Novel polypeptides

Abstract

The present invention provides an hBCMA binding polypeptide comprising at least one motif binding to hBCMA wherein said polypeptide comprises the following structure: [N-terminal moiety]-[helix 1]-[isolated moiety]-[helix 2]-[C-terminal moiety], the hBCMA binding motif being moiety [helix 1]-[isolated moiety]-[helix 2]. The invention also provides a pharmaceutical composition comprising the hBCMA binding polypeptide, and a use of the hBCMA binding polypeptide or the pharmaceutical composition as a medicament, in particular for treating or preventing cancer.

Description

Technical Field

[0001] The present invention relates to human BCMA (hBCMA) binding polypeptides. The present invention also relates to pharmaceutical compositions comprising the hBCMA binding polypeptides, and their use in treating and / or preventing cancer. Background Art

[0002] Immunotherapy has been shown to be an effective treatment for several cancers, with approved therapies including monoclonal and bispecific antibodies, immunomodulatory drugs, CAR-T therapy, and antibody-drug conjugates. However, despite the effectiveness of these treatments, some patients show very short responses or fail to respond to treatment. The side effects of some immunotherapies can be severe, especially those associated with increased cytokine release. In fact, cytokine release syndrome is one of the most common serious adverse effects of T cell engagement immunotherapeutics (Shimabukuro-Vornhagen A et al. Cytokine release syndrome. J Immunother Cancer. 2018; 6 (1): 56. doi: 10.1186 / s40425-018-0343-9). Many patients will eventually become resistant to available treatments. Therefore, despite recent progress, cancer immunotherapy still needs additional treatment options.

[0003] An obvious obstacle to current treatment modalities is the suboptimal distribution to tumor tissue. The rate of tissue distribution is inversely correlated with the size of the molecule, so larger molecules (such as antibodies) have lower tumor penetration efficiency than smaller molecules. Another problem is immune evasion by cancer cells, which often involves inhibitory immune signals in the tumor environment. Examples of such signals include: production of immunosuppressive cytokines and other molecules such as TGFβ or VEGF; cell-mediated immunosuppression, for example by tumor-derived regulatory T cells; modulation of antigen presentation and MHC I expression; or altered expression of other ligands, such as increased expression of inhibitory checkpoint ligands such as programmed death ligand 1 (PD-L1) and HLA-E, which reduces tumor cell killing by CD8+ T cells and natural killer (NK) cells (Vinay D et al., Immune evasion in cancer: Mechanistic basis and therapeutic strategies, Seminars in Cancer Biology. 2015: 35(Suppl): S185, https: / / doi.org / 10.1016 / j.semcancer.2015.03.004; Ben-Shmuel A et al., Unleashing Natural Killer Cells in the Tumor Microenvironment-The Next Generation of Immunotherapy? Front Immunol.2020;11:275,doi:10.3389 / fimmu.2020.00275).

[0004] Multiple myeloma is a hematologic malignancy that remains incurable despite recent advances in immunotherapy. Several surface proteins that are enriched on myeloma cells have been identified and targeted for therapeutic intervention. One such target is BCMA (TNFRSF17), which is highly expressed on myeloma cells but also to a lesser extent on non-malignant cells of the B-cell compartment as well as plasmacytoid dendritic cells. Two B cell stimulatory ligands, proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF), bind to BCMA and are associated with autoimmune disorders and cancer (Bossen and Schneider (2006) BAFF, APRIL and their receptors: Structure, function and signalling, Seminars in Immunology, doi: 10.1016 / j.smim.2006.04.006; Moreaux et al. (2004) BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone, Blood 103(8): 3148-3157; Samy et al. (2017) Targeting BAFF and APRIL in systemic lupus erythematosus and other antibody-associated diseases, International Reviews of Immunology, 36: 1, 3-19).

[0005] BCMA-targeted CAR-T cell therapy and antibody-based therapy have been shown in clinical practice to effectively prolong overall survival of patients. However, not all patients show a satisfactory response to treatment, and the patient's disease will eventually relapse (see, for example, Teoh, PJ, Chng, WJ CAR T-cell therapy in multiple myeloma: more room for improvement. Blood Cancer J. 11, 84 (2021). https: / / doi.org / 10.1038 / s41408-021-00469-5).

[0006] There remains a need for improved cancer immunotherapeutics that can be more effectively delivered to tumors while retaining the target specificity of antibodies and antibody-based drugs and avoiding potential side effects, such as immunogenicity.

[0007] The present invention seeks to address the above needs. Summary of the invention

[0008] The present invention provides an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide comprises the following structure:

[0009] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0010] The hBCMA binding motif is part [helix 1]-[isolated part]-[helix 2],

[0011] In particular, the present invention provides an hBCMA-binding polypeptide, wherein:

[0012] i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other,

[0013] a) X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V;

[0014] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X nAt least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0015] The present invention also provides an hBCMA-binding polypeptide, wherein:

[0016] i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other,

[0017] a) X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V;

[0018] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues;

[0019] And wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:2.

[0020] The present inventors have surprisingly found that such hBCMA-binding polypeptides based on non-antibody scaffolds are effective in binding cancer cells and triggering cytotoxic drug-mediated or antibody-dependent cellular cytotoxicity-mediated (ADCC-mediated) killing of cancer cells.

[0021] Thus, the present invention provides novel hBCMA binding polypeptides (also referred to as 'engagers') that are effective in binding to cancer cells. They are particularly useful as cancer cell binding units in conjugates and fusion proteins that can trigger cytotoxic drug-mediated or ADCC-mediated cancer cell killing, and thus they show promise in anti-cancer immunotherapy.

[0022] The present invention also provides a hBCMA-binding oligomer comprising at least two hBCMA-binding polypeptides of the present invention.

[0023] The present invention also provides an hBCMA-binding polypeptide as disclosed herein or an hBCMA-binding oligomer as disclosed herein, further comprising additional functional moieties (e.g., at least one, at least two, or at least three; e.g., 1, 2, 3, 4, or 5 additional functional moieties).

[0024] Thus, in embodiments wherein the additional functional moiety is an additional binding moiety, the invention also provides a bispecific conjugate or a multispecific conjugate comprising the additional binding moiety.

[0025] The present invention also provides an hBCMA-binding agent-drug conjugate, which comprises the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention and an additional therapeutic agent.

[0026] The present invention also provides:

[0027] - a nucleic acid molecule encoding a hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention;

[0028] - an expression vector comprising such a nucleic acid molecule;

[0029] - A host cell comprising such a nucleic acid molecule or vector.

[0030] The present invention also provides a method for preparing the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention.

[0031] The present invention also provides a pharmaceutical composition comprising the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, nucleic acid molecule or expression vector disclosed herein.

[0032] The present invention also provides an hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition as disclosed herein for use in medicine, in particular for treating cancer.

[0033] The present invention also provides use of the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition disclosed herein for the manufacture of a medicament for treating cancer.

[0034] The present invention also provides a method for treating cancer, wherein the method comprises administering to a patient in need thereof an active component comprising an hBCMA-binding polypeptide or hBCMA-binding oligomer as disclosed herein. For example, the hBCMA-binding polypeptide or hBCMA-binding oligomer can be present as a cancer cell binding unit in a conjugate or fusion protein, such as a conjugate or fusion protein that can trigger cytotoxic drug-mediated or ADCC-mediated cancer cell killing. The present invention also provides a method for treating cancer, wherein the method comprises administering to a patient in need thereof a nucleic acid molecule, expression vector or pharmaceutical composition as disclosed herein.

[0035] The present invention also provides a kit comprising an hBCMA binding polypeptide, hBCMA binding oligomer, hBCMA binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition as disclosed herein, and optionally, one or more additional therapeutic agents. Such kits are particularly useful for treating and / or preventing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shown is a schematic domain organization of the hBCMA-binding polypeptides described in the present disclosure.

[0037] Figure 2 shows the experimental setup ( Figure 2a ) and the purified hBCMA binding variant His6-Fa-G6-ABD was injected at a concentration of 200 nM on the sensor chip surface on which hBCMA-Fc had been immobilized WT The resulting sensorgram obtained after [SEQ ID 3] ( Figure 2b ).

[0038] Figure 3 Shown are sensorgrams obtained after injection of purified Fa-G6-His6 [SEQ ID 4] and Fa-G6 truncations (-2, -3, -4 and -5aa) [SEQ ID 5-8] at 20 nM concentration on the sensor chip surface immobilized with hBCMA-Fc.

[0039] Figure 4Shown are the sensorgrams obtained after injection of a panel of purified His6-Fa-G6 [SEQ ID 129] ("Fa-G6 wild type") and 14 Fa-G6 alanine substitution mutants [SEQ ID 9-22], each equipped with an N-terminal His6 tag, at a concentration of 200 nM onto the surface of a sensor chip immobilized with hBCMA-Fc.

[0040] Figure 5 Shown are the sensorgrams obtained after injection of 18 purified second generation Fa-G6 variants [SEQ ID 2 and 23-39] equipped with C-terminal His6 at 100 nM on a sensor chip surface immobilized with hBCMA-Fc.

[0041] Figure 6 shows (a) thermal melting curves of Fa-G6-His6 and 18 second generation variants and (b) thermal melting curves of Fa-G6-His6 [SEQ ID 4] also equipped with a C-terminal His6 and second generation clone 1-E6 [SEQ ID 2] as well as the measured thermal melting temperatures (Tm).

[0042] Figure 7 Shown are the receptor binding properties of 1-E6-MBP-MMAF and non-conjugated capped 1-E6 polypeptides observed in a BCMA binding ELISA assay.

[0043] Figure 8 Shown are the effects of 1-E6-MBP-MMAF on EJM cell viability in the presence of 5 μM 1-E6 or its corresponding non-hBCMA binding counterpart (inactive).

[0044] Fig. 9 Relative IFNγ secretion in the cell culture medium in co-cultures of MM is shown. IS and PBMC were exposed to dual conjugates for 4 hours. The conjugates evaluated were 1-E6-A10-His6 [SEQ ID NO. 130], 1-E6-H09-A10-His6 [SEQ ID NO. 131], 1-E6-A10-A10-His6 [SEQ ID NO. 132], 1-E6-A11-A10-His6 [SEQ ID NO. 133] and their corresponding non-hBCMA binding counterparts (containing Fa-G6 null polypeptide instead of 1-E6).

[0045] Fig.10CD 16-mediated activation in a Jurkat Lucia reporter assay is shown relative to maximal activation using the monoclonal SLAMF7 antibody Elotuzumab. Activation was monitored in the presence of MM.1S cells (compound at 20 nM and 200 nM concentrations) and in the absence of MM.1S cells (200 nM concentration). 1-E6-A10-His6 [SEQ ID NO. 130], 1-E6-1E-6-A10-His6 with three different linker lengths [SEQ ID NO. 134-136], and 1-E6-A10-1E6-His6 [SEQ ID NO. 137] and A10-1-E6-1-E6-His6 [SEQ ID NO. 138] show activation dependent on the presence of MM.1S. "Null" indicates a non-hBCMA binding counterpart (containing the Fa-G6 null polypeptide instead of 1-E6).

[0046] Fig.11 Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0047] Fig.12a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0048] Figure 12b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0049] Fig.13a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0050] Fig.13b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1s cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0051] Fig.14aShown are the results of cell killing assays using NK cells and MM.1s cells.

[0052] Fig.14b Shown are the results of an 8-hour flow cytometry-based killing assay using NK cells and MM.1s (E:T 5:1) treated with increasing concentrations of any of the three dual conjugate constructs of the invention.

[0053] Fig.15 The hBCMA target specificity of the dual conjugate construct exemplary compound 131 in an in vitro multiple myeloma cell cytotoxicity assay is demonstrated.

[0054] Fig.16 Shown is the concentration-effect curve of Fa-G6-mc-MMAF on cell viability of BCMA-positive myeloma cell lines.

[0055] Fig.17a Shown are CD16a activation responses elicited by conjugate construct example compounds of SEQ ID 156, 160 and 163 in the presence or absence of target MM.1s cells.

[0056] Fig.17b NK cell-mediated killing of MM.1s cells by exemplary compounds of SEQ ID 156, 160 and 163 in the presence or absence of target MM.1s cells is shown.

[0057] Fig.18 Relative IFNγ secretion in cell culture medium of co-cultures of MM.1S and PBMCs exposed to dual conjugates for 4 hours is shown. The conjugates evaluated were anti-BCMA-A10-His6 [SEQ ID 130] and the corresponding non-BCMA binding counterparts.

[0058] Fig.19a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0059] Fig.19b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar. DETAILED DESCRIPTION

[0060] The present inventors have discovered that the hBCMA binding polypeptides as disclosed herein are surprisingly effective in binding to cancer cells. They are therefore found to be particularly useful in the treatment and / or prevention of cancer, for example as cancer cell binding units in conjugates and fusion proteins.

[0061] As discussed in more detail below, the inventors have surprisingly discovered that hBCMA binding polypeptides based on triple-helical affibody scaffolds are effective in binding BCMA on myeloma cells and triggering strong ADCC-mediated or cytotoxic drug-mediated responses against cancer cells. Such ADCC-mediated anti-cancer responses are beneficial in the treatment of multiple myeloma.

[0062] Notably, the inventors have found that such anti-cancer responses compare favorably with those obtained using the monoclonal antibody elotuzumab, which is approved for the treatment of multiple myeloma. The inventors have also found that such anti-cancer responses compare favorably with those obtained using a biosimilar of belantamab mafodotin, which is an antibody-drug conjugate used to treat multiple myeloma, and with Daratumumab, a monoclonal antibody approved for the treatment of multiple myeloma.

[0063] In its broadest aspect, the present invention provides an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide comprises the structure:

[0064] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0065] The hBCMA binding motif is part [helix 1]-[isolated part]-[helix 2].

[0066] The various elements of the polypeptides of the present invention will now be described in more detail:

[0067] Overall structure

[0068] In one embodiment, the polypeptides of the invention may be based on a triple helical scaffold, sometimes referred to as an 'affimer'. Affibodies are small (about 6.5 kDa) engineered affinity ligands based on a Z domain polypeptide that is a mutant form of the B domain in the immunoglobulin binding region of Staphylococcal protein A (Nord K et al., Binding proteins selected from combinatorial libraries of an a-helical bacterial receptor domain, Nature Biotechnol., 1997: 15: 772, doi: 10.1038 / nbt0897-772 ). In the full-length Affibody, the C-terminal portion includes [second separation portion]-[helix 3]-[C-terminal sequence]. The general structure of the Affibody is shown in Figure 1 middle.

[0069] The portions of the molecules of the invention referred to as helix 1 and helix 2 (and when present, helix 3) are generally helical in structure. In some rare embodiments, it may be found that the structures established by sequences with specific residues may not be strictly helical. Such compounds are considered to be within the broadest aspects of the invention. More preferably, the residues in the helix 1 and helix 2 portions do cause those structures to be helical, in the sense of an alpha-helix.

[0070] Sequences of the polypeptides of the present invention

[0071] The sequences of hBCMA-binding polypeptides as disclosed herein can be expressed according to their constituent amino acids or according to nucleic acid sequences encoding polypeptides having those amino acid sequences. In the context of the present disclosure, the term "amino acid" includes any naturally occurring amino acid or non-natural amino acid. The term "non-natural amino acid" as used herein refers to a non-proteinogenic (i.e., non-encoded) amino acid that is found in nature or chemically synthesized (e.g., citrulline, hydroxyproline, β-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid, or nitroarginine). It includes α, β, γ, and δ amino acids. It includes amino acids of any chiral configuration. In particular, the amino acid may be a naturally occurring α amino acid. The amino acid may be particularly a naturally occurring L amino acid. The amino acid may be particularly a naturally occurring L-α amino acid.

[0072] Within a polypeptide chain (e.g., an hBCMA-binding polypeptide disclosed herein), amino acids are linked by peptide bonds between the carboxyl group of one amino acid and the amine group of the next amino acid in the chain. Once linked in a polypeptide chain, a single amino acid is referred to as a "residue" or "amino acid residue."

[0073] The N-terminus of the amino acid sequences herein is shown on the left, and in the case where the sequence is shown across multiple lines, the N-terminus is at the top left. Unless otherwise indicated, the amino acid residues in the sequence are L-amino acids.

[0074] Amino acid sequences listed in this application are shown using standard letter abbreviations for amino acids.

[0075] Specific sequences given herein are relevant to specific embodiments of the invention.

[0076] The present disclosure also includes derivatives of all sequences described herein (e.g., derivatives of each of the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein). Derivatives of the sequences described herein are preferably derivatives in which 1 to 5 (e.g., 1, 2, or 3) amino acid residues can be replaced by alternative residues, such as different naturally occurring amino acids or different non-natural amino acids; or different naturally occurring amino acids or different non-natural amino acids that do not include methionine. Preferably, the non-natural amino acids according to the present invention are amino acids that are isosteric with naturally occurring amino acids (e.g., norleucine).

[0077] The present disclosure also includes derivatives of the sequences described herein, wherein 1 to 5 (e.g. 1, 2 or 3) amino acid residues may be replaced by alternative residues as conservative substitutions, i.e. one residue is replaced by another residue of the same class, for example:

[0078] An aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by another aliphatic residue. A hydroxyl-, sulfur- or selenium-containing residue (serine (S), cysteine ​​(C), selenocysteine ​​(U), threonine (T) or methionine (M)) may be replaced by another hydroxyl-, sulfur- or selenium-containing residue. An aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)) may be replaced by another aromatic residue. A basic residue (histidine (H), lysine (K), arginine (R)) may be replaced by another basic residue. An acidic residue or amide (aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)) may be replaced by another acidic residue or amide.

[0079] In certain embodiments, the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) do not contain methionine. In certain embodiments, in the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein), at the positions where methionine residues are listed, the polypeptide has a sequence in which the methionine residues are independently replaced by different residues (e.g., different naturally occurring amino acids or non-natural amino acids). In certain embodiments, in the sequences described herein, at the positions where methionine residues are listed, the polypeptide has a sequence in which the methionine residues are independently replaced by amino acids selected from isoleucine (I), leucine (L), glutamine (Q) and norleucine; for example, a sequence in which isoleucine and norleucine are replaced.

[0080] In another embodiment, when one or more methionine residues are present in an hBCMA-binding polypeptide or CD16a-binding oligomer as defined herein, one, some or all of the methionine residues may be replaced by an alternative amino acid, e.g., a different naturally occurring amino acid or an unnatural amino acid, such as an amino acid selected from isoleucine (I), leucine (L), glutamine (Q) or norleucine; and in particular isoleucine (I) and norleucine.

[0081] For example, in one embodiment, one or more (e.g., each) methionine residues of the sequences described herein may be replaced by different naturally occurring amino acids or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine; and in particular isoleucine and norleucine. For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues or 1 methionine residue may be replaced by different naturally occurring amino acids or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine; and in particular isoleucine and norleucine.

[0082] For example, in embodiments where X9 can be or is methionine, the residue at X9 can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine, and norleucine; and in particular isoleucine and norleucine. 10 In embodiments where X is or is methionine, 10 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 14 In embodiments where X is or is methionine,14 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 18 In embodiments where X is or is methionine, 18 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 31 In embodiments where X is or is methionine, 31 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 35 In embodiments where X is or is methionine, 35 The residue at X may be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. In particular, in which X 31 and X 35 In embodiments where X is or is methionine, 31 and X 35 The residue at may be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine.

[0083] Alternatively or additionally, in certain embodiments, the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may contain amino acid substitutions, wherein one or more residues are replaced with a non-natural amino acid.

[0084] For example, in one embodiment, one or more residues of a sequence described herein (e.g., an hBCMA-binding polypeptide and hBCMA-binding oligomer sequence described herein) can be replaced with an unnatural amino acid (e.g., norleucine). For example, 1 to 15 residues can be replaced with an unnatural amino acid, such as 1 to 10 residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues), 1 to 5 residues (e.g., 1, 2, 3, 4, or 5), 1 to 3 residues (e.g., 1, 2, or 3), or 1 residue can be replaced with an unnatural amino acid (e.g., norleucine).

[0085] For example, in one embodiment, one or more leucine residues of a sequence described herein (e.g., an hBCMA-binding polypeptide and an hBCMA-binding oligomer sequence described herein) can be replaced with a non-natural amino acid, and preferably norleucine. For example, when present, 1 to 5 leucine residues, 1 to 3 leucine residues (e.g., 1, 2, or 3 leucine residues), or 1 or 2 leucine residues, or 1 leucine residue can be replaced with a non-natural amino acid (e.g., norleucine). For example, in certain embodiments, in a sequence described herein (e.g., an hBCMA-binding polypeptide and an hBCMA-binding oligomer sequence described herein), at the position where a leucine residue is listed, the polypeptide has a sequence in which the leucine residue is independently substituted with a non-natural amino acid, and preferably norleucine.

[0086] Alternatively or additionally, in one embodiment, one or more methionine residues of the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may be replaced with a non-natural amino acid, and preferably norleucine. For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues, or 1 methionine residue may be replaced with a non-natural amino acid (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein), at the positions where methionine residues are listed, the polypeptide has a sequence in which methionine residues are independently substituted with non-natural amino acids, and preferably norleucine.

[0087] For example, in embodiments where X9 can be or is methionine, the residue at X9 can be replaced by an unnatural amino acid (e.g., norleucine). 10 In embodiments where X is or is methionine, 10 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 14 In embodiments where X is or is methionine, 14 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 18 In embodiments where X is or is methionine, 18 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 31 In embodiments where X is or is methionine, 31 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 35 In embodiments where X is or is methionine, 35The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 31 and X 35 In embodiments where X is or is methionine, 31 and X 35 The residue at may be replaced by an unnatural amino acid (eg, norleucine).

[0088] In certain embodiments, one or more methionine residues of the sequences described herein (e.g., hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) can be oxidized, i.e., in the form of methionine sulfoxide ("Met(O)"). For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues, or 1 methionine residue can be oxidized (e.g., can be Met(O)). For example, in embodiments where X9 can be or is methionine, when present, the methionine at X9 can be oxidized (e.g., Met(O)). For example, in embodiments where X9 can be or is methionine, when present, the methionine at X9 can be oxidized (e.g., Met(O)). 10 In embodiments where X is or is methionine, when present, 10 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 14 In embodiments where X is or is methionine, when present, 14 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 18 In embodiments where X is or is methionine, when present, 18 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 31 In embodiments where X is or is methionine, when present, 31 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 35 In embodiments where X is or is methionine, when present, 35 The methionine at the position may be oxidized (e.g., Met(O)). In particular, in which X 31 and X 35 In embodiments where methionine is present, X 31 and X 35 The methionine at the residue may be oxidized (eg, Met(O)).

[0089] Alternatively or additionally, in certain embodiments, the sequences described herein (e.g., hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) comprise a peptide purification tag or moiety (e.g., a histidine tag (e.g., a polyhistidine tag) or a methionine tag (e.g., a single methionine tag or a polymethionine tag)), a signaling tag or moiety (e.g., a glycine residue or a signal peptide, e.g., a signal peptide selected from OmpA, DsbA, PhoA, and PelB), a fluorescent tag (e.g., Alexa448), or a tag or moiety that aids conjugation (a cysteine ​​tag (e.g., a single cysteine ​​at the C-terminus or N-terminus)). Such tags and / or moieties may preferably be present at the N-terminus and / or C-terminus of the BCMA-binding polypeptide and BCMA-binding oligomer sequences described herein.

[0090] Thus, the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may further comprise at least 1 histidine residue (and optionally at least 1 tyrosine residue) and / or at least 1 methionine residue; for example, at least 4, at least 5, or at least 6 histidine residues (and optionally at least 1 tyrosine residue, such as 1, 2, or 3 tyrosine residues) and / or at least 1 methionine residue. In one embodiment, the sequences described herein (e.g., hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may further comprise at least 6 histidine residues and optionally at least 1 tyrosine residue (e.g., 6 histidine residues (e.g., HHHHH) or 6 histidine residues and two tyrosine residues (e.g., YYHHHHHH)) and / or at least 1 methionine residue (e.g., 1 or 2 methionine residues). A peptide purification tag or moiety (e.g., a histidine tag or a methionine tag as described above) may preferably be present at the N-terminus and / or C-terminus of the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein. For example, an additional sequence of at least 6 histidine residues (e.g., 6 histidine residues; or 6 histidine residues and two tyrosine residues) and / or at least 1 methionine residue (e.g., 1 or 2 methionine residues) may be present at the N-terminus and / or C-terminus of the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein.

[0091] The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may also include an additional sequence of at least one cysteine ​​(e.g., one cysteine) at the N-terminus or C-terminus. The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may also include a fluorophore tag (e.g., a 448 Alexa tag) at the N-terminus or C-terminus. The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may also include a signal peptide at the N-terminus or C-terminus, preferably at the N-terminus, such as selected from OmpA, DsbA, PhoA, and PelB. The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may also include an additional sequence of at least one glycine (e.g., one glycine) at the N-terminus or C-terminus.

[0092] In certain embodiments, the hBCMA-binding polypeptide is a polypeptide wherein the hBCMA binding efficacy is at least 1%, at least 5%, or preferably at least 10% (more preferably at least 15%, 20%, 25%, or 50%) of SEQ ID NO: 2. When a hBCMA-binding polypeptide is described herein as having at least X% of the hBCMA binding efficacy of a specific peptide (e.g., SEQ ID NO: 2), it is understood that the IC50 concentration of the polypeptide for binding to the hBCMA receptor is no more than 100 / X times the IC50 concentration of the specific peptide (SEQ ID NO: 2) for the hBCMA receptor when measured under the same conditions.

[0093] For example, if the binding potency of the hBCMA binding polypeptide is at least 5%, more preferably at least 10%, 20%, 25% or 50% of the hBCMA binding potency of the specific peptide (e.g., SEQ ID NO: 2), that is, the IC value of the surrogate polypeptide for binding to the hBCMA receptor is greater than 10%, 20%, 25% or 50% when measured under the same conditions. 50 The concentration does not exceed the IC of the specific peptide (e.g., SEQ ID NO: 2) for the hBCMA receptor. 50 The concentration is 20 times, and more preferably no more than 10 times, 5 times, 4 times or 2 times.

[0094] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes for binding at hBCMA with the peptide of SEQ ID NO: 2.

[0095] The present invention provides an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide comprises the following structure:

[0096] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0097] The hBCMA binding motif is part [helix 1]-[isolated part]-[helix 2].

[0098] Preferably, the hBCMA-binding polypeptide is a polypeptide wherein:

[0099] i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein

[0100] Independently of each other,

[0101] X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V;

[0102] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0103] In one embodiment, helix 1 comprises the sequence KEX9X 10 X 11 ADX 14 EIX 17 X 18 , and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 D, for example, helix 1 contains the sequence NKEX9X 10 X11 ADX 14 EIX 17 X 18 L, and helix 2 contains the sequence NFX 25 QKWAFX 31 RX 33 LX 35 DD, where X9, X 10 , X 11 , X 14 , X 17 , X 18 , X 25 , X 31 , X 33 and X 35 As defined above.

