T cell recruiting polypeptides based on CD3 reactivity

Abstract

To provide T cell recruiting polypeptides binding CD3 on a T cell, which can be used in methods for treating cancers.SOLUTION: Provided is a polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), the first ISV having high affinity for / binding to cluster of differentiation 3 (CD3) present on a T cell, the second ISV having high affinity for / binding to a first antigen on a target cell, the first antigen being different from the CD3, and the target cell being different from the T cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Field of the Invention] The present invention provides multispecific T cell recruiting polypeptides that bind to CD3 on T cells and at least one antigen on a target cell. The present invention also relates to monovalent T cell recruiting polypeptides for use with these multispecific polypeptides. The present invention also provides methods for treatment and kits for providing the same.

[0002] [background] Cancer kills a significant number of people worldwide. Currently, cancer is the leading cause of death worldwide, followed by heart disease and stroke. Cancer is considered the leading cause of illness and death worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. The number of new cases is predicted to increase by approximately 70% over the next 20 years (Source: WHO Cancer). The total economic impact of premature death and disability due to cancer worldwide was approximately $900 billion in 2008, accounting for 1.5% of global gross domestic product.

[0003] Available treatment regimens for solid tumors typically involve a combination of surgical resection, chemotherapy, and radiation therapy. Four decades of clinical experience have shown little progress, especially in advanced stage cancers.

[0004] New cancer-fighting treatments are eagerly awaited.

[0005] Antibody therapy is now an important part of physicians' armamentarium for fighting diseases, particularly cancer. Monoclonal antibodies have been established as an important therapeutic approach for a range of diseases for several years. All currently approved antibody therapies are monospecific monoclonal antibodies (mAbs). To date, the targeting of most mAbs requires either an agonistic or antagonistic approach. While targeting cell surface antigens themselves can mediate antitumor activity by inducing apoptosis, most mAb-based activity against hematologic malignancies relies on Fc-mediated effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0006] Immunotherapy has emerged as a rapidly growing area of ​​cancer research. Immunotherapy targets the body's immune surveillance system, and in particular, T cells, against cancer cells.

[0007] Cytotoxic T cells (CTLs) are T lymphocytes that kill cancer cells, cells infected (especially by viruses), or cells that have been otherwise damaged. T lymphocytes (or T cells) express T cell receptors (TCRs) and CD3 receptors on their cell surface. The αβ TCR-CD3 complex (or "TCR complex") is composed of six type I single-spanning membrane proteins: the TCRα and TCRβ chains, which form the TCR heterodimer responsible for ligand recognition, and the noncovalently associated CD3γ, CD3δ, CD3ε, and ζ chains. These contain cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signaling components (Call et al. 2004, Molecular Immunology 40: 1295-1305).

[0008] Both the α and β chains of the T cell receptor consist of a constant domain and a variable domain. Physiologically, the αβ chains of the T cell receptor recognize peptide-loaded MHC complexes and bind upon binding to CD3 chains. These CD3 chains then transmit the binding signal to the intracellular environment.

[0009] Given the ability of natural 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 by the Fc tails of antitumor monoclonal antibodies. As an alternative, patient T cells have been engineered to carry a second TCR with known specificity for a defined tumor antigen. This adoptive cell transfer is inherently highly personalized and labor-intensive. However, a major challenge with T cell therapy remains the numerous immune escape mechanisms known to occur in cancer patients (Nagorsen et al. 2012, Pharmacology & Therapeutics 136: 334-342).

[0010] Rather than eliciting a specific T cell response by the expression by cancer cells of MHC molecules and the presence, production, delivery, and presentation of specific peptide antigens, more recent developments have attempted to combine the advantages of immunotherapy with antibody therapy by engaging all of a patient's cytotoxic T cells in a polyclonal manner using recombinant antibody-based technology: "bispecific antibodies."

[0011] Bispecific antibodies have been engineered that carry a tumor recognition moiety in one arm (the target-binding arm), while the other arm of the molecule has specificity for a T cell antigen, primarily CD3 (the effector-binding arm). The simultaneous binding of the two arms to their respective target antigens directs and activates T lymphocytes to tumor cells where they can exert their cytolytic function.

[0012] The concept of using bispecific antibodies to activate T cells against tumor cells was described more than 20 years ago, but manufacturing challenges and clinical failures have stalled the development of bispecific antibodies in smaller formats. These bispecific antibodies penetrate tissues and tumors more easily than conventional antibodies. Additionally, smaller formats are better at forming cytotoxic synapses to kill target cells. It was thought that smaller bispecific antibody formats would be easier to manufacture and less immunogenic than conventional antibodies. However, BiTE molecules, which are smaller bispecific antibodies consisting of two single-chain variable fragments (scFvs) linked by a five-amino acid peptide linker, lack stability (scFvs tend to aggregate), have low expression titers, and are poorly soluble. Furthermore, a phase 1 clinical trial of blinatumomab (a BiTE molecule) that recognizes the CD3 chain was prematurely terminated due to adverse neurological events, cytokine release syndrome, and infections, and the absence of robust signs of desired clinical responses or bioactivity. Aside from efficacy, BiTEs must be continuously infused, likely due to the lack of an Fc domain, which does not contribute to patient compliance. The same problem applies to DARTs (dual affinity retargeting molecules developed by MacroGenics), in which the heavy chain variable domain from one antibody (Ab) is linked to the light chain variable domain of another Ab. MacroGenics is currently attempting to solve this problem by fusing an Fc domain to its next-generation DARTs. Next-generation DARTs not only result in larger molecules, but also introduce manufacturing challenges and the introduction of other Fc functions. Larger formats with Fc may have better PK, but again introduce the risk of off-target activity (Garber 2014, Nature reviews 13: 799-801).

[0013] There remains a need for alternative bispecific formats. Summary of the Invention

[0014] The present invention solves this problem by providing a multispecific polypeptide comprising a first and at least one further immunoglobulin single variable domain (ISV), wherein the first ISV has high affinity for / binds to CD3 and the at least one further ISV has high affinity for / binds to an antigen present on a target cell. In certain aspects, binding of the first ISV will activate the innate cytolytic activity of T cells against the target cell, independent of MHC1.

[0015] Thus, in a first aspect, the present invention provides a polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), the first ISV has high affinity for / binds to cluster of differentiation 3 (CD3) present on T cells; the second ISV has high affinity for / binds to a first antigen on a target cell; wherein said first antigen is different from said CD3; and Wherein said target cells are distinct from said T cells, a polypeptide is provided.

[0016] In a further aspect, the present invention provides a polypeptide as described herein that directs T cells to target cells.

[0017] In a further aspect, the present invention provides a polypeptide as described herein that induces T cell activation.

[0018] In a further aspect, the present invention provides a polypeptide as described herein, wherein said T cell activation is independent of MHC recognition.

[0019] In a further aspect, the present invention provides a polypeptide as described herein, wherein said T cell activation is dependent on presentation of said polypeptide bound to said first antigen on a target cell to a T cell.

[0020] In a further aspect, the present invention provides a polypeptide as described herein, wherein said T cell activation results in one or more cellular responses of said T cell, wherein said cellular response is 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.

[0021] In a further aspect, the present invention provides a polypeptide as described herein, wherein said T cell activation results in more than about 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%, for example 70%, 80%, or even more than 90%, such as 100%, inhibition of activity of said target cells.

[0022] In a further 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 polymorphic variants or isoforms thereof.

[0023] 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 polymorphic variants or isoforms thereof.

[0024] In a further aspect, the present invention provides a method for producing a polypeptide having a nucleotide sequence of at least about 10 as measured by surface plasmon resonance. 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 The present invention provides a polypeptide as described herein having an on rate constant (K on ) for binding to said CD3 selected from the group consisting of:

[0025] In a further aspect, the present invention provides a method for producing a polypeptide having a nucleotide sequence of at most about 10 as measured by surface plasmon resonance. -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 , up to about 10 -6 s -1 , up to about 10 -7 s -1 , up to about 10 -8 s -1 , up to about 10 -9 s -1 , and up to about 10 -10 s -1 The present invention provides a polypeptide as described herein, having an off rate constant (Koff) for binding to said CD3 selected from the group consisting of:

[0026] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV binds to CD3, preferably with an EC50 value of 100 nM to 1 pM, for example with an average EC50 value of 100 nM or less, even more preferably an average EC50 value of 90 nM or less, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or less, for example less than 4, 3, 2, or 1 nM or less, for example less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or less, for example less than 4 pM, as measured by flow cytometry.

[0027] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV binds to CD3 with an average KD value of 100 nM to 10 pM, such as an average KD value of 90 nM or less, even more preferably an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or less, such as less than 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or less, such as less than 10 pM. Preferably, the KD is measured by SPR, for example as measured by Proteon.

[0028] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81 and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 101 and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 143, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 The present invention provides a polypeptide selected from the group consisting of:

[0029] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 100, or (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 81 or any of SEQ ID NOs: 81 to 100, provided that a polypeptide comprising a CDR1 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122; or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 101 or any of SEQ ID NOs: 101 to 122, provided that a polypeptide comprising a CDR2 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 143; or (f) the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to any of SEQ ID NOs: 123 to 143, provided that a polypeptide comprising a CDR3 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0030] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, - In position 8, S is changed to G, and / or A polypeptide is provided in which at position -10, G is changed to A.

[0031] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or Polypeptides are provided in which at position -9, T is changed to S, A, or P.

[0032] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or A polypeptide is provided in which at position -10, A is changed to P.

[0033] Preferably, a polypeptide comprising one or more CDRs with 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity compared to binding by a polypeptide comprising a CDR without the 3, 2, or 1 amino acid difference, when affinity is measured by surface plasmon resonance.

[0034] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, - In position 8, S is changed to G, and / or At -10th place, G has been changed to A, provided that the polypeptide comprising a CDR1 having two or one amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the two or one amino acid difference, when the affinity is measured by surface plasmon resonance; and, (ii) CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or at position -9, T is changed to S, A, or P; provided that the polypeptide comprising a CDR2 having three, two, or one amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the three, two, or one amino acid difference, when the affinity is measured by surface plasmon resonance; and, (iii) CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or At -10th place, A has been changed to P, provided that the polypeptides comprising a CDR3 having two or one amino acid difference bind to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the two or one amino acid difference, when the affinity is measured by surface plasmon resonance.

[0035] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81 and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 101 and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 The present invention provides a polypeptide selected from the group consisting of:

[0036] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 81, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 101, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 123, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0037] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0038] In a further 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.

[0039] In a further 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 NO: 1-50.

[0040] In a further 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 NO: 1-50.

[0041] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO:88.

[0042] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), and CDR2 is SEQ ID NO: 110.

[0043] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO: 128.

[0044] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 88 and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 110 and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 128 The present invention provides a polypeptide selected from the group consisting of:

[0045] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 88, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 110, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 128, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0046] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0047] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NO:51.

[0048] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first ISV cross-blocks binding to CD3 by a polypeptide having SEQ ID NO:51.

[0049] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NO:51.

[0050] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO: 90.

[0051] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: A polypeptide is provided in which at position -2, V is changed to A.

[0052] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO: 130.

[0053] Preferably, a polypeptide comprising one or more CDRs with a single amino acid difference binds to CD3 with about the same or higher affinity compared to binding by a polypeptide comprising a CDR without the single amino acid difference, when affinity is measured by surface plasmon resonance.

[0054] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is SEQ ID NO: 90; and, (ii) CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: - In the second place, V has been changed to A, provided that the polypeptide comprising a CDR2 having the single amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the single amino acid difference, when the affinity is measured by surface plasmon resonance; and, (iii) A polypeptide wherein CDR3 is SEQ ID NO: 130.

[0055] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 90 and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 112 and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 130 The present invention provides a polypeptide selected from the group consisting of:

[0056] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 90, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 112, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 130, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0057] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0058] In a further 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.

[0059] In a further 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 NO: 53-56.

[0060] In a further 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 NO: 53-56.

[0061] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO:89.

[0062] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), and CDR2 is SEQ ID NO: 111.

[0063] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO: 129.

[0064] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 89 and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 111 and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 129 The present invention provides a polypeptide selected from the group consisting of:

[0065] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 89, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 111, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 129, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0066] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0067] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is SEQ ID NO:52.

[0068] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first ISV cross-blocks binding to CD3 by a polypeptide having SEQ ID NO:52.

[0069] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first ISV is cross-blocked from binding to CD3 by a polypeptide having SEQ ID NO:52.

[0070] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or A polypeptide is provided in which at position -8, M is changed to T.

[0071] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or Polypeptides are provided in which at position -7, D is changed to A or K.

[0072] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or A polypeptide is provided in which at position -6, S is changed to V.

[0073] Preferably, a polypeptide comprising one or more CDRs with 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity compared to binding by a polypeptide comprising a CDR without the 3, 2, or 1 amino acid difference, when affinity is measured by surface plasmon resonance.

[0074] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or At -8th place, M has been changed to T, provided that the polypeptide comprising a CDR1 having three, two, or one amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the three, two, or one amino acid difference, when the affinity is measured by surface plasmon resonance; and, (ii) CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or At position -7, D is changed to A or K, provided that the polypeptide comprising a CDR2 having three, two, or one amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the three, two, or one amino acid difference, when the affinity is measured by surface plasmon resonance; and, (iii) CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or - In the 6th place, S has been changed to V, provided that the polypeptide comprising a CDR3 having the single amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the single amino acid difference, when the affinity is measured by surface plasmon resonance.

[0075] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 91 and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 114 and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 131 The present invention provides a polypeptide selected from the group consisting of:

[0076] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 91, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 114, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 131, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0077] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0078] In a further 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.

[0079] In a further 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 NO: 57-65.

[0080] In a further 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 NO: 57-65.

[0081] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or A polypeptide is provided in which at position -9, L is changed to M.

[0082] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or A polypeptide is provided in which at position -10, Y is changed to F.

[0083] In a further aspect, the invention relates to a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or A polypeptide is provided in which at position -14, V is changed to I.

[0084] Preferably, a polypeptide comprising one or more CDRs having 5, 4, 3, 2, or 1 amino acid difference(s) binds to CD3 with about the same or higher affinity compared to binding by a polypeptide comprising a CDR without the 5, 4, 3, 2, or 1 amino acid difference(s), when affinity is measured by surface plasmon resonance.

[0085] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or -At 9th place, L has been changed to M, provided that the polypeptide comprising a CDR1 having 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance; and, (ii) CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or At -10th place, Y has been changed to F, provided that the polypeptide comprising a CDR2 having three, two, or one amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the three, two, or one amino acid difference, when the affinity is measured by surface plasmon resonance; and, (iii) CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or At -14th place, V has been changed to I, provided that the polypeptides comprising a CDR3 having 5, 4, 3, 2, or 1 amino acid difference bind to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 5, 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance.

[0086] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 94 and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 118 and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 134 The present invention provides a polypeptide selected from the group consisting of:

[0087] In a further aspect, the invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 94, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 118, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 134 to 143, or (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 134, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0088] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV consists essentially of four framework 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.

[0089] In a further 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.

[0090] In a further 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 NO: 66-80.

[0091] In a further 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 NO: 66-80.

[0092] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first antigen on a target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0093] In a further aspect, the present invention provides a polypeptide as described herein, further comprising a third ISV, which has high affinity for / binds to a second antigen on a target cell, wherein said second antigen is different from said first antigen.

[0094] In a further aspect, the present invention provides a polypeptide as described herein, wherein said second antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0095] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first antigen and said second antigen are present on the same target cell.

[0096] In a further aspect, the present invention provides a polypeptide as described herein, wherein said first antigen and said second antigen are present on different target cells.

[0097] In a further aspect, the present invention provides a method for treating leukemia, the method comprising administering to a patient a therapeutically effective amount of a TAA 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, and CD3. 3. Growth factor receptors including CD36, CD45, CD52, CD147, ErbB3 and ErbB4, interleukin-2 receptor gamma chain (CD132 antigen), interleukin-10 receptor alpha chain (IL-10R-A), interleukin-10 receptor beta chain (IL-10R-B), interleukin-12 receptor beta-1 chain (IL-12R-beta1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-13 receptor alpha chain (IL-13R-beta1), interleukin-14 receptor beta chain (IL-14R-beta2), interleukin-15 receptor alpha chain (IL-15R-beta1), interleukin-16 receptor beta chain (IL-16R-beta2), interleukin-17 receptor alpha chain (IL-17R-beta1), interleukin-18 receptor beta chain (IL-18R-beta2), interleukin-19 receptor alpha chain (IL-19R-beta1), interleukin-19 receptor beta chain (IL-19 ... Interleukin-1 chain (IL-13R-alpha-1) (CD213a1 antigen), alpha-2 chain of interleukin-13 receptor (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin-21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-beta) (CDw121b ... Cytokine receptors, including interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor alpha chain (CD25 antigen), interleukin-2 receptor beta chain (CD122 antigen), interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin-6, and claudin-18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, 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, CXC Provided are polypeptides as described herein, independently selected from the group consisting of R7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA195, DR5, DR6, DcR3, and CAIX (including associated polymorphic variants and isoforms).

[0098] In a further aspect, the present invention provides a polypeptide as described herein, wherein said TAA is CD20 (Uniplot 11836), HER2 (Uniplot P04626), a polymorphic variant or isoform thereof.

[0099] In a further aspect, the present invention provides a method for detecting a genomic DNA fragment comprising the steps of: EGFR as the first antigen and CEA as the second antigen, CD19 as the first antigen and CD20 as the second antigen, CD19 as the first antigen and CD22 as the second antigen, CD123 as the first antigen and Tim-3 as the second antigen, and -CD132 as the first antigen and CD69 as the second antigen The present invention provides a polypeptide as described herein selected from the group consisting of:

[0100] In a further aspect, the present invention provides a polypeptide as described herein, further comprising a serum protein binding moiety.

[0101] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety binds to serum albumin.

[0102] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety is an ISV that binds to serum albumin.

[0103] In a further aspect, the present invention provides a polypeptide as described herein, wherein the serum albumin-binding ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein, when the CDRs are determined according to the Kabat definition, CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375); and / or wherein, when the CDRs are determined according to Kontermann 2010, CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378), and CDR3 is GGSLSR (SEQ ID NO: 375).

[0104] In a further aspect, the present invention provides a polypeptide as described herein, wherein the serum albumin-binding ISV is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG (SEQ ID NOs: 348-360).

[0105] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISVs are linked to each other directly or via a linker.

[0106] In a further aspect, the present invention provides a polypeptide as described herein, wherein the first ISV and / or the second ISV, and / or optionally the third ISV, and / or optionally the serum albumin-binding ISV are linked via a linker.

[0107] In a further aspect, the present invention provides a polypeptide as described herein, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 362-372) linkers.

[0108] In a further aspect, the present invention provides a polypeptide as described herein, wherein the serum protein binding moiety is a non-antibody based polypeptide.

[0109] In a further aspect, the present invention provides a polypeptide as described herein, further comprising PEG.

[0110] In a further aspect, the present invention provides a method for the preparation of a medicament for the preparation of a medicament for the treatment ... HH , humanized V HH , or camelization V H The polypeptide described herein is

[0111] In a further aspect, the present invention provides a polypeptide wherein the first ISV is selected from the group consisting of SEQ ID NOs: 1-80.

[0112] In a further 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 the second ISV is selected from the group consisting of SEQ ID NOs: 297 to 304.

[0113] In a further aspect, the present invention provides a polypeptide selected from the group consisting of SEQ ID NOs: 249-250, 252-253, 255-256, 258-260, 263, 265-283, 286-289, 306-307, 309-310, 312-313, 315-317, 320, 322-340, and 343-346.

[0114] In a further aspect, the invention provides a polypeptide as described herein that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 100, or (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to any of the amino acid sequences of SEQ ID NOs: 81 to 100, provided that a polypeptide comprising a CDR1 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 101 to 122, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 123 to 143, or (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 123 to 143, provided that a polypeptide comprising a CDR3 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance. The present invention provides a polypeptide selected from the group consisting of:

[0115] The present invention also provides (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 81, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 101, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 123, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0116] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, - In position 8, S is changed to G, and / or Provided herein is a polypeptide described wherein at position -10, G is changed to A.

[0117] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or The polypeptides described herein are provided wherein at position -9, T is changed to S, A, or P.

[0118] In a further aspect, the present invention provides a method for the preparation of a nucleotide sequence comprising the steps of: (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or Provided herein is a polypeptide described wherein at position -10, A is changed to P.

[0119] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:81, CDR2 is represented by SEQ ID NO:101 and CDR3 is represented by SEQ ID NO:123.

[0120] In a further aspect, the present invention provides a method for producing a composition comprising: (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 88, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 110, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 128, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0121] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is SEQ ID NO:88.

[0122] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR2 is SEQ ID NO:110.

[0123] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is SEQ ID NO:128.

[0124] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:88, CDR2 is represented by SEQ ID NO:110, and CDR3 is represented by SEQ ID NO:128.

[0125] In a further aspect, the present invention provides a method for producing a composition comprising: (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO:90, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 112, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 130, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0126] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is SEQ ID NO:90.

[0127] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: Provided herein is a polypeptide as described herein, wherein at position -2, V is changed to A.

