ID" refers to a given SEQ ID NO as used herein)

[0131]

[0132]

[0133]

[0134]

[0135] Thus, in a first aspect, the present technology relates to an ISVD that specifically binds to the constant domain of a human and / or non-human primate T cell receptor (TCR) present on T cells, wherein the ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein

[0136] a. The amino acid sequence of CDR1 (according to Kabat) is INFYG (SEQ ID NO: 79);

[0137] b. The amino acid sequence of CDR2 (according to Kabat) is HISIGDQTDYAX1SAKG (SEQ ID NO: 80); and

[0138] c. The amino acid sequence of CDR3 (according to Kabat) is LSRIX2PYDY (SEQ ID NO: 81);

[0139] in

[0140] - the amino acid residue 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; and / or

[0141] - the amino acid residue 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.

[0142] ISVD sequences can also be represented using the following AbM definition for CDRs: An ISVD that specifically binds to the constant domain of a human and / or non-human primate T cell receptor (TCR) present on T cells, wherein the ISVD consists essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein

[0143] a. The amino acid sequence of CDR1 (according to AbM) is GYVHKINFYG (SEQ ID NO: 82);

[0144] b. The amino acid sequence of CDR2 (according to AbM) is HISIGDQTD (SEQ ID NO: 83); and

[0145] c. The amino acid sequence of CDR3 (according to AbM) is LSRIX2PYDY (SEQ ID NO: 84);

[0146] And among them

[0147] - the amino acid residue at position 61 (according to Kabat) is selected from the group consisting of E, N, P, K, R, I, T, H, V, A, Y, L, Q, F and S; and / or

[0148] - the amino acid residue 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.

[0149] The inventors found that ISVDs with CDRs according to the present technology have potent TCR binding ability and no isomerization or tryptophan oxidation at the relevant positions 61 and 99 (Kabat numbering), respectively. This means that the molecule is both effective in engaging T cells and chemically stable.

[0150] In an embodiment of the present technology, the amino acid sequence of the ISVD that specifically binds to the human TCR may exhibit more than 85%, such as more than 90%, more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57, wherein the CDRs are as defined herein.

[0151] In a further embodiment, the amino acid sequence of the ISVD that specifically binds to the human TCR may exhibit more 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, such as SEQ ID NO: 37 or SEQ ID NO: 42.

[0152] In another embodiment of the present technology, the amino acid sequence of the ISVD that specifically binds to a human TCR may exhibit more than 85%, such as more than 90%, more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57, wherein the amino acid residues forming the CDR sequences are not taken into account for the purpose of determining the degree of sequence identity.

[0153] In a further embodiment, the amino acid sequence of an ISVD that specifically binds to a human TCR may exhibit more than 85%, preferably at least 90%, more preferably at least 95% sequence identity, wherein for the purpose of determining the degree of sequence identity, the amino acid residues forming the CDR sequence are not taken into account. 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, such as SEQ ID NO: 37 or SEQ ID NO: 42.

[0154] When the ISVD exhibits more than 85%, such as more than 90%, more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57, the ISVD preferably exhibits at least half the binding affinity, more preferably at least the same binding affinity, to a human TCR compared to one of the ISVDs shown in Table A-4, wherein the binding affinity is measured using the same method, such as surface plasmon resonance (SPR).

[0155] In addition, when the ISVD exhibits greater than 85%, such as greater than 90%, greater than 95% or greater than 99% sequence identity to any one of SEQ ID NOs: 2-57, the ISVD preferably exhibits at least half the T cell-mediated target cell killing potency, more preferably at least the same T cell-mediated target cell killing potency as one of the ISVDs shown in Table A-4, wherein T cell-mediated target cell killing is measured using the same method (such as a flow cytometry-based T cell-mediated cell killing assay or an impedance-based T cell-mediated killing assay).

[0156] The percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated as follows: ([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] / [the total number of amino acid residues in the first amino acid sequence])×100%, wherein 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 regarded as a difference at a single amino acid residue (i.e., at a single position).

[0157] Typically, to determine the percent "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the greatest number of amino acid residues is used as the "first" amino acid sequence, and the other amino acid sequence is used as the "second" amino acid sequence.

[0158] As used herein, "amino acid difference" refers to a deletion, insertion or substitution, and preferably a substitution, of a single amino acid residue relative to a reference sequence.

[0159] In one embodiment of the present technology, the amino acid substitution is a conservative substitution. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a)-(e) is substituted by another amino acid residue in the same group: (a) small aliphatic non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) positively charged polar 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.

[0160] In another embodiment of the present technology, the conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; 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; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0161] In an embodiment of the present invention, the entire amino acid sequence of ISVD is represented by SEQ ID NO: 85, which is

[0162] where

[0163] a.X0 is selected from E and D.

[0164] 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;

[0165] 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

[0166] 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.

[0167] In an embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is any one of Y, T, S or Q, and / or X3 is W.

[0168] In another embodiment of the present technology, X0 is E, and / or X1 is E, and / or X2 is any one of Y, T, S or Q, and / or X3 is W.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] In an embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is S, and / or X3 is W.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] In an embodiment of the present technology, X0 is D, and / or X1 is E, and / or X2 is Y, and / or X3 is W.

[0177] The inventors found that substituting the amino acid at the first position (Kabat numbering) of the sequence from E to D prevents pyroglutamate formation, while the efficacy of the ISVD is not affected.

[0178] In another embodiment of the present technology, the ISVD according to the present technology has an amino acid sequence of any one of SEQ ID NOs: 37, 42, 46, 50 and 52.

[0179] In further embodiments of the present technology, the ISVD has an amino acid sequence of SEQ ID NO:37 or SEQ ID NO:42.

[0180] The inventors observed that ISVDs according to the present technology with E at position 61 and Y, A, S or H at position 99 (Kabat numbering) had similar or higher melting temperatures (Tm), higher aggregation onset temperatures (Tagg), and a reduction in the oligomeric fraction when compared to the reference ISVD T017000700. For ISVDs with E at position 61 and Q or T at position 99 (Kabat numbering), the Tm was not significantly reduced while maintaining or having a higher Tagg and a similar or reduced oligomeric fraction (Δ% oligomer).

[0181] Thus, in embodiments, an ISVD according to the present technology or an ISVD exhibiting more than 90%, such as more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57 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.

[0182] In another embodiment, an ISVD according to the present technology or an ISVD exhibiting more than 90%, such as more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57 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.

[0183] In yet further embodiments, an ISVD according to the present technology or an ISVD exhibiting more than 90%, such as more than 95% or more than 99% sequence identity to any one of SEQ ID NOs: 2-57 has an oligomeric portion (Δ% oligomer) 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%.

[0184] A list of generated ISVDs can be found in Table A-4. Additionally, combinations of CDR sequences of generated ISVDs can be found in Table A-5.

[0185] In an embodiment of the present technology, the TCE ISVD has a CDR1, CDR2 and CDR3 sequence selected from the CDR1, CDR2 and CDR3 sequences presented in Table A-5.

[0186] In another embodiment of the present technology, the TCE ISVD has CDR1, CRD2 and CDR3 sequences selected from the combination of CDR sequences presented in the same row in Table A-5.

[0187] 5.2 Multispecific Peptides

[0188] The inventors found that introducing certain amino acid mutations and combinations thereof in the CDRs of ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969) resulted 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 ISVD sequence can further improve the chemical stability of the ISVD by minimizing or even preventing isomerization and tryptophan oxidation.

[0189] Therefore, in one aspect of the present technology, a polypeptide is provided, which polypeptide comprises a first ISVD capable of specifically binding to the 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, and wherein the target cell is different from the T cell, wherein the first and second ISVDs essentially consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), and wherein the first ISVD is an ISVD according to the present technology.

[0190] In the multispecific polypeptide, the CDR regions are as defined herein (see item 5.1). The inventors found that polypeptides comprising a first ISVD having CDRs according to the present technology have potent TCR binding ability and are free of isomerization or tryptophan oxidation at the relevant positions 61 and 99 (Kabat numbering), respectively.

[0191] In an 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 one of SEQ ID NOs: 2-57, wherein the sequences of the CDR regions are as defined herein, preferably wherein 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.

[0192] In an 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 with at least one of the amino acid sequences of any one of SEQ ID NOs: 2-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, preferably wherein 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.

[0193] 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 one of SEQ ID NOs: 32, 33 and / or 35-57, wherein the sequences of the CDR regions are as defined herein, and wherein 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.

[0194] 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 one of SEQ ID NOs: 32, 33 and / or 35-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, and wherein 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.

[0195] In further embodiments, the first ISVD comprises or consists of SEQ ID NO:37 or SEQ ID NO:42.

[0196] In yet another embodiment, the first ISVD comprises or consists of SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:52.

[0197] As shown in the examples provided further herein, substitution of specific amino acid residues at positions 61 and 99 in T017000700 (SEQ ID NO: 1) does not affect the affinity of the ISVD for the constant domain of human and / or non-human primate TCRs, while the resulting variants have better chemical stability than 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. In addition, T017000995 (SEQ ID NO: 46), T017000999 (SEQ ID NO: 50) and T017001001 (SEQ ID NO: 52) showed lower affinity for TCRs, but maintained high potency in cell killing, indicating that they may have better biodistribution and therefore have the potential to have a more target-specific effect.

[0198] In an embodiment of the present technology, the polypeptide is at least bispecific, but can also be, for example, trispecific, tetraspecific, pentaspecific, etc. In addition, the polypeptide is at least bivalent, but can also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0199] The terms "bispecific", "trispecific", "quadraspecific", "pentaspecific", etc. all belong to the term "multispecific" and refer to binding to two, three, four, five, etc. different target molecules, respectively.

[0200] The terms "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", etc. are all part of the term "multivalent" and indicate the presence of two, three, four, five, six, etc. binding units / building blocks, such as ISVDs, respectively.

[0201] For example, the polypeptide may be bispecific-bivalent, such as a polypeptide comprising or consisting of two ISVDs, wherein 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, wherein the TCR and the target antigen are preferably human.

[0202] The polypeptide may also be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVDs, wherein 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.

[0203] In another example, the polypeptide may be trispecific-trivalent, comprising or consisting of three ISVDs, wherein one ISVD specifically binds to the constant domain of a human and / or non-human primate TCR on a T cell, 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.

[0204] In yet another example, a trispecific-trivalent polypeptide comprises one ISVD that specifically binds human serum albumin adjacent to one ISVD that specifically binds a constant domain of a human and / or non-human primate TCR on a T cell and one ISVD that specifically binds a first antigen on a target cell.

[0205] Additional examples of multispecific-multivalent polypeptides will be clear to the skilled artisan based on the disclosure herein.

[0206] For example, if two ISVDs bind to two different epitopes of a target antigen, such polypeptides may be biparatopic at the same time.

[0207] The term "biparatopic" refers to binding to two different portions (eg, epitopes) of the same target molecule.

[0208] The components of the multispecific-multivalent polypeptides described herein (preferably ISVDs) may be connected to each other via one or more suitable linkers (eg, peptide linkers).

[0209] The use of linkers to connect two or more (poly)peptides is well known in the art.

[0210] One commonly used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, and typically comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 86) motif (e.g., presented as the formula (Gly-Gly-Gly-Gly-Ser) n , wherein n can be 1, 2, 3, 4, 5, 6, 7 or greater). Some commonly used examples of such GS linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 87), 15GS linkers (n=3) and 35GS linkers (n=7). For example, see Chen et al. 2013 (Adv. Drug Deliv. Rev. [Advanced Drug Delivery Review] 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. [Protein Engineering, Design and Selection] 27(10): 325-330); in one or more polypeptides disclosed herein, it is preferred to use 5GS and 9GS linkers to connect the components of the polypeptide to each other. Preferably, a linker of less than 10 amino acids is used to connect the first ISVD capable of specifically binding to the TCR to the second ISVD capable of specifically binding to a cell surface-specific target antigen.

[0211] Examples of suitable linkers are given in Table A-2 below.

