T cell recruitment polypeptides based on CD3 reactivity

Abstract

T cell recruitment polypeptides that bind to CD3 on T cells are provided. The polypeptides can be used in methods of treating cancer.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680040586.1, whose application date is May 13, 2016 and whose invention name is “T cell recruitment polypeptide based on CD3 reactivity”. Field of the Invention

[0002] The present invention provides multispecific T cell recruiting polypeptides that bind CD3 on T cells and at least one antigen on target cells. The present invention also relates to monovalent T cell recruiting polypeptides used in these multispecific polypeptides. The present invention also provides treatment methods and kits for providing them. Background Art

[0003] Cancer takes a huge toll worldwide. It is now the leading cause of death in the world, followed by heart disease and stroke. Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. The number of new cases is expected to rise by approximately 70% over the next 20 years (source: WHO Cancer). In 2008, the total economic impact of premature death and disability from cancer worldwide was approximately $900 billion, or 1.5% of global GDP.

[0004] Treatment options available for solid tumors typically include a combination of surgical resection, chemotherapy, and radiation therapy. In 40 years of clinical experience, little progress has been made, especially in the advanced stages of cancer.

[0005] New treatments to fight cancer are eagerly awaited.

[0006] Antibody therapy is now an important part of the medical equipment of doctors against diseases and especially cancer.Monoclonal antibodies have been established as the key treatment method for a series of diseases in the past few years.All antibody therapies approved in the same period rely on monospecific monoclonal antibodies (mAb).Until today, most targets of mAb need exciting or antagonistic methods.Although the targeting cell surface antigen itself can mediate anti-tumor activity by inducing apoptosis, most of the activity based on mAb for hematological malignancies relies on the effector function mediated by Fc, such as complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC).

[0007] Immunotherapy has become a rapidly developing area of ​​cancer research. Immunotherapy is directing the body's immune surveillance system, and specifically T cells, toward cancer cells.

[0008] Cytotoxic T cells (CTLs) are T lymphocytes that kill cancer cells, infected cells (particularly cells infected by viruses) or otherwise damaged cells. T lymphocytes (or T cells) express T cell receptors or TCR molecules and CD3 receptors on the cell surface. αβTCR-CD3 complexes (or "TCR complexes") consist of six different I-type single-spanning transmembrane proteins: TCRα and TCRβ chains that form TCR heterodimers responsible for ligand recognition, and non-covalently associated CD3γ, CD3δ, CD3ε and ζ chains (they have cytoplasmic sequence motifs that are phosphorylated when the receptor is activated and recruit a large number of signal transduction components) (Call et al. 2004, Molecular Immunology 40: 1295–1305).

[0009] Both the α and β chains of the T cell receptor consist of a constant domain and a variable domain. Physiologically, the αβ chains of the T cell receptor recognize the MHC complex loaded with peptides and couple when engaging with the CD3 chains. These CD3 chains then transduce the engagement signal to the intracellular environment.

[0010] Considering the potential of naturally occurring cytotoxic T lymphocytes (CTL) to mediate cell lysis, various strategies have been explored to recruit CTL to mediate tumor cell killing. Because T lymphocytes lack the expression of Fc receptors, they are not recruited to the tumor site by the Fc tail of anti-tumor monoclonal clones. As an alternative, the patient's T cells are modified with a second TCR with known specificity to a determined tumor antigen. This adoptive cell transfer is highly personalized and labor-intensive in nature. However, the main problem of T cell therapy is still the large number of immune escape mechanisms known to occur in cancer patients (Nagorsen et al. 2012, Pharmacology & Therapeutics 136: 334–342).

[0011] Newer developments have attempted to combine the advantages of immunotherapy with antibody therapy by engaging all of a patient's cytotoxic T cells in a polyclonal manner via recombinant antibody-based "bispecific antibody" technology, rather than eliciting specific T-cell responses that rely on cancer cell expression of MHC molecules and the presence, production, transport and display of specific peptide antigens.

[0012] Bispecific antibodies have been engineered to have a tumor recognition portion on one arm (the target binding arm), while the other arm of the molecule has specificity for a T cell antigen (effector binding arm), primarily CD3. By simultaneously binding both arms to their respective target antigens, T lymphocytes are directed to and activated at tumor cells, where they are able to exert their cytolytic function.

[0013] The concept of using bispecific antibodies to activate T cells against tumor cells was described more than two decades ago, but manufacturing problems and clinical failures have stalled the field. Smaller formats of bispecific antibodies have been developed that are easier to penetrate tissues and tumors than conventional antibodies. In addition, smaller formats are better at creating cytotoxic synapses (which kill target cells). It is believed that smaller formats of bispecific antibodies are easier to manufacture than conventional antibodies and have lower immunogenicity. However, smaller bispecific BiTE molecules consisting of two single-chain variable fragments (scFv) connected by a 5-amino acid peptide linker show a lack of stability (scFv tends to aggregate), low expression titers and poor solubility. In addition, the first clinical trial of blinatumomab (a BiTE molecule) that recognizes the CD3 chain was stopped prematurely due to the following: on the one hand, it was neurological adverse events, cytokine release syndrome and infection, and on the other hand, it was a lack of objective clinical response or strong signs of biological activity. In addition to efficacy, BiTE must be continuously injected-probably due to the lack of Fc domain-which will not help patients' compliance. The same problem exists for DART (dual affinity retargeting molecules developed by MacroGenics), in which the heavy variable domain from one antibody (Ab) is connected to the light variable domain of another Ab. MacroGenics is currently trying to solve this problem by fusing the Fc domain to its next generation DART, which not only makes the molecule larger, but also causes manufacturing problems and the input of other Fc functions. The larger format of Fc will have better PK, but reintroduces the risk of off-target activity. (Garber 2014, Nature reviews 13: 799-801).

[0014] There remains a need for alternative bispecific formats. SUMMARY OF THE INVENTION

[0016] The present invention solves this problem by providing a multispecific polypeptide comprising a first immunoglobulin single variable domain (ISV) and at least one further immunoglobulin single variable domain, wherein the first ISV has a high affinity for / binds to CD3; the at least one further ISV has a high affinity for / binds to an antigen present on a target cell. In particular aspects, binding of the first ISV will activate the intrinsic cytotoxic potential of T cells against target cells (independent of MHC1).

[0017] Thus, in a first aspect, the invention provides a polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), wherein

[0018] - the first ISV has a high affinity for / binds to cluster of differentiation 3 (CD3) present on T cells;

[0019] - the second ISV has a high affinity for / binds to a first antigen on a target cell;

[0020] wherein the first antigen is different from the CD3; and

[0021] wherein the target cell is different from the T cell.

[0022] In a further aspect, the invention provides a polypeptide as described herein, wherein the polypeptide directs a T cell to a target cell.

[0023] In a further aspect, the invention provides a polypeptide as described herein, wherein the polypeptide induces T cell activation.

[0024] In a further aspect, the invention provides a polypeptide as described herein, wherein the T cell activation is independent of MHC recognition.

[0025] In a further aspect, the present invention provides a 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.

[0026] In a further aspect, the invention provides a polypeptide as described herein, wherein the T cell activation causes one or more cellular responses of the T cell, wherein the 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.

[0027] In a further aspect, the invention provides a polypeptide as described herein, wherein the T cell activation results in inhibition of the activity of the target cell 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%.

[0028] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV binds to CD3γ (SEQ ID NO: 292), CD3δ (SEQ ID NO: 291) and / or CD3ε (SEQ ID NO: 293) of the TCR complex, or a polymorphic variant or isoform thereof.

[0029] Alternatively, the invention provides a polypeptide as described herein, wherein the first ISV binds CD3γ (SEQ ID NO: 379), CD3δ (SEQ ID NO: 291) and / or CD3ε (SEQ ID NO: 380) of the TCR complex, or a polymorphic variant or isoform thereof.

[0030] In a further aspect, the invention provides a polypeptide as described herein, wherein said polypeptide and / or first ISV has an association rate constant (Kon) for binding to said CD3 selected from the group consisting of: at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , 10 7 M -1 s -1 , at least about 10 8 M -1 s -1 , at least about 10 9 M -1 s -1 and at least about 10 10 M -1 s -1 , preferably as measured by surface plasmon resonance.

[0031] In a further aspect, the invention provides a polypeptide as described herein, wherein said polypeptide and / or first ISV has a dissociation rate constant (Koff) for binding to said CD3 selected from the group consisting of: at most about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 , up to about 10 -6 s -1 , up to about 10 -7 s -1 , up to about 10 -8 s -1 , up to about 10 -9 s -1 and at most about 10 -10 s -1, preferably as measured by surface plasmon resonance.

[0032] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV binds to the CD3 with an EC50 value of between 100 nM and 1 pM, such as an average EC50 value of less than 100 nM, even more preferably an average EC50 value of less than 90 nM, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM or even less, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even less, such as less than 4 pM, preferably as measured by flow cytometry.

[0033] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV binds to the CD3 with an average KD value of between 100 nM and 10 pM, such as an average KD value of 90 nM or less, even more preferably an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. Preferably, the KD is determined by SPR, for example as determined by Proteon.

[0034] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0035] (i) CDR1 is selected from the group consisting of:

[0036] (a) SEQ ID NOs: 81-100; and

[0037] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 81; and / or

[0038] (ii) CDR2 is selected from the group consisting of:

[0039] (c) SEQ ID NOs: 101-122; and

[0040] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 101; and / or

[0041] (iii) CDR3 is selected from the group consisting of:

[0042] (e) SEQ ID NOs: 123-143; and

[0043] (f) an amino acid sequence that has 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:123.

[0044] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0045] (i) CDR1 is selected from the group consisting of:

[0046] (a) SEQ ID NOs:81-100; or (b) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:81 or with any of SEQ ID NOs:81-100, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 not having said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0047] (ii) CDR2 is selected from the group consisting of:

[0048] (c) SEQ ID NOs: 101-122; or (d) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 101 or with any of SEQ ID NOs: 101-122, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or greater affinity as compared to the binding of a polypeptide comprising a CDR2 not having said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0049] (iii) CDR3 is selected from the group consisting of:

[0050] (e) SEQ ID NOs:123-143; or (f) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:123 or with any of SEQ ID NOs:123-143, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0051] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0052] (a) SEQ ID NO:81; and

[0053] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 81, wherein

[0054] - At position 1, G has changed to R;

[0055] - At position 3, T has changed to A;

[0056] - At position 4, Y has changed to F;

[0057] - S has changed to G at position 8; and / or

[0058] - At position 10 G has changed to A.

[0059] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0060] (a) SEQ ID NO: 101; and

[0061] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 101, wherein

[0062] - At position 3, V has changed to T or A;

[0063] - At position 5, S has changed to T;

[0064] - G has changed to D or E at position 6; and / or

[0065] - At position 9 T has changed to S, A or P.

[0066] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0067] (a) SEQ ID NO: 123; and

[0068] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 123, wherein

[0069] - At position 2, I has changed to T;

[0070] - I has changed to V at position 9; and / or

[0071] - At position 10 A has been changed to P.

[0072] Preferably, a polypeptide comprising one or more CDRs having 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR without said 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0073] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0074] (i) CDR1 is selected from the group consisting of:

[0075] (a) SEQ ID NO:81; and

[0076] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 81, wherein

[0077] - At position 1, G has changed to R;

[0078] - At position 3, T has changed to A;

[0079] - At position 4, Y has changed to F;

[0080] - S has changed to G at position 8; and / or

[0081] - At position 10 G has changed to A,

[0082] provided that the polypeptide comprising a CDR1 with the 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 without the 2 or 1 amino acid differences, as measured by surface plasmon resonance;

[0083] And among them

[0084] (ii) CDR2 is selected from the group consisting of:

[0085] (a) SEQ ID NO: 101; and

[0086] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 101, wherein

[0087] - At position 3, V has changed to T or A;

[0088] - At position 5, S has changed to T;

[0089] - G has changed to D or E at position 6; and / or

[0090] - T has changed to S, A or P at position 9,

[0091] provided that the polypeptide comprising a CDR2 with the 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 without the 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance;

[0092] And among them

[0093] (iii) CDR3 is selected from the group consisting of:

[0094] (a) SEQ ID NO: 123; and

[0095] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 123, wherein

[0096] - At position 2, I has changed to T;

[0097] - I has changed to V at position 9; and / or

[0098] - A has changed to P at position 10,

[0099] Provided that the polypeptide comprising a CDR3 with the 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity than the binding of a polypeptide comprising a CDR3 without the 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0100] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0101] (i) CDR1 is selected from the group consisting of:

[0102] (a) SEQ ID NOs: 81-87; and

[0103] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 81; and / or

[0104] (ii) CDR2 is selected from the group consisting of:

[0105] (c) SEQ ID NOs: 101-109; and

[0106] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 101; and / or

[0107] (iii) CDR3 is selected from the group consisting of:

[0108] (e) SEQ ID NOs: 123-127; and

[0109] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:123.

[0110] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0111] (i) CDR1 is selected from the group consisting of:

[0112] (a) SEQ ID NOs: 81-87; and

[0113] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 81, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0114] (ii) CDR2 is selected from the group consisting of:

[0115] (c) SEQ ID NOs: 101-109; and

[0116] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 101, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0117] (iii) CDR3 is selected from the group consisting of:

[0118] (e) SEQ ID NOs: 123-127; and

[0119] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 123, provided that the polypeptide comprising a CDR3 with said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without the 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0120] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO: 123.

[0121] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 1-50.

[0122] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 1-50.

[0123] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 1-50.

[0124] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO:88.

[0125] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR2 is SEQ ID NO:110.

[0126] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO:128.

[0127] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0128] (i) CDR1 is selected from the group consisting of:

[0129] (a) SEQ ID NO: 88; and

[0130] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 88; and / or

[0131] (ii) CDR2 is selected from the group consisting of:

[0132] (c) SEQ ID NO: 110; and

[0133] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 110; and / or

[0134] (iii) CDR3 is selected from the group consisting of:

[0135] (e) SEQ ID NO: 128; and

[0136] (f) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO:128.

[0137] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0138] (i) CDR1 is selected from the group consisting of:

[0139] (a) SEQ ID NOs: 88; and

[0140] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 88, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0141] (ii) CDR2 is selected from the group consisting of:

[0142] (c) SEQ ID NOs: 110; and

[0143] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 110, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0144] (iii) CDR3 is selected from the group consisting of:

[0145] (e) SEQ ID NOs: 128; and

[0146] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 128, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0147] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, and CDR3 is represented by SEQ ID NO: 128.

[0148] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NOs:51.

[0149] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NOs:51.

[0150] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NOs:51.

[0151] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO:90.

[0152] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0153] (a) SEQ ID NO: 112; and

[0154] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[0155] - At position 2 V has changed to A.

[0156] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO: 130.

[0157] Preferably, a polypeptide comprising one or more CDRs that have 1 amino acid difference binds to CD3 with about the same or higher affinity than the binding of a polypeptide comprising a CDR that does not have 1 amino acid difference, as measured by surface plasmon resonance.

[0158] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0159] (i) CDR1 is SEQ ID NO:90; and

[0160] And among them

[0161] (ii) CDR2 is selected from the group consisting of:

[0162] (a) SEQ ID NO: 112; and

[0163] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[0164] - At position 2, V has changed to A,

[0165] provided that the polypeptide comprising a CDR2 with 1 amino acid difference binds to CD3 with about the same or higher affinity as measured by surface plasmon resonance as compared to the binding of a polypeptide comprising a CDR2 without the 1 amino acid difference;

[0166] And among them

[0167] (iii) CDR3 is SEQ ID NO:130.

[0168] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0169] (i) CDR1 is selected from the group consisting of:

[0170] (a) SEQ ID NO:90; and

[0171] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 90; and / or

[0172] (ii) CDR2 is selected from the group consisting of:

[0173] (c) SEQ ID NOs: 112-113; and

[0174] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 112; and / or

[0175] (iii) CDR3 is selected from the group consisting of:

[0176] (e) SEQ ID NO: 130; and

[0177] (f) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO:130.

[0178] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0179] (i) CDR1 is selected from the group consisting of:

[0180] (a) SEQ ID NOs:90; and

[0181] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 90, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0182] (ii) CDR2 is selected from the group consisting of:

[0183] (c) SEQ ID NOs: 112-113; and

[0184] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 112, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0185] (iii) CDR3 is selected from the group consisting of:

[0186] (e) SEQ ID NOs: 130; and

[0187] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 130, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0188] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:90, CDR2 is represented by SEQ ID NO:112, and CDR3 is represented by SEQ ID NO:130.

[0189] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 53-56.

[0190] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 53-56.

[0191] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 53-56.

[0192] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO:89.

[0193] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR2 is SEQ ID NO:111.

[0194] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO:129.

[0195] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0196] (i) CDR1 is selected from the group consisting of:

[0197] (a) SEQ ID NO: 89; and

[0198] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 89; and / or

[0199] (ii) CDR2 is selected from the group consisting of:

[0200] (c) SEQ ID NO: 111; and

[0201] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 111; and / or

[0202] (iii) CDR3 is selected from the group consisting of:

[0203] (e) SEQ ID NO: 129; and

[0204] (f) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO:129.

[0205] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0206] (i) CDR1 is selected from the group consisting of:

[0207] (a) SEQ ID NOs: 89; and

[0208] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 89, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0209] (ii) CDR2 is selected from the group consisting of:

[0210] (c) SEQ ID NOs: 111; and

[0211] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 111, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0212] (iii) CDR3 is selected from the group consisting of:

[0213] (e) SEQ ID NOs: 129; and

[0214] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 129, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0215] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111, and CDR3 is represented by SEQ ID NO: 129.

[0216] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NOs:52.

[0217] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NOs:52.

[0218] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NOs:52.

[0219] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0220] (a) SEQ ID NO:91; and

[0221] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[0222] - At position 6, R has changed to N or T;

[0223] - N has been changed to H at position 7; and / or

[0224] - At position 8 the M has been changed to a T.

[0225] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0226] (a) SEQ ID NO: 114; and

[0227] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[0228] - At position 1, R has changed to Q;

[0229] - T has changed to S at position 3; and / or

[0230] - In position 7 D has changed to A or K.

[0231] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0232] (a) SEQ ID NO: 131; and

[0233] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein

[0234] - S has changed to R at position 2; and / or

[0235] - At position 6 S has changed to V.

[0236] Preferably, a polypeptide comprising one or more CDRs having 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR without said 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0237] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0238] (i) CDR1 is selected from the group consisting of:

[0239] (a) SEQ ID NO:91; and

[0240] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[0241] - At position 6, R has changed to N or T;

[0242] - N has been changed to H at position 7; and / or

[0243] - At position 8, M has changed to T,

[0244] provided that the polypeptide comprising a CDR1 with the 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 without the 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance;

[0245] And among them

[0246] (ii) CDR2 is selected from the group consisting of:

[0247] (a) SEQ ID NO: 114; and

[0248] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[0249] - At position 1, R has changed to Q;

[0250] - T has changed to S at position 3; and / or

[0251] - D has changed to A or K at position 7,

[0252] provided that the polypeptide comprising a CDR2 with the 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 without the 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance;

[0253] And among them

[0254] (iii) CDR3 is selected from the group consisting of:

[0255] (a) SEQ ID NO: 131; and

[0256] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein

[0257] - S has changed to R at position 2; and / or

[0258] - At position 6, S has changed to V,

[0259] Provided that the polypeptide comprising a CDR3 with the one amino acid difference binds to CD3 with about the same or higher affinity than the binding of a polypeptide comprising a CDR3 without the one amino acid difference, as measured by surface plasmon resonance.

[0260] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0261] (i) CDR1 is selected from the group consisting of:

[0262] (a) SEQ ID NOs:91-93; and

[0263] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 91; and / or

[0264] (ii) CDR2 is selected from the group consisting of:

[0265] (c) SEQ ID NOs: 114-117; and

[0266] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 114; and / or

[0267] (iii) CDR3 is selected from the group consisting of:

[0268] (e) SEQ ID NOs: 131-133; and

[0269] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:131.

[0270] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0271] (i) CDR1 is selected from the group consisting of:

[0272] (a) SEQ ID NOs:91-93; and

[0273] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:91, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0274] (ii) CDR2 is selected from the group consisting of:

[0275] (c) SEQ ID NOs: 114-117; and

[0276] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 114, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0277] (iii) CDR3 is selected from the group consisting of:

[0278] (e) SEQ ID NOs: 131-133; and

[0279] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 131, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0280] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:91, CDR2 is represented by SEQ ID NO:114, and CDR3 is represented by SEQ ID NO:131.

[0281] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 57-65.

[0282] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 57-65.

[0283] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 57-65.

[0284] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0285] (a) SEQ ID NO:94; and

[0286] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[0287] - At position 3, S has changed to T, A or G;

[0288] - At position 5, N has changed to S;

[0289] - M has changed to T or A at position 6; and / or

[0290] - At position 9 L has changed to M.

[0291] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0292] (a) SEQ ID NO: 118; and

[0293] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 118, wherein

[0294] - At position 2, H has changed to V;

[0295] - At position 5, S has changed to H or A;

[0296] - N has changed to S at position 8; and / or

[0297] - At position 10 Y has changed to F.

[0298] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0299] (a) SEQ ID NO: 134; and

[0300] (b) an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 134, wherein

[0301] - A has changed to S or D at position 6;

[0302] - F has changed to Y or A at position 7;

[0303] - At position 8, R has changed to H;

[0304] - At position 9, S has changed to A;

[0305] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[0306] - At position 14 V has changed to I.

