CD3-responsive T-cell recruitment peptide

Abstract

A T-cell recruitment peptide that binds to CD3 on T cells is provided. This peptide can be used in methods for treating cancer.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680040586.1, filed on May 13, 2016, entitled "CD3-Responsive T-Cell Recruiting Polypeptide". Invention Field

[0002] This invention provides multispecific T cell recruitment peptides that bind to CD3 on T cells and at least one antigen on target cells. This invention also relates to monovalent T cell recruitment peptides used in these multispecific peptides. This invention also provides treatment methods and kits providing them. Background Technology

[0003] Cancer causes enormous loss of life worldwide. It is now the leading cause of death globally, 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 projected to rise by about 70% over the next 20 years (Source: WHO Cancer). In 2008, the total economic impact of premature cancer death and disability worldwide was approximately US$900 billion, representing 1.5% of global GDP.

[0004] Treatment options 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, particularly in advanced stages of cancer.

[0005] We eagerly await new treatments to combat cancer.

[0006] Antibody therapy is now an important part of doctors' medical devices in the fight against diseases, especially cancer. Monoclonal antibodies have been established as a key treatment for a range of diseases over the past few years. All antibody therapies approved at the same time rely on monospecific monoclonal antibodies (mAbs). Until today, most mAb targets required agonist or antagonist methods. While targeting cell surface antigens can mediate antitumor activity by inducing apoptosis, most mAb-based activities against hematologic malignancies rely on Fc-mediated effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] Immunotherapy has become a rapidly developing field in cancer research. It is targeting the body's immune surveillance system, particularly T cells, towards cancer cells.

[0008] Cytotoxic T cells (CTLs) are T lymphocytes that kill cancer cells, infected cells (especially virus-infected cells), or cells damaged in other ways. T lymphocytes (or T cells) express T cell receptors or TCR molecules and CD3 receptors on their cell surface. The αβTCR-CD3 complex (or “TCR complex”) consists of six distinct type I single-spanning transmembrane proteins: the TCRα and TCRβ chains that form the TCR heterodimer responsible for ligand recognition, and the non-covalently associated CD3γ, CD3δ, CD3ε, and ζ chains (which possess cytoplasmic sequence motifs that are phosphorylated upon receptor activation 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 constant and variable domains. Physiologically, the αβ chains of the T cell receptor recognize peptide-loaded MHC complexes and couple upon binding to CD3 chains. These CD3 chains then transduce the binding signal into the intracellular environment.

[0010] Given the potential of naturally occurring cytotoxic T lymphocytes (CTLs) to mediate cell lysis, various strategies have been explored to recruit CTLs to mediate tumor cell killing. Because T lymphocytes lack Fc receptor expression, they are not recruited to tumor sites via the Fc tail of anti-tumor monoclonal antibodies. As an alternative, the patient's T cells are modified with a second TCR that has known specificity for identified tumor antigens. This adoptive cell transfer is inherently highly personalized and labor-intensive. However, a major challenge with T-cell therapy remains the prevalence 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 and antibody therapy by using a polyclonal approach to engage all of a patient’s cytotoxic T cells via a “bispecific antibody” technology based on recombinant antibodies, rather than evoking a specific T cell response that depends on the presence, production, transport, and display of MHC molecules expressed by cancer cells and specific peptide antigens.

[0012] Bispecific antibodies have been engineered to have a tumor-recognizing portion on one arm (the target-binding arm), while the other arm of the molecule is specific for T-cell antigens (the effector-binding arm) (primarily CD3). By binding both arms simultaneously to their respective target antigens, T lymphocytes are directed to tumor cells and activated there, where they can perform their cytolytic function.

[0013] The concept of using bispecific antibodies to activate T cells targeting tumor cells was described more than two decades ago, but manufacturing problems and clinical failures brought the field to a standstill. Smaller formats of bispecific antibodies have been developed, which penetrate tissues and tumors more easily than conventional antibodies. Additionally, smaller formats are better at creating cytotoxic synapses (which kill target cells). Smaller formats of bispecific antibodies are thought to be easier to manufacture than conventional antibodies and have lower immunogenicity. However, smaller bispecific BiTE molecules, composed of two single-chain variable fragments (scFvs) linked by a 5-amino acid peptide linker, exhibit a lack of stability (scFvs tend to aggregate), low expression titers, and poor solubility. Furthermore, the first clinical trial of blamintumomab (a BiTE molecule) that recognizes the CD3 chain was prematurely halted due to adverse neurological events, cytokine release syndrome, and infection, and a lack of objective clinical response or strong indications of biological activity. Besides potency, BiTEs must be continuously infused—likely due to the lack of an Fc domain—which does not contribute to patient compliance. The same problem exists for DART (an amphiphilic retargeting molecule developed by MacroGenics), where a heavy variable domain from one antibody (Ab) is linked to a light variable domain from another Ab. MacroGenics is currently attempting to address this issue by fusing the Fc domain into its next-generation DART, which not only makes the molecule larger but also introduces manufacturing problems and inputs for other Fc functions. The larger Fc format will have better pharmacokinetic activity but reintroduces the risk of off-target activity. (Garber 2014, Nature reviews 13:799-801).

[0014] An alternative bispecific format is still needed. Invention Overview

[0015] This invention addresses this problem by providing a multispecific polypeptide comprising a first immunoglobulin single variable domain (ISV) and at least one additional immunoglobulin single variable domain, wherein the first ISV has a high affinity for / binds to CD3; and the at least one additional ISV has a high affinity for / binds to antigens present on target cells. In a particular aspect, binding of the first ISV activates the inherent cytotoxic potential of T cells against target cells (independent of MHC1).

[0016] Therefore, in a first aspect, the present invention provides a polypeptide comprising first and second immunoglobulin single variable domains (ISVs), wherein

[0017] - The first ISV has a high affinity for differentiation cluster 3 (CD3) present on T cells / binds to differentiation cluster 3 (CD3) present on T cells;

[0018] - The second ISV has a high affinity for the first antigen on the target cell / binds to the first antigen on the target cell;

[0019] Wherein the first antigen is different from CD3; and

[0020] The target cells therein are different from the T cells.

[0021] In another aspect, the present invention provides a polypeptide as described herein, wherein the polypeptide directs T cells to target cells.

[0022] In another aspect, the present invention provides a polypeptide as described herein, wherein the polypeptide induces T cell activation.

[0023] In another aspect, the present invention provides a polypeptide as described herein, wherein the T cell activation is independent of MHC recognition.

[0024] In another aspect, the present invention provides a polypeptide as described herein, wherein T cell activation depends on presenting the polypeptide, which binds to the first antigen on a target cell, to the T cell.

[0025] In another aspect, the present invention provides a polypeptide as described herein, wherein activation of the T cell elicits one or more cellular responses of the T cell, wherein the cellular responses are selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis.

[0026] In another aspect, the present invention provides a polypeptide as described herein, wherein activation of the T cell causes inhibition of the target cell activity 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%.

[0027] In another aspect, the present 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.

[0028] Alternatively, the present invention provides a polypeptide as described herein, wherein the first ISV binds to CD3γ (SEQ ID NO:379), CD3δ (SEQ ID NO:291) and / or CD3ε (SEQ ID NO:380) of the TCR complex, or a polymorph or isotype thereof.

[0029] In another aspect, the present invention provides a polypeptide as described herein, wherein the polypeptide and / or the first ISV has a binding rate constant (Kon) with respect to binding the 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, measurements can be taken using surface plasmon resonance.

[0030] In another aspect, the present invention provides a polypeptide as described herein, wherein the polypeptide and / or the first ISV pair binds to CD3 having a dissociation rate constant (Koff) selected from the group consisting of: up to about 10 -3 s -1 At most about 10 -4 s -1 At most about 10 -5 s -1 At most about 10 -6 s -1 At most about 10 -7 s -1 At most about 10 -8 s -1 At most about 10 -9 s -1 and at most about 10 -10 s -1Preferably, measurements can be taken using surface plasmon resonance.

[0031] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV binds to the CD3 with an EC50 value between 100 nM and 1 pM, such as an average EC50 value below 100 nM, even more preferably an average EC50 value below 90 nM, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even smaller, such as less than 4, 3, 2, 1 nM or even smaller, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even smaller, such as less than 4 pM, preferably as measured by flow cytometry.

[0032] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV binds to the CD3 with an average KD value between 100 nM and 10 pM, such as an average KD value below 90 nM, even more preferably an average KD value below 80 nM, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even smaller, 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 smaller, such as less than 10 pM. Preferably, the KD is determined by SPR, for example as determined by Proteon.

[0033] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0034] (i) CDR1 is selected from the following groups:

[0035] (a) SEQ ID NOs: 81-100; and

[0036] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid; and / or

[0037] (ii) CDR2 is selected from the following groups:

[0038] (c)SEQ ID NOs:101-122; and

[0039] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid; and / or

[0040] (iii) CDR3 is selected from the following groups:

[0041] (e)SEQ ID NOs:123-143; and

[0042] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:123 by 3, 2 or 1 amino acid.

[0043] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0044] (i) CDR1 is selected from the following groups:

[0045] (a) SEQ ID NOs:81-100; or (b) an amino acid sequence differing from the amino acid sequence of SEQ ID NO:81 or from any of SEQ ID NOs:81-100 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0046] (ii) CDR2 is selected from the following groups:

[0047] (c) SEQ ID NOs:101-122; or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:101 or any of SEQ ID NOs:101-122, provided that the polypeptide containing CDR2 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0048] (iii) CDR3 is selected from the following groups:

[0049] (e) SEQ ID NOs:123-143; or (f) an amino acid sequence having a difference of 4, 3, 2, or 1 amino acid from the amino acid sequence of SEQ ID NO:123 or any of SEQ ID NOs:123-143, provided that the polypeptide containing CDR3 having the difference of said 4, 3, 2, or 1 amino acid binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR3 without the difference of said 4, 3, 2, or 1 amino acid.

[0050] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0051] (a) SEQ ID NO:81; and

[0052] (b) An amino acid sequence that differs from SEQ ID NO:81 by one or two amino acids, wherein

[0053] -At position 1, G has changed to R;

[0054] -At position 3, T has changed to A;

[0055] -At position 4, Y has changed to F;

[0056] - At position 8, S has changed to G; and / or

[0057] - At position 10, G has changed to A.

[0058] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0059] (a) SEQ ID NO:101; and

[0060] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, or 3 amino acids, wherein

[0061] -At position 3, V has changed to T or A;

[0062] -At position 5, S has changed to T;

[0063] - At position 6, G has changed to D or E; and / or

[0064] - At position 9, T has changed to S, A, or P.

[0065] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0066] (a) SEQ ID NO:123; and

[0067] (b) An amino acid sequence that differs from SEQ ID NO:123 by one or two amino acids, wherein

[0068] -At position 2, I has changed to T;

[0069] - At position 9, I has changed to V; and / or

[0070] - At position 10, A has changed to P.

[0071] Preferably, a polypeptide containing one or more CDRs having said 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity than a polypeptide containing a CDR that does not have a difference of 3, 2, or 1 amino acid, said affinity as measured by surface plasmon resonance.

[0072] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0073] (i) CDR1 is selected from the following groups:

[0074] (a) SEQ ID NO:81; and

[0075] (b) An amino acid sequence that differs from SEQ ID NO:81 by one or two amino acids, wherein

[0076] -At position 1, G has changed to R;

[0077] -At position 3, T has changed to A;

[0078] -At position 4, Y has changed to F;

[0079] - At position 8, S has changed to G; and / or

[0080] - At position 10, G has changed to A.

[0081] The condition is that, compared to the binding of a peptide containing a CDR1 that does not have a difference of 2 or 1 amino acid, a peptide containing a CDR1 with said 2 or 1 amino acid difference binds to CD3 with approximately the same or higher affinity, such as that measured by surface plasmon resonance.

[0082] And among them

[0083] (ii) CDR2 is selected from the following groups:

[0084] (a) SEQ ID NO:101; and

[0085] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, or 3 amino acids, wherein

[0086] -At position 3, V has changed to T or A;

[0087] -At position 5, S has changed to T;

[0088] - At position 6, G has changed to D or E; and / or

[0089] -At position 9, T has changed to S, A, or P.

[0090] The condition is that, compared to the binding of a peptide containing a CDR2 that does not have a difference of 3, 2, or 1 amino acid, a peptide containing a CDR2 with said 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity, said affinity as measured by surface plasmon resonance.

[0091] And among them

[0092] (iii) CDR3 is selected from the following groups:

[0093] (a) SEQ ID NO:123; and

[0094] (b) An amino acid sequence that differs from SEQ ID NO:123 by one or two amino acids, wherein

[0095] -At position 2, I has changed to T;

[0096] - At position 9, I has changed to V; and / or

[0097] -At position 10, A has changed to P.

[0098] The condition is that, compared to the binding of a peptide containing a CDR3 that does not have a difference of 2 or 1 amino acid, a peptide containing a CDR3 with said 2 or 1 amino acid difference binds to CD3 with approximately the same or higher affinity, said affinity as measured by surface plasmon resonance.

[0099] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0100] (i) CDR1 is selected from the following groups:

[0101] (a) SEQ ID NOs: 81-87; and

[0102] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid; and / or

[0103] (ii) CDR2 is selected from the following groups:

[0104] (c)SEQ ID NOs:101-109; and

[0105] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid; and / or

[0106] (iii) CDR3 is selected from the following groups:

[0107] (e)SEQ ID NOs:123-127; and

[0108] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:123 by 4, 3, 2 or 1 amino acid.

[0109] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0110] (i) CDR1 is selected from the following groups:

[0111] (a) SEQ ID NOs: 81-87; and

[0112] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0113] (ii) CDR2 is selected from the following groups:

[0114] (c)SEQ ID NOs:101-109; and

[0115] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0116] (iii) CDR3 is selected from the following groups:

[0117] (e)SEQ ID NOs:123-127; and

[0118] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:123, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0119] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0120] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs:1-50.

[0121] In another aspect, the present invention provides polypeptides as described herein, wherein the first ISV cross-blocks binding to CD3 by at least one polypeptide having SEQ ID NOs: 1-50.

[0122] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO: 88.

[0124] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR2 is SEQ ID NO: 110.

[0125] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO: 128.

[0126] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0127] (i) CDR1 is selected from the following groups:

[0128] (a) SEQ ID NO:88; and

[0129] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:88 by 1, 2, 3, or 4 amino acids; and / or

[0130] (ii) CDR2 is selected from the following groups:

[0131] (c)SEQ ID NO:110; and

[0132] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:110 by 1, 2, 3, or 4 amino acids; and / or

[0133] (iii) CDR3 is selected from the following groups:

[0134] (e)SEQ ID NO:128; and

[0135] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:128 by 1, 2, 3 or 4 amino acids.

[0136] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0137] (i) CDR1 is selected from the following groups:

[0138] (a) SEQ ID NOs:88; and

[0139] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:88 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0140] (ii) CDR2 is selected from the following groups:

[0141] (c)SEQ ID NOs:110; and

[0142] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:110 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0143] (iii) CDR3 is selected from the following groups:

[0144] (e)SEQ ID NOs:128; and

[0145] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:128, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0146] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0147] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NOs:51.

[0148] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV cross-blocks binding to CD3 by a polypeptide having SEQ ID NOs: 51.

[0149] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO: 90.

[0151] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0152] (a) SEQ ID NO:112; and

[0153] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0154] - At position 2, V has changed to A.

[0155] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO: 130.

[0156] Preferably, a polypeptide containing one or more CDRs with a difference of one amino acid binds to CD3 with approximately the same or higher affinity than a polypeptide containing a CDR that does not have a difference of one amino acid, such affinity as measured by surface plasmon resonance.

[0157] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0158] (i)CDR1 is SEQ ID NO:90; and

[0159] And among them

[0160] (ii) CDR2 is selected from the following groups:

[0161] (a) SEQ ID NO:112; and

[0162] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0163] - At position 2, V has changed to A.

[0164] The condition is that, compared to the binding of a peptide containing a CDR2 that does not have a difference of 1 amino acid, a peptide containing a CDR2 that has a difference of 1 amino acid binds to CD3 with approximately the same or higher affinity, such affinity as measured by surface plasmon resonance.

[0165] And among them

[0166] (iii) CDR3 is SEQ ID NO:130.

[0167] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0168] (i) CDR1 is selected from the following groups:

[0169] (a) SEQ ID NO:90; and

[0170] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:90 by 1, 2, 3, or 4 amino acids; and / or

[0171] (ii) CDR2 is selected from the following groups:

[0172] (c)SEQ ID NOs:112-113; and

[0173] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:112 by 1, 2, 3, or 4 amino acids; and / or

[0174] (iii) CDR3 is selected from the following groups:

[0175] (e)SEQ ID NO:130; and

[0176] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:130 by 1, 2, 3 or 4 amino acids.

[0177] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0178] (i) CDR1 is selected from the following groups:

[0179] (a) SEQ ID NOs:90; and

[0180] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:90 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0181] (ii) CDR2 is selected from the following groups:

[0182] (c)SEQ ID NOs:112-113; and

[0183] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:112 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0184] (iii) CDR3 is selected from the following groups:

[0185] (e)SEQ ID NOs:130; and

[0186] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:130, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0187] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0188] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs: 53-56.

[0189] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV cross-blocks binding to CD3 by at least one polypeptide having SEQ ID NOs: 53-56.

[0190] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SEQ ID NO: 89.

[0192] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR2 is SEQ ID NO: 111.

[0193] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR3 is SEQ ID NO: 129.

[0194] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0195] (i) CDR1 is selected from the following groups:

[0196] (a)SEQ ID NO:89; and

[0197] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:89 by 1, 2, 3, or 4 amino acids; and / or

[0198] (ii) CDR2 is selected from the following groups:

[0199] (c)SEQ ID NO:111; and

[0200] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:111 by 1, 2, 3, or 4 amino acids; and / or

[0201] (iii) CDR3 is selected from the following groups:

[0202] (e)SEQ ID NO:129; and

[0203] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:129 by 1, 2, 3 or 4 amino acids.

[0204] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0205] (i) CDR1 is selected from the following groups:

[0206] (a) SEQ ID NOs:89; and

[0207] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:89 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0208] (ii) CDR2 is selected from the following groups:

[0209] (c)SEQ ID NOs:111; and

[0210] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:111 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0211] (iii) CDR3 is selected from the following groups:

[0212] (e)SEQ ID NOs:129; and

[0213] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:129, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0214] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0215] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NOs:52.

[0216] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV cross-blocks binding to CD3 by a polypeptide having SEQ ID NOs:52.

[0217] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0219] (a) SEQ ID NO: 91; and

[0220] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[0221] - At position 6, R has changed to N or T;

[0222] - At position 7, N has changed to H; and / or

[0223] - At position 8, M has changed to T.

[0224] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0225] (a) SEQ ID NO: 114; and

[0226] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[0227] - At position 1, R has changed to Q;

[0228] - At position 3, T has changed to S; and / or

[0229] - At position 7, D has changed to A or K.

[0230] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0231] (a) SEQ ID NO:131; and

[0232] (b) An amino acid sequence that differs from SEQ ID NO:131 by one amino acid, wherein

[0233] - At position 2, S has changed to R; and / or

[0234] - At position 6, S has changed to V.

[0235] Preferably, a polypeptide containing one or more CDRs having said 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity than a polypeptide containing a CDR that does not have a difference of 3, 2, or 1 amino acid, said affinity as measured by surface plasmon resonance.

[0236] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0237] (i) CDR1 is selected from the following groups:

[0238] (a) SEQ ID NO: 91; and

[0239] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[0240] - At position 6, R has changed to N or T;

[0241] - At position 7, N has changed to H; and / or

[0242] - At position 8, M has changed to T.

[0243] The condition is that, compared to the binding of a peptide containing a CDR1 that does not have a difference of 3, 2, or 1 amino acid, a peptide containing a CDR1 with said 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity, said affinity as measured by surface plasmon resonance.

[0244] And among them

[0245] (ii) CDR2 is selected from the following groups:

[0246] (a) SEQ ID NO: 114; and

[0247] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[0248] - At position 1, R has changed to Q;

[0249] - At position 3, T has changed to S; and / or

[0250] - At position 7, D has changed to A or K.

[0251] The condition is that, compared to the binding of a peptide containing a CDR2 that does not have a difference of 3, 2, or 1 amino acid, a peptide containing a CDR2 with said 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity, said affinity as measured by surface plasmon resonance.

[0252] And among them

[0253] (iii) CDR3 is selected from the following groups:

[0254] (a) SEQ ID NO:131; and

[0255] (b) An amino acid sequence that differs from SEQ ID NO:131 by one amino acid, wherein

[0256] - At position 2, S has changed to R; and / or

[0257] - At position 6, S has changed to V.

[0258] The condition is that, compared to the binding of a peptide containing a CDR3 that does not have a difference of 1 amino acid, a peptide containing a CDR3 with said 1 amino acid difference binds to CD3 with approximately the same or higher affinity, said affinity as measured by surface plasmon resonance.

[0259] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0260] (i) CDR1 is selected from the following groups:

[0261] (a) SEQ ID NOs: 91-93; and

[0262] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:91 by 4, 3, 2, or 1 amino acid; and / or

[0263] (ii) CDR2 is selected from the following groups:

[0264] (c)SEQ ID NOs:114-117; and

[0265] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:114 by 4, 3, 2, or 1 amino acid; and / or

[0266] (iii) CDR3 is selected from the following groups:

[0267] (e)SEQ ID NOs:131-133; and

[0268] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:131 by 4, 3, 2 or 1 amino acid.

[0269] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0270] (i) CDR1 is selected from the following groups:

[0271] (a) SEQ ID NOs: 91-93; and

[0272] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:91 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0273] (ii) CDR2 is selected from the following groups:

[0274] (c)SEQ ID NOs:114-117; and

[0275] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:114 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0276] (iii) CDR3 is selected from the following groups:

[0277] (e)SEQ ID NOs:131-133; and

[0278] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:131, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0279] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0280] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs:57-65.

[0281] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV cross-blocks binding to CD3 by at least one polypeptide having SEQ ID NOs: 57-65.