[0212] Table A-2: Linker sequences ("ID" refers to SEQ ID NO as used herein)

[0213]

[0214]

[0215] Thus, in an embodiment, the polypeptide comprises a first ISVD capable of specifically binding to the constant domain of a human and / or non-human primate TCR according to the technology of the present invention and a second ISVD capable of specifically binding to a cell surface specific target antigen, which are connected by a 5GS and / or 9GS linker.

[0216] In another embodiment of the present technology, the polypeptide comprises a first ISVD capable of specifically binding to the constant domain of human and / or non-human primate TCR according to the present technology and a second ISVD capable of specifically binding to a cell surface specific target antigen, which are connected by a 9GS linker.

[0217] The inventors have surprisingly found that this configuration can increase the efficiency of the polypeptide in eliciting a T cell mediated cytotoxic response.

[0218] It should be understood (as demonstrated in the Examples section) that the ISVD that binds the TCR and the ISVD that binds the first antigen on the target cell can be positioned in any order in the multispecific-multivalent polypeptides of the present technology.

[0219] Therefore, one embodiment of the technology of the present invention is that the polypeptide comprises or consists of the following 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 optionally a binding unit that provides the polypeptide with an increased half-life as defined herein. In one embodiment, the ISVD is connected via a 9GS linker. The binding unit that provides the polypeptide with an increased half-life is preferably an ISVD that preferably binds to serum albumin.

[0220] Such configuration of the polypeptide may provide a potent effect with respect to treating cancer.

[0221] Furthermore, it is not excluded that other binding units / building blocks (such as other ISVDs that bind to other antigens on the target cell or to another target) may be present in the polypeptide. Furthermore, the possibility of placing other binding units / building blocks (such as ISVDs) in between is not excluded. For 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 that may even be located, for example, between the "first ISVD" and the "second ISVD".

[0222] The second ISVD of the polypeptide of the present technology specifically binds to an antigen on a target cell (preferably a cancer cell). As mentioned herein, a "target cell" is a cell that presents a specific antigen on its surface. In one embodiment, a "target cell" is a cancer cell.

[0223] Membrane (also referred to as plasma membrane or phospholipid bilayer) surrounds the cytoplasm of a cell, and membrane is the outer boundary of a cell, i.e., membrane is the surface of a cell. The membrane is used to separate a cell from its surroundings and protect the cell from the influence of its surroundings, and is mainly composed of bilayer phospholipids. Embedded in this membrane are various protein molecules, such as channels, pumps, and cell receptors. Because the membrane is a fluid, protein molecules can travel in the membrane. As used herein, the term "antigen on a target cell" represents a molecule displayed on the cell surface. In most cases, this molecule will be located in or on the plasma membrane of the cell, so that at least a portion of this molecule remains accessible from the outside of the cell in a tertiary form. The non-limiting example of a cell surface molecule located in the plasma membrane is a transmembrane protein, which comprises a hydrophilic region and a hydrophobic region with its tertiary conformation. Here, at least one hydrophobic region allows a cell surface molecule to be embedded or inserted into the hydrophobic plasma membrane of a cell, and the hydrophilic region extends to the cytoplasm and the extracellular space respectively on either side of the plasma membrane.

[0224] The antigen can be any target on a cell, such as a tumor antigen. In one embodiment, the antigen is specific to the target cell (eg, a cancer cell), such as a tumor antigen or a tumor-associated antigen (TAA) on the cancer cell.

[0225] As used herein, the term "tumor antigen" can be understood as those antigens presented on tumor cells. These antigens can present an extracellular portion on the cell surface, which is usually combined with the transmembrane and cytoplasmic portions of the molecule. These antigens can sometimes only be presented by tumor cells, but never by normal or healthy cells. Tumor antigens may be expressed only on tumor cells or may represent tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. However, this is not generally the case. More common are antigens presented by tumor cells and normal cells, and they are called "tumor-associated antigens (TAA)". Compared with normal cells, these tumor-associated antigens can be overexpressed on tumor cells, or because the structure of tumor tissue is less compact than normal tissue, it is easier to bind to antibodies in tumor cells. TAA is preferably an antigen expressed on cells of a specific tumor, but preferably an antigen not expressed in normal cells. Generally, TAA is an antigen that is normally expressed in cells only at a specific point in biological development (such as during fetal development) and is not properly expressed in the organism at the current development point, or an antigen that is not expressed in normal tissues or cells of organs that now express antigens.

[0226] In embodiments, said first antigen on the target cell is a tumor associated antigen (TAA).

[0227] In an embodiment, said first antigen on the target cell is more abundantly present on cancer cells than on normal cells.The antigen on the target cell is preferably a tumor associated antigen (TAA).

[0228] In embodiments, said first antigen on the target cell is a tumor antigen or a tumor specific antigen (TSA).

[0229] In one embodiment, a multispecific-multivalent polypeptide of the current technology comprises a second ISVD that specifically binds CD123 or Glypican-3.

[0230] In additional embodiments, the polypeptides according to the present technology further comprise a third ISVD that specifically binds a second antigen on the target cell.

[0231] The target cells bound by the polypeptides of the present technology particularly relate to mammalian cells, preferably to primate cells, and even more preferably to human cells. The target cells are preferably hyperproliferative cells, such as cancer cells.

[0232] In another embodiment of the present technology, the multispecific-multivalent polypeptides exhibit reduced binding to pre-existing antibodies in human serum. To this end, in one embodiment of the present technology, the polypeptides exhibit 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 (and preferably the ISVD at the C-terminus of the polypeptide), but preferably in each ISVD.

[0233] In another embodiment of the present technology, the polypeptide exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (A) extension. The C-terminus of an ISVD is typically VTVSS (SEQ ID NO: 88).

[0234] In another embodiment of the present technology, the polypeptide exhibits a lysine (K) or a glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD.

[0235] In another embodiment of the present technology, the ISVD exhibits a lysine (K) or a glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these 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), such 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). NO:105), preferably VTVSSA.

[0236] In another 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 the C-terminal ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and an extension of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (A) extension (such that the C-terminus of the polypeptide consists, for example, of the sequence VTVSSA, VKVSSA or VQVSSA, preferably VTVSSA). For more information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0237] As will be clear from the above and further description herein, the ISVDs of the present technology can be used as "building blocks" to form polypeptides of the present technology, for example, by appropriately combining them with other groups, residues, moieties or binding units to form compounds or constructs as described herein (such as, but not limited to, the bi / tri / tetra / multivalent and bi / tri / tetra / multispecific polypeptides of the present technology described herein) that combine one or more desired properties or biological functions in one molecule. Polypeptides with multiple ISVDs are also referred to herein as "constructs" or "ISVD formats".

[0238] 5.3 Specificity

[0239] The terms "specificity", "specifically binds" or "specific binding" refer to the number of different target molecules (such as antigens) from the same organism to which a particular binding unit (such as 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 "selectively binds". Binding units (such as ISVDs) preferably bind specifically to their designated targets.

[0240] The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is usually expressed as K expressed in moles / liter (or M). D or dissociation constant. Affinity can also be expressed as the association constant K A , the association constant is equal to 1 / K D And in (mol / L) -1 (or M -1 ) is expressed in units.

[0241] Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule: K D The lower the value of , the stronger the binding strength between the target molecule and the targeting moiety.

[0242] Typically, the binding units (such as ISVD) used in the present technology will be 10 -5 Up to 10 -12 mol / L or less, and preferably 10 -7 Up to 10 -12 mol / L or less and more preferably 10 -8 Up to 10 -12 The dissociation constant (K) in mol / L D ) bind to their target (i.e., the association constant (K A ) is 10 5 Up to 10 12 liters / mole or more, and preferably 10 7 Up to 10 12 L / mol or more and more preferably 10 8 Up to 10 12 L / mol).

[0243] Any value greater than 10 -4 mol / L K D Value (or any value less than 10 4 L / mol K A Values) are generally considered to indicate nonspecific binding.

[0244] K is considered to be specific for biological interactions (such as the binding of an immunoglobulin sequence to an antigen). D Usually in 10 -5 mol / L (10000nM or 10μM) to 10 -12 The concentration of 1 μmol / L (0.001 nM or 1 pM) or less is within the range of 1 μmol / L (0.001 nM or 1 pM) or less.

[0245] Thus, specific / selective binding may mean that using the same measurement method, such as SPR, the binding unit (or polypeptide comprising the binding unit) is 10 -5 Up to 10 -12 mol / L or less K D value combined with TCR and greater than 10 -4 mol / L K D Value binding related targets.

[0246] Thus, the ISVD preferably exhibits at least half the binding affinity, more preferably at least the same binding affinity, to human TCR as the ISVD consisting of the amino acids of SEQ ID NO: 1, wherein the binding affinity is measured using the same method (such as SPR).

[0247] Specific binding to a target from a certain species does not exclude that the binding unit can also specifically bind to a similar target from a different species. For example, specific binding to a human TCR does not exclude that the binding unit (or a polypeptide comprising the binding unit) can also specifically bind to a TCR from a cynomolgus monkey.

[0248] When an ISVD is said to exhibit "improved cross-reactivity for binding to human and non-human primate TCRs" compared to another ISVD, this means that for that ISVD, the binding activity (as measured by K) for both human TCRs and non-human primate TCRs is comparable. D or k off The ratios (denoted by ) were lower than the same ratios calculated for other ISVDs in the same assay.

[0249] Good cross-reactivity with human and non-human primate TCR binding allows the toxicity of multispecific T cell engaging polypeptides to be evaluated in preclinical studies in non-human primates.

[0250] Specific binding of a binding unit to its designated target can be determined by any suitable means known per se (including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays) and their different variants known per se in the art; as well as other techniques mentioned herein.

[0251] The dissociation constant may be an actual or apparent dissociation constant as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person and include, for example, the techniques mentioned below. In this regard, it will also be clear that it is not possible to measure more than 10 -4 mol / L or 10 -3 mol / L (e.g. 10 -2 Optionally, as will be clear to the skilled person, the (actual or apparent) dissociation constant may be based on the (actual or apparent) association constant (K A ) through the relationship [K D =1 / K A ]calculate.

[0252] The affinity of a molecular interaction between two molecules can be measured by different techniques known per se, such as 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 the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, in which one molecule is immobilized on a biosensor chip and the other molecule passes through the immobilized molecule under flow conditions, thereby generating a k on , k off The measured value, and thus K D (or K A ) value. This can be done, for example, using the well-known System (BIAcore International AB, GE Healthcare company, Uppsala, Sweden and Piscataway, New Jersey). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. [Annual Journal of Clinical Biology] 51: 19-26), Jonsson et al. (1991, Biotechniques [Biotechnology] 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. [Molecular Recognition] 8: 125-131) and Johnnson et al. (1991, Anal. Biochem. [Analytical Chemistry] 198: 268-277).

[0253] Another well-known biosensor technology 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 on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, which is reported as a wavelength shift (nm), and the magnitude of the shift is a direct measure of the number of molecules bound to the biosensor tip surface. Because the interactions can be measured in real time, association and dissociation rates as well as affinity can be determined. BLI can be used, for example, using the well-known The ELISA was performed using a ForteBio system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0254] Alternatively, you can use The affinity is measured in a kinetic exclusion assay (KinExA) using the ELISA platform (Sapidyne Instruments Inc, Boise, USA) (see, e.g., Drake et al. 2004, Anal. Biochem. [Analytical Chemistry], 328: 35-43). As used herein, the term "KinExA" refers to a solution-based method that measures the true equilibrium binding affinity and kinetics of unmodified molecules. The equilibrium solution of the antibody / antigen complex is passed through a chromatographic column with beads pre-coated with the antigen (or antibody), thereby allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished using a fluorescently labeled protein that binds to the antibody (or antigen).