[0307] Preferably, a polypeptide comprising one or more CDRs having 5, 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR without said 5, 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0308] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein

[0309] (i) CDR1 is selected from the group consisting of:

[0310] (a) SEQ ID NO:94; and

[0311] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[0312] - At position 3, S has changed to T, A or G;

[0313] - At position 5, N has changed to S;

[0314] - M has changed to T or A at position 6; and / or

[0315] - At position 9, L has changed to M,

[0316] provided that the polypeptide comprising a CDR1 with the 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 without the 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance;

[0317] And among them

[0318] (ii) CDR2 is selected from the group consisting of:

[0319] (a) SEQ ID NO: 118; and

[0320] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 118, wherein

[0321] - At position 2, H has changed to V;

[0322] - At position 5, S has changed to H or A;

[0323] - N has changed to S at position 8; and / or

[0324] - At position 10, Y has changed to F,

[0325] provided that the polypeptide comprising a CDR2 with 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as measured by surface plasmon resonance compared to the binding of a polypeptide comprising a CDR2 without the 3, 2 or 1 amino acid differences;

[0326] And among them

[0327] (iii) CDR3 is selected from the group consisting of:

[0328] (a) SEQ ID NO: 134; and

[0329] (b) an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 134, wherein

[0330] - A has changed to S or D at position 6;

[0331] - F has changed to Y or A at position 7;

[0332] - At position 8, R has changed to H;

[0333] - At position 9, S has changed to A;

[0334] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[0335] - At position 14, V has changed to I,

[0336] Provided that the polypeptide comprising a CDR3 with the 5, 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity than the binding of a polypeptide comprising a CDR3 without the 5, 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0337] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0338] (i) CDR1 is selected from the group consisting of:

[0339] (a) SEQ ID NOs: 94-100; and

[0340] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 94; and / or

[0341] (ii) CDR2 is selected from the group consisting of:

[0342] (c) SEQ ID NOs: 118-122; and

[0343] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 118; and / or

[0344] (iii) CDR3 is selected from the group consisting of:

[0345] (e) SEQ ID NOs: 134-143; and

[0346] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:134.

[0347] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0348] (i) CDR1 is selected from the group consisting of:

[0349] (a) SEQ ID NOs: 94-100; and

[0350] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 94, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0351] (ii) CDR2 is selected from the group consisting of:

[0352] (c) SEQ ID NOs: 118-122; and

[0353] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 118, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0354] (iii) CDR3 is selected from the group consisting of:

[0355] (e) SEQ ID NOs: 134-143; and

[0356] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 134, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0357] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:94, CDR2 is represented by SEQ ID NO:118, and CDR3 is represented by SEQ ID NO:134.

[0358] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 66-80.

[0359] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 66-80.

[0360] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is cross-blocked from binding to CD3 by at least one polypeptide having SEQ ID NOs: 66-80.

[0361] In a further aspect, the invention provides a polypeptide as described herein, wherein said first antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

[0362] In a further aspect, the invention provides a polypeptide as described herein, further comprising a third ISV having a high affinity for and / or binding to a second antigen on the target cell, wherein the second antigen is different from the first antigen.

[0363] In a further aspect, the invention provides a polypeptide as described herein, wherein said second antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

[0364] In a further aspect, the invention provides a polypeptide as described herein, wherein said first antigen and said second antigen are present on the same target cell.

[0365] In a further aspect, the invention provides a polypeptide as described herein, wherein the first antigen and the second antigen are present on different target cells.

[0366] In a preferred aspect, the present invention provides a polypeptide as described herein, wherein the TAA is independently selected from the group consisting of: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), epidermal growth factor receptor (EGFR), fibroblast activation protein (FAP), MART-1, carcinoembryonic antigen (CEA), gp100, MAGE-1, HER-2, Lewis YAntigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD147, growth factor receptors, including ErbB3 and ErbB4, cytokine receptors, including interleukin-2 receptor gamma chain (CD132 antigen), interleukin-10 receptor alpha chain (IL-10R-A), interleukin-10 receptor beta chain (IL-10R-B), interleukin-12 Interleukin-12 receptor β-1 chain (IL-12R-β1), interleukin-12 receptor β-2 chain (IL-12 receptor β-2), interleukin-13 receptor α-1 chain (IL-13R-α-1) (CD213a1 antigen), interleukin-13 receptor α-2 chain (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin 21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-β) (CDw121b), leukocyte Interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor alpha chain (CD25 antigen), interleukin-2 receptor beta chain (CD122 antigen), interleukin-3 receptor alpha chain (IL-3R-α) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvII I, AGS-16, CD27L, nectin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin-releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2γa, CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6, DcR3 and CAIX, including related polymorphic variants and isoforms.

[0367] In a further aspect, the invention provides a polypeptide as described herein, wherein the TAA is CD20 (UniProt11836), HER2 (Uniprot P04626), a polymorphic variant or isoform thereof.

[0368] In a further aspect, the invention provides a polypeptide as described herein, wherein the first antigen and the second antigen are selected from the group consisting of:

[0369] - EGFR as the first antigen and CEA as the second antigen;

[0370] -CD19 as the first antigen and CD20 as the second antigen;

[0371] -CD19 as the first antigen and CD22 as the second antigen;

[0372] - CD123 as the first antigen and Tim-3 as the second antigen; and

[0373] - CD132 as the first antigen and CD69 as the second antigen.

[0374] In a further aspect, the invention provides a polypeptide as described herein, further comprising a serum protein binding moiety.

[0375] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety binds to serum albumin.

[0376] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety is an ISV that binds serum albumin.

[0377] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum albumin binding ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being determined according to the Kabat definition; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being determined according to Kontermann 2010.

[0378] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum albumin binding ISV is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG and Alb82-GGG (SEQ ID NOs: 348 to 360).

[0379] In a further aspect, the invention provides a polypeptide as described herein, wherein said ISVs are directly linked to each other or via a linker.

[0380] In a further aspect, the invention provides a polypeptide as described herein, wherein said first ISV and / or said second ISV and / or possibly said third ISV and / or possibly said serum albumin binding ISV are connected via a linker.

[0381] In a further aspect, the invention provides a polypeptide as described herein, wherein the linker is selected from the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 362 to 372).

[0382] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety is a non-antibody based polypeptide.

[0383] In a further aspect, the invention provides a polypeptide as described herein, further comprising PEG.

[0384] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISV is V HH , humanized V HH or camelized V H .

[0385] In a further aspect, the invention provides a polypeptide, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 1-80.

[0386] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 1 to 80, and wherein the second ISV is selected from the group consisting of SEQ ID NOs: 297 to 304.

[0387] In a further aspect, the invention provides a polypeptide selected from the group consisting of SEQ ID NOs: 249-250, 252-253, 255-256, 258-260, 263, 265-283, 286-289, 306-307, 309-310, 312-313, 315-317, 320, 322-340 and 343-346.

[0388] In a further aspect, the invention provides a polypeptide that specifically binds to CD3 and comprises or consists essentially of: 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0389] (i) CDR1 is selected from the group consisting of:

[0390] (a) SEQ ID NOs: 81-100; or

[0391] (b) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 81-100, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 not having said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0392] (ii) CDR2 is selected from the group consisting of:

[0393] (c) SEQ ID NOs: 101-122; or

[0394] (d) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 101-122, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0395] (iii) CDR3 is selected from the group consisting of:

[0396] (e) SEQ ID NOs: 123-143; or

[0397] (f) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 123-143, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0398] The present invention also provides a polypeptide as described herein, wherein:

[0399] (i) CDR1 is selected from the group consisting of:

[0400] (a) SEQ ID NOs: 81-87; and

[0401] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 81, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0402] (ii) CDR2 is selected from the group consisting of:

[0403] (c) SEQ ID NOs: 101-109; and

[0404] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 101, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0405] (iii) CDR3 is selected from the group consisting of:

[0406] (e) SEQ ID NOs: 123-127; and

[0407] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 123, provided that the polypeptide comprising a CDR3 with said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without the 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0408] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0409] (a) SEQ ID NO:81; and

[0410] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 81, wherein

[0411] - At position 1, G has changed to R;

[0412] - At position 3, T has changed to A;

[0413] - At position 4, Y has changed to F;

[0414] - S has changed to G at position 8; and / or

[0415] - At position 10 G has changed to A.

[0416] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0417] (a) SEQ ID NO: 101; and

[0418] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 101, wherein

[0419] - At position 3, V has changed to T or A;

[0420] - At position 5, S has changed to T;

[0421] - G has changed to D or E at position 6; and / or

[0422] - At position 9 T has changed to S, A or P.

[0423] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0424] (a) SEQ ID NO: 123; and

[0425] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 123, wherein

[0426] - At position 2, I has changed to T;

[0427] - I has changed to V at position 9; and / or

[0428] - At position 10 A has been changed to P.

[0429] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:81, CDR2 is represented by SEQ ID NO:101, and CDR3 is represented by SEQ ID NO:123.

[0430] In a further aspect, the invention provides a polypeptide as described herein, wherein:

[0431] (i) CDR1 is selected from the group consisting of:

[0432] (a) SEQ ID NOs: 88; and

[0433] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 88, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0434] (ii) CDR2 is selected from the group consisting of:

[0435] (c) SEQ ID NOs: 110; and

[0436] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 110, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0437] (iii) CDR3 is selected from the group consisting of:

[0438] (e) SEQ ID NOs: 128; and

[0439] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 128, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0440] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:88.

[0441] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR2 is SEQ ID NO:110.

[0442] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:128.

[0443] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:88, CDR2 is represented by SEQ ID NO:110, and CDR3 is represented by SEQ ID NO:128.

[0444] In a further aspect, the invention provides a polypeptide as described herein, wherein:

[0445] (i) CDR1 is selected from the group consisting of:

[0446] (a) SEQ ID NO:90; and

[0447] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 90, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0448] (ii) CDR2 is selected from the group consisting of:

[0449] (c) SEQ ID NOs: 112-113; and

[0450] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 112, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0451] (iii) CDR3 is selected from the group consisting of:

[0452] (e) SEQ ID NO: 130; and

[0453] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 130, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0454] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:90.

[0455] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR2 is selected from the group consisting of:

[0456] (a) SEQ ID NO: 112; and

[0457] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[0458] - At position 2 V has changed to A.

[0459] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:130.

[0460] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:90, CDR2 is represented by SEQ ID NO:112, and CDR3 is represented by SEQ ID NO:130.

[0461] In a further aspect, the invention provides a polypeptide as described herein, wherein:

[0462] (i) CDR1 is selected from the group consisting of:

[0463] (a) SEQ ID NOs: 89; and

[0464] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 89, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0465] (ii) CDR2 is selected from the group consisting of:

[0466] (c) SEQ ID NOs: 111; and

[0467] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 111, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0468] (iii) CDR3 is selected from the group consisting of:

[0469] (e) SEQ ID NOs: 129; and

[0470] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 129, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0471] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:89.

[0472] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR2 is SEQ ID NO:111.

[0473] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:129.

[0474] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:89, CDR2 is represented by SEQ ID NO:111, and CDR3 is represented by SEQ ID NO:129.

[0475] The present invention also provides a polypeptide as described herein, wherein:

[0476] (i) CDR1 is selected from the group consisting of:

[0477] (a) SEQ ID NOs:91-93; and

[0478] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:91, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0479] (ii) CDR2 is selected from the group consisting of:

[0480] (c) SEQ ID NOs: 114-117; and

[0481] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 114, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0482] (iii) CDR3 is selected from the group consisting of:

[0483] (e) SEQ ID NOs: 131-133; and

[0484] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 131, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0485] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0486] (a) SEQ ID NO:91; and

[0487] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[0488] - At position 6, R has changed to N or T;

[0489] - N has been changed to H at position 7; and / or

[0490] - At position 8 the M has been changed to a T.

[0491] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0492] (a) SEQ ID NO: 114; and

[0493] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[0494] - At position 1, R has changed to Q;

[0495] - T has changed to S at position 3; and / or

[0496] - In position 7 D has changed to A or K.

[0497] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0498] (a) SEQ ID NO: 131; and

[0499] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein

[0500] - S has changed to R at position 2; and / or

[0501] - At position 6 S has changed to V.

[0502] In a further aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:91, CDR2 is represented by SEQ ID NO:114, and CDR3 is represented by SEQ ID NO:131.

[0503] The present invention also provides a polypeptide as described herein, wherein:

[0504] (i) CDR1 is selected from the group consisting of:

[0505] (a) SEQ ID NOs: 94-100; and

[0506] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 94, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0507] (ii) CDR2 is selected from the group consisting of:

[0508] (c) SEQ ID NOs: 118-122; and

[0509] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 118, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0510] (iii) CDR3 is selected from the group consisting of:

[0511] (e) SEQ ID NOs: 134-143; and

[0512] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 134, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0513] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0514] (a) SEQ ID NO:94; and

[0515] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[0516] - At position 3, S has changed to T, A or G;

[0517] - At position 5, N has changed to S;

[0518] - M has changed to T or A at position 6; and / or

[0519] - At position 9 L has changed to M.

[0520] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0521] (a) SEQ ID NO: 118; and

[0522] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 118, wherein

[0523] - At position 2, H has changed to V;

[0524] - At position 5, S has changed to H or A;

[0525] - N has changed to S at position 8; and / or

[0526] - At position 10 Y has changed to F.

[0527] In a further aspect, the invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0528] (a) SEQ ID NO: 134; and

[0529] (b) an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 134, wherein

[0530] - A has changed to S or D at position 6;

[0531] - F has changed to Y or A at position 7;

[0532] - At position 8, R has changed to H;

[0533] - At position 9, S has changed to A;

[0534] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[0535] - At position 14 V has changed to I.

[0536] In a further aspect, the invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:94, CDR2 is represented by SEQ ID NO:118, and CDR3 is represented by SEQ ID NO:134.

[0537] In a further aspect, the invention provides a polypeptide as described herein, which is a Nanobody, a VHH , humanized V HH or camelized V H .

[0538] In a further aspect, the invention provides a polypeptide as described herein, further comprising a serum protein binding moiety.

[0539] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety binds to serum albumin.

[0540] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety is an ISV that binds serum albumin.

[0541] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISV that binds serum albumin essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being as determined according to the Kabat definition; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being as determined according to Kontermann 2010.

[0542] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISV that binds serum albumin is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG and Alb82-GGG (SEQ ID NOs: 348 to 360).

[0543] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISV is directly linked or linked via a linker.

[0544] In a further aspect, the invention provides a polypeptide as described herein, wherein the linker is selected from the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 362 to 372).

[0545] In a further aspect, the invention provides a polypeptide as described herein, further comprising a PEG moiety.

[0546] In a further aspect, the invention provides a nucleic acid or a nucleic acid sequence encoding a polypeptide as defined herein.

[0547] In a further aspect, the invention provides a vector comprising a nucleic acid or a nucleic acid sequence as defined herein.

[0548] In a further aspect, the invention provides a host cell transformed or transfected with a nucleic acid or nucleic acid sequence as defined herein or with a vector as defined herein.

[0549] In a further aspect, the present invention provides a method of producing a polypeptide as defined herein, the method comprising culturing a host cell as defined herein under conditions allowing expression of the polypeptide as defined herein and recovering the produced polypeptide from the culture.

[0550] In a further aspect, the present invention provides a pharmaceutical composition comprising a polypeptide as described herein, or a polypeptide produced according to a method as described herein.

[0551] In a further aspect, the invention provides a polypeptide as described herein, or a polypeptide produced as described herein, for use in treating a subject in need thereof.

[0552] In a further aspect, the invention provides a method of delivering a prophylactic or therapeutic polypeptide to a specific location, tissue or cell type in vivo, the method comprising the step of administering to a subject a polypeptide as described herein or a polypeptide produced as described herein.

[0553] In a further aspect, the present invention provides a polypeptide as described herein, or a polypeptide produced as described herein, 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.

[0554] In another aspect, the present invention provides a method for preventing, treating or ameliorating a disease selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases and autoimmune diseases, comprising the step of administering a polypeptide as described herein or a polypeptide produced as described herein to a subject in need thereof.

[0555] In further aspects, the invention provides polypeptides or methods for preventing, treating or ameliorating a disease as described herein, wherein the proliferative disease is cancer.

[0556] In another aspect, the invention provides a polypeptide or method for preventing, treating or ameliorating a disease as described herein, wherein the cancer is selected from the group consisting of carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas: breast cancer, ovarian cancer, cervical cancer, glioblastomas, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer and Burkitt's lymphoma. lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penis cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary effusion lymphoma, neuroectodermal tumor, rhabdomyosarcoma; and any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis.

[0557] In further aspects, the invention provides polypeptides or methods for preventing, treating or ameliorating a disease as described herein, wherein the treatment is a combination therapy.

[0558] In a further aspect, the invention provides a kit comprising a polypeptide as defined herein, a nucleic acid or a nucleic acid sequence as defined herein, a vector as defined herein or a host cell as defined herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0559] Figure 1 ： QC of human TCR / CD3 and human CD3 transfected cell lines using 100 nM anti-human TCR α / β antibody (clone BW242 / 412) (black) and 100 nM anti-human CD3 antibody (clone OKT-3) (grey). The MCF values ​​(mean channel fluorescence) for each cell line are plotted.

[0560] Figure 2 A and 2B: Dose-dependent binding of monovalent CD3 nanobodies to human TCR / CD3 expressed on CHO-K1 cells ( Figure 2 A) and dose-dependent binding to purified primary human T cells ( Figure 2 B). MCF values ​​(mean channel fluorescence) are plotted against the concentration of Nanobodies.

[0561] Figure 3 ：Dose-dependent binding of monovalent CD3 Nanobodies to HEK293H human TCR(2IAL) / CD3 (filled circles), HEK293H human CD3 (crosses) and to the HEK293H parental cell line (open circles). MCF values ​​(mean channel fluorescence) are plotted against the concentration of Nanobodies.

[0562] Figure 4 A and 4B: T cell activation data of bead coupled monovalent CD3 nanobody ( Figure 4 A). T cell activation data of monovalent CD3 nanobodies presented in solution form ( Figure 4 B). Activation was measured by monitoring upregulation of CD69 on primary human T cells. The MCF values ​​(mean channel fluorescence) for each Nanobody are plotted.

[0563] Figure 5 A-5C: Dilution series of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific Nanobodies were compared with human TCR / CD3 ( Figure 5 A), primary human T cells ( Figure 5 B) and Ramos cells ( Figure 5 C) Binding of . MCF values ​​(mean channel fluorescence) are plotted against the concentration of Nanobodies.

[0564] Figure 6 A and 6B: In human T cell-mediated Ramos ( Figure 6 A) and Raji( Figure 6 B) Dose-dependent killing effect of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific Nanobodies in B cell killing assay. % cell death (% of TOPRO positive cells) is plotted against the concentration of Nanobodies.

[0565] Figure 7 ： Dose-dependent binding of anti-CD20 Nanobodies on human CD20 Ramos (open symbols) and Raji (closed symbols) cells. MCF values ​​(mean channel fluorescence) are plotted against the concentration of Nanobodies.

[0566] Figure 8 ： Dose-dependent killing effects of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific Nanobodies in the xCELLigence-based human T cell-mediated CHO-K1 human CD20 killing assay. CIs are plotted against the concentration of Nanobodies.

[0567] Fig. 9 ：Killing effect of 1 μM CD20xCD3 and irrelevant constructs in xCELLigence-based killing assays using CHO-K1 human CD20 cells (black bars) and using the CHO-K1 parental cell line (grey bars) to elucidate TAA-dependent killing. The Cell Index (CI) is plotted against the concentration of the Nanobody.

[0568] Fig.10 ： Dose-dependent killing effect of CD20xCD3 Nanobodies with 9GS linker (open circles - dashed line) and 35GS linker (filled squares - dashed line) and CD3xCD20 Nanobodies with 35GS linker (filled diamonds - solid line) in a flow cytometry-based killing assay using Ramos. % cell death (TORPRO-positive cells) is plotted against the concentration of the Nanobodies.

[0569] Fig.11 ： Dose-dependent killing of T017000062 in a flow cytometry-based human T cell-mediated Ramos B cell killing assay using different effector (E) to target (T) ratios (E:T ratio 10:1 - solid circles, E:T ratio 5:1 - open squares, E:T ratio 2:1 - solid triangles, and E:T ratio 1:1 - open diamonds). % cell death (% of TOPRO-positive cells) is plotted against the concentration of the nanobody.

[0570] Fig.12 ： Time-dependent cytolytic activity of CD20 / CD3 in the xCELLigence purified primary human T cell-mediated killing assay using CHO-K1 human CD20 target cells. % specific lysis is plotted against the concentration of the construct. The different curves represent the assay time after addition of T cells.

[0571] Fig.13 A-13C: Serial dilutions of the HLE construct were mixed with human TCR / CD3 ( Fig.13 A), primary human T cells ( Fig.13 B) and Ramos cells ( Fig.13 C) Binding of . MCF values ​​(mean channel fluorescence) are plotted against the concentration of Nanobodies.

[0572] Fig.14 A-14D: Dose-dependent killing of CD20xCD3 bispecific Nanobodies (solid line-diamonds) compared to CD20xCD3xALB11 constructs (solid line-filled triangles) in a flow cytometry-based human T cell-mediated Ramos B cell killing assay ( Fig.14 A, Fig.14C) and dose-dependent killing effect of the CD20xCD3xALB11 construct in the absence (solid line - filled triangles) or presence (dashed line - open triangles) of 30 μM HSA ( Fig.14 B, Fig.14 D). % cell death (% of TOPRO positive cells) is plotted against the concentration of Nanobody.

[0573] Fig.15 ： Binding of 100 nM monovalent anti-HER2 nanobody (5F07) to SK-BR-3, MCF-7 and MDA-MB-468 cell lines in flow cytometry to compare HER2 expression levels. The MCF values ​​(mean channel fluorescence) for each cell line are plotted.

[0574] Fig.16 ： Dose-dependent killing effects of bispecific CD3xHER2 nanobodies (dashed line) and bispecific HER2xCD3 (solid line) in xCELLigence-based human T cell-mediated cell killing assays. Data were analyzed using 18 hours. Cell index (CI) was plotted against the concentration of nanobodies.

[0575] Fig.17 ： Dose-dependent INF-γ production by human T cells after incubation of human CD20-positive CHO-K1 cells with bispecific CD20xCD3 nanobodies in a flow cytometry-based killing assay. Data were analyzed after 72 hours of incubation. OD at 405 nm was plotted against the concentration of nanobodies.

[0576] Fig.18 ： Study Design PBMC B cell depletion model. PBMC were injected intraperitoneally into animals on day 3 (D3). From D3 to D7, mice were treated with T017000084 (CD3 / CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (irrelevant nanobody).

[0577] Fig.19 ： Absolute PBMC-derived B cells were counted on a logarithmic scale. Individual animal results were plotted. The number of B-cells is shown for the different treatment groups.

[0578] Fig. 20 ： Study Design Ramos model. Ramos cells were injected intravenously into mice at D1. PBMCs were injected intraperitoneally into animals at D3. From D3 to D7, mice were treated with T017000084 (CD3 / CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (irrelevant nanobody).