[0282] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0284] (a) SEQ ID NO:94; and

[0285] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[0286] -At position 3, S has changed to T, A, or G;

[0287] -At position 5, N has changed to S;

[0288] - At position 6, M has changed to T or A; and / or

[0289] - At position 9, L has changed to M.

[0290] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0291] (a) SEQ ID NO: 118; and

[0292] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, or 3 amino acids, wherein

[0293] -At position 2, H has changed to V;

[0294] - At position 5, S has changed to H or A;

[0295] - At position 8, N has changed to S; and / or

[0296] - At position 10, Y has changed to F.

[0297] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0298] (a) SEQ ID NO: 134; and

[0299] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, 4, or 5 amino acids, wherein

[0300] -At position 6, A has changed to S or D;

[0301] - At position 7, F has changed to Y or A;

[0302] - At position 8, R has changed to H;

[0303] -At position 9, S has changed to A;

[0304] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[0305] - At position 14, V has changed to I.

[0306] Preferably, a polypeptide containing one or more CDRs having said 5, 4, 3, 2 or 1 amino acid difference binds to CD3 with approximately the same or higher affinity than a polypeptide containing a CDR that does not have a difference of 5, 4, 3, 2 or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0307] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein

[0308] (i) CDR1 is selected from the following groups:

[0309] (a) SEQ ID NO:94; and

[0310] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[0311] -At position 3, S has changed to T, A, or G;

[0312] -At position 5, N has changed to S;

[0313] - At position 6, M has changed to T or A; and / or

[0314] - At position 9, L has changed to M.

[0315] The condition is that, compared to the binding of a peptide containing CDR1 with a difference of 4, 3, 2, or 1 amino acid, a peptide containing CDR1 with a difference of 4, 3, 2, or 1 amino acid binds to CD3 with approximately the same or higher affinity, as measured by surface plasmon resonance.

[0316] And among them

[0317] (ii) CDR2 is selected from the following groups:

[0318] (a) SEQ ID NO: 118; and

[0319] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, or 3 amino acids, wherein

[0320] -At position 2, H has changed to V;

[0321] - At position 5, S has changed to H or A;

[0322] - At position 8, N has changed to S; and / or

[0323] - At position 10, Y has changed to F.

[0324] The condition is that, compared to the binding of a peptide containing a CDR2 that does not have a 3, 2, or 1 amino acid difference, a peptide containing a CDR2 that has ...

[0325] And among them

[0326] (iii) CDR3 is selected from the following groups:

[0327] (a) SEQ ID NO: 134; and

[0328] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, 4, or 5 amino acids, wherein

[0329] -At position 6, A has changed to S or D;

[0330] - At position 7, F has changed to Y or A;

[0331] - At position 8, R has changed to H;

[0332] -At position 9, S has changed to A;

[0333] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[0334] - At position 14, V has changed to I.

[0335] The condition is that, compared to the binding of a peptide containing a CDR3 with a difference of 5, 4, 3, 2, or 1 amino acid, a peptide containing a CDR3 with a difference of 5, 4, 3, 2, or 1 amino acid binds to CD3 with approximately the same or higher affinity, such affinity as measured by surface plasmon resonance.

[0336] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0337] (i) CDR1 is selected from the following groups:

[0338] (a) SEQ ID NOs: 94-100; and

[0339] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:94 by 4, 3, 2, or 1 amino acid; and / or

[0340] (ii) CDR2 is selected from the following groups:

[0341] (c)SEQ ID NOs:118-122; and

[0342] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:118 by 4, 3, 2, or 1 amino acid; and / or

[0343] (iii) CDR3 is selected from the following groups:

[0344] (e)SEQ ID NOs:134-143; and

[0345] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:134 by 4, 3, 2 or 1 amino acid.

[0346] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0347] (i) CDR1 is selected from the following groups:

[0348] (a) SEQ ID NOs: 94-100; and

[0349] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:94 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0350] (ii) CDR2 is selected from the following groups:

[0351] (c)SEQ ID NOs:118-122; and

[0352] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:118 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0353] (iii) CDR3 is selected from the following groups:

[0354] (e)SEQ ID NOs:134-143; and

[0355] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:134, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0356] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0357] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is selected from the group consisting of SEQ ID NOs:66-80.

[0358] In another aspect, the present invention provides peptides as described herein, wherein the first ISV cross-blocks binding to CD3 by at least one peptide having SEQ ID NOs: 66-80.

[0359] In another aspect, the present 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 another aspect, the present invention provides a polypeptide as described herein, wherein the first antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0361] In another aspect, the present invention provides a polypeptide as described herein, which further comprises a third ISV having a high affinity for and / or binding to a second antigen on a target cell, wherein the second antigen is different from the first antigen.

[0362] In another aspect, the present invention provides a polypeptide as described herein, wherein the second antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0363] In another aspect, the present invention provides a polypeptide as described herein, wherein the first antigen and the second antigen are present on the same target cells.

[0364] In another aspect, the present invention provides a polypeptide as described herein, wherein the first antigen and the second antigen are present on different target cells.

[0365] 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 γ chain (CD132 antigen), interleukin-10 receptor α chain (IL-10R-A), interleukin-10 receptor β 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), type I interleukin-1 receptor (IL-1R-1) (CD121a), type II interleukin-1 receptor (IL-1R-β) (CDw121b), leukocytes 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, 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 associated polymorphs and isotypes.

[0366] In another aspect, the present invention provides a polypeptide as described herein, wherein the TAA is CD20 (UniProt11836), HER2 (Uniprot P04626), a polymorph or isotype thereof.

[0367] In another aspect, the present invention provides a polypeptide as described herein, wherein the first antigen and the second antigen are selected from the group consisting of:

[0368] -EGFR is used as the primary antigen and CEA is used as the secondary antigen;

[0369] -CD19 is used as the first antigen and CD20 is used as the second antigen;

[0370] -CD19 is used as the first antigen and CD22 is used as the second antigen;

[0371] -CD123 is used as the primary antigen and Tim-3 is used as the secondary antigen; and

[0372] -CD132 is used as the first antigen and CD69 is used as the second antigen.

[0373] In another aspect, the present invention provides a polypeptide as described herein, which further comprises a serum protein binding portion.

[0374] In another aspect, the present invention provides a polypeptide as described herein, wherein the serum protein-binding portion binds to serum albumin.

[0375] In another aspect, the present invention provides a polypeptide as described herein, wherein the serum protein binding portion is an ISV that binds to serum albumin.

[0376] In another aspect, the present invention provides a polypeptide as described herein, wherein the serum albumin-binding ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SIGSGSDTLYADSVKG (SEQ ID NO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), and the CDRs are determined according to the Kabat definition; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SIGSGSSDTL (SEQ ID NO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), and the CDRs are determined according to Kontermann 2010.

[0377] In another aspect, the present invention provides a polypeptide as described herein, wherein the ISV that binds to 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).

[0378] In another aspect, the present invention provides polypeptides as described herein, wherein the ISVs are directly connected to each other or connected via a connector.

[0379] In another aspect, the present invention provides a polypeptide as described herein, wherein the first ISV and / or the second ISV and / or possibly the third ISV and / or possibly the serum albumin-binding ISV are connected via a connector.

[0380] In another aspect, the present invention provides a polypeptide as described herein, wherein the adapter is selected from the group consisting of adapters of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 362 to 372).

[0381] In another aspect, the present invention provides polypeptides as described herein, wherein the serum protein binding portion is a non-antibody-based polypeptide.

[0382] In another aspect, the present invention provides a polypeptide as described herein, which further comprises PEG.

[0383] In another aspect, the present invention provides a polypeptide as described herein, wherein the ISV is V HH Humanized V HH or camel-derived V H .

[0384] In another aspect, the present invention provides a polypeptide, wherein the first ISV is selected from the group consisting of SEQ ID NOs:1 to 80.

[0385] In another aspect, the present 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.

[0386] In another aspect, the present invention provides polypeptides 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.

[0387] In another aspect, the present invention provides a polypeptide that specifically binds to CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0388] (i) CDR1 is selected from the following groups:

[0389] (a) SEQ ID NOs: 81-100; or

[0390] (b) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from any of SEQ ID NOs:81-100, provided that the polypeptide containing CDR1 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0391] (ii) CDR2 is selected from the following groups:

[0392] (c)SEQ ID NOs:101-122; or

[0393] (d) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from any of SEQ ID NOs:101-122, provided that the peptide containing CDR2 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to binding to a peptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0394] (iii) CDR3 is selected from the following groups:

[0395] (e)SEQ ID NOs:123-143; or

[0396] (f) An amino acid sequence having a difference of 4, 3, 2 or 1 amino acid from any of SEQ ID NOs:123-143, provided that the polypeptide containing CDR3 having said 4, 3, 2 or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR3 without said 4, 3, 2 or 1 amino acid difference.

[0397] This invention also provides polypeptides as described herein, wherein:

[0398] (i) CDR1 is selected from the following groups:

[0399] (a) SEQ ID NOs: 81-87; and

[0400] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0401] (ii) CDR2 is selected from the following groups:

[0402] (c)SEQ ID NOs:101-109; and

[0403] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0404] (iii) CDR3 is selected from the following groups:

[0405] (e)SEQ ID NOs:123-127; and

[0406] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:123, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0407] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0408] (a) SEQ ID NO:81; and

[0409] (b) An amino acid sequence that differs from SEQ ID NO:81 by one or two amino acids, wherein

[0410] -At position 1, G has changed to R;

[0411] -At position 3, T has changed to A;

[0412] -At position 4, Y has changed to F;

[0413] - At position 8, S has changed to G; and / or

[0414] - At position 10, G has changed to A.

[0415] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0416] (a) SEQ ID NO:101; and

[0417] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, or 3 amino acids, wherein

[0418] -At position 3, V has changed to T or A;

[0419] -At position 5, S has changed to T;

[0420] - At position 6, G has changed to D or E; and / or

[0421] - At position 9, T has changed to S, A, or P.

[0422] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0423] (a) SEQ ID NO:123; and

[0424] (b) An amino acid sequence that differs from SEQ ID NO:123 by one or two amino acids, wherein

[0425] -At position 2, I has changed to T;

[0426] - At position 9, I has changed to V; and / or

[0427] - At position 10, A has changed to P.

[0428] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:81, CDR2 by SEQ ID NO:101, and CDR3 by SEQ ID NO:123.

[0429] In another aspect, the present invention provides a polypeptide as described herein, wherein:

[0430] (i) CDR1 is selected from the following groups:

[0431] (a) SEQ ID NOs:88; and

[0432] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:88 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0433] (ii) CDR2 is selected from the following groups:

[0434] (c)SEQ ID NOs:110; and

[0435] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:110 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0436] (iii) CDR3 is selected from the following groups:

[0437] (e)SEQ ID NOs:128; and

[0438] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:128, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0439] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:88.

[0440] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR2 is SEQ ID NO:110.

[0441] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:128.

[0442] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:88, CDR2 by SEQ ID NO:110, and CDR3 by SEQ ID NO:128.

[0443] In another aspect, the present invention provides a polypeptide as described herein, wherein:

[0444] (i) CDR1 is selected from the following groups:

[0445] (a) SEQ ID NO:90; and

[0446] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:90 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0447] (ii) CDR2 is selected from the following groups:

[0448] (c)SEQ ID NOs:112-113; and

[0449] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:112 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0450] (iii) CDR3 is selected from the following groups:

[0451] (e)SEQ ID NO:130; and

[0452] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:130, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0453] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:90.

[0454] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR2 is selected from the group consisting of:

[0455] (a) SEQ ID NO:112; and

[0456] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0457] - At position 2, V has changed to A.

[0458] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:130.

[0459] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:90, CDR2 by SEQ ID NO:112, and CDR3 by SEQ ID NO:130.

[0460] In another aspect, the present invention provides a polypeptide as described herein, wherein:

[0461] (i) CDR1 is selected from the following groups:

[0462] (a) SEQ ID NOs:89; and

[0463] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:89 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0464] (ii) CDR2 is selected from the following groups:

[0465] (c)SEQ ID NOs:111; and

[0466] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:111 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0467] (iii) CDR3 is selected from the following groups:

[0468] (e)SEQ ID NOs:129; and

[0469] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:129, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0470] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is SEQ ID NO:89.

[0471] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR2 is SEQ ID NO:111.

[0472] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR3 is SEQ ID NO:129.

[0473] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:89, CDR2 by SEQ ID NO:111, and CDR3 by SEQ ID NO:129.

[0474] This invention also provides polypeptides as described herein, wherein:

[0475] (i) CDR1 is selected from the following groups:

[0476] (a) SEQ ID NOs: 91-93; and

[0477] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:91 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0478] (ii) CDR2 is selected from the following groups:

[0479] (c)SEQ ID NOs:114-117; and

[0480] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:114 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0481] (iii) CDR3 is selected from the following groups:

[0482] (e)SEQ ID NOs:131-133; and

[0483] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:131, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0484] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0485] (a) SEQ ID NO: 91; and

[0486] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[0487] - At position 6, R has changed to N or T;

[0488] - At position 7, N has changed to H; and / or

[0489] - At position 8, M has changed to T.

[0490] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0491] (a) SEQ ID NO: 114; and

[0492] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[0493] - At position 1, R has changed to Q;

[0494] - At position 3, T has changed to S; and / or

[0495] - At position 7, D has changed to A or K.

[0496] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0497] (a) SEQ ID NO:131; and

[0498] (b) An amino acid sequence that differs from SEQ ID NO:131 by one amino acid, wherein

[0499] - At position 2, S has changed to R; and / or

[0500] - At position 6, S has changed to V.

[0501] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:91, CDR2 by SEQ ID NO:114, and CDR3 by SEQ ID NO:131.

[0502] This invention also provides polypeptides as described herein, wherein:

[0503] (i) CDR1 is selected from the following groups:

[0504] (a) SEQ ID NOs: 94-100; and

[0505] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:94 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0506] (ii) CDR2 is selected from the following groups:

[0507] (c)SEQ ID NOs:118-122; and

[0508] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:118 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0509] (iii) CDR3 is selected from the following groups:

[0510] (e)SEQ ID NOs:134-143; and

[0511] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:134, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0512] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is selected from the group consisting of:

[0513] (a) SEQ ID NO:94; and

[0514] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[0515] -At position 3, S has changed to T, A, or G;

[0516] -At position 5, N has changed to S;

[0517] - At position 6, M has changed to T or A; and / or

[0518] - At position 9, L has changed to M.

[0519] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR2 is selected from the group consisting of:

[0520] (a) SEQ ID NO: 118; and

[0521] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, or 3 amino acids, wherein

[0522] -At position 2, H has changed to V;

[0523] - At position 5, S has changed to H or A;

[0524] - At position 8, N has changed to S; and / or

[0525] - At position 10, Y has changed to F.

[0526] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is selected from the group consisting of:

[0527] (a) SEQ ID NO: 134; and

[0528] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, 4, or 5 amino acids, wherein

[0529] -At position 6, A has changed to S or D;

[0530] - At position 7, F has changed to Y or A;

[0531] - At position 8, R has changed to H;

[0532] -At position 9, S has changed to A;

[0533] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[0534] - At position 14, V has changed to I.

[0535] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:94, CDR2 by SEQ ID NO:118, and CDR3 by SEQ ID NO:134.

[0536] In another aspect, the present invention provides polypeptides as described herein, which are nanobodies, V...HH Humanized V HH or camel-derived V H .

[0537] In another aspect, the present invention provides a polypeptide as described herein, which further comprises a serum protein binding portion.

[0538] In another aspect, the present invention provides a polypeptide as described herein, wherein the serum protein-binding portion binds to serum albumin.

[0539] In another aspect, the present invention provides a polypeptide as described herein, wherein the serum protein binding portion is an ISV that binds to serum albumin.

[0540] In another aspect, the present invention provides a polypeptide as described herein, wherein the ISV binding to serum albumin is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SIGSGSDTLYADSVKG (SEQ ID NO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), CDRs as defined by Kabat; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SIGSGSSDTL (SEQ ID NO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), CDRs as defined by Kontermann 2010.

[0541] In another aspect, the present invention provides a polypeptide as described herein, wherein the ISV binding to 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).

[0542] In another aspect, the present invention provides polypeptides as described herein, wherein the ISVs are directly connected or connected via a connector.

[0543] In another aspect, the present invention provides a polypeptide as described herein, wherein the adapter is selected from the group consisting of adapters of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 362 to 372).

[0544] In another aspect, the present invention provides a polypeptide as described herein, which further comprises a PEG moiety.

[0545] In another aspect, the present invention provides nucleic acids or nucleic acid sequences encoding polypeptides as defined herein.

[0546] In another aspect, the present invention provides vectors comprising nucleic acids or nucleic acid sequences as defined herein.

[0547] In another aspect, the present invention provides a host cell that is transformed or transfected with a nucleic acid or nucleic acid sequence as defined herein or with a vector as defined herein.

[0548] In another aspect, the present invention provides a method for producing a polypeptide as defined herein, the method comprising culturing a host cell as defined herein under conditions that allow expression of a polypeptide as defined herein and recovering the produced polypeptide from the culture.

[0549] In another aspect, the present invention provides pharmaceutical compositions comprising a polypeptide as described herein, or a polypeptide produced according to the methods described herein.

[0550] In another aspect, the present invention provides polypeptides as described herein, or polypeptides produced as described herein, for use in treating subjects in need.

[0551] In another aspect, the present invention provides a method for delivering a preventive or therapeutic peptide to a specific location, tissue, or cell type in the body, the method comprising the step of administering a peptide as described herein or a peptide produced as described herein to a subject.

[0552] In another aspect, the present invention provides polypeptides as described herein, or polypeptides produced as described herein, for the prevention, treatment, or improvement of diseases selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases, and autoimmune diseases.

[0553] In another aspect, the present invention provides a method for preventing, treating, or improving 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.

[0554] In another aspect, the present invention provides polypeptides or methods for preventing, treating or improving diseases as described herein, wherein the proliferative disease is cancer.

[0555] In another aspect, the present invention provides polypeptides or methods for preventing, treating, or improving diseases 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 myelomas (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 bowel cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine carcinoma, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary exudative lymphoma, neuroectodermal tumors, rhabdomyosarcoma; and any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis.

[0556] In another aspect, the present invention provides peptides or methods for preventing, treating or improving diseases as described herein, wherein the treatment is a combination therapy.

[0557] In another aspect, the present invention provides a kit comprising a polypeptide as defined herein, a nucleic acid or nucleic acid sequence as defined herein, a vector as defined herein, or a host cell as defined herein. Attached Figure Description

[0558] Figure 1 ： QC of human TCR / CD3 and human CD3 transfected cell lines, using 100 nM anti-human TCRα / β antibody (clone BW242 / 412) (black) and 100 nM anti-human CD3 antibody (clone OKT-3) (gray). MCF values ​​(mean channel fluorescence) for each cell line are plotted.

[0559] Figure 2 A and 2B: The dose-dependent binding of monovalent CD3 nanobodies to human TCR / CD3 expressed on CHO-K1 cells ( Figure 2 A) and dose-dependent binding with purified primary human T cells ( Figure 2 B). Plot the MCF (mean channel fluorescence) values ​​for nanobody concentrations.

[0560] Figure 3 ：Dose-dependent binding of monovalent CD3 nanobodies to HEK293H human TCR (2IAL) / CD3 (solid circles), HEK293H human CD3 (cross), and HEK293H parental cell line (hollow circles) was demonstrated. MCF values ​​(mean channel fluorescence) were plotted for nanobody concentrations.

[0561] Figure 4 A and 4B: T cell activation data of bead-coupled monovalent CD3 nanobodies ( Figure 4 A). T cell activation data presented in solution form of monovalent CD3 nanobodies ( Figure 4 B). Activation was measured by monitoring the upregulation of CD69 in primary human T cells. The MCF value (mean channel fluorescence) of each nanobody was plotted.

[0562] Figure 5 A-5C: A series of dilutions of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies compared with human TCR / CD3 expressed on CHO-K1 cells. Figure 5 A), primary human T cells ( Figure 5 B) and Ramos cells ( Figure 5 C) binding. MCF values ​​(mean channel fluorescence) were plotted for nanobody concentrations.

[0563] Figure 6 A and 6B: In flow cytometry-based human T cell-mediated Ramos ( Figure 6 A) and Raji( Figure 6 B) The dose-dependent killing effect of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies in the B cell killing assay. % cell death (% of TOPRO-positive cells) is plotted against nanobodies concentration.

[0564] Figure 7 ： Dose-dependent binding of anti-CD20 nanobodies to human CD20 Ramos (hollow symbol) and Raji (solid symbol) cells. MCF values ​​(mean channel fluorescence) were plotted against nanobodies concentration.

[0565] Figure 8 ： The dose-dependent killing effect of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies was demonstrated in a xCELLigence-based human T cell-mediated CHO-K1 human CD20 killing assay. CI was plotted against nanobody concentration.

[0566] Figure 9 ：The cytotoxic effects of 1 μM CD20xCD3 and unrelated constructs in a xCELLigence-based killing assay used with CHO-K1 human CD20 cells (black bars) and with the CHO-K1 parental cell line (gray bars) to elucidate TAA-dependent killing. Cell index (CI) was plotted against nanobody concentrations.

[0567] Figure 10 ： The dose-dependent killing effects of CD20xCD3 nanobodies with 9GS linkers (hollow circles – dashed lines) and 35GS linkers (solid squares – dashed lines) and CD3xCD20 nanobodies with 35GS linkers (solid rhombuses – solid lines) were measured using a flow cytometry-based killing assay conducted by Ramos. % cell death (TORPRO-positive cells) was plotted against nanobody concentration.

[0568] Figure 11 ： Dose-dependent killing of T017000062 was demonstrated in a flow cytometry-based assay of human T cell-mediated Ramos B cell killing using different effector (E):T ratios (E:T ratio 10:1 – solid circles, E:T ratio 5:1 – hollow squares, E:T ratio 2:1 – solid triangles, and E:T ratio 1:1 – hollow rhombuses). % cell death (% of TOPRO-positive cells) was plotted against nanobody concentration.

[0569] Figure 12 ： Time-dependent cytolytic activity of CD20 / CD3 in a purified primary human T cell-mediated killing assay using CHO-K1 human CD20 target cells in xCELLigence. %-specific lysis was plotted against construct concentration. Different curves represent the analysis time after T cell addition.