[0128] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is SEQ ID NO:130.

[0129] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:90, CDR2 is represented by SEQ ID NO:112, and CDR3 is represented by SEQ ID NO:130.

[0130] In a further aspect, the present invention provides a method for producing a composition comprising: (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 89, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 111, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 129, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0131] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is SEQ ID NO:89.

[0132] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR2 is SEQ ID NO:111.

[0133] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR3 is SEQ ID NO:129.

[0134] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:89, CDR2 is represented by SEQ ID NO:111, and CDR3 is represented by SEQ ID NO:129.

[0135] The present invention also provides (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 91, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 114, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 131, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0136] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or Provided herein is a polypeptide described wherein at position -8, M is changed to T.

[0137] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or The polypeptides described herein are provided wherein at position -7, D is changed to A or K.

[0138] In a further aspect, the present invention provides a method for the preparation of a nucleotide sequence comprising the steps of: (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or Provided are polypeptides described herein, wherein at position -6, S is changed to V.

[0139] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:91, CDR2 is represented by SEQ ID NO:114, and CDR3 is represented by SEQ ID NO:131.

[0140] The present invention also provides (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 94, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 118, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 134, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The present invention provides a polypeptide as described herein selected from the group consisting of:

[0141] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or Provided herein is a polypeptide described wherein at position -9, L is changed to M.

[0142] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or Provided are polypeptides described herein, wherein at position -10, Y is changed to F.

[0143] In a further aspect, the present invention provides a method for the preparation of a nucleotide sequence comprising the steps of: (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or The polypeptides described herein are provided wherein at position -14, V is changed to I.

[0144] In a further aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO:94, CDR2 is represented by SEQ ID NO:118, and CDR3 is represented by SEQ ID NO:134.

[0145] In a further aspect, the present invention provides nanobodies, V HH , humanized V HH , or camelization V H The polypeptide described herein is

[0146] In a further aspect, the present invention provides a polypeptide as described herein, further comprising a serum protein binding moiety.

[0147] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety binds to serum albumin.

[0148] In a further aspect, the invention provides a polypeptide as described herein, wherein the serum protein binding moiety is an ISV that binds to serum albumin.

[0149] In a further aspect, the present invention provides a polypeptide as described herein, wherein the serum albumin-binding ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein, when the CDRs are determined according to the Kabat definition, CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375); and / or, when the CDRs are determined according to Kontermann 2010, CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378), and CDR3 is GGSLSR (SEQ ID NO: 375).

[0150] In a further aspect, the present invention provides a polypeptide as described herein, wherein the serum albumin-binding ISV is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG (SEQ ID NOs: 348-360).

[0151] In a further aspect, the invention provides a polypeptide as described herein, wherein the ISV is directly linked or linked via a linker.

[0152] In a further aspect, the present invention provides a polypeptide as described herein, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 362-372) linkers.

[0153] In a further aspect, the present invention provides a polypeptide as described herein, further comprising a PEG moiety.

[0154] In a further aspect, the present invention provides a nucleic acid or nucleic acid sequence encoding a polypeptide as defined herein.

[0155] In a further aspect, the present invention provides a vector comprising a nucleic acid or nucleic acid sequence as defined herein.

[0156] In a further aspect, the present invention provides a host cell transformed or transfected with a nucleic acid or nucleic acid sequence defined herein, or with a vector defined herein.

[0157] In a further aspect, the present invention provides a method for producing a polypeptide as defined herein, comprising culturing a host cell as defined herein under conditions that allow expression of the polypeptide as defined herein, and collecting the produced polypeptide from the culture.

[0158] In a further aspect, the present invention provides pharmaceutical compositions comprising a polypeptide described herein or a polypeptide produced by a method described herein.

[0159] In a further aspect, the present invention provides a polypeptide as described herein, or produced as described herein, for use in treating a subject in need thereof.

[0160] In a further aspect, the present invention provides a method for delivering a prophylactic or therapeutic polypeptide to a specific location, tissue, or cell type in the body, comprising administering to a subject a polypeptide described herein or produced as described herein.

[0161] In a further aspect, the present invention provides a polypeptide as described herein, or a polypeptide produced as described herein, for use in the prevention, treatment or alleviation of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease.

[0162] In a further aspect, the present invention provides a method for the prevention, treatment or alleviation of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease and an autoimmune disease, the method comprising administering to a subject in need thereof a polypeptide as described herein or a polypeptide produced as described herein.

[0163] In a further aspect, the present invention provides a polypeptide or method for use in the prevention, treatment or alleviation of a disease as described herein, wherein said proliferative disease is cancer.

[0164] In a further aspect, the present invention provides a method for treating cancer comprising administering to a patient a therapeutically effective amount of ... The present invention provides a polypeptide or method for use in the prevention, treatment, or alleviation of a disease described herein selected from the group consisting of: thyroid cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease, or AIDS-related primary effusion lymphoma, neuroectodermal tumor, rhabdomyosarcoma, and any metastasis of any of the above cancers, and non-cancerous manifestations, e.g., nasal polyps.

[0165] In a further aspect, the present invention provides a polypeptide or method for use in the prevention, treatment or alleviation of a disease described herein, wherein the treatment is a combination treatment.

[0166] In a further 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. [Brief explanation of the drawings]

[0167] [Figure 1] QC of human TCR / CD3 and human CD3 transfected cell lines using 100 mM anti-human TCR α / β antibody (clone BW242 / 412) (black) and 100 mM anti-human CD3 antibody (clone OKT-3) (gray). MCF values ​​(mean channel fluorescence) are plotted for each cell line. [Figure 2] Dose-dependent binding of monovalent CD3 nanobodies to human TCR / CD3 expressed on CHO-K1 cells (Figure 2A) and to purified primary human T cells (Figure 2B). MCF values ​​(mean channel fluorescence) are plotted against nanobody concentration. [Figure 3] Dose-dependent binding of monovalent CD3 nanobodies to HEK293H human TCR(2IAN) / CD3 (filled circles), HEK293H human CD3 (crosses), and the HEK293H parental cell line (open circles). MCF values ​​(mean channel fluorescence) are plotted against the concentration of nanobody. [Figure 4] T cell activation data for monovalent CD3 nanobodies bound to beads (Figure 4A). T cell activation data for monovalent CD3 nanobodies present in solution (Figure 4B). Activation is measured by monitoring CD69 upregulation on primary human T cells. MCF values ​​(mean channel fluorescence) are plotted for each nanobody. [Figure 5]Binding of a dilution series of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies to human TCR / CD3 expressed on CHO-K1 cells (Figure 5A), primary human T cells (Figure 5B), and Ramos cells (Figure 5C). MCF values ​​(mean channel fluorescence) are plotted against nanobody concentration. [Figure 6] Dose-dependent killing efficacy of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies in flow cytometry-based human T cell-mediated Ramos (Figure 6A) and Raji (Figure 6B) B cell killing assays. % cell death (% of TOPRO-positive cells) is plotted against nanobody concentration. [Figure 7] Dose-dependent binding of anti-CD20 nanobodies to human CD20 Ramos (open circles) and Raji (filled circles) cells. MCF values ​​(mean channel fluorescence) are plotted against nanobody concentration. [Figure 8] Dose-dependent killing efficacy of CD20xCD3 (solid line) and CD3xCD20 (dashed line) bispecific nanobodies in the xCELLigence-based human T cell-mediated CHO-K1 human CD20 killing assay. CI is plotted against nanobody concentration. [Figure 9] Killing efficacy of 1 μM CD20×CD3 and an irrelevant construct in an xCELLigence-based killing assay using CHO-K1 human CD20 cells (black bars) and the CHO-K1 parental cell line (gray bars) to illustrate TAA-dependent killing. Cell index (CI) is plotted against nanobody concentration. [Figure 10] Dose-dependent killing effect of CD20xCD3 nanobodies with a 9GS linker (open circles - dashed line) and a 35GS linker (filled squares - dashed line) and a CD3xCD20 nanobody with a 35GS linker (filled diamonds - solid line) in a flow cytometry-based killing assay using Ramos. % cell death (TOPRO-positive cells) is plotted against nanobody concentration. [Figure 11]Dose-dependent killing of T017000062 in a flow cytometry-based human T cell-mediated Ramos B cell killing assay using different effector (E) to target (T) ratios (E:T ratio 10:1 - filled circles, E:T ratio 5:1 - open squares, E:T ratio 2:1 - filled triangles, and E:T ratio 1:1 - open diamonds). % cell death (% of TOPRO-positive cells) is plotted against nanobody concentration. [Figure 12] Time-dependent cytolytic activity of CD20 / CD3 in a purified primary human T cell-mediated killing assay in xCELLigence using CHO-K1 human CD20 target cells. % specific lysis is plotted against the concentration of construct. The different curves represent the analysis time after T cell addition. [Figure 13] Binding of serially diluted HLE constructs to human TCR / CD3 expressed on CHO-K1 cells (Figure 13A), primary human T cells (Figure 13B), and Ramos cells (Figure 13C). MCF values ​​(mean channel fluorescence) are plotted against nanobody concentration. [Figure 14] Dose-dependent killing of the CD20xCD3 bispecific nanobody (solid line-diamonds) versus the CD20xCD3xALB11 construct (solid line-filled triangles) in a flow cytometry-based human T cell-mediated Ramos B cell killing assay (Figures 14A, 14C), and the dose-dependent killing effect of the CD20xCD3xALB11 construct in the absence (solid line-filled triangles) or presence (dashed line-open triangles) of 30 μM HSA (Figures 14B, 14D). % cell death (% of TOPRO-positive cells) is plotted against nanobody concentration. [Figure 15] Binding of 100 nM monovalent anti-HER2 nanobody (5F07) to SK-BR-3, MCF-7, and MDA-MB-468 cell lines in flow cytometry to compare HER2 expression levels. MCF values ​​(mean channel fluorescence) are plotted for each cell line. [Figure 16]Dose-dependent killing effect of bispecific CD3xHER2 nanobodies (dashed line) and bispecific HER2xCD3 (solid line) in the xCELLigence-based human T cell-mediated cell killing assay. Data were analyzed using 18 h data. Cell index (CI) was plotted against nanobody concentration. [Figure 17] Dose-dependent INF-γ production by human T cells after incubation of human CD20-positive CHO-K1 cells with bispecific CD20xCD3 nanobodies in a flow cytometry-based killing assay. Data were analyzed after 72 hours of incubation. OD at 450 nm was plotted against nanobody concentration. [Figure 18] Study design for the PBMC B cell depletion model. PBMCs were injected intraperitoneally into animals on day 3 (D3). Mice were treated with T017000084 (CD3 / CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (irrelevant nanobody) from D3 to D7. [Figure 19] Absolute numbers of PBMC-derived B cells on a log scale. Results for individual animals are shown. B cell numbers are shown in function of the different treatment groups. [Figure 20] Ramos model study design. Ramos cells were injected intravenously into mice on D1. PBMCs were injected intraperitoneally into animals on D3. Mice were treated with T017000084 (CD3 / CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (irrelevant nanobody) from D3 to D7. [Figure 21-1] Absolute numbers of Ramos B cells on a log scale. Results for individual animals are shown. Open circles above the graph indicate that the active dose was statistically significantly different from the unrelated NB (T017000088) based on an F-test from a mixed-effects ANOVA analysis. All effects are statistically significant at the 5% level of significance. [Figure 21-2]Absolute numbers of Ramos B cells on a log scale. Results for individual animals are shown. Open circles above the graph indicate that the active dose was statistically significantly different from the unrelated NB (T017000088) based on an F-test from a mixed-effects ANOVA analysis. All effects are statistically significant at the 5% level of significance. [Figure 22-1] Absolute numbers of PBMC-derived B cells on a log scale. Results for individual animals are shown. Open circles above the graph indicate that the active dose was statistically significantly different from the irrelevant NB (T017000088) based on an F-test from a mixed-effects ANOVA analysis. All effects are statistically significant at the 5% level of significance. [Figure 22-2] Absolute numbers of PBMC-derived B cells on a log scale. Results for individual animals are shown. Open circles above the graph indicate that the active dose was statistically significantly different from the irrelevant NB (T017000088) based on an F-test from a mixed-effects ANOVA analysis. All effects are statistically significant at the 5% level of significance. [Figure 23] Measurement of EGFR (Figure 23A; Santa Cruz, sc-120 PE) or CEACAM5 (Figure 23B; Sino Biological, 11077-MM02-P) expression levels in HER14, Hela, LoVo, and LS174-T cell lines by flow cytometry. MCF values ​​(mean channel fluorescence) are plotted for each cell line.

[0168] [Detailed Description of the Invention] The present inventors have recognized that a format for coordinating T cells and tumor cells to elicit an immune response must meet various and often conflicting requirements. The format must be broadly applicable. In particular, the format should preferably be useful for a wide range of patients and, preferably, for a wide range of tumors. The format should preferably be safe and target only the intended cells. In addition, the format should preferably be small enough to easily penetrate tissues and tumors, while also being patient-friendly. For example, the format should have a long half-life so that the format is not rapidly eliminated by renal clearance upon administration. However, a longer half-life should preferably not induce off-target activity and side effects or limit penetration into tissues and tumors. In addition, it has been recognized that tumor cells often develop evasion mechanisms that downregulate antigens targeted during therapy. Thus, in a more preferred version, the format should simultaneously target multiple antigens.

[0169] The present invention fulfills at least one of these needs.

[0170] In particular, it was hypothesized that immunoglobulin single variable domains (ISVs) would, in principle, be ideal candidates because they are small enough to easily penetrate (tumor) tissue and can be combined with other ISVs as building blocks. ISVs directed against CD3, and in particular CD3ε, would then have broad applicability.

[0171] Six clusters of related ISVs were identified. The ISVs possessed a variety of unexpected and advantageous characteristics. First, the ISVs were unexpectedly broadly applicable. Specifically, the CD3 ISVs were able to bind with high affinity to T cells from various donors. When formatted into multispecific polypeptides, the CD3 ISVs were capable of tumor cell killing via various tumor-associated antigens. Thus, the CD3 ISVs can be used against a wide range of cancers. In addition, multispecific polypeptides containing the CD3 ISVs remained active when bound to albumin. This contributes to a favorable PK profile and patient compliance while minimizing side effects. The polypeptides of the present invention were only effective when bound to both T cells and target cells, demonstrating their safety.

[0172] The inventors reasoned that simultaneous targeting of multiple antigens could reduce the likelihood of tumor escape mutants arising, thereby improving the therapeutic activity of T cell engagement strategies. Multispecific polypeptides are provided that comprise a CD3 ISV combined with immunoglobulin single variable domains (double paratopes) directed against different target antigens and / or different epitopes on a specific antigen.

[0173] Immunoglobulin sequences, such as antibodies and the 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 be achieved by immunizing laboratory animals, such as llamas, constructing phage libraries from the immune tissue, selecting phage displaying antigen-binding immunoglobulin single variable domains, and screening the domains and engineered constructs thereof for the desired specificity (WO 94 / 04678). Alternatively, analogous immunoglobulin single variable domains, such as dAbs, can be generated by directly selecting phage displaying antigen-binding immunoglobulin single variable domains from naive or synthetic libraries, followed by screening the domains and engineered constructs thereof for the desired specificity (Ward et al., Nature, 1989, 341:544-6; Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and, e.g., WO 06 / 030220, WO 06 / 003388, and other published patent applications of Domantis Ltd.). Unfortunately, the use of monoclonal and / or highly engineered antibodies also incurs high production costs and may result in suboptimal tumor penetration compared to other strategies.

[0174] The present invention provides multispecific polypeptides that specifically bind to CD3 of the T cell receptor complex, possessing a variety of unexpected and advantageous characteristics. First, the polypeptides are easy to manufacture. Furthermore, the ISVs are unexpectedly broadly applicable. Specifically, the CD3 ISVs were able to bind with high affinity to T cells from various donors. When formatted into multispecific polypeptides, the CD3 ISVs enabled tumor cell killing due to various tumor-associated antigens. In contrast, no killing was observed when the polypeptides did not bind to T cells or target cells. This highlights the safety of the polypeptides of the present invention. Therefore, the CD3 ISVs can be used against many cancers. Furthermore, the CD3 ISVs can be considered safe. In addition, multispecific polypeptides containing the CD3 ISVs remained active when bound to albumin. This may contribute to a favorable PK profile and patient compliance while minimizing side effects.

[0175] The present invention therefore relates to a polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), wherein the first ISV has high affinity for / binds to CD3 and the second ISV has high affinity for / binds to an antigen on a cell (target cell), preferably a tumor cell. The antigen is preferably specific for said target cell, such as a tumor-associated antigen (TAA). The multispecific polypeptide of the invention triggers T cell activation in order to home T cells to cells, such as tumor cells, and enable said T cells to inhibit or kill said target cells, e.g., said tumor cells.

[0176] Definition: a) Unless otherwise indicated or specified, all terms used have their ordinary meaning in the art, which will be apparent to a person skilled in the art, see for example the standard handbooks referred to in WO 08 / 020079, page 46, paragraph a).

[0177] b) The term "immunoglobulin single variable domain", which is used interchangeably with "single variable domain" and "ISV", defines a molecule in which the antigen-binding site resides on and is formed by a single immunoglobulin domain. This distinguishes it from "conventional" immunoglobulins or fragments thereof (e.g., Fab, scFv, etc.), in which two immunoglobulin domains, in particular 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 the VH and the VL will contribute to the antigen-binding site. That is, a total of six CDRs will be involved in forming the antigen-binding site. In contrast, the binding site of an immunoglobulin single variable domain is formed by one VH or VL domain. Therefore, the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0178] Thus, the terms "immunoglobulin single variable domain," "single variable domain," and "ISV" do not include conventional immunoglobulins or fragments thereof which require the interaction of at least two variable domains to form an antigen-binding site, although these terms are intended to include fragments of conventional immunoglobulins in which the antigen-binding site is formed by a single variable domain.

[0179] The term "immunoglobulin single variable domain" or "ISV" refers to an antigen-binding domain or fragment, e.g., a V HH Domain or V H Or V L The terms antigen-binding molecule or antigen-binding protein are used interchangeably and also include the term nanobody. Immunoglobulin single variable domains are those that contain a light chain variable domain sequence (e.g., V L sequence) or heavy chain variable domain sequence (e.g., V Hsequences). More particularly, 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. Thus, immunoglobulin single variable domains can be domain antibodies or immunoglobulin sequences suitable for use as domain antibodies, single domain antibodies or immunoglobulin sequences suitable for use as single domain antibodies, "dAbs" or immunoglobulin sequences suitable for use as dAbs, or nanobodies. Nanobodies are characterized by the V HH The immunoglobulin single variable domains include, but are not limited to, humanized VHH sequences, humanized VHH sequences, or camelized VH sequences. The present invention encompasses immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, and human, as well as camelized immunoglobulin sequences. Immunoglobulin single variable domains include fully human, humanized, otherwise sequence-optimized, or chimerized immunoglobulin sequences. The structure of immunoglobulin single variable domains and immunoglobulin single variable domains can be considered to be composed of, but not limited to, four framework regions or "FRs," which are 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. Between the framework regions are inserted three complementarity-determining regions or "CDRs." The complementary determining regions or "CDRs" are referred to in the art as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. It is noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx NV and therefore may also be referred to as Nanobody® or Nanobodies®, respectively.

[0180] c) Unless otherwise specified, the terms "immunoglobulin sequence", "sequence", "nucleotide sequence" and "nucleic acid" are as defined in WO 08 / 020079, page 46, paragraph b).

[0181] d) Unless otherwise indicated, all methods, steps, techniques, and operations not specifically described in detail can be performed and are carried out in a manner known per se, as would be apparent to one skilled in the art. Reference is again made, for example, to the standard handbooks and the general background art mentioned herein, as well as to the further references cited herein; and to the following reviews, for example: 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 Suppl.A: S106-112), Gonzales et al. 2005 (Tumor Biol. 26(1): 31-43), which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins, such as immunoglobulins.

[0182] e) Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. See Table A-2 on page 48 of WO 08 / 020079 (Ablynx NV), entitled "Immunoglobulin single domains directed against IL-6R and polypeptides comprising same for the treatment of diseases and disorders associated with IL-6-mediated signal transduction."

[0183] f) For purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated or determined, for example, by dividing the number of nucleotides in a first nucleotide sequence that are identical to nucleotides at corresponding positions in a second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%, as described in paragraph e) of page 49 of WO 08 / 020079 (incorporated herein by reference), where deletions, insertions, substitutions, or additions of nucleotides in a second nucleotide sequence compared to a first nucleotide sequence are each considered as differences at a single nucleotide (position), or again using an appropriate computer algorithm or technique, as described in paragraph e) of page 49 of WO 08 / 020079 (incorporated herein by reference).

[0184] g) For purposes of comparing two or more immunoglobulin single variable domains or other amino acid sequences, such as the polypeptides of the invention, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated or determined, for example, as described in paragraph f) of pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference), by dividing the number of amino acid residues in the first amino acid sequence that are identical to amino acid residues at corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%. Here, 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 as a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein, or again, using an appropriate computer algorithm or technique, as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (hereby incorporated by reference).