[0579] Fig.21 ：Absolute Ramos B cells are counted in logarithmic order. Individual animal results are plotted. The open circles above the graph indicate that the active dose is statistically significantly different from the unrelated NB (T017000088) based on the F test from the mixed effects ANOVA analysis. All effects are statistically significant at the 5% significance level.

[0580] Fig. 22 ： Absolute PBMC-derived B cells are counted in logarithmic order. Individual animal results are plotted. The open circles above the graph indicate that the active dose is statistically significantly different from the unrelated NB (T017000088) based on the F test from the mixed effects ANOVA analysis. All effects are statistically significant at the 5% significance level.

[0581] Fig.23 A and 23B: EGFR ( Fig.23 A; Santa Cruz, sc-120PE) or CEACAM5 ( Fig.23 B; Sino Biological, 11077-MM02-P) expression levels on HER14, Hela, LoVo and LS174-T cell lines. The MCF value (mean channel fluorescence) of each cell line is plotted. DETAILED DESCRIPTION OF THE INVENTION

[0583] The inventors recognize that the format that brings T cells and tumor cells together to induce an immune response should meet various and often contradictory requirements. The format should be widely applicable. In particular, the format should preferably be used for a wide range of patients, and preferably also for a wide range of tumors. The format should preferably be safe and only target the intended cells. In addition, the format should preferably be small enough to easily penetrate tissues and tumors, while on the other hand, the format should be patient-friendly. For example, the format should have an extended half-life so that the format is not immediately removed by renal clearance after administration. However, the extended half-life should preferably not introduce off-target activity and side effects or limit penetration into tissues and tumors. In addition, it is recognized that tumor cells often produce escape mechanisms by downregulation of targeted antigens within the treatment. Therefore, in another preferred form, the format should target multiple antigens simultaneously.

[0584] The present invention fulfills at least one of these needs.

[0585] In particular, it was predicted that immunoglobulin single variable domains (ISVs) would in principle be ideal candidates, as they are small enough to easily penetrate (tumor) tissue and can be combined with other ISVs as building blocks.Secondly, ISVs against CD3, in particular CD3ε, should have broad applicability.

[0586] Six clusters of related ISVs were identified, which have an unexpected range of favorable features. First, the ISVs are unexpectedly broadly applicable, i.e., the CD3 ISVs are able to bind T cells from different donors with high affinity. Formatted in multispecific polypeptides, the CD3 ISVs enable tumor cell killing of different tumor-associated antigens. Therefore, the CD3 ISVs can be used against a wide range of cancers. In addition, when bound to albumin, the multispecific polypeptides comprising the CD3 ISVs remain active. This contributes to good PK properties and patient compliance, while minimizing side effects. The polypeptides of the invention show an effect only when bound to both T cells and target cells, which indicates their safety.

[0587] The inventors believe that simultaneous targeting of multiple antigens reduces the likelihood of generating tumor escape variants, so the therapeutic activity of T cell engagement strategies is improved. Multispecific polypeptides are provided comprising a CD3 ISV combined with immunoglobulin single variable domains directed against different target antigens and / or different epitopes on a specific antigen (biparatopic).

[0588] Immunoglobulin sequences, such as antibodies and antigen-binding fragments (e.g., immunoglobulin single variable domains or ISVs) derived therefrom, are used to specifically target their respective antigens in research and therapeutic applications. The production of immunoglobulin single variable domains (such as, for example, VHHs or nanobodies) can involve immunization of experimental animals (such as llamas), construction of phage libraries from immune tissue, selection of phages displaying immunoglobulin single variable domains that bind to antigen, and screening of said domains and engineered constructs thereof for the desired specificity (WO 94 / 04678). Alternatively, similar immunoglobulin single variable domains (such as, for example, dAbs) can be produced by selecting phage displaying antigen-binding immunoglobulin single variable domains directly from natural or synthetic libraries and then screening the domains and their engineered constructs for the desired specificity (Ward et al., Nature, 1989, 341: 544-6; Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications such as WO 06 / 030220, WO 06 / 003388 and Domantis Ltd.). Unfortunately, the use of monoclonal and / or heavily engineered antibodies also carries high manufacturing costs and may result in suboptimal tumor penetration compared to other strategies.

[0589] The present invention provides multispecific polypeptides that specifically bind to CD3 of the T cell receptor complex, which have an unexpected range of advantageous features. First, the polypeptides are easy to manufacture. In addition, the ISVs are unexpectedly widely applicable, i.e., the CD3 ISVs are able to bind T cells from different donors with high affinity. Formatted in a multispecific polypeptide, the CD3 ISVs enable tumor cell killing of different tumor-associated antigens. In contrast, when the polypeptides do not bind to T cells and target cells, no killing is observed, which emphasizes the safety of the polypeptides of the present invention. Therefore, the CD3 ISVs can be used against a wide range of cancers. In addition, the CD3 ISVs can be considered safe. In addition, when bound to albumin, the multispecific polypeptides comprising the CD3 ISVs remain active. This will contribute to good PK properties and patient compliance while minimizing side effects.

[0590] Thus, the present invention relates to polypeptides comprising a first and a second immunoglobulin single variable domain (ISV), wherein the first ISV has a high affinity for / binds to CD3 and the second ISV has a high affinity for / binds to an antigen on a cell (target cell), preferably a tumor cell. The antigen is preferably specific for the target cell, such as, for example, a tumor associated antigen (TAA). The multispecific polypeptides of the invention direct T cells to cells (e.g., tumor cells) and induce T cell activation so as to allow the T cells to inhibit or kill the target cell (e.g., the tumor cell).

[0591] definition:

[0592] a) Unless otherwise stated or defined, all terms used have their ordinary meanings in the art, which are clear to a person skilled in the art. For example, see WO 08 / 020079

[0593] The manual of standards referred to in paragraph a) on page 46.

[0594] b) The term "immunoglobulin single variable domain", which can be used interchangeably with "single variable domain" and "ISV", refers to a molecule in which the antigen binding site is present in and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof (such as Fab, scFv, etc.), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will participate in the formation of the antigen binding site. In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain. Therefore, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0595] The terms "immunoglobulin single variable domain", "single variable domain" and "ISV" therefore do not include conventional immunoglobulins or fragments thereof, which require the interaction of at least two variable domains to form an antigen binding site. However, these terms do include fragments of conventional immunoglobulins, in which the antigen binding site is formed by a single variable domain.

[0596] The term "immunoglobulin single variable domain" or "ISV" includes, but is not limited to, an antigen binding domain or fragment, such as a V HH Domain or V H or V L The terms antigen binding molecule or antigen binding protein are used interchangeably and also include the term nanobody. An immunoglobulin single variable domain may be a light chain variable domain sequence (e.g., V L -sequence), or a heavy chain variable domain sequence (e.g. V H -sequences); more particularly, they may be heavy chain variable domain sequences derived from conventional four-chain antibodies or heavy chain variable domain sequences derived from heavy chain antibodies. Accordingly, the immunoglobulin single variable domain may be a domain antibody, or an immunoglobulin sequence suitable for use as a domain antibody, a single domain antibody, or an immunoglobulin sequence suitable for use as a single domain antibody, "dAbs" or an immunoglobulin sequence suitable for use as dAbs, or a nanobody, including but not limited to V HHSequences, humanized VHH sequences or camelized VH sequences. The present invention includes immunoglobulin sequences from different sources, including immunoglobulin sequences from mice, rats, rabbits, donkeys, humans and camelids. Immunoglobulin single variable domains include fully human, humanized, other sequence-optimized or chimeric immunoglobulin sequences. Immunoglobulin single variable domains and the structure of immunoglobulin single variable domains can be considered to be - but not limited to - composed of four framework regions or

[0597] The "FR's" are referred to in the art and herein as "Framework Region 1" or

[0598] "FR1"; "Framework region 2" or "FR2"; "Framework region 3" or "FR3"; "Framework region 4" or "FR4";

[0599] The framework regions are separated by three complementarity determining regions or "CDR's" which are

[0600] "CDR's" are referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. It should be noted that the term Nanobody or Nanobodies is a registered trademark of Ablynx NV and is therefore also referred to as ( or ).

[0601] c) Unless otherwise indicated, the terms "immunoglobulin sequence", "sequence", "nucleotide sequence" and "nucleic acid" are as described in WO08 / 020079, page 46, paragraph b).

[0602] d) Unless otherwise indicated, all methods, steps, techniques and operations not particularly described in detail can be carried out or have been carried out in a manner known per se, which is clear to a person skilled in the art. For example, reference is again made to the standard manuals and general background art mentioned herein and to other references cited therein; and for example the following review Presta 2006 (Adv.

[0603] Drug Deliv.Rev.58(5-6):640-656), Levin and Weiss 2006 (Mol.Biosyst.

[0604] 2(1):49-57), Irving et al. 2005 (J. Immunol. Methods 248(1-2):31-45), Schmitz et al. 2000 (Placenta 21 Suppl A:S106-112, Gonzales et al. 2005

[0605] (Tumour Biol. 26(1):31-43), which describes protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desirable properties of proteins such as immunoglobulins.

[0606] e) Amino acid residues are represented by the standard three-letter or single-letter amino acid code. Reference is made to Table A-2 on page 48 of International Application WO 08 / 020079 of Ablynx NV entitled "Immunoglobulin single variable domains directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with Il-6 mediated signalling".

[0607] f) For the purpose of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated or determined as described in WO 08 / 020079 (incorporated herein by reference), page 49, paragraph e), for example by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], wherein

[0608] The deletion, insertion, substitution or addition of each nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference of a single nucleotide (position); or using a suitable computer algorithm or technique, also as described in WO 08 / 020079 (incorporated herein by reference)

[0609] As stated in paragraph e) on page 49.

[0610] g) For the purpose of comparing two or more immunoglobulin single variable domains or other amino acid sequences, such as the polypeptides of the present invention, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be expressed as follows:

[0611] 08 / 020079 (incorporated herein by reference) pages 49 and 50, for example by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], 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 considered to be a difference of a single amino acid residue (position), i.e. an "amino acid difference" as defined herein; or by using a suitable computer algorithm or technique, also as described in paragraph f) of WO 08 / 020079 (incorporated herein by reference) pages 49 and 50.

[0612] Furthermore, when determining the degree of sequence identity between two immunoglobulin single variable domains, one skilled in the art may take into account so-called "conservative" amino acid substitutions, such as

[0613] As stated on page 50 of 08 / 020079.

[0614] It can also be based on the analysis of amino acid variation frequencies between homologous proteins of different species proposed by Schulz et al. 1978 (Principles of Protein Structure, Springer-Verlag), by Chou and Fasman 1975 (Biochemistry 13:211) and 1978 (Adv.

[0615] Enzymol. 47:45-149), and by Eisenberg et al. 1984 (Proc. Natl. Acad. Sci. USA 81:140-144), Kyte & Doolittle 1981 (J Molec. Biol. 157:105-132), and Goldman et al. 1986 (Ann. Rev. Biophys.

[0616] Chem. 15:321-353) are used to analyze the hydrophobic patterns of proteins for any amino acid substitutions of the polypeptides described herein, which are incorporated herein by reference in their entirety. Information on the primary, secondary and tertiary structures of Nanobodies is given in the specification and in the general background art cited above. Moreover, for this purpose, V of llama is given, for example, in Desmyter et al. 1996 (Nature Structural Biology, 3:803), Spinelli et al. 1996 (Natural Structural Biology 3:752-757) and Decanniere et al. 1999 (Structure, 7:361). HH The crystal structure of the domain can be found in the above-cited prior art. H V is formed at these positions in the domain H / V L Further information on the interface and possible camelizing substitutions for some amino acid residues.

[0617] h) An immunoglobulin single variable domain and a nucleic acid sequence are referred to as "identical" if they have 100% sequence identity (as defined herein) over their entire length.

[0618] i) When comparing two immunoglobulin single variable domains, the term "amino acid difference" refers to the insertion, deletion or substitution of a single amino acid residue at a certain position in the first sequence compared to the second sequence; it should be understood that two immunoglobulin single variable domains may contain one, two or more such amino acid differences.

[0619] j) When a nucleotide sequence or an amino acid sequence is referred to as "comprising" another nucleotide sequence or amino acid sequence, respectively, or as "essentially consisting of" another nucleotide sequence or amino acid sequence, this has the meaning given in paragraph i) of WO 08 / 020079, pages 51-52.

[0620] k) The term "in essentially isolated form" has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08 / 020079.

[0621] 1) The terms "domain" and "binding domain" have the meaning given to them in WO 08 / 020079, page 53, paragraph k).

[0622] m) The terms "antigenic determinant" and "epitope" are used interchangeably herein and have the same meaning as in WO 08 / 020079

[0623] has the meaning given to it in paragraph 1) on page 53.

[0624] n) As further described in paragraph m) on page 53 of WO 08 / 020079, an amino acid sequence (e.g. an antibody, polypeptide, or generally an antigen-binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, have affinity for and / or have specificity for a specific antigenic determinant, epitope, antigen or protein (or at least a part, fragment or epitope thereof) is said to be "directed against" or "directed to" said antigenic determinant, epitope, antigen or protein.

[0625] o) The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen binding molecule or antigen binding protein (such as an ISV, Nanobody or polypeptide of the invention) can bind. The specificity of an antigen binding protein can be determined based on affinity and / or avidity. Affinity (determined by the equilibrium constant (K) for the dissociation of an antigen from an antigen-binding protein) D K (or KD) is a measure of the binding strength between an antigenic determinant (i.e., target) and an antigen-binding site on an antigen-binding protein (i.e., ISV or Nanobody): D The smaller the value, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D ). As is clear to the skilled person (e.g. on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen under investigation.

[0626] Avidity is the affinity of a polypeptide, i.e., the ability of a ligand to bind via two (or more) pharmacophores (ISVs), where multiple interactions synergistically enhance the "apparent" affinity. Avidity is a measure of the strength of binding between a polypeptide of the invention and a relevant antigen. A polypeptide of the invention is able to bind to at least two targets via its two (or more) structural units, such as ISVs or nanobodies, where

[0627] Multiple interactions, e.g. a first building block, ISV or Nanobody binds a first target and a second building block, ISV, or Nanobody binds a second target, synergistically enhance the "appearance"

[0628] Affinity. Avidity is related to the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of relevant binding sites present on the antigen-binding molecule. For example, and without limitation, a polypeptide containing two or more building blocks, such as ISVs or Nanobodies directed against different targets on a cell can (and typically will) bind with higher affinity than a single monomer or single building block, such as, for example, a monovalent ISV or Nanobody, in a polypeptide comprised in the invention.

[0629] Any value greater than 10 -4mol / L K D Value (or any value below 10 4 M -1 K A Values ​​of 1 / 100 μL / mol are usually considered to be non-specific binding.

[0630] The polypeptide of the invention comprises a first and a second building block, such as a first and a second ISV, or a first and a second Nanobody. It is preferred that the affinity of each building block, such as an ISV or a Nanobody is determined separately. In other words, for a monovalent building block, ISV or Nanobody, the affinity is determined independently of the avidity effects due to other building blocks, ISVs or Nanobodies (which may or may not be present). For a monovalent building block, the affinity of an ISV or Nanobody can be determined for the monovalent building block, ISV or Nanobody itself, i.e. when the monovalent building block, ISV or Nanobody is not comprised in the polypeptide of the invention. In an alternative or in addition, the affinity of a monovalent building block, ISV or Nanobody can be determined for one target in the absence of other targets.

[0631] Binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and various variations thereof known per se in the art; as well as other techniques mentioned herein.

[0632] The dissociation constant may be an actual or apparent dissociation constant, as will be apparent to the skilled person. Methods for determining the dissociation constant will be apparent to the skilled person and include, for example, the techniques mentioned herein. In this regard, it will also be apparent that it may not be possible to measure dissociation constants greater than 10 -4 mol / L or 10 -3 mol / L (e.g., 10 -2 Optionally, as will also be apparent to the skilled person, the (actual or apparent) dissociation constant may be based on the (actual or apparent) binding constant (K A ), through the relationship [K D =1 / K A ] is calculated in this way.

[0633] Affinity indicates the strength or stability of a molecular interaction. Affinity is usually expressed as K D or dissociation constant, which has units of moles per liter (or M). Affinity can also be expressed as the binding constant, K A , which is equal to 1 / K D And has (mol / L) -1 (or M -1In this specification, the stability of the interaction between two molecules (such as an amino acid sequence, Nanobody or polypeptide of the invention and its intended target) will be mainly based on the K of their interaction. D The technician will understand that, given the relationship K A =1 / K D , through its K D The value specifies the strength of the molecular interaction and can also be used to calculate the corresponding K A Value. K D -value also characterizes the strength of molecular interaction in a thermodynamic sense, because the well-known relationship DG = RT.ln(K D )(Equivalently DG = -RT.ln(K A ))It is related to the free energy of binding (DG), where R equals the gas constant, T equals the absolute temperature and ln denotes the natural logarithm.

[0634] K for a biological interaction that is considered meaningful (e.g., specific) D Usually in 10 -10 M(0.1nM) to 10 -5 M (10000nM). The stronger the interaction, the higher the K D The lower.

[0635] K D It can also be expressed as the dissociation rate constant of the complex (expressed as k off ) and its binding rate (denoted as k on ) ratio (so K D =k off / k on and K A =k on / k off ). Dissociation rate k off With unit s -1 (where s is the SI unit symbol for seconds). The binding rate k on With unit M -1 s -1 The binding rate can be 10 2 M -1 s -1 to about 10 7 M -1 s -1 , approaching the diffusion-limited binding rate constant for biomolecular interactions. 1 / 2 =ln(2) / k off , the dissociation rate is related to the half-life of a given molecular interaction. The dissociation rate can be in the range of 10 -6 s -1 (Close to t with multiple days1 / 2 Irreversible complex) to 1s -1 (t 1 / 2 =0.69s).

[0636] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology, 13: 1551-1559). The term "surface plasmon resonance" as used herein 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 another molecule passes over the immobilized molecule under flow conditions, obtaining a k on , k off The measured value and the resulting K D (or K A ) value. This can be performed, for example, using the well-known BIAcore (R) system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. 1993 (Ann. Biol. Clin. 51: 19-26), Jonsson et al. 1991 (Biotechniques 11: 620-627), Johnsson et al. 1995 (J. Mol. Recognit. 8: 125-131) and Johnnson et al. 1991 (Anal. Biochem. 198: 268-277).

[0637] Alternatively, affinity can be determined in a kinetic exclusion analysis (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) using The KinExA platform (Sapidyne Instruments Inc, Boise, USA) is used for measurement. The term "KinExA", as used herein, refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. The equilibrium solution of the antibody / antigen complex is passed through a column with a small ball pre-coated with an antigen (or antibody), which allows free antibodies (or antigens) to bind to the coated molecules. Detection of the thus captured antibody (or antigen) is completed by binding of the antibody (or antigen) to a fluorescently labeled protein.

[0638] The skilled person will also be aware that the measured K D can correspond to the apparent K D In addition, if a molecule contains more than one recognition site for other molecules, the apparent K D In this case, the affinity measured is not affected by the avidity of the interaction of the two molecules.

[0639] Another method that can be used to assess affinity is that of Friguet et al. 1985 (J. Immunol.

[0640] Methods, 77: 305-19). This method establishes a solution binding equilibrium measurement and avoids artifacts associated with adsorption of one of the molecules on a support such as plastic.

[0641] However, K D The precise measurement of K can be extremely labor intensive and therefore, the apparent K is often determined D It should be noted that as long as all measurements are performed in a continuous manner (e.g., keeping the assay conditions unchanged), the apparent K D The measurement can be used as the true K D is an approximate value of , and therefore in this paper, K D and apparent K D should be treated with equal importance or relevance.

[0642] Finally, it should be noted that in many cases, an experienced scientist may judge that it is convenient to use to determine the binding affinity relative to some reference molecule. For example, to evaluate the binding strength between molecules A and B, one may, for example, use a reference molecule C which is known to bind B and which is suitably labeled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in ELISA or FACS (fluorescence activated cell sorting) or other formats (fluorophores for fluorescence detection, chromophores for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result, an IC corresponding to the concentration of A is obtained. 50 value at which the signal measured for C in the absence of A is halved. D ref (K of the reference molecule D ) and the total concentration of the reference molecule c ref is known, then the apparent K of the interaction AB is D It can be obtained from the following formula: KD =IC 50 / (1+c ref / K D ref ). Note that if cref< <K D ref , then K D ≈IC 50 If the IC of the compared conjugate is 50 The measurement is performed in a continuous manner (e.g. maintaining c ref fixed), the strength or stability of the molecular interaction can be measured by IC 50 evaluation, and in this paper this measurement is judged to be equal to K D or apparent K D .

[0643] p) The half-life of the amino acid sequence, compound or polypeptide of the invention can generally be as described in WO

[0644] 08 / 020079, page 57, paragraph o) and as referred to herein, refers to the time taken for the serum concentration of the sequence, compound or polypeptide in vivo to be reduced by 50%, for example due to the degradation of the amino acid sequence or compound and / or the clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of the amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques are clear to the skilled person and can generally be as described in WO 08 / 020079, page 57, paragraph o). As also mentioned in WO 08 / 020079, page 57, paragraph o), the half-life can be expressed by parameters such as t1 / 2-α, t1 / 2-β and the area under the curve (AUC). For example, reference can be made to the experimental section below, as well as standard manuals, such as Kenneth et al. 1996 (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters et al. 1996 (Pharmacokinete analysis: A Practical Approach). See also Gibaldi & Perron 1982 (Pharmacokinetics, Dekker M, 2 nd The term "increase in half-life" or "increased half-life" is as defined in WO 08 / 020079, page 57, paragraph o), and refers in particular to an increase in t1 / 2-β, with or without an increase in t1 / 2-α and / or AUC or both.

[0645] q) With respect to a target or antigen, the term "interaction site" on a target or antigen refers to a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on a target or antigen, which is a binding site, catalytic site, cleavage site, allosteric interaction site, site involved in multimerization (e.g., homomerization or heterodimerization) of the target or antigen with a ligand, receptor or other binding partner; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on a target or antigen involved in a biological action or mechanism of the target or antigen. More generally, an "interaction site" may be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention is able to bind and to which a target or antigen (and / or any pathway, interaction, signal transduction, biological mechanism or biological effect involving the target or antigen) is modulated.