[0104] Preferably, such hBCMA-binding polypeptides are polypeptides wherein the hBCMA binding efficacy is at least 1% of that of SEQ ID NO: 2. Preferably, the binding efficacy is at least 5%, such as at least 10%, 20%, 25% or 50% of the hBCMA binding efficacy of the specific peptide (e.g., SEQ ID NO: 2).

[0105] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2 for binding to hBCMA.

[0106] In one embodiment, the hBCMA-binding polypeptide is a polypeptide wherein: i) Helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other,

[0107] X9 and X 10 is any naturally occurring amino acid;

[0108] X 11 is E, F, H, Q, T or Y;

[0109] X 14 is any naturally occurring amino acid;

[0110] X 17 is A, E, Q, S, T, or V;

[0111] X18 is any naturally occurring amino acid;

[0112] X 25 is F or Y;

[0113] X 31 is I, M or V;

[0114] X 33 is K or S;

[0115] X 35 is I, L, M or V;

[0116] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues;

[0117] And wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:2.

[0118] Preferably, the binding efficacy is at least 5%, such as at least 10%, 20%, 25% or 50% of the hBCMA binding efficacy of the specific peptide (eg, SEQ ID NO: 2).

[0119] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2 for hBCMA.

[0120] As mentioned above, it is not necessary to be exactly the same as the sequence listed in (i) (although in a preferred embodiment of the invention the peptide does have the exact sequence listed). nOf the residues indicated by the label, at least 1 and no more than 8 residues may be replaced by alternative residues. The replaced residues may all be in helix 1 or helix 2, or there may be 1 replaced residue in one and 1, 2, 3, 4, 5, 6, or 7 replaced residues in the other, or there may be 2 replaced residues in one and 1, 2, 3, 4, 5, or 6 replaced residues in the other, or there may be 3 replaced residues in one and 1, 2, 3, 4, or 5 replaced residues in the other, or there may be 4 replaced residues in one and 1, 2, 3, or 4 replaced residues in the other, or there may be 5 replaced residues in one and 1, 2, or 3 replaced residues in the other, or there may be 6 replaced residues in one and 1 or 2 replaced residues in the other, or there may be 7 replaced residues in one and 1 replaced residue in the other. For example, in the case of using X n There may be 1, 2, 3, 4 or 5 substituted residues among the residues indicated by the label, for example 1, 2 or 3, for example 1. Since there are 10 residues with X n The peptide with 5 residues replaced has 50% sequence identity to the sequence. For 2 residues replaced it is 80% sequence identity and for 1 residue replaced it is 90% sequence identity.

[0121] In another embodiment, in the case of an n Of the residues in the sequence, at least 1 and no more than 5 residues may be replaced by alternative residues. The replaced residues may all be in helix 1 or helix 2, or there may be 1 replaced residue in one and 1, 2, 3, 4 or 5 replaced residues in the other, or there may be 2 replaced residues in one and 1, 2 or 3 replaced residues in the other, or there may be 3 replaced residues in one and 1 or 2 replaced residues in the other, or there may be 4 replaced residues in one and 1 replaced residue in the other. For example, in the case of an unused X n There may be 1, 2, 3, 4 or 5 substituted residues among the residues indicated by the label, for example 1, 2 or 3, for example 1. Since there are 12 residues without X n The peptide with 5 residues replaced has 58% sequence identity to the sequence labeled, for 2 residues replaced it is 83% sequence identity, and for 1 residue replaced it is 92% sequence identity.

[0122] In another embodiment, in the case of an n At least 1 and no more than 3 of the residues in the sequence may be replaced by alternative residues. The replaced residues may all be in helix 1 or helix 2, or there may be 1 replaced residue in one and 1 or 2 replaced residues in the other, or there may be 2 replaced residues in one and 1 replaced residue in the other. For example, in the sequence not using X n There may be 1, 2 or 3 substituted residues among the residues indicated by the label, for example 1 or 2, for example 1. Since there are 12 residues without X n The peptide with 3 residues replaced has 75% sequence identity to the sequence labeled, for 2 residues replaced it is 83% sequence identity, and for 1 residue replaced it is 92% sequence identity.

[0123] In one embodiment, the total number of replaced residues in the helix 1 and helix 2 portions is at least 1 and no more than 13, at least 1 and no more than 12, at least 1 and no more than 11, at least 1 and no more than 10, such as at least 1 and no more than 9, at least 1 and no more than 8, such as at least 1 and no more than 7, such as at least 1 and no more than 6, such as at least 1 and no more than 5, such as at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. In particular, there may be a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 replaced residues in those portions.

[0124] Represented as X n The residues or not indicated as X n Amino acid residue substitutions of residues of can be conservative substitutions. That is, one residue is replaced by another residue of the same class, for example:

[0125] An aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by another aliphatic residue. A hydroxyl-, sulfur- or selenium-containing residue (serine (S), cysteine ​​(C), selenocysteine ​​(U), threonine (T) or methionine (M)) may be replaced by another hydroxyl-, sulfur- or selenium-containing residue. An aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)) may be replaced by another aromatic residue. A basic residue (histidine (H), lysine (K), arginine (R)) may be replaced by another basic residue. An acidic residue or amide (aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)) may be replaced by another acidic residue or amide.

[0126] Alternatively, denoted as X n The residues or not indicated as X n Amino acid residue substitutions of a residue may be non-conservative substitutions, i.e. a residue is replaced by another residue from a different class, for example an aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by an aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)), or for example by an amino acid with opposite characteristics, for example a lysine (K) residue is replaced by an aspartic acid (D) residue.

[0127] Alternatively, denoted as X n The residues or not indicated as X n The amino acid residue replacement of the residue of can be a non-classical (also called non-natural) amino acid, i.e. an amino acid not found in a natural polypeptide chain. As mentioned above, the term "non-natural amino acid" as used herein refers to a non-proteinogenic (i.e. non-encoded) amino acid that can be found in nature or chemically synthesized (e.g. citrulline, hydroxyproline, β-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid or nitroarginine). These can be produced as secondary metabolites or chemically synthesized. Preferably, the non-natural amino acid according to the present invention is an amino acid that is isosteric with a naturally occurring amino acid (e.g., norleucine). Therefore, according to the present invention, for the amino acid represented by X n The residues or not are indicated as X n The amino acid residue replacement of the residue may be an unnatural amino acid, which is an amino acid isosteric to a naturally occurring amino acid, preferably norleucine.

[0128] In another embodiment, the hBCMA-binding polypeptide is a polypeptide wherein:

[0129] i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LM, where, independently of each other,

[0130] X9 is D, E, H, K, N, Q, S or V; 10 is A, E, F, I, K, M, N, Q, R, S, T, Y, or V; X 11 is E, F or H; X 14 is A, E, H, I, K, L, Q, R, T, or Y; X 17 is A, E, S, T, or V; X 18 is A, F, H, K, L, M, N, T, or S; X33 is K or S;

[0131] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least one and no more than three (e.g., one, two, or three) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 3 (e.g., 1, 2, or 3) of the residues in the protein are replaced by alternative residues.

[0132] Preferably, such hBCMA binding polypeptides are polypeptides wherein the hBCMA binding efficacy is at least 1% of that of SEQ ID NO. 2. For example, the binding efficacy is at least 5%, such as at least 10%, 20%, 25% or 50% of the hBCMA binding efficacy of a specific peptide (e.g., SEQ ID NO: 2).

[0133] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2 for binding to hBCMA.

[0134] As mentioned above, it is not necessary to be exactly the same as the sequence listed in (i) (although in a preferred embodiment of the invention the peptide does have the exact sequence listed). n At least one and no more than three (e.g., one, two, or three) of the residues indicated by the label may be replaced by alternative residues. The replaced residues may all be in helix 1 or helix 2, or there may be one replaced residue in one and one or two replaced residues in the other. For example, in the case of residues indicated by X n There may be 1, 2 or 3 substituted residues among the residues indicated by the label, for example 1 or 2, for example 1. Since there are 7 residues with X n The peptide with 3 residues replaced has 57% sequence identity to the sequence labeled, for 2 residues replaced it is 71% sequence identity, and for 1 residue replaced it is 86% sequence identity.

[0135] In the fields not marked with an X n At least one and no more than three (e.g., one, two, or three) of the residues in the sequence may be replaced by alternative residues. The replaced residues may all be in helix 1 or helix 2, or there may be one replaced residue in one and one or two replaced residues in the other. For example, in the sequence not containing X n There may be 1, 2 or 3 substituted residues among the residues indicated by the label, for example 1 or 2, for example 1. Since there are 15 residues without X nThe peptide with 3 residues replaced has 80% sequence identity to the sequence labeled, for 2 residues replaced it is 87% sequence identity, and for 1 residue replaced it is 93% sequence identity.

[0136] In one embodiment, the total number of replaced residues in the helix 1 and helix 2 portions is at least 1 and no more than 6, such as at least 1 and no more than 5, at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 3. In particular, there may be a total of 1, 2, 3, 4, 5 or 6 replaced residues in those portions.

[0137] Represented as X n The residues or not indicated as X n Amino acid residue substitutions of the residues in can be conservative substitutions or non-conservative substitutions, or non-classical (also called non-natural) amino acids, as described above.

[0138] In another embodiment, the hBCMA-binding polypeptide is at position X9, X 10 , X 14 , X 18 , X 31 or X 35 A polypeptide having no methionine residue at any position, such as an hBCMA-binding polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other,

[0139] X9 and X 10 is an unnatural amino acid (e.g., norleucine) or any naturally occurring amino acid except methionine;

[0140] X 11 is E, F, H, Q, T or Y;

[0141] X 14 is an unnatural amino acid (e.g., norleucine) or any naturally occurring amino acid except methionine;

[0142] X 17 is A, E, Q, S, T, or V;

[0143] X 18is an unnatural amino acid (e.g., norleucine) or any naturally occurring amino acid except methionine;

[0144] X 25 is F or Y;

[0145] X 31 is I, V, L, glutamine or an unnatural amino acid (such as norleucine) (preferably I, L, V, glutamine or norleucine);

[0146] X 33 is K or S;

[0147] X 35 is I, L, V, glutamine or an unnatural amino acid (such as norleucine) (preferably I, L, V, glutamine or norleucine); or

[0148] In another embodiment, the hBCMA-binding polypeptide is at position X9, X 10 , X 14 , X 18 , X 31 or X 35 A polypeptide having no methionine residue at any position, such as an hBCMA-binding polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other, X9 and X 10 is norleucine or any naturally occurring amino acid except methionine;

[0149] X 11 is E, F, H, Q, T or Y;

[0150] X 14 is norleucine or any naturally occurring amino acid except methionine;

[0151] X 17 is A, E, Q, S, T, or V;

[0152] X 18 is norleucine or any naturally occurring amino acid except methionine;

[0153] X 25 is F or Y;

[0154] X 31 is I, V, L, glutamine or norleucine (eg, I, V or norleucine; preferably I or norleucine);

[0155] X 33 is K or S;

[0156] X 35 is I, L, V, glutamine or norleucine (eg, I, V or norleucine; preferably I or norleucine); or

[0157] In another embodiment, the hBCMA-binding polypeptide is at position X9, X 10 , X 14 , X 18 , X 31 or X 35 A polypeptide having no methionine residue at any position, such as an hBCMA-binding polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other,

[0158] X9 and X 10 is any naturally occurring amino acid except methionine;

[0159] X 11 is E, F, H, Q, T or Y;

[0160] X 14 is any naturally occurring amino acid except methionine;

[0161] X 17 is A, E, Q, S, T, or V;

[0162] X 18 is any naturally occurring amino acid except methionine;

[0163] X 25 is F or Y;

[0164] X 31 is I or V;

[0165] X 33 is K or S;

[0166] X 35 It is I, L or V.

[0167] In another embodiment, the hBCMA-binding polypeptide is at position X9, X 10 , X 14 , X 18 , X 31 or X 35 A polypeptide having no methionine residue at any position, such as an hBCMA-binding polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LX 35 , where, independently of each other,

[0168] X9 is D, E, H, K, N, Q, S or V;

[0169] X 10 is A, E, F, I, K, N, Q, R, S, T, Y, V, L, or an unnatural amino acid (e.g., norleucine);

[0170] X 11 is E, F or H;

[0171] X 14 is A, E, H, I, K, L, Q, R, T or Y;

[0172] X 17 is A, E, S, T or V;

[0173] X 18 is A, F, H, K, L, N, T, S, I, Q or an unnatural amino acid (e.g., norleucine);

[0174] X 33 is K or S;

[0175] X 35 is a naturally occurring amino acid other than methionine (preferably a naturally occurring amino acid isomeric to methionine, such as isoleucine) or a non-natural amino acid (preferably a naturally occurring amino acid isomeric to methionine, such as norleucine); or;

[0176] In another embodiment, the hBCMA-binding polypeptide is at position X9, X 10 , X 14 , X 18 , X 31 or X 35A polypeptide having no methionine residue at any position, such as an hBCMA-binding polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LX 35 , where, independently of each other,

[0177] X9 is D, E, H, K, N, Q, S or V;

[0178] X 10 is A, E, F, I, K, N, Q, R, S, T, Y, V, L, or norleucine;

[0179] X 11 is E, F or H;

[0180] X 14 is A, E, H, I, K, L, Q, R, T or Y;

[0181] X 17 is A, E, S, T or V;

[0182] X 18 is A, F, H, K, L, N, T, S, I, Q, or norleucine;

[0183] X 33 is K or S;

[0184] X 35 is a naturally occurring amino acid isosteric to methionine (eg, isoleucine) or norleucine; or;

[0185] In another embodiment, the hBCMA-binding polypeptide is a polypeptide that does not have a methionine residue at position X9, X10, X14, X18, X31, or X35, such as an hBCMA-binding polypeptide wherein Helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LX 35 , where, independently of each other,

[0186] X9 is D, E, H, K, N, Q, S or V;

[0187] X 10is A, E, F, I, K, N, Q, R, S, T, Y, or V;

[0188] X 11 is E, F or H;

[0189] X 14 is A, E, H, I, K, L, Q, R, T or Y;

[0190] X 17 is A, E, S, T or V;

[0191] X 18 is A, F, H, K, L, N, T or S;

[0192] X 33 is K or S;

[0193] X 35 It is isoleucine or norleucine.

[0194] Preferably, in the above embodiments, the hBCMA binding efficacy is at least 1%, at least 5% or at least 10% (e.g., at least 15%, 20%, 25% or 50%) of the binding efficacy of the peptide of SEQ ID NO: 2 (i.e., the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions).

[0195] Preferably, in the above embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2.

[0196] In advantageous embodiments of the hBCMA-binding polypeptides of the invention, proline (P) and cysteine ​​(C) are not present in helix 1 or helix 2 of the hBCMA-binding polypeptides of the invention. Advantageously, glycine (G) is also not present in helix 1 or helix 2 of the hBCMA-binding polypeptides of the invention.

[0197] In certain embodiments of the invention, such as those described above, the hBCMA-binding polypeptide is a polypeptide wherein:

[0198] Helix 1 contains the sequence X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 , and / or

[0199] Helix 2 contains sequence X 23 FX 25 QKWAFX 31 RX 33 LX 35 X36 X 37 ,

[0200] In such embodiments, X6 can be any naturally occurring amino acid or not present; X7 can be any naturally occurring amino acid or not present; X8 can be any naturally occurring amino acid or not present; X 19 Can be any naturally occurring amino acid or absent; X 23 Can be any naturally occurring amino acid or absent; X 36 can be any naturally occurring amino acid or be absent; and X 37 It may be any naturally occurring amino acid or it may be absent.

[0201] More preferably, X6 can be any naturally occurring amino acid; X7 can be any naturally occurring amino acid; X8 can be any naturally occurring amino acid; X 19 Can be any naturally occurring amino acid; X 23 Can be any naturally occurring amino acid; X 36 can be any naturally occurring amino acid; and X 37 It can be any naturally occurring amino acid.

[0202] In another preferred embodiment, X6 can be D, E, N or Q, or not present; X7 can be H, K or R, or not present; X8 can be D, E, N or Q, or not present; X 19 Can be G, A, V, L, or I, or absent; X 23 Can be D, E, N, or Q, or absent; X 36 can be D, E, N, or Q, or absent; and X 37 Can be D, E, N, or Q, or absent.

[0203] In another preferred embodiment, X6 can be D, E, N or Q; X7 can be H, K or R; X8 can be D, E, N or Q; X 19 Can be G, A, V, L or I; X 23 Can be D, E, N or Q; X 36 can be D, E, N, or Q; and X 37 Can be D, E, N or Q.

[0204] In another preferred embodiment, X6 may be N or absent; X7 may be K or absent; X8 may be E or absent; 19 Can be L or absent; X 23 Can be N or not present; X 36 can be D or absent; and X 37Can be D or absent.

[0205] In a particularly preferred embodiment, X6 is N; X7 is K; and X8 is E. In another particularly preferred embodiment, X 36 is D; and X 37 It is D.

[0206] In a very particularly preferred embodiment, X6 is N; X7 is K; X8 is E; X 19 Yes L; X 23 Yes N; X 36 is D; and X 37 is D. In such embodiments,

[0207] Helix 1 contains the sequence NKEX9X 10 X 11 ADX 14 EIX 17 X 18 L, and / or helix 2 contains the sequence NFX 25 QKWAFX 31 RX 33 LX 35 DD.

[0208] In advantageous embodiments of the hBCMA-binding polypeptides of the invention, proline (P) and cysteine ​​(C) are not present in helix 1 or helix 2. Advantageously, glycine (G) is also not present in helix 1 or helix 2.

[0209] Preferably, the hBCMA binding efficacy of such hBCMA binding polypeptides is at least 1% of that of SEQ ID NO.2.

[0210] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2 for hBCMA.

[0211] In another preferred embodiment, the hBCMA-binding polypeptide is a polypeptide wherein:

[0212] i) helix 1 comprises the sequence NKEETFADLEISNL and helix 2 comprises the sequence NFYQKWAFIRSLMDD,

[0213] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, at least 1 and not more than 2 (e.g. 1 or 2) residues are replaced by alternative residues,

[0214] or (iii) at least one and no more than three (eg, one, two or three) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (eg, by an alternative residue that is a conservative substitution).

[0215] Preferably, such hBCMA binding polypeptide is a polypeptide wherein the hBCMA binding efficacy is at least 1% of that of SEQ ID NO:1.

[0216] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:1.

[0217] In another preferred embodiment, the hBCMA engaging polypeptide is a polypeptide wherein:

[0218] i) helix 1 comprises the sequence NKENQFADEEIAAL and helix 2 comprises the sequence NFYQKWAFIRKLMDD,

[0219] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, at least 1 and not more than 2 (e.g. 1 or 2) residues are replaced by alternative residues,

[0220] or (iii) at least one and no more than three (eg, one, two or three) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (eg, by an alternative residue that is a conservative substitution).

[0221] Preferably, such hBCMA binding polypeptide is a polypeptide wherein the hBCMA binding efficacy is at least 1% of that of SEQ ID NO:2.

[0222] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide is a polypeptide that competes with SEQ ID NO:2.

[0223] In one embodiment, the hBCMA-binding polypeptide is a polypeptide wherein:

[0224] a) Helix 1 comprises the sequence ETFADLEISN, and helix 2 comprises the sequence FYQKWAFIRSLM. For example, helix 1 comprises the sequence NKEETFADLEISNL, and helix 2 comprises the sequence NFYQKWAFIRSLMDD.

[0225] In another embodiment, the hBCMA-binding polypeptide is a polypeptide wherein:

[0226] b) Helix 1 comprises the sequence NQFADEEIAA, and helix 2 comprises the sequence FYQKWAFIRKLM. For example, helix 1 comprises the sequence NKENQFADEEIAAL, and helix 2 comprises the sequence NFYQKWAFIRKLMDD.

[0227] As described above, many residues can be replaced by alternative residues. For example, at least 1 and no more than 3, at least 1 and no more than 2 or 1 residue can be replaced by alternative residues, such as 3, 2 or 1 residue can be replaced by alternative residues. Amino acid residue replacement can be conservative replacement or non-conservative replacement, or non-classical (also called non-natural) amino acid, as described above.

[0228] In advantageous embodiments of the hBCMA-binding polypeptides of the invention, proline (P) and cysteine ​​(C) are not present in helix 1 or helix 2. Advantageously, glycine (G) is also not present in helix 1 or helix 2.

[0229] In one embodiment, the hBCMA binding motif that is part [helix 1]-[isolated part]-[helix 2] is (ie has the sequence):

[0230] X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 FX 25 QKWAFX 31 RX 33 LX 35 X 36 X 37

[0231] in

[0232] X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X 22 is any naturally occurring amino acid, and wherein optionally, X 20 , X 21 or X 22 One or both (eg, optionally 1) of are absent; and the other residues are as defined above.

[0233] More preferably, X6 can be D, E, N or Q, or not present; X7 can be H, K or R, or not present; X8 can be D, E, N or Q, or not present; X9, X10 , X 11 , X 14 , X 17 and X 18 As defined above; X 19 Can be G, A, V, L, or I, or absent; X 20 Can be S, T, M, P, F, Y or W (e.g. P); X 21 Can be D, E, N or Q; X 22 Can be G, A, V, L, I or absent; X 23 Can be D, E, N, or Q, or absent; X 25 , X 31 , X 33 and X 35 As defined above. 36 can be D, E, N, or Q, or absent; and X 37 Can be D, E, N, or Q, or absent.

[0234] For example, X6 can be D, E, N, or Q; X7 can be H, K, or R; X8 can be D, E, N, or Q; X9, X 10 , X 11 , X 14 , X 17 and X 18 As defined above; X 19 Can be G, A, V, L or I; X 20 Can be S, T, M, P, F, Y or W (e.g. P); X 21 Can be D, E, N or Q; X 22 Can be G, A, V, L, I; X 23 Can be D, E, N or Q; X 25 , X 31 , X 33 and X 35 As defined above. 36 can be D, E, N, or Q; and X 37 Can be D, E, N or Q.

[0235] In particularly preferred embodiments, X6 is N; X7 is K; X8 is E; X9, X 10 , X 11 , X 14 , X 17 and X 18 As defined above; X 19 Yes L; X 20 is S, T, M, P, F, Y or W (e.g. P), X 21 is D, E, N or Q (e.g. N); X 22is G, A, V, L or I (e.g. L); X 23 Yes N; X 25 , X 31 , X 33 and X 35 As defined above. 36 is D; and X 37 It is D.

[0236] For example, X9 to X 35 The portion is selected from the group consisting of:

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] In the polypeptides of the above table, optionally, 1 to 5 residues (e.g., 1, 2, 3, 4 or 5), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues. Preferably, in embodiments in which optionally 1 to 5 residues (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3 residues) in the sequence are replaced by alternative residues, the replacement residues are conservative replacements. In a preferred embodiment, the sequence is one of SEQ ID 170-275. In a preferred embodiment, the hBCMA binding motif is selected from the group consisting of:

[0243]

[0244] In the polypeptides of the above table, optionally, 1 to 5 residues (e.g., 1, 2, 3, 4 or 5), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues. Preferably, in embodiments in which optionally 1 to 5 residues (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3 residues) in the sequence are replaced by alternative residues, the replacement residues are conservative replacements.

[0245] In a preferred embodiment, the hBCMA binding motif sequence additionally has residues NKE at position X6X7X8 (ie, X6 is N; X7 is K; X8 is E). 36 X 37 In addition, there is a residue DD (ie, X 36 is D; and X37 is D). For example, the motif sequence has NKE at position X6X7X8 and 36 X 37 DD (i.e. X6 is N; X7 is K; X8 is E; X 36 is D; and X 37 Yes D).

[0246] Therefore, in a preferred embodiment, the hBCMA binding motif sequence may be selected from:

[0247] NKEX9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 FX 25 QKWAFX 31 RX 33 LX 35 X 36 X 37 ;

[0248] X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 FX 25 QKWAFX 31 RX 33 LX 35 DD; and

[0249] NKEX9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 FX 25 QKWAFX 31 RX 33 LX 35 DD.

[0250] As described above, in embodiments of the present invention, many residues may each be substituted with alternative residues as described herein. For example, in the above embodiments, even when not explicitly mentioned, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5) of the sequences of helix 1 and / or helix 2 defined above, and preferably optionally 1 to 3 (e.g., 1, 2 or 3) residues are substituted with alternative residues (e.g., substituted residues as conservative substitutions). In any such alternative polypeptides of the present invention having replacement residues at appropriate positions, binding to hBCMA is maintained. For example, the hBCMA binding efficacy is at least 1% of the binding efficacy of the peptide of SEQ ID NO: 2 (i.e., when measured under the same conditions (as described above) SEQ ID NO: 2 peptide binding efficacy to hBCMA). Alternatively or additionally, for example, in any such alternative polypeptides of the present invention having replacement residues at appropriate positions, the hBCMA binding polypeptide is a polypeptide that competes with SEQ ID NO: 2 for hBCMA binding.

[0251] More preferably, the binding efficacy is at least 5%, and more preferably at least 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 2 (i.e., the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions).

[0252] In advantageous embodiments of the hBCMA-binding polypeptides of the invention, proline (P) and cysteine ​​(C) are not present in the hBCMA-binding motif.

[0253] Advantageously, glycine (G) is also absent from the hBCMA binding motif.

[0254] As described above, the hBCMA-binding polypeptides of the present invention have an overall structure [N-terminal portion]-[helix 1]-[separating portion]-[helix 2]-[C-terminal portion].

[0255] The separating part can be a sequence of 1 to 5 (preferably 1, 2, 3, 4 or 5) naturally occurring amino acids. Preferably, the separating part is a sequence of 2 to 5 (e.g. 2, 3, 4 or 5) naturally occurring amino acids. For example, the separating part is a sequence of 3 to 5 (e.g. 3, 4 or 5) naturally occurring amino acids. Preferably, the separating part is a sequence of 3 amino acids.

[0256] In certain embodiments, the separation portion has the sequence X 20 X 21 X 22 , where X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X22 is any naturally occurring amino acid; and wherein optionally, X 20 , X 21 or X 22 One or both of the two do not exist. For example, X 20 , X 21 or X 22 None of them does not exist, X 20 , X 21 or X 22 One of the does not exist, or X 20 , X 21 or X 22 More preferably, X 20 , X 21 or X 22 None of them does not exist, or X 20 , X 21 or X 22 Most preferably, X 20 , X 21 or X 22 None of them exists, that is, the separated part has the sequence X 20 X 21 X 22 , where X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X 22 Any naturally occurring amino acid.