[0570] Figure 13 A-13C: Serial dilutions of the HLE construct and human TCR / CD3 expressed on CHO-K1 cells ( Figure 13 A), primary human T cells ( Figure 13 B) and Ramos cells ( Figure 13 C) binding. MCF values ​​(mean channel fluorescence) were plotted for nanobody concentrations.

[0571] Figure 14 A-14D: In a flow cytometry-based human T cell-mediated Ramos B cell killing assay, the CD20xCD3 bispecific nanobody (solid line – rhombus) showed dose-dependent killing compared to the CD20xCD3xALB11 construct (solid line – solid triangle). Figure 14 A, Figure 14C) and the dose-dependent killing effect of the CD20xCD3xALB11 construct in the absence of (solid line – solid triangle) or in the presence of (dashed line – hollow triangle) 30 μM HSA. Figure 14 B, Figure 14 D). Plotting % cell death (% of TOPRO-positive cells) at nanobody concentrations.

[0572] Figure 15 ： 100 nM monovalent anti-HER2 nanobody (5F07) was bound to SK-BR-3, MCF-7, and MDA-MB-468 cell lines in flow cytometry to compare HER2 expression levels. MCF values ​​(mean channel fluorescence) were plotted for each cell line.

[0573] Figure 16 ： The dose-dependent killing effects of bispecific CD3xHER2 nanobodies (dashed line) and bispecific HER2xCD3 (solid line) were demonstrated in a xCELLigence-based human T cell-mediated cell killing assay. Data were analyzed at 18 hours. Cell indices (CIs) were plotted against nanobodies concentration.

[0574] Figure 17 ： In a flow cytometry-based kill assay, human CD20-positive CHO-K1 cells were incubated with a bispecific CD20xCD3 nanobody, and INF-γ was generated via dose-dependent incubation by human T cells. Data were analyzed after 72 hours of incubation. OD at 405 nm was plotted against the nanobody concentration.

[0575] Figure 18 ： The study designed a PBMC B cell depletion model. PBMCs were injected intraperitoneally into the animals on day 3 (D3). From D3 to D7, mice were treated with either T017000084 (CD3 / CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (unrelated nanobody).

[0576] Figure 19 ： Absolute PBMC-derived B cells were counted in orders of magnitude. Results were plotted for each animal. B cell counts were shown for different treatment groups.

[0577] Figure 20 ： The study designed a Ramos model. Ramos cells were intravenously injected into mice on day 1. PBMCs were intraperitoneally injected into the animals on day 3. From day 3 to day 7, mice were treated with either T017000084(CD3 / CD20)IV Q1Dx5 or T017000088IVQ1Dx5 (unrelated nanobodies).

[0578] Figure 21 ：Absolute Ramos B cell counts were performed on an order of magnitude. Individual animal results were plotted. Hollow circles above the graphs indicate that, based on the F-test from the mixed-effects ANOVA analysis, the active dose was statistically significantly different from the unrelated NB (T017000088). All effects were statistically significant at the 5% significance level.

[0579] Figure 22 ： Absolute PBMC-derived B cells were counted in the order of magnitude. Individual animal results were plotted. Hollow circles above the graphs indicate that, based on the F-test from the mixed-effects ANOVA analysis, the active dose was statistically significantly different from the unrelated NB (T017000088). All effects were statistically significant at the 5% significance level.

[0580] Figure 23 A and 23B: Identifying EGFR in flow cytometry Figure 23 A; Santa Cruz, SC-120PE) or CEACAM5 ( Figure 23 Expression levels of B (Sino Biological, 11077-MM02-P) in HER14, HeLa, LoVo, and LS174-T cell lines. MCF values ​​(mean channel fluorescence) for each cell line are plotted. Invention Details

[0581] The inventors recognize that a formulation for combining T cells and tumor cells to induce an immune response should meet a wide variety of, and often contradictory, requirements. The formulation should be broadly applicable. Specifically, the formulation should preferably be used for a wide range of patients and, more preferably, for a wide range of tumors. The formulation should preferably be safe and target only the intended cells. Furthermore, the formulation should preferably be small enough to easily penetrate tissues and tumors, while on the other hand, the formulation should be patient-friendly. For example, the formulation should have a prolonged half-life such that it is not immediately eliminated by renal clearance after administration. However, a prolonged half-life should preferably not introduce off-target activity and side effects or limit penetration into tissues and tumors. Furthermore, it is recognized that tumor cells often develop evasion mechanisms through downregulation of the target antigen within the treatment. Therefore, in another preferred form, the formulation should simultaneously target multiple antigens.

[0582] The present invention fulfills at least one of these requirements.

[0583] Specifically, predicting immunoglobulin single variable domains (ISVs) would be ideal candidates in principle, as they are small enough to easily penetrate (tumor) tissue and can combine with other ISVs to form structural units. Secondly, ISVs targeting CD3, particularly CD3ε, should have broad applicability.

[0584] Six clusters of relevant ISVs were identified, possessing an unexpectedly wide range of advantageous features. First, the ISVs are unexpectedly broadly applicable; specifically, CD3 ISVs can bind with high affinity to T cells from various donors. Formatted in multispecific peptides, CD3 ISVs enable tumor cell killing against different tumor-associated antigens. Therefore, CD3 ISVs can be used against a wide range of cancers. Furthermore, multispecific peptides containing CD3 ISVs retain their activity when bound to albumin. This contributes to good pharmacokinetic properties and patient compliance while minimizing side effects. The peptides of the present invention only exhibit their effect when bound to both T cells and target cells, indicating their safety.

[0585] The inventors believe that simultaneously targeting multiple antigens reduces the likelihood of generating tumor escape variants, thus improving the therapeutic activity of T-cell conjugation strategies. A multispecific polypeptide is provided, comprising a CD3 ISV combined with a single variable domain of immunoglobulin targeting different epitopes (double complementary sites) on different target antigens and / or specific antigens.

[0586] Immunoglobulin sequences, such as antibodies and antigen-binding fragments derived therefrom (e.g., immunoglobulin single variable domains or ISVs), are used to specifically target their respective antigens in research and therapeutic applications. The generation of immunoglobulin single variable domains (such as VHHs or nanobodies) can involve immunization of laboratory animals (such as llamas), construction of phage libraries from immune tissues, selection of phages displaying antigen-binding immunoglobulin single variable domains, and screening for said domains and their engineered constructs for desired specificity (WO 94 / 04678). Alternatively, similar immunoglobulin monovariable domains (such as dAbs) can be generated by directly selecting phages displaying antigen-binding immunoglobulin monovariable domains from natural or synthetic libraries and subsequently screening for said domains and their engineered constructs for 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 results in high manufacturing costs and may lead to suboptimal tumor penetration compared to other strategies.

[0587] This invention provides a multispecific polypeptide that specifically binds to CD3 of the T-cell receptor complex, possessing an unexpectedly wide range of advantageous features. First, the polypeptide is easy to manufacture. Furthermore, the ISV is unexpectedly broadly applicable; that is, CD3ISV can bind to T cells from various donors with high affinity. Formatted in the multispecific polypeptide, the CD3 ISV enables the killing of tumor cells with different tumor-associated antigens. Conversely, no killing is observed when the polypeptide does not bind to T cells and target cells, highlighting the safety of the polypeptide of this invention. Therefore, CD3 ISV can be used against a wide range of cancers. Furthermore, CD3 ISV can be considered safe. Additionally, the multispecific polypeptide containing CD3 ISV retains its activity when bound to albumin. This contributes to good pharmacokinetic properties and patient compliance while minimizing side effects.

[0588] Therefore, the present invention relates to polypeptides comprising first and second immunoglobulin single variable domains (ISVs), 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 to the target cell (such as, for example, a tumor-associated antigen (TAA)). The multispecific polypeptide of the present invention directs T cells to cells (e.g., tumor cells) and induces T cell activation, thereby allowing the T cells to inhibit or kill the target cell (e.g., the tumor cell).

[0589] definition:

[0590] a) Unless otherwise stated or defined, all terms used have their common meaning in the art, which is clear to those skilled in the art. See, for example, the standard manual mentioned in paragraph a) on page 46 of WO 08 / 020079.

[0591] (b) The term "immunoglobulin single variable domain" is used interchangeably with "single variable domain" and "ISV" to refer to a molecule in which the antigen-binding site is located within a single immunoglobulin domain and is formed by that single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from "conventional" immunoglobulins or fragments thereof (such as Fab, scFv, etc.), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the formation of the antigen-binding site; that is, a total of six CDRs are involved 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.

[0592] The terms “immunoglobulin single variable domain,” “single variable domain,” and “ISV” therefore do not include conventional immunoglobulins or fragments thereof that 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.

[0593] The term "immunoglobulin single variable domain" or "ISV" includes (but is not limited to) antigen-binding domains or fragments, such as V HH Domain or V H or V L Domain. The terms antigen-binding molecule or antigen-binding protein may be used interchangeably, and the term nanobody is also included. An immunoglobulin single variable domain can be a light chain variable domain sequence (e.g., V... L -sequence), or heavy-chain variable domain sequence (e.g., V) H -sequence); more specifically, they can be heavy chain variable domain sequences derived from conventional four-chain antibodies or heavy chain variable domain sequences derived from heavy chain antibodies. Accordingly, an immunoglobulin single variable domain can 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 HHThe invention includes immunoglobulin sequences from various sources, including those from mice, rats, rabbits, donkeys, humans, and camels. The immunoglobulin single variable domain includes wholly human, humanized, other sequence-optimized, or chimeric immunoglobulin sequences. The structure of the immunoglobulin single variable domain can be considered—but is not limited to—consisting of four framework regions or “FRs,” referred to in the art and herein as “framework region 1” or “FR1”; “framework region 2” or “FR2”; “framework region 3” or “FR3”; and “framework region 4” or “FR4”, respectively. The framework regions are separated by three complementarity-determining regions or “CDRs,” 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”. It should be noted that the term nanobody (or nanobodies) is a registered trademark of Ablynx NV and is therefore also referred to as nanobody.

[0594] c) Unless otherwise stated, the terms “immunoglobulin sequence,” “sequence,” “nucleotide sequence,” and “nucleic acid” are as described in paragraph b) on page 46 of WO08 / 020079.

[0595] d) Unless otherwise stated, all methods, steps, techniques and operations not specifically described in detail may be performed or have been performed in a manner known per se, as will be clear to those skilled in the art. For example, refer again to the standard manuals and general background techniques mentioned herein and other references cited therein; and to reviews such as Presta 2006 (Adv. Drug Deliv. Rev. 58(5-6): 640-656), Levin and Weiss 2006 (Mol. Biosyst. 2(1): 49-57), Irving et al. 2005 (J. Immunol. Methods 248(1-2): 31-45), Schmitz et al. 2000 (Placenta 21 Supplement A: S106-112), Gonzales et al. 2005 (Tumour Biol. 26(1): 31-43), which describe protein engineering techniques, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0596] e) Amino acid residues are represented using standard three-letter or single-letter amino acid codes. See Table A-2 on page 48 of Ablynx NV's international application WO 08 / 020079 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".

[0597] f) For the purpose of comparing two or more nucleotide sequences, the percentage of “sequence identity” between the first and second nucleotide sequences may be calculated or determined as described in paragraph e) on page 49 of WO 08 / 020079 (which is incorporated herein by reference), for example by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], wherein each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence—compared to the first nucleotide sequence—is considered a single nucleotide (position) difference; or by using a suitable computer algorithm or technique, also as described in paragraph e) on page 49 of WO 08 / 020079 (which is incorporated herein by reference).

[0598] 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 the first and second amino acid sequences can be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference), for example by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues 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 a difference in 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) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference).

[0599] Moreover, when determining the degree of sequence identity between two immunoglobulin monovariable domains, those skilled in the art may consider so-called “conserved” amino acid substitutions, as described on page 50 of WO 08 / 020079.

[0600] The peptides described herein can also be substituted with any amino acid based on the amino acid variation frequency analysis between homologous proteins of different species proposed by Schulz et al. (1978, Principles of Protein Structure, Springer-Verlag), the structure formation potential analysis proposed by Chou and Fasman (1975, Biochemistry 13:211) and (1978, Adv. Enzymol. 47:45-149), and the protein hydrophobic pattern analysis proposed 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. Chem. 15:321-353). Their full texts are incorporated herein by reference. Information on the primary, secondary, and tertiary structures of nanobodies is provided in this specification and the general background section cited above. Furthermore, for this purpose, information on the V of the llama is provided, 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 domains. Information regarding the crystal structure in conventional V can be found in the prior art cited above. H V is formed at these locations in the structural domain H / V L Further information on the interface and possible camel-derived substitutions of some amino acid residues.

[0601] h) If an immunoglobulin single variable domain and a nucleic acid sequence have 100% sequence identity across their full length (as defined in the text), then they are referred to as “identical”.

[0602] i) When comparing two immunoglobulin monovariable domains, the term “amino acid difference” refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence; it should be understood that two immunoglobulin monovariable domains may contain one, two, or more such amino acid differences.

[0603] j) When a nucleotide sequence or amino acid sequence is referred to as “containing” another nucleotide sequence or amino acid sequence, or as “consistently” of another nucleotide or amino acid sequence, it has the meaning given in paragraph i) on pages 51-52 of WO 08 / 020079.

[0604] k) The term “in substantially separate form” has the meaning given in paragraphs j) on pages 52 and 53 of WO 08 / 020079.

[0605] l) The terms “domain” and “associative structural domain” have the meanings given in paragraph k) on page 53 of WO 08 / 020079.

[0606] m) The terms “antigenic determinant” and “epitope” are used interchangeably in this document and have the meanings given in paragraph l) on page 53 of WO 08 / 020079.

[0607] n) As further described in paragraph m) on page 53 of WO 08 / 020079, an amino acid sequence (e.g., the antibody, polypeptide of the present invention, or a common antigen-binding protein or polypeptide or fragment thereof) that can bind to a specific antigenic determinant, epitope, antigen, or protein (or at least a portion, fragment, or epitope thereto) (specifically) has affinity for and / or is specific to that antigenic determinant, epitope, antigen, or protein is referred to as “targeted” or “directed” to the said antigenic determinant, epitope, antigen, or protein.

[0608] o) The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen-binding molecule or antigen-binding protein (such as the ISV, nanobody, or peptide of this invention) can bind to. The specificity of an antigen-binding protein can be determined based on affinity and / or antibody affinity. Affinity (the equilibrium constant (K) for the dissociation of the antigen from the antigen-binding protein) is... D K (or KD) is a measure of the binding strength between an antigenic determinant (i.e., the target) and an antigen-binding site on an antigen-binding protein (i.e., an 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 the affinity constant (K)). A ), which is 1 / K D As is clear to those skilled in the art (e.g., based on further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen under study.

[0609] Affinity is the affinity of a peptide, i.e., the ability of a ligand to bind via two (or more) pharmacophores (ISVs), where multiple interactions synergistically enhance the "apparent" affinity. Affinity is a measure of the binding strength between the peptide of the present invention and the associated antigen. The peptide of the present invention can bind to at least two targets via its two (or more) structural units, such as ISVs or nanobodies, where multiple interactions, such as a first structural unit, ISV, or nanobodies binding to a first target and a second structural unit, ISV, or nanobodies binding to a second target, synergistically enhance the "apparent" affinity. Affinity is related to the affinity between the antigen determinant and its antigen-binding site on the antigen-binding molecule, and the number of associated binding sites present on the antigen-binding molecule. For example, and without limitation, peptides containing two or more structural units, such as ISVs or nanobodies targeting different targets on cells, can (and generally will) bind with a higher affinity than a single monomer or single structural unit, such as a monovalent ISV or nanobodies, contained in the peptide of the present invention.

[0610] Anything greater than 10 -4 moles / liter of K D Value (or anything below 10) 4 M -1 K A Values ​​(in liters / moles) are generally considered to be nonspecific binding.

[0611] The polypeptide of the present invention comprises first and second structural units, such as first and second ISVs, or first and second nanobodies. Preferably, the affinity of each structural unit, such as an ISV or nanobody, is determined separately. In other words, for a monovalent structural unit, ISV, or nanobody, the affinity is determined independently of the affinity effects caused by other structural units, ISVs, or nanobodies (which may or may not be present). The affinity of a monovalent structural unit, ISV, or nanobody can be determined for the monovalent structural unit, ISV, or nanobody itself, i.e., when said monovalent structural unit, ISV, or nanobody is not included in the polypeptide of the present invention. Alternatively, or further, in the absence of other targets, the affinity of a monovalent structural unit, ISV, or nanobody can be determined for a single target.

[0612] The binding of antigen-binding proteins to antigens or antigenic determinants can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, and various variations thereof known per se in the art; as well as other techniques mentioned herein.

[0613] The dissociation constant can be an actual or surface dissociation constant, as will be apparent to a person skilled in the art. The methods used to determine the dissociation constant will be apparent to a person skilled in the art, and include, for example, the techniques mentioned herein. In this respect, it will also be apparent that it may not be possible to measure values ​​greater than 10. -4 moles per liter or 10 -3 moles per liter (e.g., 10) -2 The dissociation constant (mol / L). Optionally, as will also be apparent to those skilled in the art, the (actual or apparent) dissociation constant can be based on the (actual or apparent) binding constant (K). A ), through relation [K D =1 / K A Calculated in the manner described above.

[0614] Affinity represents the strength or stability of molecular interactions. Affinity is typically expressed by K0. D Alternatively, the dissociation constant can be given, which has units of moles per liter (or M). Affinity can also be expressed as the binding constant, K. A It is equal to 1 / K D And it has (moles / liter) -1 (or M) -1 The units are as follows. In this specification, the stability of the interaction between two molecules (such as the amino acid sequence, nanobody, or peptide of the present invention and its intended target) will be determined primarily based on the K0 of their interaction. D Value expression; technicians will understand that, given relation K A =1 / K D Through its K D The value specifying the strength of molecular interactions can also be used to calculate the corresponding K. A Value. K D The - value also characterizes the strength of molecular interactions in a thermodynamic sense, because it is known from the relation DG = RT.ln(K D (Equivalent land DG = -RT.ln(K)) A It is related to the binding free energy (DG), where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0615] K values ​​considered meaningful (e.g., specific) for biological interactions D Usually in 10 -10 M(0.1nM) to 10 -5 Within the range of M (10000nM). The stronger the interaction, the greater its K. D The lower.

[0616] K D It can also be expressed as the dissociation rate constant of the complex (denoted as k). off ) and its binding rate (denoted as k)on The ratio of (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 notation for seconds). 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 The variation between these values ​​approaches the diffusion-limited binding rate constant for biomolecular interactions. This is achieved through the relationship t... 1 / 2 =ln(2) / k off The dissociation rate is related to the half-life of a given molecular interaction. The dissociation rate can range from 10... -6 s -1 (approaching a period of several days) 1 / 2 (irreversible complex) to 1s -1 (t 1 / 2 It varies between 0.69s.

[0617] The affinity of molecular interactions between two molecules can be measured using various techniques known to exist, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology, 13: 1551-1559). As used herein, “surface plasmon resonance” refers to an optical phenomenon that allows 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 flows through the immobilized molecule under flow conditions to obtain k on k off Measured value and K obtained therefrom D (or K) AThis can be done, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden, and Piscataway, NJ). For further description, see Jonsson et al. 1993 (Ann. Biol. Clin. 51:19-26), Jonsson et al. 1991 (Biotechniques 11:620-627), Johnson et al. 1995 (J. Mol. Recognit. 8:125-131) and Johnson et al. 1991 (Anal. Biochem. 198:268-277).

[0618] Alternatively, affinity can be used in kinetic exclusion analysis (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328:35-43). The platform (Sapidyne Instruments Inc, Boise, USA) measures the KinExA. The term "KinExA," as used herein, refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of the antibody / antigen complex is passed through a column containing microspheres pre-coated with antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished by the binding of a fluorescently labeled protein to the antibody (or antigen).

[0619] If the measurement process, for example, is affected to some extent by artifacts related to the coating layer on a biosensor of a molecule, thus influencing the intrinsic binding affinity of the underlying molecule, those skilled in the art will also determine that the measured K... D It can correspond to apparent K D Furthermore, if a molecule contains more than one recognition site for other molecules, the apparent K can be measured. D In this case, the measured affinity is not affected by the affinity of the interaction between the two molecules.

[0620] Another method that can be used to evaluate affinity is the 2-step ELISA (enzyme-linked immunosorbent assay) procedure described by Friguet et al. (1985) (J. Immunol. Methods, 77:305-19). This method establishes a solution-bound equilibrium measurement and avoids artifacts associated with the adsorption of one of the molecules on a support such as plastic.

[0621] However, K D Precise measurements can be extremely labor-intensive, and therefore, determining apparent K is often difficult. DThe value is used to evaluate the binding strength between two molecules. It should be noted that as long as all measurements are performed continuously (e.g., the measurement conditions remain constant), the apparent K value will remain constant. D Measurement can be used as a true K D The approximation of K, and therefore in this document, K D and apparent K D They should be treated with equal importance or relevance.

[0622] Finally, it should be noted that in many cases, experienced scientists can determine the suitability of a molecule for determining binding affinity relative to some reference molecule. For example, to evaluate the binding strength between molecules A and B, a reference molecule C, known to bind B, can be used, for instance, and appropriately labeled with a fluorophore or chromophore group or other chemical motif, such as biotin, which is readily detectable in ELISA or FACS (fluorescence-activated cell sorting) or other forms (fluorophores for fluorescence detection, chromophores for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the concentration of reference molecule C is kept constant, and the concentration of A is varied for a given concentration or amount of B. As a result, the IC50 corresponding to the concentration of A is obtained. 50 The value at which the signal measured for C is halved in the absence of A. If K... D ref (K of the reference molecule) D ) and the total concentration c of the reference molecule ref If it is known, then for the apparent K of the interaction AB D K can be obtained from the following formula: D =IC 50 / (1+c ref / K D ref Note that if c ref < <K D ref Then K D ≈IC 50 If for the IC of the comparison complex... 50 Measurements are performed continuously (e.g., maintaining c) ref If the molecular interactions are fixed, then the strength or stability of the molecular interactions can be determined by IC. 50 The evaluation is conducted, and in this paper, the measurement is determined to be equal to K. D or apparent K D .