[0646] r) when it binds to a first antigen with an affinity / avidity (as described above, suitably expressed as K) of at least 10 times, such as at least 100 times, and preferably at least 1000 times and up to 10.000 times or more than the affinity of the amino acid sequence or polypeptide for a second target or polypeptide D Value, K A Value, K off Rate and / or K on If the immunoglobulin single variable domain or polypeptide binds at a rate greater than or equal to the rate at which it binds to the first target or antigen relative to the second target or antigen, then the immunoglobulin single variable domain or polypeptide is said to be

[0647] "Specific". For example, a first antigen may bind to a target or antigen with a K that is greater than that of the amino acid sequence or polypeptide to a second target or antigen. D at least 10 times smaller, such as at least 100 times smaller,

[0648] It is preferably at least 1000 times smaller, for example 10.000 times less than K D Preferably, when an immunoglobulin single variable domain or polypeptide is "specific" for a first target or antigen compared to a second target or antigen, it is directed against said first target or antigen (as defined herein) and not against said second target or antigen.

[0649] s) The terms "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably herein to refer to the ability of an immunoglobulin single variable domain or polypeptide to interfere with the binding of a natural ligand to its one or more receptors. The extent to which an immunoglobulin single variable domain or polypeptide of the invention is able to interfere with the binding of another compound, such as a natural ligand, to its target, and therefore whether or not it can be said to cross-block according to the invention, can be determined using a competitive binding assay. A particularly suitable quantitative cross-blocking assay uses a FACS-based or ELISA-based method or Alphascreen to measure the competition between labeled (e.g., His-tagged or biotinylated) immunoglobulin single variable domain or polypeptide according to the invention and other binding agents for their binding to the target. The experimental section generally describes suitable FACS-based, ELISA-based or Alphascreen displacement-based assays for determining whether a binding molecule cross-blocks or is capable of cross-blocking an immunoglobulin single variable domain or polypeptide of the invention. It should be appreciated that the assay can be used for any immunoglobulin single variable domain or other binding agent described herein. Thus, typically, the cross-blocking amino acid sequences or other binding agents according to the invention are, for example, bound to the target in the above-mentioned cross-blocking assay so that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention, the recorded replacement of the immunoglobulin single variable domain or polypeptide according to the invention by 0.01 mM or less of a possible cross-blocking agent to be tested (the cross-blocking agent may be another conventional monoclonal antibody such as IgG, a classical monovalent antibody fragment (Fab, scFv) and engineered variants (e.g. diabodies, triabodies, miniantibodies, VHHs, dAbs, VHs, VLs)) is between 60%-100% (e.g. in an ELISA / Alphascreen-based competition assay) of the maximum theoretical replacement (e.g. replacement by a non-labeled (e.g. unlabeled) immunoglobulin single variable domain or polypeptide to be cross-blocked) (e.g. in an ELISA / Alphascreen-based competition assay) or between 80%-100% (e.g. in a FACS-based competition assay).

[0650] t) if the VHH1 type immunoglobulin single variable domain or type 1 VHH sequence has 85% identity (using the VHH1 consensus sequence as query sequence and the blast algorithm with standard settings (i.e., bloom62 scoring matrix) for the VHH1 consensus sequence (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKE

[0651] REGVSCISSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE

[0652] DTAVYYCAA) (SEQ ID NO: 361) and mandatory with cysteine ​​at position 50 (i.e., C50) (using Kabat numbering), then indicates that the amino acid sequence such as, for example, an immunoglobulin single variable domain or polypeptide according to the invention is a "VHH1 type immunoglobulin single variable domain" or a "type 1 VHH sequence".

[0653] u) If an amino acid sequence, e.g. an immunoglobulin single variable domain or a polypeptide according to the invention, is specific for two different antigens or antigenic determinants (e.g. serum albumin from two different mammalian species, e.g. human serum albumin and cynomolgus serum albumin), it is said to be "cross-reactive" towards these different antigens or antigenic determinants.

[0654] v) As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), according to V given by Kabat et al. H The amino acid residues of immunoglobulin single variable domains are numbered according to the universal numbering of domains ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication number 91), as described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods).

[0655] 240(1-2):185-195; see, for example, Figure 2 ) as applied to camelids

[0656] V HH It should be noted that - as is known in the art for V H Domain and

[0657] As is well known in VHH domains - 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 (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or

[0658] The actual sequence may contain more amino acid residues than the number allowed by 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. The total number of amino acid residues in a VH domain and a VHH domain will generally be in the range of 110 to 120, typically between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0659] The determination of CDR regions can also be carried out according to different methods. In the determination of CDR according to Kabat, the FR1 of the immunoglobulin single variable domain comprises the amino acid residues at positions 1-30, the CDR1 of the immunoglobulin single variable domain comprises the amino acid residues at positions 31-35, the FR2 of the immunoglobulin single variable domain comprises the amino acid residues at positions 36-49, the CDR2 of the immunoglobulin single variable domain comprises the amino acid residues at positions 50-65, the FR3 of the immunoglobulin single variable domain comprises the amino acid residues at positions 66-94, the CDR3 of the immunoglobulin single variable domain comprises the amino acid residues at positions 95-102, and the FR4 of the immunoglobulin single variable domain comprises the amino acid residues at positions 103-113.

[0660] In this application, unless otherwise indicated, CDR sequences are according to Kontermann and Dübel (Eds.

[0661] 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acid residues at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0662] w) The figures, sequence listing and experimental parts / examples provided are merely to further illustrate the present invention and should not be understood or interpreted as limiting the scope of the present invention and / or the appended claims in any way unless otherwise clearly stated herein.

[0663] x) The half maximal inhibitory concentration (IC50) is a measure of the effect of a compound in inhibiting a biological or biochemical function, such as a pharmacological effect. This quantitative measurement represents how many ISVs or Nanobodies (inhibitors) are needed to inhibit a given biological process (or component of a process, i.e., enzymes, cells, cell receptors, chemotaxis, degenerative changes, metastasis, invasion, etc.) by half. In other words, it is the half maximal (50%) inhibitory concentration (IC) of a substance (50% IC, or IC50). The IC50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonists, such as ISVs or Nanobodies of the invention, on the reverse agonist activity. The IC50 value can be calculated for a given antagonist, such as an ISV or Nanobody of the invention, by determining the concentration required to inhibit half of the maximal biological response of the agonist.

[0664] The term half maximal effective concentration (EC50) refers to the concentration of a compound that induces a response that is halfway between the baseline and the maximum value after a specified exposure time. It is used herein as a measure of the efficacy of a polypeptide, an ISV or a nanobody. The EC50 of a graded dose-response curve represents the concentration of a compound at which 50% of its maximum effect is observed. Concentrations are preferably expressed in molar units.

[0665] In biological systems, small changes in ligand concentrations usually result in rapid changes in responses that conform to an S-shaped curve function. The inflection point at which the response increases as the ligand concentration increases is the EC50. This can be determined mathematically by deriving the best fit line. In most cases, it is convenient to rely on charts to evaluate. In the case where the EC50 is provided in the embodiments, the experiment is designed to reflect the KD as accurately as possible. In other words, the EC50 value can therefore be considered as a KD value. The term "average KD" relates to at least 1, but preferably more than 1, such as the average KD value obtained in at least 2 experiments. The term "average" refers to the mathematical term "average" (the sum of the data divided by the number of items in the data).

[0666] Also contemplated is the IC50, which is a measure of inhibition (50% inhibition) of a compound. For both competitive binding assays and functional antagonist assays, the IC50 is the most common summary measure of dose response curves.

[0667] For agonist / stimulatory assays, the most common summary measurement is the EC50.

[0668] y) It must be noted that, as used herein, the singular forms "a", "an", and "the" include plural references unless expressly indicated otherwise. Thus, for example, reference to "agent"

[0669] One or more of the various reagents described herein are included, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can modify or substitute the methods described herein.

[0670] Unless otherwise indicated, the term "at least" preceding a series of elements is understood to refer to each element in the series. Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the present invention.

[0671] Wherever used herein, the term "and / or" includes the meanings of "and," "or," and "all or any other combination of elements connected by the term."

[0672] The term "about" or "approximately", as used herein, means within 20% of a given value or range.

[0673] Preferably within 15%, more preferably within 10%, and most preferably within 5%.

[0674] Throughout this specification and the claims that follow, unless the context requires otherwise, the words

[0675] "Comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of the stated integer or step or groups of integers or steps, but not the exclusion of any other integer or step or groups of integers or steps. When used herein, the term "comprises" can be replaced with the term "comprising" or "including", or sometimes when used herein can be replaced with the term "having".

[0676] The present invention relates to a polypeptide comprising at least a first immunoglobulin single variable domain (ISV) and at least one further immunoglobulin single variable domain, wherein the at least first ISV has a high affinity for / binds to cluster of differentiation 3 (CD3) and the at least one further ISV has a high affinity for / binds to an antigen on a target cell.

[0677] Typically, the multispecific polypeptides of the present invention combine high-affinity antigen recognition on target cells with T cell activation, which results in activation independent of the natural specificity of the T cell. The mode of action of binding molecules that bind to both cell surface molecules on target cells (such as tumor antigens) and to T cell co-receptor CD3 is well known. Bringing a T cell into close proximity with a target cell, i.e., engaging the T cell, causes the T cell to kill the target cell. In the present invention, the method is utilized in combating proliferative diseases, inflammatory diseases, infectious diseases, and autoimmune diseases. Typically, T cells are equipped with particles containing a lethal combination of pore-forming proteins (called pore-forming proteins) and cell death-inducing proteases (called granzymes). Preferably, these proteins are delivered to the target cell via a cytolytic synapse, which is formed when the T cell is in close proximity to the target cell to be killed. Normally, the close proximity between the T cell and the target cell is achieved by the T cell using its matching T cell receptor to bind to the MHC / peptide complex. The polypeptides of the present invention bring the T cell into close proximity with the target cell in the absence of a T cell receptor / MHC interaction.

[0678] Therefore, the present invention relates to a polypeptide as described herein, wherein said polypeptide directs T cells to a target cell.

[0679] Through one arm (first ISV), the multispecific polypeptide has a high affinity for / binds to CD3 (a protein component of the T cell receptor signaling complex on T cells). Through another arm (second ISV and / or third ISV, etc.), the multispecific polypeptide recognizes an antigen on a target cell and has a high affinity for / binds to an antigen on a target cell. Preferably, T cell activation is only seen on the surface of the target cell when the multispecific polypeptide is presented to the T cell. The dependence on the antigen on the activated target cell results in a favorable safety attribute. In one embodiment, the multispecific polypeptide transiently links the T cell and the target cell. Preferably, the multispecific polypeptide is capable of binding a resting polyclonal T cell (such as CD4 + and / or CD8 + T cells) are induced to activation for efficient redirected lysis of target cells. Preferably, after the first target cell is lysed, the T cells are directed to the next target cell.

[0680] Proteins and polypeptides comprising or essentially consisting of two or more immunoglobulin single variable domains (such as at least two immunoglobulin single variable domains of the invention) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs". Some non-limiting examples of such multivalent constructs will become clear from the further description herein. The polypeptides of the invention are "multivalent", i.e. comprise two or more building blocks or ISVs, wherein at least a first building block, ISV or Nanobody and a second building block, ISV or Nanobody are different and are directed against different targets, such as antigens or antigenic determinants. A polypeptide of the invention containing at least two building blocks, ISVs or Nanobodies, wherein at least one building block, ISV or Nanobody is directed against a first antigen (i.e. against a first target, such as, for example, CD3) and at least one building block, ISV or Nanobody is directed against a second antigen (i.e. against a second target different from the first target, such as, for example, a TAA, such as CD20 or HER2) will also be referred to as a "multispecific" polypeptide of the invention, and the building blocks, ISVs or Nanobodies present in the polypeptide will also be referred to herein as being in a "multivalent format" or "multispecific format". Thus, for example, a "bispecific" peptide of the invention is a polypeptide comprising at least one structural unit, ISV or Nanobody directed against a first target (e.g. CD3) and at least one other structural unit, ISV or Nanobody directed against a second target (i.e., against a second target different from the first target, such as for example a TAA, such as CD20 or HER2), while a "trispecific" polypeptide of the invention is a polypeptide comprising at least one structural unit, ISV or Nanobody directed against a first target (e.g. CD3), a second structural unit, ISV or Nanobody directed against a second target different from the first target (e.g. a TAA, such as CD20 or HER2) and at least one other structural unit, ISV or Nanobody directed against a third antigen (i.e., different from both the first and second targets, such as another TAA), such as, for example, serum albumin, etc.; etc. As will be clear from the description, the invention is not limited to bispecific polypeptides in the sense that the multispecific polypeptides of the invention may comprise at least a first building block, ISV or Nanobody directed against a first target, a second building block, ISV or Nanobody directed against a second target and any number of building blocks, ISVs or Nanobodies directed against one or more targets which may be the same or different from the first and / or second target, respectively. The building blocks, ISVs or Nanobodies may optionally be linked via a linker sequence.

[0681] The terms bispecific polypeptide, bispecific format, bispecific construct, bispecific Nanobody construct, bispecific and bispecific antibody are used interchangeably herein.

[0682] As becomes clear from the above and the further description herein, the immunoglobulin single variable domains of the invention can be used as "building blocks" to form polypeptides of the invention, 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 invention described herein), which combine one or more desired properties or biological functions in one molecule.

[0683] It will be appreciated (as also demonstrated in the Examples section) that the ISV that binds CD3 and the ISV that binds an antigen on a target cell can be placed in any order in the polypeptide of the invention. More specifically, in one embodiment, the ISV that binds CD3 is placed at the N-terminus and the ISV that binds an antigen on a target cell is placed at the C-terminus. In another embodiment, the ISV that binds an antigen on a target cell is placed at the N-terminus and the ISV that binds CD3 is placed at the C-terminus.

[0684] In a preferred aspect, the polypeptide of the invention comprises at least a first immunoglobulin single variable domain (ISV), at least a second immunoglobulin single variable domain, and at least a third immunoglobulin single variable domain, wherein the at least first ISV has a high affinity for / binds to CD3; the at least second ISV has a high affinity for / binds to a first antigen on a target cell, and the at least third ISV has a high affinity for / binds to a second antigen on a target cell, wherein the second antigen is different from the first antigen. The first antigen and the second antigen can be on the same target cell or on different target cells.

[0685] It will be appreciated (as also demonstrated in the Examples section) that the ISV that binds CD3 and the ISV that binds a first antigen and a second antigen on a target cell can be placed in any order in the polypeptides of the invention. More specifically, in one embodiment, the ISV that binds CD3 is placed at the N-terminus, the ISV that binds a first antigen on a target cell is placed centrally, and the ISV that binds a second antigen on a target cell is placed at the C-terminus. In another embodiment, the ISV that binds CD3 is placed at the N-terminus, the ISV that binds a second antigen on a target cell is placed centrally, and the ISV that binds the first antigen on a target cell is placed at the C-terminus. In another embodiment, the ISV that binds a first antigen on a target cell is placed at the N-terminus, the ISV that binds a second antigen on a target cell is placed centrally, and the ISV that binds CD3 is placed at the C-terminus. In another embodiment, the ISV that binds a first antigen on a target cell is placed at the N-terminus, the ISV that binds CD3 is placed centrally, and the ISV that binds a second antigen on a target cell is placed at the C-terminus. In another embodiment, the ISV that binds a second antigen on the target cell is placed at the N-terminus, the ISV that binds CD3 is placed centrally, and the ISV that binds a first antigen on the target cell is placed at the C-terminus. In another embodiment, the ISV that binds a second antigen on the target cell is placed at the N-terminus, the ISV that binds a first antigen on the target cell is placed centrally, and the ISV that binds CD3 is placed at the C-terminus.

[0686] The present invention also relates to compounds or constructs, and in particular proteins or polypeptides, comprising or essentially consisting of one or more ISV's or polypeptides of the present invention, and optionally further comprising one or more other groups, residues, parts or binding units. It will be clear to the skilled person from the further disclosure herein that said other groups, residues, parts, binding units or amino acid sequences may or may not provide additional functionality to the polypeptide of the present invention (and / or to the compound or construct in which said polypeptide is present), and may or may not alter the properties of the polypeptide of the present invention.

[0687] The compounds, constructs or polypeptides of the invention can generally be prepared by a method comprising at least one step of suitably linking one or more immunoglobulin single variable domains of the invention to one or more additional groups, residues, moieties or binding units, optionally via one or more suitable linkers, to provide a compound, construct or polypeptide of the invention. The polypeptides of the invention can also be prepared by a method comprising generally providing a nucleic acid encoding a polypeptide of the invention, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such a method can be implemented in a manner known per se, which will be clear to the skilled person, for example based on the methods and techniques further described herein.

[0688] Starting from the amino acid sequence of the invention, the process of designing / selecting and / or preparing the compound, construct or polypeptide of the invention is also referred to herein as the "formatting" of the amino acid sequence of the invention; the amino acids of the invention as part of the compound, construct or polypeptide of the invention are referred to as "formatted" or "formatted" compounds, constructs or polypeptides of the invention. Examples of the way in which the amino acid sequence of the invention is formatted and examples of such formats will be clear to the skilled person on the basis of the disclosure herein; such formatted immunoglobulin single variable domains or polypeptides form another aspect of the invention.

[0689] For example, such additional groups, residues, moieties or binding units may be one or more additional immunoglobulin single variable domains, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, the one or more other groups, residues, moieties or binding units are immunoglobulin sequences. More preferably, the one or more other groups, residues, moieties or binding units are selected from the group consisting of domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains suitable for use as single domain antibodies (ISV), "dAb"'s, immunoglobulin single variable domains suitable for use as dAbs, or nanobodies. Alternatively, such groups, residues, moieties or binding units may, for example, be chemical groups, residues, moieties, which themselves may or may not have biological and / or pharmacological activity. For example, but not limited thereto, such groups may be linked to one or more immunoglobulin single variable domains or polypeptides of the invention to provide a "derivative" of the ISV or polypeptide of the invention, as further described herein.

[0690] Compounds or constructs comprising or consisting essentially of one or more derivatives described herein, and optionally further comprising one or more other groups, residues, moieties or binding units optionally connected via one or more linkers are also within the scope of the invention. Preferably, the one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains. In the above-mentioned compounds or constructs, the one or more immunoglobulin single variable domains and the one or more groups, residues, moieties or binding units of the invention may be directly connected to each other and / or connected via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are immunoglobulin single variable domains, the linker may also be an immunoglobulin single variable domain so that the obtained compound or construct is a fusion (protein) or fusion (polypeptide).

[0691] In some embodiments, the polypeptide comprises at least two or more immunoglobulin single variable domains as disclosed herein. In some embodiments, the polypeptide consists essentially of two or more immunoglobulin single variable domains as disclosed herein. A polypeptide "consisting essentially of" two or more immunoglobulin single variable domains is a polypeptide that does not have additional immunoglobulin single variable domains in addition to the two or more immunoglobulin single variable domains as disclosed herein. For example, a polypeptide consisting essentially of two immunoglobulin single variable domains does not include any additional immunoglobulin single variable domains. However, it should be understood that the polypeptide consisting essentially of two or more immunoglobulin single variable domains may include additional functionality, such as tags, toxins, one or more linkers, binding sequences, etc. These additional functionalities include both amino acid-based groups and non-amino acid-based groups. In some embodiments, the polypeptide consists of one or more immunoglobulin single variable domains as disclosed herein. It should be understood that the terms "polypeptide construct" and "polypeptide" are used interchangeably herein (unless the context clearly indicates otherwise).

[0692] In some embodiments, the polypeptide comprises a multivalent or multispecific construct comprising an immunoglobulin single variable domain as disclosed herein. In some embodiments, the polypeptide comprises one or more antibody-based scaffolds and / or non-antibody-based scaffolds as disclosed herein. In some embodiments, the polypeptide comprises a serum binding protein portion. In some embodiments, the serum binding protein portion is an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain is

[0693] It will be appreciated that the order (orientation) of building blocks (such as, for example, the first building block, the second building block, the third building block, etc.) on the polypeptide can be selected according to the needs of the skilled person, and according to the relative affinities that may depend on the position of these building blocks in the polypeptide. Whether the polypeptide comprises a linker is a matter of design choice. However, some orientations (with or without a linker) may provide preferred binding characteristics compared to other orientations. For example, the order of the first and second building blocks in the polypeptide of the invention may be (from N-terminus to C-terminus): (i) first building block (e.g., first ISV, such as first Nanobody)-[linker]-second building block (e.g., second ISV, such as second Nanobody); or (ii) second building block (e.g., second ISV, such as second Nanobody)-[linker]-first building block (e.g., first ISV, such as first Nanobody); (wherein the linker is optional). All orientations are included in the present invention. Polypeptides containing building block orientations that provide the desired (binding) properties can be easily identified by routine screening, for example as exemplified in the experimental section.

[0694] The first immunoglobulin single variable domain (ISV) of the polypeptide of the invention has a high affinity for / binds to an effector cell (preferably the TCR complex of said effector cell, and even more preferably CD3).

[0695] Effector cells are cells containing a TCR complex, preferably immune cells such as T helper cells, monocytes, macrophages or dendritic cells, preferably CD4 + T helper cells (also called CD4 cells, T helper cells or T4 cells), more preferably cytotoxic T cells (also called T C cells, CTL or CD8 + In some embodiments, the cells are present in vivo. In some embodiments, the cells are present in vitro. The effector cells of the present invention particularly relate to mammalian cells, preferably to primate cells, and even more preferably to human cells.

[0696] As used herein, the term "TCR complex" or "αβTCR-CD3 complex" refers to the T cell receptor complex presented on the surface of T cells (see Kuhns et al. 2006, Immunity 24: 133-139). The TCR complex consists of six different type I single-span transmembrane proteins: TCRα and TCRβ chains that form TCR heterodimers responsible for ligand recognition, and non-covalently associated CD3γ, CD3δ, CD3ε and ζ chains (which have cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signaling components). The α chain and β chain of the T cell receptor are both composed of constant domains and variable domains. The sequences of human CD3 and human TCR α / β construct domains are provided in Table A-10 (SEQ ID NOs: 291-296; see UniProtKB: CD3δ: P04234, CD3γ: P09693, CD3ε: P07766, CD3ζ: P20963, TCRα: P01848 and TCRβ: P01850).