[0257] Preferably, X 20 is S, T, M, P, F, Y, or W (e.g., P or T), X 21 is D, E, N, Q, and X 22 is G, A, V, L, I; wherein optionally, X 20 , X 21 or X 22 In one embodiment, X 20 is P, S, C, U, T or M, X 21 is D, E, N, or Q, and X 22 is G, A, V, L, I. More preferably, X 20 Is P or T; X 21 is N; and X 22 is L. For example, the isolated portion has the sequence PNL or TNL.

[0258] The N-terminal portion may be absent or may be a sequence of 1 to 15 naturally occurring amino acids. For example, the N-terminal portion may be absent or may be a sequence of 1 to 10, 1 to 8, 1 to 6, or 1 to 5 naturally occurring amino acids (e.g., 1, 2, 3, 4, or 5 naturally occurring amino acids).

[0259] In certain embodiments, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a is any naturally occurring amino acid (eg, M) or is absent; X b is any naturally occurring amino acid (e.g., M) or is absent; X1 is any naturally occurring amino acid or is absent; X2 is any naturally occurring amino acid or is absent; X3 is any naturally occurring amino acid or is absent; X4 is any naturally occurring amino acid or is absent; and X5 is any naturally occurring amino acid or is absent.

[0260] Preferably, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a M or not present; X b is M or absent; X1 is G, A, V, L, I or absent, X2 is D, E, N or Q or absent, X3 is D, E, N or Q or absent, X4 is H, K, R or absent, and X5 is F, Y, W or absent. More preferably, X a M or not present; X b is M or absent; X1 is V, G or absent (e.g., V or absent); X2 is D or absent; X3 is N or absent; X4 is K or absent; and X5 is F or absent. More preferably, X a M or not present; X b is M or absent; X1 is V or absent; X2 is D or absent; X3 is N or absent; X4 is K or absent; and X5 is F or absent.

[0261] In certain embodiments, preferably, X a , X b , none of X1, X2, X3, X4, and X5 does not exist; or X a and / or X b is absent and the other residues are not absent; or X a , X b and X1 is absent and the other residues are not absent; or X a , X b , X1 and X2 are absent and the other residues are not absent; or X a, X b , X1, X2 and X3 are absent and the other residues are not absent; X a , X b , X1, X2, X3 and X4 are absent and the other residues are not absent; or X a , X b , X1, X2, X3, X4 and X5 all do not exist.

[0262] In certain embodiments, preferably, none of X1, X2, X3, X4, and X5 is absent; X1 is absent and the other residues are not absent; X1 and X2 are absent and the other residues are not absent; X1, X2, and X3 are absent and the other residues are not absent; X1, X2, X3, and X4 are absent and the other residues are not absent; or X1, X2, X3, X4, and X5 are all absent. In such embodiments, optionally, X a and / or X b Does not exist.

[0263] In certain particularly preferred embodiments, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where: X a and X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a and X b absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a and X b does not exist, X1 does not exist, X2 is D, X3 is N, X4 is K, and X5 is F; or X a and X b does not exist, X1 does not exist, X2 does not exist, X3 is N, X4 is K, and X5 is F; or X a and X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 is K, and X5 is F; X a and X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F; or X a and X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist.

[0264] In certain embodiments, the N-terminal portion has the sequence XXX, wherein: X1 is any naturally occurring amino acid (preferably G, A, V, L, or I; preferably G, A, V, L, or I; more preferably V or G) or is absent; X2 is any naturally occurring amino acid (preferably D, E, N, or Q; more preferably D) or is absent; X3 is any naturally occurring amino acid (preferably D, E, N, or Q; more preferably N) or is absent; X4 is any naturally occurring amino acid (preferably H, K, or R; more preferably K) or is absent; X5 is any naturally occurring amino acid (preferably F, Y, or W; more preferably F) or is absent.

[0265] In certain embodiments, preferably, none of X1, X2, X3, X4 and X5 is absent; or X a and / or X b is absent and the other residues are not absent; or X a , X b and X1 is absent and the other residues are not absent; or X a , X b , X1 and X2 are absent and the other residues are not absent; or X a , X b , X1, X2 and X3 are absent and the other residues are not absent; X a , X b , X1, X2, X3 and X4 are absent and the other residues are not absent; or X a , X b , X1, X2, X3, X4 and X5 all do not exist.

[0266] For example, the N-terminal portion has the sequence X1X2X3X4X5, wherein: X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent. For example, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, that is, the N-terminal portion has the sequence VDNKF, GDNKF or does not exist; and more preferably has the sequence VDNKF or does not exist.

[0267] In a preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, ie the N-terminal portion has the sequence VDNKF or GDNKF; and more preferably the N-terminal portion has the sequence VDNKF.

[0268] In a preferred embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, ie, the N-terminal portion is absent.

[0269] The C-terminal portion may be absent or may be a sequence of 1 to 50 naturally occurring amino acids. For example, the C-terminal portion may be absent or may be a sequence of 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 22, or 1 to 21 naturally occurring amino acids. Preferably, the C-terminal portion may be absent or may be a sequence of 10 to 35, 10 to 30, 15 to 25, 18 to 22, 1 to 21 naturally occurring amino acids (e.g., 18, 19, 20, 21, or 22 naturally occurring amino acids). Preferably, the C-terminal portion has a sequence such that it enhances target binding through the hBCMA binding motifs of helix 1 and helix 2.

[0270] In certain embodiments, the C-terminal portion is absent or has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is a sequence of 1 to 14 naturally occurring amino acids, X 39 is any naturally occurring amino acid, X 56 is any naturally occurring amino acid or is absent, X 57 is any naturally occurring amino acid or is absent; and X 58 is any naturally occurring amino acid or is absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example as conservative substitutions.

[0271] In certain embodiments, X 38 is a sequence of 1 to 9, 1 to 7 or 1 to 5 naturally occurring amino acids. More preferably, X 38 is a sequence of 1 to 4, for example 1, 2, 3 or 4. In a particularly preferred embodiment, X 38 is any naturally occurring amino acid (ie any single (one) naturally occurring amino acid residue).

[0272] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W, X 56 G, A, V, L, I or not present, X 57 is P or not present, and X 58 is H, K, R or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. In another preferred embodiment, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQ X 56 X 57 X 58 , where X 56 G, A, V, L, I or not present, X 57 is P or not present, and X 58 is H, K, R or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example as conservative substitutions.

[0273] In a particularly preferred embodiment, the C-terminal portion has X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 It is S, X 56 A or not, X 57 is P or not present, and X 58 is K or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues as conservative substitutions. In another particularly preferred embodiment, the C-terminal portion has PSQSANLLAEAKKLNDAQX 56 X 57 X 58, where X 56 A or not, X 57 is P or not present, and X 58 is K or absent, and optionally wherein 1 to 5 (eg 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0274] For example, in certain embodiments, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is P, and X 39 is S; and:

[0275] X 56 It's A, X 57 is P, and X 58 is K; or X 56 A, or X 57 is P, and X 58 does not exist; or X 56 It's A, X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. Alternatively, for example, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 It's A, X 57 is P, and X 58 is K; or X 56 A, or X 57 is P, and X 58 does not exist; or X 56 It's A, X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0276] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S; and X 56 It's A, X 57 is P, and X 58 is K. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. Alternatively, for example, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 It's A, X 57 is P, and X 58 is K. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0277] In an alternative preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is P, and X 39 is S; and:

[0278] X 56 Does not exist, X 57 does not exist, and X 58In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. Alternatively, for example, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0279] In one embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a It is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F; or X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist (e.g., X a It is M, X bis M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b , X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent); and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W, X 56 G, A, V, L, I or not present, X 57 is P, and X 58 is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions; and more preferably X 38 Yes P, X 39 It is S, X 56 It's A, X 57 is P, and X 58 is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0280] In another embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5X a X b X1X2X3X4X5, where X a It is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 is D, X3 is N, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 is N, X4 is K, and X5 is F; X a Does not exist, Xb does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 is K, and X5 is F; X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F; or X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist (e.g., X a It is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X a Does not exist, X b not present, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X a Does not exist, X b absent, X1 absent, X2 absent, X3 absent, X4 absent, and X5 absent); and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 G, A, V, L, I or not present, X 57 is P, and X 58 is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative substitutions; and more preferably X 56 It's A, X 57 is P, and X 58 is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0281] In one embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a It is M, X b is M, X1 is G, A, V, L or I, X2 is D, E, N or Q, X3 is D, E, N or Q, X4 is H, K or R, and X5 is F, Y or W (e.g., X a It is M, X bis M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F); and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W (preferably S); and X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A); X 57 is P, and X 58 Does not exist; X 56 is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions.

[0282] In one embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a It is M, X b is M, X1 is G, A, V, L or I, X2 is D, E, N or Q, X3 is D, E, N or Q, X4 is H, K or R, and X5 is F, Y or W (e.g., X a It is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F); and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A); X 57 is P, and X58 Does not exist; X 56 is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0283] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X a Does not exist, X b X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent); and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W, X 56 G, A, V, L, I or not present, X 57 is P, and X 58 is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions; and more preferably X 38 Yes P, X 39 It is S, X 56 It's A, X 57 is P, and X 58is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0284] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X a Does not exist, X b X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent); and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 G, A, V, L, I or not present, X 57 is P, and X 58 is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions; and more preferably X 56 It's A, X 57 is P, and X 58 is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are substituted by alternative residues, eg, as conservative substitutions.

[0285] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is G, A, V, L or I, X2 is D, E, N or Q, X3 is D, E, N or Q, X4 is H, K or R, and X5 is F, Y or W (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F); and the C-terminal portion has the sequence X 38 X39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W (preferably S); and X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A); X 57 is P, and X 58 Does not exist; X 56 is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 More preferably, X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions.

[0286] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is G, A, V, L or I, X2 is D, E, N or Q, X3 is D, E, N or Q, X4 is H, K or R, and X5 is F, Y or W (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F); and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A); X 57 is P, and X 58 Does not exist; X 56is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 More preferably, X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0287] In a very preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; and X 56 Does not exist, X 57 does not exist, and X 58 Does not exist.

[0288] In an alternative highly preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K).

[0289] In an alternative highly preferred embodiment, X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist; X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K).

[0290] In certain embodiments, (i) the separation portion has a sequence X 20 X 21 X 22 ; and / or the N-terminal portion has the sequence X a X b X1X2X3X4X5; and / or the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X57 X 58 ;

[0291] Wherein, in the separation part, X 20 It is P; X 21 Yes N; X 22 It is L;

[0292] wherein in the N-terminal portion, X a M or not present; X b is M or absent; X1 is V or G (preferably V), or absent; X2 is D or absent; X3 is N or absent; X4 is K or absent; X5 is F or absent;

[0293] and wherein in the C-terminal portion, X 56 A or not; X 57 Is P or does not exist; X 58 It is K or not present;

[0294] or (ii) the separating portion, the N-terminal portion and the C-terminal portion are as defined in (i), wherein optionally

[0295] (a) within each portion, 1, 2 or 3 residues are replaced by alternative residues; or

[0296] (b) within those portions, at least 1 and no more than 10 (eg no more than 5, such as 1, 2, 3, 4 or 5) residues are collectively replaced by the alternative residues.

[0297] In such embodiments, preferably the isolated portion has the sequence PNL.

[0298] Alternatively or additionally, in such embodiments, the N-terminal portion has the sequence MMVDNKF or VDNKF.

[0299] In another embodiment, the N-terminal portion may comprise the sequence X1X2X3X4X5, wherein: X1 is G, V or absent; X2 is D or absent; X3 is N or absent; X4 is K or absent; X5 is F or absent. For example, the N-terminal portion may comprise the sequence VDNKF, the sequence DNKF, the sequence NKF, the sequence KF or the sequence F.

[0300] The isolated portion may comprise the sequence PNL.

[0301] The C-terminal portion may have the structure [second separation portion]-[helix 3]-[C-terminal sequence]. For example:

[0302] - the second separated part comprises the sequence PS;

[0303] - Helix 3 contains the sequence QSANLLAEAKKLNDAQ;

[0304] - and the C-terminal sequence comprises the sequence APK.

[0305] For example, the C-terminal portion may comprise the sequence PSQSANLLAEAKKLNDAQAPK.

[0306] In those five parts (N-terminal part, separation part, second separation part, helix 3 and C-terminal sequence), at least 1 and no more than 5 residues can be replaced together by alternative residues. For example, the number of replaced residues is at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. For example, there can be 1, 2, 3, 4 or 5 replacement residues in total in those parts.

[0307] In an advantageous embodiment of the hBCMA-binding polypeptide of the invention, wherein the C-terminal portion comprises a helical or substantially helical region (e.g., Helix 3 described immediately above), proline (P) and cysteine ​​(C) are not present in the helical or substantially helical region. In another embodiment, additionally, glycine (G) is also not present in the helical or substantially helical region.

[0308] Thus, in the entire hBCMA-binding polypeptide of the present invention (i.e., in the entire overall structure [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]), at least 1 and no more than 18 residues can be replaced by alternative residues. For example, the number of replaced residues is at least 1 and no more than 18, at least 1 and no more than 17, at least 1 and no more than 16, at least 1 and no more than 15, at least 1 and no more than 14, at least 1 and no more than 13, at least 1 and no more than 12, at least 1 and no more than 11, at least 1 and no more than 10, at least 1 and no more than 9, at least 1 and no more than 8, at least 1 and no more than 7, or at least 1 and no more than 6. In particular, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 substituted residues in total throughout the overall structure [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]. Amino acid residue substitutions may be conservative substitutions or non-conservative substitutions, or non-classical (also called non-natural) amino acids, as described above.

[0309] Preferably, in the entire hBCMA-binding polypeptide of the present invention (i.e., in the entire overall structure [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]), at least 1 and no more than 15 residues can be replaced by alternative residues. For example, the number of replaced residues is at least 1 and no more than 13, at least 1 and no more than 12, at least 1 and no more than 11, at least 1 and no more than 10, at least 1 and no more than 9, at least 1 and no more than 8, at least 1 and no more than 7, or at least 1 and no more than 6.

[0310] In particular, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substituted residues in total throughout the overall structure [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]. Amino acid residue substitutions may be conservative substitutions or non-conservative substitutions, or non-classical (also called non-natural) amino acids, as described above.

[0311] In one aspect of the invention, the hBCMA binding polypeptide comprises a sequence selected from SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128, as shown in Table 1. In such sequences, optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservatively replaced. In one embodiment, the hBCMA binding polypeptide comprises a sequence selected from SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128. In one embodiment, the hBCMA binding polypeptide has a sequence selected from the group consisting of SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128.

[0312] In one aspect of the invention, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NO. 2 and 23-39, as shown in Table 1. In such sequences, optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservatively replaced. In one embodiment, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NO. 2 and 23-39. In one embodiment, the hBCMA-binding polypeptide has a sequence selected from the group consisting of SEQ ID NO. 2 and 23-39.

[0313] In one aspect of the invention, the hBCMA-binding polypeptide comprises the sequence:

[0314] a) VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK[SEQ IDNO:1].

[0315] In another aspect of the invention, the hBCMA-binding polypeptide comprises the sequence:

[0316] b) VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 2].

[0317] In another aspect of the invention, the hBCMA-binding polypeptide comprises the sequence

[0318] c) VDNKFNKEEIFADREIAFLPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 23].

[0319] In another aspect of the invention, the hBCMA-binding polypeptide comprises the sequence

[0320] c) VDNKFNKEHQFADYEIAMLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 33].

[0321] As described above, many residues in the polypeptide of the present invention can be replaced by alternative residues. For example, in the polypeptide listed in a), b), c) or d) above, at least 1 and no more than 5 residues can be replaced by alternative residues. For example, the number of replaced residues is at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. In particular, there can be 1, 2, 3, 4 or 5 replacement residues in total in those parts. Since there are 58 residues in the polypeptide of this aspect of the present invention, the peptide in which 5 residues are replaced has a sequence identity of 91% (91.4%) with the sequence. For a 4 residue replacement, there is 93% sequence identity (93.1%), for a 3 residue replacement, there is 95% sequence identity (94.8%), for a 2 residue replacement, there is 97% sequence identity (96.6%), and for a 1 residue replacement, there is 98% sequence identity (98.3%).

[0322] As disclosed above, in one embodiment, helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other, X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V. In this embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptides of the invention having alternative residues in appropriate positions, binding to hBCMA is maintained. For example, the hBCMA binding efficacy is at least 0.1% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions.

[0323] At 0.1% binding efficacy, it is understood that the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 1000 times the concentration.

[0324] More preferably, the binding efficacy is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions. That is, the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50%. 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 500 times, 100 times, 50 times, 25 times, 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0325] As disclosed above, in another embodiment, helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18[SEQID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LM, wherein, independently of one another, X9 is D, E, H, K, N, Q, S or V; X10 is A, E, F, I, K, M, N, Q, R, S, T, Y or V; Xu is E, F or H; X 14 is A, E, H, I, K, L, Q, R, T, or Y; X 17 is A, E, S, T, or V; X 18 is A, F, H, K, L, M, N, T, or S; X 33 is K or S. In this embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptides of the invention having the replacement residues in the appropriate positions, binding to hBCMA is maintained. For example, the hBCMA binding efficacy is at least 0.1% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions.

[0326] At 0.1% binding efficacy, it is understood that the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 1000 times the concentration.

[0327] More preferably, the binding efficacy is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions. That is, the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50%. 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 500 times, 100 times, 50 times, 25 times, 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0328] As disclosed above, in another embodiment, Helix 1 comprises the sequence ETFADLEISN and Helix 2 comprises the sequence FYQKWAFIRSLM. In this embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptides of the invention with the replacement residues in the appropriate positions, binding to hBCMA is maintained.

[0329] For example, the hBCMA binding efficacy is at least 0.1% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions.

[0330] At 0.1% binding efficacy, it is understood that the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 1000 times the concentration.

[0331] More preferably, the binding efficacy is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions. That is, the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50%. 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 500 times, 100 times, 50 times, 25 times, 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0332] As also disclosed above, in another embodiment, Helix 1 comprises the sequence NQFADEEIAA and Helix 2 comprises the sequence FYQKWAFIRKLM. In this embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptide of the invention having the replacement residues in the appropriate positions, binding to hBCMA is maintained. For example, the hBCMA binding efficacy is at least 0.1% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions.

[0333] At 0.1% binding efficacy, it is understood that the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 1000 times the concentration.

[0334] More preferably, the binding efficacy is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions. That is, the EC of the surrogate polypeptide for binding to hBCMA when measured under the same conditions is at least 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, 25% or 50%. 50 The concentration of peptide SEQ ID NO: 2 does not exceed the EC of hBCMA 50 500 times, 100 times, 50 times, 25 times, 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0335] The present invention further provides an hBCMA-binding polypeptide, wherein the hBCMA-binding polypeptide consists of a motif that binds to hBCMA, wherein the polypeptide consists of the following structure:

[0336] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0337] The hBCMA binding motif is the portion [helix 1]-[separating portion]-[helix 2] (and optionally, the peptide further comprises one or more additional functional portions as described below (e.g. 1, 2, 3, 4, 5, 6 or more); e.g. one, two or three additional functional portions).

[0338] The present invention also provides an hBCMA-binding polypeptide, which consists of the CD16a-binding polypeptide of the present invention; and optionally comprises an additional binding portion. The present invention also provides an hBCMA-binding polypeptide of the present invention, wherein the hBCMA-binding polypeptide consists of the hBCMA-binding polypeptide (and optionally further comprises one or more additional functional portions as described below (e.g., 1, 2, 3, 4, 5, 6 or more); e.g., one, two or three additional functional portions).

[0339] In one embodiment, a hBCMA-binding polypeptide (e.g., an hBCMA-binding polypeptide of the invention described above or below) consists of a motif that binds to hBCMA, wherein the polypeptide consists of the following structure:

[0340] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0341] The hBCMA binding motif is the portion [helix 1]-[separating portion]-[helix 2] (and optionally further comprises one or more additional functional portions as described below (e.g. 1, 2, 3, 4, 5, 6 or more); for example one, two or three additional functional portions).

[0342] In one embodiment, the hBCMA-binding polypeptide consists of an hBCMA-binding polypeptide of the invention (and optionally further comprises one or more additional functional moieties as described below (e.g., 1, 2, 3, 4, 5, 6 or more); such as one, two or three additional functional moieties).

[0343] For the avoidance of doubt, in embodiments wherein the hBCMA-binding motif and / or hBCMA-binding polypeptide consists of the hBCMA-binding motif and / or hBCMA-binding polypeptide of the present invention, the hBCMA-binding polypeptide is not linked to another hBCMA-binding polypeptide, i.e., the hBCMA-binding polypeptide is not part of an hBCMA-binding oligomer of the present invention.

[0344] In another embodiment, the hBCMA-binding polypeptide (e.g., the hBCMA-binding polypeptide of the invention described above or below) consists of a motif that binds to hBCMA, wherein the polypeptide consists of the following structure:

[0345] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0346] The hBCMA binding motif is the portion [helix 1]-[separating portion]-[helix 2], and wherein the polypeptide further comprises one or more additional functional portions as described below (e.g., 1, 2, 3, 4, 5, 6 or more; such as one, two or three additional functional portions). In another very preferred embodiment, the hBCMA binding polypeptide consists of the hBCMA binding polypeptide of the invention, and further comprises one or more additional functional portions as described below (e.g., 1, 2, 3, 4, 5, 6 or more); such as 1, 2 or 3 additional functional portions (preferably 1 or 2 additional functional portions).

[0347] Multimeric hBCMA-binding peptides: hBCMA-binding oligomers

[0348] The present invention also provides hBCMA-binding oligomers comprising at least two (i.e., two or more, such as 2, 3, 4, 5, 6 or more; preferably 2, 3 or 4) hBCMA-binding polypeptides of the present invention. In a preferred embodiment, the hBCMA-binding oligomer of the present invention comprises two hBCMA-binding polypeptides of the present invention. In another embodiment, the hBCMA-binding oligomer of the present invention comprises at least three, at least four, at least 5 or at least 6 hBCMA-binding polypeptides of the present invention, such as 3, 4, 5 or 6 or more hBCMA-binding polypeptides of the present invention.

[0349] This aspect of the invention may also be defined such that the hBCMA-binding polypeptide constitutes two or more hBCMA-binding moieties or hBCMA-binding peptides, such as two, three, four, five, six or more hBCMA-binding moieties or hBCMA-binding peptides, such as two, three or four hBCMA-binding moieties or hBCMA-binding peptides, such as two ... oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers, such as hBCMA-binding oligomers

[0350] The hBCMA-binding oligomer of the present invention comprises at least a first hBCMA-binding polypeptide as the hBCMA-binding polypeptide of the present invention and a second hBCMA-binding polypeptide as the hBCMA-binding polypeptide of the present invention. The first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide may have the same sequence. Alternatively, the first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide may have different sequences. The hBCMA-binding oligomer of the present invention may optionally further comprise a third hBCMA-binding polypeptide as the hBCMA-binding polypeptide of the present invention. The third hBCMA-binding polypeptide may have the same sequence as the first hBCMA-binding polypeptide sequence and / or the second hBCMA-binding polypeptide sequence. Alternatively, the third hBCMA-binding polypeptide may have a different sequence from the first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide. The hBCMA-binding oligomer of the present invention may optionally further comprise a fourth hBCMA-binding polypeptide as the hBCMA-binding polypeptide of the present invention. The fourth hBCMA-binding polypeptide may have the same sequence as the first hBCMA-binding polypeptide sequence and / or the second hBCMA-binding polypeptide sequence and / or the third hBCMA-binding polypeptide sequence. Alternatively the fourth hBCMA-binding polypeptide may have a different sequence than the first hBCMA-binding polypeptide, the second hBCMA-binding polypeptide, and the third hBCMA-binding polypeptide.

[0351] In a preferred embodiment, the hBCMA-binding oligomer of the present invention comprises a first hBCMA-binding polypeptide comprising a first binding motif selected from the group consisting of SEQ ID NOs. 170 to 275 (preferably selected from the group consisting of SEQ ID NOs. 170-188) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservatively replaced); and a second hBCMA-binding polypeptide comprising a second binding motif selected from the group consisting of SEQ ID NOs. 170 to 275 (preferably selected from the group consisting of SEQ ID NOs. 170-188) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservatively replaced). The first hBCMA-binding motif and the second hBCMA-binding motif may have the same sequence or different sequences.

[0352] In a preferred embodiment, the hBCMA-binding oligomer of the present invention comprises a first hBCMA-binding polypeptide having a sequence selected from the group consisting of SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128 (preferably SEQ ID NO. 2 and 23-39) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by a substituted residue, and preferably a conservatively replaced residue); and a second hBCMA-binding polypeptide having a sequence selected from the group consisting of SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128 (preferably SEQ ID NO. 2 and 23-39) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by a substituted residue, and preferably a conservatively replaced residue). The first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide may have the same sequence or different sequences.

[0353] The hBCMA-binding polypeptides in the hBCMA-binding oligomers of the present invention may be separated by a linker. For example, each hBCMA-binding polypeptide in the hBCMA-binding oligomers of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein, such as a flexible amino acid linker, a rigid amino acid linker, or a cleavable amino acid linker or a non-amino acid linker. In the case where the hBCMA-binding oligomers of the present invention contain more than one linker, the linkers may be the same, or may be different. Preferably, the linker used in the hBCMA-binding oligomer of the present invention comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0354] In a preferred embodiment, the linker used in the hBCMA-binding oligomer of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSG GGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0355] In a preferred embodiment, the linker used in the hBCMA-binding oligomer of the present invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker of the hBCMA-binding oligomer of the present invention comprises or has the sequence GGGGS; for example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS.

[0356] Alternatively, the hBCMA-binding polypeptides in the hBCMA-binding oligomers of the invention may not be separated by a linker (ie, they may be directly attached to each other).

[0357] In a preferred embodiment, the hBCMA binding oligomer does not comprise a linker.

[0358] In one embodiment, the hBCMA-binding oligomer of the present invention comprises at least two (e.g., two) hBCMA-binding polypeptides, and the hBCMA-binding oligomer comprises the following structure:

[0359] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0360] wherein each N-terminal portion in the oligomer can have the same sequence or a different sequence; each C-terminal portion in the oligomer can have the same sequence or a different sequence; each isolated portion in the oligomer can have the same sequence or a different sequence; each helix 1 portion in the oligomer can have the same sequence or a different sequence; and each helix 2 portion in the oligomer can have the same sequence or a different sequence.

[0361] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS GGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0362] Alternatively, the linker may be absent, ie, the hBCMA binding oligomer comprises the following structure:

[0363] [N-terminal part]-[helix 1]-[detached part]-[helix 2]-[C-terminal part]-[N-terminal part]-[helix 1]-[detached part]-[helix 2]-[C-terminal part].