[0623] p) The half-life of the amino acid sequence, compound, or peptide of the present invention can generally be defined as described in paragraph o) on page 57 of WO 08 / 020079 and as mentioned herein, referring to the time taken for the serum concentration of said sequence, compound, or peptide to decrease by 50% in vivo, for example, due to degradation of the amino acid sequence or compound and / or clearance or isolation of the sequence or compound by natural mechanisms. The in vivo half-life of the amino acid sequence, compound, or peptide of the present invention can be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and can generally be as described in paragraph o) on page 57 of WO 08 / 020079. As also mentioned in paragraph o) on page 57 of WO 08 / 020079, the half-life can be expressed by parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC). For example, you can refer 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 (Pharmacokinetics Analysis: A Practical Approach). You can also refer to Gibaldi & Perron 1982 (Pharmacokinetics, Dekker M, 2). nd (Revised version). The term “increase in half-life” or “increased half-life” as defined in paragraph o) on page 57 of WO08 / 020079 specifically refers to an increase in t1 / 2-β, with or without an increase in t1 / 2-α and / or AUC or both.

[0624] q) Regarding targets or antigens, the term "interacting 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, or polymerization (e.g., homopolymerization 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 the target or antigen involved in the biological action or mechanism of the target or antigen. More generally, "interacting site" can be any site, epitope, antigenic determinant, part, domain, or stretch of amino acid residues on the target or antigen that the amino acid sequence or polypeptide of the present invention can bind to and cause the target or antigen (and / or any pathway, interaction, signal transduction, biological mechanism, or biological effect involving said target or antigen) to be regulated.

[0625] r) When it has an affinity for the first antigen 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 (as described above, suitably expressed as K) for binding to the second target or polypeptide, it exhibits an affinity / antibody affinity (as described above). D Value, K A Value, K off Rate and / or K on If the binding rate is specified, then the immunoglobulin monovariable domain or polypeptide is considered "specific" to the first target or antigen relative to the second target or antigen. For example, the first antigen may bind to the target or antigen at a rate greater than the binding rate of the amino acid sequence or polypeptide to the second target or antigen. D At least 10 times smaller, for example, at least 100 times smaller, preferably at least 1000 times smaller, for example, less than 10,000 times smaller of K. D Value binding. Preferably, when an immunoglobulin monovariable domain or polypeptide is "specific" to a first target or antigen compared to a second target or antigen, it targets (as defined herein) the first target or antigen, but not the second target or antigen.

[0626] The terms “cross-block,” “cross-blocked,” and “cross-blocking” are used interchangeably herein to refer to the ability of an immunoglobulin monovariable domain or polypeptide to interfere with the binding of a natural ligand to one or more receptors. The immunoglobulin monovariable domains or polypeptides of the present invention are capable of interfering with the binding of another compound, such as a natural ligand, to its target, and therefore can be determined using competitive binding assays, regardless of whether they can be called cross-blockers according to the invention. A particularly suitable quantitative cross-blocking assay uses FACS- or ELISA-based methods or Alphascreen to measure the competition between labeled (e.g., His-labeled or biotinylated) immunoglobulin monovariable domains or polypeptides of the present invention and other binding agents with respect to their binding to the target. The experimental section generally describes suitable FACS-, ELISA-, or Alphascreen-based replacement assays for determining whether binding molecules cross-block or are capable of cross-blocking the immunoglobulin monovariable domains or polypeptides of the present invention. It should be understood that the assays can be used with any immunoglobulin monovariable domain or other binding agent described herein. Therefore, typically, the cross-blocking amino acid sequence or other binding agent according to the invention is bound, for example, to the target in the aforementioned cross-blocking detection, such that during detection and in the presence of the second amino acid sequence or other binding agent of the invention, the recorded immunoglobulin monovariable domain or polypeptide according to the invention is replaced by 0.01 mM or less of the possible cross-blocking reagent to be detected (the cross-blocking reagent can be another conventional monoclonal antibody such as IgG, a classic monovalent antibody fragment (Fab, scFv), and a modified variant (e.g., a biantibody, a triantibody, a microantibody, VHHs, dAbs, VHs, VLs)) at 60%-100% (e.g., in competitive detection based on ELISA / Alphascreen) or 80%-100% (e.g., in competitive detection based on FACS).

[0627] t) If the VHH1 type immunoglobulin single variable domain or type 1 VHH sequence has 85% identity (using the VHH1 common sequence as the query sequence and using a blast algorithm with standard settings (i.e., a brosom62 score matrix) for the VHH1 common sequence (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) (SEQ ID NO:361) and is forced to have a cysteine ​​residue at position 50 (i.e., C50) (using Kabat numbering), then the amino acid sequence, such as, for example, the immunoglobulin single variable domain or polypeptide according to the invention, is a "VHH1 type immunoglobulin single variable domain" or a "type 1 VHH sequence".

[0628] u) If an amino acid sequence, such as an immunoglobulin monovariable domain or polypeptide according to the invention, is specific to two different antigens or antigenic determinants (e.g., serum albumins from two different mammalian species, such as human serum albumin and macaque serum albumin), then it is said to be “cross-reactive” to these different antigens or antigenic determinants.

[0629] v) As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079 (incorporated hereby by reference), according to V given by Kabat et al. H The generalized numbering system for domains (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91) assigns the amino acid residues of a single variable domain to immunoglobulins, as in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, this publication) Figure 2 V applied to camelids in ) HH As with structural domains. It should be noted—as is the case with V in this field—that... HAs is well known with the VHH domain, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. The total number of amino acid residues in the VH and VHH domains will typically be in the range of 110 to 120, and usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein. The determination of the CDR region can also be performed using different methods. According to Kabat's CDR determination, FR1 of the immunoglobulin monovariable domain contains amino acid residues 1-30, CDR1 of the immunoglobulin monovariable domain contains amino acid residues 31-35, FR2 of the immunoglobulin monovariable domain contains amino acids 36-49, CDR2 of the immunoglobulin monovariable domain contains amino acid residues 50-65, FR3 of the immunoglobulin monovariable domain contains amino acid residues 66-94, CDR3 of the immunoglobulin monovariable domain contains amino acid residues 95-102, and FR4 of the immunoglobulin monovariable domain contains amino acid residues 103-113.

[0630] In this application, unless otherwise stated, the CDR sequence is determined according to Kontermann and Dübel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acid residues at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0631] The accompanying drawings, sequence listings, and experimental sections / exemplary embodiments provided in this document are merely illustrative of the invention and should not be construed as limiting the scope of the invention and / or the appended claims in any way, unless otherwise expressly stated herein.

[0632] x) The half-maximal inhibitory concentration (IC50) is a measure of a compound's effect in inhibiting biological or biochemical functions, such as pharmacological effects. This quantitative measurement indicates how many ISVs or nanobodies (inhibitors) are required to inhibit a given biological process (or a component of that process, i.e., enzymes, cells, cell receptors, chemotaxis, degenerative changes, metastasis, invasion, etc.). 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 dose-response curves and examining the effect of different concentrations of antagonists, such as the ISVs or nanobodies of the present invention, on reversing agonist activity. The IC50 value can be calculated for a given antagonist, such as the ISV or nanobodies of the present invention, by determining the concentration required for the maximum biological response of the agonist that inhibits half-maximal inhibitory concentration.

[0633] The term "half-maximal effective concentration" (EC50) refers to the concentration of a compound that induces half the response between baseline and maximum after a specified exposure time. In this document, it is used as a measure of the potency of peptides, ISVs, or nanobodies. The EC50 of a graded dose-response curve represents the concentration of the compound at which 50% of its maximum effect is observed. Concentrations are preferably expressed in molar units.

[0634] In biological systems, small changes in ligand concentration typically result in a rapid change in the response, which follows an S-shaped curve. The inflection point where the increase in response with increasing ligand concentration begins to slow down is the EC50. This can be mathematically determined by differentiating the best-fit line. In most cases, relying on a graph for evaluation is convenient. Where EC50 is provided in the Examples section, experiments are designed to reflect KD as accurately as possible. In other words, the EC50 value can therefore be considered as the KD value. The term "average KD" refers to the average KD value obtained from at least one, but preferably more than one, such as at least two experiments. The term "average" refers to the mathematical term "average" (the sum of data divided by the number of terms in the data).

[0635] Also relevant is IC50, which is a measure of a compound's inhibition (50% inhibition). For competitive binding assays and functional antagonist assays, IC50 is the most common profiling measure of dose-response curves. For agonist / stimulant assays, the most common profiling measure is EC50.

[0636] It must be noted that, as used herein, unless otherwise expressly indicated, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “reagent” includes one or more of the different reagents described herein, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art that can modify or substitute for the methods described herein. Unless otherwise stated, 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 can determine, using only conventional experiments, numerous equivalents of specific embodiments of the invention described herein. These equivalents are intended to be encompassed within the scope of this invention.

[0637] Wherever used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of the elements connected by the term.”

[0638] The term “about” or “approximately”, as used herein, means within 20% of a given value or range, preferably within 15%, more preferably within 10%, and most preferably within 5%.

[0639] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and its variations (such as “comprises” and “comprising”) shall be understood to imply the inclusion of the indicated integer or step or a group of integers or steps, but not to exclude any other integer or step or a group of integers or steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “including”, or sometimes by the term “having”.

[0640] The present invention relates to a polypeptide comprising at least a first immunoglobulin monovariable domain (ISV) and at least one additional immunoglobulin monovariable domain, wherein the at least first ISV has a high affinity for / binds to differentiation cluster 3 (CD3), and the at least one additional ISV has a high affinity for / binds to an antigen on a target cell.

[0641] Typically, the multispecific polypeptides of the present invention combine high-affinity antigen recognition on target cells with T cell activation, resulting 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 (such as tumor antigens) on target cells and the T cell co-receptor CD3 is well known. This brings the T cell into close proximity to the target cell, i.e., binds the T cell, causing the T cell to kill the target cell. In the present invention, this method is utilized in combating proliferative diseases, inflammatory diseases, infectious diseases, and autoimmune diseases. Typically, T cells are equipped with granules 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 formed when the T cell is in close proximity to the target cell intended to be killed. Normally, close proximity between T cells and target cells is achieved by the T cell binding to an MHC / peptide complex using its matching T cell receptor. The polypeptides of the present invention bring the T cell into such close proximity to the target cell in the absence of T cell receptor / MHC interaction.

[0642] Therefore, the present invention relates to polypeptides as described herein, wherein the polypeptides direct T cells to target cells.

[0643] Through one arm (first ISV), the multispecific polypeptide has a high affinity for / binds to CD3 (a protein component of the T cell receptor signal transduction complex on T cells). Through another arm (second ISV and / or third ISV, etc.), the multispecific polypeptide recognizes an antigen on the target cell and has a high affinity for / binds to the antigen on the target cell. Preferably, T cell activation is only observed on the surface of the target cell when the multispecific polypeptide is presented to the T cell. This antigen-dependent activation on the activated target cell results in advantageous safety properties. In one embodiment, the multispecific polypeptide transiently links the T cell and the target cell. Preferably, the multispecific polypeptide is capable of binding resting polyclonal T cells (such as CD4+) to the target cell. + and / or CD8 + T cells are induced to activate for efficient redirection and lysis of target cells. Preferably, after the first target cell is lysed, the T cells are directed to the next target cell.

[0644] Proteins and peptides comprising or substantially composed of two or more immunoglobulin monovariable domains (such as at least two immunoglobulin monovariable domains of the present invention) will be referred to herein as “multivalent” proteins or peptides or “multivalent constructs.” From the further description herein, some non-limiting examples of such multivalent constructs will become apparent. The peptides of the present invention are “multivalent,” i.e., comprising two or more structural units or ISVs, wherein at least the first structural unit, ISV, or nanobody is different from the second structural unit, ISV, or nanobody, and targets different targets, such as antigens or antigenic determinants. A peptide of the present invention containing at least two structural units, ISVs, or nanobodies (where at least one structural unit, ISV, or nanobody targets a first antigen (i.e., a first target, such as, for example, CD3) and at least one structural unit, ISV, or nanobody targets a second antigen (i.e., a second target different from the first target, such as, for example, TAA, CD20, or HER2)) will also be referred to herein as a “multispecific” peptide of the present invention, and the structural units, ISVs, or nanobodies present in such peptides will also be referred to herein as “multivalent” or “multispecific”. Therefore, for example, the "bispecific" peptide of the present invention is a polypeptide comprising at least one structural unit, ISV or nanobody targeting a first target (e.g., CD3) and at least one other structural unit, ISV or nanobody targeting a second target (i.e., a second target different from the first target, such as, for example, a TAA, such as CD20 or HER2), while the "trispecific" peptide of the present invention is a polypeptide comprising at least one structural unit, ISV or nanobody targeting a first target (e.g., CD3), a second structural unit, ISV or nanobody targeting 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 targeting 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 specification, the present invention is not limited to bispecific peptides in the sense that the multispecific peptides of the present invention may comprise at least a first structural unit, ISV, or nanobody targeting a first target, a second structural unit, ISV, or nanobody targeting a second target, and any number of structural units, ISVs, or nanobodies targeting one or more targets that may be the same as or different from the first and / or second targets, respectively. The structural units, ISVs, or nanobodies may optionally be linked via linker sequences.

[0645] The terms bispecific peptide, bispecific format, bispecific construct, bispecific nanobody construct, bispecific and bispecific antibody may be used interchangeably in this document.

[0646] As will become clear from the foregoing and further description herein, the immunoglobulin monovariable domains of the present invention can be used as “structural units” to form the polypeptides of the present invention, for example by suitably combining them with other groups, residues, structural moieties or binding units to form compounds or constructs described herein (e.g., but not limited to the bivalent / trivalent / tetravalent and bivalent / trivalent / tetravalent multispecific polypeptides of the present invention described herein), which combine one or more desired properties or biological functions within a molecule.

[0647] It will be understood (as also demonstrated in the Examples section) that the CD3-binding ISV and the target cell-binding ISV can be placed in any order in the polypeptide of the present invention. More specifically, in one embodiment, the CD3-binding ISV is located at the N-terminus, and the target cell-binding ISV is located at the C-terminus. In another embodiment, the target cell-binding ISV is located at the N-terminus, and the CD3-binding ISV is located at the C-terminus.

[0648] In a preferred aspect, the polypeptide of the present invention comprises at least a first immunoglobulin monovariable domain (ISV), at least a second immunoglobulin monovariable domain, and at least a third immunoglobulin monovariable 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.

[0649] It will be understood (as also demonstrated in the Examples section) that the CD3-binding ISV and the ISVs binding to the first and second antigens on target cells can be positioned in any order in the polypeptide of the present invention. More specifically, in one embodiment, the CD3-binding ISV is located at the N-terminus, the ISV binding to the first antigen on target cells is located at the center, and the ISV binding to the second antigen on target cells is located at the C-terminus. In another embodiment, the CD3-binding ISV is located at the N-terminus, the ISV binding to the second antigen on target cells is located at the center, and the ISV binding to the first antigen on target cells is located at the C-terminus. In another embodiment, the ISV binding to the first antigen on target cells is located at the N-terminus, the ISV binding to the second antigen on target cells is located at the center, and the ISV binding to CD3 is located at the C-terminus. In yet another embodiment, the ISV binding to the first antigen on target cells is located at the N-terminus, the ISV binding to CD3 is located at the center, and the ISV binding to the second antigen on target cells is located at the C-terminus. In another embodiment, the ISV that binds to the second antigen on the target cell is located at the N-terminus, the ISV that binds to CD3 is located at the center, and the ISV that binds to the first antigen on the target cell is located at the C-terminus. In yet another embodiment, the ISV that binds to the second antigen on the target cell is located at the N-terminus, the ISV that binds to the first antigen on the target cell is located at the center, and the ISV that binds to CD3 is located at the C-terminus.

[0650] This invention also relates to compounds or constructs, and particularly to proteins or polypeptides comprising or substantially consisting of one or more ISVs or polypeptides of this invention, and optionally further comprising one or more other groups, residues, portions, or binding units. Those skilled in the art will appreciate from the further disclosure herein that such other groups, residues, portions, binding units, or amino acid sequences may or may not provide additional functionality to the polypeptides of this invention (and / or to the compounds or constructs in which said polypeptides are present), and may or may not alter the properties of the polypeptides of this invention.

[0651] The compounds, constructs, or peptides of the present invention can generally be prepared by methods comprising at least one step of suitably linking one or more immunoglobulin monovariable domains of the present invention to one or more additional groups, residues, structural moieties, or binding units via one or more suitable linkers to provide the compounds, constructs, or peptides of the present invention. The peptides of the present invention can also be prepared by methods generally comprising providing a nucleic acid encoding the peptide of the present invention, expressing the nucleic acid in a suitable manner, and recovering the expressed peptide of the present invention. These methods can be carried out in ways known per se, as will be apparent to those skilled in the art, for example, based on the methods and techniques further described herein.

[0652] The process of designing / selecting and / or preparing the compounds, constructs, or peptides of the present invention, starting from the amino acid sequence of the present invention, is also referred to herein as the “formatting” of the amino acid sequence of the present invention; the amino acids of the present invention that are part of the compounds, constructs, or peptides of the present invention are referred to as “formatted” or “formatted” compounds, constructs, or peptides of the present invention. Examples of the amino acid sequence formatting methods of the present invention, and examples of such formats, will be clear to those skilled in the art based on the disclosure herein; such formatted immunoglobulin single variable domains or peptides form another aspect of the present invention.

[0653] For example, such additional groups, residues, structural moieties, or binding units may be one or more additional immunoglobulin monovariable 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 additional groups, residues, structural moieties, or binding units are immunoglobulin sequences. More preferably, the one or more additional groups, residues, structural moieties, or binding units are selected from the group consisting of domain antibodies, immunoglobulin monovariable domains suitable for use as domain antibodies, single-domain antibodies, immunoglobulin monovariable domains (ISVs) suitable for use as single-domain antibodies, "dAbs", immunoglobulin monovariable domains suitable for use as dAbs, or nanobodies. Alternatively, such groups, residues, structural moieties, or binding units may be, for example, chemical groups, residues, or structural moieties that 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 of the immunoglobulin monovariable domains or polypeptides of the present invention to provide "derivatives" of the ISVs or polypeptides of the present invention, as further described herein.

[0654] Compounds or constructs comprising or substantially consisting of one or more derivatives described herein, and optionally further comprising one or more other groups, residues, structural moieties, or binding units optionally linked via one or more linkers, are also within the scope of this invention. Preferably, the one or more other groups, residues, structural moieties, or binding units are immunoglobulin monovariable domains. In the compounds or constructs described above, one or more immunoglobulin monovariable domains and one or more groups, residues, structural moieties, or binding units of this invention can be directly linked to each other and / or linked via one or more suitable linkers or spacers. For example, when one or more groups, residues, structural moieties, or binding units are immunoglobulin monovariable domains, the linker may also be an immunoglobulin monovariable domain, so that the resulting compound or construct is a fusion (protein) or a fusion (peptide).

[0655] In some embodiments, the polypeptide comprises at least two or more immunoglobulin monovariable domains as disclosed herein. In some embodiments, the polypeptide is substantially composed of two or more immunoglobulin monovariable domains as disclosed herein. A polypeptide “substantially composed of” two or more immunoglobulin monovariable domains is a polypeptide that does not have any other immunoglobulin monovariable domains besides the two or more immunoglobulin monovariable domains disclosed herein. For example, a polypeptide substantially composed of two immunoglobulin monovariable domains does not include any other immunoglobulin monovariable domains. However, it should be understood that a polypeptide substantially composed of two or more immunoglobulin monovariable domains may include additional functionalities, such as labels, 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 comprises one or more immunoglobulin monovariable 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).

[0656] In some embodiments, the polypeptide comprises a multivalent or multispecific construct including an immunoglobulin monovariable 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 moiety. In some embodiments, the serum-binding protein moiety is an immunoglobulin monovariable domain. In some embodiments, the immunoglobulin monovariable domain is...

[0657] It will be understood that the order (orientation) of structural units (such as, for example, a first structural unit, a second structural unit, a third structural unit, etc.) on the polypeptide can be selected according to the needs of those skilled in the art and according to the relative affinity that may depend on the position of these structural units in the polypeptide. Whether the polypeptide contains a linker is a matter of design choice. However, some orientations (with or without a linker) can provide preferred binding characteristics compared to others. For example, the order of the first and second structural units in the polypeptide of the present invention can be (from N-terminus to C-terminus): (i) first structural unit (e.g., a first ISV, such as a first nanobody) - [linker] - second structural unit (e.g., a second ISV, such as a second nanobody); or (ii) second structural unit (e.g., a second ISV, such as a second nanobody) - [linker] - first structural unit (e.g., a first ISV, such as a first nanobody); (wherein the linker is optional). All orientations are included in the present invention. Polypeptides containing structural unit orientations that provide the desired (binding) properties can be readily identified by conventional screening, for example as illustrated in the experimental section.

[0658] The first immunoglobulin single variable domain (ISV) of the polypeptide of the present invention has a high affinity for effector cells (preferably the TCR complex of the effector cells, and even more preferably CD3) and binds to effector cells (preferably the TCR complex of the effector cells, and even more preferably CD3).

[0659] Effector cells are cells containing the TCR complex, preferably immune cells such as T helper cells, monocytes, macrophages, or dendritic cells, preferably CD4+. + T helper cells (also known as CD4 cells, T helper cells, or T4 cells), preferably cytotoxic T cells (also known as T cells). C Cells, CTLs, or CD8 + T cells, natural killer T cells (NKT cells), or natural killer cells (NK cells). In some embodiments, the cells are present in vivo. In some embodiments, the cells are present in vitro. The effector cells of the present invention particularly relate to mammalian cells, preferably primate cells, and even more preferably human cells.

[0660] 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 distinct type I single-transmembrane proteins: the TCRα and TCRβ chains that form the TCR heterodimer responsible for ligand recognition, and the non-covalently associated CD3γ, CD3δ, CD3ε, and ζ chains (which possess cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signal transduction components). Both the α and β chains of the T cell receptor consist of constant and variable domains. The sequences of the human CD3 and human TCRα / 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).

[0661] In one embodiment, the present 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 polymorph or isotype thereof.