[0697] In one embodiment, the invention relates to a polypeptide as described herein, wherein the first ISV binds to CD3γ (SEQ ID NO: 292), CD3δ (SEQ ID NO: 291) and / or CD3ε (SEQ ID NO: 293) of the TCR complex, or a polymorphic variant or isoform thereof.

[0698] Alternatively, the invention provides a polypeptide as described herein, wherein the first ISV binds CD3γ (SEQ ID NO: 379), CD3δ (SEQ ID NO: 291) and / or CD3ε (SEQ ID NO: 380) of the TCR complex, or a polymorphic variant or isoform thereof.

[0699] 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 as known in the art.

[0700] 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 polypeptides of the present invention are capable of inducing T cell activation. As described herein, suitable assays for measuring T cell activation are known in the art, such as described in WO 99 / 54440 or Schlereth et al. 2005 (Cancer Immunol. Immunother. 20: 1-12), or as illustrated in the Examples or below.

[0701] In one embodiment the invention relates to a polypeptide as described herein, wherein said polypeptide induces T cell activation. Preferably, the polypeptide of the invention induces T cell activation only when said second ISV and / or further ISV binds to an antigen on a target cell.

[0702] In one embodiment, the invention relates to a 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.

[0703] T cell activation via the polypeptides of the invention can be monitored by re-expression and / or release of CD69, CD25 and various cell adhesion molecules, cytokines (e.g., IFN-γ, TNF-α, IL-6, IL-2, IL-4 and IL-10), up-regulation 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 via the multispecific polypeptides is independent of T cell receptor specificity, the presence of class I MHC and / or β2 microglobulin, and / or the presence of any co-stimulatory stimuli.

[0704] In one embodiment, the invention relates to a polypeptide as described herein, wherein said T cell activation is independent of MHC recognition.

[0705] The polypeptides of the present invention have been shown to activate previously unstimulated peripheral polyclonal CD8 + - and CD4 + - Redirected lysis of positive T cells. Redirected lysis of target cells through recruitment of T cells by the polypeptide of the present invention involves cytolytic synapse formation and delivery of perforin and granzyme. Cytolysis via T cells has been described in, for example, Atkinson and Bleackley 1995 (Crit. Rev. Immunol 15 (3-4): 359-384). Preferably, the engaged T cells are capable of continuous target cell lysis and are not affected by immune escape mechanisms (which interfere with peptide antigen processing and presentation, or cloned T cell differentiation) (see, for example, WO 2007 / 042261). In vitro, redirected lysis is visible at low picomolar concentrations, which indicates that a very low number of polypeptides of the present invention need to be combined with target cells to trigger T cells. As demonstrated in the examples, a low ratio of effector to target can indicate continuous target cell lysis. Therefore, the present invention relates to effective polypeptides. Preferably, the polypeptide of the present invention mediates the killing of target cells (e.g., cancer cells), such as stimulating T cells to form pores and delivering pro-apoptotic components of cytotoxic T cell granules.

[0706] In one embodiment, the present invention relates to a 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.

[0707] As used herein, the term "potency" is a measure of the biological activity of an agent, such as a polypeptide, ISV or Nanobody. The potency of an agent can be determined by any suitable method known in the art, such as, for example, the methods described in the Experimental section. Cell culture-based potency assays are often the preferred format for determining biological activity because they measure the physiological response induced by the agent and can produce results in a relatively short period of time. Depending on the mechanism of action of the product, various types of cell-based assays 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 measuring the induction / inhibition of functionally critical proteins or other signaling molecules (such as phosphorylated proteins, enzymes, cytokines, cAMP, etc.), Ramos B cell depletion model, T cell-mediated tumor cell killing assays (e.g., as shown in the Examples section), all of which are known in the art. The results from a cell-based potency assay can be expressed as "relative potency", as determined by comparing the response obtained for a multispecific polypeptide of the invention with a corresponding reference monovalent ISV (e.g., a polypeptide comprising only one ISV or one Nanobody, optionally further comprising an unrelated Nanobody (see the Experimental section)).

[0708] In one embodiment, the invention relates to a polypeptide as described herein, wherein the T cell activation causes inhibition of the target cell activity, such as delaying or minimizing the spread of the target cells, inhibiting or delaying the growth and / or proliferation of the target cells and / or killing the 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%.

[0709] The first building block, ISV, Nanobody or VHH of the invention has a high affinity for its target, i.e. CD3. The first building block, ISV or Nanobody of the invention may, for example, be directed against an antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of said first target. The first building block, such as a first ISV, Nanobody or VHH, is preferably selected for its high affinity for its target itself, without taking into account the influence of any avidity effect.

[0710] Thus, the present invention relates to a polypeptide as described herein, wherein the first ISV binds to CD3 with an average KD value of between 100 nM and 10 pM, such as an average KD value of below 90 nM, even more preferably an average KD value of below 80 nM, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. Preferably, the KD is determined by Kinexa or SPR, e.g. as determined by Proteon. For example, the KD is determined as shown in the Examples section.

[0711] Therefore, the present invention relates to a polypeptide as described herein, wherein said first ISV has a high affinity when measured monovalently.Preferably, said average KD is measured by surface plasmon resonance (SPR) on a recombinant protein.

[0712] Therefore, the present invention relates to a polypeptide as described herein, wherein said polypeptide has a dissociation constant (K) for said CD3 (or for binding to said CD3) selected from the group consisting of: D ): up to about 10 -5 M, up to about 10 -6 M, up to about 10 -7 M, up to about 10 -8 M, up to about 10 -9 M, up to about 10 -10 M, up to about 10 -11 M and at most about 10 -12 M, is preferably as measured by surface plasmon resonance.

[0713] The present invention also relates to a polypeptide as described herein, wherein said first ISV binds to said CD3 with an EC50 value of between 100 nM and 1 pM, such as an average EC50 value of less than 100 nM, even more preferably an average EC50 value of less than 90 nM, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM or even less, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even less, such as less than 4 pM.

[0714] Therefore, the present invention relates to a polypeptide as described herein, wherein said average KD is determined by FACS, Biacore, ELISA on a monovalent first ISV (such as a Nanobody) or a polypeptide comprising a monovalent first ISV (such as a Nanobody), e.g. said EC50 is determined as shown in the Examples section.

[0715] It has been shown in the Examples that KD correlates well with EC50.

[0716] In one embodiment, the invention relates to a polypeptide as described herein, wherein said polypeptide has an association rate constant (Kon) for said CD3 (or for binding to said CD3) selected from the group consisting of: at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , 10 7 M 1 s -1 , at least about 10 8 M -1 s -1 , at least about 10 9 M -1 s -1 and at least about 10 10 M -1 s -1 , preferably as measured by surface plasmon resonance or as performed in the Examples section.

[0717] In one embodiment, the invention relates to a polypeptide as described herein, wherein said polypeptide has a dissociation rate constant (Koff) for said CD3 (or for binding to said CD3) selected from the group consisting of: up to about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 , up to about 10 -6 s -1 , up to about 10 -7 s -1 , up to about 10 -8 s -1, up to about 10 -9 s -1 and at most about 10 -10 s -1 , preferably as measured by surface plasmon resonance or as performed in the Examples section.

[0718] Amino acid sequence modifications of the binding molecules, ISV or polypeptides described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or ISV. Amino acid sequence variants of binding molecules, ISV or polypeptides are prepared by introducing appropriate nucleotide changes into the nucleic acid of the binding molecule, ISV or polypeptide or by peptide synthesis.

[0719] The modification includes, for example, residue deletions from the amino acid sequence of the binding molecule, ISV or polypeptide, and / or residue insertions into the amino acid sequence of the binding molecule, ISV or polypeptide, and / or residue replacements in the amino acid sequence of the binding molecule, ISV or polypeptide. Any combination of deletions, insertions and replacements can be produced to achieve the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also change the post-translational processing of the binding molecule, such as changing the number or position of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids can be substituted in the CDR, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be substituted in the framework region (FR). The substitution is preferably a conservative substitution as described herein. Additionally or alternatively, 1, 2, 3, 4, 5 or 6 amino acids may be inserted or deleted in each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be inserted or deleted in each FR.

[0720] A useful method for identifying certain residues or regions of a binding molecule, ISV or polypeptide that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells 1989 (Science 244: 1081-1085). Thus, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) within a binding molecule is identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the epitope. Those amino acid positions that show functional sensitivity to the substitution are then completed by introducing additional or other variants at or for the substituted site. Thus, while the site for introducing an amino acid sequence variant is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed binding molecule variants are screened for the desired activity.

[0721] Preferably, amino acid sequence insertions comprise amino-terminal fusions and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing more than a hundred residues.

[0722] Another type of variant is an amino acid substitution variant. These variants preferably have at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in a binding molecule, ISV or polypeptide that are replaced by different residues. The sites of greatest interest for substitution mutagenesis include CDRs, particularly hypervariable regions, but FR changes are also contemplated. For example, if a CDR sequence includes 6 amino acids, it is envisioned that one, two or three of these amino acids are replaced. Similarly, if a CDR sequence includes 15 amino acids, it is envisioned that one, two, three, four, five or six of these amino acids are replaced.

[0723] In general, if an amino acid is substituted in one or more or all CDRs, it is preferred that the subsequently obtained "substituted" sequence is at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, more particularly preferably 80% or even more than 90% identical to the "original" CDR sequence. This means that the length of the CDR depends on the extent to which it is identical to the "substituted" sequence. For example, a CDR with 5 amino acids is preferably 80% identical to the sequence it replaces, so as to have at least one substituted amino acid. Thus, the CDRs of a binding molecule may have different degrees of identity with the sequences they replace, for example, CDR1 may have 80%, while CDR3 may have 90%.

[0724] Preferred substitutions (or replacements) are conservative substitutions. However, any substitutions (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table B-1 below) are contemplated, as long as the polypeptide retains its ability to bind to CD3 present on T cells through the first ISV and to the first antigen on the target cell through the second ISV and / or its CDR has an identity with the subsequently substituted sequence (at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, more particularly preferably 80% identical to the "original" CDR sequence).

[0725] Conservative substitutions are shown below in Table B-1.

[0726] Table 8-1: Amino Acid Substitution

[0727] source Exemplary substitutions Preferred substitutions Ala(A) val,leu,ile val Arg(R) lys,gln,asn lys Asn(N) gln,his,asp,lys,arg gln Asp(D) glu,asn glu Cys(C) ser,ala ser Gln(Q) asn,glu asn Glu(E) asp,gln asp Gly(G) ala ala His(H) asn, gln, lys, arg arg lle(I) leu, val, met, ala, phe leu Leu(L) norleucine,ile,val,met,ala ile Lys(K) arg, gln, asn arg Met(M) leu, phe, ile leu Phe(F) leu, val, ile, ala, tyr tyr Pro(P) ala ala Ser(S) thr thr Thr(T) ser Ser Trp(W) Tyr, phe tyr Tyr(Y) trp, phe, th r, ser phe Val(V) ile, leu, met, phe, ala leu

[0728] Sequence analysis also revealed only a limited number of sequence changes in the CDRs (see Example 4.2 and Tables A-1 to A-6).

[0729] Therefore, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0730] (i) CDR1 is selected from the group consisting of:

[0731] (a) SEQ ID NOs: 81-100; and

[0732] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 81 or from any one of SEQ ID NOs: 81-100; and / or

[0733] (ii) CDR2 is selected from the group consisting of:

[0734] (c) SEQ ID NOs: 101-122; and

[0735] (d) an amino acid sequence having 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 101 or from any one of SEQ ID NOs: 101-122; and / or

[0736] (iii) CDR3 is selected from the group consisting of:

[0737] (e) SEQ ID NOs: 123-143; and

[0738] (f) an amino acid sequence that has 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 123 or from any one of SEQ ID NOs: 123-143.

[0739] Additional preferred CDR sequences are described in Table A-8.

[0740] Generally, combinations of CDRs listed in Table A-8 (i.e. those mentioned in the same row of Table A-4) are preferred. Thus, it is generally preferred that when a CDR in an ISV is a CDR sequence mentioned in Table A-8 or is suitably chosen from the group consisting of CDR sequences that have 4, 3, 2 or only 1 amino acid difference with a CDR sequence listed in Table A-8, at least one and preferably both of the other CDRs are suitably chosen from the CDR sequences belonging to the same combination in Table A-8 (i.e. mentioned in the same row of Table A-8) or is suitably chosen from the group consisting of CDR sequences that have 4, 3, 2 or only 1 amino acid difference with a CDR sequence belonging to said same combination.

[0741] Sequence analysis of the resulting binders further led to the identification of 6 different clusters. Corresponding comparisons are provided (see Table A-1, Table A-2, Table A-3, Table A-4, Table A-5 and Table A-6). Clustering is based on sequence similarities and differences in CDR2 and CDR3. Cluster A is the most prominent, including 50 clones (SEQ ID NO:s 1-50), clusters B and D are represented by only 1 clone (SEQ ID NO:51 and SEQ ID NO:52, respectively), cluster C includes 4 clones (SEQ ID NO:s 53-56), cluster E includes 9 clones (SEQ ID NO:s 57-65) and cluster F includes 15 clones (SEQ ID NO:s 66-80). Clustering based on structural similarities and differences in amino acid sequences is converted into functional similarities and differences as disclosed in the examples. Representatives of all clusters are separated based on high affinity for binding to CD3 (Examples 3 & 4) and human T cell activation (Example 4.2). Generally, cluster A representatives show the best EC50 values. Although cluster C representatives had slightly worse EC50 values ​​than cluster B representatives, cluster C representatives had lower IC50 values ​​in a flow cytometry-based T cell-mediated Ramos killing assay (see Example 10).

[0742] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is chosen from the group consisting of:

[0743] (a) SEQ ID NO:81; and

[0744] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 81, wherein

[0745] - At position 1, G has changed to R;

[0746] - At position 3, T has changed to A;

[0747] - At position 4, Y has changed to F;

[0748] - S has changed to G at position 8; and / or

[0749] - At position 10 G has changed to A.

[0750] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is chosen from the group consisting of:

[0751] (a) SEQ ID NO: 101; and

[0752] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 101, wherein

[0753] - At position 3, V has changed to T or A;

[0754] - At position 5, S has changed to T;

[0755] - G has changed to D or E at position 6; and / or

[0756] - At position 9 T has changed to S, A or P.

[0757] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is chosen from the group consisting of:

[0758] (a) SEQ ID NO: 123; and

[0759] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 123, wherein

[0760] - At position 2, I has changed to T;

[0761] - I has changed to V at position 9; and / or

[0762] - At position 10 A has been changed to P.

[0763] In one embodiment, the invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0764] (i) CDR1 is selected from the group consisting of:

[0765] (a) SEQ ID NOs: 81-87; and

[0766] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 81; and / or

[0767] (ii) CDR2 is selected from the group consisting of:

[0768] (c) SEQ ID NOs: 101-109; and

[0769] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 101; and / or

[0770] (iii) CDR3 is selected from the group consisting of:

[0771] (e) SEQ ID NOs: 123-127; and

[0772] (f) an amino acid sequence that has 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:123.

[0773] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO: 123.

[0774] Nanobodies belonging to cluster B are represented by 1 clone.

[0775] Therefore, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0776] (i) CDR1 is selected from the group consisting of:

[0777] (a) SEQ ID NO: 88; and

[0778] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 88; and / or

[0779] (ii) CDR2 is selected from the group consisting of:

[0780] (c) SEQ ID NO: 110; and

[0781] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 110; and / or

[0782] (iii) CDR3 is selected from the group consisting of:

[0783] (e) SEQ ID NO: 128; and

[0784] (f) an amino acid sequence that has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO:128.

[0785] Therefore, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, and CDR3 is represented by SEQ ID NO: 128.

[0786] Nanobodies of cluster C show very limited sequence variability in the CDRs.

[0787] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is chosen from the group consisting of:

[0788] (a) SEQ ID NO: 112; and

[0789] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[0790] - At position 2 V has changed to A.

[0791] Therefore, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0792] (i) CDR1 is selected from the group consisting of:

[0793] (a) SEQ ID NO:90; and

[0794] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 90; and / or

[0795] (ii) CDR2 is selected from the group consisting of:

[0796] (c) SEQ ID NOs: 112-113; and

[0797] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 112; and / or

[0798] (iii) CDR3 is selected from the group consisting of:

[0799] (e) SEQ ID NO: 130; and

[0800] (f) an amino acid sequence that has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO:130.

[0801] In one aspect, the invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:90, CDR2 is represented by SEQ ID NO:112, and CDR3 is represented by SEQ ID NO:130.

[0802] Nanobodies belonging to cluster D are represented by 1 clone.

[0803] Therefore, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0804] (i) CDR1 is selected from the group consisting of:

[0805] (a) SEQ ID NO: 89; and

[0806] (b) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 89; and / or

[0807] (ii) CDR2 is selected from the group consisting of:

[0808] (c) SEQ ID NO: 111; and

[0809] (d) an amino acid sequence that has 1, 2, 3 or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 111; and / or

[0810] (iii) CDR3 is selected from the group consisting of:

[0811] (e) SEQ ID NO: 129; and

[0812] (f) an amino acid sequence that has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO:129.

[0813] Therefore, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:89, CDR2 is represented by SEQ ID NO:111, and CDR3 is represented by SEQ ID NO:129.

[0814] Cluster E included 9 clones.

[0815] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is chosen from the group consisting of:

[0816] (a) SEQ ID NO:91; and

[0817] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[0818] - At position 6, R has changed to N or T;

[0819] - N has been changed to H at position 7; and / or

[0820] - At position 8 the M has been changed to a T.

[0821] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is chosen from the group consisting of:

[0822] (a) SEQ ID NO: 114; and

[0823] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[0824] - At position 1, R has changed to Q;

[0825] - T has changed to S at position 3; and / or

[0826] - In position 7 D has changed to A or K.

[0827] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is chosen from the group consisting of:

[0828] (a) SEQ ID NO: 131; and

[0829] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 131, wherein

[0830] - S has changed to R at position 2; and / or

[0831] - At position 6 S has changed to V.

[0832] In one embodiment, the invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0833] (i) CDR1 is selected from the group consisting of:

[0834] (a) SEQ ID NOs:91-93; and

[0835] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 91; and / or

[0836] (ii) CDR2 is selected from the group consisting of:

[0837] (c) SEQ ID NOs: 114-117; and

[0838] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 114; and / or

[0839] (iii) CDR3 is selected from the group consisting of:

[0840] (e) SEQ ID NOs: 131-133; and

[0841] (f) an amino acid sequence that has 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 131.

[0842] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:91, CDR2 is represented by SEQ ID NO:114, and CDR3 is represented by SEQ ID NO:131.

[0843] Nanobodies belonging to cluster F were represented by 15 clones.

[0844] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is chosen from the group consisting of:

[0845] (a) SEQ ID NO:94; and

[0846] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[0847] - At position 3, S has changed to T, A or G;

[0848] - At position 5, N has changed to S;

[0849] - M has changed to T or A at position 6; and / or

[0850] - At position 9 L has changed to M.

[0851] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is chosen from the group consisting of:

[0852] (a) SEQ ID NO: 118; and

[0853] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 118, wherein

[0854] - At position 2, H has changed to V;

[0855] - At position 5, S has changed to H or A;

[0856] - N has changed to S at position 8; and / or

[0857] - At position 10 Y has changed to F.

[0858] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is chosen from the group consisting of:

[0859] (a) SEQ ID NO: 134; and

[0860] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 134, wherein

[0861] - A has changed to S or D at position 6;

[0862] - F has changed to Y or A at position 7;

[0863] - At position 8, R has changed to H;

[0864] - At position 9, S has changed to A;

[0865] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[0866] - At position 14 V has changed to I.

[0867] In one embodiment, the invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0868] (i) CDR1 is selected from the group consisting of:

[0869] (a) SEQ ID NOs: 94-100; and

[0870] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 94; and / or

[0871] (ii) CDR2 is selected from the group consisting of:

[0872] (c) SEQ ID NOs: 118-122; and

[0873] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO: 118; and / or

[0874] (iii) CDR3 is selected from the group consisting of:

[0875] (e) SEQ ID NOs: 134-143; and

[0876] (f) an amino acid sequence that has 3, 2 or 1 amino acid differences from the amino acid sequence of SEQ ID NO:134.

[0877] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO: 94, CDR2 is represented by SEQ ID NO: 118, and CDR3 is represented by SEQ ID NO: 134.

[0878] The second immunoglobulin single variable domain (ISV) of the polypeptide of the present invention has a high affinity for / binds to an antigen on a target cell (preferably a cancer cell). A "target cell" as used herein refers to a cell that has a specific antigen on its surface. In a preferred aspect, a "target cell" is a cancer cell.

[0879] Membrane (also referred to as plasma membrane or phospholipid bilayer) surrounds the cytoplasm of cell, it is the outer boundary of cell, i.e. membrane is the surface of cell. The membrane is used to separate and protect cell from external environment and is mostly made of phospholipid bilayer. Embedded in the membrane are various protein molecules, such as channels, pumps and cell receptors. Because membrane is fluid, protein molecules can move in membrane. As used herein, term "antigen on target cell" means the molecule presented on the surface of cell. In most cases, the molecule will be located in the plasma membrane of cell or on the plasma membrane of cell, so that at least a part of the molecule remains accessible from the cell in tertiary form. Non-limiting examples of cell surface molecules located in plasma membrane are transmembrane proteins that include hydrophilic and hydrophobic regions in its tertiary conformation. Here, at least one hydrophobic region allows cell surface molecules to be embedded or inserted into the hydrophobic plasma membrane of cell, and hydrophilic region extends to cytoplasm and extracellular space respectively on either side of plasma membrane.

[0880] The antigen can be any target on a cell, for example, a tumor antigen. In a preferred embodiment, the antigen is specific to the target cell (eg, a cancer cell), such as a tumor associated antigen (TAA) on the cancer cell.