[0255] Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0256] In one embodiment, the ISVD of the present technology has a selected from 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 The rate constant (k) of the group consisting of on), the rate constant is preferably as measured by SPR, preferably the SPR being performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0257] In one embodiment, the ISVD of the present technology has a selected from 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 Composed of panels combining non-human primate TCR k on , the k on Preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0258] In one embodiment, the ISVD of the present technology has a selected from the group consisting of at most about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 and at most about 10 -4 s -1 Composed of a panel of binding human TCR k off , the k off Preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0259] In one embodiment, the ISVD of the present technology has a selected from the group consisting of at most about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 and at most about 10 -4 s -1 Composed of panels combining non-human primate TCR k off , the k off Preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0260] ISVDs with Y, F, H, K, L, or R at position 99 showed particularly favorable k compared to ISVDs with different amino acids at position 99. off. Therefore, 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.

[0261] ISVDs with W, A, E, F, H, I, K, L, Q, R, S, T, V, or Y at position 103 (Kabat numbering) show particularly favorable koff compared to ISVDs with another amino acid at position 103. Thus, in one embodiment, the ISVD has W, A, E, F, H, I, K, L, Q, R, S, T, V, or Y at position 103. In one embodiment, the ISVD has W, A, E, F, H, I, K, L, Q, S, T, or V at position 103.

[0262] ISVDs with A, E, F, H, I, K, L, N, P, Q, R, S, T, V, or Y at position 61 (Kabat numbering) showed k as good as the reference TCE ISVD with D at position 61 off Since isomerization is observed at D61 in the reference ISVD, it is desirable to obtain ISVDs with at least similar off-rates having 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, preferably the ISVD has E at position 61.

[0263] In one embodiment, the ISVD of the present technology has a selected from the group consisting of at most about 10 -6 M, up to about 10 -7 M, up to about 10 -8 M and at most about 10 -9 The affinity of the group consisting of M for binding to human TCR (K D ), the affinity is preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0264] In one embodiment, the ISVD of the present technology has a selected from the group consisting of at most about 10 -5 M, up to about 10 -6 M, up to about 10 -7 M and at most about 10 -8 The K of the group consisting of M binding to non-human primate TCR D , the K D Preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0265] In some embodiments, the TCR-binding ISVD of the present technology has the same or lower dissociation rate constant (k) compared to SEQ ID NO: 1. off ) binds to human TCR. In some embodiments, the ISVD of the present technology has the same or lower k compared to the ISVD of SEQ ID NO: 1. off Binding to non-human primate TCR. In some embodiments, the TCR-binding ISVD of the present technology has the same or lower dissociation rate constant (k) compared to ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). off ) binds to human TCR.

[0266] In some embodiments, the ISVDs of the present technology have the same or lower k compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). off Binding to non-human primate TCR. Dissociation rate (k off ) can be measured by any method known to the skilled person. In one embodiment, the dissociation rate (k off ) is measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0267] The inventors found that the same or higher affinity (K D ) of the present technology also have similar or higher potency than the reference ISVD when tested in a cell killing assay.

[0268] Surprisingly, the inventors found that a subset of ISVDs of the present technology bind to human TCRs with affinities (K D ) was more than 40-fold lower than the reference ISVD T017000700 (SEQ ID NO: 1), but maintained high potency in the cell killing assay. These specific ISVDs with E at position 61 and Q, S or T at position 99 (Kabat numbering) offer interesting opportunities in generating biotherapeutics with better biodistribution.

[0269] 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 WO 2016180969). Thus, in certain embodiments, the k of the ISVD of the present technology for binding to non-human primate TCRs is off In binding to human TCR k off within 5 times of the range.

[0270] In another embodiment, the ISVD of the present technology has the same or lower K compared to SEQ ID NO: 1. D Binding to human TCR, the K D Preferably as measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. In another embodiment, the ISVD of the present technology has the same or lower K compared to SEQ ID NO: 1. D In combination with nonhuman primate TCR, the K D Preferably as measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. In another embodiment, the ISVD of the present technology has the same or lower K compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). D Binding to human TCR, the K D Preferably as measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C. In another embodiment, the ISVD of the present technology has the same or lower K compared to ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). D In combination with nonhuman primate TCR, the K D Preferably as measured by surface plasmon resonance (SPR), preferably performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0271] In one embodiment, the ISVD of the present technology has (i) a selected from the group consisting of at most about 10 -6 M, up to about 10 -7 M, up to about 10 -8 M, up to about 10 -8 M and at most about 10 -9The affinity of the group consisting of M for binding to human TCR (K D ), and (ii) having a selected from up to about 10 -5 M, up to about 10 -6 M, up to about 10 -7 M and at most about 10 -8 The K of the group consisting of M binding non-human primate TCR D , the K D Preferably as measured by SPR, preferably the SPR is performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0272] In one embodiment, the ISVDs of the present technology exhibit improved cross-reactivity, i.e., the ratio of binding activity (as measured by K) to binding to human and non-human primate TCRs. D or K off ) is lower than the same ratio of ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO2016180969), the K D or K off Preferably it is determined by SPR, preferably performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0273] For example, the ISVD of the present invention technology shows that the K D The difference in human-cynomolgus monkey cross-reactivity of ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969) is lower than that of human-cynomolgus monkey cross-reactivity of ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). D It is determined by SPR, preferably performed on a ProteOn XPR36 instrument, preferably at 25°C.

[0274] 5.4 (In vivo) Half-life extension

[0275] As disclosed above, the polypeptide according to the present technology may further include one or more other groups, residues, parts or binding units (optionally connected via one or more peptide linkers), wherein the one or more other groups, residues, parts or binding units provide the polypeptide with an increased (in vivo) half-life compared to a corresponding polypeptide without the one or more other groups, residues, parts or binding units. In vivo half-life extension means, for example, that the polypeptide exhibits an increased half-life in a mammal (such as a human subject) after administration. The half-life can be expressed as, for example, t1 / 2β.

[0276] The type of group, residue, part or binding unit is generally not limited and can for example be selected from the group consisting of: a polyethylene glycol molecule, a serum protein or a fragment thereof, a binding unit that can bind to a serum protein, an Fc part and a small protein or peptide that can bind to a serum protein.

[0277] More specifically, the one or more other groups, residues, moieties or binding units that provide the polypeptide with an increased half-life can be selected from the group consisting of: a binding unit that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG), and preferably a binding unit that can bind to human serum albumin. The binding unit is preferably an ISVD.

[0278] For example, WO 04 / 041865 describes ISVDs that bind to serum albumin (and in particular human serum albumin) which may be linked to other proteins (such as one or more other ISVDs that bind to a desired target) in order to increase the half-life of the protein.

[0279] International application WO 06 / 122787 describes a number of ISVDs against (human) serum albumin. These ISVDs include the ISVD known as 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). Again, these can be used to extend the half-life of therapeutic proteins and polypeptides and other therapeutic entities or moieties.

[0280] Furthermore, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0281] In an embodiment of the present technology, the polypeptide comprises a serum albumin binding portion selected from the following: 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 shown on pages 7-9 of WO 2012 / 175400; and albumin binders described in WO2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, WO2018 / 134234.

[0282] In another embodiment, the polypeptide comprises a serum albumin binding portion selected from Table A-3.

[0283] Table A-3: ISVD sequences that bind serum albumin ("ID" refers to SEQ ID NO as used herein)

[0284]

[0285]

[0286]

[0287]

[0288] When such an ISVD that binds to human serum albumin is at the C-terminal position, it may exhibit a C-terminal alanine (A) or glycine (G) extension (preferably A). In some embodiments of the present technology, the ISVD that binds to human serum albumin is at another position other than the C-terminal position (i.e., not the C-terminal ISVD of the polypeptide).

[0289] 5.5 Nucleic Acid Molecules

[0290] Also provided are nucleic acid molecules encoding an ISVD or polypeptide as disclosed herein.

[0291] "Nucleic acid molecule" (interchangeably used with "nucleic acid") is a chain of nucleotide monomers connected to each other via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, for example, for expression and / or production of polypeptides. Suitable hosts or host cells for preparation purposes will be clear to the skilled person, and can be, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms. The present technology also encompasses (non-human) hosts or host cells comprising nucleic acids encoding the polypeptides.

[0292] The nucleic acid may be, for example, DNA, RNA or a hybrid thereof, and may also contain (e.g. chemically) modified nucleotides, such as PNA. It may be single-stranded or double-stranded, and is preferably in the form of double-stranded DNA. For example, the nucleotide sequence may be genomic DNA or cDNA.

[0293] Nucleic acid can be prepared or obtained in a manner known per se, and / or can be separated from a suitable natural source. The nucleotide sequence encoding a naturally occurring (poly)peptide can for example be subjected to site-directed mutagenesis to provide a nucleic acid molecule encoding a polypeptide with sequence variation. In addition, it will be clear to the technician that in order to prepare nucleic acid, several nucleotide sequences (such as at least one nucleotide sequence encoding the targeting moiety) and for example a nucleic acid encoding one or more joints can also be linked together in a suitable manner.

[0294] Techniques for producing nucleic acids will be clear to the skilled person and may, for example, 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 the expression of a truncated expression product; introducing one or more restriction sites (e.g. to form cassettes and / or regions that can be easily digested and / or ligated using appropriate restriction enzymes) and / or introducing mutations by a PCR reaction using one or more "mismatched" primers.

[0295] 5.6 Carrier

[0296] Also provided are vectors comprising a nucleic acid molecule encoding an ISVD or polypeptide as disclosed herein.

[0297] As used herein, "vector" is a vehicle suitable for carrying genetic material into a cell. Vectors include naked nucleic acid (eg, plasmids or mRNA) or nucleic acid embedded in a larger structure (eg, liposomes or viral vectors).

[0298] The vector generally comprises at least one nucleic acid optionally linked to one or more regulatory elements (such as, for example, one or more suitable promoters, enhancers, terminators, etc.). The vector is preferably an expression vector, i.e. a vector suitable for expressing the encoded polypeptide or construct under appropriate conditions (e.g., when the vector is introduced into (e.g., human) cells). For DNA-based vectors, this generally includes the presence of elements for transcription (e.g., promoters and poly A signals) and translation (e.g., Kozak sequences).

[0299] Preferably, in the 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. For example, if a promoter is able to initiate or otherwise control / regulate the transcription and / or expression of a coding sequence, the promoter is considered to be "operably linked" to a coding sequence (wherein the coding sequence is understood to be "under the control" of the promoter). Typically, when two nucleotide sequences are operably linked, the two nucleotide sequences will be in the same orientation and generally also in the same reading frame. The two nucleotide sequences are also generally substantially contiguous, but this may not be required.

[0300] Preferably, any regulatory elements of the vector are such that they are able to provide their intended biological function in the intended host cell or host organism.

[0301] For example, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, which means, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked.

[0302] 5.7 Composition

[0303] The present technology also provides a composition comprising at least one ISVD or polypeptide as disclosed herein, at least one nucleic acid molecule encoding an ISVD or polypeptide as disclosed herein, or at least one vector comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition 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.

[0304] 5.8 Host Organisms

[0305] The present technology also relates to host cells or host organisms comprising an ISVD or polypeptide as disclosed herein, a nucleic acid encoding an ISVD or polypeptide as disclosed herein, and / or a vector comprising a nucleic acid molecule encoding an ISVD or polypeptide as disclosed herein.

[0306] Suitable host cells or host organisms are clear to those skilled in the art and are, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

[0307] 5.9 Methods and Uses of ISVD and Peptides

[0308] The present technology also provides a method for producing an ISVD or polypeptide as disclosed herein. The method 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 separation and / or purification steps. Specifically, the method may include:

[0309] a) expressing a nucleic acid sequence encoding an ISVD or a polypeptide according to the technology of the present invention in a suitable host cell or (non-human) host organism or in another suitable expression system; optionally followed by:

[0310] b) isolating and / or purifying the polypeptide.

[0311] Suitable host cells or host organisms for the purpose of preparation will be clear to the skilled person and may be, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

[0312] Typically, multispecific-multivalent polypeptides of the current technology combine high affinity antigen recognition on target cells with T cell activation, resulting in activation that is independent of the natural specificity of the T cells. The first ISVD of the polypeptide of the present technology has high affinity / specific binding to / with effector cells (preferably the TCR of the effector cells, and even more preferably the constant domain of the TCR).