[0662] Alternatively, the present invention provides a polypeptide as described herein, wherein the first ISV binds to CD3γ (SEQ ID NO:379), CD3δ (SEQ ID NO:291) and / or CD3ε (SEQ ID NO:380) of the TCR complex, or a polymorph or isotype thereof.

[0663] A homotype is an alternative protein sequence that can be generated from the same gene by a single biological event or by a combination of biological events, such as selective promoter use, selective splicing, selective initiation, and ribosome frameshift, all as known in the art.

[0664] As used herein, “T cell activation” refers to one or more cellular responses of T cells (e.g., cytotoxic T cells), such as those selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis. The peptides of the present invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art, for example 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.

[0665] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the polypeptide induces T cell activation. Preferably, the polypeptide of the present invention induces T cell activation only when the second ISV and / or additional ISV binds to an antigen on the target cell.

[0666] In one embodiment, the present invention relates to a polypeptide as described herein, wherein T cell activation depends on the presentation of the polypeptide, which binds to a first antigen on a target cell, to the T cell.

[0667] T cell activation via the peptides of the present invention can be monitored by the following: 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), upregulation of granzyme and perforin expression, and / or cell proliferation, membrane blebbing, activation of caspaseogen 3 and / or 7, fragmentation of nuclear DNA, and / or cleavage of caspase substrate poly(ADP-ribose) polymerase. Preferably, the retargeting lysis of target cells via the multispecific peptides 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.

[0668] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the T cell activation is independent of MHC recognition.

[0669] The peptides of the present invention demonstrate the ability to activate previously unstimulated peripheral polyclonal CD8 in vitro. + -and CD4 + - Redirected lysis of positive T cells. Redirected lysis of target cells recruited by T cells via the peptides of the present invention involves cytolytic synapse formation and delivery of perforin and granzymes. Cytolysis via T cells has been described, for example, in Atkinson and Bleackley 1995 (Crit. Rev. Immunol 15(3-4):359-384). Preferably, the conjugated T cells are capable of continuous target cell lysis and are unaffected by immune escape mechanisms (which interfere with peptide antigen processing and presentation, or clonal T cell differentiation) (see, for example, WO 2007 / 042261). In vitro, redirected lysis is visible at low picomolar concentrations, indicating that very low amounts of the peptides of the present invention are required to bind to target cells to trigger T cell lysis. As demonstrated in the examples, a low ratio of effectors to targets can indicate continuous target cell lysis. Therefore, the present invention relates to potent peptides. Preferably, the peptides of the present invention mediate the killing of target cells (e.g., cancer cells), such as stimulating T cells to form pores and deliver pro-apoptotic components of cytotoxic T cell granules.

[0670] In one embodiment, the present invention relates to a polypeptide as described herein, wherein T cell activation elicits one or more cellular responses of the T cell, wherein the cellular responses are selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis.

[0671] As used herein, the term "potency" refers to a measure of the biological activity of an agent, such as a peptide, ISV, or nanobody. The potency of an agent can be determined by any suitable method known in the art, such as those described in the Experimental section, for example. Cell culture-based potency assays are often the preferred form for determining biological activity because they measure the physiological response induced by the agent and can produce results in a relatively short period. Based on the mechanism of action of the product, assays based on various cell types 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 models, and T cell-mediated tumor cell killing assays (e.g., as shown in the Examples section), all of which are known in the art. Results from cell-based potency assays can be expressed as "relative potency," as determined by comparing the response of the multispecific peptide of the present invention to a corresponding reference monovalent ISV (e.g., a peptide containing only one ISV or one nanobody, optionally further comprising an unrelated nanobody (see Experimental section)).

[0672] In one embodiment, the present invention relates to a polypeptide as described herein, wherein T cell activation causes inhibition of the activity of the target cells, 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 symptom and / or symptom relief) 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%.

[0673] The first structural unit, ISV, nanobody, or VHH of the present invention has high affinity for its target (i.e., CD3). The first structural unit, ISV, or nanobody of the present invention may, for example, target an antigenic determinant, epitope, portion, domain, subunit, or conformation of the first target (where applicable). The first structural unit, such as the first ISV, nanobody, or VHH, is preferably chosen for its high affinity for its target itself, regardless of any affinity effects.

[0674] Therefore, the present invention relates to peptides as described herein, wherein the first ISV binds to CD3 with an average KD value between 100 nM and 10 pM, such as an average KD value below 90 nM, even more preferably an average KD value below 80 nM, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even smaller, 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 smaller, such as less than 10 pM. Preferably, the KD is determined by Kinexa or SPR, for example, as determined by Proteon. For example, the KD is determined as shown in the Examples section.

[0675] Therefore, the present invention relates to peptides as described herein, wherein the first ISV has high affinity when measured in monovalent form. Preferably, the average KD is measured on the recombinant protein by surface plasmon resonance (SPR).

[0676] Therefore, the present invention relates to polypeptides as described herein, wherein the polypeptide has a dissociation constant (K) selected from the group consisting of said CD3 (or the group binding said CD3). D ): At most about 10 -5 M, at most about 10 -6 M, at most about 10 -7 M, at most about 10 -8 M, at most about 10 -9 M, at most about 10 -10 M, at most about 10 -11 M and at most about 10 -12 M, preferably as measured by surface plasmon resonance.

[0677] The present invention also relates to peptides as described herein, wherein the first ISV binds to the CD3 with an EC50 value between 100 nM and 1 pM, such as an average EC50 value below 100 nM, even more preferably an average EC50 value below 90 nM, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even smaller, such as less than 4, 3, 2, 1 nM or even smaller, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even smaller, such as less than 4 pM.

[0678] Therefore, the present invention relates to peptides as described herein, wherein the average KD is determined by FACS, Biacore, ELISA on a monovalent first ISV (such as a nanobody) or a peptide containing a monovalent first ISV (such as a nanobody), for example, the EC50 is determined as shown in the Examples section.

[0679] As demonstrated in the examples, KD correlates well with EC50.

[0680] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the polypeptide has a binding rate constant (Kon) for CD3 (or for binding to 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, it is measured by surface plasmon resonance or as described in the Examples section.

[0681] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the polypeptide has a dissociation rate constant (Koff) for CD3 (or for binding to CD3) selected from the group consisting of at most about 10 -3 s -1 At most about 10 -4 s -1 At most about 10 -5 s -1 At most about 10 -6 s -1 At most about 10 -7 s -1 At most about 10 -8 s -1At most about 10 -9 s -1 and at most about 10 -10 s -1 Preferably, it is measured by surface plasmon resonance or as described in the Examples section.

[0682] Amino acid sequence modifications of the binding molecules, ISVs, or peptides described herein may be considered. For example, improving the binding affinity and / or other biological properties of antibodies or ISVs may be desired. Amino acid sequence variants of the binding molecules, ISVs, or peptides are prepared by introducing appropriate nucleotide changes into the nucleic acids of said binding molecules, ISVs, or peptides or by peptide synthesis.

[0683] The modifications include, for example, residue deletions from the amino acid sequence of the binding molecule, ISV, or peptide, and / or insertions into the amino acid sequence of the binding molecule, ISV, or peptide, and / or substitutions of residues within the amino acid sequence of the binding molecule, ISV, or peptide. Any combination of deletions, insertions, and substitutions can be produced to achieve the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter 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 frame region (FR). Substitutions are preferably conservative substitutions as described herein. Alternatively or alternatively, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or omitted in each CDR (depending on their length, of course), 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 omitted in each FR.

[0684] A useful method for identifying specific residues or regions of binding molecules, ISVs, or peptides as preferred sites for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells 1989 (Science 244:1081-1085). Thus, residues within the binding molecule or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanines) to influence the interaction between the amino acid and the epitope. Those amino acid sites that show functional sensitivity to the substitution are then perfected by introducing additional or different variants at or for the substituted site. Therefore, while the site for introducing amino acid sequence variants is predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scan or random mutagenesis is performed at the target codon or region, and the expressed binding molecule variants are screened for the desired activity.

[0685] Preferably, the amino acid sequence insertion includes amino-terminal fusion and / or carboxyl-terminal fusion, with a length ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing more than one hundred residues.

[0686] Another type of variant is the 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 the binding molecule, ISV, or polypeptide that are substituted with different residues. The sites of most interest for substitution mutagenesis include the CDR, particularly the hypervariable region, but FR alterations are also considered. For example, if the CDR sequence includes 6 amino acids, it is conceivable that one, two, or three of these amino acids are substituted. Similarly, if the CDR sequence includes 15 amino acids, it is conceivable that one, two, three, four, five, or six of these amino acids are substituted.

[0687] Generally, if an amino acid is substituted in one or more or all of the CDRs, it is preferred that the subsequently obtained "substituted" sequence is at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, even 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 degree to which it is identical to the "substituted" sequence. For example, a CDR having 5 amino acids is preferably 80% identical to its substituted sequence so as to have at least one substituted amino acid. Therefore, the CDRs of the bound molecule can have different degrees of identity with the sequences to which they are substituted; for example, CDR1 can have 80%, while CDR3 can have 90%.

[0688] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the “exemplary substitutions” listed in Table B-1 below) is contemplated, provided that the polypeptide retains its ability to bind to CD3 present on T cells via a first ISV and to bind to a first antigen on target cells via a second ISV and / or its CDR has identity with the subsequently substituted sequence (at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and more particularly preferably 80% identical to the “original” CDR sequence).

[0689] Conservative substitutions are shown in Table B-1 below.

[0690] Table 8-1: Amino Acid Substitutions

[0691] source Exemplary replacement Preferred replacement 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 Ile(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, thr, ser phe Val(V) iIe,leu,met,phe,ala leu

[0692] Sequence analysis also showed that only a limited number of sequence changes existed in the CDR (see Example 4.2 and Tables A-1 to A-6).

[0693] Therefore, the present invention relates to peptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0694] (i) CDR1 is selected from the following groups:

[0695] (a) SEQ ID NOs: 81-100; and

[0696] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:81 or from any of SEQ ID NOs:81-100 by 4, 3, 2 or 1 amino acid; and / or

[0697] (ii) CDR2 is selected from the following groups:

[0698] (c)SEQ ID NOs:101-122; and

[0699] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:101 or from any of SEQ ID NOs:101-122 by 4, 3, 2 or 1 amino acid; and / or

[0700] (iii) CDR3 is selected from the following groups:

[0701] (e)SEQ ID NOs:123-143; and

[0702] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:123 or from any of SEQ ID NOs:123-143 by 3, 2 or 1 amino acid.

[0703] Other preferred CDR sequences are described in Table A-8.

[0704] Generally, combinations of CDRs listed in Table A-8 (i.e., those mentioned in the same row of Table A-4) are preferred. Therefore, it is generally preferred that, when the CDR in the ISV is a CDR sequence mentioned in Table A-8 or suitably selected from the group consisting of CDR sequences differing from the CDR sequences listed in Table A-8 by 4, 3, 2, or only 1 amino acid, at least one and preferably both of the other CDRs are suitably selected from the group consisting of CDR sequences belonging to the same combination in Table A-8 (i.e., those mentioned in the same row of Table A-8) or suitably selected from the group consisting of CDR sequences belonging to the same combination by 4, 3, 2, or only 1 amino acid.

[0705] Sequence analysis of the resulting conjugates further led to the identification of six distinct clusters. Corresponding alignments are provided (see Tables A-1, A-2, A-3, A-4, A-5, and A-6). Clustering was based on sequence similarities and differences in CDR2 and CDR3. Cluster A was the most prominent, comprising 50 clones (SEQ ID NO:s 1-50), clusters B and D were represented by only one clone (SEQ ID NO:51 and SEQ ID NO:52, respectively), cluster C comprised four clones (SEQ ID NO:s 53-56), cluster E comprised nine clones (SEQ ID NO:s 57-65), and cluster F comprised 15 clones (SEQ ID NO:s 66-80). Clustering based on structural similarities and differences in amino acid sequences was converted to functional similarities and differences as revealed in the examples. Representatives of all clusters were isolated based on high affinity for CD3 binding (Examples 3 & 4) and human T cell activation (Example 4.2). Generally, the representative of cluster A exhibited the best EC50 values. Although the C-cluster representative has a slightly worse EC50 value than the B-cluster representative, the C-cluster representative has a lower IC50 value in flow cytometry-based T-cell-mediated Ramos killing assay (see Example 10).

[0706] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0707] (a) SEQ ID NO:81; and

[0708] (b) An amino acid sequence that differs from SEQ ID NO:81 by 1, 2, 3, or 4 amino acids, wherein

[0709] -At position 1, G has changed to R;

[0710] -At position 3, T has changed to A;

[0711] -At position 4, Y has changed to F;

[0712] - At position 8, S has changed to G; and / or

[0713] - At position 10, G has changed to A.

[0714] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0715] (a) SEQ ID NO:101; and

[0716] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, 3, or 4 amino acids, wherein

[0717] -At position 3, V has changed to T or A;

[0718] -At position 5, S has changed to T;

[0719] - At position 6, G has changed to D or E; and / or

[0720] - At position 9, T has changed to S, A, or P.

[0721] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0722] (a) SEQ ID NO:123; and

[0723] (b) An amino acid sequence that differs from SEQ ID NO:123 by 1, 2, or 3 amino acids, wherein

[0724] -At position 2, I has changed to T;

[0725] - At position 9, I has changed to V; and / or

[0726] - At position 10, A has changed to P.

[0727] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0728] (i) CDR1 is selected from the following groups:

[0729] (a) SEQ ID NOs: 81-87; and

[0730] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid; and / or

[0731] (ii) CDR2 is selected from the following groups:

[0732] (c)SEQ ID NOs:101-109; and

[0733] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid; and / or

[0734] (iii) CDR3 is selected from the following groups:

[0735] (e)SEQ ID NOs:123-127; and

[0736] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:123 by 3, 2 or 1 amino acid.

[0737] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0738] Nanobodies belonging to cluster B are represented by a single clone.

[0739] Therefore, the present invention relates to peptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0740] (i) CDR1 is selected from the following groups:

[0741] (a) SEQ ID NO:88; and

[0742] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:88 by 1, 2, 3, or 4 amino acids; and / or

[0743] (ii) CDR2 is selected from the following groups:

[0744] (c)SEQ ID NO:110; and

[0745] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:110 by 1, 2, 3, or 4 amino acids; and / or

[0746] (iii) CDR3 is selected from the following groups:

[0747] (e)SEQ ID NO:128; and

[0748] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:128 by 1, 2 or 3 amino acids.

[0749] Therefore, the present invention relates to the polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0750] C-cluster nanobodies exhibit very limited sequence variability in CDR.

[0751] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0752] (a) SEQ ID NO:112; and

[0753] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0754] - At position 2, V has changed to A.

[0755] Therefore, the present invention relates to peptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0756] (i) CDR1 is selected from the following groups:

[0757] (a) SEQ ID NO:90; and

[0758] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:90 by 1, 2, 3, or 4 amino acids; and / or

[0759] (ii) CDR2 is selected from the following groups:

[0760] (c)SEQ ID NOs:112-113; and

[0761] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:112 by 1, 2, 3, or 4 amino acids; and / or

[0762] (iii) CDR3 is selected from the following groups:

[0763] (e)SEQ ID NO:130; and

[0764] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:130 by 1, 2 or 3 amino acids.

[0765] In one aspect, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0766] Nanobodies belonging to the D cluster are represented by a single clone.

[0767] Therefore, the present invention relates to peptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0768] (i) CDR1 is selected from the following groups:

[0769] (a)SEQ ID NO:89; and

[0770] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:89 by 1, 2, 3, or 4 amino acids; and / or

[0771] (ii) CDR2 is selected from the following groups:

[0772] (c)SEQ ID NO:111; and

[0773] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:111 by 1, 2, 3, or 4 amino acids; and / or

[0774] (iii) CDR3 is selected from the following groups:

[0775] (e)SEQ ID NO:129; and

[0776] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:129 by 1, 2 or 3 amino acids.

[0777] Therefore, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0778] The E cluster comprises 9 clones.

[0779] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0780] (a) SEQ ID NO: 91; and

[0781] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[0782] - At position 6, R has changed to N or T;

[0783] - At position 7, N has changed to H; and / or

[0784] - At position 8, M has changed to T.

[0785] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0786] (a) SEQ ID NO: 114; and

[0787] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[0788] - At position 1, R has changed to Q;

[0789] - At position 3, T has changed to S; and / or

[0790] - At position 7, D has changed to A or K.

[0791] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0792] (a) SEQ ID NO:131; and

[0793] (b) An amino acid sequence that differs from SEQ ID NO:131 by one or two amino acids, wherein

[0794] - At position 2, S has changed to R; and / or

[0795] - At position 6, S has changed to V.

[0796] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0797] (i) CDR1 is selected from the following groups:

[0798] (a) SEQ ID NOs: 91-93; and

[0799] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:91 by 4, 3, 2, or 1 amino acid; and / or

[0800] (ii) CDR2 is selected from the following groups:

[0801] (c)SEQ ID NOs:114-117; and

[0802] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:114 by 4, 3, 2, or 1 amino acid; and / or

[0803] (iii) CDR3 is selected from the following groups:

[0804] (e)SEQ ID NOs:131-133; and

[0805] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:131 by 3, 2 or 1 amino acid.

[0806] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0807] Nanobodies belonging to the F cluster are represented by 15 clones.

[0808] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of:

[0809] (a) SEQ ID NO:94; and

[0810] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[0811] -At position 3, S has changed to T, A, or G;

[0812] -At position 5, N has changed to S;

[0813] - At position 6, M has changed to T or A; and / or

[0814] - At position 9, L has changed to M.

[0815] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR2 is selected from the group consisting of:

[0816] (a) SEQ ID NO: 118; and

[0817] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, 3, or 4 amino acids, wherein

[0818] -At position 2, H has changed to V;

[0819] - At position 5, S has changed to H or A;

[0820] - At position 8, N has changed to S; and / or

[0821] - At position 10, Y has changed to F.

[0822] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR3 is selected from the group consisting of:

[0823] (a) SEQ ID NO: 134; and

[0824] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, or 4 amino acids, wherein

[0825] -At position 6, A has changed to S or D;

[0826] - At position 7, F has changed to Y or A;

[0827] - At position 8, R has changed to H;

[0828] -At position 9, S has changed to A;

[0829] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[0830] - At position 14, V has changed to I.

[0831] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0832] (i) CDR1 is selected from the following groups:

[0833] (a) SEQ ID NOs: 94-100; and

[0834] (b) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:94 by 4, 3, 2, or 1 amino acid; and / or

[0835] (ii) CDR2 is selected from the following groups:

[0836] (c)SEQ ID NOs:118-122; and

[0837] (d) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:118 by 4, 3, 2, or 1 amino acid; and / or

[0838] (iii) CDR3 is selected from the following groups:

[0839] (e)SEQ ID NOs:134-143; and

[0840] (f) An amino acid sequence that differs from the amino acid sequence of SEQ ID NO:134 by 3, 2 or 1 amino acid.

[0841] In one embodiment, the present invention relates to a polypeptide as described herein, wherein the first ISV is substantially composed of four frame regions (FR1 to FR4, respectively) and three 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.

[0842] The second immunoglobulin single variable domain (ISV) of the polypeptide of the present invention has a high affinity for antigens on target cells (preferably cancer cells) and binds to antigens on target cells (preferably cancer cells). "Target cell" herein refers to a cell on which a specific antigen is present. In a preferred aspect, the "target cell" is a cancer cell.

[0843] A membrane (also called the plasma membrane or phospholipid bilayer) surrounds the cytoplasm of a cell and forms the outer boundary of the cell; that is, the membrane is the surface of the cell. This membrane serves to separate and protect the cell from its external environment and is mostly composed of a phospholipid bilayer. Embedded within this membrane are various protein molecules, such as channels, pumps, and cell receptors. Because the membrane is fluid, protein molecules can move within it. As used herein, the term "antigen on a target cell" refers to a molecule exhibited on the surface of the cell. In most cases, this molecule will be located within or on the cell's plasma membrane, such that at least a portion of the molecule remains accessible from the outside of the cell in a tertiary conformation. Non-limiting examples of cell surface molecules located in the plasma membrane are transmembrane proteins that contain both hydrophilic and hydrophobic regions in their tertiary conformation. Here, at least one hydrophobic region allows the cell surface molecule to be embedded in or inserted into the cell's hydrophobic plasma membrane, while the hydrophilic region extends to the cytoplasm and extracellular space on either side of the plasma membrane, respectively.

[0844] The antigen can be any target on the cell, such as a tumor antigen. In a preferred embodiment, the antigen is specific to the target cell (e.g., a cancer cell), such as a tumor-associated antigen (TAA) on the cancer cell.

[0845] As used herein, the term "tumor antigen" can be understood as those antigens presented on tumor cells. These antigens are capable of presenting an extracellular portion on the cell surface, which is typically combined with transmembrane and cytoplasmic portions of the molecule. These antigens can sometimes be presented solely 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 called tumor-specific antigens. However, this is not usually the case. More commonly, antigens are presented by both tumor cells and normal cells, and these are called "tumor-associated antigens (TAAs)." These tumor-associated antigens can be overexpressed on tumor cells compared to normal cells, or because the tumor tissue structure is less compact, making antibody binding in tumor cells more readily available compared to normal cells. TAAs are preferably antigens expressed on cells of a specific tumor but preferably not expressed in normal cells. Typically, TAAs are antigens that are normally expressed in cells only at a specific point in the organism's development (such as during fetal development) and are inappropriately expressed in the organism at the current point of development, or antigens that are not expressed in normal tissues or cells of the organ currently expressing the antigen.

[0846] In one embodiment, the first antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0847] In one embodiment, the second antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0848] In one embodiment, the antigen is present more abundantly on cancer cells than on normal cells. The antigen on the target cells 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 activating protein (FAP), CD19, and CD33.

[0849] 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.

[0850] Other tumor-associated antigens suitable as antigens on target cells for binding to the second ISV within the peptide of the present invention include: TAG-72, Ep-CAM, PSMA, PSA, and glycolipids such as GD2 and GD3.