[0185] In determining the degree of sequence identity between two immunoglobulin single variable domains, the skilled person can also take into account so-called "conservative" amino acid substitutions, as described on page 50 of WO 08 / 020079.

[0186] Any amino acid substitutions applied to the polypeptides described herein may be based on the frequency analysis of amino acid mutations between homologous proteins of different species developed by Schulz et al. 1978 (Principles of Protein Structure, Springer-Verlag), the structure-forming potential analysis developed by Chou and Fasman 1975 (Biochemistry 13: 211) and 1978 (Adv. Enzymol. 47: 45-149), and the structure-forming potential analysis developed 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: The analysis of hydrophobicity patterns in proteins developed by [End Page 109] and [End Page 110] (pp. 321-353), all of which are incorporated herein by reference in their entirety. Information on the primary, secondary, and tertiary structure of nanobodies is provided in the description herein and in the general background art cited above. For this purpose, V obtained from llamas has also been used. HH Crystal structures of domains are provided, for example, by Desmyter et al. 1996 (Nature Structural Biology, 3: 803), Spinelli et al. 1996 (Natural Structural Biology 3: 752-757), and Decaniere et al. 1999 (Structure, 7: 361). H In the domain, V H / V LFurther information on some of the amino acid residues that form the interface and potential camelizing substitutions at these positions can be found in the prior art cited above.

[0187] h) Immunoglobulin single variable domains and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length.

[0188] i) When comparing two immunoglobulin single variable domains, the term "amino acid difference" refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence. It is understood that two immunoglobulin single variable domains can contain one, two, or more such amino acid differences.

[0189] j) When a nucleotide sequence or an amino acid sequence is said to "comprise" or "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, this has the meaning given in paragraph i) of pages 51-52 of WO 08 / 020079.

[0190] k) The term "essentially isolated from" has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08 / 020079.

[0191] l) The terms "domain" and "binding domain" have the meaning given in paragraph k) on page 53 of WO 08 / 020079.

[0192] m) The terms "antigenic determinant" and "epitope", which may be used interchangeably herein, have the meaning given in paragraph l) of page 53 of WO 08 / 020079.

[0193] n) As further described in paragraph m) of page 53 of WO 08 / 020079, an amino acid sequence (e.g. an antibody, a polypeptide of the invention, or generally an antigen-binding protein or polypeptide, or fragment thereof) that is able to (specifically) bind to, has affinity for and / or has specificity for a particular antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or "directed against" said antigenic determinant, epitope, antigen or protein.

[0194] o) The term "specificity" refers to the number of different antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (e.g., an ISV, Nanobody, or polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity.

[0195] The equilibrium constant for dissociation of the antigen from the antigen-binding protein (K D Affinity, expressed as the affinity (KD) of an antigen, is a measure of the binding strength between an antigenic determinant, i.e., a target, and an antigen-binding site on an antigen-binding protein, i.e., an ISV or nanobody. D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (or affinity is expressed as 1 / K D The affinity constant (K A )). As will be clear to the skilled artisan (e.g., based on the further disclosure herein), affinity can be determined in a manner known per se depending on the specific antigen of interest.

[0196] Avidity is the affinity of a polypeptide. That is, a ligand can bind via two (or more) pharmacophores (ISVs). In this binding, multiple interactions cooperate to improve the "apparent" affinity. Avidity is a measure of the binding strength between a polypeptide of the present invention and an appropriate antigen. A polypeptide of the present invention can bind to at least two targets via its two (or more) building blocks, e.g., ISVs or nanobodies. In this binding, multiple interactions, e.g., a first building block, ISV, or nanobody binding to a first target and a second building block, ISV, or nanobody binding to a second target, cooperate to improve the "apparent" affinity. Avidity is related to both the affinity between an antigenic determinant and an antigen-binding site on the antigen-binding molecule and the number of appropriate binding sites present in the antigen-binding molecule. For example, but not limited to, a polypeptide containing two or more building blocks, e.g., ISVs or Nanobodies, directed against different targets on a cell, can (and typically will) bind with higher avidity than each of the individual monomers or individual building blocks, e.g., monovalent ISVs or Nanobodies, contained in a polypeptide of the invention.

[0197] 10 -4 Any K greater than mol / liter D value (or 10 4 M -1 Any K less than (liters / mol) A values) are generally considered to represent nonspecific binding.

[0198] The polypeptides of the invention comprise a first and a second building block, e.g., a first and a second ISV or a first and a second Nanobody. Preferably, the affinity of each building block, e.g., an ISV or Nanobody, is determined individually. In other words, the affinity is determined for a monovalent building block, ISV, or Nanobody independently of the avidity effects of other building blocks, ISVs, or Nanobodies, which may or may not be present. The affinity for a monovalent building block, ISV, or Nanobody can be determined for the monovalent building block, ISV, or Nanobody itself, i.e., when said monovalent building block, ISV, or Nanobody is not comprised in a polypeptide of the invention. Alternatively or additionally, the affinity for a monovalent building block, ISV, or Nanobody can be determined for one target in the absence of other targets.

[0199] The binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, scatter analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as their various modifications known per se in the art, and other techniques mentioned herein.

[0200] As will be apparent to one skilled in the art, the dissociation constant can be an actual or apparent dissociation constant. Methods for determining dissociation constants will be apparent to one skilled in the art and include, for example, the techniques mentioned herein. In this regard, -4 moles / liter or 10 -3 More than 10 moles per liter (e.g., 10 -2 It will also be apparent that dissociation constants in moles / liter (K) cannot be measured. Also, as will be apparent to those skilled in the art, in some cases, the (actual or apparent) dissociation constant can be calculated using the (actual or apparent) association constant (K A ) based on the correlation [K D =1 / K A ] can be calculated as follows.

[0201] Affinity refers to the strength or stability of an intermolecular interaction. Affinity is generally defined as K D or dissociation constant, which has units of mol / liter (or M). Affinity is determined by the association constant K A It can also be expressed as 1 / K D is equal to (mol / liter) -1 (or M -1 ) units. As used herein, the stability of an interaction between two molecules (e.g., an amino acid sequence, Nanobody, or polypeptide of the invention and its intended target) is measured by the K D It will be primarily expressed in terms of the value of the correlation K A =1 / K D From the perspective of K D Specifying the strength of a molecular interaction by its value is the corresponding K A It will be apparent to those skilled in the art that KD values ​​can also be calculated using the well-known correlation DG = RT.In(K D )(DG=RT.In(K A It also characterizes the strength of intermolecular interactions in a thermodynamic sense, since it is related to the free energy of binding (DG) by ∑ R = ∑ ∑ T = ∑ ...

[0202] K for biological interactions that are considered meaningful (e.g., specific) D is typically 10 -10 M(0.1nM)~10 -5 The stronger the interaction, the higher its K D becomes smaller.

[0203] K D can also be expressed as the ratio of the dissociation rate constant of the complex (denoted koff) to its association rate (denoted kon). (K D =k off / k on and K. A =kon / k off (The off rate k off is, s -1 (where s is the SI unit for second). on is M -1 s -1 From the diffusion-limited association rate constant approach for biomolecular interactions, the on rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 The off rate can be varied by the correlation t 1 / 2 =In(2) / k off The off rate is related to the half-life of a given molecular interaction by -6 s -1 (A few days t 1 / 2 Near-irreversible complex with ~1s -1 (t 1 / 2 =0.69s).

[0204] The affinity of an intermolecular interaction between two molecules can be measured by various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology, 13: 1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, in which one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions, resulting in k on , k off Measurement value of K D (or K A) values ​​are obtained. For example, this can be done using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, 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).

[0205] Alternatively, affinity can be measured in a Kinetic Exclusion Assay (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem., 328: 35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method that measures the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrated solution of antibody / antigen complexes is passed through a column containing beads pre-coated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecules. Detection of the antibody (or antigen) captured in this way is achieved by binding a fluorescently labeled protein to the antibody (or antigen).

[0206] If the measurement method somehow affects the intrinsic binding affinity of the molecule in question, for example, due to artifacts associated with coating one molecule on the biosensor, the measured K D is an apparent K DIt will also be apparent to those skilled in the art that the apparent KD can correspond to the affinity of the two molecules. Also, when one molecule contains more than one recognition site for another molecule, the apparent KD can be measured. In such a situation, the measured affinity may be affected by the avidity of the interaction of the two molecules.

[0207] Another approach that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) method of Friguet et al. 1985 (J. Immunol. Methods, 77: 305-19). This method establishes a liquid-phase binding equilibrium measurement and avoids possible artifacts associated with adsorption of one of the molecules onto a support, e.g., plastic.

[0208] However, K D Accurate measurement of K can be laborious and often results in an apparent K D The apparent K value is measured to assess the binding strength of two molecules. Only if all measurements are performed in a consistent manner (e.g., without changing assay conditions) will the apparent K be obtained. D The measured value is the true K D can be used as an approximation of K D and apparent K D It should be noted that should be treated with equal importance or relevance.

[0209] Finally, in many situations, experienced scientists will use K to determine binding affinity for some reference molecule. DIt should be noted that, for example, to assess the binding strength between molecules A and B, one can use, for example, a reference molecule C known to bind to B and appropriately labeled with a fluorophore or chromophore group or other chemical moiety, e.g., biotin for easy detection in ELISA or FACS (fluorescence activated cell sorter), or other formats (fluorophore for fluorescence detection, chromophore for absorbance detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is maintained at a fixed concentration, and the concentration of A is varied for a given concentration or amount of B. As a result, an IC corresponds to the concentration of A at which the signal measured for C in the absence of A is halved. 50 The value of K of the reference molecule is obtained. D K D ref and the total concentration of the reference molecule, C ref is known, the apparent K for the interaction AB D is expressed by the following formula:K D =IC 50 / (1+c ref / K D ref ) can be obtained from ref < <K D ref If K D ≒IC 50 It is important to note whether IC 50 Measurements of the binders being compared (e.g., C ref The strength or stability of intermolecular interactions can be measured using IC, provided that the measurements are performed in a consistent manner (i.e., the IC is fixed and maintained). 50 This measurement will be referred to as K throughout this text. D Or apparent K D is judged to be equal to

[0210] p) The half-life of an amino acid sequence, compound, or polypeptide of the present invention can generally be defined as described in paragraph o) on page 57 of WO 08 / 020079, and as mentioned therein, refers to the time it takes for the serum concentration of the amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound by natural mechanisms and / or clearance or sequestration of the sequence or compound. The in vivo half-life of an amino acid sequence, compound, or polypeptide of the present invention can be determined by any method known per se, for example, by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and can, for example, generally be those described in paragraph o) on page 57 of WO 08 / 020079. Furthermore, as mentioned in paragraph o) on page 57 of WO 08 / 020079, half-life can be expressed using parameters such as t½-alpha, t½-beta, and area under the curve (AUC). See, for example, the experimental section below, and standard handbooks, such as Kenneth et al. 1996 (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters et al. 1996 (Pharmacokinete analysis: A Practical Approach). See also Gibaldi & Perron 1982 (Pharmacokinetics, Dekker M, 2nd Rev. edition). The term "prolonged half-life" or "prolonged half-life" is as defined in WO 08 / 020079, page 57, paragraph o), and refers in particular to an increase in t½-beta, where t½-alpha and / or AUC, or both, may or may not be increased.

[0211] q) With respect to a target or antigen, the term "interaction site" on the target or antigen means a site, epitope, antigenic determinant, portion, domain, or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor, or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerization (such as homomultimerization or heterodimerization) of the target or antigen, or any other site, epitope, antigenic determinant, portion, domain, or stretch of amino acid residues on the target or antigen that is involved in the biological action or mechanism of the target or antigen. More generally, an "interaction site" can be any site, epitope, antigenic determinant, portion, domain, or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and / or any pathway, interaction, signal transduction, biological mechanism, or biological action in which the target or antigen is involved) is modulated.

[0212] r) the immunoglobulin single variable domain or polypeptide has at least 10-fold, for example at least 100-fold, and preferably at least 1000-fold, and up to 10,000-fold or more better affinity / avidity (as defined above, K) for binding to a second target or antigen than the affinity of said amino acid sequence or polypeptide for binding to the second target or polypeptide; D value, K A value, K off Rate and / or K on A target or antigen is said to be "specific" for a first target or antigen if it binds to the first antigen at a K (suitably expressed as a K rate) for that amino acid sequence or polypeptide to bind to a second target or polypeptide. D K is at least 10 times smaller, e.g., at least 100 times smaller, and preferably at least 1000 times smaller, e.g., less than 10,000 times smaller than DPreferably, when an immunoglobulin single variable domain or polypeptide is "specific" for a first target or antigen compared to a second target or antigen, the immunoglobulin single variable domain or polypeptide is directed against said first target or antigen (as defined herein) but not against said second target or antigen.

[0213] The terms "cross-block," "cross-blocked," and "cross-blocking" are used interchangeably herein to refer to the ability of an immunoglobulin single variable domain or polypeptide to prevent a natural ligand from binding to its receptor. The extent to which an immunoglobulin single variable domain or polypeptide of the invention can prevent another compound, e.g., a natural ligand, from binding to its target, and therefore whether an immunoglobulin single variable domain or polypeptide can be said to cross-block according to the invention, can be determined using a competitive binding assay. One particularly suitable quantitative cross-blocking assay uses a FACS- or ELISA-based approach or Alphascreen to measure competition for binding to a target between a labeled (e.g., His-tagged or biotinylated) immunoglobulin single variable domain or polypeptide of the invention and another binding agent. The experimental section generally describes FACS-, ELISA-, or Alphascreen-displacement-based assays suitable for determining whether a binding molecule cross-blocks or is able to cross-block an immunoglobulin single variable domain or polypeptide of the invention. It will be appreciated that the assay can be used with any of the immunoglobulin single variable domains or other binding agents described herein.Thus, in general, a cross-blocking amino acid sequence or other binding agent of the invention is one that will bind to a target in a cross-blocking assay such that, for example, in the assay and in the presence of a second amino acid sequence or other binding agent of the invention, the recorded displacement of an immunoglobulin single variable domain or polypeptide of the invention is 60% to 100% (e.g., in an ELISA / Alphascreen-based competitive assay) or 80% to 100% (e.g., in a FACS-based competitive assay) of the maximum theoretical displacement (e.g., displacement by a cold (e.g., unlabeled) immunoglobulin single variable domain or polypeptide that needs to be cross-blocked) by the potential cross-blocking agent to be tested (which can be another conventional monoclonal antibody, e.g., IgG, classical monovalent antibody fragments (Fab, scFv), and engineered variants (e.g., diabodies, triabodies, minibodies, VHH, dAb, VH, VL)) present in an amount of 0.01 mM or less.

[0214] t) An amino acid sequence, such as an immunoglobulin single variable domain or polypeptide of the invention, is said to be a "VHH1-type immunoglobulin single variable domain" or a "VHH1-type sequence" if the VHH1-type immunoglobulin single variable domain or VHH1-type sequence has 85% identity (using the BLAST algorithm with standard settings, i.e., the blosom62 scoring matrix, using the VHH1 consensus sequence as the query sequence) to the VHH1 consensus sequence (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) (SEQ ID NO: 361), and enforces a cysteine ​​at position 50, i.e., C50 (using Kabat numbering).

[0215] u) An amino acid sequence, such as an immunoglobulin single variable domain or polypeptide of the invention, is said to be "cross-reactive" with two different antigens or antigenic determinants (e.g., serum albumin from two different mammalian species, e.g., human serum albumin and cynomolgus serum albumin) if it is specific (as defined herein) for both of these different antigens or antigenic determinants.

[0216] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of immunoglobulin single variable domains are similar to those of camelid-derived V in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see e.g., Figure 2 of this publication). HH As applied to the domain, the V provided by Kabat et al. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, Publication No. 91) H Numbered according to the common numbering for domains. V HIt should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains will usually be in the range of 110-120, often 112-115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0217] CDR regions can also be determined by various methods. In the CDR determination according to Kabat, FR1 of an immunoglobulin single variable domain comprises amino acid residues 1 to 30. CDR1 of an immunoglobulin single variable domain comprises amino acid residues 31 to 35. FR2 of an immunoglobulin single variable domain comprises amino acid residues 36 to 49. CDR2 of an immunoglobulin single variable domain comprises amino acid residues 50 to 65. FR3 of an immunoglobulin single variable domain comprises amino acid residues 66 to 94. CDR3 of an immunoglobulin single variable domain comprises amino acid residues 95 to 102. FR4 of an immunoglobulin single variable domain comprises amino acid residues 103 to 113.

[0218] In the present application, unless otherwise specified, the CDR sequences were determined according to Kontermann and Dubel (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 1 to 25. CDR1 contains amino acid residues 26 to 35. FR2 contains amino acid residues 36 to 49. CDR2 contains amino acid residues 50 to 58. FR3 contains amino acid residues 59 to 94. CDR3 contains amino acid residues 95 to 102. FR4 contains amino acid residues 103 to 113.

[0219] w) The Figures, Sequence Listing, and Experimental Section / Examples are provided solely to further illustrate the present invention and should not be construed or deemed to in any way limit the scope of the present invention and / or the appended claims, unless otherwise expressly indicated herein.

[0220] x) The half maximal inhibitory concentration (IC50) is a measure of a compound's effectiveness in inhibiting a biological or biochemical function, e.g., pharmacological effect. This quantitative measurement indicates how much of an ISV or Nanobody (inhibitor) is required to inhibit half of a given biological process (or component of a process, i.e., enzyme, cell, cell receptor, chemotaxis, anaplasia, metastasis, invasiveness, 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 a dose-response curve and testing the effect of various concentrations of an antagonist, e.g., an ISV or Nanobody of the invention, on reversing agonist activity in inverse agonist activity. The IC50 value can be calculated for a given antagonist, e.g., an ISV or Nanobody of the invention, by determining the concentration required to inhibit half of the maximal biological response of the agonist.

[0221] The term half-maximal effective concentration (EC50) refers to the concentration of a compound that elicits a response halfway between baseline and maximum after a specific exposure time. In this context, EC50 is used as a measure of polypeptide, ISV, or nanobody potency. The EC50 of a step-wise dose-response curve represents the concentration of a compound at which 50% of its maximum effect is observed. Concentrations are preferably expressed in molar units.

[0222] In biological systems, small changes in ligand concentration typically result in rapid changes in response, following a sigmoid function. The inflection point at which the increase in response with increasing ligand concentration begins to decline is the EC50. This can be mathematically determined by deriving a line of best fit. It is often convenient to rely on a graph for estimation. Where an EC50 is provided in the Examples section, the experiment was designed to reflect the KD as accurately as possible. In other words, the EC50 value can then be considered the KD value. The term "average KD" refers to the average KD value obtained in at least one, but preferably more than one, e.g., at least two experiments. The term "average" refers to the mathematical term "mean" (the sum of the data divided by the number of items in the data).

[0223] It also relates to IC50, a measure of compound inhibition (50% inhibition). For competitive binding assays and functional antagonist assays, IC50 is the most common summary measure of the dose-response curve. For agonist / stimulator assays, the most common summary measure is EC50.

[0224] y) As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, it should be noted that reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the methods described herein.

[0225] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain without undue experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0226] As used herein, the term "and / or" includes the meaning of "and," "or," and "all or any other combination of the elements connected by said term."

[0227] As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range.

[0228] Throughout this specification and the claims that follow, unless otherwise required, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced by "containing" or "including," or optionally, as used herein, by the term "having."

[0229] The present invention relates to a polypeptide comprising at least a first immunoglobulin single variable domain (ISV) and at least one further immunoglobulin single variable domain (ISV), wherein said at least first ISV has high affinity for / binds to cluster of differentiation 3 (CD3) and said at least one further ISV has high affinity for / binds to an antigen on a target cell.

[0230] Typically, the multispecific polypeptides of the present invention combine high-affinity antigen recognition for target cells with T cell activation, resulting in activation independent of the T cell's native specificity. The mode of action of binding molecules that bind both a cell surface molecule on a target cell, such as a tumor antigen, and the T cell co-receptor CD3, is generally known. Close proximity of a T cell to a target cell, i.e., binding of the T cell, results in killing of the target cell by the T cell. In the present invention, this process is utilized to combat proliferative, inflammatory, infectious, and autoimmune diseases. Typically, T cells are equipped with granules containing a lethal combination of a pore-forming protein, known as perforin, and a death-inducing protease, known as granzyme. Preferably, these proteins are delivered into the target cell via a cytolytic synapse, which is formed when the T cell approaches the target cell it intends to kill. Approach between the T cell and the target cell is usually achieved by the T cell binding to an MHC / peptide complex using its matching T cell receptor. The polypeptides of the present invention provide such access of T cells to target cells in the absence of T cell receptor / MHC interactions.

[0231] Thus, the present invention relates to the polypeptides described herein that direct T cells to target cells.