[0313] Effector cells are cells comprising a TCR complex, preferably immune cells, such as T cells, preferably CD4+T helper cells (also referred to as CD4 cells, T helper cells or T4 cells), more preferably cytotoxic T cells (also referred to as TC cells, CTLs or CD8+T cells) or natural killer T cells (NKT cells). In some embodiments, the cells are present in vivo. In some embodiments, the cells are present in vitro. The effector cells of the present technology particularly relate to mammalian cells, preferably primate cells, and even more preferably human cells.

[0314] As used herein, "T cell activation" refers to one or more cellular responses of T cells (e.g., cytotoxic T cells), such as selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, expression of activation markers, and redirected target cell lysis. The multispecific-multivalent polypeptides of the current technology are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art as described herein, for example, as described in WO 99 / 54440 or Schlereth et al. 2005 (Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy] 20: 1-12), or as illustrated in the Examples or below.

[0315] In an embodiment, the present technology relates to a multispecific-multivalent polypeptide as 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 other ISVDs bind to an antigen on a target cell.

[0316] In embodiments, the present technology relates to a multispecific-multivalent polypeptide as described herein, wherein said T cell activation is dependent on presentation of said polypeptide bound to said first antigen on a target cell to the T cell.

[0317] 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 procaspase 3 and / or 7, fragmentation of nuclear DNA and / or cleavage of the caspase substrate poly (ADP ribose) polymerase. Preferably, redirected lysis of target cells by the multispecific-multivalent polypeptides is independent of T cell receptor specificity, the presence of MHC class I and / or β2 microglobulin, and / or any co-stimulatory stimulation.

[0318] In embodiments, the present technology relates to multispecific-multivalent polypeptides as described herein, wherein the T cell activation is independent of MHC recognition.

[0319] The multispecific-multivalent polypeptides of the present invention have been shown to activate previously unstimulated peripheral polyclonal CD8 + and CD4 + Redirected lysis of positive T cells. Redirecting lysis of target cells by recruiting T cells via the polypeptides of the present invention involves cytolytic synapse formation and the delivery of perforins and granzymes. Cell lysis by T cells has been described in, for example, Atkinson and Bleackley, 1995 (Crit. Rev. Immunol [Review of Immunology] 15 (3-4): 359-384). Preferably, the engaged T cells are able to continuously lyse target cells and are not affected by immune escape mechanisms that interfere with peptide antigen processing and presentation or clone T cell differentiation (see, for example, WO 2007 / 042261). In vitro, redirected lysis was observed at low picomolar concentrations, indicating that very small amounts of polypeptides of the present invention need to be bound to target cells to trigger T cells. Therefore, the present invention relates to effective polypeptides. Preferably, the multispecific-multivalent polypeptides of the current technology mediate the killing of target cells (e.g., cancer cells), such as stimulating T cells to form holes and deliver pro-apoptotic components of cytotoxic T cell granules.

[0320] In an embodiment, the present technology relates to a multispecific-multivalent polypeptide as described herein, wherein said T cell activation causes one or more cellular responses of said T cell, wherein said cellular response is 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.

[0321] As used herein, the term "efficacy" is a measure of the biological activity of an agent such as a polypeptide or ISVD. The efficacy of an agent can be determined by any suitable method known in the art, such as described in the experimental section. Cell culture-based efficacy assays are generally preferred formats for determining biological activity because they measure the physiological response triggered by the agent and can generate results in a relatively short period of 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, and assays for measuring the induction / inhibition of functional essential proteins or other signaling molecules (such as phosphorylated proteins, enzymes, cytokines, cAMP, etc.), Ramos B cell exhaustion models, T cell-mediated tumor cell killing assays (e.g., shown in the Examples section), all of which are well known in the art.

[0322] In one embodiment, the multispecific-multivalent polypeptides of the present technology show improved potency and efficacy in both human and cynomolgus monkey T cell-mediated killing assays compared to the same format in which the first ISVD is replaced by T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969).

[0323] "Efficacy" (of a polypeptide of the present technology) measures the maximum intensity of the effect itself at saturating polypeptide concentration. Efficacy indicates the maximum response that can be achieved by a polypeptide of the present technology. It refers to the ability of a polypeptide to produce a desired (therapeutic) effect.

[0324] Thus, in embodiments, the present technology relates to multispecific-multivalent polypeptides as described herein, wherein said T cell activation causes inhibition of said target cell activity, e.g., thereby delaying or minimizing the spread of target cells, inhibiting or delaying the growth and / or proliferation of target cells, and / or killing target cells (e.g., causing regression of disorders and / or symptoms) by more than about 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%, such as 70%, 80%, or even more than 90%, such as 100%. In a specific embodiment, T cell activation causes target cell lysis by more than about 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%.

[0325] In one embodiment, a multispecific-multivalent polypeptide as described herein is selected from at most about 10 -9 M, up to about 10 -10 M, and at most about 10 -11The EC50 value of the group consisting of M causes human T cells to lyse target cells, as determined in a T cell-mediated killing assay. For example, EC50 is determined in a flow cytometry-based assay as shown in Example 8 or 9 or in an impedance-based cytotoxicity assay as shown in Example 10.

[0326] In one embodiment, a multispecific-multivalent polypeptide as described herein is selected from at most about 10 -9 M, and at most about 10 -10 M, and at most about 10 -11 The EC50 value of the group consisting of M causes non-human primate T cells to lyse target cells, as determined in a T cell-mediated killing assay. For example, EC50 is determined in a flow cytometry-based assay as shown in Example 8 or 9 or in an impedance-based cytotoxicity assay as shown in Example 10.

[0327] In a flow cytometry-based T cell-mediated killing assay, multispecific-multivalent polypeptides having a TCR-binding ISVD according to the present technology having E at position 61 and Y or S at position 99 showed particularly high potency. Thus, in one embodiment, the multispecific-multivalent polypeptide comprises a TCR-binding ISVD having E at position 61 and Y or S at position 99. In one embodiment, the multispecific-multivalent polypeptide has at most about 5.10 -10 M, up to about 10 -10 M, such as 5.10 -11 M causes human T cells to lyse target cells as determined in a flow cytometry-based T cell-mediated killing assay or an impedance-based T cell-mediated killing assay. In one embodiment, the multispecific-multivalent polypeptide comprises an ISVD that binds a TCR with E at position 61 and Y, Q, T, or S at position 99, and has an EC50 value of at most about 5.10 -10 M, up to about 10 -10 M, such as up to 5.10 -11 M causes human T cells to lyse target cells with an EC50 value as determined in a flow cytometry-based T cell-mediated killing assay or an impedance-based T cell-mediated killing assay.

[0328] In one embodiment, the multispecific-multivalent polypeptide as 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 wherein the first ISVD is replaced by ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969).

[0329] In one embodiment, the multispecific-multivalent polypeptides of the present technology cause human T cells to lyse target cells with an improved (lower) EC50 value as determined in a T cell-mediated killing assay compared to the same polypeptide in which the first ISVD is replaced by ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). For example, the EC50 is determined as described in the Examples section.

[0330] In one embodiment, the multispecific-multivalent polypeptides of the present technology cause non-human primate T cells to lyse target cells with an improved (lower) EC50 value as determined in a T cell-mediated killing assay compared to the same polypeptide in which the first ISVD is replaced with ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). For example, the EC50 is determined as described in the Examples section.

[0331] In one embodiment, the multispecific-multivalent polypeptides comprising a first ISVD of the present technology as described herein show improved binding to the constant domains of human and / or non-human primate TCRs compared to the same polypeptide in which the first ISVD is replaced with ISVD T0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969) (i.e., a polypeptide comprising as the first ISVD an ISVD having the CDR sequence of T0170056G05).

[0332] The binding characteristics of the ISVDs of the present technology are discussed in more detail below (Section 5.3; "Specificity").

[0333] In some embodiments, the multispecific-multivalent polypeptides of the present technology have an association rate constant (k) for binding to a human TCR selected from the group consisting of: on ): 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 .

[0334] In some embodiments, a multispecific-multivalent polypeptide as described herein has a polypeptide selected from at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 105 M -1 s -1 and at least about 10 6 M -1 s -1 Composed of panels combining non-human primate TCR k on .

[0335] In some embodiments, the multispecific-multivalent polypeptides of the present technology have a member selected from at most about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 and at most about 10 -4 s -1 The dissociation rate constant (k off ).

[0336] In some embodiments, a multispecific-multivalent polypeptide as described herein has a polypeptide selected from at most about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 and at most about 10 -4 s -1 Composed of panels combining non-human primate TCR k off .

[0337] In some embodiments, the multispecific-multivalent polypeptides of the present technology have a member selected from at most about 10 -5 M, up to about 10 -6 M, up to about 10 -7 M, up to about 10 -8 M and at most about 10 -9 The affinity of the group consisting of M for binding to human TCR (K D ).

[0338] In some embodiments, the multispecific-multivalent polypeptides of the present technology have a member selected from at most about 10 -5 M, up to about 10 -6 M, up to about 10 -7 M and at most about 10 -8 The K of the group consisting of M binding non-human primate TCR D .

[0339] In one embodiment, k is measured by surface plasmon resonance (SPR). on , k off or K DFor example, k is determined as shown in the Examples section on , k off or K D .

[0340] In another embodiment, k is measured by bio-layer interferometry (BLI). on , k off or K D .

[0341] In one embodiment, the multispecific-multivalent polypeptides of the present technology have an improved (lower) K compared to the same polypeptide wherein the first ISVD is replaced by ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). D Binds to human TCR.

[0342] In one embodiment, the multispecific-multivalent polypeptides of the present technology have an improved (lower) K compared to the same polypeptide wherein the first ISVD is replaced by ISVDT0170056G05 (disclosed as SEQ ID NO: 50 in WO 2016180969). D Binding to non-human primate TCR.

[0343] The polypeptide, nucleic acid molecule or vector as described, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule or vector, preferably the polypeptide or a composition comprising the polypeptide, can be used as a medicament.

[0344] Therefore, the present technology provides a polypeptide, a nucleic acid molecule or a vector as described, or a composition comprising an ISVD or a polypeptide, a nucleic acid molecule or a vector for use as a medicament.

[0345] Also provided is a polypeptide, nucleic acid molecule or vector as 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.

[0346] Additionally, provided are polypeptides, nucleic acid molecules or vectors as described herein, or compositions comprising an ISVD or polypeptide, nucleic acid molecule or vector for use in treating cancer.

[0347] Also provided is a method for preventing, treating or ameliorating a disease, wherein the method comprises administering a pharmaceutically active amount of a polypeptide, nucleic acid molecule or vector as described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule or vector to a subject in need thereof.

[0348] 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 a pharmaceutically active amount of a polypeptide, a nucleic acid molecule or a vector as described herein, or a composition comprising an ISVD or a polypeptide, a nucleic acid molecule or a vector to a subject in need thereof.

[0349] In addition, a method for treating cancer is provided, wherein the method comprises administering a pharmaceutically active amount of a polypeptide, nucleic acid molecule or vector as described herein, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule or vector to a subject in need thereof.

[0350] Further provided is the use of a polypeptide, a nucleic acid molecule or a vector as described herein, or a composition comprising the polypeptide, a nucleic acid molecule or a vector in the preparation of a medicament.

[0351] Also provided is the use of a polypeptide, nucleic acid molecule or vector as described herein, or a composition comprising the polypeptide, nucleic acid molecule or vector, in the preparation of a medicament for preventing, treating or ameliorating a disease selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases and autoimmune diseases.

[0352] Further provided is the use of a polypeptide, a nucleic acid molecule or a vector as described herein, or a composition comprising the polypeptide, nucleic acid molecule or vector, in the preparation of a pharmaceutical composition, which is preferably for the treatment of cancer.

[0353] The "subject" referred to in the context of the present technology can be any animal, preferably a mammal. Among mammals, humans and non-human mammals can be distinguished. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cattle, horses, sheep, goats or porcine animals) or animals generally used for research purposes and / or for producing antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates such as cynomolgus monkeys, or camelids such as llamas or alpacas).