[0851] The TAA of this invention also includes hematopoietic differentiation antigens, namely glycoproteins commonly associated with cluster differentiation (CD) grouping, such as CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD69, and CD147; growth factor receptors, including HER2, ErbB3, and ErbB4; and cytokine receptors, including the interleukin-2 receptor γ chain (CD132 antigen), the interleukin-10 receptor α chain (IL-10R-A), the interleukin-10 receptor β chain (IL-10R-B), and the interleukin-12 receptor β-1 chain (IL-12R-β). 1) 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), type I interleukin-1 receptor (IL-1R-1) (CD121a), type II interleukin-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); and others such as CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endothelial sialic acid protein), CD44v6, gpA33, Ron, Trop2, PSCA, dense protein 6, dense protein 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC1 6, EGFRvIII, AGS-16, CD27L, stalkin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, phosphatidylinositol glycan-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, CA195, DR5, DR6, DcR3, and CAIX.

[0852] Therefore, this invention relates to polypeptides 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 γ chain (CD132 antigen), interleukin-10 receptor α chain (IL-10R-A), and interleukin-10 receptor β chain (IL-10R-A). 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), type I interleukin-1 receptor (IL-1R-1) (CD121a), type II interleukin-1 receptor (IL-1R-1) (CD121a), ... Interleukin-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 (endothelial sialic acid protein), CD44v6, gpA33, Ron, Trop2, PSCA, dense protein 6, dense protein 18.2, CLEC12A, CD38, ephA2, c-Met, CD 56, MUC16, EGFRvIII, AGS-16, CD27L, stalkin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, phosphatidylinositol glycan-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 polymorphs and isotypes, preferably the TAA is CD20 (UniProt 11836), HER2 (Uniprot P04626), its polymorphs and / or isotypes.

[0853] The second structural unit, ISV, nanobody, or VHH of the present invention has high affinity for its antigen. The second structural unit, ISV, or nanobody of the present invention may, for example, target an antigenic determinant, epitope, portion, domain, subunit, or conformation (where applicable) of the antigen on a target cell.

[0854] The target cells of the present invention particularly relate to mammalian cells, and preferably to primate cells, and even more preferably to human cells. Target cells are preferably hyperproliferating cells, such as, for example, cancer cells.

[0855] This invention relates to polypeptides as described herein, wherein the second or additional ISV binds to an antigen on a target cell with an average KD value between 100 nM and 10 pM, such as an average KD value below 90 nM, even more preferably an average KD value below 80 nM, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even smaller, 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 smaller, such as less than 10 pM. Preferably, the KD is determined by KinExA or SPR, for example, as determined by Proteon.

[0856] Therefore, the present invention relates to polypeptides as described herein, wherein the second ISV or additional ISV has high affinity for its antigen when measured in monovalent form.

[0857] Therefore, the present invention relates to polypeptides as described herein, wherein the average KD is measured by surface plasmon resonance (SPR) and / or KinExA or Proteon, for example on recombinant proteins, as described in the Examples section.

[0858] The present invention also relates to peptides as described herein, wherein the second ISV or additional ISV binds to an antigen on a target cell with an EC50 value between 100 nM and 1 pM, such as an average EC50 value below 100 nM, even more preferably an average EC50 value below 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.

[0859] Therefore, the present invention relates to polypeptides as described herein, wherein the average EC50 is determined by FACS or ELISA on a monovalent second ISV (such as a nanobody) or a polypeptide containing a monovalent second ISV (such as a nanobody).

[0860] As demonstrated in the examples, KD correlates well with EC50.

[0861] Simultaneous targeting of multiple antigens reduces the likelihood of tumor escape variants, thus improving the therapeutic activity of T-cell conjugation strategies. This invention provides multispecific peptides comprising CD3 ISVs (see Example 19) combined with immunoglobulin single variable domains targeting different (target) antigens. Preferred combinations of first and second antigens are provided below (it will be understood that ISVs binding to said antigens can be placed in any order within the peptides of this invention):

[0862] 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)

[0863] Similarly, simultaneously targeting multiple epitopes, antigenic determinants, portions, domains, subunits, or conformations of proteins or antigens on target cells can reduce the likelihood of generating tumor escape variants, thus improving the therapeutic activity of T-cell conjugation strategies (see Example 20). This invention provides peptides comprising an anti-CD3 ISV (also known as a double-complementary-site construct) combined with an immunoglobulin monovariable domain targeting different epitopes, antigenic determinants, portions, domains, subunits, or conformations of antigens on target cells. Preferred combinations of first and second TAA ISVs are provided below (it will be understood that ISVs binding said antigens can be placed in any order in the peptides of this invention):

[0864]

[0865]

[0866] The polypeptides and compositions of the present invention can be used to prevent and / or treat the diseases and conditions of the present invention (also referred to herein as "diseases and conditions of the present invention"), which include, but are not limited to, cancer. The term "cancer" refers to a pathological condition in mammals 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 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 bowel cancer, rectal cancer, and more. Kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine carcinoma, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary exudative lymphoma, neuroectodermal tumors, rhabdomyosarcoma (for other cancers see, for example, Cancer, Principles and Practice (DeVita et al., ed. 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.

[0867] For a general description of immunoglobulin single variable domains, further descriptions are provided below, along with references to the prior art cited herein. However, it should be noted that this description and the prior art primarily describe the so-called "V" domain. H Type 3 immunoglobulin single variable domain (i.e., with V) H Type 3 human germline sequences such as DP-47, DP-51, or DP-29 (which have highly sequence-homological immunoglobulin monovariable domains) form a preferred aspect of the invention. However, it should be noted that the invention covers any type of immunoglobulin monovariable domain in its broadest sense and, for example, also encompasses those belonging to the so-called "V" class. H The "type 4" immunoglobulin single variable domain (i.e., with V) H Type 4 human germline sequences, such as DP-78, have highly sequence-homological immunoglobulin single variable domains, as described, for example, in WO 07 / 118670.

[0868] Typically, immunoglobulin monovariable domains (especially V) HH Sequences and sequence-optimized immunoglobulin monovariable domains can be particularly characterized by the presence of the above-mentioned “characteristic residues” (as described herein) in more than one frame region sequence (as described, for example, in Table B-2).

[0869] Table B-2: Characteristic residues in VHHs

[0870]

[0871]

[0872] The immunoglobulins of the present 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 (preferably naturally occurring) an amino acid residue independently selected and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), relating to WO 12 / 175741 and WO 15 / 060643.

[0873] In addition and / or additionally, the immunoglobulins of the present invention may have certain preferred amino acid residues at positions 11, 89, 110 and / or 112, as further described in detail in WO 15 / 060643 (which is incorporated herein by reference).

[0874] Moreover, such immunoglobulin single variable domains can be derived from any suitable source in any appropriate manner, such as naturally occurring V. HH The immunoglobulin single variable domain can be a sequence (i.e., derived from a suitable camel species, such as a llama) or a synthetic or semi-synthetic VHs or VLs (e.g., derived from humans). Such immunoglobulin single variable domains can include “humanized” or other “sequence-optimized” VHHs modified by techniques such as affinity maturation (e.g., starting with a synthetic, random, or naturally occurring immunoglobulin sequence), CDR transplantation, mosaicking, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to those skilled in the art involving modified immunoglobulin sequences, “camel-derived” immunoglobulin sequences (particularly camel-derived heavy chain variable domain sequences, i.e., camel-derived VHs), and human VHs, human VLs, camel-derived VHHs; or any suitable combination of the foregoing, as further described herein. As described herein, particularly preferred types of immunoglobulin single variable domains of the present invention include those having a similar structure to naturally occurring VHs. HH The amino acid sequence of the domain corresponds to, but is "humanized," the amino acid sequence of the immunoglobulin monovariable domain. This humanization is achieved by using V-type immunoglobulin monovariable domains derived from conventional 4-chain antibodies from humans (e.g., as mentioned above). H One or more amino acid residues appearing at one or more corresponding positions in the domain replace the naturally occurring V. HHThe amino acid sequence of the sequence (and especially in the framework sequence) contains more than one amino acid residue. This can be done in a manner known to those skilled in the art, for example based on the prior art in humanization as further described herein and cited herein. Again, it should be noted that such humanized immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known herein and are therefore not strictly limited to the use of naturally occurring V HH Peptides obtained by using peptides with structural domains as starting materials.

[0875] Another particularly preferred type of immunoglobulin monovariable domain of the present invention includes those having a domain corresponding to naturally occurring V. H The amino acid sequence of the domain, but an immunoglobulin monovariable domain whose amino acid sequence has been "camelized," the camelization being achieved by using a heavy chain antibody V. HH One or more amino acid residues present at one or more corresponding positions in the domain are substituted from the naturally occurring V of conventional 4-chain antibodies. H The amino acid sequence of the domain contains one or more amino acid residues. This can be done in a manner known to those skilled in the art, for example, based on what is described herein. Such a "camelization" substitution is preferably inserted into V. H -V L At the amino acid sites formed and / or present at the interface, and / or at so-called camelid features, 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)). Preferably, V is used as the starting material or starting point for generating or designing camelidized immunoglobulin single variable domains. H The preferred sequence is V from mammals. H Sequence, more preferably human V H Sequences, such as V H 3. Sequence. However, it should be noted that such camel-like 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 naturally occurring V. H Peptides obtained by using peptides with structural domains as starting materials.

[0876] For example, as further described in this paper, "humanization" and "camelization" can be achieved by providing separately encoded naturally occurring V... HH Domain or V HThe nucleotide sequence of the domain is modified, and then, in a manner known per se, one or more codons in the nucleotide sequence are altered such that the new nucleotide sequences respectively encode the "humanized" or "camelized" immunoglobulin monovariable domain of the present invention. This nucleic acid can then be expressed in a manner known per se, thereby providing the immunoglobulin monovariable domain required by the present invention. Alternatively, based on naturally occurring V... HH Domain or V H The amino acid sequence of the domain can be designed to represent the humanized or camel-derived immunoglobulin single variable domain required by this invention, and then synthesized de novo using known peptide synthesis techniques. Furthermore, based on naturally occurring V... HH Domain or V H The amino acid or nucleotide sequence of the domain can be designed to encode the desired humanized or camelized immunoglobulin single variable domain of the present invention, and then synthesized de novo using techniques known per se for nucleic acid synthesis. After this, the resulting nucleic acid can be expressed in a manner known per se to provide the immunoglobulin single variable domain required by the present invention.

[0877] Therefore, this invention relates to polypeptides as described herein, wherein the ISV is a nanobody, V HH Humanized V HH or camel-derived V H .

[0878] Generally, proteins or peptides that comprise or consist essentially of a single structural unit, a single immunoglobulin monovariable domain, or a single nanobody will be referred to in this document as “monovalent” proteins or peptides, “monovalent constructs,” “monovalent structural units,” “monovalent immunoglobulin monovariable domains,” or “monovalent nanobodies,” respectively.

[0879] In this respect, the present invention also relates to the monovalent structural units of the polypeptides constituting the present invention.

[0880] Therefore, the present invention relates to an ISV or polypeptide that specifically binds to a constant domain of CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0881] (i) CDR1 is selected from the following groups:

[0882] (a) SEQ ID NOs: 81-100; or

[0883] (b) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from any of SEQ ID NOs:81-100, provided that the polypeptide containing CDR1 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0884] (ii) CDR2 is selected from the following groups:

[0885] (c)SEQ ID NOs:101-122; or

[0886] (d) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from any of SEQ ID NOs:101-122, provided that the peptide containing CDR2 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to binding to a peptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0887] (iii) CDR3 is selected from the following groups:

[0888] (e)SEQ ID NOs:123-143; or

[0889] (f) An amino acid sequence having a difference of 4, 3, 2 or 1 amino acid from any of SEQ ID NOs:123-143, provided that the polypeptide containing CDR3 having said 4, 3, 2 or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR3 without said 4, 3, 2 or 1 amino acid difference.

[0890] As discussed above, ISVs belonging to different clusters are separated based on the structural similarities and differences in CDR2 and CDR3.

[0891] Immunoglobulin monovariable domains belonging to cluster A are represented by polypeptides according to the following:

[0892] (i) CDR1 is selected from the following groups:

[0893] (a) SEQ ID NOs: 81-87; and

[0894] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:81 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0895] (ii) CDR2 is selected from the following groups:

[0896] (c)SEQ ID NOs:101-109; and

[0897] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:101 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0898] (iii) CDR3 is selected from the following groups:

[0899] (e)SEQ ID NOs:123-127; and

[0900] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:123, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0901] In another respect, among polypeptides belonging to cluster A, CDR1 is selected from the group consisting of:

[0902] (a) SEQ ID NO:81; and

[0903] (b) An amino acid sequence that differs from SEQ ID NO:81 by one or two amino acids, wherein

[0904] -At position 1, G has changed to R;

[0905] -At position 3, T has changed to A;

[0906] -At position 4, Y has changed to F;

[0907] - At position 8, S has changed to G; and / or

[0908] - At position 10, G has changed to A.

[0909] In another respect, among polypeptides belonging to cluster A, CDR2 is selected from the group consisting of:

[0910] (a) SEQ ID NO:101; and

[0911] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, or 3 amino acids, wherein

[0912] -At position 3, V has changed to T or A;

[0913] -At position 5, S has changed to T;

[0914] - At position 6, G has changed to D or E; and / or

[0915] - At position 9, T has changed to S, A, or P.

[0916] In another respect, among polypeptides belonging to cluster A, CDR3 is selected from the group consisting of:

[0917] (a) SEQ ID NO:123; and

[0918] (b) An amino acid sequence that differs from SEQ ID NO:123 by one or two amino acids, wherein

[0919] -At position 2, I has changed to T;

[0920] - At position 9, I has changed to V; and / or

[0921] - At position 10, A has changed to P.

[0922] Therefore, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0923] (i) CDR1 is selected from the following groups:

[0924] (a) SEQ ID NO:81; and

[0925] (b) An amino acid sequence that differs from SEQ ID NO:81 by one or two amino acids, wherein

[0926] -At position 1, G has changed to R;

[0927] -At position 3, T has changed to A;

[0928] -At position 4, Y has changed to F;

[0929] - At position 8, S has changed to G; and / or

[0930] - At position 10, G has changed to A.

[0931] And among them

[0932] (ii) CDR2 is selected from the following groups:

[0933] (a) SEQ ID NO:101; and

[0934] (b) An amino acid sequence that differs from SEQ ID NO:101 by 1, 2, or 3 amino acids, wherein

[0935] -At position 3, V has changed to T or A;

[0936] -At position 5, S has changed to T;

[0937] - At position 6, G has changed to D or E; and / or

[0938] -At position 9, T has changed to S, A, or P.

[0939] And among them

[0940] (iii) CDR3 is selected from the following groups:

[0941] (a) SEQ ID NO:123; and

[0942] (b) An amino acid sequence that differs from SEQ ID NO:123 by one or two amino acids, wherein

[0943] -At position 2, I has changed to T;

[0944] - At position 9, I has changed to V; and / or

[0945] - At position 10, A has changed to P.

[0946] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:81, CDR2 by SEQ ID NO:101, and CDR3 by SEQ ID NO:123. Preferably, the polypeptide is selected from any one of SEQ ID NOs:1 to 50.

[0947] Immunoglobulin single variable domains belonging to cluster B are represented by polypeptides according to the following:

[0948] (i) CDR1 is selected from the following groups:

[0949] (a) SEQ ID NOs:88; and

[0950] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:88 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0951] (ii) CDR2 is selected from the following groups:

[0952] (c)SEQ ID NOs:110; and

[0953] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:110 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0954] (iii) CDR3 is selected from the following groups:

[0955] (e)SEQ ID NOs:128; and

[0956] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:128, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0957] In another respect, among peptides belonging to the B cluster, CDR1 is SEQ ID NO:88.

[0958] In another respect, among the peptides belonging to the B cluster, CDR2 is SEQ ID NO:110.

[0959] In another respect, among peptides belonging to the B cluster, CDR3 is SEQ ID NO:128.

[0960] In another aspect, the present invention relates to a polypeptide wherein: CDR1 is represented by SEQ ID NO:88, CDR2 by SEQ ID NO:110, and CDR3 by SEQ ID NO:128. Preferably, the polypeptide is SEQ ID NO:51.

[0961] The single variable domain of immunoglobulins belonging to cluster C is represented by a polypeptide according to the following:

[0962] (i) CDR1 is selected from the following groups:

[0963] (a) SEQ ID NO:90; and

[0964] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:90 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0965] (ii) CDR2 is selected from the following groups:

[0966] (c)SEQ ID NOs:112-113; and

[0967] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:112 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0968] (iii) CDR3 is selected from the following groups:

[0969] (e)SEQ ID NO:130; and

[0970] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:130, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0971] In another respect, among the peptides belonging to the C cluster, CDR1 is SEQ ID NO:90.

[0972] In another respect, among peptides belonging to the C cluster, CDR2 is selected from the group consisting of:

[0973] (a) SEQ ID NO:112; and

[0974] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0975] - At position 2, V has changed to A.

[0976] In another respect, among the peptides belonging to the C cluster, CDR3 is SEQ ID NO:130.

[0977] Therefore, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[0978] (i)CDR1 is SEQ ID NO:90; and

[0979] And among them

[0980] (ii) CDR2 is selected from the following groups:

[0981] (a) SEQ ID NO:112; and

[0982] (b) An amino acid sequence that differs from SEQ ID NO:112 by one amino acid, wherein

[0983] - At position 2, V has changed to A.

[0984] And among them

[0985] (iii) CDR3 is SEQ ID NO:130.

[0986] In another aspect, the present invention relates to a polypeptide wherein: CDR1 is represented by SEQ ID NO:90, CDR2 by SEQ ID NO:112, and CDR3 by SEQ ID NO:130. Preferably, the polypeptide is selected from any one of SEQ ID NOs:53-56.

[0987] The single variable domain of immunoglobulins belonging to cluster D is represented by a polypeptide according to the following:

[0988] (i) CDR1 is selected from the following groups:

[0989] (a) SEQ ID NOs:89; and

[0990] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:89 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[0991] (ii) CDR2 is selected from the following groups:

[0992] (c)SEQ ID NOs:111; and

[0993] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:111 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[0994] (iii) CDR3 is selected from the following groups:

[0995] (e)SEQ ID NOs:129; and

[0996] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:129, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[0997] In another respect, among the peptides belonging to the D cluster, CDR1 is SEQ ID NO:89.

[0998] In another respect, among the peptides belonging to the D cluster, CDR2 is SEQ ID NO:111.

[0999] In another respect, among the peptides belonging to the D cluster, CDR3 is SEQ ID NO:129.

[1000] In another aspect, the present invention relates to a polypeptide wherein: CDR1 is represented by SEQ ID NO:89, CDR2 by SEQ ID NO:111, and CDR3 by SEQ ID NO:129. Preferably, the polypeptide is SEQ ID NO:52.

[1001] The immunoglobulin single variable domain belonging to cluster E is represented by a polypeptide according to the following:

[1002] (i) CDR1 is selected from the following groups:

[1003] (a) SEQ ID NOs: 91-93; and

[1004] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:91 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[1005] (ii) CDR2 is selected from the following groups:

[1006] (c)SEQ ID NOs:114-117; and

[1007] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:114 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[1008] (iii) CDR3 is selected from the following groups:

[1009] (e)SEQ ID NOs:131-133; and

[1010] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:131, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[1011] In another respect, among peptides belonging to the E cluster, CDR1 is selected from the group consisting of:

[1012] (a) SEQ ID NO: 91; and

[1013] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[1014] - At position 6, R has changed to N or T;

[1015] - At position 7, N has changed to H; and / or

[1016] - At position 8, M has changed to T.

[1017] In another respect, among peptides belonging to the E cluster, CDR2 is selected from the group consisting of:

[1018] (a) SEQ ID NO: 114; and

[1019] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[1020] - At position 1, R has changed to Q;

[1021] - At position 3, T has changed to S; and / or

[1022] - At position 7, D has changed to A or K.

[1023] In another respect, among peptides belonging to the E cluster, CDR3 is selected from the group consisting of:

[1024] (a) SEQ ID NO:131; and

[1025] (b) An amino acid sequence that differs from SEQ ID NO:131 by one amino acid, wherein

[1026] - At position 2, S has changed to R; and / or

[1027] - At position 6, S has changed to V.

[1028] Therefore, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[1029] (i) CDR1 is selected from the following groups:

[1030] (a) SEQ ID NO: 91; and

[1031] (b) An amino acid sequence that differs from SEQ ID NO:91 by 1, 2, or 3 amino acids, wherein

[1032] - At position 6, R has changed to N or T;

[1033] - At position 7, N has changed to H; and / or

[1034] - At position 8, M has changed to T.

[1035] And among them

[1036] (ii) CDR2 is selected from the following groups:

[1037] (a) SEQ ID NO: 114; and

[1038] (b) An amino acid sequence that differs from SEQ ID NO:114 by 1, 2, or 3 amino acids, wherein

[1039] - At position 1, R has changed to Q;

[1040] - At position 3, T has changed to S; and / or

[1041] - At position 7, D has changed to A or K.

[1042] And among them

[1043] (iii) CDR3 is selected from the following groups:

[1044] (a) SEQ ID NO:131; and

[1045] (b) An amino acid sequence that differs from SEQ ID NO:131 by one amino acid, wherein

[1046] - At position 2, S has changed to R; and / or

[1047] - At position 6, S has changed to V.

[1048] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:91, CDR2 by SEQ ID NO:114, and CDR3 by SEQ ID NO:131. Preferably, the polypeptide is selected from any of SEQ ID NOs:57-65.

[1049] The immunoglobulin single variable domain belonging to cluster F is represented by a polypeptide according to the following:

[1050] (i) CDR1 is selected from the following groups:

[1051] (a) SEQ ID NOs: 94-100; and

[1052] (b) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:94 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR1 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR1 without said 4, 3, 2, or 1 amino acid difference; and / or

[1053] (ii) CDR2 is selected from the following groups:

[1054] (c)SEQ ID NOs:118-122; and

[1055] (d) An amino acid sequence differing from the amino acid sequence of SEQ ID NO:118 by 4, 3, 2, or 1 amino acid, provided that the polypeptide containing CDR2 with said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as, as measured by, surface plasmon resonance, compared to the binding of a polypeptide containing CDR2 without said 4, 3, 2, or 1 amino acid difference; and / or

[1056] (iii) CDR3 is selected from the following groups:

[1057] (e)SEQ ID NOs:134-143; and

[1058] (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO:134, provided that the polypeptide containing CDR3 having said 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as the polypeptide containing CDR3 without said 4, 3, 2, or 1 amino acid difference, said affinity as measured by surface plasmon resonance.