[0364] In one embodiment, the hBCMA-binding oligomer of the present invention comprises at least 3 (e.g., 3) hBCMA-binding polypeptides, and the hBCMA-binding oligomer comprises the following structure:

[0365] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0366] and wherein each linker portion in the oligomer can have the same sequence or have a different sequence; each N-terminal portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each C-terminal portion can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each separation portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each helix 1 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; and each helix 2 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence.

[0367] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S, and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. Further, for example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS, GGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGSGG, GGS GGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG or SGGGGSGGGGSGGGGSGGGG.

[0368] Alternatively, one or both linkers may be absent, i.e. the hBCMA binding oligomer comprises the following structure:

[0369] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0370] [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]-[linker]-[N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]; or

[0371] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[helix 1]-[isolated part]-[helix 2]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part].

[0372] In some aspects of the invention, the hBCMA-binding oligomer constitutes two or more hBCMA-binding polypeptides, optionally linked by one or more linkers, e.g., two, three, four, five, six or more hBCMA-binding polypeptides, optionally linked by one or more linkers, e.g., two, three or four hBCMA-binding polypeptides, optionally linked by one or more linkers, e.g., two hBCMA-binding polypeptides, optionally linked by one or more linkers.

[0373] For example, such an oligomer may comprise two hBCMA-binding polypeptides, each of which has a sequence in which Helix 1 comprises the sequence NKEETFADLEISNL and Helix 2 comprises the sequence NFYQKWAFIRSLMDD. As described elsewhere, such portions may have multiple residues substituted with alternative residues. For example, each of the two polypeptides may have the sequence:

[0374] VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLN DAQAPK[SEQ IDNO:1].

[0375] In an alternative example, such an oligomer may comprise two hBCMA-binding polypeptides, each of which has a sequence in which Helix 1 comprises the sequence NKENQFADEEIAAL and Helix 2 comprises the sequence NFYQKWAFIRKLMDD. As described elsewhere, such portions may have multiple residues substituted with alternative residues. For example, each of the two polypeptides may have the sequence:

[0376] VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK[SEQ ID NO:2].

[0377] In an alternative example, such an oligomer may comprise two hBCMA-binding polypeptides, one of which has a sequence in which Helix 1 comprises the sequence NKEETFADLEISNL and Helix 2 comprises the sequence NFYQKWAFIRSLMDD, and the other of which has a sequence in which Helix 1 comprises the sequence NKENQFADEEIAAL and Helix 2 comprises the sequence NFYQKWAFIRKLMDD. As described elsewhere, such portions may have multiple residues substituted with alternative residues. For example, one of the two polypeptides may have the sequence:

[0378] VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLN DAQAPK[SEQ IDNO:1], and

[0379] Another can have a sequence:

[0380] VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK[SEQ ID NO:2].

[0381] Connectors

[0382] When present, a linker connects two or more functional moieties (defined further below) of an hBCMA-binding polypeptide of the invention (e.g., in an hBCMA-binding oligomer of the invention), or a linker can connect an hBCMA-binding polypeptide of the invention with an additional functional moiety of the invention, or a linker can connect two or more functional moieties of an hBCMA-binding polypeptide-drug conjugate of the invention. In embodiments where more than one additional functional moiety is present, a linker can also connect an additional functional moiety of the invention with an additional functional moiety of the invention. One skilled in the art will appreciate that different types of linkers with different properties are known in the art, and will be able to select a suitable linker based on the desired properties. Types of linkers include, for example, flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers; non-amino acid linkers may also be used. For example, a linker may be selected to increase stability or improve folding, to increase expression, to improve biological activity, to enable targeting, or to alter pharmacokinetics. Non-amino acid linkers may be referred to as synthetic linkers.

[0383] For the avoidance of doubt, in certain embodiments, a linker may not connect two hBCMA-binding polypeptides of the invention together (e.g., in an hBCMA-binding oligomer of the invention), and / or a linker may not connect an hBCMA-binding polypeptide of the invention and an additional functional moiety of the invention together; and / or in embodiments where there is more than one additional functional moiety, a linker may not connect an additional functional moiety of the invention and an additional functional moiety of the invention together. For example, in certain embodiments, two hBCMA-binding polypeptides of the invention (e.g., in an hBCMA-binding oligomer of the invention) may be directly linked; and / or an hBCMA-binding polypeptide or oligomer of the invention may be directly linked to an additional functional moiety of the invention; and / or an additional functional moiety of the invention may be directly linked to an additional functional moiety of the invention.

[0384] In one embodiment, the hBCMA-binding polypeptide, hBCMA-binding oligomer, or hBCMA-binding agent-drug conjugate according to any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, and a synthetic linker. In one embodiment, the linker is between two or more hBCMA-binding polypeptides, such as in an hBCMA-binding oligomer of the invention, or between a hBCMA-binding polypeptide or hBCMA-binding oligomer portion, such as an immune signaling molecule or an additional binding portion (e.g., as described in further detail below). In another embodiment, the linker is between an hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention and a therapeutic agent (e.g., as described in further detail below).

[0385] In one embodiment, the hBCMA-binding oligomer according to any aspect disclosed herein comprises at least one linker, such as at least one linker selected from a flexible amino acid linker, a rigid amino acid linker, and a cleavable amino acid linker. The linker is between two or more hBCMA-binding polypeptides. An additional linker between a hBCMA-binding polypeptide or hBCMA-binding oligomer and an additional functional portion (e.g., an immune signaling molecule or an additional binding portion (e.g., as described in further detail below)) may also be present in the hBCMA-binding oligomer according to any aspect disclosed herein. An additional functional portion (e.g., an immune signaling molecule or an additional binding portion (e.g., as described in further detail below)) and an additional functional portion (e.g., an immune signaling molecule or an additional binding portion (e.g., as described in further detail below)) may also be present in the hBCMA-binding oligomer according to any aspect disclosed herein.

[0386] In embodiments and aspects of the invention comprising more than one linker, each linker can be the same, or can be different, or some linkers can be the same and some can be different (e.g., in embodiments having 3 or more linkers (e.g., 3, 4, 5, 6, 7, 8 or more linkers)).

[0387] When the domain of connection needs some distance and conformational freedom, flexible amino acid linkers can be used, and can be advantageous in some embodiments of the present invention. Such linkers are usually composed of small non-polar (e.g., G) or polar (e.g., S or T) amino acids. Some flexible linkers are mainly composed of segments of G and S residues, such as (GGGGS)p or (GGGSG)p. Other examples include (GGSGG)p, (GSGGG)p or (SGGGG)p.

[0388] Adjusting the copy number "p" allows optimization of the linker to achieve appropriate separation between functional parts, or to maintain necessary inter-part interactions. In one embodiment, the linker is (GGGSG)p, such as (GGGSG)1, (GGGSG)2, or (GGGSG)3, such as (GGGSG)3. In one embodiment, the linker is (GGGGS)p, such as (GGGSG)1, (GGGGS)2, or (GGGGS)3, such as (GGGGS)3. In one embodiment, the linker is (GGSGG)p, such as (GGGSG)1, (GGSGG)2, or (GGSGG)3, such as (GGSGG)3. In one embodiment, the linker is (GSGGG)p, such as (GGGSG)1, (GGSGG)2, or (GGSGG)3, such as (GGSGG)3. In one embodiment, the linker is (GSGGG)p, such as (GGGSG)1, (GSGGG)2, or (GSGGG)3, such as (GSGGG)3. In one embodiment, the linker is (SGGGG)p, such as (GGGSG)1, (SGGGG)2, or (SGGGG)3, such as (SGGGG)3. In another embodiment, the linker is G.

[0389] In one embodiment, the linker of the invention comprises or has 1 to 50 (e.g., 1 to 40, 1 to 30 or 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T. In one embodiment, the linker of the invention comprises or has 1 to 50 (e.g., 1 to 25, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; or 1 to 20, such as 1, 5, 10, 15, or 20; or 1 to 15, such as 1, 5, 10, or 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G and S. For example, the linker of the invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG.

[0390] Furthermore, for example, the linker of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGG GGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0391] In a preferred embodiment, the linker of the invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker used in the hBCMA-binding oligomer of the invention comprises or has the sequence GGGGS; for example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS.

[0392] In addition to G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues (such as T and A) to maintain flexibility and containing polar amino acid residues to improve solubility. In general, it is known in the art that linker sequence and length can affect the characteristics of the connected parts, so the skilled person will be able to select a suitable linker for use in binding polypeptides as described herein.

[0393] Other types of linkers (such as rigid and / or cleavable linkers) can also be used to connect domains in multi-domain constructs to improve or control their biological activity. Such linkers are known in the art (Chen X et al., Fusion protein linkers: Property, design and functionality, Adv Drug Deliv Rev 2013: 65: 10: 1357, doi: 10.1016 / j.addr.2012.09.039 ).

[0394] Alternatively, different portions of a binding polypeptide or oligomer as described herein (e.g., two or more hBCMA binding polypeptides, an hBCMA binding polypeptide and an additional functional moiety, such as an immune signaling molecule or an additional binding moiety, or an hBCMA binding polypeptide or hBCMA binding oligomer and a therapeutic agent) can be covalently linked by a chemical linker. Such chemical linkers can be generated, for example, by maleimide or 'click' chemistry. Such chemical linkers can be cleavable, such as a MC-Val-Ala-PAB linker, or non-cleavable, such as a MPB linker. One skilled in the art will be aware of other linkers suitable for use with binding polypeptides as described herein.

[0395] With respect to the above description of binding polypeptides comprising hBCMA-binding polypeptides according to the present disclosure, it should be noted that the designation of the first part, the second part and the further part or functional part is for reasons of clarity in order to distinguish, on the one hand, the hBCMA-binding polypeptides according to the present invention and, on the other hand, the binding parts or functional parts that exhibit other functions. These designations are not intended to refer to the actual order of the different regions of the binding polypeptide. Similarly, the designation of the first part and the second part or functional part (or monomeric unit) is for reasons of clarity in order to distinguish between the units. Thus, for example, the first part or functional part (or monomeric unit) may be present at the N-terminus, the middle or the C-terminus of the binding polypeptide without limitation.

[0396] The hBCMA-binding polypeptides in the hBCMA-binding oligomers of the present invention may be separated by a linker. For example, each hBCMA-binding polypeptide in the hBCMA-binding oligomers of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein. In the case where the hBCMA-binding oligomers of the present invention comprise more than one linker, the linkers may be the same, or may be different.

[0397] Preferably, the linker used in the hBCMA-binding oligomer of the present invention comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0398] In a preferred embodiment, the linker used in the hBCMA-binding oligomer of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker used in the hBCMA-binding oligomer of the present invention comprises or has the sequence GGGGS; for example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0399] In embodiments of the invention comprising an additional functional moiety, the hBCMA-binding polypeptide of the invention and the additional functional moiety in the hBCMA-binding polypeptide or hBCMA-binding oligomer can be separated by a linker. For example, each additional functional moiety in the hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention and the hBCMA-binding polypeptide can be separated by a linker. Preferably, the linker is a linker as defined herein. In embodiments of the invention comprising two or more additional functional moieties, the additional functional moieties can be separated by a linker. Preferably, the linker is a linker as defined herein.

[0400] In cases where an hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention comprises more than one linker (i.e., where there are at least two hBCMA-binding polypeptides and at least one additional functional moiety; or where there are at least one hBCMA-binding polypeptide and at least two additional functional moieties), the linkers may be the same, or may be different. Preferably, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional functional moiety comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0401] In a preferred embodiment, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional functional moiety is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the hBCMA-binding oligomer of the present invention comprises or has the sequence GGGGS; for example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0402] Additional features section

[0403] The hBCMA-binding polypeptides as disclosed herein may be attached to one or more additional functional moieties, such as binding partners that recognize immune cell surface molecules. The hBCMA-binding oligomers as disclosed herein may be attached to one or more functional moieties, such as binding partners that recognize immune cell surface molecules. Thus, in one embodiment, at least one hBCMA-binding polypeptide (or hBCMA-binding oligomer) is attached to one or more additional functional moieties, optionally via a linker as described above.

[0404] As used herein, 'functional portion' refers to a component or 'part' that possesses a specific desired biological activity.

[0405] The one or more additional components (ie one or more additional functional parts) may for example be a signalling molecule or a derivative thereof. Examples of suitable signalling molecules include immune signalling molecules or derivatives thereof, such as cytokines or derivatives thereof, such as IL-15 and derivatives thereof.

[0406] The one or more additional components (i.e., one or more additional functional parts) can be one or more additional binding parts, such as one or more binding partners that recognize cell surface proteins or antigens, such as immune cell surface proteins or cell surface tumor antigens (also known as cancer cell surface antigens or cancer cell surface targets). Cell surface tumor antigens can be, for example, tumor-associated antigens or tumor-specific antigens.

[0407] Such additional binding moieties may, for example, be specific for immune cell surface proteins, such as NK cell activation receptors, such as CD16a. In particular, such CD16a binding moieties may, for example, be immune cell binding polypeptides as disclosed in UK Patent Application No. 2208027.9 (which may be obtained at the time of publication or from the public documents of other patent applications publicly available, such as international patent applications claiming priority). Such 'dual conjugate' binding polypeptides may, for example, comprise at least one hBCMA binding polypeptide as disclosed herein, such as one, two, three or more hBCMA binding polypeptides as disclosed herein, or at least one hBCMA binding oligomer as disclosed herein. At least one (e.g., one, two, three or more) hBCMA binding polypeptides as disclosed herein or at least one hBCMA binding oligomer as disclosed herein may, for example, be attached to at least one CD16a binding polypeptide as described above, such as one, two, three or more CD16a binding polypeptides as described above. In such 'dual binder' binding polypeptides, an additional binding moiety (e.g., a binding moiety with specificity for an immune cell surface molecule such as CD16a, e.g., at least one CD16a binding polypeptide as described above) may be attached at the N-terminus or C-terminus of a hBCMA binding moiety (e.g., at least one hBCMA binding polypeptide as disclosed herein or at least one hBCMA binding oligomer as disclosed herein), optionally via one or more linkers as described above. An additional binding moiety (e.g., a binding moiety with specificity for an immune cell surface molecule such as CD16a, e.g., at least one CD16a binding polypeptide as described above) may alternatively be attached between two hBCMA binding moieties, optionally separated by one or more linkers as described above. Non-limiting examples of such 'dual conjugate' binding polypeptides comprising one hBCMA binding polypeptide and one or more CD16a binding polypeptides are given in Table 1; the sequences of these dual conjugates comprising a C-terminal His6 tag are given as [SEQ ID NO. 130-133], and the sequences without a C-terminal His6 tag are given as [SEQ ID NO. 139-142]. Additional non-limiting examples of 'dual conjugate' polypeptides comprising two hBCMA binding polypeptides and a portion targeting CD16a are given in Table 1; the sequences of these dual conjugates comprising a C-terminal His6 tag are given as [SEQ ID NO. 134-138], and the sequences without a C-terminal His6 tag are given as [SEQ ID NO. 143-147].Also provided are non-limiting examples of such 'dual conjugate' binding polypeptides comprising one hBCMA binding polypeptide and one or more CD16a binding polypeptides; the sequences of these dual conjugates comprising a C-terminal His6 tag are given as SEQ ID NO. 154-163, and the sequence without a C-terminal His6 tag is given as SEQ ID NO. 166. Also provided are further non-limiting examples of 'dual conjugate' polypeptides comprising two hBCMA binding polypeptides and a portion targeting CD16a; the sequences of such dual conjugates comprising a C-terminal His6 tag include SEQ ID NO. 149, 150, 151, 152, 153, and the sequence without a C-terminal His6 tag includes SEQ ID NO. 167.

[0408] In certain embodiments, hBCMA-binding polypeptides of the invention (e.g., hBCMA-binding polypeptides incorporated into 'dual conjugate' polypeptides, e.g., dual conjugate polypeptides further comprising at least one moiety that targets a NK cell surface target (e.g., CD16a)) are particularly active in functional assays (e.g., CD16 reporter assays and / or cell killing assays). Exemplary sequences of such dual conjugate polypeptides include SEQ ID NOs. 149, 150, 151, 158, 160, and 161 (also disclosed in the Examples below).

[0409] Those skilled in the art will appreciate that such non-limiting examples of dual conjugates (as well as other dual conjugates comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer disclosed herein attached to a CD16a binding moiety, and other molecules comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer attached to a different functional moiety (e.g., additional binding moieties as described below)) may optionally comprise one or more additional N-terminal, C-terminal or other modifications, such as a C-terminal His6 tag, depending on the intended use of the conjugate or the requirements for expression, purification, etc. Non-limiting examples of such N-terminal, C-terminal or other modifications may include a peptide purification tag or moiety (e.g., a histidine tag (e.g., a polyhistidine tag) or a methionine tag (e.g., a single methionine tag or a polymethionine tag)), a signaling tag or moiety (e.g., a glycine residue or a signal peptide, e.g., selected from OmpA, DsbA, PhoA, and PelB), a fluorescent tag (e.g., Alexa448), or a tag or moiety that aids conjugation (a cysteine ​​tag (e.g., a single cysteine ​​at the C-terminus or N-terminus)). Such tags and / or moieties may preferably be present at the N-terminus and / or C-terminus of a dual conjugate comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer as described herein.

[0410] Further examples of additional components (i.e., additional functional moieties) include additional binding moieties that are binding partners that recognize: B cell maturation antigen (BCMA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), a disintegrin and metalloproteinase 17 (ADAMI 7), programmed death ligand 1 (PD-L1), SLAM family member 7 (SLAMF7), epithelial cell adhesion molecule (EPCAM), epidermal growth factor receptor (EGFR / ErbB-1), epidermal growth factor receptor variant 3 (EGFRvIII), erb-b2 tyrosine kinase 2 (ERBB2 / HER2 / CD340), prostate specific membrane antigen (PSMA), Claudin8.2 (CLDN18.2), delta-like protein 3 (DLL3), mucin 16 (MUC16), mucin 17 (MUC17), mucin 1 (MUC1), trophoblast glycoprotein (TPBG / 5T4 / WAIF1), V-set domain-containing T cell activation inhibitor 1 (B7-H4 / VTCN1 / B7x / B7S1), cluster of differentiation 20 (CD20), B lymphocyte surface antigen B4 (CD 19), sialic acid-binding Ig-like lectin 2 (CD22), TNF receptor superfamily member 8 (CD30), natural cytotoxicity triggering receptor 1 (NKp46) and NKG2D.

[0411] Examples of additional components (i.e., additional functional portions) include additional binding portions that are binding partners that recognize cell surface tumor antigens or cancer cell surface targets selected from the group consisting of: BCMA, ADAM 17, SLAMF7, PD-L1, EPCAM, EGFR / ErbB-1, EGFRvIII, ERBB2 / HER2 / CD340, PSMA, CLDN18.2, DLL3, MUC16, MUC17, MUC1, TPBG / 5T4 / WAIF1 and B7-H4 / VTCN1 / B7x / B7S1.

[0412] Examples of additional components (i.e., additional functional moieties) include additional binding moieties that are binding partners that recognize immune cell surface proteins or immune cell surface targets selected from the group consisting of: CTLA-4, PD-1, CD20, CD19, CD22, and CD30. Examples of additional components (i.e., additional functional moieties) include additional binding moieties that are binding partners that recognize cell surface tumor antigens or cancer cell surface targets expressed in hematological malignancies (e.g., BCMA, CD20, CD19, CD22, or CD30).

[0413] Alternatively or additionally, such additional binding moieties may be specific for a different target on multiple myeloma cells that is not hBCMA, for example. For example, the additional binding moiety may bind to a different target on the same multiple myeloma cell.

[0414] For the avoidance of doubt, examples of additional binding moieties (e.g., binding partners that recognize cell surface proteins or antigens) are non-limiting and are described in detail herein by way of illustration. The additional binding moieties referred to herein are not hBCMA-binding polypeptides of the present invention.

[0415] In one embodiment, the hBCMA-binding polypeptides or hBCMA-binding oligomers of the invention are attached to one or more additional functional moieties (e.g., one or more additional binding moieties and / or signaling molecules), optionally via a linker as described above. For example, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is attached to one, two, three, four, or more additional functional moieties (e.g., one, two, three, four, or more additional binding moieties and / or signaling molecules). In certain embodiments, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is attached to one or two additional functional moieties (e.g., one or two additional binding moieties or signaling molecules). In certain embodiments, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is attached to one additional functional moiety (e.g., one additional binding moiety or signaling molecule).

[0416] hBCMA-binding polypeptides or hBCMA-binding oligomers of the present invention comprising one or more (e.g., 1, 2, 3, 4, 5, or 6 or more) additional functional moieties may be referred to as heteromultimers. hBCMA-binding polypeptides of the present invention consisting of only one hBCMA-binding polypeptide and one additional functional moiety and no other functional moieties (i.e., no additional hBCMA-binding polypeptide functional moieties or additional functional moieties) may be referred to as heterodimers. hBCMA-binding polypeptides of the present invention consisting of only one hBCMA-binding polypeptide and two additional functional moieties and no other functional moieties may be referred to as heterotrimers. hBCMA-binding oligomers of the present invention consisting of only two hBCMA-binding polypeptide functional moieties and one additional functional moiety and no other functional moieties may be referred to as heterotrimers.

[0417] In certain preferred embodiments, the additional functional moiety is a signaling molecule. A signaling molecule (e.g., an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof) may be optionally attached to the N-terminus or C-terminus of an hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above. Alternatively, one or more signaling molecules may be attached between two hBCMA-binding polypeptides in an hBCMA-binding oligomer, optionally separated by one or more linkers or a connecting sequence as described above. Preferably, a signaling molecule may be optionally attached to the N-terminus or C-terminus of an hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above. Preferably, a signaling molecule may be optionally attached to the C-terminus of an hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above.

[0418] In certain preferred embodiments, the additional functional part is an additional binding part. For example, the additional functional part is an additional binding part that identifies a binding partner of one of the following: CTLA-4, PD-1, BCMA, ADAMI 7, PD-L1, SLAMF7, EPC AM, EGFR / ErbB-1, EGFRvIII, ERBB2 / HER2 / CD340, PSMA, CLDN18.2, DLL3, MUC16, MUC17, MUC1, TPBG / 5T4 / WAIF1, B7-H4 / VTCN1 / B7x / B7S1, CD20, CD19, CD22 or CD30. For example, the additional functional portion is an additional binding portion specific for one of the following: CTLA-4, PD-1, BCMA, ADAMI 7, PD-L1, SLAMF7, EPCAM, EGFR / ErbB-1, EGFRvIII, ERBB2 / HER2 / CD340, PSMA, CLDN18.2, DLL3, MUC16, MUC17, MUC1, TPBG / 5T4 / WAIF1, B7-H4 / VTCN1 / B7x / B7S1, CD20, CD19, CD22 or CD30.

[0419] In certain preferred embodiments, the additional binding moiety is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In certain preferred embodiments, the additional binding moiety is specific for an immune cell surface protein (e.g., a NK cell surface protein, such as CD16a, NKp46, NKG2D, PD-1, particularly CD16a).

[0420] As described above, an additional binding moiety (e.g., a binding partner that recognizes the NK cell surface protein CD16a) may be optionally attached to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above. Alternatively, one or more additional binding moieties may be attached between two hBCMA-binding polypeptides in the hBCMA-binding oligomer, optionally separated by one or more connecting sequences as described above. Preferably, an additional binding moiety (e.g., a binding partner that recognizes the NK cell surface protein CD16a) may be optionally attached to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above. More preferably, an additional binding moiety (e.g., a binding partner that recognizes the NK cell surface protein CD16a) may be optionally attached to the C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer via a linker as described above.

[0421] The present inventors have surprisingly found that a 'dual binder' polypeptide comprising an hBCMA binding polypeptide or hBCMA binding oligomer as disclosed herein can retain its hBCMA binding ability when fused to an additional binding moiety that targets the NK cell surface protein CD16a. The 'dual binder' polypeptide comprising an hBCMA binding polypeptide as disclosed herein fused to a CD16a binding moiety is also surprisingly capable of activating NK cells in the presence of tumor cells expressing BCMA.

[0422] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention further comprises one or more additional functional moieties, such as at least one, at least two, at least three, or at least four additional functional moieties. For example, in a specific embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention further comprises 1, 2, 3, 4, or 5 additional functional moieties.

[0423] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two, at least three, or at least four additional functional moieties (e.g., 2, 3, 4, or 5 additional functional moieties), each additional functional moiety may be the same, or may be different, or some additional functional moieties may be the same while some additional functional moieties are different (e.g., in embodiments having at least 3 or at least 4 additional functional moieties (e.g., 3, 4, 5, 6, or more additional functional moieties)).

[0424] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two, at least three, or at least four additional functional moieties (e.g., 2, 3, 4, or 5 additional functional moieties), each additional functional moiety may have the same function, or may have a different function, or some additional functional moieties may have the same function and some may have different functions (e.g., in embodiments having at least 3 or at least 4 additional functional moieties (e.g., 3, 4, 5, 6, or more additional functional moieties)).

[0425] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two, at least three, or at least four additional functional moieties (e.g., 2, 3, 4, or 5 additional functional moieties), the first additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not hBCMA, or an additional binding moiety specific for an immune cell surface, such as an NK cell surface protein, such as CD16a); and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof. For example, in a preferred embodiment, the first additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA), and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof. In particular, the first additional functional moiety may comprise an additional binding moiety specific for a myeloma cell surface antigen that is not BCMA; and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof. Alternatively, for example, in a preferred embodiment, the first additional functional portion may include an additional binding portion specific for an immune cell target (e.g., an NK cell target, such as CD16a), and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof. In particular, the first additional functional portion may include an additional binding portion specific for an NK cell target (e.g., CD16a); and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof.

[0426] In another preferred embodiment, the first additional functional portion may include an additional binding portion (e.g., an additional binding portion specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA, or an additional binding portion specific for an immune cell target, such as an NK cell target, such as CD16a); and the second additional functional portion may include an additional binding portion (e.g., an additional binding portion specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA, or an additional binding portion specific for an immune cell target, such as an NK cell target, such as CD16a). For example, the first additional functional portion may include an additional binding portion specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA); and the second additional functional portion may include an additional binding portion specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA). In particular, the first additional functional moiety may comprise an additional binding moiety that is specific for a myeloma cell surface antigen that is not BCMA; and the second additional functional moiety may comprise an additional binding moiety that is specific for a myeloma cell surface antigen that is not BCMA.

[0427] Alternatively, for example, the first additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA); and the second additional functional portion may include an additional binding portion that is specific for an immune cell surface target (e.g., an NK cell target, such as CD16a). In particular, the first additional functional portion may include an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA; and the second additional functional portion may include an additional binding portion that is specific for a NK cell target (e.g., CD16a).