[0354] In the context of prophylactic and / or therapeutic purposes, the subject may be any animal, and more particularly any mammal, but is preferably a human subject.

[0355] As used herein, the terms "treat," "treatment," and "treating," in the context of administering one or more therapies to a subject, refer to a reduction in the progression, severity, and / or duration or improvement of a disorder associated with a hyperproliferative cellular disorder (e.g., cancer) and / or an improvement in one or more symptoms thereof resulting from 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 improvement in the progression, severity, and / or duration of a hyperproliferative cellular disorder (e.g., cancer), referring to a reduction in cancer cells by 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%, relative to a control (e.g., a negative control such as phosphate-buffered saline). In other embodiments, the terms "treat," "treatment," and "treating," in the context of administering one or more therapies to a subject, refer to a reduction or improvement in the progression, severity, and / or duration of a hyperproliferative cellular disorder (e.g., cancer), and refer to no change in the number of cancer cells, a reduction in hospital stay, a decrease in mortality, or an increase in survival time in a subject with cancer.

[0356] Substances (including polypeptides, nucleic acid molecules and vectors) or compositions can be administered to a subject by any suitable route of administration, for example, enteral (such as oral or rectal) or parenteral (such as epidermal, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal or transmucosal). Parenteral administration, such as intramuscular, subcutaneous or intradermal administration, is preferred. Most preferred is subcutaneous administration.

[0357] An effective amount of a polypeptide, nucleic acid molecule or vector as described, or a composition comprising an ISVD or a polypeptide, nucleic acid molecule or vector, can be administered to a subject to provide the desired therapeutic effect.

[0358] One dose or multiple doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0359] Table A-4: Sequences of ISVD

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] Table A-5: Sequences of the CDR regions of ISVD variants (according to Kabat definition)

[0374]

[0375]

[0376]

[0377]

[0378] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to the skilled person. For example, reference is made to standard manuals such as Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, (2nd 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, (2nd ed.) University of California Press, Berkeley, CA. California Press), Roitt et al. (2001, Immunology (6th ed.) Mosby / Elsevier, Edinburgh), Roitt et al. (2001, Roitt's Essential Immunology (10th ed.) Blackwell Publishing, UK), and Janeway et al. (2005, Immunobiology (6th ed.) Garland Science Publishing / Churchill Livingstone, New York), as well as the general background technology cited in this article.

[0379] As will be clear to a skilled person, unless otherwise stated, all methods, steps, techniques, and operations not specifically described in detail can be performed and have been performed in a manner known per se. For example, reference is again made to the standard handbooks and general background art mentioned herein and to the further references cited therein; and to reviews such as 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, Tumour Biol. 26(1):31-43), which describe techniques for protein engineering (such as affinity maturation) and other techniques for improving the specificity and other desired properties of proteins (such as immunoglobulins).

[0380] As used herein, the term "sequence" (for example in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the relevant amino acid sequence, unless the context requires a more limited interpretation.

[0381] An "amino acid sequence" should be interpreted as referring to a single amino acid or an unbranched sequence of two or more amino acids, depending on the context. A nucleotide sequence should be interpreted as meaning an unbranched sequence of 3 or more nucleotides.

[0382] The amino acids are those L-amino acids commonly found in naturally occurring proteins and are listed in Table B-1. Those amino acid sequences containing D-amino acids are not intended to be covered by this definition. Any amino acid sequence containing post-translationally modified amino acids can be described as an amino acid sequence with modified positions (e.g., hydroxylation or glycosylation) as originally translated using the symbols shown in Table B-1; but these modifications are not explicitly shown in the amino acid sequence. This definition includes any peptide or protein that can be expressed as sequence-modified linkages, crosslinks and end caps, non-peptidyl bonds, etc.

[0383] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure and each has the same meaning for the purposes of this disclosure. Each term refers to an organic compound composed of a linear chain of two or more amino acids. The compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. The skilled artisan will understand that, although there is no art-recognized dividing point in the number of amino acids that distinguishes polypeptides from proteins, polypeptides typically contain fewer amino acids than proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; and proteins are typically prepared in vitro or in vivo by recombinant methods known in the art.

[0384] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," etc. should be construed in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0385] For example, when a nucleotide sequence, an amino acid sequence or a polypeptide is referred to as "comprising" another nucleotide sequence, an amino acid sequence or a polypeptide, respectively, or as "consisting essentially of" another nucleotide sequence, an amino acid sequence or a polypeptide, this may mean that the latter nucleotide sequence, an amino acid sequence or a polypeptide, respectively, has been incorporated into the first-mentioned nucleotide sequence, an amino acid sequence or a polypeptide, respectively, but more generally, this generally means that the first-mentioned nucleotide sequence, an amino acid sequence or a polypeptide, respectively, contains within its sequence a stretch of nucleotides or amino acid residues having the same nucleotide sequence or amino acid sequence as the latter sequence, respectively, regardless of how the first-mentioned sequence has actually been generated or obtained (this may be, for example, by any suitable method described herein). By way of non-limiting example, when a polypeptide of the present invention is referred to as comprising an immunoglobulin single variable domain, this may mean that the immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide of the present invention, but more generally, this generally means that the polypeptide of the present invention contains within its sequence the sequence of these immunoglobulin single variable domains, regardless of how the polypeptide of the present invention has been generated or obtained. Furthermore, when a nucleic acid or nucleotide sequence is referred to as comprising another nucleotide sequence, the first-mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of the 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).

[0386] When an amino acid sequence or a polypeptide is said to "essentially consist of" an immunoglobulin single variable domain, it means that the amino acid sequence or polypeptide is identical to the immunoglobulin single variable domain or corresponds to a polypeptide or amino acid sequence with the addition of a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, at the amino terminus, carboxyl terminus, or both the amino terminus and carboxyl terminus of the immunoglobulin single variable domain. When "consisting of" is used, it means that the amino acid sequence or polypeptide is identical to the immunoglobulin single variable domain.

[0387] It must 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 "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or substitute the methods described herein.

[0388] Unless otherwise indicated, the term "at least" in front of a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalent forms of the specific embodiments of the technology of the present invention described herein using only routine experiments. Such equivalent forms are intended to be covered by the technology of the present invention.

[0389] The term "and / or" wherever used herein includes the meanings of "and", "or" and "all or any other combinations of the elements connected by the term".

[0390] 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.

[0391] 6 Examples

[0392] 6.1 Example 1: Generation and dissociation rate determination of T cell engagement (TCE) ISVD Generation of variants

[0393] Amino acids in positions 61, 99, and 103 (Kabat numbering) in ISVD T017000700 (SEQ ID NO: 1) were substituted to generate different variants that maintained function and overall developability but had reduced or no isomerization at position 61 and reduced or no oxidation at positions 99 and / or 103 (Kabat numbering).

[0394] ISVD T017000700 and the generated variant ISVDs are shown in Table A-4 above. All ISVDs were tagged with a FLAG-HIS-tag (FLAG3-HIS6, SEQ ID NO: 77) so that they could be analyzed in further experiments.

[0395] Dissociation rate assay for human TCR αβ-zipper protein

[0396] Binding of the TCE ISVD variants to TCRαβ was assessed by determining the dissociation rate constant (kd) against recombinant human TCRαβ-zipper protein using SPR-based assays on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.) or an SPR-32 instrument (Bruker Daltonics SPR).

[0397] Setup on the ProteOn XPR36

[0398] Binding of crude extract monovalent TCE-FLAG3-HIS6 ISVD constructs to human TCRαβ-zipper protein (huTCR(2XN9)-zipper, produced in-house) was probed by surface plasmon resonance (SPR) (Bio-rad Laboratories, Inc., ProteOn XPR36). Targets were immobilized on a GLC sensor chip (short matrix, regular capacity) using standard amine coupling chemistry. Crude extracts of ISVD constructs were injected (in duplicate) at a dilution of 1:10 to assess binding parameters. Each ISVD was injected for 120 s and dissociation was assessed during 600 s.

[0399] The data were double referenced by subtracting the reference analyte lane and blank buffer injections. The dissociation rate constant (kd) for each interaction was calculated using ProteOn Manager 3.1.0 (Bio-rad Laboratories, Inc., version 3.1.0.6) applying the Langmuir 1:1 interaction model.

[0400] Settings on SPR-32

[0401] Binding of crude extract monovalent TCE-FLAG3-HIS6 ISVD constructs to human TCRαβ-zipper protein (huTCR(2XN9)-zipper, produced in-house) was probed by surface plasmon resonance (SPR) (Bruker Daltonics, 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 dilution of 1:10 to assess binding parameters. Each ISVD was injected for 120 s and dissociation was assessed during 600 s.

[0402] Data were double referenced by subtracting reference points and blank buffer injections. Dissociation rate constants (kd) for each interaction were calculated using the SPR-32 Analyser software (Bruker Daltonics, version 3.2.0.19) applying the Langmuir 1:1 interaction model.

[0403] The measurement results can be seen in Tables 1-3.

[0404] Table 1 SPR-based off-rate assay of TCE ISVD variants and reference TCE ISVD constructs at position 61 (Kabat numbering) against human TCR αβ-zipper protein

[0405]

[0406] As can be seen in Table 1, binding of the TCE ISVD variants with a substitution at position 61 (Kabat numbering) to human TCR αβ-zipper protein was maintained at a very similar level compared to the reference (T017000700) for all constructs tested (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 dissociation rate assays.

[0407] Table 2: SPR-based off-rate assays of TCE ISVD variants and reference TCE ISVD constructs at position 99 and position 61 fixed to E (Kabat numbering) against human TCR αβ-zipper protein

[0408]

[0409] As can be seen in Table 2, TCE ISVD variants with substitutions at position 99 (Kabat numbering) were tested for binding to human TCR α β-zipper protein in a construct where residue 61 was fixed to glutamic acid (E). In an SPR-based dissociation rate assay, all tested variants (i.e., with residues Y, A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V at position 99) maintained detectable levels of binding to human TCR α β-zipper protein, however, the dissociation rate ranged from up to about 100 times faster when compared to reference T017000700.

[0410] Table 3: SPR-based off-rate assays of TCE ISVD variants and reference TCE ISVD constructs at position 103 (Kabat numbering) against human TCR αβ-zipper protein

[0411]

[0412] As can be seen in Table 3, as assessed by SPR-based dissociation rate assays, for constructs with amino acid residues A, E, F, H, I, K, L, Q, R, S, T, V, Y at position 103, the binding of the TCEISVD variants with substitutions at Kabat position 103 to human TCR αβ-zipper protein was maintained at a very similar level compared to the reference T017000700, while no binding was observed for constructs with amino acid residues G or P at position 103.

[0413] Based on the results of the SPR analysis, it can be concluded that when the amino acids at one or more of positions 61, 99 and 103 (Kabat numbering) in the sequence of reference T017000700 are substituted, a functional ISVD binding to TCR can be obtained.

[0414] Several of the generated ISVDs were selected and further analyzed in additional experiments, which are discussed below.

[0415] 6.2 Example 2: Determination of biophysical properties (Tm, Tagg and oligomerization) of TCE ISVD variants

[0416] Determination of melting temperature in thermal shift assay (TSA)

[0417] Thermal shift assays (TSA) were performed in 96-well plates on a LightCycler 480II machine (Roche). One ISVD was analyzed per row at the following pH: 4, 5, 6, 7, 8, and 9. 5 μL of ISVD sample (0.8 mg / mL in PBS) was added to 5 μL of Sypro Orange (40x in MilliQ water; Invitrogen catalog number S6551) and 10 μL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, and 115 mM NaCl, pH range 3.5 to 9) in each well. The applied temperature gradient (from 37°C to 99°C at a rate of 0.03°C / s) induced the unfolding of the ISVD, thereby exposing the hydrophobic patches. Sypro Orange binds to those hydrophobic patches, resulting in an increase in fluorescence intensity (Ex / Em=465 / 580nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 was used as a measure of the melting temperature.