[0232] For one arm (first ISV), the multispecific polypeptide has high affinity for / binds to CD3, a protein component of the signaling complex of the T cell receptor on T cells. For another arm (e.g., second and / or third ISV), the multispecific polypeptide recognizes and binds to an antigen on a target cell with high affinity. Preferably, T cell activation is observed only when the multispecific polypeptide is presented to the T cell on the surface of the target cell. The antigen dependence of the target cell for activation results in a favorable safety profile. In one embodiment, the multispecific polypeptide transiently links the T cell to the target cell. Preferably, the multispecific polypeptide can attract resting polyclonal T cells, e.g., CD4+ and / or CD8+ T cells, to an activated state for highly potent redirected lysis of the target cell. Preferably, the T cell is homing to a subsequent target cell after lysis of the first target cell.

[0233] Proteins and polypeptides comprising or consisting essentially of two or more immunoglobulin single variable domains (e.g., at least two immunoglobulin single variable domains of the invention) will be referred to herein as "multivalent" proteins or polypeptides or "multivalent constructs." Some non-limiting examples of such multivalent constructs will become clear from the further description herein. The polypeptides of the invention are "multivalent," i.e., comprise two or more building blocks or ISVs, of which at least a first building block, ISV, or Nanobody and a second building block, ISV, or Nanobody are different and directed against different targets, e.g., antigens or antigenic determinants. Polypeptides of the invention that contain at least two building blocks, ISVs or Nanobodies, of which at least one building block, ISV or Nanobody is directed against a first antigen (i.e. a first target, such as, for example, CD3) and at least one building block, ISV or Nanobody is directed against a second antigen (i.e. a second target different from the first target, such as, for example, a TAA, for example, CD20 or HER2), will also be referred to as "multispecific" polypeptides of the invention. Building blocks, ISVs or Nanobodies present in such polypeptides will also be referred to herein as being in a "multivalent format" or "multispecific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide comprising at least one building block, ISV or Nanobody directed against a first target (e.g., CD3) and at least one further building block, ISV or Nanobody directed against a second target (i.e. a second target different from said first target, such as, for example, a TAA, for example, CD20 or HER2).On the other hand, a "trispecific" polypeptide of the invention is a polypeptide comprising at least one building block, ISV or nanobody directed against a first target (e.g., CD3), a second building block, ISV or nanobody directed against a second target different from said first target (e.g., a TAA, such as CD20 or HER2), and at least one further building block, ISV or nanobody directed against a third antigen (i.e., different from both the first and second target, e.g., another TAA); etc. As will be clear from the description, the invention is not limited to bispecific polypeptides in the sense that the multispecific polypeptide of the invention can comprise at least a first building block, ISV or nanobody directed against a first target, a second building block, ISV or nanobody directed against a second target, and any number of building blocks, ISVs or nanobodies directed against one or more targets which can be the same as or different from the first and / or second targets, respectively. The building blocks, ISVs or nanobodies can optionally be linked via linker sequences.

[0234] The terms bispecific polypeptide, bispecific format, bispecific construct, bispecific nanobody construct, bispecific, and bispecific antibody are used interchangeably herein.

[0235] As will be clear from the above and further description herein, the immunoglobulin single variable domains of the invention can be used as "building blocks" to form, e.g., by appropriately combining them with other groups, residues, moieties, or binding units, the polypeptides of the invention to form compounds or constructs described herein (including, but not limited to, the bivalent / trivalent / tetravalent / multivalent and bi / tri / quadruplex / multispecific polypeptides of the invention described herein) that combine one or more desired properties or biological functions in one molecule.

[0236] It will be understood that the ISV that binds to CD3 and the ISV that binds to an antigen on a target cell can be arranged in any order in the polypeptide of the present invention (as also explained in the Examples section). In particular, in one embodiment, the ISV that binds to CD3 is arranged at the N-terminus and the ISV that binds to an antigen on a target cell is arranged at the C-terminus. In another embodiment, the ISV that binds to an antigen on a target cell is arranged at the N-terminus and the ISV that binds to CD3 is arranged at the C-terminus.

[0237] In a preferred embodiment, the polypeptide of the invention comprises at least a first, at least a second, and at least a third immunoglobulin single variable domain (ISV), wherein said at least a first ISV has high affinity for / binds to CD3, said at least a second ISV has high affinity for / binds to a first antigen on a target cell, and said at least a third ISV has high affinity for / binds to a second antigen on a target cell, wherein said second antigen is different from said first antigen. The first and second antigens can be present on the same or different target cells.

[0238] It will be understood that the ISVs that bind to CD3 and the ISVs that bind to the first and second antigens on the target cell can be arranged in any order in the polypeptide of the invention (as also explained in the Examples section). In particular, in one embodiment, the ISV that binds to CD3 is arranged at the N-terminus, the ISV that binds to the first antigen on the target cell is arranged in the center, and the ISV that binds to the second antigen on the target cell is arranged at the C-terminus. In another embodiment, the ISV that binds to CD3 is arranged at the N-terminus, the ISV that binds to the second antigen on the target cell is arranged in the center, and the ISV that binds to the first antigen on the target cell is arranged at the C-terminus. In another embodiment, the ISV that binds to the first antigen on the target cell is arranged at the N-terminus, the ISV that binds to the second antigen on the target cell is arranged in the center, and the ISV that binds CD3 is arranged at the C-terminus. In another embodiment, the ISV that binds to the first antigen on the target cell is arranged at the N-terminus, the ISV that binds to the second antigen on the target cell is arranged in the center, and the ISV that binds CD3 is arranged at the C-terminus. In another embodiment, the ISV that binds to the second antigen on the target cell is positioned at the N-terminus, the ISV that binds to CD3 is positioned in the center, and the ISV that binds to the first antigen on the target cell is positioned at the C-terminus. In another embodiment, the ISV that binds to the second antigen on the target cell is positioned at the N-terminus, the ISV that binds to the first antigen on the target cell is positioned in the center, and the ISV that binds to CD3 is positioned at the C-terminus.

[0239] The present invention further relates to compounds or constructs, in particular proteins or polypeptides, comprising or consisting essentially of one or more ISVs or polypeptides of the invention, optionally further comprising one or more other groups, residues, moieties, or binding units. As will become apparent to those skilled in the art from the further disclosure herein, such further groups, residues, moieties, binding units, or amino acid sequences may or may not provide additional functionality to the polypeptide of the invention (and / or to the compound or construct in which it is present), and may or may not modify the properties of the polypeptide of the invention.

[0240] The compounds, constructs or polypeptides of the invention can generally be prepared by a method comprising at least one step of suitably linking one or more immunoglobulin single variable domains of the invention to one or more further groups, residues, moieties or binding units, optionally via one or more suitable linkers, to provide the compounds, constructs or polypeptides of the invention. The polypeptides of the invention can also generally be prepared by a method comprising at least the steps of providing a nucleic acid encoding a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such a method can be carried out in a manner known per se. The method will be clear to the skilled person, for example, based on the methods and techniques further described herein.

[0241] Methods for designing / selecting and / or preparing compounds, constructs or polypeptides of the invention starting from an amino acid sequence of the invention are also referred to herein as "formatting" said amino acid sequence of the invention. Amino acids of the invention that constitute part of a compound, construct or polypeptide of the invention are also said to be "formatted" into or in a formatted state as said compound, construct or polypeptide of the invention. Examples of ways in which amino acid sequences of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein. Such formatted immunoglobulin single variable domains or polypeptides constitute a further aspect of the invention.

[0242] For example, such further groups, residues, moieties or binding units can be one or more further immunoglobulin single variable domains. This makes the compound or construct a (fusion) protein or (fusion) polypeptide. In a preferred, but non-limiting aspect, the one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, the one or more other groups, residues, moieties or binding units are selected from the group consisting of domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains (ISVs) suitable for use as single domain antibodies, "dAbs", immunoglobulin single variable domains suitable for use as dAbs, or nanobodies. Alternatively, such groups, residues, moieties or binding units can be, for example, chemical groups, residues, moieties which may or may not themselves be biologically and / or pharmacologically active. For example, but not limited to, such groups can be attached to one or more immunoglobulin single variable domains or polypeptides of the invention to provide "derivatives" of the ISVs or polypeptides of the invention, as further described herein.

[0243] Also within the scope of the present invention are compounds or constructs that comprise or consist essentially of one or more of the derivatives described herein, and that further comprise one or more other groups, residues, moieties, or binding units, optionally linked via one or more linkers. Preferably, the one or more other groups, residues, moieties, or binding units are immunoglobulin single variable domains. In the above-mentioned compounds or constructs, the one or more immunoglobulin single variable domains of the invention and the one or more groups, residues, moieties, or binding units can be linked to each other directly and / or via one or more suitable linkers or spacers. For example, if the one or more groups, residues, moieties, or binding units are immunoglobulin single variable domains, the linker can also be an immunoglobulin single variable domain. The resulting compound or construct is a fusion protein or fusion polypeptide.

[0244] In some embodiments, the polypeptide comprises at least two or more immunoglobulin single variable domains disclosed herein. In some embodiments, the polypeptide consists essentially of two or more immunoglobulin single variable domains disclosed herein. A polypeptide "consisting essentially of" two or more immunoglobulin single variable domains is a polypeptide that, in addition to two or more immunoglobulin single variable domains disclosed herein, does not have any further immunoglobulin single variable domains. For example, a polypeptide consisting essentially of two immunoglobulin single variable domains does not contain any further immunoglobulin single variable domains. However, it should be understood that a polypeptide consisting essentially of two or more immunoglobulin single variable domains can comprise further functionality, such as a label, a toxin, one or more linkers, binding sequences, etc. These further functionality includes both amino acid-based and non-amino acid-based groups. In some embodiments, the polypeptide consists of one or more immunoglobulin single variable domains disclosed herein. It should be understood that the terms "polypeptide construct" and "polypeptide" can be used interchangeably herein (unless the context clearly indicates otherwise).

[0245] In some embodiments, the polypeptide comprises a multivalent or multispecific construct comprising an immunoglobulin single variable domain disclosed herein. In some embodiments, the polypeptide comprises one or more antibody-based and / or non-antibody-based scaffolds disclosed herein. In some embodiments, the polypeptide comprises a serum-binding protein moiety. In some embodiments, the serum-binding protein moiety is an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain is a Nanobody®.

[0246] It will be understood that the order (orientation) of building blocks, e.g., first building block, second building block, third building block, etc., on a polypeptide can be selected according to the needs of the skilled artisan and the relative affinities, which may depend on the positions of these building blocks in the polypeptide. Whether the polypeptide includes a linker is a matter of design choice. However, some orientations, with or without a linker, may provide preferred binding properties compared to other orientations. For example, the order of the first and second building blocks in a polypeptide of the invention can be (from N-terminus to C-terminus): (i) first building block (e.g., first ISV, e.g., first Nanobody)-[linker]-second building block (e.g., second ISV, e.g., second Nanobody); or (ii) second building block (e.g., second ISV, e.g., second Nanobody)-[linker]-first building block (e.g., first ISV, e.g., first Nanobody); (wherein the linker is optional). All orientations are encompassed by the present invention. Polypeptides containing building block orientations that provide desired (binding) properties can be readily identified, for example by routine screening as exemplified in the experimental section.

[0247] The first immunoglobulin single variable domain (ISV) of the polypeptide of the invention binds with high affinity to effector cells, preferably to the TCR complex of said effector cells, and even more preferably to CD3.

[0248] Effector cells are cells containing a TCR complex, preferably immune cells, such as T helper cells, monocytes, macrophages, or dendritic cells, preferably CD4 + T helper cells (also known as CD4 cells, T helper cells, or T4 cells), more preferably cytotoxic T cells (T c cells, CTL, or CD8 +In some embodiments, the effector cells are mammalian cells, preferably primate cells, and even more preferably human cells.

[0249] As used herein, the terms "TCR complex" or "αβ TCR-CD3 complex" refer to the T cell receptor complex displayed on the surface of T cells (see Kuhns et al. 2006, Immunity 24:133-139). The TCR complex is composed of six type I single-spanning membrane proteins: the TCRα and TCRβ chains, which form the TCR heterodimer responsible for ligand recognition, and the non-covalently associated CD3γ, CD3δ, CD3ε, and ζ chains. These contain cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signaling components. Both the α and β chains of the T cell receptor consist of constant and variable domains. Sequences for human CD3 and human TCR α / β constant domains are provided in Table A-10 (SEQ ID NOs: 291-296; see UniProtKB: CD3 delta: P04234, CD3 gamma: P09693, CD3 epsilon: P07766, CD3 zeta: P20963, TCR alpha: P01848, and TCR beta: P01850).

[0250] In one embodiment, the invention relates to a polypeptide as described herein, wherein the first ISV binds to CD3γ (SEQ ID NO: 292), CD3δ (SEQ ID NO: 291), and / or CD3ε (SEQ ID NO: 293) of the TCR complex, or a polymorphic variant or isoform thereof.

[0251] 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 polymorphic variants or isoforms thereof.

[0252] Isoforms are alternative protein sequences that can arise from the same gene due to one or a combination of biological events, such as alternative promoter usage, alternative splicing, alternative initiation, and ribosomal frameshifting (all known in the art).

[0253] As used herein, "T cell activation" refers to one or more cellular responses of T cells, e.g., cytotoxic T cells, selected from, for example, proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, and redirected target cell lysis. The polypeptides of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art, as described herein, e.g., in WO 99 / 54440 or Schlereth et al. 2005 (Cancer Immunol. Immunother. 20: 1-12), or as exemplified in the Examples or below.

[0254] In one embodiment, the invention relates to a polypeptide as described herein that triggers T cell activation. Preferably, the polypeptide of the invention triggers T cell activation only when the second and / or further ISV binds to an antigen on a target cell.

[0255] In one embodiment, the present invention relates to a polypeptide described herein, wherein said T cell activation depends on presentation of said polypeptide bound to said first antigen on a target cell to a T cell.

[0256] T cell activation by the polypeptides of the invention can be monitored by upregulation of CD69, CD25, and various cell adhesion molecules, de novo expression and / or release of cytokines (e.g., IFN-γ, TNF-α, IL-6, IL-2, IL-4, and IL-10), upregulation of granzymes and expression of perforin, and / or cell proliferation, membrane blebbing, activation of procaspases 3 and / or 7, nuclear DNA fragmentation, and / or cleavage of poly(ADP-ribose) polymerase, a substrate for caspases. Preferably, redirected lysis of target cells by multispecific polypeptides is independent of T cell receptor specificity, the presence of MHC class I and / or β2-microglobulin, and / or any costimulatory stimuli.

[0257] In certain embodiments, the present invention relates to a polypeptide as described herein, wherein said T cell activation is independent of MHC recognition.

[0258] The polypeptides of the present invention are derived from non-prestimulated peripheral polyclonal CD8 + and CD4 +The polypeptides of the present invention exhibit in vitro redirected lysis of target cells by T cells recruited by the polypeptides of the present invention, accompanied by cytotoxic synapse formation and the transport of perforin and granzymes. Cytolysis by T cells is described, for example, in Atkinson and Bleackley 1995 (Crit. Rev. Immunol 15(3-4):359-384). Preferably, the engaged T cells are capable of continuous target cell lysis and are not affected by immune evasion mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation (see, for example, WO 2007 / 042261). In vitro, redirected lysis is observed at low picomolar concentrations, suggesting that very few polypeptides of the present invention are required to bind to target cells to trigger T cells. As demonstrated in the examples, a low target-to-effector ratio may exhibit continuous target cell lysis. Thus, the present invention relates to potent polypeptides. Preferably, the polypeptides of the invention mediate killing of target cells, eg, cancer cells, eg, stimulation of T cells in membrane pit formation and transport of pro-apoptotic components of cytotoxic T cell granules.

[0259] In certain embodiments, the invention relates to a polypeptide described herein, wherein said T cell activation results in one or more cellular responses of said T cell, wherein said cellular response is selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, expression of activation markers, and redirected target cell lysis.

[0260] As used herein, the term "potency" is a measure of the biological activity of an agent, e.g., a polypeptide, ISV, or nanobody. The potency of an agent can be measured by any suitable method known in the art, e.g., as described in the Experimental Section. Cell culture-based potency assays are often the preferred format for measuring biological activity because they measure physiological responses elicited by the agent and can provide results in a relatively short period of time. Various types of cell-based assays based on the mechanism of action of the product can be used, including, but not limited to, proliferation assays, cytotoxicity assays, cell killing assays, reporter gene assays, cell surface receptor binding assays, assays measuring the attraction / inhibition of functionally essential proteins or other signaling molecules (e.g., phosphoproteins, enzymes, cytokines, cAMP, etc.), the Ramos B cell depletion model, and T cell-mediated tumor cell killing assays (e.g., as shown in the Examples Section), all of which are well known in the art. The results from cell-based potency assays can be expressed as "relative potency" determined by comparing the response obtained for a multispecific polypeptide of the invention with a corresponding reference monovalent ISV, e.g. a polypeptide comprising only one ISV or one Nanobody, optionally further comprising an irrelevant Nanobody (see Experimental section).

[0261] In certain embodiments, the invention relates to a polypeptide as described herein, wherein said T cell activation results in inhibition of the activity of said target cells (e.g., regressing the disorder and / or symptoms), e.g., by more than about 10%, e.g., 20%, 30%, or 40%, or even more than 50%, e.g., more than 60%, e.g., 70%, 80%, or even more than 90%, e.g., 100%, to delay or minimize the spread of target cells, inhibit or delay the growth and / or proliferation of target cells, and / or kill target cells.

[0262] The first building block, ISV, nanobody or VHH of the invention has high affinity for its target, i.e. CD3. The first building block, ISV or nanobody of the invention can be, for example, directed against an antigenic determinant, epitope, part, domain, subunit or conformation (if applicable) of said first target. The first building block, e.g., the first ISV, nanobody or VHH, is preferably selected for its high affinity for its target itself, regardless of the influence of any avidity effects.

[0263] Thus, the present invention relates to a polypeptide as described herein, wherein the first ISV binds to CD3 with an average KD value of 100 nM to 10 pM, for example, an average KD value of 90 nM or less, even more preferably, an average KD value of 80 nM or less, for example, less than 70, 60, 50, 40, 30, 20, 10, 5 nM or less, for example, less than 4, 3, 2, or 1 nM, for example, less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or less, for example, less than 10 pM. Preferably, the KD is measured by Kinexa or SPR, e.g., measured by Proteon. For example, the KD is measured as provided in the Examples section.

[0264] Thus, the present invention relates to a polypeptide as described herein, wherein said first ISV has high affinity when measured monovalently, preferably said average KD is measured by surface plasmon resonance (SPR) on a recombinant protein.

[0265] Thus, the present invention preferably provides a method for producing a polymer having a molecular weight of up to about 10 as measured by surface plasmon resonance. -5 M, maximum about 10 -6 M, maximum about 10 -7 M, maximum about 10 -8 M, maximum about 10 -9 M, maximum about 10 -10 M, maximum about 10 -11 M, and up to about 10 -12M.

[0266] The present invention also relates to a polypeptide as described herein, wherein the first ISV binds to CD3 with an EC50 value of 100 nM to 1 pM, for example with an average EC50 value of 100 nM or less, even more preferably with an average EC50 value of 90 nM or less, for example less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or less, for example less than 4, 3, 2, or 1 nM or less, for example less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or less, for example less than 4 pM.

[0267] Thus, the present invention relates to a polypeptide as described herein, wherein said average KD is measured by FACS, Biacore, ELISA with a monovalent first ISV, e.g., Nanobody, or a polypeptide comprising a monovalent first ISV, e.g., Nanobody. For example, said EC50 is measured as provided in the Examples section.

[0268] It is shown in the Examples that the KD correlates well with the EC50.

[0269] In certain embodiments, the present invention preferably provides a plasmon resonance (SPR) signal of at least about 10 Hz, as measured by surface plasmon resonance or as measured as performed in the Examples section. 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 The present invention relates to a polypeptide as described herein, having an on rate constant (K on ) for (or for binding to) said CD3 selected from the group consisting of:

[0270] In one embodiment, the present invention preferably provides a plasmon resonance (SPR) signal of up to about 10 Hz, as measured by surface plasmon resonance or as measured as performed in the Examples section. -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 , up to about 10 -6 s -1 , up to about 10 -7 s -1 , up to about 10 -8 s -1 , up to about 10 -9 s -1 , and up to about 10 -10 s -1 The present invention relates to a polypeptide as described herein, having an off-rate constant (Koff) for (or for binding to) said CD3 selected from the group consisting of:

[0271] Modifications to the amino acid sequence of the binding molecules, ISVs, or polypeptides described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or ISV. Amino acid sequence variants of a binding molecule, ISV, or polypeptide are prepared by introducing appropriate nucleotide changes into the binding molecule, ISV, or polypeptide nucleic acid, or by peptide synthesis.

[0272] Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the binding molecule, ISV, or polypeptide. Any combination of deletions, insertions, and substitutions may be made to produce the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also alter post-translational processing of the binding molecule, for example, changing the number or location of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDRs, 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 substituted in the framework regions (FRs). These substitutions are preferably conservative substitutions as described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acids can be inserted or deleted in each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be inserted or deleted in each FR.