[0428] Alternatively, in another embodiment, the first additional functional part may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; and the second additional functional part may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0429] When present (e.g., in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least three or at least four additional functional moieties (e.g., 3, 4, or 5)), the third additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety that is specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen that is not BCMA, or an additional binding moiety that is specific for an immune cell target, e.g., a NK cell target, e.g., CD16a), or comprises an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0430] When present (e.g., in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least four additional functional moieties (e.g., 4 or 5)), the fourth additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety that is specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen that is not BCMA, or an additional binding moiety that is specific for an immune cell target, e.g., a NK cell target, e.g., CD16a), or comprises an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0431] When present (e.g., in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least five additional functional moieties (e.g., 5 or 6)), the fifth additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety that is specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen that is not BCMA, or an additional binding moiety that is specific for an immune cell target, e.g., a NK cell target, e.g., CD16a), or comprises an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0432] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least three (e.g., 3, 4, or 5) additional functional moieties, the first additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety that is specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA, or an additional binding moiety that is specific for an immune cell target, such as an NK cell target, such as CD16a); and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; and the third additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety that is specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA, or an additional binding moiety that is specific for an immune cell target, such as an NK cell target, such as CD16a).

[0433] For example, in one embodiment, the first additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA) or an immune cell target (e.g., an NK cell target). In particular, the first additional functional portion may include an additional binding portion that is specific for BCMA; and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In another embodiment, the first additional functional portion may include an additional binding portion that is specific for BCMA; and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional portion may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target).

[0434] For example, the first additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA); and the second additional functional portion may be an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA); and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA) or an immune cell target (e.g., an NK cell target, such as CD16a). In particular, the first additional functional portion may include an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA; and the second additional functional portion may include an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA; and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA). In another embodiment, the first additional functional portion may comprise an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA; and the second additional functional portion may comprise an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA; and the third additional functional portion may comprise an additional binding portion that is specific for an immune cell target (e.g., a NK cell target, such as CD16a).

[0435] Alternatively, the first additional functional portion may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the second additional functional portion may be an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as a myeloma cell surface antigen that is not BCMA) or an immune cell target (e.g., an NK cell target, such as CD16a). In particular, the first additional functional portion may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the second additional functional portion may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the third additional functional portion may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as an immune cell target (e.g., an NK cell target, such as CD16a)). In another embodiment, the first additional functional portion may comprise an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the second additional functional portion may comprise an additional binding portion that is specific for an immune cell target (e.g., an NK cell target, such as CD16a); and the third additional functional portion may comprise an additional binding portion that is specific for a myeloma cell surface antigen that is not BCMA.

[0436] In one embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety of the invention has the additional binding moiety separated from the hBCMA-binding polypeptide or hBCMA-binding oligomer by a linker. The linker can be any linker as defined above. For example, the linker is selected from a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, and a non-amino acid linker.

[0437] Preferably, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0438] In a preferred embodiment, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety of the invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS GS, GGGGSGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GGSGGGGSGGG, GGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0439] In a preferred embodiment, the linker used in the hBCMA-binding oligomer of the present invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker used in the hBCMA-binding oligomer of the present invention comprises or has the sequence GGGGS; for example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

[0440] Alternatively, in one embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety of the invention has the additional binding moiety not separated from the hBCMA-binding polypeptide or hBCMA-binding oligomer by a linker (i.e., the hBCMA-binding polypeptide or CD16a-binding oligomer is directly attached to the additional binding moiety).

[0441] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety of the invention does not comprise a linker. In a preferred embodiment, the hBCMA-binding polypeptide or CD16a-binding oligomer is directly attached to the additional binding moiety.

[0442] In certain embodiments, the hBCMA-binding polypeptides of the invention comprising additional functional moieties comprise the following structure:

[0443] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part];

[0444] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part];

[0445] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0446] [additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

[0447] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part];

[0448] Wherein, when there is more than one additional functional part, each additional function may be the same, or may be different.

[0449] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS S, GGSGG, GGSGGGSGGGSGG, GGSGGGSGGGGGSGGGGSGGGGSGG, GSGGG, GGSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGGSGGGGSGGGGSGGGG.

[0450] Alternatively, the linker may be absent, ie, the CD16a binding polypeptide or CD16a binding oligomer comprises the following structure:

[0451] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part];

[0452] [Additional functional part]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0453] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part]-[additional functional part];

[0454] [Additional functional part]-[Additional functional part]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]; or

[0455] [Additional functional part]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Additional functional part].

[0456] In such embodiments, more preferably, the hBCMA-binding polypeptide comprises the following structure:

[0457] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part];

[0458] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0459] (or [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part];

[0460] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part]-[additional functional part]).

[0461] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a NK cell target (e.g. CD16a).Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0462] In certain embodiments, the hBCMA-binding oligomers of the invention comprising additional functional moieties comprise the following structure:

[0463] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part];

[0464] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0465] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part];

[0466] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0467] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0468] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part];

[0469] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0470] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]; or

[0471] [N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part];

[0472] wherein each linker portion in the oligomer can have the same sequence or a different sequence; each N-terminal portion in the oligomer can have the same sequence or a different sequence; each C-terminal portion in the oligomer can have the same sequence or a different sequence; each separation portion in the oligomer can have the same sequence or a different sequence; each helix 1 portion in the oligomer can have the same sequence or a different sequence; and each helix 2 portion in the oligomer can have the same sequence or a different sequence.

[0473] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a NK cell target (e.g. CD16a).Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0474] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S, and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. Additionally, for example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGGGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS, GGGSGGGGSGGGGSGGGGS, GGSGG, GGGSGGGGSGG, GGS GGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG or SGGGGSGGGGSGGGGSGGGG.

[0475] In such embodiments, one or more linkers may be absent.

[0476] In such embodiments, more preferably, the hBCMA-binding oligomer comprises the following structure:

[0477] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]; or

[0478] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

[0479] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0480] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]; or

[0481] [N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part];

[0482] And more preferably

[0483] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]; or

[0484] [N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Additional functional part].

[0485] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a NK cell target (e.g. CD16a).Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0486] In such embodiments, one or more linkers may be absent.

[0487] In a particularly preferred embodiment, the hBCMA-binding oligomer comprises the following structure:

[0488] [N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part].

[0489] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a NK cell target (e.g., CD16a).Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0490] In certain embodiments, the hBCMA-binding oligomer of the invention comprising additional functional moieties comprises at least three hBCMA-binding polypeptides and comprises the following structure:

[0491] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[additional functional part];

[0492] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0493] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0494] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0495] wherein each linker portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each N-terminal portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each C-terminal portion can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each separation portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each helix 1 portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; and each helix 2 portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence.

[0496] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a NK cell target (e.g. CD16a).Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0497] In such embodiments, the linker is preferably a linker as defined above. In such embodiments, one or more linkers may be absent.

[0498] In such embodiments, more preferably, the hBCMA-binding oligomer comprises the following structure:

[0499] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part]; or

[0500] [N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Additional functional part].

[0501] In such embodiments, more preferably, each additional functional moiety is an additional binding moiety specific for NK cell CD16a.Most preferably, each additional functional moiety is an additional binding moiety specific for CD16a.

[0502] In the above embodiments defining the structure of a CD16a binding polypeptide or CD16a binding oligomer comprising an additional functional moiety, each additional functional moiety may be any one of those described herein. For example, each additional functional moiety may be independently selected from

[0503] Additional binding moieties (e.g., additional binding moieties specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen that is not BCMA, or an immune cell target, e.g., a NK cell target, e.g., CD16a); and

[0504] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0505] For example, each additional functional moiety can be independently selected from

[0506] an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen that is not BCMA);

[0507] additional binding moieties specific for immune cell targets (e.g., NK cell targets such as CD16a); and

[0508] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0509] For example, each additional functional moiety can be independently selected from

[0510] an additional binding moiety that is specific for a myeloma cell surface antigen that is not BCMA;

[0511] Additional binding moieties specific for NK cell targets (e.g., CD16a); and

[0512] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0513] For example, each additional functional moiety can be independently selected from

[0514] Additional binding moieties that are specific for a myeloma cell surface antigen that is not BCMA; and

[0515] Additional binding moieties specific for NK cell targets (e.g., CD16a); and

[0516] Cytokines, such as IL-15 or its derivatives.

[0517] For example, each additional functional moiety can be independently selected from

[0518] Additional binding moieties that are specific for a myeloma cell surface antigen that is not BCMA; and

[0519] Additional binding moieties specific for NK cell targets (e.g., CD16a); and

[0520] IL-15 or its derivatives.

[0521] In a preferred embodiment, each additional functional moiety can be independently selected from

[0522] Additional binding moieties that are specific for a myeloma cell surface antigen that is not BCMA; and

[0523] an additional binding moiety specific for CD16a; and

[0524] IL-15 or its derivatives.

[0525] In a preferred embodiment, each additional functional moiety can be independently selected from

[0526] an additional binding moiety specific for CD16a; and

[0527] Cytokines, such as IL-15 or its derivatives.

[0528] In a preferred embodiment, each additional functional moiety can be independently selected from

[0529] Additional binding moieties that are specific for a myeloma cell surface antigen that is not BCMA; and

[0530] Additional binding moieties specific for CD16a.

[0531] In a preferred embodiment, each additional functional moiety is an additional binding moiety specific for CD16a.

[0532] In the above embodiments defining the structure of an hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional functional moiety, each additional functional moiety can be an additional binding moiety (e.g., an additional binding moiety specific for an immune cell surface molecule, such as an NK cell surface target, such as CD16a). For example, each additional functional moiety can be an additional binding moiety for CD16a.

[0533] Further non-limiting examples of additional binding moieties include aptamers, monomers, nanobodies, small molecules and antibodies. In particular, the additional binding moiety can be an antibody, such as a monoclonal antibody, such as a monoclonal antibody specific for hBCMA, such as a monoclonal antibody specific for CD3 (e.g., foralumab, otelizumab, teplizumab, visilizumab, OKT3, UCHT1, SP34 or F2B), a monoclonal antibody specific for CD38 (e.g., daratumumab or isatuximab), or, for example, a monoclonal antibody specific for SLAMF7 (e.g., elotuzumab). The additional binding moiety can also be a T cell engager.

[0534] The additional binding moiety may be attached at the N-terminus or C-terminus of at least one hBCMA-binding polypeptide, optionally via a linker as described above. The additional binding moiety may alternatively be attached between two hBCMA-binding polypeptides, optionally separated by a linker as described above.

[0535] hBCMA binder-drug conjugates

[0536] Alternatively or additionally, an hBCMA-binding polypeptide or hBCMA-binding oligomer as disclosed herein may be attached to a therapeutic agent to form an hBCMA-binding agent-drug conjugate. Thus, in one embodiment, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is typically covalently attached to one or more therapeutic agents, optionally via one or more linkers as described above. Non-limiting examples of such therapeutic agents include cytotoxic drugs such as mitomycin C, desmethyltopotecan, SN-38, MMAE, MMAF, doxorubicin, pyrrolobenzodiazepines, Amanitin, maytansinoids (e.g., maytansinoid DM1 or maytansinoid DM4), or duostatins (e.g., dolastatin 5.2). Other non-limiting examples of therapeutic agents include radioisotopes or radiolabeled compounds that can be attached to the hBCMA-binding polypeptides or hBCMA-binding oligomers of the invention (e.g., to form a radioligand). The hBCMA-binding polypeptides or hBCMA oligomers of the invention attached to radioisotopes or radiolabeled compounds can be used for diagnosis (e.g., by imaging) or treatment (e.g., by radiotherapy) of cancer.

[0537] For the avoidance of doubt, during the production of hBCMA binder-drug conjugates according to the present invention, one or more therapeutic agents (e.g., MMAF) must be modified by reaction between a functional group at the point of attachment (e.g., -NH, -OH, or -SH (e.g., in Cys)) and any attachment group or linker used. Thus, one skilled in the art will understand that the therapeutic agent in the hBCMA binder-drug conjugate (e.g., hBCMA binder-MMAF conjugate) comprises such modifications.

[0538] Peptide production

[0539] The hBCMA-binding polypeptides of the present invention can be made using methods known in the art. For example, they can be prepared by chemical synthesis methods, by recombinant protein production techniques in, for example, bacteria, yeast, insect, fungi, plants or mammalian cells, or by cell-free in vitro protein expression methods using cell lysates and / or purified cell extracts. For example, the hBCMA-binding polypeptides used in the hBCMA-binding agent-drug conjugates of the present invention can be produced by chemical synthesis or recombinant protein production methods.

[0540] The hBCMA-binding polypeptides of the present invention can also be fused with different molecules with therapeutic potential, such as immunoglobulins or polypeptides with therapeutic potential, by the above-mentioned recombinant or chemical synthesis techniques.

[0541] Modulation of peptide properties

[0542] The pharmacokinetic properties of the hBCMA-binding polypeptides of the invention can be modulated by methods known in the art. For example, they can be linked to moieties that extend plasma half-life, such as polyethylene glycol polymers, unstructured polypeptides (such as XTEN or PAS), FcRn binding ligands, such as serum albumin or the Fc domain of an immunoglobulin, or polypeptides capable of binding to serum proteins with long in vivo half-lives, including, for example, serum albumin or immunoglobulins.

[0543] preparation

[0544] The hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly an hBCMA-binding agent-drug conjugate) according to the present invention may be present in a formulation, and in particular in a pharmaceutical formulation.

[0545] In certain embodiments, the present invention provides nucleic acid molecules encoding hBCMA-binding polypeptides or hBCMA-binding oligomers of the present invention. Nucleic acid molecules encoding hBCMA-binding polypeptides or hBCMA-binding oligomers of the present invention can be used as drugs, for example, for the treatment of cancer. Nucleic acid molecules can be, for example, DNA or RNA molecules, such as mRNA molecules. Nucleic acid molecules according to the present invention can be present in a formulation, and in particular in a pharmaceutical formulation. Therefore, the present invention also provides formulations of nucleic acid molecules (e.g., DNA or RNA, and in particular mRNA molecules) encoding hBCMA-binding polypeptides or hBCMA-binding oligomers of the present invention, and in particular pharmaceutical formulations.

[0546] Pharmaceutical formulations include, for example, those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary) or intraperitoneal administration, although the most appropriate route may depend, for example, on the condition and disorder of the subject being treated.

[0547] In one embodiment of the invention, an hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly an hBCMA-binding agent-drug conjugate) or nucleic acid (e.g., a DNA or RNA molecule, e.g., an mRNA molecule of the invention) according to the invention is administered as a pharmaceutical formulation suitable for oral or parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary) administration.

[0548] Pharmaceutical formulations suitable for oral administration may be in the form of discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of active ingredient; powders or granules; solutions or suspensions in aqueous liquids or non-aqueous liquids; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. The peptides of the present invention may also be in the form of boluses, electuaries or pastes. Various pharmaceutically acceptable carriers and their formulations are described in, for example, EW Martin's Remington's Pharmaceutical Sciences standard formulations. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supplement. 42: 2S, 1988.

[0549] Preparations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may include antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Preferably, the preparation may be present in a unit dose or separate dose container, such as a sealed ampoule and vial. The preparation can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as saline, a physiologically acceptable solution, or water for injection, immediately before use.

[0550] Ready-to-use injection and infusion solutions and suspensions can be prepared from sterile powders, granules or other dry compositions. Exemplary compositions for parenteral administration include injectable solutions or suspensions that may contain, for example, suitable nontoxic, parenterally acceptable diluents or solvents (such as mannitol, 1,3-butylene glycol, water, Ringer's solution, isotonic sodium chloride solution); or other suitable dispersants or wetting agents and suspending agents (including synthetic monoglycerides or diglycerides) and fatty acids (including oleic acid or Cremaphor).

[0551] The hBCMA-binding polypeptides or hBCMA-binding oligomers of the invention (particularly hBCMA-binding agent-drug conjugates), and pharmaceutical preparations comprising such polypeptides, oligomers or binding agent-drug conjugates or nucleic acid molecules of the invention (e.g., DNA or RNA molecules of the invention, such as mRNA molecules of the invention), and pharmaceutical preparations comprising those nucleic acid molecules, can be used to treat and / or prevent cancer, such as multiple myeloma.

[0552] The amount of hBCMA binding polypeptide or hBCMA binding oligomer (particularly hBCMA binding agent-drug conjugate) or nucleic acid molecule required to achieve a therapeutic effect will vary with the specific route of administration and the characteristics of the subject being treated, such as species, age, weight, sex, medical condition, specific disease and its severity, and other relevant medical and physical factors. One of ordinary skill can readily determine and administer the effective amount of binding polypeptide, oligomer, binding agent-drug conjugate and / or compositions comprising the same, or nucleic acid molecule and / or compositions comprising the same, required for the treatment and / or prevention of cancer.

[0553] Depending on the subject to be treated and the severity of the cancer, the hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules of the invention, or pharmaceutical formulations thereof, can be administered, for example, daily, weekly, every two weeks, every three weeks or every four weeks, or even as a high single dose.

[0554] The hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules of the present invention or their pharmaceutical preparations can be administered, for example, as a parenteral or oral dose. Parenteral administration includes intravenous (into a vein, such as a central or peripheral vein, bolus or infusion), intraarterial (into an artery, such as a central or peripheral artery), intraosseous infusion (into the bone marrow), intramuscular (into the muscle), intradermal (into the dermis) and subcutaneous (under the skin) administration. In a preferred embodiment, the dose of the present invention is administered intravenously or intraarterially, and more preferably by intravenous infusion (e.g., central vein infusion or peripheral vein infusion). In another preferred embodiment, the dose of the present invention is administered by subcutaneous injection. Therefore, pharmaceutical preparations particularly useful in the present invention are those suitable for intravenous administration, more particularly intravenous infusion or subcutaneous administration.

[0555] The hBCMA binding polypeptides, hBCMA binding oligomers (particularly hBCMA binding agent-drug conjugates) or nucleic acid molecules of the present invention or their pharmaceutical preparations can be administered as part of a treatment cycle. In a treatment cycle, the polypeptide can be administered on day 1 of the cycle, wherein the cycle lasts for X days, and no polypeptide of the present invention is administered on the following X-1 day. X can be, for example, 1 day to 42 days, such as 2 days to 28 days, such as 2 days to 21 days, such as 2 days to 14 days. Alternatively, the binding polypeptide can be administered in divided doses, such as on days 1 and 2 of the cycle.

[0556] Depending on the type, category or stage of the cancer to be treated, the cycle can be repeated once or several times. For example, the cycle can be repeated 1 to 100 times, such as 2 to 50 times, such as 8 to 40 times, such as 8 or 16 times. For example, the hBCMA-binding polypeptide (e.g., in the form of an hBCMA-binding agent-drug conjugate) can be administered 8 times of a 7-day cycle, followed by 16 times of a 14-day cycle, optionally followed by a further repeated 28-day cycle. For example, it can be administered in a 21-day cycle (i.e., once every 3 weeks) until disease progression or unacceptable toxicity. An ordinary skilled physician or clinician can easily determine the number of cycles of the hBCMA-binding polypeptide (e.g., hBCMA-binding agent-drug conjugate) required to prevent, combat or prevent cancer progression.

[0557] Combination therapy

[0558] Although the hBCMA-binding polypeptides or hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules disclosed herein can be used as the sole active ingredient in the present invention, they can also be used in combination with one or more additional therapeutic agents, and the use of such combinations provides an embodiment of the present invention. Such additional therapeutic agents may be agents or other pharmaceutically active materials suitable for treating and / or preventing cancer. Such agents are known in the art.

[0559] Non-limiting examples of additional therapeutic agents for use in the present invention may include proteasome inhibitors (PIs) (e.g., carfilzomib, bortezomib, or ixazomib), immunomodulatory agents (IMiDs) (e.g., lenalidomide, thalidomide, or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, melflufen, or bendamustine), anthracyclines (e.g., Such as doxorubicin), steroids (such as dexamethasone, prednisone or prednisolone), BCL-2 inhibitors (such as venetoclax), histone deacetylase (HDAC) inhibitors (such as panobinostat), anti-CD38 agents (such as daratumumab or isatuximab), anti-SLAMF7 agents (such as elotuzumab), immune checkpoint inhibitors (such as CTLA-4 inhibitors, PD-1 inhibitors or PD-L1 inhibitors) or ADAM17 inhibitors. For example, the additional therapeutic agent can be selected from a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflurane), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

[0560] One or more additional therapeutic agents may be used simultaneously, sequentially, or separately with the administration of a dose of an hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, or nucleic acid molecule of the invention. The individual components of such combinations may be administered separately at different times during the course of treatment, or simultaneously in divided or single combination forms.

[0561] In one embodiment of the invention, one or more additional therapeutic agents are selected from PI, IMiD and steroids. For example, one or more additional therapeutic agents are PI (e.g., bortezomib or carfilzomib), IMiD (e.g., lenalidomide, thalidomide and pomalidomide) and steroids (e.g., prednisone, prednisolone and dexamethasone). For example, one or more therapeutic agents are bortezomib, thalidomide and dexamethasone.

[0562] In one embodiment of the invention, one or more additional therapeutic agents are selected from PIs, alkylating agents, and steroids. For example, one or more additional therapeutic agents are PIs (e.g., bortezomib or carfilzomib), alkylating agents (e.g., cyclophosphamide, melphalan, melflufen, or bendamustine), and steroids (e.g., prednisone, prednisolone, and dexamethasone). For example, one or more therapeutic agents are bortezomib, melphalan, melflufen, and prednisone.

[0563] In one embodiment of the invention, the one or more additional therapeutic agents are selected from a PI and a steroid. For example, the one or more therapeutic agents are a PI (e.g., bortezomib or carfilzomib) and a steroid (e.g., prednisone, prednisolone, and dexamethasone). For example, the PI is bortezomib and the steroid is dexamethasone.

[0564] In one embodiment of the invention, one or more additional therapeutic agents are selected from IMiDs and steroids. For example, one or more therapeutic agents are IMiDs (e.g., lenalidomide, thalidomide, and pomalidomide) and steroids (e.g., prednisone, prednisolone, and dexamethasone). For example, the IMiD is lenalidomide, and the steroid is dexamethasone.

[0565] In another embodiment of the invention, one or more therapeutic agents may be selected from NK cell-based or T cell-based therapies. For example, allogeneic NK cells may be administered where the hBCMA-binding polypeptides of the invention are attached to additional binding moieties that are specific for NK cell surface proteins, such as NK cell binding polypeptides. For example, those NK cells may be co-injected with the polypeptides.

[0566] Alternatively, the hBCMA-binding polypeptides or hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules according to the invention can be combined with a therapeutic method, such as a stem cell transplantation procedure, e.g., an autologous stem cell transplantation or an allogeneic stem cell transplantation. Thus, in one embodiment of the invention, the hBCMA-binding polypeptides disclosed herein can be combined with an autologous stem cell transplantation procedure. In another embodiment of the invention, the hBCMA-binding polypeptides disclosed herein can be combined with an allogeneic stem cell transplantation procedure.

[0567] Simultaneous, sequential or separate administration of one or more additional therapeutic agents or therapeutic procedures with the hBCMA-binding polypeptides, hBCMA-binding oligomers, hBCMA-binding agent-drug conjugates or nucleic acid molecules of the invention further enhances their effectiveness in treating and / or preventing cancer.

[0568] Reagent test kit

[0569] The present invention provides a kit comprising an hBCMA-binding polypeptide, an hBCMA-binding oligomer (particularly an hBCMA-binding agent-drug conjugate) or a nucleic acid molecule as disclosed herein, and one or more additional therapeutic agents useful for treating and / or preventing cancer.

[0570] Non-limiting examples of additional therapeutic agents for use in the kits of the invention may include a proteasome inhibitor (PI) (e.g., carfilzomib, bortezomib, or ixazomib), an immunomodulator (IMiD) (e.g., lenalidomide, thalidomide, or pomalidomide), an alkylating agent (e.g., cyclophosphamide, melphalan, melflufenamic acid, or bendamustine), an anthracycline (e.g., doxorubicin), a steroid (e.g., dexamethasone, prednisone, or prednisolone), a BCL-2 inhibitor (e.g., venetoclax), a histone deacetylase (HD AC) inhibitor (e.g., panobinostat), an anti-CD38 agent (e.g., daratumumab or isatuximab), an anti-SLAMF7 agent (e.g., elotuzumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), or an ADAM17 inhibitor.

[0571] For example, the one or more additional therapeutic agents can be selected from a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflurane), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

[0572] The one or more additional therapeutic agents can, for example, be selected from a proteasome inhibitor (eg, carfilzomib or bortezomib), an immunomodulator (eg, lenalidomide or thalidomide), an alkylating agent (eg, melphalan or melflufenamic acid), and a steroid (eg, dexamethasone or prednisone).

[0573] In one embodiment of the present invention, the kit of the present invention can be used for treating and / or preventing cancer.

[0574] For the avoidance of doubt, the hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules disclosed herein are present in a kit according to the invention in a form and amount suitable for use according to the invention. Suitable pharmaceutical formulations are described herein. One skilled in the art can readily determine the amount of hBCMA-binding polypeptides or oligomers (e.g., in the form of hBCMA-binding agent-drug conjugates) or nucleic acid molecules disclosed herein suitable for use according to the invention.

[0575] Uses of the polypeptides of the present invention

[0576] The hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules of the present invention, and the pharmaceutical preparations or kits of the present invention comprising the hBCMA-binding polypeptides, hBCMA-binding oligomers, hBCMA-binding agent-drug conjugates or nucleic acid molecules can be used in medicine, for example, for treating and / or preventing cancer in a subject. Non-limiting examples of cancer include lung cancer (e.g., non-small cell lung cancer), breast cancer, or blood cancer. Blood cancers may include leukemias (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia), lymphomas (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, small lymphocytic lymphoma, and other high-grade B-cell lymphomas), or plasmacytomas and myelomas (e.g., MGUS, plasmacytomas, latent myeloma, multiple myeloma, light chain myeloma, or non-secretory myeloma).

[0577] Preferably, the hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly hBCMA-binding agent-drug conjugate) of the present invention or the nucleic acid molecule of the present invention, and the pharmaceutical preparation or kit of the present invention comprising the hBCMA-binding polypeptide, oligomer, binding agent-drug conjugate or nucleic acid molecule can be used to treat and / or prevent myeloma (e.g., MGUS, plasmacytoma, latent myeloma, multiple myeloma, light chain myeloma or non-secretory myeloma). Most preferably, the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate or nucleic acid molecule of the present invention, and the pharmaceutical preparation or kit of the present invention comprising the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate or nucleic acid molecule can be used to treat and / or prevent multiple myeloma.

[0578] Alternatively or additionally, the hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules of the present invention, and the pharmaceutical preparations or kits of the present invention comprising the same can also be used to treat and / or prevent autoimmune disorders in a subject. Non-limiting examples of autoimmune disorders include Addison's disease, celiac disease, dermatomyositis, Graves disease, Hashimoto's thyroiditis, multiple sclerosis and optic neuritis, myasthenia gravis, pernicious anemia, reactive arthritis or rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus or type I diabetes, particularly autoimmune disorders in which hBCMA signaling is involved, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and optic neuritis and Sjogren's syndrome.