[1059] In another respect, among peptides belonging to the F cluster, CDR1 is selected from the group consisting of:

[1060] (a) SEQ ID NO:94; and

[1061] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[1062] -At position 3, S has changed to T, A, or G;

[1063] -At position 5, N has changed to S;

[1064] - At position 6, M has changed to T or A; and / or

[1065] - At position 9, L has changed to M.

[1066] In another respect, among peptides belonging to the F cluster, CDR2 is selected from the group consisting of:

[1067] (a) SEQ ID NO: 118; and

[1068] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, or 3 amino acids, wherein

[1069] -At position 2, H has changed to V;

[1070] - At position 5, S has changed to H or A;

[1071] - At position 8, N has changed to S; and / or

[1072] - At position 10, Y has changed to F.

[1073] In another respect, among peptides belonging to the F cluster, CDR3 is selected from the group consisting of:

[1074] (a) SEQ ID NO: 134; and

[1075] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, 4, or 5 amino acids, wherein

[1076] -At position 6, A has changed to S or D;

[1077] - At position 7, F has changed to Y or A;

[1078] - At position 8, R has changed to H;

[1079] -At position 9, S has changed to A;

[1080] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[1081] - At position 14, V has changed to I.

[1082] Therefore, the present invention relates to an ISV or polypeptide that specifically binds to CD3 and comprises or substantially consists of: four frame regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein:

[1083] (i) CDR1 is selected from the following groups:

[1084] (a) SEQ ID NO:94; and

[1085] (b) An amino acid sequence that differs from SEQ ID NO:94 by 1, 2, 3, or 4 amino acids, wherein

[1086] -At position 3, S has changed to T, A, or G;

[1087] -At position 5, N has changed to S;

[1088] - At position 6, M has changed to T or A; and / or

[1089] - At position 9, L has changed to M.

[1090] And among them

[1091] (ii) CDR2 is selected from the following groups:

[1092] (a) SEQ ID NO: 118; and

[1093] (b) An amino acid sequence that differs from SEQ ID NO:118 by 1, 2, or 3 amino acids, wherein

[1094] -At position 2, H has changed to V;

[1095] - At position 5, S has changed to H or A;

[1096] - At position 8, N has changed to S; and / or

[1097] - At position 10, Y has changed to F.

[1098] And among them

[1099] (iii) CDR3 is selected from the following groups:

[1100] (a) SEQ ID NO: 134; and

[1101] (b) An amino acid sequence that differs from SEQ ID NO:134 by 1, 2, 3, 4, or 5 amino acids, wherein

[1102] -At position 6, A has changed to S or D;

[1103] - At position 7, F has changed to Y or A;

[1104] - At position 8, R has changed to H;

[1105] -At position 9, S has changed to A;

[1106] - At position 11, G has changed to D, T, N, S, K, or R; and / or

[1107] - At position 14, V has changed to I.

[1108] In another aspect, the present invention provides a polypeptide as described herein, wherein: CDR1 is represented by SEQ ID NO:94, CDR2 by SEQ ID NO:118, and CDR3 by SEQ ID NO:134. Preferably, the polypeptide is selected from any of SEQ ID NOs:66-80.

[1109] In another aspect, the present invention relates to polypeptides that cross-block the binding of CD3 by at least one of an ISV or polypeptide belonging to a cluster of A, B, C, D, E or F.

[1110] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by at least one of an ISV or polypeptide having SEQ ID NOs:1-50.

[1111] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by an ISV or polypeptide having SEQ ID NO:51.

[1112] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by at least one of an ISV having SEQ ID NOs:53-56 or a polypeptide.

[1113] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by an ISV or polypeptide having SEQ ID NO:52.

[1114] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by at least one of an ISV having SEQ ID NOs:57-65 or a polypeptide.

[1115] Therefore, the present invention relates to polypeptides that cross-block the binding of CD3 by at least one of an ISV having SEQ ID NOs:66-80 or a polypeptide.

[1116] In another aspect, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of an ISV or polypeptide belonging to a cluster of A, B, C, D, E or F.

[1117] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides belonging to SEQ ID NOs:1-50.

[1118] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by an ISV or polypeptide having SEQ ID NO:51.

[1119] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides belonging to SEQ ID NOs:53-56.

[1120] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by an ISV or polypeptide having SEQ ID NO:52.

[1121] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides belonging to SEQ ID NOs:57-65.

[1122] Therefore, the present invention relates to polypeptides whose binding to CD3 is cross-blocked by at least one of the ISVs or polypeptides belonging to SEQ ID NOs:66-80.

[1123] This invention also relates to compounds or constructs, and particularly to proteins or polypeptides comprising or substantially consisting of one or more ISVs or polypeptides of this invention, and optionally further comprising one or more other groups, residues, portions, or binding units. Those skilled in the art will appreciate from the further disclosure herein that such other groups, residues, portions, binding units, or amino acid sequences may or may not provide additional functionality to the polypeptides of this invention (and / or to the compounds or constructs in which said polypeptides are present), and may or may not alter the properties of the polypeptides of this invention.

[1124] In a particular but non-limiting aspect of the invention, which will be further described herein, the ISVs and peptides of the invention may have an increased half-life in serum compared to immunoglobulin monovariable domains or peptides from which they are derived (as further described herein). For example, the immunoglobulin monovariable domains or peptides of the invention may be linked (chemically or otherwise) to one or more groups or structural moieties that extend the half-life to provide derivatives of the ISVs or peptides of the invention having an increased half-life.

[1125] In one particular aspect of the invention, the compounds or constructs of the invention, or the peptides of the invention, may have an increased half-life compared to the corresponding ISVs or peptides of the invention. Some preferred, but non-limiting, examples of such compounds, constructs, and peptides will become apparent to those skilled in the art based on further disclosure herein, such as immunoglobulin monovariable domains or peptides of the invention that have been chemically modified to increase their half-life (e.g., by PEGylation); immunoglobulin monovariable domains or peptides of the invention containing at least one additional binding site for binding serum proteins (e.g., serum albumin); or constructs or peptides of the invention containing at least one structural motif (particularly at least one amino acid sequence) linked to an ISV or peptide of the invention that increases the half-life of the invention. Examples of ISVs or peptides of the present invention that include such extended half-life structural portions or immunoglobulin monovariable domains will become apparent to those skilled in the art based on the further disclosure herein; for example, including but not limited to peptides in which one or more immunoglobulin monovariable domains or peptides of the present 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 serum proteins (e.g., domain antibodies capable of binding serum proteins such as serum albumin (e.g., human serum albumin), serum immunoglobulins such as IgG, or transferrin, immunoglobulin monovariable domains suitable for use as domain antibodies, monodomain antibodies, immunoglobulin monovariable domains suitable for use as monodomain antibodies, “dAbs”’s, immunoglobulin monovariable domains suitable for use as dAbs, or nanobodies; see further description herein and referenced references); ISVs or peptides in which the present invention are linked to an Fc portion (e.g., human Fc) or suitable portions or fragments thereof; or peptides in which one or more immunoglobulin monovariable domains or peptides of the present invention are suitably linked to one or more small proteins or peptides capable of binding serum proteins (e.g., but not limited to WO). Polypeptides (proteins and peptides) linked together as described in WO 91 / 01743, WO 01 / 45746, WO02 / 076489, WO08 / 068280, WO 09 / 127691 and WO 11 / 095545.

[1126] Generally, the half-life of the compounds, constructs, or peptides of the present invention having an increased half-life is preferably at least 1.5 times, more preferably at least 2 times, for example at least 5 times, for example at least 10 times, or greater than 20 times, the half-life of the corresponding ISV or peptide itself of the present invention. For example, the compounds, constructs, or peptides of the present invention having an increased half-life may, for example in humans, have a half-life greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, for example greater than 12 hours, or even greater than 24, 48, or 72 hours.

[1127] In a preferred but non-limiting aspect of the invention, such compounds, constructs or peptides of the invention have, for example in humans, a serum half-life greater than that of the corresponding ISV or peptide of the invention by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours, or even more than 24, 48 or 72 hours.

[1128] In another preferred but non-limiting aspect of the invention, such compounds, constructs, or peptides of the invention exhibit a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or longer. For example, the compounds, constructs, or peptides of the invention may have a half-life of 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 greater than 14 days (e.g., about 14-19 days).

[1129] This invention demonstrates that the inclusion of a binding unit targeting albumin in the construct itself does not significantly affect the efficacy or effectiveness obtained. Although a slight loss of efficacy / potency was observed in the presence of HSA, the CD3 multispecific peptide with extended half-life remained effective in tumor cell killing. Albumin-based drug delivery has been shown to be useful for achieving improved cancer therapy, largely due to its passive targeting of tumors through enhanced permeability and retention effects, as well as the increased demand of tumor cells for albumin as an energy and amino acid source. However, albumin lacks not only an active mechanism to overcome cell membrane barriers but also the ability to penetrate tumor tissue (Qianqian Guo et al., Polym. Chem., 2013, 4, 4584-4587).

[1130] In a particularly preferred but non-limiting aspect of the invention, the present invention provides a polypeptide comprising first and second immunoglobulin single variable domains (ISVs); and further comprising one or more (preferably one) serum albumin-binding immunoglobulin single variable domains as described herein, such 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).

[1131] Table B-3: Immunoglobulin monovariable domains in the HLE of the ISV and peptides used in this invention

[1132]

[1133]

[1134] Therefore, the present invention relates to polypeptides as described herein, further comprising a serum protein-binding portion.

[1135] This invention relates to polypeptides as described herein, wherein the serum protein-binding portion binds to serum albumin.

[1136] This invention relates to polypeptides as described herein, wherein the serum protein binding portion is an immunoglobulin monovariable domain that binds serum albumin.

[1137] This invention relates to the polypeptide as described herein, wherein the ISV that binds serum albumin is substantially composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein: CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SIGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375), with CDRs determined according to the Kabat definition; and / or wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SIGSGSSDTL (SEQ ID NO: 378), and CDR3 is GGSLSR (SEQ ID NO: 375), with CDRs determined according to Kontermann 2010.

[1138] This invention relates to polypeptides as described herein, wherein the ISVs that bind serum albumin include Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG (Table B-3).

[1139] In the polypeptides of the present invention, two or more structural units, ISVs or nanobodies and optionally one or more polypeptides, one or more other groups, drugs, agents, residues, portions 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 through one or more suitable spacer regions or linkers, or any combination thereof.

[1140] Suitable spacer regions or linkers for multivalent and multispecific peptides will be clear to those skilled in the art and can generally be any linker or spacer region in the art used for linking amino acid sequences. Preferably, the linker or spacer region is adapted for constructing proteins or peptides intended for pharmaceutical use.

[1141] Some particularly preferred spacer regions include spacer regions and linkers used in the art for linking antibody fragments or antibody domains. These include linkers mentioned in the general background cited above, as well as linkers used in the art for constructing diabodies or ScFv fragments (however, in this respect, it should be noted that in diabodies and ScFv fragments, the linker sequence used should have a sequence that allows for the relevant V... H and V L The length, flexibility, and other properties of the domains that come together to form a complete antigen-binding site are not specifically limited to the length or flexibility of the linker for the peptides used in this invention, since each ISV or nanobody itself forms a complete antigen-binding site.

[1142] For example, the linker can be a suitable amino acid sequence, and especially between 1 and 50, preferably between 1 and 30, such as an amino acid sequence of 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, such as type (gly x ser y ) zThe preferred adapters include, for example, (gly4ser)3 or (gly3ser2)3, as described in WO 99 / 42077, and the GS30, GS15, GS9, and GS7 adapters described by Ablynx in the application mentioned herein (see, for example, WO06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as hinge regions of naturally occurring heavy chain antibodies or similar sequences (as described in WO 94 / 04678). Preferred adapters are described in Table B-4.

[1143] Table B-4: Connectors

[1144] 5GS GGGGS 7GS SGGSGGS 9GS GGGGSGGGS 10GS GGGGSGGGGS 15GS GGGGSGGGGSGGGGS 18GS GGGGSGGGGSGGGGGGGGS 20GS GGGGSGGGGSGGGGSGGGGS 25GS GGGGSGGGGSGGGGSGGGGSGGGGS 30GS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Poly A AAA

[1145] Some other particularly preferred connectors are polyalanine (such as AAA), as well as connectors GS30 (SEQ ID NO:85 in WO06 / 122825) and GS9 (SEQ ID NO:84 in WO 06 / 122825).

[1146] Other suitable linkers typically contain organic compounds or polymers, especially those suitable for use in pharmaceutical proteins. For example, poly(ethylene glycol) moieties have been used to link antibody domains, see, for example, WO 04 / 081026.

[1147] Including within the scope of this invention, the length, flexibility, and / or other properties of one or more adapters used (though not critical, as they are typically used in ScFv fragments) can have some influence on the properties of the final peptide of this invention, including, but not limited to, affinity, specificity, or affinity for CD3, or for more than one other antigen. Based on the disclosure herein, those skilled in the art will be able to determine one or more optimal adapters for a particular peptide of this invention, optionally after some limited routine experiments.

[1148] For example, in the multivalent peptides of the present invention comprising structural units, ISVs, or nanobodies targeting first and second targets, the length and flexibility of the linker are preferably such that they allow the various structural units, ISVs, or nanobodies of the present invention present in the peptide to bind to their homologous targets, such as antigenic determinants on the respective targets. Again, based on the disclosure herein, optionally after some limited routine experiments, those skilled in the art will be able to determine one or more optimal linkers for a particular peptide of the present invention.

[1149] Within the scope of this invention, the use of one or more linkers endows the polypeptides of the invention with more than one other advantageous property or functionality, and / or provides more than one site for forming derivatives and / or for attaching functional groups (e.g., derivatives of ISVs, nanobodies, or polypeptides as described herein). For example, linkers containing more than one charged amino acid residue can provide improved hydrophilicity, while linkers forming or containing small epitopes or tags can be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, those skilled in the art will be able to determine the optimal linker for a particular polypeptide of the invention, optionally after some limited routine experiments.

[1150] Finally, when more than two linkers are used for the peptides of the present invention, these linkers may be the same or different. Again, based on the disclosure herein, optionally after some limited routine experiments, those skilled in the art will be able to determine the optimal linker for a particular peptide of the present invention.

[1151] Typically, for ease of expression and production, the peptides of this invention will be linear peptides. However, in its broadest sense, the invention is not limited thereto. For example, when the peptides of this invention comprise three or more structural units, ISVs, or nanobodies, a "star" construct may be provided by using a connector having three or more "arms," ​​each "arm" connected to a structural unit, ISV, or nanobodies. Although generally not preferred, cyclic constructs may also be used.

[1152] Therefore, the present invention relates to polypeptides as described herein, wherein the first ISV and the second ISV, as well as the possible third ISV and / or the serum albumin-binding ISV, are directly connected to each other or connected via a linker.

[1153] This invention relates to peptides as described herein, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS linkers.

[1154] This invention relates to polypeptides as described herein, wherein the serum protein binding portion is based on a non-antibody polypeptide (e.g., PEG).

[1155] This invention also relates to methods for preparing the ISVs, peptides, and constructs described herein. The ISVs, peptides, and constructs of this invention can be prepared in ways known per se, as will become apparent to those skilled in the art from the further description herein. For example, the ISVs, peptides, and constructs of this 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 peptides and constructs include the methods and techniques described herein.

[1156] The method for producing the ISV, polypeptide, or protein construct of the present invention may include the following steps:

[1157] - Expression may be performed in a suitable host cell or host organism (also referred to herein as "the host of the invention") or in another suitable expression system encoding the nucleic acid of the IVS, polypeptide, or protein construct of the invention.

[1158] Optional, followed by:

[1159] - Isolate and / or purify the ISV, polypeptide or protein constructs of the present invention thus obtained.

[1160] Specifically, the method may include the following steps:

[1161] - The host of the present invention is cultured and / or maintained under conditions that enable the host of the present invention to express and / or produce at least one ISV, polypeptide or protein construct of the present invention;

[1162] Optional, followed by:

[1163] - Isolate and / or purify the ISV, polypeptide or protein constructs of the present invention thus obtained.

[1164] Therefore, the present invention also relates to nucleic acid or nucleotide sequences (also referred to as "nucleic acids of the present invention" or "nucleotide sequences of the present invention") encoding the ISV, polypeptide, or protein constructs of the present invention. The nucleic acids of the present 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 present invention may be genomic DNA, cDNA, or synthetic DNA (such as DNA having codons already specifically adapted for expression in the intended host cell or host organism).

[1165] According to one embodiment of the invention, the nucleic acid of the invention is in a substantially isolated form, as defined herein. The nucleic acid of the invention can also be in the form of a vector, present in a vector, and / or part of a vector, such as, for example, a plasmid, granule, or YAC, which can also be in a substantially isolated form.

[1166] Based on the information provided herein regarding the polypeptide or protein constructs of the present invention, the nucleic acids of the present invention can be prepared or obtained in a manner known per se, and / or isolated from suitable natural sources. Furthermore, as will be apparent to those skilled in the art, in order to prepare the nucleic acids of the present invention, several nucleotide sequences (such as at least one nucleotide sequence encoding the immunoglobulin monovariable domain of the present invention) and nucleic acids, for example, encoding one or more adapters, can also be linked together in a suitable manner.

[1167] The techniques used to generate the nucleic acids of this invention will be clear to those skilled in the art and may include, for example, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof) to introduce mutations that result in truncated expression products; introducing one or more restriction sites (e.g., to generate cassettes and / or regions that can be readily cleaved and / or linked using suitable restriction enzymes); and / or introducing mutations via PCR reactions using one or more “mismatched” primers. These and other techniques will be clear to those skilled in the art and may also be found in standard manuals, such as those mentioned herein by Sambrook et al. and Ausubel et al., and in the examples below.

[1168] As will be apparent to those skilled in the art, the nucleic acids of the present invention can also be in the form of genetic constructs, present in genetic constructs, and / or part of genetic constructs. Such genetic constructs typically comprise at least one nucleic acid of the present invention, optionally linked to one or more elements of genetic constructs known per se, such as, for example, one or more suitable regulatory elements (such as suitable promoters, enhancers, terminators, etc.) and other elements of genetic constructs as described herein. Such genetic constructs comprising at least one nucleic acid of the present invention will also be referred to herein as “genetic constructs of the present invention.”

[1169] The genetic constructs of the present invention can be DNA or RNA, and preferably double-stranded DNA. The genetic constructs of the present invention can also be in forms suitable for transformation of a intended host cell or host organism, in forms suitable for integration into the genomic DNA of an intended host cell, or in forms suitable for independent replication, maintenance, and / or inheritance in an intended host organism. For example, the genetic constructs of the present invention can be in the form of vectors, such as plasmids, granules, YACs, viral vectors, or transposons. In particular, the vector can be an expression vector, i.e., a vector capable of providing in vitro and / or in vivo expression (e.g., in suitable host cells, host organisms, and / or expression systems).

[1170] In a preferred but non-limiting embodiment, the genetic construct of the present invention comprises

[1171] a) At least one nucleic acid of the present invention; operably ligated to

[1172] b) One or more control elements, such as a promoter and optionally a suitable terminator;

[1173] And also optional

[1174] c) One or more other elements of a genetic construct that are known in themselves;

[1175] The terms “regulatory element,” “promoter,” “terminator,” and “operably linked” have their usual meanings in the art (as further described herein): and the “other elements” present in the genetic construct can be, for example, 3' or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or elements that can promote or increase the efficiency of transformation or integration. These and other suitable elements of the genetic construct will be apparent to those skilled in the art and can, 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 inducible 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) can be used in substantially a similar manner.

[1176] Preferably, in the genetic constructs of the present invention, the at least one nucleic acid and the regulatory element, and optionally one or more other elements, are “operably linked” to each other, generally meaning that they have a functional relationship with each other in this way. For example, a promoter is considered “operably linked” to a coding sequence if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of a coding sequence (where the coding sequence should be understood as being “under” the “control” of the promoter). Typically, when two nucleotide sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They will also typically be substantially contiguous, although this may not be necessary.

[1177] The nucleic acids and / or genetic constructs of the present invention can be used to transform host cells or host organisms, i.e., to express and / or produce the polypeptide or protein constructs of the present invention. The host is preferably a non-human host. Suitable hosts or host cells will be apparent to those skilled in the art, and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism, such as:

[1178] - Bacterial strains, including but not limited to Gram-negative strains such as strains of *Escherichia coli*; strains of the genus *Proteus*, such as *Proteus mirabilis*; strains of the genus *Pseudomonas*, such as *Pseudomonas fluorescens*; and Gram-positive strains such as strains of the genus *Bacillus*, such as *Bacillus subtilis* or *Bacillus brevis*; strains of the genus *Streptomyces*, such as *Streptomyces lividans*; strains of the genus *Staphylococcus*, such as *Staphylococcus carnosus*; and strains of the genus *Lactococcus*, such as *Lactococcus lactis*.

[1179] - Fungal cells, including but not limited to cells from the genus *Trichoderma*, such as *Trichoderma reesei*; cells from the genus *Neurospora*, such as *Neurospora crassa*; cells from the genus *Sordaria*, such as *Sordaria macrospora*; cells from the genus *Aspergillus*, such as *Aspergillus niger* or *Aspergillus sojae*; or cells from other filamentous fungi;

[1180] - Yeast cells, including but not limited to cells from the genera *Saccharomyces*, such as *Saccharomyces cerevisiae*; cells from the genera *Schizosaccharomyces*, such as *Schizosaccharomyces pombe*; cells from the genera *Pichia*, such as *Pichia pastoris* or *Pichiamethanolica*; cells from the genera *Hansenula*, such as *Hansenula polymorpha*; cells from the genera *Kluyveromyces*, such as *Kluyveromyces lactis*; cells from the genera *Arxula*, such as *Arxula adeninivorans*; and cells from the genera *Yarrowia*, such as *Yarrowialipolytica*.