[0418] Aggregation onset temperature determination (Tagg assay)

[0419] The temperature at which the ISVD protein begins to aggregate (=aggregation starting temperature=Tagg) is determined by dynamic light scattering (DLS) using a DynaPro plate reader (Wyatt). To this end, an ISVD with a 3xFLAG-HIS6 tag (SEQ ID NO: 77) produced in Escherichia coli (E. coli), purified by preparative SEC via IMAC, filtered (0.22 μm), and a concentration of 1 mg / mL (D-PBS) was used. After thawing, the sample was filtered through a 0.1 μm membrane and centrifuged at 14000 rpm for 5 min. 30 μL of the sample (4 replicates) was heated from 40°C to 80°C at a constant rate of 0.25°C / min, and the light scattering intensity was continuously recorded. The hydrodynamic radius derived from the measured intensity was plotted against temperature to determine the temperature (=Tagg, °C) at which the radius began to increase.

[0420] Oligomerization assay

[0421] The oligomerization tendency of monovalent ISVDs under stress conditions (1 week at 45°C) was investigated by analytical size exclusion chromatography (SE-HPLC). For this purpose, ISVDs with a 3xFLAG-HIS6 tag produced in E. coli, purified via IMAC followed by preparative SEC, filtered (0.22 μm), at a concentration of 1 mg / mL (DPBS) were used. The SE-HPLC profiles of two 100 μL aliquots were compared: one sample was incubated at -20°C for 1 week and the other sample was incubated at 45°C for 1 week. The samples were cleared by centrifugation at 20000 RCF for 5 minutes and then analyzed on an Acquity UPLC BEH200 SEC (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 stressed samples (+45°C) and unstressed samples (-20°C) was calculated and reported as Δ% Oligomer (=% Oligomer 1W 45°C - % Oligomer T0).

[0422] result

[0423] Purified variants of TCE ISVD with selected amino acid substitutions at positions 61, 99 and 103 (Kabat numbering) (see Table A-4) were characterized with respect to melting temperature (Tm), aggregation initiation (Tagg) and oligomerization after 1 week at 45°C as shown in Tables 4 and 5.

[0424] At position 61 of TCE ISVD, the amino acid residue variants A, E, P, Q, R, S, V were selected because these are the most frequently occurring residues at this position in human VH genes.

[0425] 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 least affected the off-rate of binding of each ISVD variant to TCRαβ (Table 2). Residues A, H, Q, S, T were selected because each TCE ISVD represented a range of different off-rates for binding to TCRαβ.

[0426] In approximately 95% of human VH genes and naturally occurring ISVDs [internal analysis of sequences], tryptophan (W) is found at kabat position 103. Other residues found at position 103 of naturally occurring ISVD are Y, R and S. Therefore, Y, R and S are selected for the amino acid residue variant at position 103.

[0427] Table 4: Biophysical properties (Tm, Tagg and oligomerization) of TCE ISVD variants at positions 61 and 99 and reference TC017000700

[0428]

[0429]

[0430] Table 5: Biophysical properties (Tm, Tagg and oligomerization) of TCE ISVD variants at position 103 and reference TC017000700

[0431]

[0432] As can be seen from Table 4, T017000978, T017000992, T017000999 and T017001002 have maintained or improved all three properties compared to reference T017000700. For Tm, an increase of 0°C to 1°C was observed, for Tagg, an increase of 1°C to 5°C was observed, and a decrease in oligomer fraction from 0.2% of the reference to 0.0% or 0.1% of the ISVD according to the present technology was observed.

[0433] Surprisingly, as can be seen in Table 5, although TCR binding of the ISVD variants at position 103 was not affected, substitution at this position resulted in a decrease in melting temperature by 7.3°C to 15.1°C and a decrease in aggregation onset temperature by 7°C to 20°C compared to the reference ISVD. This suggests that W103 may be important for the biophysical stability of the TCE ISVD.

[0434] Therefore, the next step was to retain the tryptophan at position 103 while changing the amino acid residues at positions 61 and 99.

[0435] 6.3 Example 3: Determination of the affinity of TCE ISVD variants for human and cynomolgus monkey TCRαβ proteins

[0436] Purified monovalent TCE-FLAG3-HIS6 and TCE-HIS6 ISVD constructs were probed for binding to human TCRαβ-zipper protein (huTCR(2XN9)-zipper, produced in-house) and cynomolgus monkey TCRαβ-zipper protein (cyTCR(AEA41865)-zipper, produced in-house) by surface plasmon resonance (SPR) (Bio-Rad Laboratories, Inc., ProteOn XPR36). Both targets were immobilized on a GLC sensor chip (short matrix, regular capacity) using standard amine coupling chemistry. ISVD constructs were injected at 6 different concentrations in a multi-cycle kinetic (MCK) experiment. Each concentration of ISVD was injected for 120 s and dissociation was assessed during 600 s.

[0437] The data were double referenced by subtracting the reference analyte lane and blank buffer injection. Affinity constants (ka, kd, and K) were calculated using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc., version 3.1.0.6) using the Langmuir 1:1 interaction model. D ).

[0438] The results of the affinity measurements of the TCE ISVD amino acid variants for human and cynomolgus monkey TCR αβ-zipper proteins are summarized below in Tables 6 and 7. T017000700 was used as a reference.

[0439] Table 6: SPR-based kinetic assay of TCE ISVD variants and reference T017000700 binding to human TCR αβ-zipper fusion protein

[0440]

[0441]

[0442] Table 7: SPR-based kinetic assay of TCE ISVD variants and reference T017000700 binding to cynomolgus monkey TCR αβ-zipper fusion protein

[0443]

[0444] As can be seen in Tables 6 and 7, the affinities of the TCE ISVD variants remained comparable to the reference TCE ISVD for all constructs tested, except T017000995, T017000999 and T017001001 (K D value within 2.5 times).

[0445] Each ISVD showed a K of 1.1% for cynomolgus monkey TCRαβ protein and 1.1% for human TCRαβ protein. D 2 to 4 times of K D This suggests that ISVDs according to the present technology have the potential to be developed and used in human therapeutic applications.

[0446] 6.4 Example 4: Binding of monovalent TCE ISVD to purified primary human T cells

[0447] TCE ISVDs according to the present technology were tested for binding to human T cells in flow cytometry. The following ISVDs were tested: T017000700 (reference), T017000978, T017000995, T017000999, and T017001001. Negative controls were also added. These included unstained cells (US), cells stained with GaM-PE only, and cells stained with Cells stained with M2 antibody and GaM-PE (a-FLAG+GaM-PE) served as negative controls.

[0448] Briefly, cells were harvested and transferred to a V-bottom 96-well plate (5x1E4 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)) at 4°C for 3.5 hours.

[0449] Next, cells were washed three times with FACS buffer and incubated with 1 μg / mL monoclonal The M2 antibody (Sigma F1804) was incubated at 4°C for 30 min, washed again, and incubated at 4°C with a 1 / 100 dilution of R-Phycoerythrin-conjugated AffiniPure F(ab')2Fragment Goat Anti-mouse IgG, Fc y FragmentSpecific (Jackson Immunoresearch, 115-116-071; referred to as GaM-PE) for 30 min.

[0450] Subsequently, the cells were resuspended in culture medium supplemented with 5 nM -3 iodide (642 / 661) (Life Technologies-Molecular Probes, T3605) in FACS buffer to distinguish live cells from dead cells. Cells were analyzed by flow cytometry (Miltenyi) using FlowLogic software. First, the P1 population, which accounted for more than 80% of the total cell population, was selected based on the FSC-SSC distribution. -3 iodide-positive (dead) cells, and the mean fluorescence intensity PE value was calculated.

[0451] The measurement results can be found in Figure 1As expected, no staining of human T cells was observed for the control condition without TCE ISVD. In addition, it can be seen from this figure that T017000978 has comparable staining to the reference T017000700 based on the measured mean fluorescence intensity. Variants T017000995, T017000999 and T017001001 have much lower staining, which corresponds to the binding affinity determined on the zipper protein as shown in Table 6.

[0452] 6.5 Example 5: Generation of TCE-CD123-ALB construct

[0453] The TCE ISVD variants are formatted as multi-specific ISVD constructs together with tumor anchoring ISVD building blocks for CD123 and ISVD building blocks for human serum albumin (connected by 9GS linkers). The TCE variant building block is placed at the N-terminal position (position 1) or the second position (position 2); then the anti-CD123 building block is placed at position 2 or position 1, respectively. In all constructs, the anti-human serum albumin building block is placed at C-terminal position 3. In addition to the substitutions at positions 61 and 99 (Kabat numbering) in the TCE building block, when the TCE building block is located at position 1 in the multi-specific construct, the first residue of the TCE building block is maintained as E or changed to D. E1D mutations are usually introduced to avoid pyroglutamic acid formation. The generated constructs are listed in Table 8 below. Reference construct T017001017 includes reference ISVDT017000700 that binds to TCR.

[0454] Table 8: List of TCE-CD123-ALB and CD123-TCE-ALB constructs

[0455]

[0456]

[0457] The following TCE ISVD building blocks were used in the constructs listed above:

[0458] For T017001017 and T017001027, the reference TCE ISVD T017000700 (SEQ ID NO: 1) was used.

[0459] For T017001018 and T017001028, T017000978 (SEQ ID NO: 37) was used.

[0460] For T017001019, T017001024 and T017001029, T017001001 (SEQ ID NO: 52) was used. T017001024 further contained an E1D mutation in TCE ISVD.

[0461] For T017001020, T017001025 and T017001030, T017000999 (SEQ ID NO: 50) was used. T017001025 further contained an E1D mutation in TCE ISVD.

[0462] For T017001021, T017001026 and T017001031, T017000995 (SEQ ID NO 46) was used. T017001026 further contained an E1D mutation in TCE ISVD.

[0463] For T017001022, T017000975 (SEQ ID NO: 34) was used. This is T017000700 with an E1D mutation.

[0464] For T017001023, T017000991 (SEQ ID NO: 42) was used. This is T017000978 with an E1D mutation.

[0465] 6.6 Example 6: Determination of the affinity of TCE-CD123-ALB constructs for human and cynomolgus monkey TCR αβ-zipper proteins

[0466] Purified TCE-CD123-ALB and CD123-TCE-ALB ISVD constructs were probed for binding to human TCRαβ-zipper protein (huTCR(2XN9)-zipper, produced in-house) and cynomolgus monkey TCRαβ-zipper protein (cyTCR(AEA41865)-zipper, produced in-house) by surface plasmon resonance (SPR) (Cytiva, Biacore 8K+). Both targets were immobilized on a CM5 sensor chip using standard amine coupling chemistry. In a multi-cycle kinetic experiment, a total of 15 ISVD constructs (Table 8) were injected at 12 different concentrations (serial dilutions from 20 μM to 0.84 nM). Each concentration of ISVD was injected for 180 s, and dissociation was assessed during 600 s.

[0467] The data were double referenced by subtracting the reference flow cell (FC) and blank buffer injections. Affinity constants (ka, kd and K) were calculated using the Langmuir 1:1 interaction model using Biacore Insight Evaluation software (Science, Inc., version 3.0.12.15655). D The affinity measurements of the 15 multivalent ISVD constructs (Table 8) to human TCRαβ protein are summarized in Table 9, and the affinity measurements to cynomolgus monkey TCRαβ protein are summarized in Table 10.

[0468] Table 9: Determination of affinity of TCE-CD123-ALB constructs for human TCR αβ-zipper protein

[0469]

[0470] Table 10: Determination of affinity of TCE-CD123-ALB constructs for cynomolgus monkey TCR αβ-zipper protein

[0471]

[0472]

[0473] Tables 9 and 10 show that the TCE ISVD variants according to the present invention retain their ability to bind to TCR when used in constructs. This shows that the TCE ISVD variants according to the present invention can be used in combination with targeted ISVD. Therefore, they are suitable for being developed and used in target-specific therapeutic applications.