[0579] The hBCMA-binding polypeptides, hBCMA-binding oligomers (particularly hBCMA-binding agent-drug conjugates) or nucleic acid molecules of the invention can also be used in biotechnology and research applications, such as detection of hBCMA in biological samples. The hBCMA-binding polypeptides, hBCMA-binding oligomers, hBCMA-binding agent-drug conjugates or nucleic acid molecules of the invention can, for example, be used in enzyme-linked immunosorbent assays (ELIS) ​​or attached to reporters such as fluorophores, for example, for flow cytometry or immunohistochemistry.

[0580] Example

[0581] The following examples illustrate the invention.

[0582] Preparation Example 1: Selection of binders against hBCMA using phage display

[0583] In this example, human BCMA (hBCMA) was used as a target in phage display selections using a phage-based affinity library. Individual clones obtained after four selection cycles were assayed for binding to hBCMA in a monoclonal phage-ELISA (enzyme-linked immunosorbent assay), and ELISA-positive clones were subjected to DNA sequencing.

[0584] Materials and methods

[0585] Affibody library.

[0586] An M13 phage display library of affibody molecules was prepared based on the phagemid vector pAffi-1 (Grbnwall et al. (2007) J. Biotechnol. 128:162-183). This phagemid containing the lac promoter and OmpA signal peptide was designed for phage display of the encoded affibody library members as binding sites to the albumin binding domain (ABD WT ), an amber stop codon and an in-frame fusion of a truncated form of M13 bacteriophage coat protein 3 (residues 249-406). A synthetic 121 base long oligonucleotide (5'-GCGCTTTGGCTTGGGTCATCXXXTAAACTXXXYYYGAAGGCXXXXXXTTGXXXXXXGTTCAGGTTCGGCAGXXXXXXGATCTCXXXXXXCGCXXXXXXXXXTTCTTTGTTGAATTTGTTGT-3') encoding amino acids 3-41 of the Z domain (reverse complementary strand) [SEQ ID NO. 280] (Nilsson et al. (1987) Protein Eng. 1: 107-113) (wherein a mixture of trinucleotide codons were used to build the blocks during synthesis (XXX = an equal mixture of codons for all 20 amino acids except Cys and Pro, YYY = 60% Ile and 10% each for His, Tyr, Lys and Asp) to randomize the wild-type codons at 14 positions in the domain) was used as a template for PCR amplification using the following: Forward primer:

[0587] (5-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3') [SEQ ID NO.281] and reverse primer:

[0588] (5-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3') [SEQ ID NO. 282], approximately 6.4 μg of Xhol and Nhel double cut and gel purified (Qiagen, Germany) PCR product was ligated with 35 μg of Xhol and Nhel double cut and gel purified pAffi-1 phagemid vector using T4 DNA ligase. The resulting ligation mixture was desalted using column purification (Qiagen, Germany), divided into 24 portions and used for electroporation (0.1 cm BioRad cuvette) 25 μl electrocompetent ER2738 Escherichia coli (E. coli) cells (F', glnV amber suppressor) (Lucigen, USA).

[0589] 970 μl recovery medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) was added to the electroporated cells, which were then combined (six electroporations per pool) and incubated at 37°C for 1 hour under shaking, after which the cell pool was titrated by spreading a dilution series on an Amp (ampicillin) plate and transferred to four 5-liter shake flasks, each containing one liter of tryptic soy broth + yeast extract medium (30 g / l tryptic soy broth, 5 g / l yeast extract; TSB+Y) supplemented with 2% glucose and 100 μg / ml Amp. Overnight cell cultures were pelleted by centrifugation and resuspended in 50 ml of cold 40% glycerol, then distributed to 29 tubes, approximately 3.5 ml of cells / glycerol solution per tube. According to the OD titration after electroporation and overnight culture 600 The library size (diversity) was calculated to be about 3*10 10 , and each 3.5 ml cell aliquot contains a cell number corresponding to approximately 10,9x library size. The tubes containing cells were stored at -80 °C until used for phage stock preparation using M13KO7 helper phage.

[0590] Naive phage library stock production.

[0591] To produce phage stocks displaying affimers, 2 ml of the library glycerol stock was distributed and inoculated into three E-flasks containing 750 ml TSB+Y, 1% (w / v) glucose, 10 μg / ml Tet (tetracycline) and 100 μg / ml Carb (carbenicillin) and grown at 37°C with shaking at 150 rpm until the culture reached an OD 600 =1. Infect cells with 600 μl (infection multiplicity (MOI) of 5) of M13KO7 helper phage (New England Biolabs) / E flask, gently swirl and incubate at 37°C without shaking for 15 minutes, then incubate at 37°C at 70 rpm for 15 minutes. Centrifuge cells of each culture and resuspend in 670 ml TSB+Y, 100 μg / ml Carb and 1 mM IPTG (isopropyl β-D-1-thiogalactoside). Add 25 μg / ml Kan (kanamycin) 2 hours after inoculation. Incubate the culture at 37°C with shaking at 200 rpm for 16 to 18 hours. Harvest phage library stock solution by precipitating twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titer of the stock solution is measured by point titration, and the percentage of phage particles carrying phagemids with affinity body inserts is analyzed using polymerase chain reaction-screening.

[0592] Phage display selection of hBCMA binding candidates.

[0593] Four cycles of panning were performed using biotinylated hBCMA-Fc (human TNFRSF17 / BCMA / CD269 protein (His and human IgG1 Fc tags)), biotinylated (Sino Biological Inc., Eschborn, Germany, catalog number 10620-H03H-B, corresponding to residues 1-54 of Uniprot entry Q02223) at concentrations of 150 nM (cycle 1), 100 nM (cycle 2), and 50 nM (cycles 3-4). SA (streptavidin) coated paramagnetic beads (Dynabeads M-280 streptavidin, catalog number 11205D, Invitrogen, Waltham, Massachusetts, USA) were washed twice with PBS (150 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4H2O, pH 7.4). To avoid selection of nonspecific binders, all tubes used for biopanning were pretreated with 1% (w / v) bovine serum albumin (BSA) in PBS-T (PBS supplemented with 0.05% (v / v) Tween-20, pH 7.4). In addition, in cycles 2-4, negative selection was performed by preincubating the phage stock in PBS-T with 0.1% (w / v) BSA and SA beads containing biotinylated trastuzumab mAb (containing Fc region) (Herceptin, Roche, Basel, Germany) for 30 minutes at room temperature (RT) under constant end-to-end (eoe) rotation to remove phage carrying binders to SA and Fc. In cycle 1 and subsequent cycles, the amount of phage stock used was approximately 8*10 11 Colony forming units (cfu). The biotinylated target protein was immobilized on 1.5 mg beads at RT and eoe for 1 hour. The beads containing the target were incubated with PBS-T containing 1% (w / v) BSA at RT for 30 minutes, then washed with PBS-T, and then the pre-incubated phage stock solution was added for selection. The selection step was performed at RT, and the incubation time for selection was 3 hours (cycle 1), followed by washing with PBS-T 1, 3, 6 or 10 times at eoe, RT in subsequent cycles. The final wash volume was transferred to a new 1% (w / v) BSA pretreated tube to remove sticky binders attached to the tube wall. Antigen-bound phages were eluted by incubating with 0.3M acetic acid (pH 2.8) at eoe, RT for 15 minutes, and the eluate was then transferred to a new pretreated tube and neutralized with an equal volume of 1M Tris-HCl (pH 8).

[0594] Amplification of phage stocks.

[0595] New phage stocks were generated by infecting E. coli XL-1 Blue cells (Agilent) grown in TSB+Y with 10 μg / ml Tet at 37°C with shaking at 150 rpm until the OD of the phage eluate was 600 =0.5-0.8 (total volume after cycle 1, half volume after cycle 2-3). 15 ml of bacterial culture was used in cycle 1-2, and 7.5 ml of bacterial culture was used in cycle 3-4. The infected culture was gently vortexed, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was then centrifuged and resuspended in TSB+Y, then plated on blood agar plates (40 g / l blood agar) with 100 μg / ml Carb and 1% (w / v) glucose and incubated at 37°C for 16 to 18 hours. Bacterial colonies were collected into TSB+Y by scraping and inoculated into 200 ml TSB+Y containing 100 μg / ml Carb, and grown at 37°C with shaking at 150 rpm until OD 600 =0.5-0.8. 30 ml after cycle 1, 20 ml after cycle 2, or 10 ml after cycle 1 were superinfected with M13KO7 helper phage (MOI of approximately 10), gently swirled, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was centrifuged and resuspended in 150 ml TSB+Y containing 100 μg / ml Carb and 1 mM IPTG. 25 μg / ml Kan was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 150 rpm for 16 to 18 hours. The phage library stock solution was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titer of the stock solution was measured by spot titration on Carb plates, and the percentage of colonies carrying phagemids with affimer inserts of the correct size was analyzed using polymerase chain reaction screening.

[0596] Monoclonal phage supernatant preparation.

[0597] After cycle four, 48 bacterial colonies that produced PCR products of the correct size were grown in 500 μl TSB+Y / Carb in a 96-well deep-well plate at 30°C with 250 rpm shaking for 16 to 18 hours. 30 μl overnight culture was inoculated into 720 μl TSB+Y / Carb in a new 96-well deep-well plate and incubated at 37°C, 250 rpm for 2 hours, and then superinfected by adding an excess of M13K07 helper phage in 100 ul TSB+Y / Carb per well. The plate was incubated at 37°C without shaking for 30 minutes. Finally, 150 μl TSB+Y / Carb / IPTG / Kan was added to each well. The final concentrations were 100 μg / ml Carb, 1 mM IPTG, and 25 μg / ml Kan. The deep-well plate was incubated at 37°C, 250 rpm for 16 to 18 hours. The phage supernatant was collected by centrifugation the next day.

[0598] Monoclonal phage-ELISA screening of candidates.

[0599] MaxiSorp ELISA plates (clear flat bottom immunonon-sterile 384-well plates, catalog number 464718, ThermoFisher Scientific) were incubated at 4°C with slow shaking with 30 μl of 5 μg / ml hBCMA-Fc (Sino Biological, catalog number 10620-H15H, corresponding to residues 1-54 of NCBI entry NP_001183.2 fused to rabbit Fc), 15 μg / ml human serum albumin (HSA; Sigma product number SRP6182) (for assessing the correct display of the expression cassette containing the tripartite fusion protein including the affibody, the affibody albumin binding domain and the truncated protein 3), 10 μg / ml trastuzumab (as a control for binding to the Fc tag), or 5 μg / ml irrelevant control protein (human CD38, His tag, Aerosol). Biosystems, catalog number CD8-H5224, corresponding to residues 43-300 of NCBI entry NP_001766.2) was coated in 100mM sodium carbonate buffer pH 8.5 / well (1 / 4 of the well with each coating protein) for 16 to 18 hours. The coating solution was removed and the plate was washed twice with PBS-T and blocked with PBS supplemented with 1% (w / v) BSA for 1 hour at RT under slow shaking. The blocking solution was discarded and the wells were incubated with 10μl phage supernatant diluted with 20μl PBS-T 1:3 at RT under slow shaking for 1 hour. The supernatant was removed and the plate was washed three times with PBS-T. After adding 30μl PBS-T containing 1:5000 α-M13-HRP (Sigma-Aldrich, Stockholm, Sweden) and incubated for 30 minutes at RT under slow shaking, the plate was washed twice with PBS-T and once with PBS. 30 μl of TMB substrate (TMB substrate kit, Thermo Fisher Scientific) was added to each well. The reaction was terminated by adding 30 μl of 2M H2SO4 after 15 to 25 minutes. The absorbance at 450 nm was measured using a CLARIOstar microplate reader (BMGLabtech, Ortenberg, Germany). Candidates considered to be ELISA-positive clones had high HSA signals and relatively high signals for hBCMA-Fc compared to the signals observed for trastuzumab (Fc control) and other unrelated target controls.

[0600] DNA Sequencing After ELISA screening, six ELISA-positive clones were sent for DNA sequencing by Sanger sequencing (Microsynth Seqlab Sanger Sequencing Service, Microsynth, Balgach, Switzerland).

[0601] result

[0602] To identify polypeptides that bind to hBCMA, an affibody phage library was constructed and used as input for the first cycle of a four-cycle phage display selection campaign as described in Materials and Methods (above). After the fourth cycle of selection, phage eluates from the acid and trypsin selection tracks were used to infect E. coli cells to obtain single colonies on carbenicillin plates. Using a phage-ELISA experiment, 48 colonies (clones) with the correct size of the affibody gene insert were randomly selected from each track and subjected to binding analysis using microtiter plates coated with the four test antigens (HSA, hBCMA-Fc, trastuzumab (Fc), and CD38). One clone that showed strong ELISA signals for HSA and hBCMA-Fc and low signals for trastuzumab (Fc) and CD38 controls was subsequently subjected to DNA sequencing. This clone is denoted Fa-G6, and its deduced amino acid sequence is in Table 1 and is listed as SEQ ID NO. 1 in the sequence listing. Table 1 The remaining sequences of the disclosure are also listed along with the corresponding SEQ ID references. Throughout this Examples section, the exemplified compound numbered X is a polypeptide having the same numbered amino acid sequence (ie, SEQ ID X).

[0603] Biological Example 2: Initial Biosensor Characterization of Candidate hBCMA Binding Clone Fa-G6

[0604] In this example, the hBCMA binding polypeptide Fa-G6 corresponding to SEQ ID NO: 1 was subcloned, expressed, and purified as His6-Affibody-ABD WT The fusion protein [SEQ ID NO:3] was initially analyzed for binding to hBCMA-Fc by surface plasmon resonance.

[0605] Materials and methods

[0606] Subcloning of Fa-G6 polypeptide variants.

[0607] A DNA fragment encoding a candidate variant of the Fa-G6 binding polypeptide [SEQ ID 1] was amplified from the library phagemid vector pAffi-1 by PCR using specific primers that introduced restriction sites for Xhol and AscI. The fragment was cut and ligated to a T7 promoter-based E. coli expression vector prepared using the same restriction enzymes. This generated a candidate variant of the Fa-G6 binding polypeptide with an N-terminal His6 tag and a C-terminal ABD WTThe DNA construct was sequence verified using Sanger sequencing (Microsynth). The amino acid sequence of this Affibody variant is listed in Table 1 and in the sequence listing as SEQ ID NO. 3.

[0608] Expression and purification.

[0609] E. coli BL21 (DE3) cells were transformed with plasmids containing the expression DNA constructs and grown in 10 ml TSB + Y containing 25 μg / ml Kan at 37°C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated 1:100 into 200 ml TSB + Y containing 25 μg / ml Kan and grown at 37°C with shaking at 150 rpm until OD 600 =0.6-1.

[0610] IPTG is added to a final concentration of 1mM to induce protein expression. The culture is cultured at 25°C with 150rpm shaking for 16 to 18 hours and harvested by centrifugation. The cell pellet is resuspended in denaturing lysis buffer (7M guanidine hydrochloride, 47mMNa2HPO4, 2.65mM NaH2PO4, 10mM TRIS-HCl, 100mM NaCl, pH 8). After incubation at 37°C with 150rpm shaking for 2 hours, the cell is centrifuged and the denatured protein from the supernatant of the cell lysate is added to the tube containing 3mlHisPur Cobalt IMAC resin (Cat. No. 89966, Thermo Scientific). The supernatant is incubated with resin at RT for 30 minutes and rotated end to end. Contaminants were removed by washing three times with wash buffer (7 M guanidinium chloride, 46.6 mM Na2HPO4, 3.4 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), and the hBCMA binding affibody Fa-G6-ABD was subsequently eluted with elution buffer (6 M urea, 50 mM NaH2PO4, 100 mM NaCl, 30 mM glacial acetic acid, 70 mM sodium acetate, pH 5) by incubation at RT for 10 min with end-over-end rotation. WTFusion protein. After IMAC purification, the protein buffer was replaced with PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA), which was measured by the absorbance at 280 nm using an extinction coefficient calculated based on the amino acid sequence. Low molecular weight markers were from Cytiva (Product No. 17044601).

[0611] With N-terminal His6 tag and C-terminal ABD WT Biosensor analysis of the hBCMA binding variant Fa-G6.

[0612] Biacore T200 instrument (Cytiva) was used to analyze His6-Fa-G6-ABD WT Real-time interaction of fusion protein [SEQ ID NO.3] with hBCMA. Protein ligand hBCMA-Fc (human TNFRSF17 / BCMA / CD269 protein (Fc tag), Sino Biological Inc., catalog number 10620-H15H, corresponding to residues 1-54 of Uniprot entry Q02223) and HSA as a positive control were diluted in 10 mM NaOAc (pH 4.5) and immobilized on a Series S CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated to serve as a reference cell. His6-Fa-G6-ABD WT The fusion protein [SEQ ID NO:3] was diluted in running buffer PBS-T and then subjected to binding analysis at 25°C and a flow rate of 30 μl / min. After each injection series, the flow cell was regenerated by injection of 10 mM HCl. In the first experiment, the hBCMA binding variant was injected at a concentration of 200 nM onto the hBCMA-Fc surface and the trastuzumab (containing Fc) control surface.

[0613] result

[0614] To investigate whether the anti-hBCMA binding polypeptide candidate clone Fa-G6 identified by phage-ELISA and DNA sequencing [SEQ ID NO. 1] exhibits target binding when expressed as a soluble protein, it was subcloned, expressed in the cytoplasm of E. coli and purified as His6-polypeptide-ABD WT Fusion protein [SEQ ID NO.3]. ABD WTThe moiety is a small serum albumin binding protein that can be used for affinity chromatography purification and / or immobilization using human serum albumin as a ligand, if desired. The fusion protein purified by immobilized metal ion affinity chromatography (IMAC) was analyzed by SDS-PAGE using the hexa-histidyl (His6) genetic fusion partner in the protein, showing that the protein is highly pure and has an approximate molecular weight consistent with its amino acid sequence. The fusion protein was injected at a concentration of 200 nM onto the surface of a sensor chip containing immobilized hBCMA-Fc, showing that the fusion protein binds to hBCMA-Fc. Figure 2a A typical binding experiment is shown in cartoon form in Figure 1. The binding experiment trace obtained is shown in Figure 2b No binding was observed with the trastuzumab (Fc) control (data not shown).

[0615] Biological Example 3: Biosensor Evaluation of N-terminally Truncated Variants of hBCMA Binding Clone Fa-G6

[0616] In this example, residues 1-5 at the N-terminus of the hBCMA binding clone Fa-G6 [SEQ ID NO: 1] were serially truncated to investigate the effect on the binding interaction between Fa-G6 and hBCMA.

[0617] Materials and methods

[0618] hBCMA binding clones were cloned to construct truncation mutants.

[0619] DNA fragments encoding hBCMA binding polypeptide candidates [SEQ ID NO. 1] were amplified from the library vector pAffi-1 with specific primers designed to introduce overhangs to the complete coding sequence, or to the coding sequence with deletions of up to five residues spanning the N-terminus, with ends complementary to the ends of the linearized expression vector (one for each truncated variant). The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to clone five monomeric Affibody constructs (wild-type Fa-G6 variant and four truncated mutants with deletions corresponding to -2, -3, -4 and -5 residues of the Affibody N-terminus, respectively) with a C-terminal His6 tag. The DNA constructs were sequence verified using Sanger sequencing (Microsynth). The amino acid sequences of these five hBCMA binding polypeptide variants are listed in Table 1 and the sequence listing as SEQ ID NOs 4-8.

[0620] Expression and purification of truncated variants.

[0621] E. coli BL21 (DE3) cells were transformed with plasmids containing the five constructs, all with a C-terminal His6 tag, and grown in 10 ml TSB+Y containing 25 μg / ml Kan at 37°C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated 1:100 in 200 ml TSB+Y containing 25 μg / ml Kan and grown at 37°C with shaking at 150 rpm until OD 600 =0.6-1. Add IPTG to a final concentration of 1 mM to induce protein expression. The culture was shaken at 150 rpm at 25 ° C for 16 to 18 hours and the cells were harvested by centrifugation. The cell pellet was resuspended in denaturing lysis buffer. After incubation at 37 ° C with 150 rpm shaking for 2 hours, the cells were centrifuged and the denatured protein from the supernatant of the cell lysate was added to a tube containing 3 ml of HisPur Cobalt IMAC resin (Catalog No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT for 30 minutes and rotated end to end. Contaminants were removed by washing three times with wash buffer, and then the Fa-G6 affibody variant was eluted with elution buffer by incubating for 10 minutes under end-to-end rotation at RT.

[0622] After IMAC purification, the protein buffer was replaced with PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen), which was measured by the absorbance at 280 nm using an extinction coefficient calculated from the amino acid sequence. Low molecular weight markers were from Cytiva (Cat. No. 17044601).

[0623] Biosensor analysis of truncation mutants.

[0624] The interaction between the four truncation mutants [exemplary compounds 5-8 with SEQ ID NOs: 5-8] and hBCMA was analyzed in real time using a Biacore T200 instrument (Cytiva), and compared with the wild-type hBCMA binder Fa-G6 [SEQ ID NO: 4], the protein ligand hBCMA-Fc (Sino Biological Inc., catalog number 10620-H15H) was immobilized (1000RU) on a S series CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated to serve as a reference cell. The analytes were diluted in running buffer PBS-T and then subjected to binding analysis at 25°C and a flow rate of 30 μl / min. After each injection series, the flow cell was regenerated by injecting 10 mM HCl. The wild-type Fa-G6-His6 construct and the four truncation mutants were injected onto the hBCMA-Fc surface at a concentration of 20 nM.

[0625] result

[0626] To investigate whether truncation of the N-terminal residues has an effect on the binding of Fa-G6 clones to hBCMA, truncations corresponding to -2, -3, -4 and -5 residues of the N-terminus of the polypeptide were performed to obtain four truncation mutants, which were expressed in the E. coli cytoplasm together with wild-type Fa-G6 and purified as His6 fusion proteins [SEQ ID NO: 4-8]. Analysis of the IMAC-purified fusion proteins by SDS-PAGE using the His6 genetic fusion partner in the protein showed that all five proteins were highly pure and had an approximate molecular weight consistent with their amino acid sequence. Injection of the four truncation mutants and wild-type binders with successively smaller molecular weights on the surface of a sensor chip containing immobilized hBCMA-Fc showed that truncation of up to five residues at the N-terminus did not impair the binding response curve to hBCMA-Fc compared to the parental full-length Fa-G6 ( Figure 3 ).

[0627] Biological Example 4: Alanine scanning of hBCMA binding clone Fa-G6

[0628] The Fa-G6 clones that bind to hBCMA indicate relatively low solubility and / or thermal stability properties, facilitating the development of second generation hBCMA binders based on the parent Fa-G6 binder, which have potentially different biophysical properties compared to Fa-G6. In this example, alanine scanning of Fa-G6 was performed to investigate which positions are important for the binding interaction of Fa-G6 with hBCMA, which was used as the basis for the design of a second generation library based on Fa-G6.

[0629] Materials and methods

[0630] Site-directed mutagenesis of the hBCMA-binding clone Fa-G6.

[0631] Using specific primers that introduce restriction sites, a DNA fragment encoding Fa-G6 binding polypeptide candidate variants [SEQ ID 1] was amplified from the library phagemid vector pAffi-1 by PCR. The fragment was cut and connected to the E. coli expression vector based on the T7 promoter prepared using the same restriction enzyme. This produced a monomeric polypeptide construct with an N-terminal His6 tag. This expression plasmid containing the His6-Fa-G6 construct (SEQ ID NO.129) was sequence verified using Sanger sequencing (Microsynth) and then used as a DNA template in a separate mutagenesis PCR reaction to replace the original codons in 14 variable positions with alanine codons using primers designed for each position. 14 alanine substitution variants were sequence verified using Sanger sequencing (Microsynth), and their amino acid sequences are listed as SEQ ID NO 9-22 in Table 1 and the sequence table.

[0632] Expression and purification of alanine variants.

[0633] E. coli BL21 (DE3) cells were transformed with individual plasmids encoding 14 alanine variants [SEQ ID NO 9-22] (each equipped with an N-terminal His6 tag) and the wild-type His6-Fa-G6 construct [SEQ ID NO. 129] and grown in 10 ml TSB+Y containing 25 μg / ml Kan at 37°C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated at 1:100 in 200 ml TSB+Y containing 25 μg / ml Kan and grown at 37°C with shaking at 150 rpm until OD 600=0.6-1. Add IPTG to a final concentration of 1mM to induce protein expression. The culture was shaken at 150rpm at 25°C for 16 to 18 hours and the cells were harvested by centrifugation. The cell pellet was resuspended in a denaturing lysis buffer. After incubation at 37°C with 150rpm shaking for 2 hours, the cells were centrifuged and the denatured protein from the supernatant of the cell lysate was added to a tube containing 3ml HisPur Cobalt IMAC resin (Cat. No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT for 30 minutes and rotated end to end. Contaminants were removed by washing three times with washing buffer, and then the hFa-G6-His6 variant was eluted with elution buffer by incubating for 10 minutes under end-to-end rotation at RT. After IMAC purification, the protein buffer was replaced with PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen) as measured by absorbance at 280 nm using an extinction coefficient calculated from the amino acid sequence. Low molecular weight markers were from Cytiva (Cat. No. 17044601).

[0634] Biosensor analysis of alanine variants.

[0635] The Biacore T200 instrument (Cytiva) was used to analyze the interaction between the His6-tagged wild-type Fa-G6 polypeptide [SEQ ID NO. 129] and 14 alanine variants [SEQ ID 9-22] and hBCMA in real time. The protein ligand hBCMA-Fc (Sino Biological Inc., catalog number 10620-H15H) was immobilized (3500RU) on a S series CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated to serve as a reference cell. The analytes were diluted in running buffer PBS-T and then subjected to binding analysis at 25°C and a flow rate of 30 μl / min. After each injection series, the flow cell was regenerated by injecting 10 mM HCl. The wild-type His6-Fa-G6 construct and the 14 alanine variants were injected onto the hBCMA-Fc surface at a common concentration of 200 nM.

[0636] result

[0637] To investigate which amino acids occupying the 14 variable positions in the Fa-G6 clone are most important for the binding interaction with hBCMA, site-directed alanine scanning mutagenesis was performed to obtain 14 alanine variants for the subsequent design of a second generation library based on Fa-G6, which were expressed in the E. coli cytoplasm along with wild-type Fa-G6 (His6-Fa-G6, [SEQ ID 129]) and purified as His6-affimer fusion proteins [SEQ ID 9-22]. Using the His6 gene fusion partner in the protein, IMAC-purified fusion proteins were analyzed by SDS-PAGE, showing that all 15 proteins were highly pure and had an approximate molecular weight consistent with their amino acid sequences.