[0881] 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 be presented only by tumor cells and never by normal or healthy cells. Tumor antigens can be expressed exclusively on tumor cells, or can represent tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. However, this is usually not the case. More common are antigens presented by tumor cells and normal cells, and they are referred to as "tumor-associated antigens (TAA)". Compared with normal cells, these tumor-associated antigens can be overexpressed on tumor cells, or because the tumor tissue structure is less compact, it is easier to obtain antibody binding in tumor cells compared to normal cells. TAA is preferably an antigen expressed on cells of a specific tumor but preferably not expressed in normal cells. Generally, TAA is an antigen that is normally expressed in cells only at a specific point in the development of an organism (such as during fetal development) and is inappropriately expressed in an organism at the current development point, or an antigen that is not expressed in normal tissues or cells of an organ that now expresses the antigen.

[0882] In one embodiment, the first antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

[0883] In one embodiment, the second antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

[0884] In one embodiment, the antigen is present more on cancer cells than on normal cells. The antigen on the target cell is preferably a tumor associated antigen (TAA). Preferred TAAs include MART-1, carcinoembryonic antigen ("CEA"), gp100, MAGE-1, HER-2, CD20, Lewis Y Antigens, melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), fibroblast activation protein (FAP), CD19 and CD33.

[0885] Cell surface antigens that are preferentially expressed on AML LSCs compared to normal hematopoietic stem cells and are therefore preferred as TAAs include CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, and CD25.

[0886] Other tumor-associated antigens suitable as antigens on target cells for binding to the second ISV within the polypeptide of the invention include: TAG-72, Ep-CAM, PSMA, PSA, glycolipids such as GD2 and GD3.

[0887] TAAs of the present invention also include hematopoietic differentiation antigens, i.e., glycoproteins generally associated with cluster differentiation (CD) groupings, such as CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD69, and CD147; growth factor receptors, including HER2, ErbB3, and ErbB4; cytokine receptors, including interleukin-2 receptor gamma chain (CD132 antigen), interleukin-10 receptor alpha chain (IL-10R-A), interleukin-10 receptor beta chain (IL-10R-B), interleukin-12 receptor beta-1 chain (IL-12R-β1), and interleukin-12 receptor beta-2 chain (IL-12R-β2). 1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), interleukin-13 receptor alpha-2 chain (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin 21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-beta) (CDw121b ), interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor alpha chain (CD25 antigen), interleukin-2 receptor beta chain (CD122 antigen), interleukin-3 receptor alpha chain (IL-3R-α) (CD123 antigen); and others such as CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin-6, claudin-18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC1 6, EGFRvIII, AGS-16, CD27L, shank-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin-releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2γa, CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6, DcR3 and CAIX.

[0888] Thus, the present invention relates to a polypeptide as described herein, wherein the TAA is selected from the group consisting of: melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), fibroblast activation protein (FAP), MART-1, carcinoembryonic antigen ("CEA"), gp100, MAGE-1, HER-2, Lewis YAntigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD147; growth factor receptors, including ErbB3 and ErbB4; cytokine receptors, including interleukin-2 receptor gamma chain (CD132 antigen), interleukin-10 receptor alpha chain (IL-10R-A), interleukin-10 receptor beta chain (IL-10 R-B), interleukin-12 receptor beta-1 chain (IL-12R-β1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-13 receptor alpha-1 chain (IL-13R-α-1) (CD213a1 antigen), interleukin-13 receptor alpha-2 chain (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin 21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-2 receptor type II (IL-21R-2) 1 receptor (IL-1R-β) (CDw121b), interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor α chain (CD25 antigen), interleukin-2 receptor β chain (CD122 antigen), interleukin-3 receptor α chain (IL-3R-α) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD 56,MUC16,EGFRvIII,AGS-16,CD27L,Shantin-4,SLITRK6,mesothelin,folate receptor,tissue factor,axl,glypican-3,CA9,Cripto,CD138,CD37,MUC1,CD70,gastrin-releasing peptide receptor,PAP,CEACAM5,CEACAM6,CXCR7,N-cadherin,FXYD2γa,CD21,CD133,Na / K-ATPase,mIgM (membrane-bound IgM),mIgA (membrane-bound IgA),Mer,Tyro2,CD120,CD95,CA 195, DR5, DR6, DcR3 and CAIX, and related polymorphic variants and isoforms, preferably the TAA is CD20 (UniProt 11836), HER2 (Uniprot P04626), polymorphic variants and / or isoforms thereof.

[0889] The second building block, ISV, Nanobody or VHH of the invention has a high affinity for its antigen. The second building block, ISV or Nanobody of the invention may, for example, be directed against an antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of said antigen on a target cell.

[0890] The target cells of the present invention particularly relate to mammalian cells, and preferably to primate cells, and even more preferably to human cells. The target cells are preferably hyperproliferative cells, such as for example cancer cells.

[0891] The present invention relates to a polypeptide as described herein, wherein the second ISV or further ISV binds to an antigen on a target cell with an average KD value of between 100 nM and 10 pM, such as an average KD value of 90 nM or less, even more preferably an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. Preferably, the KD is determined by KinExA or SPR, for example as determined by Proteon.

[0892] Accordingly, the present invention relates to a polypeptide as described herein, wherein the second ISV or further ISV has a high affinity for its antigen when measured monovalently.

[0893] Therefore, the present invention relates to a polypeptide as described herein, wherein said average KD is measured by surface plasmon resonance (SPR) and / or KinExA or Proteon, e.g. on a recombinant protein, such as described in the Examples section.

[0894] The invention also relates to a polypeptide as described herein, wherein the second ISV or additional ISV binds to an antigen on a target cell with an EC50 value of between 100 nM and 1 pM, such as an average EC50 value of less than 100 nM, even more preferably an average EC50 value of less than 90 nM, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, 1 nM or even less, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even less, such as less than 4 pM.

[0895] Therefore, the present invention relates to a polypeptide as described herein, wherein said average EC50 is determined by FACS or ELISA on a monovalent second ISV (such as a Nanobody) or a polypeptide comprising a monovalent second ISV (such as a Nanobody).

[0896] It has been shown in the Examples that KD correlates well with EC50.

[0897] Targeting multiple antigens simultaneously can reduce the likelihood of generating tumor escape variants, so the therapeutic activity of the T cell engagement strategy is improved. The present invention provides multispecific polypeptides comprising a CD3 ISV in combination with an immunoglobulin single variable domain directed against different (target) antigens (on target cells) (see Example 19). A preferred combination of a first and a second antigen is provided as follows (it will be understood that the ISVs binding to the antigens can be placed in any order in the polypeptides of the present invention):

[0898] First Antigen Second antigen EGFR (OMIM:131550) CD20 (OMIM:112210) EGFR (OMIM:131550) CEA (OMIM:114890) EGFR (OMIM:131550) HER2 (OMIM:164870) HER2 (OMIM:164870) CD20 (OMIM:112210) HER2 (OMIM:164870) CEA (OMIM:114890) CD20 (OMIM:112210) CEA (OMIM:114890)

[0899] Similarly, simultaneous targeting of multiple epitopes, antigenic determinants, parts, domains, subunits or conformations of a protein or antigen on a target cell can reduce the likelihood of generating tumor escape variants, so the therapeutic activity of the T cell engagement strategy is improved (see Example 20). The present invention provides polypeptides comprising an anti-CD3 ISV (also referred to as a biparatopic construct) in combination with an immunoglobulin single variable domain directed to a different epitope, antigenic determinant, part, domain, subunit or conformation of an antigen on a target cell. Preferred combinations of a first and a second TAA ISV are provided as follows (it will be understood that the ISVs binding to the antigens can be placed in any order in the polypeptides of the invention):

[0900]

[0901]

[0902] The polypeptides and compositions of the invention can be used to prevent and / or treat the diseases and disorders of the invention (also referred to herein as "diseases and disorders of the invention"), including, but not limited to, cancer. The term "cancer" refers to a pathological condition in mammals that is typically characterized by abnormally regulated cell proliferation or survival. Examples of cancer include, but are not limited to, carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma: breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, Renal cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma (for other cancers see, e.g., Cancer, Principles and practice (DeVita et al., eds. 1997)); and any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis; and other conditions and diseases described herein.

[0903] For a general description of immunoglobulin single variable domains, reference is made to the further description below, as well as to the prior art cited herein. At this point, however, it should be noted that the present description and the prior art mainly describe so-called "V H Type 3 immunoglobulin single variable domain (i.e., with V H 3 type human germline sequences such as DP-47, DP-51 or DP-29) which form a preferred aspect of the present invention. However, it should be noted that the present invention in its broadest sense covers any type of immunoglobulin single variable domain and for example also covers those belonging to the so-called "V H 4 type" immunoglobulin single variable domain (i.e., with V H 4 human germline sequences such as DP-78 (immunoglobulin single variable domains with a high degree of sequence homology), as described, for example, in WO 07 / 118670.

[0904] Typically, immunoglobulin single variable domains (especially V HH Sequences and sequence-optimized immunoglobulin single variable domains) may be particularly characterized by the presence of the above "signature residues" (as described herein) in one or more framework region sequences (as described, for example, in Table B-2).

[0905] Table B-2: Characteristic residues in VHHs

[0906]

[0907]

[0908] The immunoglobulins of the invention may also contain a C-terminal extension (X)n (wherein n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably naturally occurring) amino acid residue independently selected from, and preferably independently selected from, the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), which relates to WO 12 / 175741 and WO 15 / 060643.

[0909] Additionally and / or additionally, the immunoglobulins of the invention may have certain preferred amino acid residues at positions 11, 89, 110 and / or 112, as described in further detail in WO 15 / 060643 (which is incorporated herein by reference).

[0910] Furthermore, such immunoglobulin single variable domains may be derived from any suitable source in any suitable manner, for example, they may be naturally occurring V HH Sequences (i.e., from a suitable camelid species, such as a llama) or synthetic or semi-synthetic VHs or VLs (e.g., from a human). Such immunoglobulin single variable domains may include "humanized" or other "sequence-optimized" VHHs, "camelized" immunoglobulin sequences (particularly camelized heavy chain variable domain sequences, i.e., camelized VHs), as well as human VHs, human VLs, VHHs of camelids; or any suitable combination of any of the foregoing, as further described herein. As described herein, particularly preferred types of immunoglobulin single variable domains of the invention comprise sequences having sequences similar to those of naturally occurring V HH The amino acid sequence of the immunoglobulin single variable domain corresponds to that of the amino acid sequence of the 4-chain antibody, but is "humanized", i.e., by using the V sequences of a conventional 4-chain antibody from a human (e.g., as indicated above) H One or more of the amino acid residues occurring at the corresponding one or more positions in the structural domain replace the naturally occurring V HHThis can be done in a manner known per se as will be apparent to the skilled person, for example based on the further description herein and the prior art in humanization cited herein. Again, it should be noted that such a humanized immunoglobulin single variable domain of the invention can be obtained in any suitable manner known per se and is therefore not strictly limited to the use of single variable domains comprising naturally occurring V HH A polypeptide having a structural domain is a polypeptide obtained as a starting material.

[0911] Another particularly preferred type of immunoglobulin single variable domain of the invention comprises a V domain having a V corresponding to a naturally occurring H The amino acid sequence of the immunoglobulin single variable domain is similar to that of the heavy chain antibody V domain, but is "camelized", that is, by replacing the amino acid sequence of the heavy chain antibody V domain with that of the heavy chain antibody V domain. HH One or more of the amino acid residues present at one or more corresponding positions in the structural domain are substituted for the naturally occurring V residues from a conventional 4-chain antibody. H This can be done in a manner known per se as will be apparent to the skilled person, for example based on the description herein. Such "camelizing" substitutions are preferably inserted between V H -V L The V sequences used as starting material or starting point for the generation or design of camelid immunoglobulin single variable domains are preferably at amino acid positions that are formed and / or present at the camelid interface, and / or at the so-called camelid signature, as defined herein (see also, for example, WO 94 / 04678 and Davies and Riechmann 1994 (FEBS letters 339:285-290) and 1996 (Protein Engineering 9:531-537)). H The sequence is preferably from a mammalian V H Sequence, more preferably human V H Sequence, such as V H 3 sequences. However, it should be noted that such camelized immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known per se and are therefore not strictly limited to the use of single variable domains comprising naturally occurring V H A polypeptide having a structural domain is a polypeptide obtained as a starting material.

[0912] For example, as further described herein, both "humanization" and "camelization" can be achieved by providing a naturally occurring V HH Domain or V HThe nucleic acid can then be expressed in a manner known per se, thereby providing the immunoglobulin single variable domains required for the present invention. Alternatively, the nucleic acid can be expressed in a manner known per se, respectively, based on the naturally occurring V HH Domain or V H The amino acid sequence of the humanized or camelized immunoglobulin single variable domain required by the present invention can be designed, respectively, and then synthesized de novo using a peptide synthesis technique known per se. HH Domain or V H The amino acid sequence or nucleotide sequence of the structural domain can be respectively designed to encode the nucleotide sequence of the desired humanized or camelized immunoglobulin single variable domain of the present invention, and then synthesized de novo using a technique known per se for nucleic acid synthesis, after which the nucleic acid thus obtained can be expressed in a manner known per se, thereby providing the desired immunoglobulin single variable domain of the present invention.

[0913] Accordingly, the present invention relates to a polypeptide as described herein, wherein the ISV is a Nanobody, a V HH , humanized V HH or camelized V H .

[0914] Generally, a protein or polypeptide that comprises or essentially consists of a single building block, a single immunoglobulin single variable domain or a single Nanobody will be referred to herein as a "monovalent" protein or polypeptide, as a "monovalent construct", as a "monovalent building block", as a "monovalent immunoglobulin single variable domain", or as a "monovalent Nanobody", respectively.

[0915] In this respect, the present invention also relates to monovalent structural units constituting the polypeptides of the present invention.

[0916] Thus, the present invention relates to an ISV or polypeptide that specifically binds to the constant domain of CD3 and comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0917] (i) CDR1 is selected from the group consisting of:

[0918] (a) SEQ ID NOs: 81-100; or

[0919] (b) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 81-100, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 not having said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0920] (ii) CDR2 is selected from the group consisting of:

[0921] (c) SEQ ID NOs: 101-122; or

[0922] (d) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 101-122, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0923] (iii) CDR3 is selected from the group consisting of:

[0924] (e) SEQ ID NOs: 123-143; or

[0925] (f) an amino acid sequence having 4, 3, 2 or 1 amino acid differences with the amino acid sequence of any one of SEQ ID NOs: 123-143, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0926] As discussed above, ISVs belonging to different clusters were separated based on structural similarities and differences in CDR2 and CDR3.

[0927] An immunoglobulin single variable domain belonging to cluster A is represented by a polypeptide according to the following, wherein:

[0928] (i) CDR1 is selected from the group consisting of:

[0929] (a) SEQ ID NOs: 81-87; and

[0930] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 81, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0931] (ii) CDR2 is selected from the group consisting of:

[0932] (c) SEQ ID NOs: 101-109; and

[0933] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 101, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0934] (iii) CDR3 is selected from the group consisting of:

[0935] (e) SEQ ID NOs: 123-127; and

[0936] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 123, provided that the polypeptide comprising a CDR3 with said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without the 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0937] In another aspect, in the polypeptides belonging to cluster A, CDR1 is selected from the group consisting of:

[0938] (a) SEQ ID NO:81; and

[0939] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 81, wherein

[0940] - At position 1, G has changed to R;

[0941] - At position 3, T has changed to A;

[0942] - At position 4, Y has changed to F;

[0943] - S has changed to G at position 8; and / or

[0944] - At position 10 G has changed to A.

[0945] In another aspect, in the polypeptides belonging to cluster A, CDR2 is selected from the group consisting of:

[0946] (a) SEQ ID NO: 101; and

[0947] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 101, wherein

[0948] - At position 3, V has changed to T or A;

[0949] - At position 5, S has changed to T;

[0950] - G has changed to D or E at position 6; and / or

[0951] - At position 9 T has changed to S, A or P.

[0952] In another aspect, in the polypeptides belonging to cluster A, CDR3 is selected from the group consisting of:

[0953] (a) SEQ ID NO: 123; and

[0954] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 123, wherein

[0955] - At position 2, I has changed to T;

[0956] - I has changed to V at position 9; and / or

[0957] - At position 10 A has been changed to P.

[0958] Thus, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0959] (i) CDR1 is selected from the group consisting of:

[0960] (a) SEQ ID NO:81; and

[0961] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 81, wherein

[0962] - At position 1, G has changed to R;

[0963] - At position 3, T has changed to A;

[0964] - At position 4, Y has changed to F;

[0965] - S has changed to G at position 8; and / or

[0966] - At position 10 G has changed to A,

[0967] And among them

[0968] (ii) CDR2 is selected from the group consisting of:

[0969] (a) SEQ ID NO: 101; and

[0970] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 101, wherein

[0971] - At position 3, V has changed to T or A;

[0972] - At position 5, S has changed to T;

[0973] - G has changed to D or E at position 6; and / or

[0974] - T has changed to S, A or P at position 9,

[0975] And among them

[0976] (iii) CDR3 is selected from the group consisting of:

[0977] (a) SEQ ID NO: 123; and

[0978] (b) an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 123, wherein

[0979] - At position 2, I has changed to T;

[0980] - I has changed to V at position 9; and / or

[0981] - At position 10 A has been changed to P.

[0982] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO: 123. Preferably, the polypeptide is selected from any one of SEQ ID NOs: 1-50.

[0983] An immunoglobulin single variable domain belonging to cluster B is represented by a polypeptide according to the following, wherein:

[0984] (i) CDR1 is selected from the group consisting of:

[0985] (a) SEQ ID NOs: 88; and

[0986] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 88, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0987] (ii) CDR2 is selected from the group consisting of:

[0988] (c) SEQ ID NOs: 110; and

[0989] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 110, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[0990] (iii) CDR3 is selected from the group consisting of:

[0991] (e) SEQ ID NOs: 128; and

[0992] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 128, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0993] In another aspect, in the polypeptide belonging to cluster B, CDR1 is SEQ ID NO:88.

[0994] In another aspect, in the polypeptide belonging to cluster B, CDR2 is SEQ ID NO:110.

[0995] In another aspect, in the polypeptide belonging to cluster B, CDR3 is SEQ ID NO:128.

[0996] In another aspect, the invention relates to a polypeptide, wherein: CDR1 is represented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, and CDR3 is represented by SEQ ID NO: 128. Preferably, the polypeptide is SEQ ID NOs:51.

[0997] An immunoglobulin single variable domain belonging to cluster C is represented by a polypeptide according to the following, wherein:

[0998] (i) CDR1 is selected from the group consisting of:

[0999] (a) SEQ ID NO:90; and

[1000] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 90, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1001] (ii) CDR2 is selected from the group consisting of:

[1002] (c) SEQ ID NOs: 112-113; and

[1003] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 112, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1004] (iii) CDR3 is selected from the group consisting of:

[1005] (e) SEQ ID NO: 130; and

[1006] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 130, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[1007] In another aspect, in the polypeptide belonging to cluster C, CDR1 is SEQ ID NO:90.

[1008] In another aspect, in the polypeptide belonging to cluster C, CDR2 is selected from the group consisting of:

[1009] (a) SEQ ID NO: 112; and

[1010] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[1011] - At position 2 V has changed to A.

[1012] In another aspect, in the polypeptide belonging to cluster C, CDR3 is SEQ ID NO:130.

[1013] Thus, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[1014] (i) CDR1 is SEQ ID NO:90; and

[1015] And among them

[1016] (ii) CDR2 is selected from the group consisting of:

[1017] (a) SEQ ID NO: 112; and

[1018] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 112, wherein

[1019] - At position 2, V has changed to A,

[1020] And among them

[1021] (iii) CDR3 is SEQ ID NO:130.

[1022] In another aspect, the present invention relates to a polypeptide, wherein: CDR1 is represented by SEQ ID NO: 90, CDR2 is represented by SEQ ID NO: 112, and CDR3 is represented by SEQ ID NO: 130. Preferably, the polypeptide is selected from any one of SEQ ID NOs: 53-56.

[1023] An immunoglobulin single variable domain belonging to cluster D is represented by a polypeptide according to the following, wherein:

[1024] (i) CDR1 is selected from the group consisting of:

[1025] (a) SEQ ID NOs: 89; and

[1026] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 89, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1027] (ii) CDR2 is selected from the group consisting of:

[1028] (c) SEQ ID NOs: 111; and

[1029] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 111, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1030] (iii) CDR3 is selected from the group consisting of:

[1031] (e) SEQ ID NOs: 129; and

[1032] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 129, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[1033] In another aspect, in the polypeptide belonging to cluster D, CDR1 is SEQ ID NO:89.

[1034] In another aspect, in the polypeptide belonging to cluster D, CDR2 is SEQ ID NO:111.

[1035] In another aspect, in the polypeptide belonging to cluster D, CDR3 is SEQ ID NO:129.

[1036] In another aspect, the present invention relates to a polypeptide, wherein: CDR1 is represented by SEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111, and CDR3 is represented by SEQ ID NO: 129. Preferably, the polypeptide is SEQ ID NOs:52.

[1037] An immunoglobulin single variable domain belonging to cluster E is represented by a polypeptide according to the following, wherein:

[1038] (i) CDR1 is selected from the group consisting of:

[1039] (a) SEQ ID NOs:91-93; and

[1040] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:91, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1041] (ii) CDR2 is selected from the group consisting of:

[1042] (c) SEQ ID NOs: 114-117; and

[1043] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 114, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1044] (iii) CDR3 is selected from the group consisting of:

[1045] (e) SEQ ID NOs: 131-133; and

[1046] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 131, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[1047] In another aspect, in the polypeptide belonging to cluster E, CDR1 is selected from the group consisting of:

[1048] (a) SEQ ID NO:91; and

[1049] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[1050] - At position 6, R has changed to N or T;

[1051] - N has been changed to H at position 7; and / or

[1052] - At position 8 the M has been changed to a T.

[1053] In another aspect, in the polypeptide belonging to cluster E, CDR2 is selected from the group consisting of:

[1054] (a) SEQ ID NO: 114; and

[1055] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[1056] - At position 1, R has changed to Q;

[1057] - T has changed to S at position 3; and / or

[1058] - In position 7 D has changed to A or K.

[1059] In another aspect, in the polypeptide belonging to cluster E, CDR3 is selected from the group consisting of:

[1060] (a) SEQ ID NO: 131; and

[1061] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein

[1062] - S has changed to R at position 2; and / or

[1063] - At position 6 S has changed to V.