[0273] A useful method for identifying specific residues or regions of a binding molecule, ISV, or polypeptide that are preferred locations for mutagenesis is described in Cunningham and Wells 1989 (Science 244: 1081-1085) and is called "alanine scanning mutagenesis." In this method, a residue or group of target residues within the binding molecule is identified (e.g., a charged residue, e.g., Arg, Asp, His, Lys, and Glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) that affects the interaction of the amino acid with the epitope. Amino acid locations that demonstrate functional sensitivity to the substitution are then refined by introducing further or other variants at or for the substitution site. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, ala scanning or random mutagenesis is conducted at the target codon or region to analyze the performance of a mutation at a given site. The expressed binding molecule variants are screened for the desired activity.

[0274] Preferably, amino acid sequence insertions comprise amino- and / or carboxy-terminal fusions ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length for polypeptides containing several hundred or more residues.

[0275] Another type of variant is an amino acid substitution variant. These variants preferably have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the binding molecule, ISV, or polypeptide replaced by another residue. The most popular sites for substitutional mutagenesis include the CDRs, particularly the hypervariable regions, although FR mutations are also contemplated. For example, if a CDR sequence includes six amino acids, one, two, or three of these amino acids may be substituted. Similarly, if a CDR sequence includes 15 amino acids, one, two, three, four, five, or six of these amino acids may be substituted.

[0276] Generally, when amino acids are substituted in one or more or all CDRs, it is preferred that the resulting "replacement" sequence is at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, especially preferably 80%, or even more than 90% identical to the "original" CDR sequence. This means that the degree of identity to the "replacement" sequence depends on the length of the CDR. For example, a CDR having five amino acids is preferably 80% identical to its replacement sequence because it has at least one amino acid replaced. Thus, the CDRs of a binding molecule can have different degrees of identity to their replacement sequences. For example, CDR1 can have 80%, while CDR3 can have 90%.

[0277] Preferred substitutions (or replacements) are conservative, although any substitution (including non-conservative substitutions or one or more from the "exemplary substitutions" listed in Table B-1 below) is envisioned, provided that the polypeptide retains its ability to bind to CD3 present on T cells via the first ISV and to bind to a first antigen on a target cell via the second ISV, and / or its CDRs have identity to the replaced sequences (at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and especially preferably 80% identical to the "original" CDR sequences).

[0278] Conservative substitutions are shown in Table B-1 below.

[0279] [Table 1]

[0280] Sequence analysis further demonstrated that there are only a limited number of sequence variations in the CDRs (see Example 4.2 and Tables A-1 to A-6).

[0281] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81 or any of SEQ ID NOs: 81 to 100 and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, and (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 to 122 and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 143, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 or any of SEQ ID NOs: 123 to 143 The present invention relates to a polypeptide selected from the group consisting of:

[0282] Further preferred CDR sequences are shown in Table A-8.

[0283] Generally, the CDR combinations listed in Table A-8 (i.e., those mentioned in the same column in Table A-4) are preferred. Thus, if a CDR in an ISV is a CDR sequence mentioned in Table A-8, or a CDR sequence suitably selected from the group consisting of CDR sequences that have only 4, 3, 2, or 1 amino acid difference from a CDR sequence listed in Table A-8, it is generally preferred that at least one, and preferably both, of the other CDRs are suitably selected from the CDR sequences belonging to the same combination in Table A-8 (i.e., mentioned in the same column in Table A-8), or suitably selected from the group consisting of CDR sequences that have only 4, 3, 2, or 1 amino acid difference from a CDR sequence belonging to the same combination.

[0284] Sequence analysis of the obtained binders 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 is the most prominent, containing 50 clones (SEQ ID NOs: 1-50). Cluster B and Cluster D are each represented by only one clone (SEQ ID NOs: 51 and 52, respectively). Cluster C contains four clones (SEQ ID NOs: 53-56). Cluster E contains nine clones (SEQ ID NOs: 57-65). And Cluster F contains 15 clones (SEQ ID NOs: 66-80). Clustering based on structural similarities and differences in amino acid sequences was translated into functional similarities and differences, as demonstrated by the examples. Representatives of all clusters were isolated based on their high affinity binding to CD3 (Examples 3 and 4) and human T cell activation (Example 4.2). In general, representatives of cluster A exhibited the best EC50 values. Representatives of cluster C had somewhat less favorable EC50 values ​​than representatives of cluster B, but representatives of cluster C also had lower IC50 values ​​in a flow cytometry-based T cell-mediated Ramos killing assay (see Example 10).

[0285] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, - In position 8, S is changed to G, and / or At position -10, G is changed to A.

[0286] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or - The present invention relates to a polypeptide in which at position -9, T is changed to S, A, or P.

[0287] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or At position -10, A is changed to P.

[0288] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81 and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 101 and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 The present invention relates to a polypeptide selected from the group consisting of:

[0289] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV essentially consists of four framework 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.

[0290] Nanobodies belonging to cluster B are represented by one clone.

[0291] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 88 and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 110 and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 128 The present invention relates to a polypeptide selected from the group consisting of:

[0292] Thus, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of four framework 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.

[0293] Nanobodies of cluster C show very limited sequence variability in the CDRs.

[0294] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: -2 position V is changed to A.

[0295] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 90 and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 112 and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 130 The present invention relates to a polypeptide selected from the group consisting of:

[0296] In one aspect, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of four framework 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.

[0297] Nanobodies belonging to cluster D are represented by one clone.

[0298] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 89 and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 111 and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 129 The present invention relates to a polypeptide selected from the group consisting of:

[0299] Thus, the present invention relates to a polypeptide as described herein, wherein the first ISV essentially consists of four framework 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.

[0300] Cluster E contains 9 clones.

[0301] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or At position -8, M is changed to T.

[0302] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or - The present invention relates to polypeptides in which at position -7, D is changed to A or K.

[0303] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or -5. The polypeptide wherein at position -6, S is changed to V.

[0304] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 91 and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 114 and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 131 The present invention relates to a polypeptide selected from the group consisting of:

[0305] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV essentially consists of four framework 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.

[0306] Nanobodies belonging to cluster F are represented by 15 clones.

[0307] Therefore, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or - The polypeptide wherein at position 9, L is changed to M.

[0308] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or At position -10, Y is changed to F.

[0309] Thus, the present invention provides a polypeptide as described herein, wherein the first ISV essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or At position -14, V is changed to I.

[0310] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 94 and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 118 and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 134 The present invention relates to a polypeptide selected from the group consisting of:

[0311] In one embodiment, the present invention relates to a polypeptide described herein, wherein the first ISV essentially consists of four framework 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.

[0312] The second immunoglobulin single variable domain (ISV) of the polypeptide of the invention has high affinity for / binds to an antigen on a target cell, preferably a cancer cell. As referred to herein, a "target cell" is a cell that displays a particular antigen on its surface. In a preferred embodiment, the "target cell" is a cancer cell.

[0313] The membrane (also called the plasma membrane or phospholipid bilayer) surrounds the cell cytoplasm and is the outer boundary of the cell. In other words, the membrane is the cell's surface. This membrane functions to separate and protect the cell from its surrounding environment and is primarily composed of a bilayer of phospholipids. Various protein molecules, such as channels, pumps, and cell receptors, are embedded within this membrane. Because the membrane is fluid, protein molecules can move within the membrane. As used herein, the term "antigen on a target cell" refers to a molecule displayed on the surface of a cell. In most cases, this molecule will be located in or on the plasma membrane of the cell. This ensures that at least a portion of the molecule is accessible from the outside of the cell in its tertiary structure. A non-limiting example of a cell surface molecule located in the plasma membrane is a transmembrane protein that contains hydrophilic and hydrophobic regions in its tertiary conformation. Here, at least one hydrophobic region allows the cell surface molecule to be embedded or inserted into the hydrophobic plasma membrane of the cell, while hydrophilic regions extend into the cytoplasm and extracellular space, respectively, on either side of the plasma membrane.

[0314] The antigen can be any target on a cell, for example, a tumor antigen. In a preferred embodiment, the antigen is specific to the target cell, for example, a cancer cell, for example, a tumor-associated antigen (TAA) on the cancer cell.

[0315] As used herein, the term "tumor antigen" can be understood to mean an antigen presented on tumor cells. These antigens may often be presented on the cell surface in an extracellular portion associated with the transmembrane and cytoplasmic portions of the molecule, and these antigens may be presented only by tumor cells and not by normal or healthy cells. Tumor antigens may be expressed only on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. However, this is not common. More commonly, antigens are presented by tumor cells and normal cells and are called "tumor-associated antigens (TAA)." These tumor-associated antigens may be overexpressed on tumor cells compared to normal cells, or may be more accessible to antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. TAAs are antigens that are preferably expressed on cells of a specific tumor but preferably not expressed on normal cells. In many cases, TAAs are antigens that are normally expressed in cells only at specific times in an organism's development (e.g., during fetal development) and are inappropriately expressed in the organism at the current point in development, or are antigens that are not expressed in normal tissues or cells of the organ that currently expresses the antigen.

[0316] In certain embodiments, said first antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0317] In certain embodiments, said second antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

[0318] In one embodiment, the antigen is more abundant on cancer cells than on normal cells. The antigen on the target cell is preferably a tumor-associated antigen (TAA). Preferred TAAs include MART-1, carcinoembryonic antigen ("CEA"), gp100, MAGE-1, HER-2, CD20, Lewis Y antigen, melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), fibroblast activation protein (FAP), CD19, and CD33.

[0319] Preferred cell surface antigens as TAAs because they are preferentially expressed on AML LSCs compared to normal hematopoietic stem cells include CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, and CD25.

[0320] Other tumor-associated antigens suitable as antigens on target cells for binding by a second ISV in a polypeptide of the invention include TAG-72, Ep-CAM, PSMA, PSA, glycolipids such as GD2 and GD3.

[0321] The TAAs of the present invention are directed to hematopoietic differentiation antigens, i.e., glycoproteins commonly associated with the Cluster of Differentiation (CD) group, such as CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD69, and CD147; growth factor receptors, including HER2, ErbB3, and ErbB4; the gamma chain of the interleukin-2 receptor (CD132 antigen), the alpha chain of the interleukin-10 receptor (IL-10R-A), the interleukin-11 receptor (IL-11R-B), the interleukin-12 receptor (IL-12R-C), the interleukin-13 receptor (IL-13R-D), the interleukin-14 receptor (IL-14R-E), the interleukin-15 receptor (IL-14R-F), the interleukin-16 receptor (IL-16R-F), the interleukin-17 receptor (IL-17R-F), the interleukin-18 receptor (IL-18R-F), the interleukin-19 receptor (IL-19R-F), the interleukin-20 receptor (IL-19R-F), the interleukin-19 ... Interleukin-10 receptor beta chain (IL-10R-B), interleukin-12 receptor beta-1 chain (IL-12R-beta1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), interleukin-13 receptor alpha-2 chain (interleukin-13 binding protein ...1 chain (IL-13R-alpha-1) (CD213a1 antigen), interleukin-13 receptor alpha-1 chain (IL-13 binding protein), interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), interleukin-13 receptor alpha-1 chain (IL-13 binding protein), interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), interleukin-13 receptor alpha-1 chain (IL-13 binding protein), interleukin-13 receptor alpha-1 chain (IL-13 binding protein Interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin-21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-beta) (CD121b), interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor Cytokine receptors, including the alpha chain of interleukin-2 receptor (CD25 antigen), the beta chain of interleukin-2 receptor (CD122 antigen), the alpha chain of interleukin-3 receptor (IL-3R-alpha) (CD123 antigen); as well as others, such as CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin-6, and claudin-18.Also included are 2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, 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, FXYD2 gamma a, CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA195, DR5, DR6, DcR3, and CAIX.

[0322] Thus, the present invention provides a method for treating tumors in which 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 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, CD3 3, CD36, CD45, CD52, CD147; growth factor receptors including ErbB3 and ErbB4; interleukin-2 receptor gamma chain (CD132 antigen), interleukin-10 receptor alpha chain (IL-10R-A), interleukin-10 receptor beta chain (IL-10R-B), interleukin-12 receptor beta-1 chain (IL-12R-beta1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-13 receptor alpha chain (IL-13R-beta1), interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), interleukin-14 receptor alpha chain (IL-14R-beta1), interleukin-15 receptor alpha chain (IL-14R-beta1), interleukin-16 receptor beta-2 chain (IL-14R-beta2), interleukin-17 receptor alpha chain (IL-14R-beta1), interleukin-18 receptor alpha chain (IL-14R-beta1), interleukin-19 receptor alpha chain (IL-14R-beta1), interleukin-19 receptor beta-2 chain (IL-14R-beta2), interleukin-19 ... alpha chain (IL-14R-beta1), interleukin-19 receptor alpha chain (IL-14R-beta1), interleukin-19 receptor alpha chain Interleukin-1 chain (IL-13R-alpha-1) (CD213a1 antigen), alpha-2 chain of interleukin-13 receptor (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin-21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-beta) (CDw121b ... Cytokine receptors, including interleukin-1 receptor antagonist protein (IL-1ra), interleukin-2 receptor alpha chain (CD25 antigen), interleukin-2 receptor beta chain (CD122 antigen), interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin-6, and claudin-18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, 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, CX The present invention relates to a polypeptide as described herein, wherein the TAA is selected from the group consisting of CR7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA195, DR5, DR6, DcR3, and CAIX, and related polymorphic variants and isoforms, preferably CD20 (Uniplot 11836), HER2 (Uniplot P04626), polymorphic variants and / or isoforms thereof.

[0323] The second building blocks, ISVs, Nanobodies or VHHs of the invention have high affinity for their antigen. They can for example be directed against an antigenic determinant, epitope, part, domain, subunit or conformation (if applicable) of said antigen on a target cell.

[0324] The target cells of the present invention particularly relate to mammalian cells, preferably primate cells, even more preferably human cells. The target cells are preferably hyperproliferative cells, such as cancer cells.

[0325] The present invention relates to a polypeptide as described herein, wherein the second or further ISV binds to an antigen on a target cell with an average KD value of 100 nM to 10 pM, such as an average KD value of 90 nM or less, even more preferably an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or less, such as less than 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or less, such as less than 10 pM. Preferably, the KD is measured by KinExA or SPR, e.g. as measured by Proteon.

[0326] Thus, the present invention relates to a polypeptide as described herein, wherein said second or further ISV has a high affinity for its antigen when measured monovalently.

[0327] Thus, in the present invention, the average KD is measured, for example, by surface plasmon resonance (SPR) and / or KinExA or Proteon for recombinant proteins, as described, for example, in the Examples section.

[0328] The invention also relates to a polypeptide as described herein, wherein the second or further ISV binds to an antigen on a target cell with an EC50 value of between 100 nM and 1 pM, for example with an average EC50 value of 100 nM or less, even more preferably with an average EC50 value of 90 nM or less, for example less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or less, for example less than 4, 3, 2, or 1 nM or less, for example less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or less, for example less than 4 pM.

[0329] Thus, the present invention relates to a polypeptide as described herein, wherein said mean EC50 is measured by FACS or ELISA for a monovalent second ISV, such as a Nanobody, or a polypeptide comprising a monovalent second ISV, such as a Nanobody.

[0330] It is shown in the Examples that the KD correlates well with the EC50.

[0331] Simultaneous targeting of multiple antigens can reduce the likelihood of tumor escape mutants arising, thus improving the therapeutic activity of T cell engagement strategies. The present invention provides multispecific polypeptides comprising a CD3 ISV combined with immunoglobulin single variable domains directed against different (target) antigens (on target cells) (see Example 19). Preferred combinations of first and second antigens are provided below (it will be understood that the ISVs binding to said antigens can be arranged in any order in the polypeptides of the invention).

[0332] [Table 2]

[0333] Similarly, simultaneous targeting of multiple epitopes, antigenic determinants, portions, domains, subunits, or conformations of a protein or antigen on a target cell can reduce the likelihood of tumor escape mutants, thereby improving the therapeutic activity of T cell engagement strategies (see Example 20). The present invention provides polypeptides (also called double-paratope constructs) comprising anti-CD3 ISVs combined with immunoglobulin single variable domains directed against different epitopes, antigenic determinants, portions, domains, subunits, or conformations of an antigen on a target cell. Preferred combinations of a first TAA ISV and a second TAA ISV are provided below (it will be understood that the ISVs binding to the antigens can be arranged in any order in the polypeptides of the invention).

[0334] [Table 3]

[0335] The polypeptides and compositions of the present invention can be used to prevent and / or treat diseases and disorders of the present invention (also referred to herein as "diseases and disorders of the present invention"). Such diseases and disorders include, but are not limited to, cancer. The term "cancer" refers to a pathological condition in mammals that is typically characterized by uncontrolled cell proliferation or survival. Examples of cancer include carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, and rectal cancer. Cancers include, but are not limited to, bowel 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 cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease, or AIDS-related primary effusion lymphoma, neuroectodermal tumor, rhabdomyosarcoma (e.g., for additional cancers, see Cancer, Principles and Practice (DeVita, et al. eds 1997)); and any metastasis of any of the above cancers, as well as non-cancerous manifestations, such as nasal polyps; and other disorders and diseases described herein.

[0336] For a general description of immunoglobulin single variable domains, reference is made to the further description below and to the prior art cited therein. In this regard, however, the description and prior art refer to the so-called "V H 3 classes of immunoglobulin single variable domains (i.e., V HIt should be noted that the present invention primarily describes immunoglobulin single variable domains with a high degree of sequence homology to three classes of human germline sequences, such as DP-47, DP-51, or DP-29, which constitute preferred aspects of the invention. However, in its broadest sense, the invention generally covers any type of immunoglobulin single variable domain, including, for example, the so-called "V" immunoglobulins described in WO 07 / 118670. H Immunoglobulin variable domains (i.e., V H Note that four classes of immunoglobulin single variable domains with a high degree of sequence homology to human germline sequences (eg, DP-78) are also covered.

[0337] Generally, immunoglobulin single variable domains (especially V HH Sequence-optimized immunoglobulin single variable domains) can be characterized, inter alia, by the presence of one or more "hallmark residues" (e.g., as set forth in Table B-2) in one or more framework sequences (again, as further described herein).

[0338] [Table 4] TIFF2025186357000005.tif130169

[0339] The immunoglobulins of the invention may also contain a C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, for example, 1, 2, 3, 4, or 5 (preferably 1 or 2, for example, 1), and each X is a (preferably naturally occurring) amino acid residue, which amino acid residues are independently selected, preferably independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I). For such C-terminal extensions, see WO 12 / 175741 and WO 15 / 060643.

[0340] Alternatively and / or additionally, the immunoglobulins of the invention can have certain preferred amino acid residues at positions 11, 89, 110, and / or 112, as described in more detail in WO 15 / 060643 (hereby incorporated by reference).

[0341] Again, such immunoglobulin single variable domains can be obtained in any suitable manner and from any suitable source, for example, naturally occurring V HH sequences (i.e., from a suitable species of Camelidae, e.g., llama), or synthetic or semi-synthetic VH or VL (e.g., of human origin). Such immunoglobulin single variable domains can include "humanized" or otherwise "sequence-optimized" VHH, "camelized" immunoglobulin sequences (in particular camelized heavy chain variable domain sequences, i.e., camelized VH), as well as human VH, human VL, Camelid VHH that have been modified by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR-grafting, veneering, combining fragments obtained from various immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art, or any suitable combination of any of the foregoing as further described herein. As mentioned herein, a particularly preferred class of immunoglobulin single variable domains of the invention are naturally occurring VHH, VL, and VL. HH corresponding to the amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the sequence may be replaced by a V nucleotide sequence from a conventional four-chain antibody of human origin (e.g., as shown above). HThe present invention also encompasses immunoglobulin single variable domains having amino acid sequences that have been "humanized" by replacing one or more amino acid residues occurring at the corresponding positions in the domain with one or more amino acid residues occurring at the corresponding positions in the domain. This can be done in a manner known per se, which will be clear to the skilled person, for example, on the basis of the further description herein and the prior art on humanization referred to herein. Again, such humanized immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known per se, and therefore can be obtained from, as starting material, a naturally occurring V HH It should be noted that there is no strict limitation on the polypeptides obtained using polypeptides containing the domains.

[0342] Another particularly preferred class of immunoglobulin single variable domains of the invention are naturally occurring V H corresponding to the amino acid sequence of the natural V domain from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the V domain of a heavy chain antibody HH The term "camelized" includes immunoglobulin single variable domains having amino acid sequences that have been "camelized" by replacing one or more amino acid residues occurring at the corresponding positions in the V domain. This can be done in a manner known per se, which will be clear to the skilled person, for example, based on the description herein. Such "camelizing" substitutions are preferably H -V L The amino acid sequences are inserted at amino acid positions that form and / or are present at the interface and / or at the so-called camel hallmarks defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 (FEBS letters 339: 285-290) and 1996 (Protein Engineering 9: 531-537)). Preferably, the V domains used as starting material or starting points for generating or designing camelized immunoglobulin single variable domains are inserted at amino acid positions that form and / or are present at the interface and / or at so-called camel hallmarks as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 (FEBS letters 339: 285-290) and 1996 (Protein Engineering 9: 531-537)). H The sequence is preferably a V from a mammal H sequence, more preferably a human VH Sequence, e.g., V H 3 sequences. However, such camelized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se, and therefore may be obtained using as starting material the naturally occurring V H It should be noted that there is no strict limitation on the polypeptides obtained using polypeptides containing the domains.