[0638] Figure 4 The 14 alanine variants and wild-type Fa-G6 binder were injected onto a sensor chip surface containing immobilized hBCMA-Fc. The resulting sensorgrams show that substitutions to alanine at positions 9, 10, 11, 14, and 18 reduced binding of these variants to hBCMA, while substitutions to alanine at positions 13, 24, 25, 27, 28, 31, 32, and 35 impaired binding. Substitution to alanine at position 17 showed improved hBCMA-Fc binding.

[0639] Preparation Example 5: Second Generation Library Construction, Selection and Phage ELISA

[0640] In this example, two different polypeptide libraries were constructed based on certain variable positions and different degrees of re-randomization of the Fa-G6 clone. These two libraries were used to select new hBCMA binders with diverse biophysical properties and potential for different application purposes compared to the existing Fa-G6. Individual clones obtained after three phage display selection cycles were assayed for binding to hBCMA in a monoclonal phage ELISA, and ELISA-positive clones were subjected to DNA sequencing.

[0641] Materials and methods

[0642] hBCMA Affibody Fa-G6 second generation library.

[0643] Two M13 phage display second generation selection libraries were prepared based on the Fa-G6 clone. The phagemid vector used was pAffi-1 (Grbnwall et al., (2007) J. Biotechnol. 128: 162-183) and its variant denoted as pAffi-100. The pAffi-1 phagemid containing the lac promoter and the OmpA signal peptide was designed for phage display of the encoded affinity library members as in-frame fusions with the albumin binding domain (ABD), an amber stop codon, and a truncated form of the M13 phage coat protein 3 (residues 249-406). The pAffi-100 phagemid also contains the lac promoter, the OmpA signal peptide, the affinity library members as in-frame fusions with the ABD, but additionally contains a trypsin protease cleavage site before the amber stop codon, followed by the full-length form of the M13 phage coat protein 3.

[0644] Two synthetic 121 base oligonucleotides based on the Fa-G6 sequence: Oligo A (5'-ACAACAAATTCAACAAAGAA X01 X01 X03 GCG X04 X01 GAGATC X06X06 CTGCCGAACCTGAACX03 X09 CAA X10 X11GCCTTC X12 X13 X14TTA X15 GATGACCCAAGCCAAAGCGC-3') [SEQ ID NO. 283] and Oligo B (5'-ACAACAAATTCAACAAAGAA X01 X01 Y03 GCG Y04 X01 GAGATC X06X06 CTGCCGAACCTGAAC Y03 Y09 CAA Y10 Y11 GCCTTC Y12 Y13X14 TTA Y15GATGACCCAAGCCAAAGCGC-3') [SEQ ID NO. NO.284] (which encodes amino acid positions 3-41 (reverse complementary strand) corresponding to the Z domain numbering) (Nilsson et al., (1987) Protein Eng.1:107-113), in which the wild-type codons at 14 positions in the domain have been randomized using a mixture of trinucleotide codon building blocks (amino acid codons excluding Cys, Pro, and Gly; X01 = an equal mixture of codons for all 17 amino acids, X03 = 90% Phe and the remaining 10% is evenly distributed among the remaining amino acids, X04 = 90% Asp and the remaining 10% is evenly distributed among the remaining amino acids, X06 = 20% Ala and the remaining 80% is evenly distributed among the remaining amino acids, X09 = 90% Tyr and the remaining 10% is evenly distributed among the remaining amino acids, X10 = 90% Lys and the remaining 10% is evenly distributed among the remaining amino acids, X11 = 90% Trp and the remaining 10% is evenly distributed among the remaining amino acids, X12 = 90% He and the remaining 10% is evenly distributed among the remaining amino acids, X13 = 90% Arg and the remaining 10% is evenly distributed among the remaining amino acids, X14 = 90% Ser and 10% Lys, X15 = 90% Met and the remaining 10% is evenly distributed among the remaining amino acids, Y03 = 60% Phe and the remaining 40% is evenly distributed among the remaining amino acids, Y04 = 60% Asp and the remaining 40% is evenly distributed among the remaining amino acids, Y09 = 60% Tyr and the remaining 40% is evenly distributed among the remaining amino acids, Y10 = 60% Lys and the remaining 40% is evenly distributed among the remaining amino acids, Y11 = 60% Trp and the remaining 40% is evenly distributed among the remaining amino acids, Y12 = 60% He and the remaining 40% is evenly distributed among the remaining amino acids, Y13 = 60% Arg and the remaining 40% is evenly distributed among the remaining amino acids, Y15 = 60% Met and the remaining 40% is evenly distributed among the remaining amino acids); used as a template during synthesis for use with a forward primer.

[0645] (5-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3') [SEQ ID NO. 285] and reverse primer

[0646] (5-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3′) (SEQ ID NO. 286) was amplified by PCR.

[0647] About 5.4 μg of Xhol and Nhel double cut and gel purified (Qiagen, Germany) PCR products of Oligo A and Oligo B were ligated to 30 μg of Xhol and Nhel double cut and gel purified pAffi-1 and pAffi-100 phagemid vectors, respectively, using T4 DNA ligase.

[0648] Each resulting ligation mixture was desalted using column purification (Qiagen, Germany), divided into 24 portions and used to electroporate (0.1 cm BioRad cuvette) 25 μl of electrocompetent ER2738 E. coli cells (F′, glnV amber suppressor) (Lucigen, USA). 970 μl recovery medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) was added to the electroporated cells, which were then combined (six electroporations per pool) and incubated at 37°C for 1 hour under shaking, after which the cell pools were titrated by spreading a dilution series on ampicillin plates and transferred to four 5-liter shake flasks, each containing 500 ml of tryptic soy broth + yeast extract medium (30 g / l tryptic soy broth, 5 g / l yeast extract; TSB+Y) supplemented with 1.5% glucose and 100 μg / ml ampicillin. Overnight cell cultures were pelleted by centrifugation and resuspended in 50 ml of cold 40% glycerol, then distributed to approximately 29 tubes, approximately 2 ml of cells / glycerol solution per tube. According to the post-electroporation titration and OD after overnight culture 600 The library size (diversity) of Oligo A in pAffi-1 was calculated to be approximately 3.4×10 7 , and each 2 ml cell aliquot contains a cell number corresponding to approximately 147x the library size. For Oligo B in pAffi-100, the calculated library size (diversity) is approximately 5.9 × 10 7 , and each 2 ml cell aliquot contained a cell number corresponding to approximately 84x the library size. The tubes containing the cells were stored at -80 °C until used for phage stock preparation using M13KO7 helper phage.

[0649] Phage stock preparation.

[0650] To generate phage stocks displaying affimers, 0.5 ml of each library glycerol stock was dispensed and inoculated into an unbaffled E flask containing 500 ml TSB+Y, 1% (w / v) glucose, 10 μg / ml Tet (tetracycline) and 100 μg / ml Carb (carbenicillin) and grown at 37°C with shaking at 150 rpm until the culture reached an OD of 600 =1. 25 ml cells / culture were infected with M13KO7 helper phage (New England Biolabs) with an infection efficiency (MOI) of 5, gently swirled and incubated at 37°C without shaking for 15 minutes, followed by incubation at 37°C at 70 rpm for 15 minutes. Cells from each culture were centrifuged and resuspended in two baffled E flasks for each library containing 700 ml TSB+Y, 100 μg / ml Carb and 1 mM IPTG (isopropyl β-D-1-thiogalactoside). 25 μg / ml Kan (kanamycin) was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 200 rpm for 16 to 18 hours. The phage library stock solution was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titers of the stocks were measured by spot titration and polymerase chain reaction screening was used to analyze the percentage of clones carrying phagemids with the affibody gene insert of the correct size.

[0651] Selection from the secondary library of the BCMA-binding clone Fa-G6.

[0652] Three rounds of panning with two optimized selection library stocks were performed in six separate selection tracks using recombinant biotinylated hBCMA-Fc at concentrations of 140 nM in cycle 1, 40 nM in cycle 2, and 25 nM in cycle 3. In three additional selection tracks, which included competition with non-biotinylated targets, the concentration of biotinylated hBCMA-Fc was 30 nM in cycle 1 and 10 nM in cycles 2-3, and the concentration of recombinant non-biotinylated hBCMA (BCMA-His, 10620-H08H, Sino Biological) was 3 μM in cycle 1 and 1 μM in cycles 2-3. Simultaneous negative selection was performed using trastuzumab containing human IgG1 Fc at 300 nM in all selection cycles. The paramagnetic beads (Dynabeads M-280 Streptavidin, catalog number 11205D, Invitrogen, Waltham, Massachusetts, USA) coated with SA (streptavidin) were washed twice with PBS (150 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4H2O, pH 7.4). To avoid nonspecific binding agents, all tubes used for bio-panning were pretreated with 1% (w / v) bovine serum albumin (BSA) in PBS-T (PBS supplemented with 0.05% (v / v) Tween-20, pH 7.4). In addition, the phage stock solution in PBS-T was pre-incubated with 0.1% (w / v) BSA and beads at room temperature (RT) under constant end-to-end (eoe) rotation for 30 minutes to remove phages carrying binding agents for SA. The amount of phage stock solution used in cycle 1 was 10 12 Colony forming units (cfu), 10 in cycle 2 13 cfu, 10 in cycle 3 11-13 cfu, it depends on the trajectory.

[0653] For the three tracks with solid phase selection, the biotinylated target protein was immobilized on 0.5 mg beads at RT and end-to-end (eoe) for 1 hour. The beads containing the target were incubated with PBS-T containing 1% (w / v) BSA at RT and eoe for 30 minutes, then washed with PBS-T, and then pre-incubated phage stock and trastuzumab were added for selection at RT and eoe for 2 hours. For the six tracks with liquid phase selection, pre-incubated phage stock and trastuzumab were added to the biotinylated target protein for selection in solution. The selection step was performed at RT, and the incubation time for selection was 2 hours in cycles 1 and 3, or 1.5 hours in cycle 2. Three of these tracks included competition with non-biotinylated targets (dissociation rate selection), which was added after the above incubation and incubated for an additional 1 hour. The phage-antigen complex was captured by incubating with 0.5 mg SA beads at RT and eoe for 30 minutes.

[0654] For each subsequent cycle, the phage antigen complex captured by the SA beads was washed with PBS-T enzyme at RT for a total of 5 minutes, 10 minutes or 20 minutes. For three tracks including competition with non-biotinylated targets, PBS-T containing 100nM non-biotinylated hBCMA was used to wash in cycles 2-3 for 4 minutes at RT and eoe. The final wash volume was transferred to a new 1% (w / v) BSA pretreated tube to remove sticky binders attached to the tube wall. For six selection tracks, antigen-bound phages were eluted by incubating with 0.5M acetic acid (pH 2.8) at eoe, RT for 15 minutes, and the eluate was subsequently transferred to a new pretreated tube and neutralized with an equal volume of 1M Tris-HCl (pH 8). For the remaining three selection tracks, antigen-bound phages were eluted by incubation with 0.25 mg / ml trypsin (Gibco Life Technologies) in TBS-T (TRIS-buffered saline, 0.1% (v / v) Tween-20) supplemented with 1 mM CaCl2 for 30 min at eoe, RT, and the eluate was then transferred to a new pre-treated tube. After cycles 1-2, E. coli XL-1 Blue cells (Agilent) were grown in TSB+Y with 10 μg / ml Tet at 37°C with shaking at 150 rpm until OD 600=0.5-0.8, and then infect them with phage eluate (100 ml after cycle 1, and 50 ml after cycle 2) to produce new phage stock (total volume after cycle 1, half volume after cycle 2). The infected culture was gently vortexed, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was then centrifuged and resuspended in TSB+Y, and then plated on blood agar plates (40 g / l blood agar) with 100 μg / ml Carb and 1% (w / v) glucose and incubated at 37°C for 16 to 18 hours. Bacterial colonies were collected into TSB+Y by scraping and inoculated into 200 ml TSB+Y containing 100 μg / ml Carb, and grown at 37°C with shaking at 150 rpm until OD 600 = 0.5. 30 ml were superinfected with M13KO7 helper phage (MOI 8) or KM13 helper phage (MOI 33 in cycle 1 and MOI 5 in cycle 2), vortexed gently, incubated at 37°C without shaking for 25 minutes, followed by shaking at 70 rpm for 15 minutes at 37°C. The culture was centrifuged and resuspended in 150 ml TSB+Y containing 100 μg / ml Carb and 1 mM IPTG.

[0655] 25 μg / ml Kan was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 150 rpm for 16 to 18 hours. The phage library stock was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titer of the stock was measured by spot titration, and the percentage of phage particles carrying phagemids with affibody inserts was analyzed using polymerase chain reaction screening.

[0656] Monoclonal phage ELISA.

[0657] After cycle 3, 20-21 bacterial colonies that produce PCR products of the correct size and equally represent nine selection tracks are grown in 500 μl TSB+Y / Carb in a 96-well deep-well plate at 30°C with 250 rpm shaking for 16 to 18 hours. 30 μl overnight culture is inoculated into 720 μl TSB+Y / Carb in a new 96-well deep-well plate and incubated at 37°C, 250 rpm for 2 hours, and then superinfected by adding M13K07 helper phage (MOI8) in 100 ul TSB+Y / Carb per well. The plate is incubated at 37°C without shaking for 30 minutes. Finally, 150 μl TSB+Y / Carb / IPTG / Kan is added to each well. The final concentration is 100 μg / ml Carb, 1 mM IPTG and 25 μg / ml Kan. The deep well plates were incubated at 37°C, 250 rpm for 16 to 18 hours. The next day, the phage supernatant was collected by centrifugation.

[0658] Monoclonal phage-ELISA screening of optimization candidates.

[0659] MaxiSorp ELISA plates (clear flat bottom immunonon-sterile 384-well plates, catalog number 464718, ThermoFisher Scientific) were coated with 30 μl of 1 μg / ml biotinylated hBCMA-Fc, 20 μg / ml HSA (for evaluation of the correct display of the expression cassette containing the triple fusion protein including the affibody, the albumin binding domain and the truncated (pAffi-1) or full-length (pAffi-100) protein 3), 10 μg / ml SA or 10 μg / ml trastuzumab (as a control for binding to the Fc tag) in 100 mM sodium carbonate buffer pH 9.6 / well (1 / 4 of the well with each coating protein) for 16 to 18 hours at 4°C with slow shaking. The coating solution was removed and the plates were washed twice with PBS-T and blocked with PBS supplemented with 1% (w / v) BSA for 1 hour at RT with slow shaking. Blocking solution is discarded and the wells are incubated with 10 μl phage supernatants diluted with 20 μl PBS-T 1:3 at RT under slow shaking for 1 hour. The supernatant is removed and the plate is washed three times with PBS-T. After adding 30 μl PBS-T containing 1:5000 α-M13-HRP (Sigma-Aldrich, Stockholm, Sweden) and incubating for 30 minutes under slow shaking at RT, the plate is washed twice with PBS-T and washed once with PBS. 30 μl TMB substrate (TMB substrate kit, Thermo Fisher Scientific) is added to each well. 30 μl 2M H2SO4 is added to terminate the reaction after 15 to 25 minutes. The absorbance at 450 nm is measured using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). Candidates considered as ELISA-positive clones had high HSA signal and relatively high signal for hBCMA compared to SA and trastuzumab controls.

[0660] DNA Sequencing After ELISA screening, 120 ELISA-positive clones were sent for sequencing by Sanger sequencing (Microsynth).

[0661] result

[0662] In order to isolate the second generation polypeptides that bind to hBCMA, two affinity phage libraries were constructed mainly based on the hBCMA binding clone Fa-G6 and the results from alanine scanning. As described in Materials and Methods (above), the two libraries were used as inputs for the first cycle of three-cycle selection activities. After the third cycle of selection, the eluates from the nine selection tracks were used to infect E. coli cells to obtain single colonies on Carb plates. 20-21 clones from each track were randomly selected for binding analysis using phage-ELISA experiments. Most of the clones analyzed showed strong ELISA signals for HSA and hBCMA, as well as low signals for the controls used, and were subjected to DNA sequencing. DNA sequencing of phage ELISA positive clones identified 107 unique variants. The amino acid sequences of these 107 hBCMA binding polypeptide variants are listed in Table 1 and in the sequence table as SEQ ID NO 2 (clone 1-E6) and 23-128.

[0663] Biological Example 6: Characterization of Second Generation Candidate hBCMA Binding Peptides

[0664] In this example, a subset of 18 second generation polypeptide variants corresponding to SEQ ID 2 and 23-39 (designated 1-E6, 1-E1, 1-A5, 1-A7, 1-F9, 1-F10, 2-B1, 2-C4, 2-D3, 2-H2, 2-A7, 2-E5, 2-E6, 2-G5, 2-A10, 2-B10, 2-C5 and 2-D10) were subcloned, expressed, purified as affibody-His6 fusion proteins and initially evaluated for their binding to hBCMA-Fc by surface plasmon resonance. Circular dichroism (CD) spectroscopy was used to assess the secondary structure content and their thermal denaturation profiles.

[0665] Materials and methods

[0666] Cloning of second generation hBCMA binding clones.

[0667] DNA fragments encoding a subset of 18 new candidate hBCMA binders [SEQ ID 2 and 23-39], also representing nine selection tracks, were amplified from the library vector pAffi-1 with specific primers designed to introduce overhangs complementary to the ends of the linearized expression vector. The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to clone 18 monomeric polypeptide constructs with a C-terminal His6 tag. The 18 DNA constructs containing SEQ ID 2 and 23-39 in the form [polypeptide sequence]-YYHHHHHH were sequence verified using Sanger sequencing (Microsynth).

[0668] Expression and purification.

[0669] E. coli BL21 (DE3) cells were transformed with plasmids containing DNA constructs encoding 18 polypeptide variants each equipped with a C-terminal His6 tag and the parent Fa-G6-His6 construct and cultured in 10 ml TSB+Y with 25 μg / ml Kan at 37°C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated 1:100 in 200 ml TSB+Y with 25 μg / ml Kan and grown at 37°C with shaking at 150 rpm until OD 600 =0.6-1. IPTG was added to a final concentration of 1 mM to induce protein expression. The culture was shaken at 150 rpm at 25 ° C for 16 to 18 hours and the cells were harvested by centrifugation. The cell pellet was resuspended in denaturing lysis buffer. After incubation at 37 ° C with shaking at 150 rpm for 2 hours, the cells were centrifuged and the denatured protein from the supernatant of the cell lysate was added to a tube containing 3 ml of HisPur Cobalt IMAC resin (Catalog No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT for 30 minutes and rotated end to end. Contaminants were removed by washing three times with wash buffer, and then the hBCMA binding polypeptide was eluted with elution buffer by incubating for 10 minutes at RT under end-to-end rotation.

[0670] After IMAC purification, the protein buffer was replaced with PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen), which was measured by the absorbance at 280 nm using an extinction coefficient calculated from the amino acid sequence. Low molecular weight markers were from Cytiva (Cat. No. 17044601).

[0671] Biosensor analysis of second-generation Fa-G6 variants. The real-time interaction of 18 second-generation variants expressed and purified as polypeptide-His6 (SEQ ID 2 and 23-39 in the form of [peptide sequence]-YYHHHHHH) with hBCMA was analyzed using a Biacore T200 instrument (Cytiva). The protein ligand hBCMA-Fc (Sino Biological Inc., catalog number 10620-H15H) was immobilized on an S series CM5 sensor chip (Cytiva) by amine coupling (3500RU) using the manufacturer's instructions. One flow cell was activated and deactivated to serve as a reference cell. The analyte was diluted in running buffer PBS-T and then subjected to binding analysis at 25°C and a flow rate of 30μl / min. After each injection series, the flow cell was regenerated by injecting 10mM HCl. 18 second-generation variants and parent Fa-G6 binders were injected onto the hBCMA-Fc surface at 100nM.

[0672] CD analysis. A Chirascan CD spectrometer (Applied Photophysics, Leatherhead, United Kingdom) was used to determine the secondary structure content of Fa-G6 binder and second generation binder by recording CD spectra in the range of 195 nm-260 nm at 20°C, and their thermal denaturation curves by recording CD ellipticity at 221 nm during heating from 20°C to 92°C.

[0673] result

[0674] To investigate whether a subset of new candidate hBCMA binding variants that emerged from the selection of the second generation library and identified by phage-ELISA and DNA sequencing exhibited hBCMA target binding when expressed as soluble proteins, 18 new variants [SEQ ID 2 and 23-39] selected to equally represent the nine selection trajectories were subcloned, cytoplasmically expressed in E. coli and purified as Affibody-His6 fusion proteins. Analysis of the IMAC-purified fusion proteins by SDS-PAGE using the His6 gene fusion partner in the protein showed that all 18 proteins were highly pure and had an approximate molecular weight consistent with their amino acid sequences. The 18 candidate optimized variants were injected separately at a single concentration of 100 nM onto a sensor chip immobilized with hBCMA-Fc. The resulting sensorgrams ( Figure 5 ) showed that all 18 new variants bound to hBCMA. The obtained CD spectra showed that the analyzed binders all had high α-helical secondary structure content at 20°C, and according to the thermal denaturation curve of the second-generation hBCMA binder ( Figure 6a), improved resistance to thermal denaturation could be observed for most of these binders compared to the parental Fa-G6 clone also included in the analysis. One of the new variants (clone 1-E6) showed a melting temperature (Tm) of 63°C determined by variable temperature measurements, which is 11°C higher than the melting temperature observed for the parental Fa-G6 clone (Tm of 52°C). Figure 6b Shown in.

[0675] In summary, the results show that a new library of hBCMA-binding peptides based on the parental Fa-G6 binder has been generated, with diverse biophysical properties compared to Fa-G6.

[0676] Biological Example 7: Anti-hBCMA Drug Conjugates to BCMA + Multiple myeloma cells are cytotoxic

[0677] In this example, hBCMA binder-drug conjugates were synthesized and tested for their cell binding and cancer cell killing abilities using multiple myeloma cells.

[0678] MMAF conjugation to generate hBCMA binder-drug conjugates

[0679] hBCMA binder 1-E6 SEQ ID 2 (6756.5Da) was produced synthetically at Almac Science Limited (Scotland). The protein consists of 59 natural amino acids, including a free terminal cysteine ​​residue carrying a thiol group. The selected toxin is MMAF with a non-cleavable MPB linker. The unbound cysteine ​​residue thiol functional group will be utilized to the electrophilic maleimide portion of the linker through a thiol-Michael addition reaction. Buffer exchange and purification were performed in an Amicon Ultra centrifugal filter (3kDa cutoff). The reaction progress was analyzed by HPLC-MS using an Agilent 1100 series liquid chromatography / mass selective detector (MSD) (single quadrupole) equipped with an electrospray interface and a UV diode array detector. ACE 3C8 (3.0mm×50mm) column was used for analysis with a gradient of acetonitrile / 0.1% TFA in water over 3 minutes and a flow rate of 1mL / min. Purity and ID analysis were performed using an Agilent 1290 ultra-high performance liquid chromatography / quadrupole time-of-flight (Q-ToF) mass spectrometer. Analysis was performed using ACE 3C8 5μ (3.0 mm × 50 mm), UV detection was performed at 280 nm, and the gradient was acetonitrile / 0.1% formic acid aqueous solution, and the flow rate was 0.8 mL / min. The Fa-G6 deletion variant of the polypeptide lacking binding ability was also synthesized using the same method as above.

[0680] TCEP HCl (8.47mM) tris (2-carboxyethyl) phosphine HCl (4.24mg, 14.8μM) was dissolved in PBS pH 7.4 (2mL, 10mM) and vortexed gently. MPB-MMAF solution (2.06mM) MPB-MMAF (4.00mg, 4.11μmol) was dissolved in anhydrous DMF (2.0mL) and vortexed gently. Both solutions were freshly prepared before use. hBCMA binder 1-E6 (10mg, 1.48μmol) was weighed into a glass vial and dissolved in PBS buffer (4mL, pH 7.4, 10mM) and TCEP (2.0mL, 10 molar equivalents) was carefully added. The reaction mixture was gently stirred at room temperature for 30 minutes. The solution was then divided into two centrifugal filtration units and centrifuged at 5000rpm (6°C) for 25 minutes. Each concentrate (0.5 mL) was diluted with PBS pH 7.4 (3.5 mL) and the centrifugation step was repeated. The reaction mixture was transferred from the filtration unit to a 10 mL glass bottle, followed by the addition of PBS pH 7.4 (4 mL) and anhydrous DMF (2 mL, 2.78 molar equivalents) containing MPB-MMAF. The reaction mixture was gently stirred at room temperature for 2 hours. The reaction mixture was diluted with PBS (2.0 mL, pH 7.4) containing 10% DMF and the resulting solution was centrifuged at 5000 rpm for 25 minutes in a filtration device. Eight cycles of washing were performed by this filtration method, so that no trace of non-conjugated toxins was detected by LCMS. Then, the buffer was replaced with a buffer pH 7.8 (50 mM Arg, 75 mM NaCl, 2% (w / w) sucrose, 0.01% (w / w) polysorbate 20) containing arginine. Buffer exchange was performed at 5000 rpm (6 ° C) for 25 minutes in a centrifugal filter device. The buffer exchange step was repeated 7 times to obtain material with UPLC UV purity 87% and HRMS (deconvoluted): 7729.

[0681] Capping of 1-E6 and Fa-G6 null variant polypeptides

[0682] The protocol for generating the capped non-toxic form of the peptide was essentially the same as that for MMAF conjugation, with the only major difference being that the free thiol-bearing terminal cysteine ​​residue was reacted with 1-ethylpyrrole-2,5-dione at 1.50 molar equivalents compared to the linker drug. This procedure yielded capped forms with UPLC UV purity of 99% and HRMS (deconvoluted): 6681 for 1-E6 and 6637 for Fa-G6.

[0683] BCMA binding ELISA

[0684] 96-well assay plates (3690, Costar) were coated with 1 μg / ml BCMA (193-BC-050, R&D) and incubated overnight at 4°C. After blocking, serial dilutions of 1-E6-capped and 1-E6-MPB-MMAF in blocking buffer (37528, Thermo Scientific) were added and incubated. Anti-affinity antibody (20.1000.01.0005, Affibody AB) and anti-goat antibody-HRP (PAI-28664, Invitrogen) in blocking buffer were then used. 1-Step TM Ultra TMB-ELISA substrate solution (34028, Thermo Scientific) was reacted and the reaction was terminated by adding 2M sulfuric acid. The absorbance was read at 450nm using a SpectraMax plate reader. All incubation steps were performed at room temperature for 1 hour, and the assay plate was washed 4 times with PBST after each incubation.

[0685] MMAF conjugation reduced the affinity of 1E6 for BCMA to some extent ( Figure 7 ). It can still be demonstrated that 1-E6-MPB-MMAF shows binding affinity to BCMA.