[1181] -Amphibian cells or cell lines, such as Xenopus oocytes;

[1182] - Cells or cell lines derived from insects, such as cells / cell lines derived from Lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or cells / cell lines derived from Drosophila, such as Schneider and Kc cells;

[1183] - Plants or plant cells, such as in tobacco plants; and / or

[1184] - Mammalian cells or cell lines, such as human cells or cell lines, 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) and PER.C6 cells;

[1185] And all other hosts or host cells known to be used 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 those skilled in the art. Reference may also be made to the general background fields cited above herein, as well as, 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 references cited herein.

[1186] For the expression of ISVs, peptides, or constructs in cells, they can also be expressed as so-called “intracellular antibodies,” 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).

[1187] According to a preferred but non-limiting embodiment of the invention, the ISV, polypeptide or protein construct of the invention is produced in bacterial cells, particularly bacterial cells suitable for large-scale drug production (such as cells of the strains mentioned above).

[1188] According to another preferred but non-limiting embodiment of the invention, the ISV, polypeptide or protein construct of the invention is produced in yeast cells, particularly yeast cells suitable for large-scale drug production (such as cells of the species mentioned above).

[1189] According to yet another preferred but non-limiting embodiment of the invention, the ISV, polypeptide, or construct of the invention is produced in mammalian cells, particularly in human cells or in human cell lines, and even more particularly in human cells or human cell lines suitable for large-scale drug production (such as the cell lines mentioned above).

[1190] Suitable techniques for transforming the host or host cells of this invention will be clear to those skilled in the art and may depend on the intended host cell / host organism and the genetic construct to be used. See again the manual and patent application mentioned above.

[1191] Following transformation, steps can be performed to detect and select those host cells or host organisms that have been successfully transformed with the nucleotide sequence / genetic construct of the present invention. This could be, for example, a selection step based on selective markers present in the genetic construct of the present invention, or a step involving, for example, detecting the polypeptide of the present invention using a specific antibody.

[1192] 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 another aspect of the invention.

[1193] Preferably, these host cells or host organisms are such that they express or (at least) are capable of expressing (e.g., under suitable conditions) the ISV, polypeptide, or protein construct of the present invention (and in the case of the host organism: in at least one of its cells, portions, tissues, or organs). The present invention also includes the next generation, offspring, and / or progeny of the host cells or host organisms of the present invention, obtained, for example, through cell division or through sexual or asexual reproduction.

[1194] Therefore, in another aspect, the present invention relates to a host or host cell that expresses (or is capable of expressing, where appropriate) the ISV, polypeptide, or protein construct of the present invention; and / or a host or host cell containing nucleic acids encoding the ISV, polypeptide, or protein construct of the present invention. Some preferred but non-limiting examples of such hosts or host cells may be as commonly described in WO 04 / 041867, WO 04 / 041865, or WO 09 / 068627. For example, the ISV, polypeptide, and protein construct of the present invention can advantageously be expressed, produced, or manufactured in yeast strains such as Pichia pastoris. Also refer to WO 04 / 25591, WO 10 / 125187, WO 11 / 003622, and WO 12 / 056000, which also describe the expression / production of immunoglobulin single variable domains and polypeptides containing them in Pichia pastoris and other host / host cells.

[1195] To generate / obtain the expression of the ISV, peptide, or protein construct of the present invention, the transformed host cell or transformed host organism can generally be maintained, sustained, and / or cultured under conditions that enable the expression / generation of the (desired) ISV, peptide, or protein construct of the present invention. Suitable conditions will be apparent to those skilled in the art and will generally depend on the host cell / host organism used and the regulatory elements controlling the expression of the (related) nucleotide sequences of the present invention. Refer again to the manual and patent application mentioned above in the paragraph concerning the genetic constructs of the present invention.

[1196] 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 present invention is under the control of an inducible promoter); all of these can be selected by those skilled in the art. Similarly, under such conditions, the ISV, peptide, or protein construct of the present invention may be expressed constitutively, transiently, or only when properly induced.

[1197] Those skilled in the art will also appreciate that the ISVs, peptides, or protein constructs of the present invention can be generated (first) in an immature form (as described above) and then post-translational modified, depending on the host cell / host organism used. Furthermore, the ISVs, peptides, or protein constructs of the present invention can be glycosylated, also depending on the host cell / host organism used.

[1198] The ISV, peptide, or protein construct of the present invention can then be isolated from host cells / host organisms and / or from the culture medium in which said host cells or host organisms are cultured, using protein isolation and / or purification techniques known per se, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific cleavable amino acid sequence fused with the peptide or construct of the present invention) and / or preparative immunoassay techniques (i.e., using an antibody against the amino acid sequence to be isolated), using host cells / host organisms and / or the culture medium in which said host cells or host organisms are cultured.

[1199] The constructs of the present invention can generally be prepared by a method comprising at least one step of suitably linking the ISV or polypeptide of the present invention to one or more additional groups, residues, structural moieties, or binding units via one or more suitable linkers to provide the constructs of the present invention. The ISV, polypeptide, and constructs of the present invention can then be further modified, and in particular by chemical and / or biological (e.g., enzymatic) modification of one or more amino acid residues forming the polypeptide or construct of the present invention to obtain derivatives of the polypeptide or construct of the present invention.

[1200] This invention also relates to pharmaceutical compositions comprising the ISV, polypeptide, compound, or construct of this invention.

[1201] In the above methods, the amino acid sequences, ISVs, nanobodies, peptides, compounds, or constructs of the present invention and / or compositions comprising them can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, for example, the amino acid sequences, ISVs, nanobodies, peptides, compounds, or constructs of the present invention and / or compositions comprising them can be administered orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly, or by any other route of administration bypassing the gastrointestinal tract), intranasally, percutaneously, topically, via suppositories, or by inhalation, also depending on the specific pharmaceutical formulation or composition to be used. Clinicians will be able to select the appropriate route of administration and the appropriate pharmaceutical formulation or composition for said administration, depending on the disease or condition to be prevented or treated and other factors known to clinicians.

[1202] As used herein, the term "therapeutic agent" refers to any agent capable of treating and / or managing conditions of hyperproliferating cells (e.g., cancer) or one or more symptoms thereof. In some embodiments, the term "therapeutic agent" refers to the multispecific polypeptide of the present invention. Preferably, the therapeutic agent is an agent known to be, or already used, or currently used for treating, preventing, and / or managing conditions of hyperproliferating cells (e.g., cancer) or one or more symptoms thereof.

[1203] As used herein, in the context of cancer, a “therapeuticly effective amount” refers to a single therapeutic amount or a therapeutic amount in combination with other therapies that provides therapeutic benefit in the treatment and / or management of cancer. In one aspect, a therapeutically effective amount is a therapeutic amount sufficient to destroy, alter, control, or remove primary, regional, or metastatic cancerous tissue. In another aspect, a therapeutically effective amount is a therapeutic amount sufficient to alleviate cancer symptoms. In yet another aspect, a therapeutically effective amount is a therapeutic amount sufficient to delay or minimize the spread of cancer. In a specific implementation, a therapeutically effective amount of a therapy is a therapeutic amount sufficient to inhibit the growth or proliferation of cancer cells, kill existing cancer cells (e.g., induce cancer regression), and / or prevent the spread of cancer cells to other tissues or regions (e.g., prevent metastasis). In another specific embodiment, the therapeutically effective amount of the therapy is 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 combination with a certain amount of the multispecific polypeptide of the present invention, the term may cover the amount that improves the overall therapy, reduces or avoids unwanted effects, or enhances or synergizes with the therapeutic efficacy of another therapy. In one embodiment, the therapeutically effective amount of the therapy reduces or avoids unwanted effects relative to a control (e.g., a negative control such as phosphate-buffered saline) in assays known in the art or described herein, or enhances or synergizes 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%.

[1204] As used herein, a “therapeuticly effective amount” in the context of a non-cancerous hyperproliferative cell disease refers to a single therapeutic amount or a therapeutic amount in combination with other therapies that provides therapeutic benefit in the treatment and / or management of said disease. In one aspect, a therapeutically effective amount is a therapeutic amount sufficient to destroy, alter, control, or remove cells affected by the non-cancerous hyperproliferative cell disease. In another aspect, a therapeutically effective amount is a therapeutic amount sufficient to alleviate the symptoms of the non-cancerous hyperproliferative cell disease. In yet another aspect, a therapeutically effective amount is a therapeutic amount sufficient to delay or minimize the spread of the non-cancerous hyperproliferative cell disease. In a specific embodiment, a therapeutically effective amount of a therapy is a therapeutic amount sufficient to inhibit the growth or proliferation of the non-cancerous hyperproliferative cell disease, or to kill existing non-cancerous hyperproliferative cells (e.g., causing disease regression). In another specific embodiment, the therapeutically effective amount of the therapy is sufficient to inhibit the growth of non-cancerous, overproliferating 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 combination with a certain amount of the multispecific polypeptide of the present invention, the term may cover amounts that improve the overall therapy, reduce or avoid unwanted effects, or enhance the therapeutic efficacy or synergistic effect with another therapy. In one embodiment, the therapeutically effective dose of the therapy reduces or avoids unwanted effects relative to a control (e.g., a negative control such as phosphate-buffered saline) in assays known in the art, or enhances or synergizes 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%.

[1205] As used herein, the term "therapy" means any regimen, method, and / or agent capable of treating, preventing, and / or managing a condition of excessively proliferating cells (e.g., cancer). In some embodiments, the term "therapy" means a biological therapy, supportive therapy, and / or other therapy for treating, preventing, and / or managing a condition of excessively proliferating cells (e.g., cancer) or one or more symptoms of such a condition known to those skilled in the art (such as medical personnel).

[1206] As used herein, in the context of administering a therapy to a subject, the terms "treatment" ("treat", "treatment", and "treating") mean a reduction or improvement in the progression, severity, and / or duration of a condition associated with a hyperproliferating cell disorder (e.g., cancer), and / or an improvement in one or more symptoms thereof, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In a specific implementation, in the context of administering a therapy to a subject, the term "treatment" means a reduction or improvement in the progression, severity, and / or duration of a hyperproliferating cell disorder (e.g., cancer) that is defined as a reduction of cancer cells by at least 5% relative to a control (e.g., a negative control such as phosphate-buffered saline), 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%. In other implementations, in the context of administering a therapy to a subject, the term "treatment" refers to reducing or improving the progression, severity, and / or duration of a disease with excessively proliferating cells (e.g., cancer), meaning no change in the number of cancer cells, a reduction in hospitalization time, a reduction in mortality, or an increase in the survival time of a subject with cancer.

[1207] The amino acid sequences, ISVs, nanobodies, peptides, compounds, and / or constructs and / or compositions comprising the present invention are administered according to a treatment regimen suitable for the prevention and / or treatment of a hyperproliferative cell condition (e.g., cancer) to be prevented or treated. Clinicians will generally be able to determine an appropriate treatment regimen based on factors such as the stage of the hyperproliferative cell condition (e.g., cancer) to be treated, the severity of the hyperproliferative cell condition (e.g., cancer) to be treated and / or the severity of its symptoms, the specific amino acid sequence, ISV, nanobodies, peptides, compounds, and / or constructs of the present invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the patient's age, sex, weight, diet, overall condition, and similar factors known to clinicians.

[1208] Generally, the treatment regimen will include administering one or more amino acid sequences, ISVs, nanobodies, peptides, compounds and / or constructs of the present invention, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. Based on the factors described above, clinicians can determine the specific amount or dose to be administered.

[1209] Typically, for the prevention and / or treatment of the hyperproliferative cell disorders (e.g., cancer) mentioned herein, and depending on the type and stage of the hyperproliferative cell disorder (e.g., cancer) to be treated, the potency of the specific amino acid sequence, ISV, nanobody, peptide, compound, or construct of the present invention to be used, the specific route of administration used, and the specific pharmaceutical formulation or composition, the amino acid sequence, ISV, nanobody, peptide, compound, or construct of the present invention will generally be administered at a dose 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 of subject body weight; or as a single daily dose or as multiple fractions throughout the day (e.g., by infusion). Clinicians will generally be able to determine the appropriate daily dose, depending on the factors mentioned herein. It should also be understood that, in specific circumstances, clinicians may choose to deviate from these doses, for example based on the factors cited above and their professional judgment. Generally, given the differences in affinity / affinity, efficacy, biodistribution, half-life, and similar factors known to those skilled in the art, some guidelines regarding dosage can be obtained from the amount of a conventional antibody or antibody fragment targeting the same target that is typically administered via substantially the same route.

[1210] Typically, the methods described above utilize a single amino acid sequence, ISV, nanobody, peptide, compound, or construct of the present invention. However, the use of a combination of two or more amino acid sequences, ISVs, nanobodies, peptide compounds, and / or constructs of the present invention is within the scope of the present invention.

[1211] The ISVs, nanobodies, amino acid sequences, peptides, compounds, and / or constructs of the present invention can also be used in combination with one or more other pharmaceutically active compounds or components, i.e., as a combination therapy, which may or may not induce a synergistic effect. Furthermore, based on the factors cited above and their professional judgment, clinicians will be able to select the other compounds or components mentioned above, as well as appropriate combination therapy regimens.

[1212] In particular, the amino acid sequences, ISVs, nanobodies, peptides, compounds, and / or constructs of the present invention can be used in combination with other pharmaceutically active compounds or components that are or can be used to prevent and / or treat the hyperproliferating cell diseases (e.g., cancer), conditions, and / or symptoms cited herein, with or without a synergistic effect. Examples of the compounds and components, as well as the routes, methods of administration, and pharmaceutical formulations or compositions thereof, are known to clinicians.

[1213] When two or more substances or ingredients are used as part of a combination therapy, they may be administered via the same route of administration or via different routes of administration at substantially the same time or at different times (e.g., substantially simultaneously, consecutively, or according to alternative regimens). When said substances or ingredients are administered simultaneously via the same route of administration, they may be administered as different pharmaceutical preparations or compositions or as part of a combination of pharmaceutical preparations or compositions, as will be apparent to those skilled in the art.

[1214] In one aspect, the present invention provides a method for administering an immunoglobulin monovariable domain and a polypeptide construct, polypeptide, compound, and / or construct comprising one or more immunoglobulin monovariable domains. In some embodiments, the immunoglobulin monovariable domain, polypeptide, compound, and / or construct is administered as a pharmaceutical composition. In addition to the immunoglobulin monovariable domain and its polypeptide construct, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[1215] As described in detail, the pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as drench (aqueous or non-aqueous solution or suspension), tablets (e.g. those targeting oral, sublingual, and systemic absorption), bolus, powder, granule, or paste for application to the tongue; parenteral administration, such as as a sterile solution or suspension or sustained-release formulation administered via subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, such as as a cream, ointment, patch, or spray applied to the skin, lungs, or mouth; intravaginal or rectal administration, such as as a vaginal suppository, cream, or foam; sublingual; ocularly; transdermal; or via the nose, lungs, and other mucosal surfaces.

[1216] The phrase “pharmaceutically acceptable” as used in this article refers to compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[1217] The phrase “pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or carrier, 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”, meaning it is compatible with other components of the formulation and harmless 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 carboxymethyl cellulose, ethyl cellulose, 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 buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[1218] The formulations of the present invention include those suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal, and / or parenteral administration. The formulations can be conveniently present in unit dosage forms and can be prepared by any method known in the pharmaceutical industry. The amount of active ingredient (e.g., an immunoglobulin monovariable domain or a polypeptide construct thereof) that can be combined with a carrier material to produce a single dosage form will vary depending on the host to be treated and the specific mode of administration. The amount of active ingredient 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, this amount will be in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[1219] In some embodiments, the formulation comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micellar forming agents (e.g., bile acids), and polymerization carriers (e.g., polyesters and polyanhydrides). In some embodiments, the aforementioned formulation provides an orally bioavailable immunoglobulin monovariable domain or polypeptide construct.

[1220] Methods for preparing these formulations or compositions include the steps of combining an immunoglobulin monovariable domain or polypeptide construct with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and tightly binding an immunoglobulin monovariable domain or polypeptide construct to a liquid carrier or a finely fragmented solid carrier, or both, and then shaping the product (if desired).

[1221] Formulations suitable for oral administration may be in the following forms: capsules, cachets, pills, tablets, lozenges (using a flavoring matrix, typically sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions of aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions, or as elixirs or syrups, or as pasteurees (using an inert matrix, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes, each containing a predetermined amount of an immunoglobulin monovariable domain or polypeptide construct as the active ingredient. Immunoglobulin monovariable domain or polypeptide constructs may also be administered as large pills, electuals, or pastes.

[1222] In solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) intended for oral administration, 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 silica; binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; humectants, such as glycerin; disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; solution retarders, such as paraffin; absorption enhancers, such as quaternary ammonium compounds; humectants, 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 glycol, sodium lauryl sulfate, and mixtures thereof; and colorants. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions can also be used as fillers in soft-shell and hard-shell gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[1223] Tablets can optionally be prepared with one or more excipients by compression or molding. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium glycolate starch or cross-linked sodium carboxymethyl cellulose), surfactants, or dispersants. Molded tablets can be prepared in suitable machinery, wherein the mixture of powdered compounds is wetted with an inert liquid diluent.

[1224] Other solid dosage forms of tablets and pharmaceutical compositions (such as sugar-coated pills, capsules, pellets, and granules) may optionally be scored or prepared with coatings and shells (such as enteric coatings and other coatings known in the pharmaceutical formulation field). They may also be formulated to provide a slow or controlled release of the active ingredient therein, for example using different proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide a desired release profile. They may be formulated for rapid release, for example, by lyophilization. They may be sterilized, for example, by filtration through a bacteria-retaining filter, which is sterilized by incorporating a sterilizing agent in the form of a sterile solid composition, which may be immediately dissolved in sterile water or some other sterile injectable medium 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 portion of the gastrointestinal tract, optionally in a delayed manner. Examples of encapsulation compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, having one or more of the excipients described above, if suitable.

[1225] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms 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-butanediol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofuranol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof.

[1226] In addition to inert diluents, oral compositions may also include adjuvants (such as humectants), emulsifiers and suspending agents, sweeteners, flavorings, colorings, flavorings and preservatives.

[1227] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth gum, and mixtures thereof.

[1228] Formulations of pharmaceutical compositions for rectal or vaginal administration can be presented as suppositories, which can be prepared by mixing an immunoglobulin monovariable domain or polypeptide construct with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glyco...

Claims

1. A polypeptide comprising a first immunoglobulin monovariable domain (ISV) and a second immunoglobulin monovariable domain, wherein... - The first ISV has a high affinity for differentiation cluster 3 (CD3) present on T cells / binds to differentiation cluster 3 (CD3) present on T cells; - The second ISV has a high affinity for the first antigen on the target cell / binds to the first antigen on the target cell; Wherein the first antigen is different from CD3; and The target cells described therein are different from the T cells; and The first ISV consists of four frame regions, FR1 to FR4, and three complementary determinant regions, CDR1 to CDR3, respectively. (i) CDR1 is a) SEQ ID NO: 82, or b) SEQ ID NO: 81 (ii) CDR2 is SEQ ID NO:102, and (iii) CDR3 is SEQ ID NO:

123.

2. The polypeptide according to claim 1, wherein the first antigen on the target cell is a tumor antigen.

3. The polypeptide according to claim 1, wherein the first antigen on the target cell is a tumor-associated antigen (TAA).

4. The polypeptide according to any one of claims 1 to 3, further comprising a third ISV, said third ISV having a high affinity for and / or binding to a second antigen on the target cell, wherein said 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.

6. The polypeptide of claim 5, wherein the second antigen on the target cell is a tumor-associated antigen (TAA).

7. The polypeptide according to claim 3 or 6, 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 Y antigen, 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, and CD147, growth factor receptor, cytokine receptor, CD30, IL23R, IGF-1R, IL5R, IgE, CD24 8 (endothelial sialic acid protein), CD44v6, gpA33, Ron, Trop2, PSCA, micrin 6, micrin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, phosphatidylinositol glycan-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 isotypes.

8. The polypeptide of claim 7, wherein the growth factor receptor comprises ErbB3 and ErbB4.

9. The polypeptide of claim 7, wherein the cytokine receptor comprises interleukin-2 receptor γ chain (CD132 antigen), interleukin-10 receptor α chain (IL-10R-A), interleukin-10 receptor β chain (IL-10R-B), 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), and interleukin-13 receptor α-2 chain (IL-13R-α-1). Interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin-21 receptor precursor (IL-21R), type I interleukin-1 receptor (IL-1R-1), type II interleukin-1 receptor (IL-1R-β), interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor α chain (CD25 antigen), interleukin-2 receptor β chain (CD122 antigen), and interleukin-3 receptor α chain (IL-3R-α).

10. The polypeptide of claim 6, wherein the first antigen and the second antigen are selected from the group consisting of: -EGFR is used as the primary antigen and CEA is used as the secondary antigen; -CD19 is used as the first antigen and CD20 is used as the second antigen; -CD19 is used as the first antigen and CD22 is used as the second antigen; -CD123 is used as the primary antigen and Tim-3 is used as the secondary antigen; and -CD132 is used as the first antigen and CD69 is used as the second antigen.

11. The polypeptide according to any one of claims 1 to 3, further comprising a serum protein binding portion.

12. The polypeptide of claim 11, wherein the serum protein binding portion is an ISV that binds to serum albumin.

13. A polypeptide that specifically binds to CD3 and comprises or is composed of: four framework regions of FR1 to FR4 and three complementarity-determining regions of CDR1 to CDR3, wherein: (i) CDR1 is a) SEQ ID NO: 82, or b) SEQ ID NO: 81 (ii) CDR2 is SEQ ID NO:102, and (iii) CDR3 is SEQ ID NO:

123.

14. The polypeptide of claim 13, further comprising a serum protein binding portion.

15. The polypeptide of claim 14, wherein the serum protein binding portion is an ISV that binds to serum albumin.

16. A nucleic acid or nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 15, or a vector containing said nucleic acid or nucleic acid sequence.

17. A host cell, said host cell having been transformed or transfected with the nucleic acid or nucleic acid sequence as defined in claim 16 or the vector.

18. A method for producing a polypeptide according to any one of claims 1 to 15, the 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 a polypeptide according to any one of claims 1 to 15 or a polypeptide produced by the method according to claim 18.

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