[0343] For example, again as further described herein, "humanized" and "camelized" both refer to naturally occurring V HH Domain or V H This can be done by providing a nucleotide sequence encoding each of the domains and then, in a manner known per se, changing one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes each of the "humanized" or "camelized" immunoglobulin single variable domains of the invention. This nucleic acid can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domains of the invention. Alternatively, the naturally occurring V HH Domain or V H Based on the amino acid sequence of each of the domains, the amino acid sequence of each of the desired humanized or camelized immunoglobulin single variable domains of the invention can be designed and then synthesized de novo using techniques for peptide synthesis known per se. HH Domain or V H Based on the amino acid or nucleotide sequence of each of the domains, a nucleotide sequence encoding each of the desired humanized or camelized immunoglobulin single variable domains of the invention can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se. The nucleic acids thus obtained can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domains of the invention.

[0344] Thus, the present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a subject the medicament the ISV, a nanobody, a V HH , humanized VHH , or camelization V H The present invention relates to a polypeptide as described herein, wherein

[0345] Generally, a protein or polypeptide that comprises or consists essentially of one building block, one immunoglobulin single variable domain or one Nanobody will be referred to herein as a "monovalent" protein or polypeptide, a "monovalent construct," a "monovalent building block," a "monovalent immunoglobulin single variable domain," or a "monovalent Nanobody," respectively.

[0346] In this aspect, the invention also relates to the monovalent building blocks that make up the polypeptides of the invention.

[0347] Thus, the present invention provides an ISV or polypeptide that specifically binds to the constant domain of CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NOs: 81 to 100, or (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to any of the amino acid sequences of SEQ ID NOs: 81 to 100, provided that a polypeptide comprising a CDR1 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 101 to 122, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 123 to 143, or (f) An amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 123 to 143, provided that a polypeptide comprising a CDR3 having 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without 4, 3, 2, or 1 amino acid difference, when the affinity is measured by surface plasmon resonance. The present invention relates to an ISV or polypeptide selected from the group consisting of:

[0348] As described above, ISVs belonging to different clusters were isolated based on structural similarities and differences in CDR2 and CDR3.

[0349] Immunoglobulin single variable domains belonging to cluster A are: (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 81, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 101, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 123, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0350] In another embodiment, in the polypeptide belonging to Cluster A, CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, - In position 8, S is changed to G, and / or At -10th place, G has been changed to A.

[0351] In another embodiment, in the polypeptide belonging to Cluster A, CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or At position -9, T is changed to S, A, or P.

[0352] In another embodiment, in the polypeptide belonging to Cluster A, the CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or At -10th place, A has been changed to P.

[0353] Accordingly, the present invention provides an ISV or polypeptide that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: - In the first place, G has been changed to R, - In the third place, T has been changed to A, - In the fourth place, Y has been changed to F, In position -8, S is changed to G, and / or At -10th place, G has been changed to A, And here, (ii) CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101 selected from the group consisting of where: at position -3, V is changed to T or A; - In the 5th place, S has been changed to T, At position -6, G is changed to D or E, and / or at position -9, T is changed to S, A, or P; And here, (iii) CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 123 selected from the group consisting of where: - In the second place, I has been changed to T, In position -9, I is changed to V, and / or At position -10, A is changed to P, or the ISV or polypeptide.

[0354] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO: 123. Preferably, the polypeptide is selected from any of SEQ ID NOs: 1 to 50.

[0355] Immunoglobulin single variable domains belonging to cluster B are: (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 88, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 110, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 128, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0356] In another embodiment, in the polypeptide belonging to cluster B, CDR1 is SEQ ID NO:88.

[0357] In another embodiment, in the polypeptide belonging to cluster B, CDR2 is SEQ ID NO:110.

[0358] In another embodiment, in the polypeptide belonging to cluster B, the CDR3 is SEQ ID NO:128.

[0359] In another aspect, the present invention relates to a polypeptide wherein CDR1 is represented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, and CDR3 is represented by SEQ ID NO: 128. Preferably, the polypeptide is SEQ ID NO: 51.

[0360] Immunoglobulin single variable domains belonging to cluster C are: (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO:90, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 112, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 130, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0361] In another embodiment, in the polypeptide belonging to cluster C, CDR1 is SEQ ID NO:90.

[0362] In another embodiment, in the polypeptide belonging to cluster C, CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: - In second place, V has been changed to A.

[0363] In another embodiment, in the polypeptide belonging to cluster C, the CDR3 is SEQ ID NO:130.

[0364] Accordingly, the present invention provides an ISV or polypeptide that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is SEQ ID NO: 90; And here, (ii) CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 112 selected from the group consisting of where: - In the second place, V has been changed to A, And here, (iii) An ISV or polypeptide in which CDR3 is SEQ ID NO: 130.

[0365] In another aspect, the present invention relates to a polypeptide in which CDR1 is represented by SEQ ID NO: 90, CDR2 is represented by SEQ ID NO: 112, and CDR3 is represented by SEQ ID NO: 130. Preferably, the polypeptide is selected from any of SEQ ID NOs: 53 to 56.

[0366] Immunoglobulin single variable domains belonging to cluster D are: (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 89, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 111, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 129, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0367] In another embodiment, in the polypeptide belonging to cluster D, CDR1 is SEQ ID NO:89.

[0368] In another embodiment, in the polypeptide belonging to cluster D, CDR2 is SEQ ID NO:111.

[0369] In another embodiment, in the polypeptide belonging to cluster D, the CDR3 is SEQ ID NO:129.

[0370] In another aspect, the present invention relates to a polypeptide wherein CDR1 is represented by SEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111, and CDR3 is represented by SEQ ID NO: 129. Preferably, the polypeptide is SEQ ID NO: 52.

[0371] Immunoglobulin single variable domains belonging to cluster E are: (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 91, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 114, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 131, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0372] In another embodiment, in the polypeptide belonging to Cluster E, CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or At -8th place, M has been changed to T.

[0373] In another embodiment, in the polypeptide belonging to Cluster E, CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or At position -7, D is changed to A or K.

[0374] In another embodiment, in the polypeptide belonging to cluster E, the CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or - In 6th place, S has been changed to V.

[0375] Accordingly, the present invention provides an ISV or polypeptide that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or At -8th place, M has been changed to T, And here, (ii) CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: - In the first place, R has been changed to Q, - in position 3, T is changed to S, and / or At position -7, D is changed to A or K, And here, (iii) CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO: 131 selected from the group consisting of where: - In position 2, S is changed to R, and / or - The ISV or polypeptide wherein at position 6, S is changed to V.

[0376] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO: 91, CDR2 is represented by SEQ ID NO: 114, and CDR3 is represented by SEQ ID NO: 131. Preferably, the polypeptide is selected from any of SEQ ID NOs: 57 to 65.

[0377] Immunoglobulin single variable domains belonging to cluster F are: (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 94, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 118, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of SEQ ID NO: 134, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. The polypeptide is represented by a polypeptide selected from the group consisting of:

[0378] In another aspect, in the polypeptide belonging to cluster F, CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or At -9th place, L has been changed to M.

[0379] In another aspect, in the polypeptide belonging to cluster F, CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or At -10th place, Y has been changed to F.

[0380] In another aspect, in the polypeptide belonging to cluster F, the CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or At -14th place, V has been changed to I.

[0381] Accordingly, the present invention provides an ISV or polypeptide that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: - at position 3, S is changed to T, A, or G; - In the 5th place, N has been changed to S, In position -6, M is changed to T or A, and / or -At 9th place, L has been changed to M, And here, (ii) CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: - In the second position, H is changed to V, At position -5, S is changed to H or A, In position -8, N is changed to S, and / or At -10th place, Y has been changed to F, And here, (iii) CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: - In position 6, A is changed to S or D, At position -7, F is changed to Y or A, At position -8, R is changed to H, - In the 9th place, S has been changed to A, at position -11, G is changed to D, T, N, S, K, or R, and / or In an ISV or polypeptide, at position -14, V is changed to I.

[0382] In another aspect, the present invention provides a polypeptide as described herein, wherein CDR1 is represented by SEQ ID NO: 94, CDR2 is represented by SEQ ID NO: 118, and CDR3 is represented by SEQ ID NO: 134. Preferably, the polypeptide is selected from any of SEQ ID NOs: 66 to 80.

[0383] In a further aspect, the invention relates to a polypeptide that cross-blocks binding to CD3 by at least one ISV or polypeptide belonging to cluster A, B, C, D, E, or F.

[0384] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by at least one ISV or polypeptide having SEQ ID NO: 1-50.

[0385] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by an ISV or polypeptide having SEQ ID NO:51.

[0386] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by at least one ISV or polypeptide having SEQ ID NO: 53-56.

[0387] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by an ISV or polypeptide having SEQ ID NO:52.

[0388] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by at least one ISV or polypeptide having SEQ ID NO: 57-65.

[0389] Thus, the present invention relates to polypeptides that cross-block binding to CD3 by at least one ISV or polypeptide having SEQ ID NO: 66-80.

[0390] In a further aspect, the invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one ISV or polypeptide belonging to cluster A, B, C, D, E, or F.

[0391] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one ISV or polypeptide belonging to SEQ ID NOs: 1-50.

[0392] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by an ISV or polypeptide having SEQ ID NO:51.

[0393] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one ISV or polypeptide belonging to SEQ ID NOs: 53-56.

[0394] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by an ISV or polypeptide having SEQ ID NO:52.

[0395] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one ISV or polypeptide belonging to SEQ ID NOs: 57-65.

[0396] Thus, the present invention relates to polypeptides that are cross-blocked from binding to CD3 by at least one ISV or polypeptide belonging to SEQ ID NOs: 66-80.

[0397] Furthermore, the invention relates to compounds or constructs, in particular proteins or polypeptides, comprising or consisting essentially of one or more ISVs or polypeptides of the invention, optionally further comprising one or more other groups, residues, moieties, or binding units. As will become apparent to those skilled in the art from the further disclosure herein, such further groups, residues, moieties, binding units, or amino acid sequences may or may not provide further functionality to the polypeptide of the invention (and / or the compound or construct in which it is present), and may or may not modify the properties of the polypeptide of the invention.

[0398] In specific non-limiting aspects of the invention, as will be further described herein, the ISVs and polypeptides of the invention may have an extended half-life in serum (as further described herein) compared to the immunoglobulin single variable domain or polypeptide from which they are derived. For example, to provide derivatives of an ISV or polypeptide of the invention with an extended half-life, the immunoglobulin single variable domain or polypeptide of the invention may be conjugated (chemically or otherwise) to one or more half-life enhancing groups or moieties (e.g., PEG).

[0399] In specific aspects of the invention, a compound or construct of the invention or a polypeptide of the invention may have an increased half-life compared to a corresponding ISV or polypeptide of the invention. Some preferred, non-limiting examples of such compounds, constructs, and polypeptides will be apparent to the skilled artisan based on the further disclosure herein, and include, for example, an immunoglobulin single variable domain or polypeptide of the invention that has been chemically modified to increase its half-life (e.g., by means of pegylation); an immunoglobulin single variable domain or polypeptide of the invention that comprises at least one additional binding site for binding to a serum protein (e.g., serum albumin); or a construct or polypeptide of the invention that comprises at least an ISV or polypeptide of the invention linked to at least one moiety (in particular, at least one amino acid sequence) that increases the half-life of the ISV or polypeptide of the invention.Examples of ISVs or polypeptides of the invention comprising such half-life extending moieties or immunoglobulin single variable domains will be clear to the skilled person based on the further disclosure herein, and may for example be those in which one or more immunoglobulin single variable domains or polypeptides of the invention bind to one or more serum proteins or fragments thereof (e.g., (human) serum albumin or a suitable fragment thereof), or one or more binding units capable of binding to a serum protein (e.g., domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains suitable for use as single domain antibodies, "dAbs", immunoglobulin single variable domains suitable for use as dAbs, serum proteins, e.g., serum albumin (e.g., human serum albumin), serum immunoglobulin single variable domains, or polypeptides in which one or more immunoglobulin single variable domains or polypeptides of the invention are suitably linked to one or more small proteins or peptides capable of binding to serum proteins (including, but not limited to, the proteins and peptides described in WO 91 / 01743, WO 01 / 45746, WO 02 / 076489, WO 08 / 068280, WO 09 / 127691, and WO 11 / 095545).

[0400] Generally, a compound, construct, or polypeptide of the invention having an extended half-life preferably has a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times, or more than 20 times longer than the half-life of the corresponding ISV or polypeptide of the invention per se. For example, a compound, construct, or polypeptide of the invention having an extended half-life may have a half-life that is extended 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, for example, in humans, compared to the corresponding ISV or polypeptide of the invention per se.

[0401] In preferred, non-limiting aspects of the invention, such compounds, constructs or polypeptides of the invention have a serum half-life, e.g., in humans, that is extended by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48, or 72 hours, compared to the corresponding ISV or polypeptide of the invention itself.

[0402] In another preferred, non-limiting embodiment of the present invention, such compounds, constructs, or polypeptides of the present 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 more. For example, the compounds, constructs, or polypeptides of the present invention can 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 more), or more than 14 days (e.g., about 14-19 days).

[0403] In the present invention, it has been demonstrated that the inclusion of an albumin-targeting binding unit in the construct does not substantially affect the resulting potency or efficacy. Although a small loss of potency / efficacy was observed in the presence of HSA, the CD3 multispecific polypeptide with extended half-life was still effective in killing tumor cells. Albumin-based drug delivery has proven useful in achieving improved cancer therapy, primarily due to its passive targeting of tumors due to enhanced permeability and retention, as well as the increased demand for albumin by tumor cells as a source of energy and amino acids. However, albumin lacks not only the active mechanism for overcoming cell membrane barriers, but also the ability to penetrate tumor tissues (Qianqian Guo et al. Polym. Chem., 2013, 4, 4584-4587).

[0404] In particularly preferred, non-limiting aspects of the invention, the invention provides polypeptides of the invention comprising a first and a second immunoglobulin single variable domain (ISV), and further comprising one or more (preferably one) serum albumin-binding immunoglobulin single variable domains described herein, e.g., the serum albumin-binding immunoglobulin single variable domains designated 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).

[0405] [Table 5]

[0406] Thus, the present invention relates to a polypeptide as described herein that further comprises a serum protein binding moiety.

[0407] The present invention relates to a polypeptide as described herein, wherein said serum protein binding moiety binds to serum albumin.

[0408] The present invention relates to a polypeptide as described herein, wherein said serum protein binding moiety is an immunoglobulin single variable domain that binds to serum albumin.

[0409] The present invention relates to a polypeptide as described herein, wherein the ISV that binds to serum albumin essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein, when the CDRs are determined according to the Kabat definition, CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375), and / or, when the CDRs are determined according to Kontermann 2010, CDR1 is GFTFSSFGMS (SEQ ID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378), and CDR3 is GGSLSR (SEQ ID NO: 375).

[0410] The present 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).

[0411] In a polypeptide of the invention, two or more building blocks, ISVs or Nanobodies, and optionally one or more polypeptides, one or more other groups, drugs, agents, residues, moieties or binding units, can be directly linked to each other (e.g., as described in WO 99 / 23221) and / or can be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

[0412] Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and can generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in the construction of proteins or polypeptides intended for pharmaceutical use.

[0413] Some particularly preferred spacers include spacers and linkers used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, and linkers used in the art to construct, for example, diabodies or ScFv fragments (provided that in this regard, the linker sequences used in diabodies and ScFv fragments are those that are suitable V H and V L It should be noted that there is no particular limitation on the length or flexibility of the linker used in the polypeptides of the invention, although it should have a length, some flexibility, and other properties that allow the domains to come together to form the complete antigen-binding site (since each ISV or nanobody itself forms the complete antigen-binding site).

[0414] For example, the linker can be a suitable amino acid sequence, particularly an amino acid sequence of 1 to 50, preferably 1 to 30, e.g., 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include the gly-ser linker. Examples thereof include the (gly)-ser linker described in WO 99 / 42077. x ser y ) zlinkers of the type (e.g., (gly4ser)3 or (gly3ser2)3), as well as the GS30, GS15, GS9, and GS7 linkers described in the Ablynx applications referenced herein (see, e.g., WO 06 / 040153 and WO 06 / 122825), and hinge-like regions, such as the hinge region of a natural heavy chain antibody or similar sequences (e.g., as described in WO 94 / 04678). Preferred linkers are shown in Table B-4.

[0415] [Table 6]

[0416] Some other particularly preferred linkers are poly-alanine (eg, AAA), and linkers GS30 (SEQ ID NO: 85 in WO 06 / 122825) and GS9 (SEQ ID NO: 84 in WO 06 / 122825).

[0417] Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use with proteins for pharmaceutical applications. For example, poly(ethylene glycol) moieties have been used to link antibody domains. See, e.g., WO 04 / 081026.

[0418] It is within the scope of the present invention that the length, degree of flexibility, and / or other properties (not critical, but typical for linkers used in ScFv fragments) of the linker used may have some effect on the properties of the final polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for CD3 or one or more other antigens. Based on the disclosure herein, one skilled in the art will be able to determine, possibly after some limited routine trial and error, the optimal linker for use in a particular polypeptide of the invention.

[0419] For example, in a multivalent polypeptide of the invention comprising building blocks, ISVs or Nanobodies directed against a first and a second target, the length and flexibility of the linker is preferably such that each building block, ISV or Nanobody of the invention present in the polypeptide is able to bind to its cognate target, e.g., to its respective antigenic determinant. Again, based on the disclosure herein, a person skilled in the art will be able to determine, possibly after some limited routine trial and error, the optimal linker to use for a particular polypeptide of the invention.

[0420] It is also within the scope of the present invention that the linker used may impart one or more other desirable properties or functionalities to the polypeptide of the invention and / or provide one or more sites for the formation of derivatives and / or attachment of functional groups (e.g., as described herein for derivatives of ISVs, Nanobodies, or polypeptides of the invention). For example, a linker containing one or more charged amino acid residues may provide improved hydrophilicity, while a linker forming or containing a small epitope or tag may be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, one skilled in the art will be able to determine, optionally after some limited routine trial and error, the optimal linker for use with a particular polypeptide of the invention.

[0421] Finally, when two or more linkers are used in a polypeptide of the invention, these linkers can be the same or different. Again, based on the disclosure herein, one of skill in the art will be able to determine, possibly after some limited routine trial and error, the optimal linker for use in a particular polypeptide of the invention.

[0422] Typically, for ease of expression and production, the polypeptides of the invention will be linear polypeptides, although the invention, in its broadest sense, is not so limited. For example, when a polypeptide of the invention comprises three or more building blocks, ISVs, or Nanobodies, they can be joined by the use of a linker having three or more "arms," ​​each of which is attached to a building block, ISV, or Nanobody to provide a "star" construct. Circular constructs can also be used, although these are generally less preferred.

[0423] Thus, the present invention relates to a polypeptide as described herein, wherein the first ISV and the second ISV, and optionally the third ISV and / or the ISV that binds to serum albumin, are directly linked to each other or linked via a linker.

[0424] The present invention relates to a polypeptide as described herein, wherein the linker is selected from the group consisting of a 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS linker.

[0425] The present invention relates to the polypeptides described herein, wherein the serum protein binding moiety is a non-antibody-based polypeptide (eg, PEG).

[0426] The present invention also relates to methods for preparing the ISVs, polypeptides, and constructs described herein. The ISVs, polypeptides, and constructs of the present invention can be prepared in any manner known per se, as will be clear to those skilled in the art from the further description herein. For example, the ISVs, polypeptides, and constructs of the present invention can be prepared in any manner known per se for the preparation of antibodies, in particular antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments). Some preferred, non-limiting methods for preparing polypeptides and constructs include the methods and techniques described herein.

[0427] The method for producing an ISV, polypeptide, or protein construct of the invention comprises the steps of: - expression of a nucleic acid encoding said ISV, polypeptide or protein construct of the invention in a suitable host cell or host organism (herein also referred to as "host of the invention") or in another suitable expression system; If necessary, continue - isolating and / or purifying the ISV, polypeptide or protein construct of the invention thus obtained. may include:

[0428] In particular, such a method - culturing and / or maintaining a host of the invention under conditions such that said host of the invention expresses and / or produces at least one ISV, polypeptide or protein construct of the invention, If necessary, continue Isolating and / or purifying the ISV, polypeptide or protein construct of the invention thus obtained. may include:

[0429] Thus, the present invention also relates to nucleic acids or nucleotide sequences (also referred to as "nucleic acids of the invention" or "nucleotide sequences of the invention") that encode the ISVs, polypeptides, or protein constructs of the invention. Nucleic acids of the invention can 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, nucleotide sequences of the invention can be genomic DNA, cDNA, or synthetic DNA (e.g., DNA with codon usage specifically adapted for expression in the intended host cell or host organism).

[0430] According to one embodiment of the invention, the nucleic acid of the invention is in substantially isolated form, as defined herein. The nucleic acid of the invention may also be in the form of, present in, and / or be part of a vector, such as a plasmid, cosmid, or YAC. Again, the vector may be in substantially isolated form.