[0474] 6.7 Example 7: Determination of Binding of TCE-CD123-ALB ISVD Constructs to Purified Primary Human T Cells

[0475] Flow cytometry was used to determine the dose-dependent binding of the TCE-CD123-ALB ISVD constructs according to the technology of the present invention to primary human T cells. The following constructs were used: T017001017 (reference), T017001018, T017001019, T017001020, and T017001021. Unstained cells (US), cells stained with GaM-PE only, and cells stained with ABH0074 and GaM-PE only (ABH0074+GaM-PE) were included as negative controls.

[0476] Briefly, cells were harvested and transferred to a V-bottom 96-well plate (5x1E4 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)) at 4°C for 2.5 hours.

[0477] Next, the cells were washed three times with FACS buffer and incubated with 10 μg / mL anti-VHH mAb (prepared in-house) at 4°C for 30 min, washed again, and incubated with 1 / 100 diluted R-phycoerythrin-conjugated AffiniPure F(ab')2 fragment goat-specific anti-mouse IgG Fcγ fragment (Jackson ImmunoResearch, 115-116-071) at 4°C for 30 min. Subsequently, the cells were resuspended in 4% paraformaldehyde supplemented with 5 nM -3 iodide (642 / 661) (Life Technologies-Molecular Probes, T3605) in FACS buffer to distinguish live cells from dead cells. Cells were analyzed by flow cytometry (Miltenyi Biotec) using FlowLogic software. First, the P1 population, which accounted for more than 80% of the total cell population, was selected based on the FSC-SSC distribution. -3 iodide-positive (dead) cells, and the mean fluorescence intensity PE value was calculated.

[0478] The measurement results are shown in Figure 2 As expected, no staining of human T cells was observed for the control condition without TCE ISVD. In addition, as can be seen from the figure, T017001018 (containing TCE ISVD T017000978) and reference T017001017 (containing TCE ISVD T017000700) bind to human T cells with similar EC50. On the other hand, 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 on the zipper protein as shown in Table 9.

[0479] 6.8 Example 8: Multispecific TCE-CD123-ALB construct induces human T cell-mediated killing of target cells

[0480] Flow cytometry-based cytotoxicity assay

[0481] In the cytotoxicity assay based on flow cytometry, human primary T cells are used as effector cells and non-adhesive target cells, and the ISVD constructs according to the technology of the present invention are further characterized for redirecting T cell-mediated killing. According to the manufacturer's instructions, PKH26 red fluorescence cell linker kit (Sigma, PKH26GL-1KT) is used to mark target cells with 4 μM PKH26 membrane dyes. By effector cells (2.5x 105 cells / well) and PKH26-labeled target cells (2.5x104 cells / well) in 96-well V-bottom plates (Greiner Bio-one, #651 180) in the assay culture medium of target cell line (with 1% penicillin / streptomycin (Life Technologies, 15140) and 30 μM Alburex HSA (CSL Behring, 2160-679) target growth culture medium) co-incubated (effector and target ratio is 10:1). To analyze concentration-dependent cell lysis, serial dilutions of ISVD constructs in target assay medium were added to cells 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) containing 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros Organics, 19038)). Subsequently, cells were resuspended in 5 μM PBS supplemented with 5 μM PBS. -3 iodide (642 / 661) (ThermoFisher Scientific, T3605) in 100 μL FACS buffer to distinguish live cells from dead cells. Cells were analyzed using a MACSQuant X flow cytometer (Miltenyi Biotec). Each sample was obtained with a total sample volume of 70 μL. A gate was set for PKH26-positive cells, and the number of cells within this population was determined. -3 positive cells. Percent specific lysis = ((%TO-PRO-3 + no construct - %TO-PRO-3 + with construct) / %TO-PRO-3 + no construct)) x 100. The assay was performed in the presence of excess HSA so that the ISVD was completely saturated with HSA as described above.

[0482] result

[0483] To evaluate the functionality of the TCE ISVD variants as T cell engagers, the TCE-CD123-ALB formats listed in Table 8 were evaluated using human primary T cells (two donors) and a human MOLM-13 target cell line expressing CD123 in the presence of 30 μM HSA as described above in a flow cytometry-based T cell-mediated MOLM-13 cell killing assay. A graphical representation of these results is shown in Figure 3A-Figure 3F The calculated EC50 values ​​for target cell killing are shown in Table 11.

[0484] Table 11: EC50 (M) of TCE-CD123-ALB constructs 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

[0485]

[0486]

[0487] As can be seen from Table 11 and Figure 3, all constructs in which the TCE building block is in the N-terminal position (position 1) mediate effective cell killing of target cells. In addition, the mutation from glutamic acid to aspartic acid in the first position of the TCE ISVD does not significantly affect the killing efficacy of the constructs. In addition, having the TCE ISVD in the N-terminal position of the construct appears to result in increased efficacy when compared to placing the same TCE ISVD in the second position of the construct.

[0488] Surprisingly, ISVD constructs T017001019, T017001020 and T017001021 (which contain TCE ISVDs T017001001, T01700999 and T017000995, respectively, which have lower affinity for TCR zipper protein and human T cells than other constructs) have high potency in target cell killing. In fact, even though the affinity observed on human TCR zipper protein and human T cells is more than 40 times lower than the reference value (see Tables 6 and 9 and Figure 1 and Figure 2 ), and there was no significant difference in the efficacy of target cell killing.

[0489] It has been reported that low affinity for T cell receptors compared to affinity for tumor-associated targets is important for distribution to tumor tissues compared to secondary lymphoid tissues rich in T cells. It is desirable to preferentially target tumor tissues to reduce T cell-mediated target elimination.

[0490] Therefore, these specific ISVD constructs with low affinity for TCRs and high potency in 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, which in turn may reduce the risk of toxicity.

[0491] In summary, the TCE ISVD variants according to the present technology are very suitable for use in constructs for target-specific therapeutic applications in humans.

[0492] 6.9 Example 9: Human and cynomolgus monkey T cell-mediated killing of KG-1a cells induced by multispecific TCE-CD123-ALB constructs

[0493] To evaluate whether the TCE-CD123-ALB ISVD construct can 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 contained anti-TCR ISVD T017000975 and albumin binding ISVD, but did not contain anti-CD123 ISVD.

[0494] In a flow cytometry-based assay, the knockdown of CD123 target cells by the TCE-CD123-ALB construct was Directed T cell-mediated killing

[0495] Human T cells were collected from buffy coat blood from healthy volunteers (Blood bank Gent) using RosetteSep (StemCell Technologies, 15061) and subsequently enriched on 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 in flow cytometry assays 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. The cells were frozen in liquid nitrogen.

[0496] T cells from cynomolgus monkeys (Macaca fascicularis) were isolated in-house (Sanofi, Montpellier, France) from PBMCs (separated via Ficoll density centrifugation) using the pan T cell isolation kit (Miltenyi Biotec, 130-091-993) according to the manufacturer's instructions. The cells were frozen in liquid nitrogen.

[0497] According to the manufacturer's instructions, human KG1a cells expressing CD123 were labeled with 4 μM PKH-26 membrane dye using PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) and used as target cells. 2.5x1E5 effectors (i.e., human or cynomolgus monkey primary T cells) and 2.5x1E4 target cells (i.e., PKH-labeled KG1a cells) were co-incubated in 96-well V-bottom plates (effector to target ratio of 10: 1). In order to measure concentration-dependent cell lysis, serial dilutions of TCE-CD123-ALB constructs in assay culture medium with 30 μM HSA (CSL Behring, Alburex 20 human serum albumin) were added to cells and incubated at 37 ° C in a 5% CO2 atmosphere for 18 hours.

[0498] After incubation, cells were pelleted by centrifugation and resuspended in 5 nM -3 iodide (642 / 661) in FACS buffer (Life Technologies-Molecular Probes, T3605) to distinguish live cells from dead cells. Cells were analyzed by flow cytometry (Miltenyi Biotec). A gate was set for PKH26-positive target cells, and the expression of -3The percentage of propidium-3-positive cells.

[0499] result

[0500] The results can be seen in Table 12 below and Figure 4.

[0501] Table 12: TCE-CD123-ALB constructs in flow cytometry-based T cell-mediated KG-1a cell killing assay

[0502] Sample ID Potency (M) - Human T cells Potency (M) - Cynomolgus monkey T cells T017001017 2.1E-10 3.9E-11 T017001018 2.7E-10 4.0E-11 T017001019 2.3E-09 7.4E-10 T017001020 7.6E-10 2.3E-10 T017001021 5.7E-09 9.4E-10 T017000689 - -

[0503] As can be seen in Table 12 and Figure 4, all TCE ISVD constructs were potent in killing KG-1a cells using both human and cynomolgus monkey T cells. As expected, the negative control construct T017000689 showed no cell killing.

[0504] The low affinity TCE ISVDs T017001019, T017001020 and T017001021 also had high potency in the KG1-a cell killing assay. This further supports that these TCE constructs with low affinity anti-TCR building blocks have significant potential as T cell engaging biotherapeutics, as mentioned in Example 8.

[0505] Furthermore, for all TCE-CD123-ALB formats tested, the mediated cytotoxic activity observed for each format using human T cells as effector cells was very similar to that mediated by cynomolgus macaque T cells.

[0506] Therefore, this cell killing assay further supports that the TCE ISVD variants according to the present technology are well suited for use in constructs for target-specific therapeutic applications in humans.

[0507] 6.10 Example 10: Functionality of TCE-GPC3-GPC3-ALB Fusion

[0508] Generation of TCE-GPC3-GPC3-ALB fusion

[0509] The TCE ISVD variant was formatted as a multispecific ISVD construct with two tumor-anchored ISVD building blocks for Glypican-3 (GPC3) and one ISVD building block for human serum albumin (connected by 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 ISVD T017000975 (T017000700 with E1D mutation; SEQ ID NO: 34) binding to TCR, two tumor-anchored ISVD building blocks for Glypican-3 (GPC3) and one ISVD binding to human serum albumin. The negative control (T017000698) did not contain the anti-GPC3 ISVD building block.

[0510] Impedance-based cytotoxicity assay

[0511] In an impedance-based cytotoxicity assay (e.g., as described in WO 2018091606A1), human or cynomolgus monkey primary effector T cells and adherent target cells were used to characterize ISVD constructs for redirected T cell-mediated killing. The impedance changes induced by target cell adhesion to the electrode surface were measured using an xCELLigence instrument (Roche). T cells are non-adhesive and therefore do not affect impedance measurements. The RTCA MP instrument quantifies the changes in electrical impedance and displays them as a dimensionless parameter called the cell index, which is proportional to the total area of ​​the tissue culture well covered by the cells. 50 μL of 120 μM Alburex HSA (CSL Behring, 2160-679) was added to each well of a 96E plate (ACEA Biosciences; 05 232368 001) to have a final concentration of 30 μM in assay culture medium (target cell growth medium + 1% penicillin / streptomycin (Life Technologies catalog number 15140)). The outer wells were not used and filled with 200 μL of culture medium or D-PBS. The 96E plate was placed in a 96E plate. Station (in an incubator at 37°C, 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 (2x 1E4 cells / well) in assay medium were seeded onto 96E plates, and 50 μL of serially diluted ISVD construct solution (4X concentration) in assay medium were added. (Final volume = 200 μL). After 30 min at room temperature, 50 μL of primary T 5 cells (3x 1E5 cells / well) in assay medium were added per well to achieve an effector to target ratio of 15:1. The plate was placed on The cells were placed in a station and impedance was measured every 15 min for 4 days. Data were analyzed at fixed time points (60 h).

[0512] The resulting constructs are shown in Table 13 below.

[0513] Table 13: List of TCE-GPC3-GPC3-ALB and control constructs

[0514] Construct ID TCE ISVD ID SEQ ID A022600427 T017000975 73 A022600462 T017000991 74 T017000698 T017000975 75

[0515] result

[0516] The TCE ISVD variant T017000991 (SEQ ID NO: 42) was compared to the reference T017000975 (SEQ ID NO: 34) in a fusion construct with two ISVD building blocks against the tumor anchor GPC3 (Glypican-3) and one ISVD building block against albumin connected by a GS linker. As a negative control, a construct without an ISVD binding to GPC3 was generated (T017000698, Table 13). As a positive control, a bispecific antibody binding to GPC3 was generated. One arm of this antibody binds to GPC3 and the other arm binds to CD3. A graphical representation of the results is shown in Figure 5A-5D The calculated IC50 values ​​for target cell killing are shown in Table 14.