[1064] Thus, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[1065] (i) CDR1 is selected from the group consisting of:

[1066] (a) SEQ ID NO:91; and

[1067] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein

[1068] - At position 6, R has changed to N or T;

[1069] - N has been changed to H at position 7; and / or

[1070] - At position 8, M has changed to T,

[1071] And among them

[1072] (ii) CDR2 is selected from the group consisting of:

[1073] (a) SEQ ID NO: 114; and

[1074] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein

[1075] - At position 1, R has changed to Q;

[1076] - T has changed to S at position 3; and / or

[1077] - D has changed to A or K at position 7,

[1078] And among them

[1079] (iii) CDR3 is selected from the group consisting of:

[1080] (a) SEQ ID NO: 131; and

[1081] (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein

[1082] - S has changed to R at position 2; and / or

[1083] - At position 6 S has changed to V.

[1084] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO: 91, CDR2 is represented by SEQ ID NO: 114, and CDR3 is represented by SEQ ID NO: 131. Preferably, the polypeptide is selected from any one of SEQ ID NOs: 57-65.

[1085] An immunoglobulin single variable domain belonging to cluster F is represented by a polypeptide according to the following, wherein:

[1086] (i) CDR1 is selected from the group consisting of:

[1087] (a) SEQ ID NOs: 94-100; and

[1088] (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 94, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1089] (ii) CDR2 is selected from the group consisting of:

[1090] (c) SEQ ID NOs: 118-122; and

[1091] (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 118, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or

[1092] (iii) CDR3 is selected from the group consisting of:

[1093] (e) SEQ ID NOs: 134-143; and

[1094] (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 134, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[1095] In another aspect, in the polypeptide belonging to cluster F, CDR1 is selected from the group consisting of:

[1096] (a) SEQ ID NO:94; and

[1097] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[1098] - At position 3, S has changed to T, A or G;

[1099] - At position 5, N has changed to S;

[1100] - M has changed to T or A at position 6; and / or

[1101] - At position 9 L has changed to M.

[1102] In another aspect, in the polypeptide belonging to cluster F, CDR2 is selected from the group consisting of:

[1103] (a) SEQ ID NO: 118; and

[1104] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 118, wherein

[1105] - At position 2, H has changed to V;

[1106] - At position 5, S has changed to H or A;

[1107] - N has changed to S at position 8; and / or

[1108] - At position 10 Y has changed to F.

[1109] In another aspect, in the polypeptide belonging to cluster F, CDR3 is selected from the group consisting of:

[1110] (a) SEQ ID NO: 134; and

[1111] (b) an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 134, wherein

[1112] - A has changed to S or D at position 6;

[1113] - F has changed to Y or A at position 7;

[1114] - At position 8, R has changed to H;

[1115] - At position 9, S has changed to A;

[1116] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[1117] - At position 14 V has changed to I.

[1118] Thus, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[1119] (i) CDR1 is selected from the group consisting of:

[1120] (a) SEQ ID NO:94; and

[1121] (b) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 94, wherein

[1122] - At position 3, S has changed to T, A or G;

[1123] - At position 5, N has changed to S;

[1124] - M has changed to T or A at position 6; and / or

[1125] - At position 9, L has changed to M,

[1126] And among them

[1127] (ii) CDR2 is selected from the group consisting of:

[1128] (a) SEQ ID NO: 118; and

[1129] (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 118, wherein

[1130] - At position 2, H has changed to V;

[1131] - At position 5, S has changed to H or A;

[1132] - N has changed to S at position 8; and / or

[1133] - At position 10, Y has changed to F,

[1134] And among them

[1135] (iii) CDR3 is selected from the group consisting of:

[1136] (a) SEQ ID NO: 134; and

[1137] (b) an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 134, wherein

[1138] - A has changed to S or D at position 6;

[1139] - F has changed to Y or A at position 7;

[1140] - At position 8, R has changed to H;

[1141] - At position 9, S has changed to A;

[1142] - at position 11 G has been changed to D, T, N, S, K or R; and / or

[1143] - At position 14 V has changed to I.

[1144] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO: 94, CDR2 is represented by SEQ ID NO: 118, and CDR3 is represented by SEQ ID NO: 134. Preferably, the polypeptide is selected from any one of SEQ ID NOs: 66-80.

[1145] In a further aspect, the invention relates to a polypeptide whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides belonging to clusters A, B, C, D, E or F.

[1146] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides having SEQ ID NOs: 1-50.

[1147] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by an ISV or a polypeptide having SEQ ID NO: 51.

[1148] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides having SEQ ID NOs: 53-56.

[1149] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by an ISV or a polypeptide having SEQ ID NO: 52.

[1150] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides having SEQ ID NOs: 57-65.

[1151] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides having SEQ ID NOs: 66-80.

[1152] In a further aspect, the invention relates to a polypeptide that is cross-blocked from binding to CD3 by at least one of the ISVs or polypeptides belonging to clusters A, B, C, D, E or F.

[1153] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one of the ISVs or polypeptides belonging to SEQ ID NOs: 1-50.

[1154] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by an ISV or a polypeptide having SEQ ID NO: 51.

[1155] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one of the ISVs or polypeptides belonging to SEQ ID NOs: 53-56.

[1156] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by an ISV or a polypeptide having SEQ ID NO: 52.

[1157] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one of the ISVs or polypeptides belonging to SEQ ID NOs: 57-65.

[1158] Therefore, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one of the ISVs or polypeptides belonging to SEQ ID NOs: 66-80.

[1159] The present invention also relates to compounds or constructs, and in particular proteins or polypeptides, comprising or essentially consisting of one or more ISV's or polypeptides of the present invention, and optionally further comprising one or more other groups, residues, parts or binding units. It will be clear to the skilled person from the further disclosure herein that said other groups, residues, parts, binding units or amino acid sequences may or may not provide additional functionality to the polypeptide of the present invention (and / or to the compound or construct in which said polypeptide is present), and may or may not alter the properties of the polypeptide of the present invention.

[1160] In one specific but non-limiting aspect of the invention, which will be further described herein, the ISV's and polypeptides of the invention may have an increased half-life in serum compared to the immunoglobulin single variable domain or polypeptide from which they are derived (as further described herein). For example, an immunoglobulin single variable domain or polypeptide of the invention may be linked (chemically or otherwise) to one or more half-life extending groups or moieties (e.g., PEG) to provide a derivative of the ISV's or polypeptides of the invention having an increased half-life.

[1161] In a particular aspect of the invention, the compound or construct of the invention or the polypeptide of the invention may have an increased half-life compared to the corresponding ISV or polypeptide of the invention. Some preferred but non-limiting examples of such compounds, constructs and polypeptides will become clear to the skilled person on the basis of the further disclosure herein, for example comprising an immunoglobulin single variable domain or polypeptide of the invention that has been chemically modified to increase its half-life (e.g. by pegylation); an immunoglobulin single variable domain or polypeptide of the invention comprising at least one additional binding site for binding to a serum protein (e.g. serum albumin); or a construct or polypeptide of the invention comprising at least one ISV or polypeptide of the invention linked to a structural moiety (particularly at least one amino acid sequence) that increases the half-life of the ISV or polypeptide of the invention. Examples of ISV's or polypeptides of the invention comprising such half-life extending moieties or immunoglobulin single variable domains will become clear to the skilled person on the basis of the further disclosure herein; for example include but are not limited to polypeptides wherein one or more immunoglobulin single variable domains or polypeptides of the invention are suitably linked to one or more serum proteins or fragments thereof (e.g. (human) serum albumin or suitable fragments thereof) or to one or more binding units capable of binding to serum proteins (e.g. domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains suitable for use as single domain antibodies, "dAb's", immunoglobulin single variable domains suitable for use as dAbs, or Nanobodies capable of binding to serum proteins such as serum albumin (e.g. human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description herein and to the references mentioned); ISV's or polypeptides of the invention are linked to an Fc portion (e.g. human Fc) or a suitable portion or fragment thereof; or ISV's or polypeptides wherein one or more immunoglobulin single variable domains or polypeptides of the invention are suitably linked to one or more small proteins or peptides capable of binding to serum proteins (e.g. but not limited to WO 2011 / 036654). 91 / 01743, WO 01 / 45746, WO 02 / 076489, WO 08 / 068280, WO 09 / 127691 and WO 11 / 095545).

[1162] Typically, the half-life of a compound, construct or polypeptide of the invention with an increased half-life is preferably at least 1.5 times, preferably at least 2 times, such as at least 5 times, such as at least 10 times or more than 20 times, greater than the half-life of the corresponding ISV or polypeptide of the invention itself. For example, a compound, construct or polypeptide of the invention with an increased half-life may have a half-life, for example in humans, that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours.

[1163] In a preferred but non-limiting aspect of the invention, such a compound, construct or polypeptide of the invention has a serum half-life in humans, e.g., greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24, 48 or 72 hours, compared to the corresponding ISV or polypeptide of the invention itself.

[1164] In another preferred but non-limiting aspect of the present invention, this compound, construct or polypeptide of the present invention exhibits a serum half-life of at least about 12 hours in humans, preferably at least 24 hours, more preferably at least 48 hours, more preferably at least 72 hours or longer. For example, the compound, construct or polypeptide of the present invention can have at least 5 days (e.g., about 5-10 days), preferably at least 9 days (e.g., about 9-14 days), more preferably at least about 10 days (e.g., about 10-15 days), or at least about 11 days (e.g., about 11-16 days), more preferably at least about 12 days (e.g., about 12-18 days or longer), or a half-life greater than 14 days (e.g., about 14-19 days).

[1165] In the present invention, it is demonstrated that the inclusion of a binding unit targeting albumin in the construct itself does not have a significant impact on the potency or efficacy obtained. Although a slight loss of potency / efficacy was observed in the presence of HSA, the CD3 multispecific polypeptides with extended half-life are still effective in tumor cell killing. Albumin-based drug delivery has been demonstrated to be useful for achieving improved cancer therapy, largely due to its passive targeting of tumors through enhanced permeability and retention effects and the increased demand of tumor cells for albumin as a source of energy and amino acids. However, albumin not only lacks an active mechanism to overcome the cell membrane barrier, but also lacks the ability to penetrate tumor tissue (Qianqian Guo et al. Polym. Chem., 2013, 4, 4584-4587).

[1166] In a particularly preferred but non-limiting aspect of the invention, the invention provides a polypeptide of the invention comprising a first and a second immunoglobulin single variable domain (ISV); and further comprising one or more (preferably one) serum albumin binding immunoglobulin single variable domains as described herein, for example, the serum albumin binding immunoglobulin single variable domains designated as Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (Table B-3).

[1167] Table B-3: Immunoglobulin single variable domains in HLE for use in ISVs and polypeptides of the invention

[1168]

[1169]

[1170] Therefore, the present invention relates to a polypeptide as described herein, further comprising a serum protein binding moiety.

[1171] The present invention relates to a polypeptide as described herein, wherein said serum protein binding moiety binds to serum albumin.

[1172] The present invention relates to a polypeptide as described herein, wherein the serum protein binding moiety is an immunoglobulin single variable domain that binds serum albumin.

[1173] The present invention relates to a polypeptide as described herein, wherein the serum albumin binding ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and wherein CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being determined according to the Kabat definition; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), the CDRs being determined according to Kontermann 2010.

[1174] The present invention relates to a polypeptide as described herein, wherein the ISV that binds serum albumin comprises Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (Table B-3).

[1175] In the polypeptides of the invention, two or more building blocks, ISVs or Nanobodies and optionally one or more polypeptides, one or more other groups, drugs, agents, residues, moieties or binding units may be directly linked to each other (as described for example in WO 99 / 23221) and / or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

[1176] Suitable spacers or linkers for multivalent and multispecific polypeptides will be clear to the skilled person and may generally be any linker or spacer used in the art for linking amino acid sequences. Preferably, the linker or spacer is suitable for use in constructing a protein or polypeptide intended for pharmaceutical use.

[1177] Some particularly preferred spacers include spacers and linkers used in the art to connect antibody fragments or antibody domains. These include linkers mentioned in the general background cited above, as well as linkers used in the art to construct diabodies or ScFv fragments (however, in this regard, it should be noted that in diabodies and ScFv fragments, the linker sequences used should have a sequence that allows the relevant V H and V L The length, degree of flexibility and other properties of the domains brought together to form a complete antigen-binding site, there is no particular limitation on the length or flexibility of the linker used in the polypeptides of the invention, since each ISV or Nanobody itself forms a complete antigen-binding site).

[1178] For example, the linker may be a suitable amino acid sequence, and in particular an amino acid sequence of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, such as the type (gly x ser y ) z, such as (gly4ser)3 or (gly3ser2)3, as described in WO 99 / 42077, and the GS30, GS15, GS9 and GS7 linkers described by Ablynx in applications referred to herein (see, for example, WO06 / 040153 and WO 06 / 122825), and a hinge-like region, such as the hinge region of a naturally occurring heavy chain antibody or similar sequence (as described in WO 94 / 04678). Preferred linkers are described in Table B-4.

[1179] Table B-4: Connectors

[1180] 5GS GGGGS 7GS SGGSGGS 9GS GGGGSGGGS 10GS GGGGSGGGGS 15GS GGGGSGGGGSGGGGS 18GS GGGGSGGGGSGGGGGGGS 20GS GGGGSGGGGSGGGGSGGGGS 25GS GGGGSGGGGSGGGGSGGGGSGGGGS 30GS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Poly A AAA

[1181] Some other particularly preferred linkers are polyalanine (eg, AAA), and linkers GS30 (SEQ ID NO: 85 in WO 06 / 122825) and GS9 (SEQ ID NO: 84 in WO 06 / 122825).

[1182] Other suitable linkers generally comprise organic compounds or polymers, especially those suitable for use in proteins for pharmaceutical use. For example, poly(ethylene glycol) moieties have been used to link antibody domains, see for example WO 04 / 081026.

[1183] It is within the scope of the invention that the length, degree of flexibility and / or other properties of the linker(s) used (although not critical, as they are typically used for linkers used in ScFv fragments) may have some effect on the properties of the final polypeptide of the invention, including but not limited to affinity, specificity or avidity for CD3, or for one or more other antigens. Based on the disclosure herein, optionally after some limited routine experimentation, a skilled person will be able to determine the optimal linker(s) for a particular polypeptide of the invention.

[1184] For example, in a multivalent polypeptide of the invention comprising building blocks, ISV's or Nanobodies directed against a first and a second target, the length and flexibility of the linker is preferably such that it allows each building block, ISV's or Nanobody of the invention present in the polypeptide to bind to its cognate target, e.g. an antigenic determinant on each target. Again, based on the disclosure herein, optionally after some limited routine experimentation, the skilled person will be able to determine the optimal linker or links for a particular polypeptide of the invention.

[1185] It is also within the scope of the invention that the one or more linkers used confer one or more other advantageous properties or functionalities to the polypeptides of the invention and / or provide one or more sites for forming derivatives and / or for attaching functional groups (e.g. derivatives of the ISV's, Nanobodies or polypeptides of the invention as described herein). For example, linkers containing one or more charged amino acid residues may provide improved hydrophilic properties, while linkers forming or containing small epitopes or tags may be used for detection, identification and / or purification purposes. Again, based on the disclosure herein, optionally after some limited routine experimentation, the skilled person will be able to determine the best linker for a particular polypeptide of the invention.

[1186] Finally, when more than two linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, optionally after some limited routine experimentation, a skilled person will be able to determine the best linker for a particular polypeptide of the invention.

[1187] Typically, for ease of expression and production, the polypeptides of the invention will be linear polypeptides. However, in its broadest sense, the invention is not limited thereto. For example, when the polypeptides of the invention comprise three or more building blocks, ISVs or nanobodies, they may be connected by using a linker with three or more "arms", each of which is connected to a building block, ISV or nanobody, thereby providing a "star-shaped" construct. Although generally not preferred, it is also possible to use a circular construct.

[1188] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV and said second ISV and possibly said third ISV and / or said serum albumin binding ISV are directly linked to each other or via a linker.

[1189] The present invention relates to a polypeptide as described herein, wherein the linker is selected from the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS.

[1190] The present invention relates to a polypeptide as described herein, wherein the serum protein binding moiety is a non-antibody based polypeptide (eg PEG).

[1191] The present invention also relates to methods for preparing the ISV's, polypeptides and constructs described herein. The ISV's, polypeptides and constructs of the present invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the ISV's, polypeptides and constructs of the present invention can be prepared in any manner known per se for preparing antibodies and in particular for preparing antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred but non-limiting methods for preparing polypeptides and constructs include the methods and techniques described herein.

[1192] A method of producing an ISV, polypeptide or protein construct of the invention may comprise the following steps:

[1193] - expressing in a suitable host cell or host organism (also referred to herein as "host of the invention") or in another suitable expression system of a nucleic acid encoding said IVS, polypeptide or protein construct of the invention,

[1194] Optionally, followed by:

[1195] - isolating and / or purifying the ISV, polypeptide or protein construct of the invention thus obtained.

[1196] In particular, the method may comprise the following steps:

[1197] - cultivating and / or maintaining a host of the invention under conditions such that said host of the invention expresses and / or produces at least one ISV, polypeptide or protein construct of the invention;

[1198] Optionally, followed by:

[1199] - isolating and / or purifying the ISV, polypeptide or protein construct of the invention thus obtained.

[1200] Thus, the present invention also relates to nucleic acids or nucleotide sequences encoding an ISV, polypeptide or protein construct of the invention (also referred to as "nucleic acids of the invention" or "nucleotide sequences of the invention"). Nucleic acids of the invention may be in the form of single-stranded or double-stranded DNA or RNA, and are preferably in the form of double-stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA, or synthetic DNA (such as DNA with codon usage that has been specifically adapted for expression in an intended host cell or host organism).

[1201] According to one embodiment of the present invention, nucleic acid of the present invention is in a substantially isolated form, as defined herein. Nucleic acid of the present invention can also be in the form of a vector, be present in a vector and / or be a part of a vector, such as, for example, a plasmid, a cosmid or a YAC, which can also be in a substantially isolated form.

[1202] Based on the information given herein about the polypeptides or protein constructs of the invention, the nucleic acids of the invention can be prepared or obtained in a manner known per se and / or can be isolated from a suitable natural source. Moreover, as will be clear to the skilled person, in order to prepare the nucleic acids of the invention, several nucleotide sequences (such as at least one nucleotide sequence encoding the immunoglobulin single variable domain of the invention) and, for example, nucleic acids encoding one or more linkers can also be linked together in a suitable manner.

[1203] The techniques for producing nucleic acids of the present invention will be clear to those skilled in the art, 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 expression of truncated expression products; introducing one or more restriction sites (e.g., to produce cassettes and / or regions that can be easily cut and / or ligated using suitable restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatched" primers. These and other techniques will be clear to the skilled person, and reference may also be made to standard manuals, such as Sambrook et al. and Ausubel et al., mentioned herein, and the following examples.

[1204] As will be clear to those skilled in the art, nucleic acid of the present invention can also be the form of genetic construct, be present in the genetic construct and / or be a part of genetic construct.Described genetic construct comprises at least one nucleic acid of the present invention usually, and it is optionally connected to one or more elements of genetic construct known per se, such as for example other elements of one or more suitable regulating and controlling elements (such as suitable promotor, enhancer, terminator etc.) and the genetic construct related to this paper.Described genetic construct comprising at least one nucleic acid of the present invention will also be referred to as " genetic construct of the present invention " in this article.

[1205] Genetic construct of the present invention can be DNA or RNA, and preferably double-stranded DNA.Genetic construct of the present invention can also be the form that is suitable for transforming the expected host cell or host organism, the form that is suitable for being integrated into the genomic DNA of the expected host cell, or the form that is suitable for independent replication, maintenance and / or inheritance in the expected host organism.For example, genetic construct of the present invention can be the form of carrier, such as for example plasmid, cosmid, YAC, viral vector or transposon.Especially, carrier can be expression vector, promptly can provide the carrier of external and / or internal expression (for example in suitable host cell, host organism and / or expression system).

[1206] In a preferred but non-limiting embodiment, the genetic construct of the present invention comprises

[1207] a) at least one nucleic acid of the present invention; operably linked to

[1208] b) one or more regulatory elements, such as a promoter and optionally a suitable terminator;

[1209] and optionally

[1210] c) one or more other elements of a genetic construct known per se;

[1211] wherein the terms "regulatory element", "promoter", "terminator" and "operably linked" have their usual meaning in the art (as further described herein): and wherein the "other elements" present in the genetic construct may be, for example, 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or elements that may facilitate or increase transformation or integration (efficiency). These and other suitable elements of the genetic construct will be clear to the skilled person and may, for example, depend on the type of construct used; the intended host cell or host organism; the mode of expression of the nucleotide sequence of the invention of interest (e.g. by constitutive, transient or induced expression); and / or the transformation technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in a substantially similar manner.

[1212] Preferably, in the genetic construct of the present invention, the at least one nucleic acid of the present invention and the regulatory element, and optionally the one or more other elements are "operably connected" to each other, generally meaning that they have a functional relationship with each other in this way. For example, if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of a coding sequence (wherein the coding sequence should be understood to be "under the control" of the promoter), the promoter is considered to be "operably connected" to the coding sequence. Generally, when two nucleotide sequences are operably connected, they will be in the same orientation and generally also in the same reading frame. They will also generally be substantially continuous, although this may not be necessary.