[0431] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information of the polypeptide or protein constructs of the invention provided herein, and / or can be isolated from suitable natural sources. As will be clear to the skilled person, several nucleotide sequences, e.g., at least one nucleotide sequence encoding an immunoglobulin single variable domain of the invention and, e.g., nucleic acids encoding one or more linkers, can also be linked to each other in a suitable manner to prepare the nucleic acids of the invention.

[0432] Techniques for generating the nucleic acids of the invention will be clear to those skilled in the art and may include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more natural and / or synthetic sequences (or two or more portions thereof); introducing mutations that result in expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using appropriate restriction enzymes); and / or introducing mutations by PCR reactions using one or more "mismatched" primers. These and other techniques will be clear to those skilled in the art. Again, reference is made to the standard handbooks mentioned herein, e.g., Sambrook et al. and Ausubel et al., and to the Examples below.

[0433] Furthermore, as will be apparent to those skilled in the art, the nucleic acids of the present invention can be in the form of, present in, and / or be part of a genetic construct. Such a genetic construct generally comprises at least one nucleic acid of the present invention, which may be linked to one or more elements of a genetic construct known per se, such as one or more suitable regulatory elements (e.g., suitable promoters, enhancers, terminators, etc.) and further elements of a genetic construct as mentioned herein. Such a genetic construct comprising at least one nucleic acid of the present invention will also be referred to herein as a "genetic construct of the present invention."

[0434] The genetic constructs of the present invention can be DNA or RNA, preferably double-stranded DNA. The genetic constructs of the present invention can be in a form suitable for transformation of an intended host cell or host organism, for integration into the genomic DNA of the intended host cell, or for independent replication, maintenance, and / or inheritance in the intended host organism. For example, the genetic constructs of the present invention can be in the form of a vector, such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i.e., a vector capable of providing expression in vitro and / or in vivo (e.g., in a suitable host cell, host organism, and / or expression system).

[0435] In a preferred, non-limiting embodiment, the genetic construct of the present invention comprises: a) comprises at least one nucleic acid of the invention; b) operably linked to one or more regulatory elements, for example, a promoter and, optionally, a suitable terminator; Depending on the situation, c) also containing one or more further elements of the genetic construct known per se, Here, the terms "regulatory element," "promoter," "terminator," and "operably linked" have their usual meaning in the art (as further described herein). Here, said "additional 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 facilitate or improve (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to those skilled in the art and may depend, for example, on the type of construct used, the intended host cell or host organism, the way in which the nucleotide sequence of the present invention of interest is expressed (e.g., by constitutive, transient, or inducible expression), and / or the transformation technique 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 an essentially similar manner.

[0436] Preferably, in a genetic construct of the present invention, the at least one nucleic acid of the present invention, the regulatory element, and optionally the one or more additional elements are "operably linked" to each other. "Operably linked" generally means being in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence if it is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (wherein the coding sequence is understood to be "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They will also usually be essentially contiguous, although this may not be required.

[0437] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform a host cell or host organism, i.e. for the expression and / or production of the polypeptide or protein construct of the 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 fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism, such as bacterial strains (including, but not limited to, gram-negative strains, such as Escherichia coli strains; Proteus strains, such as Proteus mirabilis strains; Pseudomonas strains, such as Pseudomonas fluorescens strains; and gram-positive strains, such as Bacillus strains, such as Bacillus subtilis strains or Bacillus brevis strains; Streptomyces strains, such as Streptomyces lividans strains; Staphylococcus strains, such as Staphylococcus carnosus strains; and Lactococcus strains, such as Lactococcus lactis strains); - fungal cells (including, but not limited to, cells from Trichoderma species, such as Trichoderma reesei; cells from Neurospora species, such as Neurospora crassa; cells from Sordaria species, such as Sordaria macrospora; cells from Aspergillus species, such as Aspergillus niger or Aspergillus sojae; or cells from other filamentous fungi); yeast cells (including, but not limited to, cells from Saccharomyces species, such as Saccharomyces cerevisiae; cells from Schizosaccharomyces species, such as Schizosaccharomyces pombe; cells from Pichia species, such as Pichia pastoris or Pichia methanolica; cells from Hansenula species, such as Hansenula polymorpha; cells from Kluyveromyces species, such as Kluyveromyces lactis; cells from Arxula species, such as Arxula adeninivorans; cells from Yarrowia species, such as Yarrowia lipolytica); - Amphibian cells or cell lines, for example, Xenopus oocytes; - insect-derived cells or cell lines, such as cells / cell lines from Lepidoptera (including but not limited to Spodoptera SF9 and Sf21 cells) or Drosophila-derived cells / cell lines, such as Schneider and Kc cells; plants or plant cells, for example tobacco plants; and / or - mammalian cells or cell lines, e.g., cells or cell lines of human origin, cells or cell lines of mammalian origin (including, but not limited to, CHO cells, BHK cells (e.g., BHK-21 cells) and human cells or cell lines, e.g., HeLa, COS (e.g., COS-7), and REP.C6 cells); and all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments) that will be clear to a person skilled in the art. Reference is also made to the general background art cited above, 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 further references cited therein.

[0438] Regarding the expression of ISVs, polypeptides 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 U.S. Pat. No. 7,004,940; WO 03 / 014960; Cattaneo and Biocca 1997 (Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag); and Kontermann 2004 (Methods 34: 163-170).

[0439] According to one preferred, non-limiting embodiment of the invention, the ISV, polypeptide or protein construct of the invention is produced in bacterial cells, in particular bacterial cells suitable for large-scale pharmaceutical production, such as cells of the strains mentioned above.

[0440] According to another preferred, non-limiting embodiment of the invention, the ISV, polypeptide, or protein construct of the invention is produced in yeast cells, in particular yeast cells suitable for large-scale pharmaceutical production, such as cells of the species mentioned above.

[0441] According to yet another preferred, non-limiting embodiment of the invention, the ISV, polypeptide or construct of the invention is produced in mammalian cells, in particular human cells or cells of a human cell line, especially human cells or cells of a human cell line suitable for large-scale pharmaceutical production, such as the cell lines mentioned above.

[0442] Suitable techniques for transforming the hosts or host cells of the present invention will be apparent to those skilled in the art and may depend on the intended host cell / host organism and the genetic construct used. Again, reference is made to the handbooks and patent applications mentioned above.

[0443] After transformation, steps can be taken to detect and select those host cells or host organisms which have been successfully transformed with a nucleotide sequence / gene construct of the invention. This step can be, for example, a selection step based on a selectable marker present in the genetic construct of the invention, or a step involving the detection of a polypeptide of the invention using, for example, a specific antibody.

[0444] 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) constitute further aspects of the present invention.

[0445] Preferably, these host cells or host organisms are such that they express or are (at least) capable of expressing (e.g., under appropriate conditions) the IVS, polypeptide, or protein construct of the invention (in the case of a host organism, in at least one cell, part, tissue, or organ thereof). The invention also includes further generations, progeny, and / or offspring of the host cells or host organisms of the invention, e.g., obtained by cell division or by sexual or asexual reproduction.

[0446] Thus, in another aspect, the invention relates to a host or host cell that expresses (or is capable of expressing, under appropriate circumstances, the ISV, polypeptide, or protein construct of the invention and / or contains nucleic acid encoding same. Some preferred, non-limiting examples of such hosts or host cells can be generally described in WO 04 / 041867, WO 04 / 041865, or WO 09 / 068627. For example, the ISV, polypeptide, or protein construct of the invention can advantageously be expressed, produced, or manufactured in yeast strains, such as Pichia pastoris strains. Reference is also made 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 comprising same in Pichia and other hosts / host cells.

[0447] To produce / obtain expression of an ISV, polypeptide, or protein construct of the invention, the transformed host cell or transformed host organism can generally be kept, maintained, and / or cultured under conditions such that the (desired) ISV, polypeptide, or protein construct of the invention is expressed / produced. Appropriate conditions will be clear to the skilled artisan and will usually depend on the host cell / host organism used and the regulatory elements controlling the expression of the (relevant) nucleotide sequence of the invention. Again, in the section on the genetic constructs of the invention, reference is made to the handbooks and patent applications mentioned above.

[0448] Generally, appropriate conditions can include the use of an appropriate medium, the presence of an appropriate food source and / or appropriate nutrients, the use of an appropriate temperature, and, optionally, the presence of an appropriate attractant or compound (e.g., when the nucleotide sequence of the invention is under the control of an inducible promoter), all of which can be selected by one of skill in the art. Again, under such conditions, the ISV, polypeptide, or protein construct of the invention can be expressed in a constitutive manner, transiently, or only when appropriately induced.

[0449] It will also be apparent to one skilled in the art that the ISV, polypeptide or protein construct of the invention may be (initially) produced in an immature state (as mentioned above) and then be subject to post-translational modifications depending on the host cell / host organism used. The ISV, polypeptide or protein construct of the invention may also be glycosylated, again depending on the host cell / host organism used.

[0450] The ISV, polypeptide or protein construct of the invention can then be isolated from the host cell / host organism and / or from the medium in which said host cell or host organism has been cultured using protein isolation and / or purification techniques known per se, such as (preparative) chromatographic and / or electrophoretic techniques, differential precipitation techniques, affinity techniques (using, for example, specific cleavable amino acid sequences fused to the polypeptide or construct of the invention) and / or preparative immunoglobulin techniques (i.e., using antibodies against the amino acid sequence to be isolated).

[0451] Constructs of the invention can generally be prepared by a method which at least comprises the step of suitably linking an ISV or polypeptide of the invention to one or more further groups, residues, moieties or binding units, optionally via one or more suitable linkers, to provide a construct of the invention. The ISVs, polypeptides and constructs of the 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 comprising the polypeptide or construct of the invention, to obtain derivatives of the polypeptide or construct of the invention.

[0452] The invention also relates to pharmaceutical compositions comprising an ISV, polypeptide, compound or construct of the invention.

[0453] In the above methods, the amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, or constructs of the invention and / or compositions comprising them can be administered in any suitable manner, depending on the particular pharmaceutical formulation or composition used. Thus, the amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, or constructs of the invention and / or compositions comprising them can be administered, for example, orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly, or via any other route of administration that avoids the digestive tract), intranasally, transdermally, topically, by suppository, by inhalation, again depending on the particular pharmaceutical formulation or composition used. A physician will be able to select an appropriate route of administration and a suitable pharmaceutical formulation or composition to be used for such administration depending on the disease or disorder to be prevented or treated and other factors known to the physician.

[0454] As used herein, the term "therapeutic agent" refers to any agent that can be used in the treatment and / or management of a hyperproliferative cell disorder, e.g., cancer, or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to a multispecific polypeptide of the invention. Preferably, a therapeutic agent is an agent that is known to be useful, has been used, or is currently being used in the treatment, prevention, and / or management of a hyperproliferative cell disorder, e.g., cancer, or one or more symptoms thereof.

[0455] As used herein, a "therapeutically effective amount," with respect to cancer, refers to a therapeutic amount, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment and / or management of cancer. In one aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to destroy, modify, inhibit, or eliminate primarily primary, regional, or metastatic cancer tissue. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to reduce the symptoms of cancer. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to slow or minimize the spread of cancer. In specific embodiments, a therapeutically effective amount of a treatment is a therapeutic amount sufficient to inhibit the growth or proliferation of cancer cells, kill existing cancer cells (e.g., regress cancer), and / or prevent the spread of cancer cells to other tissues or regions (prevent metastasis). In another specific embodiment, a therapeutically effective amount of a treatment is a therapeutic amount sufficient to inhibit tumor growth by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, as measured by standard methods known in the art. When used in reference to the amount of a multispecific polypeptide of the invention, this term can encompass an amount that improves overall treatment, reduces or avoids undesirable effects, or improves the therapeutic efficacy of or is synergistic with another treatment.In one embodiment, a therapeutically effective amount of a treatment reduces or avoids an undesirable effect, improves the therapeutic effect of, or is synergistic with another treatment by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (e.g., a negative control, e.g., phosphate buffered saline) in an assay known in the art or described herein.

[0456] As used herein, a "therapeutically effective amount" with respect to a non-cancerous hyperproliferative cell disorder refers to a therapeutic amount, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment and / or management of the disorder. In one aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to destroy, modify, inhibit, or eliminate cells affected by the non-cancerous hyperproliferative cell disorder. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to reduce the symptoms of the non-cancerous hyperproliferative cell disorder. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to slow or minimize the spread of the non-cancerous hyperproliferative cell disorder. In a specific embodiment, a therapeutically effective amount of a treatment is a therapeutic amount sufficient to inhibit the growth or proliferation of non-cancerous hyperproliferative cells and kill existing non-cancerous hyperproliferative cells (e.g., regress the disorder). In another specific embodiment, a therapeutically effective amount of a treatment is a therapeutic amount sufficient to inhibit the proliferation of non-cancerous hyperproliferative cells by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, as measured by standard methods known in the art. When used in reference to the amount of a multispecific polypeptide of the invention, this term can encompass an amount that improves overall treatment, reduces or avoids undesirable effects, or improves the therapeutic efficacy of or is synergistic with another treatment.In one embodiment, a therapeutically effective amount of a treatment reduces or avoids an undesirable effect, or improves the therapeutic effect of, or is synergistic with, another treatment by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (e.g., a negative control, e.g., phosphate buffered saline) in assays known in the art.

[0457] As used herein, the term "treatment" refers to any protocol, method, and / or agent that can be used in the treatment, prevention, and / or management of a hyperproliferative cell disorder, e.g., cancer. In certain embodiments, the terms "therapies" and "therapy" refer to biological treatments, supportive treatments, and / or other treatments known to those of skill in the art, e.g., medical professionals, that are useful in the treatment, prevention, and / or management of a hyperproliferative cell disorder, e.g., cancer, or one or more symptoms thereof.

[0458] As used herein, the terms "treat," "treatment," and "treating," in reference to administering a therapy to a subject, refer to the reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., a disorder associated with cancer, and / or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In specific embodiments, the terms "treat," "treatment," and "treating," in reference to administering therapy to a subject, refer to a reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, and refers to a reduction in cancer cells by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, relative to a control (e.g., a negative control, e.g., phosphate buffered saline). In other embodiments, the terms "treat," "treatment," and "treating," in reference to administering therapy to a subject, refer to a reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, and do not refer to a change in cancer cell count, a reduction in length of hospital stay, a decrease in mortality, or an increase in survival of a subject with cancer.

[0459] The amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, and / or constructs of the invention, and / or compositions comprising them, are administered according to a treatment regimen appropriate for preventing and / or treating the hyperproliferative cell disorder, e.g., cancer, being prevented or treated. A physician will generally be able to determine an appropriate treatment regimen depending on factors such as the stage of the hyperproliferative cell disorder, e.g., cancer, being treated, the severity of the hyperproliferative cell disorder, e.g., cancer, being treated, and / or the severity of its symptoms, the particular amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, and / or constructs of the invention used, the particular route of administration and pharmaceutical formulation or composition used, the patient's age, sex, weight, diet, general condition, and similar factors well known to physicians.

[0460] Generally, a treatment regimen will involve the administration of one or more pharmaceutically effective amounts or doses of one or more amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, and / or constructs of the invention, or one or more compositions comprising same. The specific amounts or doses to be administered can be determined by a physician, again based on the factors cited above.

[0461] Generally, for the prevention and / or treatment of the hyperproliferative cell disorders mentioned herein, e.g., cancer, and depending on the type of hyperproliferative cell disorder, e.g., cancer, and the stage of the disease being treated, the potency of the particular amino acid sequence, ISV, Nanobody, polypeptide, compound, or construct of the invention used, the particular route of administration, and the particular pharmaceutical formulation or composition used, the amino acid sequence, ISV, Nanobody, polypeptide, compound, or construct of the invention will generally be administered in an amount of 1 gram to 0.01 milligram per kg of body weight per day, preferably 0.1 gram to 0.01 milligram per kg of body weight per day, e.g., about 0.1, 1, 10, 100, or 1000 milligrams per kg of body weight per day, e.g., 0.1 mg to 25 mg per kg of the subject's body weight, either continuously (e.g., by infusion), in a single daily dose, or as multiple divided doses throughout the day. A physician will generally be able to determine an appropriate daily dose depending on the factors mentioned herein. It will also be apparent that in particular cases, a physician may select deviations from these amounts based, for example, on the factors cited above and the physician's professional judgment. Generally, some guidance in the amount to be administered can be obtained from the amount normally administered for a comparable conventional antibody or antibody fragment against the same target administered via substantially the same route, taking into account differences in affinity / avidity, efficacy, biodistribution, half-life, and similar factors well known to those of skill in the art.

[0462] Typically, one amino acid sequence, ISV, Nanobody, polypeptide, compound or construct of the invention will be used in the above methods, although it is within the scope of the invention to use two or more amino acid sequences, ISVs, Nanobodies, polypeptides, compounds and / or constructs of the invention in combination.

[0463] The ISVs, Nanobodies, amino acid sequences, polypeptides, compounds, and / or constructs of the invention can also be used in combination with one or more additional pharmaceutically active compounds or ingredients, i.e., as a combined treatment regimen. Such treatment regimens may or may not result in a synergistic effect. Again, a physician will be able to select such additional compounds or ingredients and an appropriate combined treatment regimen based on the above-cited factors and their professional judgment.

[0464] In particular, the amino acid sequences, ISVs, Nanobodies, polypeptides, compounds, and / or constructs of the invention can be used in combination with other pharmaceutically active compounds or ingredients that are or can be used in the prevention and / or treatment of hyperproliferative disorders, such as cancer, the diseases and / or disorders cited herein, which may or may not result in synergistic effects. Examples of such compounds and ingredients, as well as routes, methods, and pharmaceutical formulations or compositions for their administration, will be clear to the physician.

[0465] When two or more substances or components are used as part of a combined treatment regimen, they can 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., according to a substantially simultaneous, sequential, or alternating schedule). When the same substances or components are administered simultaneously via the same route of administration, they can be administered in different pharmaceutical formulations or compositions or as part of a combined pharmaceutical formulation or composition, as will be apparent to one of skill in the art.

[0466] In one aspect, the disclosure provides methods for administering immunoglobulin single variable domains and polypeptide constructs comprising one or more immunoglobulin single variable domains, polypeptides, compounds, and / or constructs. In some embodiments, the immunoglobulin single variable domains, polypeptides, compounds, and / or constructs are administered as pharmaceutical compositions. The pharmaceutical composition comprises the immunoglobulin single variable domains and polypeptide constructs thereof, as well as a pharmaceutically acceptable carrier.

[0467] As detailed above, the pharmaceutical compositions of the present disclosure can be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained-release formulation; topical application, e.g., as a cream, ointment, or sustained-release patch, or as a spray applied to the skin, lungs, or oral cavity; vaginally or rectally, e.g., as a suppository, cream, or foam; sublingually; ophthalmically; transdermally; or adapted for nasal, pulmonary, and other mucosal surfaces.

[0468] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0469] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent-containing material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; 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 glycerin, 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; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances utilized in pharmaceutical formulations.

[0470] Formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., immunoglobulin single variable domain or polypeptide construct thereof) which may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient which may be combined with the carrier materials to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99%, preferably from about 5% to about 70%, and most preferably from about 10% to about 30% of the active ingredient.

[0471] In certain embodiments, the formulation comprises an excipient selected from the group consisting of cyclodextran, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides, hi certain embodiments, the above formulations render the immunoglobulin single variable domain or polypeptide construct orally bioavailable.

[0472] Methods of preparing these formulations or compositions include the step of bringing into association the immunoglobulin single variable domain or polypeptide construct with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the immunoglobulin single variable domain or polypeptide construct with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0473] Formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (with an inert base, for example, gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each of which contains a predetermined amount of the immunoglobulin single variable domain or polypeptide construct as an active ingredient. The immunoglobulin single variable domain or polypeptide constructs of the invention can also be administered as a bolus, electuary, or paste.

[0474] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient may be incorporated into one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or one or more of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, such as glycerol; disintegrants, such as agar-agar. , calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarders such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; absorbents such as kaolin and bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard-shelled gelatin capsules, using, for example, excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0475] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersants. Molded tablets can be made in a suitable machine. In this case, a mixture of powdered compounds is moistened with an inert liquid diluent.

[0476] Tablets and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules, pills, and granules, may optionally be scored and prepared with coatings and envelopes, such as enteric coatings and other coatings well known in the pharmaceutical arts. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres, in varying proportions to provide the desired release profile. They may be formulated for rapid release, for example, lyophilized. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a bactericide in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient only, or preferentially, in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if possible, with one or more of the above-mentioned excipients.

[0477] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0478] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0479] Suspensions may contain, in addition to the active ingredient, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0480] Pharmaceutical compositions for rectal or vaginal administration can be formulated as suppositories. The suppositories can be prepared by mixing the immunoglobulin single variable domain or polypeptide construct with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and will melt in the rectum or vaginal cavity and release the active ingredient.

[0481] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0482] Dosage forms for topical or transdermal administration of an immunoglobulin single variable domain or polypeptide construct include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants which may be required.