[0517] Table 14. IC50 (M) of TCE-GPC3-GPC3-ALB constructs in impedance-based human and cynomolgus T cell-mediated HuH-7 cell killing assay in the presence of 30 μM HSA using an effector to target ratio of 15 to 1

[0518]

[0519] When using human or cynomolgus monkey T cells, the IC50 values ​​of the two constructs in which the TCE building block was fused to the ISVD binding to GPC3 were comparable. As expected, the construct without the ISVD binding to GPC3, i.e., the TCRαβ binding ISVD fused only to the ISVD binding to human serum albumin, did not mediate any level of cell killing or only mediated a low level of cell killing.

[0520] 6.11 Example 11: Chemical Stability Evaluation of TCE ISVD

[0521] To evaluate the chemical stability of TCE ISVD according to the present technology, forced degradation studies were performed with T017000991 (SEQ ID NO:42) and compared with the reference T017000975 (SEQ ID NO:34), both of which had a HIS6-tag (SEQ ID NO:78) attached.

[0522] The study consisted of forced oxidation with 10 mM H2O2 and a four-week incubation period at 25°C and 40°C, simulating accelerated and stress conditions, respectively. After the stress test, the stability of T017000991 was analyzed by peptide mapping using: trypsin digestion, peptide separation by reverse phase chromatography, and mass spectrometry MS / MS detection. The molecules were screened for the following modifications: deamidation, isomerization / racemization, and methionine or tryptophan oxidation. To evaluate the value of sequence optimization of T017000991, the chemical stability of the ISVD with chemical modifications was compared with the reference T017000975.

[0523] result

[0524] The results of the peptide mapping analysis of different temperature conditions are presented in Table 15 below.

[0525] Table 15: Comparison of tryptic peptide mapping analysis of T017000991 and T017000975 (control) after temperature stress.

[0526]

[0527] No significant changes were observed under forced oxidation conditions. Sequence coverage for both molecules was approximately 85%. In combination with AspN digestion, 100% sequence coverage was obtained for both molecules. No additional modifications were detected.

[0528] Two chemical tendencies were identified in T017000975: D61 is susceptible to isomerization, and W99 is susceptible to oxidation (Kabat numbering). As previously described, these two sensitive amino acids were substituted (D61E, W99Y) in T017000991, resulting in complete elimination of these two tendencies in TCE ISVD according to the technology of the present invention under the evaluation conditions.

[0529] Therefore, the chemical stability of the TCE ISVD according to the present technology has been improved relative to the reference TCE ISVD.

[0530] 6.12 Example 12: Chemical stability of TCE ISVD as part of a formatted ISVD construct

[0531] To further demonstrate the improved stability of TCE ISVDs according to the present technology, the chemical stability of T017000991 as part of the formatted ISVD construct was analyzed.

[0532] Short term storage studies were performed. T017000991 and T017000975 (as a control) were formatted with two anti-GPC3 ISVDs and one ISVD that binds human serum albumin. The constructs are presented in Table 16 below.

[0533] Table 16

[0534] ISVD construct ID SEQ ID NO Format A022600424 76 T017000975-GPC3-GPC3-ALB A022600462 74 T017000991-GPC3-GPC3-ALB

[0535] When formatted molecules are stored as liquids with an appropriate formulation buffer, tryptophan oxidation and aspartate isomerization have been observed during storage.

[0536] At this time, the tryptophan oxidation rate was found to be dependent on pH, temperature, ISVD concentration, transition metals, and polysorbate 20. Therefore, in order to accelerate any potential oxidation events, stress studies were performed. The stress conditions consisted of incubation in plastic tubes at 40°C / 75% relative humidity (RH) ± 5% RH and the addition of 100 ppm of Fe(II) as an oxidant for 3 months.

[0537] For 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 also monitored for stability at 25°C / 60% RH ± 5% RH for 3 months.

[0538] The stability profile was monitored by reverse phase chromatography following peaks with relative retention time (RRT) < 1.0 (as an indicator of tryptophan oxidation) and by peptide mapping after trypsin digestion and reverse phase LC-MS / MS.

[0539] result

[0540] A graphical representation of the results can be found in Figure 6 , which shows the stability curve at 25° C. obtained by following the peak with RRT<1.0 by reverse phase chromatography. A reduced oxidation rate was observed for A022600462 compared to A022600424, with a 5-fold reduction in tryptophan oxidation.

[0541] A similar trend can be observed during the evaluation of stability curves under stress conditions ( Figure 7). Addition of Fe(II) to the formulated solution catalyzed the oxidation rate of the ISVD constructs in the presence of polysorbate 20 and histidine. After one day of incubation at 40°C, the oxidation rate of A022600462 was approximately four times lower compared to the control A022600424. Even lower oxidation rates were seen after 14 days of forced degradation conditions.

[0542] Peptide mapping was used to precisely determine the position at which different oxidation events occurred in the amino acid sequence. For this purpose, the temperature stress stability of the samples was analyzed and compared. After trypsin digestion, almost 85% of the sequence could be covered by the generated peptides. Using the Biopharma Finder software (BPF; version 3.2) on a mass spectrometer (MS), the different peptides could be identified and the chemical modifications on them elucidated. The results showed that there were different tryptophan oxidation sites on the selected format, some of which were on the TCE ISVD located in peptide number 13, more precisely on W99 (A022600424) and W103 (A022600424 and A022600462). The results are shown in Figure 8 middle.

[0543] Peptide mapping analysis of samples from the storage stability study showed that the overall oxidation rate on A022600462 was much lower when compared to A0022600424, since tryptophan W99 was removed from the sequence of the former. The oxidation rate on the second tryptophan appeared to remain stable, while tryptophan oxidation on other building blocks present in this format did not appear to be affected by the introduced amino acid substitutions. As expected, removal of the D61 residue resulted in the elimination of the isomerization tendency of the final format.

[0544] In conclusion, the problems associated with tryptophan oxidation and isomerization in ISVD have been overcome by substituting amino acids at the relevant positions while maintaining the functionality of ISVD.

[0545] 7 Industrial Applicability

[0546] The ISVDs, polypeptides, nucleic acid molecules encoding them, vectors comprising nucleic acids, and compositions described herein can be used, for example, to treat a subject having cancer.

[0547] Table A-6: Sequence Listing

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

Claims

1. An immunoglobulin single variable domain (ISVD) that specifically binds to the constant domain of a human and / or non-human primate T cell receptor (TCR) present on T cells, wherein the ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein 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); in - the amino acid residue 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; and / or - the amino acid residue 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.

2. An immunoglobulin single variable domain (ISVD) that specifically binds to the constant domain of a human and / or non-human primate T cell receptor (TCR) present on T cells, wherein the ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein 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); And among them - the amino acid residue at position 61 (according to Kabat) is selected from the group consisting of 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 the group consisting of Y, A, P, D, Q, E, R, F, S, G, T, H, V, K, L and I.

3. The ISVD of claim 1, wherein X1 is E or D.

4. The ISVD of claim 1, wherein X1 is E.

5. The ISVD according to claim 2, wherein the amino acid residue at position 61 (according to Kabat) is E or D.

6. The ISVD of claim 2, wherein the amino acid residue at position 61 (according to Kabat) is E.

7. The ISVD of any one of claims 1-6, wherein X2 is Y, A, Q, F, S, T, or H.

8. The ISVD of any one of claims 1-6, wherein X2 is Y, Q, S or T.

9. The ISVD of any one of claims 1-6, wherein X2 is Y.

10. The ISVD according to any one of claims 1-9, wherein 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.

11. The ISVD according to any one of claims 1-9, wherein the amino acid residue at position 103 (Kabat numbering) in the ISVD is W.

12. The ISVD of claim 1, wherein X1 is E, X2 is Y, and the amino acid residue at position 103 (Kabat numbering) is W.

13. The ISVD of claim 2, wherein 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.

14. The ISVD according to any one of claims 1 to 13, wherein the amino acid residue at position 1 (Kabat numbering) is selected from E and D.

15. The ISVD according to any one of claims 1-14, wherein the ISVD is a heavy chain ISVD.

16. The ISVD according to claim 15, wherein the heavy chain ISVD is selected from V HH , humanized V HH , Camelization V H , domain antibodies, single domain antibodies and dAbs.

17. The ISVD according to any one of the preceding claims, which has a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of any one of SEQ ID NOs: 2-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, and wherein preferably the sequence is SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50 or SEQ ID NO:

52.

18. The ISVD according to any one of the preceding claims, which has a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO: 32, 33 and / or 35-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, and wherein 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.

19. The ISVD according to claim 17 or 18, wherein the ISVD comprises or consists of SEQ ID NO: 37 or SEQ ID NO:

42.

20. The ISVD of claim 17 or 18, wherein the ISVD comprises or consists of SEQ ID NO:46, SEQ ID NO:50 or SEQ ID NO:

52.

21. An immunoglobulin single variable domain (ISVD) comprising or consisting of the following sequence: X0VQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKER EKVAHISIGDQTDYA X1SAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIX2PYDY X3GQGTLVTVSS(SEQ ID NO:85), in 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.

22. The ISVD of claim 21, wherein X0 is D.

23. The ISVD according to claim 21 or 22, wherein: X1 is selected from the group consisting of D or E.

24. The ISVD of claim 21 or 22, wherein X1 is E.

25. The ISVD of any one of claims 21-24, wherein X2 is selected from the group consisting of Y, T, S and Q.

26. The ISVD of any one of claims 21-24, wherein X2 is Y.

27. The ISVD of any one of claims 21-26, wherein X3 is W.

28. 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, and wherein the target cell is different from the T cell, wherein the first ISVD and the second ISVD essentially consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), and wherein the first ISVD is an ISVD according to any one of claims 1-27.

29. The polypeptide according to claim 28, wherein 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 with at least one of the amino acid sequences of any one of SEQ ID NOs: 2-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, preferably wherein 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.

30. The polypeptide according to claim 28 or 29, wherein the first ISVD has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 32, 33 and / or 35-57, wherein for the purpose of determining the degree of sequence identity the amino acid residues forming the CDR sequences are not taken into account, and wherein 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.

31. The polypeptide of claim 28 or 29, wherein the first ISVD comprises or consists of SEQ ID NO: 37 or SEQ ID NO:

42.

32. The polypeptide of claim 28 or 29, wherein the first ISVD comprises or consists of SEQ ID NO:46, SEQ ID NO:50 or SEQ ID NO:

52.

33. The polypeptide of any one of claims 28-32, further comprising a third ISVD that specifically binds to a second antigen on a target cell.

34. The polypeptide according to any one of claims 28 to 33, wherein the polypeptide further comprises one or more other groups, residues, parts or binding units, optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, parts or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, parts or binding units.

35. The polypeptide according to claim 34, wherein the one or more other groups, residues, moieties or binding units are ISVDs that can bind to human serum albumin.

36. A method of producing an ISVD according to any one of claims 1-27 or a polypeptide according to any one of claims 28-35, wherein the method comprises: a. expressing the nucleic acid sequence encoding the ISVD or polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b. Isolating and / or purifying the ISVD or polypeptide.

37. A nucleic acid encoding the ISVD of any one of claims 1-27 or the polypeptide of any one of claims 28-35.

38. A vector comprising the nucleic acid according to claim 37.

39. A non-human host or host cell transformed or transfected with the nucleic acid according to claim 37 or the vector according to claim 38.

40. A composition comprising the ISVD of any one of claims 1-27 or the polypeptide of any one of claims 28-35.

41. The composition of claim 40, wherein the composition is a pharmaceutical composition.

42. A polypeptide according to any one of claims 28 to 35 or a composition according to claim 40 or 41 for use as a medicament.

43. The polypeptide according to any one of claims 28-35 or the composition according to claim 40 or 41, 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.

44. The polypeptide or composition for use according to claim 43, wherein the disease is cancer.

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