[1213] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e., to express and / or produce polypeptides or protein constructs of the invention. The host is preferably a non-human host. Suitable hosts or host cells will be clear to those skilled in the art and may be, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms, for example:

[1214] - bacterial strains, including but not limited to Gram-negative strains, such as strains of Escherichia coli; strains of Proteus, for example Proteus mirabilis; strains of Pseudomonas, for example Pseudomonas fluorescens; and Gram-positive strains, such as strains of Bacillus, for example Bacillus subtilis or Bacillus brevis; strains of Streptomyces, for example Streptomyces lividans; strains of Staphylococcus, for example Staphylococcus carnosus; and strains of Lactococcus, for example Lactococcus lactis;

[1215] - fungal cells, including but not limited to cells from Trichoderma species,

[1216] For example from Trichoderma reesei; from cells of Neurospora species, for example from Neurospora crassa; from cells of Sordaria species, for example from Sordaria macrospora; from cells of Aspergillus species, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi;

[1217] - yeast cells, including but not limited to cells from the species Saccharomyces,

[1218] For example, Saccharomyces cerevisiae; cells from species of Schizosaccharomyces, such as Schizosaccharomyces pombe; cells from species of Pichia, such as Pichia pastoris or Pichia methanolica; cells from species of Hansenula, such as Hansenula polymorpha; cells from species of Kluyveromyces, such as Kluyveromyces lactis; cells from species of Arxula, such as Arxula adeninivorans; cells from species of Yarrowia, such as Yarrowia lipolytica;

[1219] - amphibian cells or cell lines, such as Xenopus oocytes;

[1220] - cells or cell lines derived from insects, such as cells / cell lines derived from Lepidoptera,

[1221] Including but not limited to Spodoptera SF9 and Sf21 cells or cells / cell lines derived from Drosophila, such as Schneider and Kc cells;

[1222] - a plant or plant cell, for example in a tobacco plant; and / or

[1223] - mammalian cells or cell lines, e.g. cells or cell lines derived from humans, cells or cell lines from mammals, including but not limited to CHO cells, BHK cells (e.g. BHK-21 cells) and human cells or cell lines such as HeLa, COS (e.g. COS-7)

[1224] and PER.C6 cells;

[1225] As well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference may also be made to the general background art cited above herein, as well as to, for example, WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. 1998 (Res. Immunol. 149: 589-99); Riechmann and Muyldermans 1999 (J. Immunol. Met. 231: 25-38); van der Linden 2000 (J. Biotechnol. 80: 261-70); Joosten et al. 2003 (Microb. Cell Fact. 2: 1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. 66: 384-92); and other documents cited herein.

[1226] For expression of ISVs, polypeptides or constructs in cells, they may also be expressed as so called "intrabodies", as described, for example, in WO 94 / 02610, WO 95 / 22618 and US 7004940; WO 03 / 014960; Cattaneo and Biocca 1997 (Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag) and Kontermann 2004 (Methods 34: 163-170).

[1227] According to a preferred, but non-limiting embodiment of the present invention, the ISV, polypeptide or protein construct of the present invention is produced in bacterial cells, in particular bacterial cells suitable for large-scale pharmaceutical production, such as cells of the strains mentioned above.

[1228] According to another preferred, but non-limiting embodiment of the present invention, the ISV, polypeptide or protein construct of the present invention is produced in yeast cells, in particular yeast cells suitable for large-scale pharmaceutical production, such as cells of the species mentioned above.

[1229] According to yet another preferred, but non-limiting embodiment of the present invention, the ISV, polypeptide or construct of the present invention is produced in mammalian cells, in particular in human cells or in cells of a human cell line, more in particular in human cells or cells of a human cell line suitable for large-scale pharmaceutical production, such as the cell lines mentioned above.

[1230] Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell / host organism and the genetic construct to be used. Reference is again made to the manuals and patent applications mentioned above.

[1231] After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with nucleotide sequence / genetic construct of the present invention may be performed. This may for example be a selection step based on a selectable marker present in a genetic construct of the present invention, or may involve a step for example using a specific antibody to detect a polypeptide of the present invention.

[1232] Transformed host cells (which may be in the form of stable cell lines) or host organisms (which may be in the form of stable mutant lines or strains) form a further aspect of the invention.

[1233] Preferably, these host cells or host organisms are such that they express or are (at least) capable of expressing (e.g. under suitable conditions) an ISV, polypeptide or protein construct of the invention (and in the case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes the next generation, progeny and / or daughter generations of a host cell or host organism of the invention, e.g. obtained by cell division or by sexual or asexual reproduction.

[1234] Thus, in another aspect, the invention relates to a host or host cell expressing (or capable of expressing, where appropriate) an ISV, polypeptide or protein construct of the invention; and / or a host or host cell containing a nucleic acid encoding an ISV, polypeptide or protein construct of the invention. Some preferred but non-limiting examples of such hosts or host cells may be as generally described in WO 04 / 041867, WO 04 / 041865 or WO 09 / 068627. For example, the ISV, polypeptide and protein constructs of the invention may advantageously be expressed, produced or manufactured in a yeast strain such as a strain of Pichia pastoris. Reference is also made to WO04 / 25591, WO 10 / 125187, WO 11 / 003622 and WO 12 / 056000, which also describe the expression / production of immunoglobulin single variable domains and polypeptides comprising the same in Pichia pastoris and other hosts / host cells.

[1235] In order to produce / obtain expression of the ISV, polypeptide or protein construct of the invention, the transformed host cell or transformed host organism may typically be kept, maintained and / or cultured under conditions such that the (desired) ISV, polypeptide or protein construct of the invention is expressed / produced. Suitable conditions will be clear to the skilled person and will typically depend on the host cell / host organism used, and on the regulatory elements controlling the expression of the (relevant) nucleotide sequence of the invention. Reference is again made to the manuals and patent applications mentioned above in the paragraph on the genetic constructs of the invention.

[1236] Typically, suitable conditions may include the use of a suitable culture medium, the presence of a suitable food source and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequence of the invention is under the control of an inducible promoter); all of which may be selected by the skilled artisan. Likewise, under such conditions, the ISV, polypeptide or protein construct of the invention may be expressed in a constitutive manner, in a transient manner, or only when appropriately induced.

[1237] It will also be clear to the skilled person that the ISV, polypeptide or protein construct of the invention may be (first) produced in an immature form (as described above) and may then be post-translationally modified, depending on the host cell / host organism used. Furthermore, the ISV, polypeptide or protein construct of the invention may be glycosylated, also depending on the host cell / host organism used.

[1238] The ISV, polypeptide or protein construct of the invention can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism is cultivated using protein separation and / or purification techniques known per se, such as (preparative) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using specifically cleavable amino acid sequences fused to the polypeptide or construct of the invention) and / or preparative immunological techniques (i.e. using antibodies directed against the amino acid sequence to be isolated).

[1239] The constructs of the invention can generally be prepared by a process comprising at least one step of suitably linking an ISV or polypeptide of the invention to one or more further groups, residues, moieties or binding units, optionally via one or more suitable linkers, to provide a construct of the invention. The ISV, polypeptides and constructs of the invention may then be further modified, and in particular by chemically and / or biologically (e.g. enzymatically) modifying one or more amino acid residues forming the polypeptide or construct of the invention to obtain derivatives of the polypeptide or construct of the invention.

[1240] The present invention also relates to pharmaceutical compositions comprising the ISV, polypeptide, compound or construct of the present invention.

[1241] In the above methods, the amino acid sequences, ISV's, Nanobodies, polypeptides, compounds or constructs of the invention and / or compositions comprising the same may be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, for example, the amino acid sequences, ISV's, Nanobodies, polypeptides, compounds or constructs of the invention and / or compositions comprising the same may be administered orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly or by any other route of administration that avoids the gastrointestinal tract), intranasally, transdermally, topically, by suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select an appropriate route of administration and an appropriate pharmaceutical formulation or composition for said administration, depending on the disease or condition to be prevented or treated and other factors well known to the clinician.

[1242] As used herein, the term "therapeutic agent" refers to any agent that can be used to treat and / or manage a hyperproliferative cell disorder (e.g., cancer) or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to a multispecific polypeptide of the invention. Preferably, the therapeutic agent is an agent that is known to be useful or has been used or is currently being used to treat, prevent and / or manage a hyperproliferative cell disorder (e.g., cancer) or one or more symptoms thereof.

[1243] As used herein, "therapeutically effective amount" in the context of cancer refers to a single therapeutic amount or a therapeutic amount in combination with other therapies that provides a therapeutic benefit in treating and / or processing cancer. In one aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to destroy, change, control or remove primary, regional or metastatic cancer tissue. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to alleviate the symptoms of cancer. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to delay or minimize the spread of cancer. In a specific embodiment, the therapeutically effective amount of therapy is a therapeutic amount sufficient to inhibit the growth or proliferation of cancer cells, kill existing cancer cells (e.g., cause cancer regression) and / or prevent cancer cells from spreading to other tissues or regions (e.g., prevent metastasis). In another specific embodiment, a therapeutically effective amount of a therapy is a therapeutic amount sufficient to inhibit tumor growth 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% (as measured by standard methods known in the art). When used in conjunction with an amount of a multispecific polypeptide of the invention, the term can encompass an amount that improves the overall therapy, reduces or avoids an undesired effect, or enhances the therapeutic efficacy or synergy with another therapy. In one embodiment, a therapeutically effective amount of a therapy reduces or avoids an unwanted effect, or enhances the therapeutic efficacy or synergy with another therapy 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 an assay known in the art or described herein.

[1244] As used herein, "therapeutically effective amount" in the context of non-cancer hyperproliferative cell disorders refers to a therapeutic amount alone or in combination with other therapies that provides a therapeutic benefit in treating and / or handling the disorder. In one aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to destroy, change, control or remove cells affected by a non-cancer hyperproliferative cell disorder. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to alleviate the symptoms of a non-cancer hyperproliferative cell disorder. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to delay or minimize the spread of a non-cancer hyperproliferative cell disorder. In a specific embodiment, the therapeutically effective amount of therapy is a therapeutic amount sufficient to inhibit the growth or proliferation of a non-cancer hyperproliferative cell disorder, kill existing non-cancer hyperproliferative cells (e.g., cause regression of the disorder). In another specific embodiment, a therapeutically effective amount of a therapy is a therapeutic amount sufficient to inhibit the growth of non-cancer hyperproliferative 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% (as measured by standard methods known in the art). When used in conjunction with an amount of a multispecific polypeptide of the invention, the term can encompass an amount that improves the overall therapy, reduces or avoids an unwanted effect, or enhances the therapeutic efficacy or synergy with another therapy. In one embodiment, a therapeutically effective amount of a therapy reduces or avoids an unwanted effect, or enhances the therapeutic efficacy or synergy with another therapy 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 an assay known in the art.

[1245] As used herein, the term "therapy" refers to any regimen, method, and / or agent that can be used to treat, prevent, and / or manage a hyperproliferative cell disorder (e.g., cancer). In certain embodiments, the term "therapy" refers to biological therapy, supportive therapy, and / or other therapy used to treat, prevent, and / or manage a hyperproliferative cell disorder (e.g., cancer) or one or more symptoms thereof known to those skilled in the art (e.g., medical personnel).

[1246] As used herein, the terms "treat," "treatment," and "treating" in the context of administering a therapy to a subject refer to a reduction in the progression, severity, and / or duration of a condition associated with a hyperproliferative cell disorder (e.g., cancer) or an improvement in one or more symptoms thereof resulting from the administration of one or more therapies (including, but not limited to, administration of one or more prophylactic or therapeutic agents). In specific embodiments, the term "treat," in the context of administering a therapy to a subject, refers to a reduction or improvement in the progression, severity, and / or duration of a hyperproliferative cell disorder (e.g., cancer) 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% reduction in cancer cells relative to a control (e.g., a negative control such as phosphate buffered saline). In other embodiments, the term "treat," "treat," "treat," or "treating," in the context of administering a therapy to a subject, refers to reducing or ameliorating the progression, severity, and / or duration of a hyperproliferative cell disorder (e.g., cancer), refers to no change in the number of cancer cells, reduced hospital stays, reduced mortality, or increased survival of subjects with cancer.

[1247] The amino acid sequences, ISV's, Nanobodies, polypeptides, compounds and / or constructs of the invention and / or compositions comprising the same are administered according to a treatment regimen that is suitable for the prevention and / or treatment of the hyperproliferative cell disorder (e.g. cancer) to be prevented or treated. The clinician will generally be able to determine an appropriate treatment regimen, depending on factors such as the stage of the hyperproliferative cell disorder (e.g. cancer) to be treated, the severity of the hyperproliferative cell disorder (e.g. cancer) to be treated and / or the severity of its symptoms, the specific amino acid sequence, ISV's, Nanobodies, polypeptides, compounds and / or constructs of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, sex, weight, diet, general condition of the patient, and similar factors well known to the clinician.

[1248] Generally, the treatment regimen will comprise administering one or more amino acid sequences, ISV's, Nanobodies, polypeptides, compounds and / or constructs of the invention, or one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. Still based on the above factors, the clinician can determine the specific amount or dose to be administered.

[1249] In general, for the prevention and / or treatment of the hyperproliferative cell disorders (e.g. cancer) mentioned herein, and depending on the type of hyperproliferative cell disorder (e.g. cancer) to be treated and the stage of the disease, the efficacy of the specific amino acid sequence, ISV, Nanobody, polypeptide, compound or construct of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequence, ISV, Nanobody, polypeptide, compound or construct of the invention will generally be administered in an amount of 1 gram to 0.01 mg per kg body weight per day, preferably 0.1 gram to 0.01 mg per kg body weight per day, such as about 0.1, 1, 10, 100 or 1000 mg per kg body weight per day, for example 0.1 mg to 25 mg per kg body weight of the subject; either as a single daily dose or continuously (e.g. by infusion) as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases the clinician may choose to deviate from these amounts, for example based on the factors cited above and his professional judgment. In general, some guidance as to the amount to be administered can be obtained from the amount of a comparable conventional antibody or antibody fragment directed to the same target of administration that is typically administered by essentially the same route, taking into account differences in affinity / avidity, efficacy, biodistribution, half-life, and similar factors known to the skilled artisan.

[1250] Typically, in the above methods, a single amino acid sequence, ISV, Nanobody, polypeptide, compound or construct of the invention will be used. However, it is within the scope of the invention to use two or more amino acid sequences, ISV, Nanobody, polypeptide compounds and / or constructs of the invention in combination.

[1251] The ISV's, Nanobodies, amino acid sequences, polypeptides, compounds and / or constructs of the invention may also be used in combination with one or more other pharmaceutically active compounds or ingredients, i.e. as a combination therapy, which may or may not give rise to a synergistic effect. And, based on the factors cited above and their professional judgment, the clinician will be able to select such other compounds or ingredients, as well as a suitable combination therapy.

[1252] In particular, the amino acid sequences, ISV's, Nanobodies, polypeptides, compounds and / or constructs of the invention may be used in combination with other pharmaceutically active compounds or ingredients that are or can be used to prevent and / or treat the hyperproliferative cell disorders (e.g. cancer), diseases and / or conditions cited herein, with the result that a synergistic effect may or may not be obtained. Examples of such compounds and ingredients, as well as routes, methods and pharmaceutical formulations or compositions for their administration will be clear to the clinician.

[1253] When two or more substances or ingredients are used as part of a combined treatment regimen, they may be administered by the same route of administration or by different routes of administration at substantially the same time or at different times (e.g., substantially simultaneously, sequentially, or according to an alternative regimen). When the substances or ingredients are administered simultaneously by the same route of administration, they may be administered as different pharmaceutical preparations or compositions or as part of a combined pharmaceutical preparation or composition, as will be apparent to the skilled artisan.

[1254] In one aspect, the invention provides methods for administering immunoglobulin single variable domains and polypeptide constructs, polypeptides, compounds and / or constructs comprising one or more immunoglobulin single variable domains. In some embodiments, the immunoglobulin single variable domains, polypeptides, compounds and / or constructs are administered as pharmaceutical compositions. In addition to the immunoglobulin single variable domains and polypeptide constructs thereof, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[1255] As described in detail, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid forms, including those suitable for the following: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (such as those targeted for oral, sublingual and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as parenteral administration by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension or a sustained release formulation; topical administration, such as a cream, ointment or controlled release patch or spray for application to the skin, lungs or oral cavity; intravaginally or intrarectally, such as as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, lungs and to other mucosal surfaces.

[1256] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[1257] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that participates in the delivery or transport of the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[1258] Preparations of the present invention include those suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal and / or parenteral administration. Preparations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredients (e.g., immunoglobulin single variable domain or its polypeptide construct) that can be combined with a carrier material to produce a single dosage form will vary according to the host to be treated and a specific mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, the amount will be in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, most preferably about 10% to about 30%.

[1259] In certain embodiments, the formulation comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle formers (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides). In certain embodiments, the aforementioned formulation provides an orally bioavailable immunoglobulin single variable domain or polypeptide construct.

[1260] The method for preparing these preparations or compositions comprises the step of combining the immunoglobulin single variable domain or polypeptide construct with a carrier and optionally one or more auxiliary components. Generally, the preparation is prepared by combining the immunoglobulin single variable domain or polypeptide construct uniformly and closely with a liquid carrier or a finely divided solid carrier or both, and then shaping the product (if necessary).

[1261] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and gum arabic) and / or as a mouthwash, etc., each containing a predetermined amount of the immunoglobulin single variable domain or polypeptide construct as the active ingredient. The immunoglobulin single variable domain or polypeptide construct invention may also be administered as a bolus, electuary or paste.

[1262] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; humectants, such as glycerol; disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; solution retarders, such as paraffin; absorption promoters, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glyceryl monostearate and nonionic surfactants; adsorbents, such as kaolin and bentonite; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and colorants. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be employed as fillers in soft and hard shell gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[1263] Tablets can be prepared optionally with one or more auxiliary ingredients by compression or molding. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant, or a dispersant. Molded tablets can be prepared in a suitable machine, wherein the mixture of the powdered compound is moistened with an inert liquid diluent.

[1264] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the art of pharmaceutical formulation. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example using hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions to provide the desired release profile. They may be formulated for rapid release, such as freeze drying. They may be sterilized, for example, by filtration through a filter that retains bacteria, the latter being sterilized by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain an opacifying agent, and may be compositions that release the active ingredient only or preferably in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[1265] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (such as, for example, water or other solvents), solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[1266] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[1267] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[1268] Formulations of the pharmaceutical composition for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing the immunoglobulin single variable domain or polypeptide construct with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which are solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active compound.

[1269] Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[1270] Dosage forms for topical or transdermal administration of ...

Claims

1. A polypeptide comprising a first immunoglobulin single variable domain (ISV) and a second immunoglobulin single variable domain, wherein - the first ISV has a high affinity for / binds to cluster of differentiation 3 (CD3) present on T cells; - the second ISV has a high affinity for / binds to a first antigen on a target cell; wherein the first antigen is different from the CD3; wherein the target cell is different from the T cell; and The first ISV consists essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is selected from the group consisting of: (a) SEQ ID NOs:91-93; and (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:91, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or (ii) CDR2 is selected from the group consisting of: (c) SEQ ID NOs: 114-117; and (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 114, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or (iii) CDR3 is selected from the group consisting of: (e) SEQ ID NOs: 131-133; and (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 131, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

2. The polypeptide according to claim 1, wherein the first ISV consists essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is represented by SEQ ID NO:91, CDR2 is represented by SEQ ID NO:114, and CDR3 is represented by SEQ ID NO:

131.

3. The polypeptide according to claim 1 or 2, wherein the first antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

4. The polypeptide according to any one of claims 1 to 3, further comprising a third ISV having a high affinity for and / or binding to a second antigen on the target cell, wherein the second antigen is different from the first antigen.

5. The polypeptide according to claim 4, wherein the second antigen on the target cell is a tumor antigen, preferably a tumor associated antigen (TAA).

6. The polypeptide according to any one of claims 1 to 5, further comprising a serum protein binding moiety.

7. The polypeptide of claim 6, wherein the serum protein binding moiety is an ISV that binds serum albumin.

8. A polypeptide that specifically binds to CD3 and comprises or consists essentially of: 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is selected from the group consisting of: (a) SEQ ID NOs:91-93; and (b) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO:91, provided that the polypeptide comprising a CDR1 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR1 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or (ii) CDR2 is selected from the group consisting of: (c) SEQ ID NOs: 114-117; and (d) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 114, provided that the polypeptide comprising a CDR2 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR2 that does not have said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance; and / or (iii) CDR3 is selected from the group consisting of: (e) SEQ ID NOs: 131-133; and (f) an amino acid sequence that has 4, 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 131, provided that the polypeptide comprising a CDR3 having said 4, 3, 2 or 1 amino acid differences binds to CD3 with about the same or higher affinity as compared to the binding of a polypeptide comprising a CDR3 without said 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

9. The polypeptide according to claim 8, wherein CDR1 is selected from the group consisting of: (a) SEQ ID NO:91; and (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 91, wherein - At position 6, R has changed to N or T; - N has been changed to H at position 7; and / or - At position 8 the M has been changed to a T.

10. The polypeptide according to any one of claims 8 or 9, wherein CDR2 is selected from the group consisting of: (a) SEQ ID NO: 114; and (b) an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 114, wherein - At position 1, R has changed to Q; - T has changed to S at position 3; and / or - In position 7 D has changed to A or K.

11. The polypeptide according to any one of claims 8 to 10, wherein CDR3 is selected from the group consisting of: (a) SEQ ID NO: 131; and (b) an amino acid sequence having 1 amino acid difference from SEQ ID NO: 131, wherein - S has changed to R at position 2; and / or - At position 6 S has changed to V. 12 . The polypeptide according to claim 8 , wherein CDR1 is represented by SEQ ID NO: 91, CDR2 is represented by SEQ ID NO: 114, and CDR3 is represented by SEQ ID NO:

131.

13. A polypeptide according to any one of claims 8 to 12, which is a nanobody, a V HH , humanized V HH or camelized V H .

14. The polypeptide according to any one of claims 8 to 13, further comprising a serum protein binding moiety.

15. The polypeptide of claim 14, wherein the serum protein binding moiety is an ISV that binds serum albumin.

16. A nucleic acid or a nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 15, or a vector comprising said nucleic acid or nucleic acid sequence.

17. A host cell transformed or transfected with a nucleic acid or a nucleic acid sequence as defined in claim 16 or with a vector as defined in claim 16.

18. A method for producing a polypeptide according to any one of claims 1 to 15, said method comprising culturing a host cell as defined in claim 17 under conditions allowing expression of a polypeptide as defined in any one of claims 1 to 15 and recovering the produced polypeptide from the culture.

19. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 15 or a polypeptide produced according to the method of claim 18.

20. A polypeptide according to any one of claims 1 to 15 or a polypeptide produced according to the method of claim 18 for use in treating a subject in need thereof.

21. The polypeptide according to any one of claims 1 to 15 or the polypeptide produced according to the method of claim 18, for use in preventing, treating or ameliorating a disease selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases and autoimmune diseases.

22. A polypeptide for use according to claim 21, wherein the proliferative disease is cancer.

23. The polypeptide for use according to claim 22, wherein the cancer is selected from the group consisting of carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma: breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, Small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penis cancer, urethra cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma; and any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis.

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