[0483] Ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0484] Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0485] Transdermal patches have the added advantage of providing controlled delivery of immunoglobulin single variable domains or polypeptide constructs to the body. Such dosage forms can be constructed by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0486] Ophthalmic formulations, eye ointments, powders, solutions, and the like, are also contemplated as being within the scope of this disclosure.

[0487] Pharmaceutical compositions suitable for parenteral administration comprise one or more immunoglobulin single variable domains or polypeptide constructs in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may ...

Claims

1. 1. A polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), the first ISV has high affinity for / binds to cluster of differentiation 3 (CD3) present on T cells; the second ISV has high affinity for / binds to a first antigen on a target cell; wherein said first antigen is different from said CD3; and wherein the target cells are different from the T cells. Polypeptide.

2. The polypeptide of claim 1, which directs T cells to target cells.

3. The polypeptide according to claim 1 or 2, which induces T cell activation.

4. The polypeptide of claim 3 , wherein the T cell activation is independent of MHC recognition.

5. The polypeptide according to claim 3 or 4, wherein the T cell activation depends on presenting the polypeptide bound to the first antigen on a target cell to the T cell.

6. 6. The polypeptide of any one of claims 3 to 5, wherein the T cell activation results in 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 lysis of target cells.

7. 6. The polypeptide of any one of claims 3 to 5, wherein said T cell activation results in inhibition of the activity of said target cells by more than about 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%, for example 70%, 80%, or even more than 90%, such as 100%.

8. 8. The polypeptide of any one of claims 1 to 7, 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 polymorphic variants or isoforms thereof.

9. The polypeptide and / or first ISV preferably has a molecular weight of at least about 10 as measured by surface plasmon resonance. 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 9. The polypeptide of claim 1, having an on rate constant (Kon) for binding to CD3 selected from the group consisting of:

10. The polypeptide and / or first ISV preferably has a molecular weight of up to about 10 as measured by surface plasmon resonance. －３ s -1 , up to about 10 －４ s -1 , up to about 10 －５ s -1 , up to about 10 －６ s -1 , up to about 10 －７ s -1 , up to about 10 －８ s -1 , up to about 10 －９ s -1 , and up to about 10 －１ 0s -1 10. The polypeptide of claim 1, having an off rate constant (Koff) for binding to CD3 selected from the group consisting of:

11. 11. The polypeptide of any one of claims 1 to 10, wherein the first ISV binds to CD3, preferably with an EC50 value of 100nM to 1pM, such as with an average EC50 value of 100nM or less, even more preferably with an average EC50 value of 90nM or less, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5nM or less, such as less than 4, 3, 2, or 1nM or less, for example less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5pM or less, such as less than 4pM, as measured by flow cytometry.

12. 12. The polypeptide of any one of claims 1 to 11, wherein the first ISV binds to CD3 with an average KD value of 100nM to 10pM, such as an average KD value of 90nM or less, even more preferably an average KD value of 80nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5nM or less, such as less than 4, 3, 2, or 1nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20pM or less, such as less than 10pM, preferably as measured by SPR, e.g. when measured by Proteon.

13. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NOs: 81 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81 or any of SEQ ID NOs: 81 to 100; and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, and (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 to 122 and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 143, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 or any of SEQ ID NOs: 123 to 143 The polypeptide according to any one of claims 1 to 12, selected from the group consisting of:

14. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from SEQ ID NO:81 selected from the group consisting of where: At position -1, G is changed to R, - At position 3, T is changed to A, At position -4, Y is changed to F, At position -8, S is changed to G, and / or 14. The polypeptide of claim 13, wherein at position -10, G is changed to A.

15. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO: 101; selected from the group consisting of where: At position -3, V is changed to T or A; At position -5, S is changed to T, At position -6, G is changed to D or E, and / or The polypeptide of claim 13 or 14, wherein at position -9, T is changed to S, A, or P.

16. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second position, I is changed to T, In position -9, I is changed to V, and / or The polypeptide of any one of claims 13 to 15, wherein at position -10, A is changed to P.

17. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 81; and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 101; and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 123 14. The polypeptide of claim 13, selected from the group consisting of:

18. The polypeptide of any one of claims 13 to 17, wherein the first ISV consists essentially of four framework 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.

19. The polypeptide of claim 13, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO:

88.

20. The polypeptide of claim 13 or 19, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is SEQ ID NO:

110.

21. 21. The polypeptide of any one of claims 13, 19, or 20, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO:

128.

22. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 88 and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 110; and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 128 14. The polypeptide of claim 13, selected from the group consisting of:

23. 23. The polypeptide of claim 13 or 22, wherein the first ISV consists essentially of four framework 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.

24. The polypeptide of claim 13, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO:

90.

25. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO:112 selected from the group consisting of where:

25. The polypeptide of claim 13 or 24, wherein at position -2, V is changed to A.

26. 26. The polypeptide of any one of claims 13, 24, or 25, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO:

130.

27. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 90; and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 112; and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 130; 14. The polypeptide of claim 13, selected from the group consisting of:

28. 28. The polypeptide of claim 13 or 27, wherein the first ISV consists essentially of four framework 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.

29. The polypeptide of claim 13, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO:

89.

30. The polypeptide of claim 13 or 29, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is SEQ ID NO:

111.

31. 31. The polypeptide of any one of claims 13, 29, or 30, wherein the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is SEQ ID NO:

129.

32. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to the amino acid sequence of SEQ ID NO: 89; and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from the amino acid sequence of SEQ ID NO: 111; and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having one, two, or three amino acid differences from the amino acid sequence of SEQ ID NO: 129 14. The polypeptide of claim 13, selected from the group consisting of:

33. 33. The polypeptide of claim 13 or 32, wherein the first ISV consists essentially of four framework 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.

34. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or The polypeptide of claim 13, wherein at position -8, M is changed to T.

35. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:114 selected from the group consisting of where: At position -1, R is changed to Q, In position -3, T is changed to S, and / or 35. The polypeptide of claim 13 or 34, wherein at position -7, D is changed to A or K.

36. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 131 selected from the group consisting of where: In position -2, S is changed to R, and / or 36. The polypeptide of any one of claims 13, 34, or 35, wherein at position -6, S is changed to V.

37. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 91; and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 114; and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 131 14. The polypeptide of claim 13, selected from the group consisting of:

38. 38. The polypeptide of claim 13 or 37, wherein the first ISV consists essentially of four framework 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.

39. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: At position -3, S is changed to T, A, or G; At position -5, N is changed to S, In position -6, M is changed to T or A, and / or The polypeptide of claim 13, wherein L is changed to M at position -9.

40. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from SEQ ID NO: 118 selected from the group consisting of where: At position -2, H is changed to V; At position -5, S is changed to H or A; In position -8, N is changed to S, and / or 40. The polypeptide of claim 13 or 39, wherein at position -10, Y is changed to F.

41. The first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: At position -6, A is changed to S or D, At position -7, F is changed to Y or A; At position -8, R is changed to H; At position -9, S is changed to A, At position -11, G is changed to D, T, N, S, K, or R; and / or 41. The polypeptide of any one of claims 13, 39, or 40, wherein at position -14, V is changed to I.

42. the first ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 94 and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 118 and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 134 14. The polypeptide of claim 13, selected from the group consisting of:

43. 43. The polypeptide of claim 13 or 42, wherein the first ISV consists essentially of four framework 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.

44. The polypeptide of any one of claims 1 to 43, wherein said first antigen on a target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

45. 45. The polypeptide of any one of claims 1 to 44, further comprising a third ISV, which has high affinity for / binds to a second antigen on a target cell, wherein the second antigen is different from the first antigen.

46. 46. ​​The polypeptide of claim 45, wherein the second antigen on the target cell is a tumor antigen, preferably a tumor-associated antigen (TAA).

47. The polypeptide of any one of claims 44 to 46, wherein the first antigen and the second antigen are present on the same target cell.

48. The polypeptide of any one of claims 44 to 46, wherein the first antigen and the second antigen are present on different target cells.

49. The TAA may be 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, and 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 and growth factor receptors, including CD147, ErbB3, and ErbB4, as well as the gamma chain of the interleukin-2 receptor (CD132 antigen), the alpha chain of the interleukin-10 receptor (IL-10R-A), the beta chain of the interleukin-10 receptor (IL-10R-B), the beta-1 chain of the interleukin-12 receptor (IL-12R-beta1), the beta-2 chain of the interleukin-12 receptor (IL-12R-beta-2), the alpha-1 chain of the interleukin-13 receptor (IL-13R-alpha-1), ) (CD213a1 antigen), alpha-2 chain of interleukin-13 receptor (interleukin-13 binding protein), interleukin-17 receptor (IL-17 receptor), interleukin-17B receptor (IL-17B receptor), interleukin-21 receptor precursor (IL-21R), interleukin-1 receptor type I (IL-1R-1) (CD121a), interleukin-1 receptor type II (IL-1R-beta) (CDw121b), interleukin-1 receptor antagonist protein (I cytokine receptors, including the alpha chain of the interleukin-2 receptor (IL-1ra) (CD25 antigen), the beta chain of the interleukin-2 receptor (CD122 antigen), and the alpha chain of the interleukin-3 receptor (IL-3R-alpha) (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,EGFRvIII, 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, FXYD2 gamma a, The polypeptide of any one of claims 44 to 48, which is independently selected from the group consisting of CD21, CD133, Na / K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA195, DR5, DR6, DcR3, and CAIX, and associated polymorphic variants and isoforms.

50. 50. The polypeptide of claim 49, wherein the TAA is CD20 (Uniplot 11836), HER2 (Uniplot P04626), a polymorphic variant or isoform thereof.

51. The first antigen and the second antigen are EGFR as the first antigen and CEA as the second antigen, - CD19 as the first antigen and CD20 as the second antigen, CD19 as the first antigen and CD22 as the second antigen, - CD123 as the first antigen and Tim-3 as the second antigen, and - CD132 as the first antigen and CD69 as the second antigen 50. The polypeptide of any one of claims 44 to 49, selected from the group consisting of:

52. 52. The polypeptide of any one of claims 1 to 51, further comprising a serum protein binding moiety.

53. 53. The polypeptide of claim 52, wherein the serum protein binding moiety binds to serum albumin.

54. 54. The polypeptide of claim 52 or 53, wherein the serum protein binding moiety is an ISV that binds to serum albumin.

55. 55. The polypeptide of claim 54, wherein the ISV that binds to serum albumin consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein, as determined by the Kabat definition, CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375).

56. 56. The polypeptide of claim 55, wherein the ISVs that bind serum albumin comprise 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: 348-360).

57. 57. The polypeptide of any one of claims 1 to 56, wherein the ISVs are directly linked to each other or linked via a linker.

58. 57. The polypeptide of any one of claims 1 to 56, wherein the first ISV and / or the second ISV, and / or optionally the third ISV, and / or optionally the ISV that binds serum albumin are linked via a linker.

59. 51. The polypeptide of claim 50, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 362-372) linkers.

60. 54. The polypeptide of claim 52 or 53, wherein the serum protein-binding moiety is a non-antibody polypeptide.

61. 53. The polypeptide of any one of claims 1 to 52, further comprising PEG.

62. The ISV may be a nanobody, ＨＨ , humanized V ＨＨ , or camelized V Ｈ 62. The polypeptide of any one of claims 1 to 61,

63. 63. The polypeptide of any one of claims 1 to 62, 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.

64. A polypeptide selected from the group consisting of SEQ ID NOs: 249-250, 252-253, 255-256, 258-260, 263, 265-283, 286-289, 306-307, 309-310, 312-313, 315-317, 320, 322-340, and 343-346.

65. A polypeptide that specifically binds to CD3 and comprises or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), (i) CDR1 is (a) SEQ ID NOs: 81 to 100, or (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 81 to 100, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 122, or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 101 to 122, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 143, or (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference with respect to the amino acid sequence of any of SEQ ID NOs: 123 to 143, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. A polypeptide selected from the group consisting of:

66. (i) CDR1 is (a) SEQ ID NOs: 81 to 87, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 81, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 101 to 109, or (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 101, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 123 to 127, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 123, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

67. CDR1 is (a) SEQ ID NO: 81, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 81 selected from the group consisting of where: At position -1, G is changed to R, - At position 3, T is changed to A, At position -4, Y is changed to F, At position -8, S is changed to G, and / or 67. The polypeptide of claim 65 or 66, wherein at position -10, G is changed to A.

68. CDR2 is (a) SEQ ID NO: 101, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 101; selected from the group consisting of where: At position -3, V is changed to T or A; At position -5, S is changed to T, At position -6, G is changed to D or E, and / or 68. The polypeptide of any one of claims 65 to 67, wherein at position -9, T is changed to S, A, or P.

69. CDR3 is (a) SEQ ID NO: 123, and (b) an amino acid sequence having one or two amino acid differences relative to SEQ ID NO: 123 selected from the group consisting of where: - In the second position, I is changed to T, In position -9, I is changed to V, and / or 69. The polypeptide of any one of claims 65 to 68, wherein at position -10, A is changed to P.

70. 70. The polypeptide of any one of claims 65 to 69, 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.

71. (i) CDR1 is (a) SEQ ID NO: 88, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 88, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 110, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 110, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 128, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 128, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

72. 72. The polypeptide of any one of claims 65 or 71, wherein CDR1 is SEQ ID NO:

88.

73. 73. The polypeptide of any one of claims 65, 71, or 72, wherein CDR2 is SEQ ID NO:

110.

74. The polypeptide of any one of claims 65, 71 to 73, wherein CDR3 is SEQ ID NO:

128.

75. 75. The polypeptide of any one of claims 65, 71 to 74, 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.

76. (i) CDR1 is (a) SEQ ID NO: 90, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO:90, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 112 to 113, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 112, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 130, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 130, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or a higher affinity than the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

77. 77. The polypeptide of any one of claims 65 or 76, wherein CDR1 is SEQ ID NO:

90.

78. CDR2 is (a) SEQ ID NO: 112, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO:112 selected from the group consisting of where:

80. The polypeptide of any one of claims 65, 76, or 77, wherein at position -2, V is changed to A.

79. The polypeptide of any one of claims 65, 76 to 78, wherein CDR3 is SEQ ID NO:

130.

80. 80. The polypeptide of any one of claims 65, 76 to 79, 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.

81. (i) CDR1 is (a) SEQ ID NO: 89, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 89, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NO: 111, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 111, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NO: 129, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 129, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

82. 82. The polypeptide of any one of claims 65 or 81, wherein CDR1 is SEQ ID NO:

89.

83. 83. The polypeptide of any one of claims 65, 81, or 82, wherein CDR2 is SEQ ID NO:

111.

84. The polypeptide of any one of claims 65, 81 to 83, wherein CDR3 is SEQ ID NO:

129.

85. 85. The polypeptide of any one of claims 65, 81 to 84, 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.

86. (i) CDR1 is (a) SEQ ID NOs: 91 to 93, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO:91, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 114 to 117, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 114, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 131 to 133, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 131, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with about the same or a higher affinity than the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

87. CDR1 is (a) SEQ ID NO: 91, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO:91 selected from the group consisting of where: At position -6, R is changed to N or T; In position -7, N is changed to H, and / or 87. The polypeptide of any one of claims 65 or 86, wherein at position -8, M is changed to T.

88. CDR2 is (a) SEQ ID NO: 114, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 114 selected from the group consisting of where: At position -1, R is changed to Q, In position -3, T is changed to S, and / or 88. The polypeptide of any one of claims 65, 86, or 87, wherein at position -7, D is changed to A or K.

89. CDR3 is (a) SEQ ID NO: 131, and (b) an amino acid sequence having one amino acid difference relative to SEQ ID NO:131 selected from the group consisting of where: In position -2, S is changed to R, and / or 89. The polypeptide of any one of claims 65, 86 to 88, wherein at position -6, S is changed to V.

90. 90. The polypeptide of any one of claims 65, 86 to 89, 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.

91. (i) CDR1 is (a) SEQ ID NOs: 94 to 100, and (b) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO:94, provided that a polypeptide comprising a CDR1 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR1 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (ii) CDR2 is (c) SEQ ID NOs: 118 to 122, and (d) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 118, provided that a polypeptide comprising a CDR2 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or a higher affinity than the binding by a polypeptide comprising a CDR2 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance. and / or (iii) CDR3 is (e) SEQ ID NOs: 134 to 143, and (f) an amino acid sequence having 4, 3, 2, or 1 amino acid difference relative to the amino acid sequence of SEQ ID NO: 134, provided that a polypeptide comprising a CDR3 having the 4, 3, 2, or 1 amino acid difference binds to CD3 with approximately the same or higher affinity as compared to the binding by a polypeptide comprising a CDR3 without the 4, 3, 2, or 1 amino acid difference, as measured by surface plasmon resonance.

66. The polypeptide of claim 65, selected from the group consisting of:

92. CDR1 is (a) SEQ ID NO: 94, and (b) an amino acid sequence having 1, 2, 3, or 4 amino acid differences relative to SEQ ID NO:94 selected from the group consisting of where: At position -3, S is changed to T, A, or G; At position -5, N is changed to S, In position -6, M is changed to T or A, and / or 92. The polypeptide of any one of claims 65 or 91, wherein at position -9, L is changed to M.

93. CDR2 is (a) SEQ ID NO: 118, and (b) an amino acid sequence having one, two, or three amino acid differences relative to SEQ ID NO: 118 selected from the group consisting of where: At position -2, H is changed to V; At position -5, S is changed to H or A; In position -8, N is changed to S, and / or 93. The polypeptide of any one of claims 65, 91, or 92, wherein at position -10, Y is changed to F.

94. CDR3 is (a) SEQ ID NO: 134, and (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences from SEQ ID NO: 134 selected from the group consisting of where: At position -6, A is changed to S or D, At position -7, F is changed to Y or A; At position -8, R is changed to H; At position -9, S is changed to A, At position -11, G is changed to D, T, N, S, K, or R; and / or 94. The polypeptide of any one of claims 65, 91 to 93, wherein at position -14, V is changed to I.

95. 65. The polypeptide of any one of claims 91 to 94, 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.

96. Nanobody, V ＨＨ , humanized V ＨＨ , or camelized V Ｈ The polypeptide of any one of claims 65 to 95,

97. 97. The polypeptide of any one of claims 65 to 96, further comprising a serum protein binding moiety.

98. 98. The polypeptide of claim 97, wherein the serum protein binding moiety binds to serum albumin.

99. 99. The polypeptide of any one of claims 97 or 98, wherein the serum protein binding moiety is an ISV that binds to serum albumin.

100. 100. The polypeptide of claim 99, wherein the ISV that binds to serum albumin consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein, as determined by the Kabat definition, CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and CDR3 is GGSLSR (SEQ ID NO: 375).

101. 100. The polypeptide of claim 99, wherein the ISV that binds serum albumin is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG (SEQ ID NOs: 348-360).

102. The polypeptide of any one of claims 97 to 101, wherein the ISV is directly linked or linked via a linker.

103. 103. The polypeptide of claim 102, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 362-372).

104. 97. The polypeptide of any one of claims 65 to 96, further comprising a PEG moiety.

105. A nucleic acid or nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 104.

106. A vector comprising a nucleic acid or nucleic acid sequence as defined in claim 105.

107. A host cell transformed or transfected with a nucleic acid or nucleic acid sequence as defined in claim 105 or a vector as defined in claim 106.

108. A method for producing a polypeptide according to any one of claims 1 to 104, comprising culturing a host cell as defined in claim 107 under conditions which allow expression of the polypeptide as defined in any one of claims 1 to 104, and collecting the produced polypeptide from the culture.

109. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 104 or a polypeptide produced by the method according to claim 108.

110. A polypeptide according to any one of claims 1 to 104, or a polypeptide produced by the method according to claim 108, for use in treating a subject in need thereof.

111. 108. A method for delivering a prophylactic or therapeutic polypeptide to a specific location, tissue, or cell type in the body, comprising administering to a subject a polypeptide according to any one of claims 1 to 104, or a polypeptide produced by the method of claim 108.

112. 109. A polypeptide according to any one of claims 1 to 104, or a polypeptide produced by the method according to claim 108, for use in the prevention, treatment or alleviation of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease and an autoimmune disease.

113. 108. A method for the treatment or alleviation of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease, the method comprising the step of administering to a subject in need thereof a polypeptide according to any one of claims 1 to 104, or a polypeptide produced by the method according to claim 108.

114. 114. The polypeptide for use according to claim 112 or the method according to claim 113, wherein the proliferative disease is cancer.

115. The cancer may be carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, stomach cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, 114. The polypeptide or method for use according to claim 112 or 113, wherein the cancer is selected from the group consisting of: cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease, or AIDS-related primary effusion lymphoma, neuroectodermal tumor, rhabdomyosarcoma, and any metastasis of any of the above cancers, and non-cancerous indications, such as nasal polyps.

116. 116. A polypeptide or method for use according to claim 114 or 115 in a combination treatment.

117. A kit comprising a polypeptide as defined in any one of claims 1 to 104, a nucleic acid or nucleic acid sequence as defined in claim 105, a vector as defined in claim 106, or a host cell as defined in claim 107.

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