[0686] Cell viability of BCMA+MM.1S cells

[0687] 4×10 cells were treated with different concentrations of 1-E6-MPB-MMAF in the presence of 5 μM 1-E6 or Fa-G6_W24A (F28A capped). 5 MM.1s cells (CRL-2974, ATCC) were added for 72 hours. The wells treated with DMSO and the wells treated with 200 μM chlorpromazine hydrochloride (C8138, Sigma-Aldrich) were used as controls for live and dead cells, respectively. After cultivation, CellTiter-Glo (G924B, Promega) was dispensed into the cells and luminescence was read using a PerkinElmer EnVision Multilabel plate reader.

[0688] 1-E6-MPB-MMAF is cytotoxic to BCMA+ cells in a dose-dependent manner ( Figure 8 ). Compared to the non-competitive Fa-G6 deletion variant of the polypeptide, the efficacy was lower in the presence of competing 1-E6. Therefore, it can be demonstrated that drug conjugates of 1-E6 can be used to target the proliferation of BCMA+ myeloma cells.

[0689] Biological Example 8: hBCMA conjugates of the present invention combined with hCD16a binders induce Induce immune cell activation.

[0690] Experiments were performed to evaluate the propensity of the anti-hBCMA conjugates of the invention attached to hCD16a binders to elicit an IFN□□ response in co-cultures of human PBMCs and BCMA-positive multiple myeloma cells. Thawed and overnight rested PBMCs (4W-270, Lonza) were co-cultured with MM.1s cells (CRL-2974, ATCC) in V-shaped 96-well plates (249935, Thermo Scientific) at an effector to target ratio (E:T) of 25:1. A total of 4×10 5 Cells were treated with 200 nM hBCMA x hCD16a dual conjugate or 200 nM elotuzumab (300 mg Empliciti, Bristol-Myers Squibb) in a humidified 5% CO2 incubator at 37°C for 4 hours.

[0691] The supernatant was harvested and then centrifuged at 600 g for 5 minutes. Human IFN-γ ELISA MAX TM IFN-γ levels were measured by enzyme-linked immunosorbent assay (ELISA) using the Bio-Rad Biosystems Deluxe Set (430104, BioLegend). Data were processed using Excel and graphed using GraphPad Prism 9.

[0692] hBCMA×hCD16a dual conjugate constructs were investigated, containing the hBCMA binding polypeptide of the invention (1-E6, SEQ ID 2) genetically fused to hCD16a binding arms consisting of CD16a binding polypeptides in monomeric or dimeric form, referred to herein as A10 (SEQ ID 167), H09 (SEQ ID 276), and A11 (SEQ ID 277). The double conjugates tested were: monomer A10 (1-E6-A10-His6 [SEQ ID NO. 130]); heterodimer H09-A10 (1-E6-H09-A10-His6 [SEQ ID NO. 131]); homodimer A10 (1-E6-A10-A10-His6 [SEQ ID NO. 132]; and heterodimer A11-A10 (1-E6-A11-A10-His6 [SEQ ID NO. 133]). Sequence information and SEQ ID NO. 1 for each of the four double conjugate constructs are provided. ID references are given in Table 1 below. Corresponding constructs containing an inactive non-hBCMA binding polypeptide (Fa-G6 inactive) were also evaluated. The anti-SLAMF7 monoclonal antibody elotuzumab was used as a positive control, and a single culture of PBMCs and MM.1s was used as a negative control. Dual conjugates containing BCMA binding polypeptides all elicited IFND responses in co-cultures of PBMCs and MM.1s cells that were greater than the response of the positive control elotuzumab at a concentration of 200 nM for all conjugates ( Fig. 9 ). Dual conjugate constructs lacking hBCMA binding showed no or limited IFND responses in co-cultures of PBMC and MM.1s cells. These experiments demonstrate hBCMA-dependent activation of PBMCs against BCMA-positive MM.1s myeloma cell lines induced by anti-hBCMA×CD16a NK cell conjugates of the invention.

[0693] The CD16a binding polypeptides referred to herein as A10, H09 and A11 have the sequences:

[0694]

[0695] Biological Example 9: hBCMA conjugates of the present invention combined with hCD16a binders induce CD16-mediated activation.

[0696] Experiments were performed using the commercially available Jurkat-Lucia NFAT cell line from Invivogen to evaluate the propensity of the anti-hBCMA conjugates of the present invention attached to the hCD16a binder A10 to elicit CD16-dependent responses in reporter assays. Jurkat-Lucia NFAT cell line at 100,000 cells / mL and MM.IS at 50,000 cells / mL were cultured according to the manufacturer's instructions. Cells were incubated with the conjugate constructs for 24 hours.

[0697] Anti-hBCMA dual conjugate constructs containing one or two hBCMA binding polypeptides of the invention (1-E6) genetically fused to the hCD16a binding arm A10 were evaluated. The sequence information and SEQ ID references for these dual conjugate constructs are given in Table 1 below [SEQ ID NOs 130, 134-138]. Conjugates containing ineffective non-hBCMA binding polypeptides (Fa-G6 ineffective, Fig.10 The anti-SLAMF7 monoclonal antibody elotuzumab was used as a positive control. Both double conjugates containing BCMA binding polypeptides elicited a CD16-mediated response in the presence of MM.1s cells ( Fig.10 ). The dual conjugate construct without hBCMA binding showed no response. These experiments demonstrate hBCMA-dependent activation by the anti-hBCMA×CD16a NK cell conjugates of the invention.

[0698] Table 1

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706] Table 1 shows the sequences of the polypeptides disclosed in this application, including some double conjugates [SEQ ID No 130-147]

[0707] Table 2:

[0708]

[0709]

[0710] Table 2 shows the sequences of primers and synthetic oligonucleotides used in the preparation examples of the present application.

[0711] Biological Example 10:

[0712] hCD16a Activation by hBCMA×hCD16a Dual Conjugate Carrying IL-15 Cytokine Peptide:

[0713] Materials and methods:

[0714] The cDNA encoding the polypeptide of the present invention containing a stop codon was synthesized and connected to the NdeXhol restriction site of the pET29 vector. Escherichia coli BL21 (DE3) was transformed with the vector under kanamycin selection. The compound (referred to as example compound 148 (SEQ ID 148) in this article) was expressed by inducing IPTG at an OD of 0.6 and harvested after 16 hours. Example compound 148 was purified from inclusion bodies by being dissolved in 1×PBS supplemented with 30mM imidazole, 6M urea, 10mM glutathione (reduced). Soluble materials were purified by IMAC using 1×PBS supplemented with 500mM imidazole, 6M urea as elution buffer. Example compound 148 was then refolded by dialysis against 1×PBS.

[0715] According to the method of Example 11, in the Lucia luciferase reporter assay Jurkat-Lucia TM The propensity of compounds to stimulate CD16a activation was assessed in NFAT-CD16 cells (InvivoGen) and compared to the antibody-dependent cellular cytotoxicity (ADCC) CD16a activation of elotuzumab (Empliciti, Bristol Myers Squibb, a clinical-grade α-SLAMF7 monoclonal antibody).

[0716] result

[0717] A hBCMA×hCD16a dual conjugate carrying the IL-15 cytokine sequence was constructed (Example Compound 148 = SEQ ID 148) and expressed as a soluble gene product in E. coli (DE3). The CD16a activation response of the conjugate construct in the presence and absence of target MM.1s cells is shown in Fig.11 middle.

[0718] As can be seen from the figure, affibody-based IL-15 containing dual conjugates can activate CD16a in a target-specific manner.

[0719]

[0720] Biological Example 11:

[0721] CD16a activation and expression of hBCMA × hCD16a dual conjugate constructs with different domain orders and linkers NK cell-mediated cell killing

[0722] Compounds with different distances and targeting domain orderings in the dual conjugate construct were prepared. The propensity of the hBCMA×hCD16a dual conjugates to stimulate CD16a activation and enhance NK cell-mediated killing was assessed. In each case, the hCD16a binding polypeptide was a polypeptide having SEQ ID 167. In each case, the hBCMA binding polypeptide was a polypeptide having SEQ ID 2.

[0723]

[0724]

[0725] Design, construction, expression and protein purification of hBCMA×hCD16a binding polypeptide conjugate constructs.

[0726] The hBCMA×hCD16a binding construct was designed for expression and purification as follows: cDNA encoding each hBCMA×hCD16a binding construct containing a stop codon was synthesized and ligated to the Ndel Xhol restriction site of the pET29 vector. The vector was transformed into E. coli BL21 (DE3) under kanamycin selection and the construct was expressed by inducing IPTG at an OD of 0.6 and harvested after 16 hours. The soluble cytoplasmic product was harvested and resuspended in 1×PBS supplemented with 30mM imidazole. Purification was achieved by IMAC using 1×PBS supplemented with 500mM imidazole as the elution buffer. Further purification was achieved by RP-HPLC, and identity / purity was identified by SDS-PAGE and analyzed by LC-MS / MS.

[0727] Heterodimeric hBCMA x hCD16a dual binding constructs (exemplary compounds 130, 154, 155, 156, and 157) and heterotrimeric hBCMA x hCD16a binding constructs containing two BCMA binding polypeptides (exemplary compounds 149, 150, 151, 152, and 153) were evaluated.

[0728] The propensity of the hBCMA×hCD16a dual conjugate to stimulate CD16a activation and enhance NK cell-mediated killing was assessed:

[0729] CD16 activated Jurkat-Lucia TM Luciferase reporter assay

[0730] The propensity of the hBCMA×hCD16a dual conjugate to stimulate CD16 activation was assessed in a Lucia luciferase reporter assay and compared to the antibody-dependent cellular cytotoxicity (ADCC) effect of elotuzumab (a clinical-grade α-SLAMF7 monoclonal antibody) by evaluating CD16a activation. TM NFAT-CD16 cells (Invivogen) were seeded with MM.1s cells at an effector to target (E:T) ratio of 2:1 in a 96-well flat-bottom plate. 3 × 10 5 Cells were treated with 20 nM or 200 nM of the agent of interest for 24 h. Supernatants were collected and analyzed using QU ANTI Luc TM Lucia luciferase activity, reflecting the induced ADCC response, was assessed using luciferase Gold (InvivoGen). Responses were normalized to the maximal response of elotuzumab.

[0731] In vitro cytotoxicity assay based on calcein release

[0732] NK cell cytotoxic activity was assessed in a calcein release-based cytotoxicity assay previously described by (Gauthier et al., 2023) PMID 36635380. In brief, MM1.S target cells were pre-labeled with calcein-AM (Biolegend) and co-cultured with NK cells in a 96-well v-bottom plate at a ratio of 1: 1. After the effector and target cells, the conjugate was finally added to the co-culture. After 4 hours of co-culture, the supernatant was collected and transferred to a black flat-bottom 96-well plate. The fluorescent signal of calcein released by dead MM1.S cells was quantified in SpectraMax i3x (Molecular Devices). The specific lysis percentage was calculated in Microsoft Excel (Microsoft), spontaneous release was performed using target cells alone, and maximum release was performed using target cells containing detergent. The cytotoxic activity of NK cells in the presence of the conjugate was compared with the cytotoxic activity of antibody-dependent cells of belantuzumab biosimilars (purchased from IchorBio).

[0733] result

[0734] hBCMA x hCD16a dual conjugate constructs containing one or two hBCMA binding polypeptides of SEQ ID 2 genetically fused to an hCD16a binding arm consisting of the polypeptide of SEQ ID 167 and a C-terminal His6 tag were constructed and expressed as soluble gene products in E. coli (DE3). Fig.12a and Figure 12bShown are the hCD16a activation responses of the engager constructs in the presence or absence of target MM.1s cells.

[0735] The results show that affimer-based heterodimeric hBCMA×hCD16a dual binding constructs and heterotrimeric hBCMA×hCD16a binding constructs (here consisting of polypeptides of SEQ ID 2 (for hBCMA) and SEQ ID 167 (for hCD16a)) can activate hCD16a in a target-specific manner when genetically fused together in either order. Good activation was observed for conjugates with linker lengths varying from 0 to 15 amino acids.

[0736] Biological Example 12:

[0737] Evaluation of hBCMA × hCD16a using the Jurkat CD16a activation reporter assay and NK-mediated killing cell assay Domain boundaries in dual conjugate constructs.

[0738] hBCMA×hCD16a dual binder constructs with truncations at both the N- and C-termini of the BCMA binding polypeptide and the CD16a binding A10 Affibody were generated and evaluated in hCD16a-mediated activated cell-based reporter assays and cell killing. This enabled the assessment of the domain boundaries of the targeting domains in the dual binder constructs.

[0739]

[0740] Design, construction, expression and protein purification of hBCMA×hCD16a CD16a binding polypeptide dual conjugate constructs change.

[0741] The hBCMA x hCD16a dual binding construct was designed for expression and purification as described above in Biological Example 11. The heterodimeric hBCMA x hCD16a dual binding construct prepared is listed above (SEQ ID 156 and 158-163).

[0742] The propensity of the hBCMA x hCD16a dual conjugate to stimulate CD16a activation and enhance NK cell-mediated killing was assessed according to the protocol described in Biological Example 11 above.

[0743] result

[0744] A hBCMA x hCD16a dual conjugate construct containing one BCMA binding polypeptide of SEQ ID 2 genetically fused to an hCD16a binding arm consisting of the polypeptide of SEQ ID 167 (with truncations in some cases) and a C-terminal His6 tag was constructed and expressed as a soluble gene product in E. coli (DE3).

[0745] Fig.13aThe CD16a activation response of the conjugate constructs in the presence or absence of target MM.1s cells is shown. In addition, the conjugates induced NK cell-mediated killing of MM.1s cells ( Fig.13b ).

[0746] The results show that the affibody-based heterodimeric hBCMA×hCD16a dual binding construct (here composed of the polypeptides of SEQ ID 2 (for hBCMA) and SEQ ID 167 (for hCD16a)) can activate hCD16a in a target-specific manner when genetically fused together, including with truncations at certain positions.

[0747] Biological Example 13:

[0748] NK cell-mediated killing of multiple myeloma cells

[0749] The ability of hBCMA×hCD16a dual conjugates to promote NK cell-mediated lysis and killing of cells was evaluated by flow cytometry assays and Incucyte-based assays. The compounds studied are shown in the following table:

[0750]

[0751] The compounds were produced according to the preparation method described in Biological Example 11.

[0752] In vitro flow cytometry-based assay.

[0753] NK cell function was assessed in a flow cytometry assay previously described (Bryceson, Fauriat et al. 2010). In brief, NK cells were co-cultured with target cells at an E:T ratio of 1:1 for 6 hours, followed by staining of degranulation markers CD107a and interferon-γ. BD GolgiStop (BD Biosciences) was added at a concentration of 1 / 1500 after 1 hour. For cytotoxicity assays, based on the kinetics from live cell imaging assays, target cells were pre-stained with CellTrace Violet (Invitrogen) and co-incubated with NK cells at different E:T ratios for 8 to 12 hours. Samples were obtained on BD LSR II or FACSymphony A5 (BD Biosciences). Data were analyzed using FlowJo version 10 (BD Biosciences).

[0754] Incucyte-based in vitro cytotoxicity assay.

[0755] Real-time tumor cell killing was measured on the Incucyte S3 platform (Essen Biosciences). Depending on the type of assay, NK cells were co-cultured with MM.1s cells labeled with red fluorescent protein mCherry on 96-well plates pre-coated with poly-L-ornithine 0.01% (Sigma-Aldrich) at different E:T ratios. For assays involving reagents (e.g., affibody constructs), these were added after effectors and target cells. Scanning was performed every 60 minutes using a 10X objective lens for at least 24 hours and images were analyzed using the Incucyte controller v2020A (Essen Biosciences). The same masking was used for repeated analysis. In the experiments performed, cells were treated with 100nM dual conjugates, invalid variants (not in combination with hBCMA) or daratumumab.

[0756] result

[0757] The ability of the dual conjugate example compound 131 to induce lysis of MM.1s cells was tested. The results are shown in Fig.14a The results are from one representative experiment (n=4). In a 24-hour killing assay, Example Compound 131 caused rapid and superior lysis of MM.1s cells compared to Daratumumab and the control null variant anti-hBCMA-null-H09-A10-His6. The overall cytotoxic effect of Example Compound 131 was observed within the first 8 to 12 hours after the start of co-culture.

[0758] Finally, flow cytometry-based killing assays were performed to establish functional EC 50 and the EC of three constructs of example compounds 131, 132, and 130 90 The frequency of dead target cells was calculated based on the staining of dead cell markers on target cells pre-incubated with cell tracer violet (CTV). The results are shown in Fig.14b Results are from two independent experiments (n=8), where spontaneous target cell death was subtracted. The plateau cytotoxicity of Example Compound 131 was the highest, and that of Example Compound 132 was the lowest, consistent with the degranulation response, showing that the latter construct had a higher fratricidal effect. Compared with Example Compound 130, Example Compound 131 reached plateau cytotoxicity at a lower dose. EC 50 The values ​​were 0.4 nM for example compound 131 and example compound 132, and 1.8 nM for example compound 130, while the EC90 values ​​were 2.0 nM, 3.8 nM, and 14.7 nM, respectively. The data demonstrate that these dual conjugates elicited an effective response.

[0759] Biological Example 14:

[0760] Enhances NK cell-mediated lysis of multiple myeloma cells in an hBCMA-selective manner.

[0761] The ability of the hBCMA×hCD16a dual conjugate to enhance NK cell-mediated lysis of multiple myeloma cells in a BCMA-selective manner was investigated. The compound tested was Exemplary Compound 131.

[0762] Genetic cell engineering

[0763] BCMA knockout variants of the cell line MM.1s were prepared using CRISPR-Cas9. BCMA overexpression variants of the cell line MM.1s and variants expressing the red fluorescent protein mCherry were performed by lentiviral transfection, and the vectors were designed and ordered from VectorBuilder (VectorBuilder Inc.). MM.1s expressing nuclear red fluorescent protein mCherry: MM.1s cells were transduced with 0.3MOI lentiviral particles. After transduction, approximately 30% of the cells appeared red under a fluorescence microscope. The cells were then sorted by fluorescence activated cell sorting (FACS) on a SonySH800 cell sorter (Sony Biotechnology). MM.1s with BCMA gene overexpression: On day 1, 1 million MM.1s cells were transduced with 10MOI lentiviral particles (denoted as SK-V93 and shown in Figure S6A) and seeded on 24-well plates. On day 4, the cells were washed in PBS and seeded at 0.5 million cells / ml in fresh culture medium. After sufficient amplification, the cells were subjected to FACS. MM.1s knocking out the BCMA gene: The following sgRNA sequences were used: sgRNA #0, gacgagtttaaaaacac (SEQ ID 287); sgRNA #1, gagcttaataatttctt (SEQ ID 288); and sgRNA #2, gtgaccaattcagtgaa (SEQ ID 289). sgRNA and Cas9 were assembled in a sterile 96-well U-bottom plate at a ratio of 9:1.

[0764] 300.000 MM.1s cells were used for each nucleofection. Knockout efficacy was confirmed five days later by PCR and Sanger sequencing, analyzed using Synthego Performance Analysis (ICE) (Synthego). After sufficient expansion, cells were subjected to FACS.

[0765] In vitro flow cytometry-based assay.

[0766] NK cell function was assessed in a flow cytometry-based assay as previously described (Bryceson, Fauriat et al. 2010). Briefly, NK cells were co-cultured with target cells at a 1:1 E:T ratio for 6 hours, followed by staining for degranulation markers CD107a and interferon-γ. BD GolgiStop (BD Biosciences) was added 1 hour later at a concentration of 1 / 1500.

[0767] result

[0768] Using engineered MM.1s target cells in which BCMA was knocked out or overexpressed, a clear correlation between BCMA expression levels and NK cell activation was observed (see Fig.15 ). The data demonstrate that dual conjugate example compound 131 at a concentration of 100 nM elicits a response specific for BCMA. The figure shows the results for MM.1s wild type (WT), BCMA knockout (KO), and BCMA overexpression (OE). The results are from one representative experiment (n=4).

[0769] Biological Example 15:

[0770] Another anti-hBCMA drug conjugate against BCMA + Multiple myeloma cells are cytotoxic.

[0771] An exemplary compound having the sequence of Fa-G6 (SEQ ID 1) with a c-terminal Cys was produced by conventional solid peptide synthesis. This compound has SEQ ID 164:

[0772]

[0773] The MC-MMAF linker toxin was dissolved in DMF organic solvent and conjugated at a molar ratio of 2: 1. The product containing one mc-MMAF was separated by RP-HPLC and prepared in PBS buffer containing 10% IPA.

[0774] By using Cell viability assay, the anticancer activity of the affibody drug conjugate Fa-G6-MMAF was investigated in five multiple myeloma cancer cell lines, namely EJM, MOLP-2, NCI-H929, OPM-2, and U-266. Fa-G6-MMAF was evaluated in a concentration range of 1 ng / mL to 30 μg / mL.

[0775] result

[0776] The results are shown in Fig.16 The Fa-G6-MMAF affibody-drug conjugate showed anticancer activity in all multiple myeloma cell lines studied, with EC50 The values ​​ranged from 0.19 μg / mL to 3.5 μg / mL. Thus, Fa-G6-MMAF showed activity in several BCMA+ multiple myeloma cell lines.

[0777] Biological Example 16A:

[0778] Compounds containing unnatural amino acid norleucine

[0779] experiment

[0780] The polypeptide (aBCMA-1-E6-(6-55)M35nL) having the sequence of SEQ ID 165 was synthesized by solid phase peptide synthesis (SPSS), purified by RP-HPLC and lyophilized. The correct Mw was confirmed by LC-MS / MS.

[0781]

[0782] The binding affinity to the extracellular domain of BCMA was studied by SPR in Biacore T200. BCMA was immobilized on a CM5 chip (Cytiva) as a ligand with 1300Ru. The example compound with SEQ ID 165 was used as an analyte and the response was monitored in a concentration range of 1 nM to 100 nM. The data were fitted to a 1:1 binding mode.

[0783] result

[0784] The exemplary compound having SEQ ID 165 was observed to bind BCMA with an apparent dissociation constant of 10 nM.

[0785] Biological Example 16B:

[0786] Evaluation of BCMA × hCD16a using the Jurkat CD16a activation reporter assay and NK-mediated cell killing assay Domain boundaries in dual conjugate constructs.

[0787] hBCMA x hCD16a dual binding constructs were generated and evaluated in hCD16a mediated activated cell based reporter assays and cell killing as described above in Biological Example 11. Conjugates with the full length aBCMA 1E6 domain (SEQ ID 156) were compared to conjugates carrying truncations at the N- and C-termini (SEQ ID 160) and 1E6

[0788] Activity of the conjugate with the VI G point mutation (SEQ ID 163).

[0789]

[0790] Design, construction, expression and protein purification of hBCMA×hCD16a CD16a binding polypeptide dual conjugate constructs change.

[0791] The hBCMA x hCD16a dual binding construct was designed for expression and purification according to Biological Example 11. The heterodimeric hBCMA x hCD16a dual binding construct prepared is listed above (SEQ ID 156, 160 and 163).

[0792] The propensity of the hBCMA x hCD16a dual conjugate to stimulate CD16a activation and enhance NK cell-mediated killing was assessed according to the protocol described in Biological Example 11.

[0793] result

[0794] A hBCMA×hCD16a dual conjugate construct containing one BCMA binding polypeptide genetically fused to the hCD16a binding arm and a C-terminal His6 tag was constructed and expressed as a soluble gene product in E. coli (DE3). Fig.17a The CD16a activation response of the conjugate constructs in the presence or absence of target MM.1s cells is shown. Fig.17b As shown, the conjugate induced NK cell-mediated killing of MM.1s cells.

[0795] The results show that the hBCMA binding arm consisting of the polypeptide of SEQ ID 2 (1E6) can be shortened and still retain hCD16a activation. In addition, valine 1 can replace glycine without loss of activity.

[0796] Biological Example 17:

[0797] Anti-BCMA conjugates induce immune cell activation in the presence of tumor cells.

[0798] Experiments were performed to evaluate the propensity of the anti-BCMA conjugate example compound having SEQ ID No. 130 to elicit an IFND□□ response in co-cultures of human PBMCs and BCMA-positive multiple myeloma cell line MM.IS. Thawed and overnight resting PBMCs (4W-270, Lonza) were co-cultured with MM.1s cells (CRL-2974, ATCC) in a V-shaped 96-well plate (249935, ThermoScientific) at an effector to target ratio of 25:1. A total of 4×10 5 Cells were treated with 200 nM anti-BCMA conjugate or 200 nM elotuzumab (300 mg Empliciti, Bristol-Myers Squibb) for 4 hours at 37°C in a humidified 5% CO2 incubator.

[0799] The supernatant was harvested and then centrifuged at 600 g for 5 minutes. Human IFN-γ ELISA MAX TM IFN-γ levels were measured by enzyme-linked immunosorbent assay (ELISA) using the Bio-Rad Biosystems Deluxe Set (430104, BioLegend). Data were processed using Excel and graphed using GraphPad Prism 9.

[0800] Anti-BCMA conjugate constructs having SEQ ID 130 containing a BCMA binding polypeptide (referred to herein as 1-E6 (SEQ ID 2)) genetically fused to an hCD16a binding polypeptide (referred to herein as A10 (SEQ ID 167)) were evaluated. The SLAMF7 monoclonal antibody elotuzumab was used as a positive control, and a single culture of PBMCs and MM.1s was used as a negative control. The dual conjugate containing the BCMA binding polypeptide elicited an IFND response in a co-culture of PBMCs and MM.1s cells, and the response was greater than that of the positive control elotuzumab ( Fig.18 ). The dual conjugate construct lacking BCMA binding showed no IFND response in co-cultures of PBMC and MM.1s cells. This experiment demonstrates BCMA-dependent activation of PBMCs against BCMA-positive MM.1s myeloma cell lines induced by the anti-BCMA conjugates described herein.

[0801] Biological Example 18:

[0802] CD16a activation and NK cell activation by hBCMA×hCD16a dual conjugate with hCD16a binding peptide Cytokine-mediated cell killing

[0803] hBCMA x hCD16a dual conjugates comprising hBCMA binding polypeptides having SEQ ID Nos. 166 and 144 were expressed in E. coli (DE3) and thereafter characterized for their CD16a activation properties.

[0804] The cDNA encoding each hBCMA×hCD16a double conjugate containing a stop codon was synthesized and ligated to the Ndel Xhol restriction site of the pET29 vector. The vector was transformed into E. coli BL21 (DE3) under kanamycin selection and the construct was expressed by inducing IPTG at an OD of 0.6 and harvested after 16 hours. The soluble cytoplasmic product was harvested and resuspended in 1×PBS. Heat denaturation at 95°C for 7 minutes was applied and the precipitated protein was precipitated by centrifugation at 20,000×g. Further purification was achieved by RP-HPLC and identity / purity was confirmed by SDS-PAGE and LC / MS / MS analysis. The tendency of hBCMA×hCD16a double conjugates to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated according to the protocol described in Biological Example 11.

[0805]

[0806]

[0807] result

[0808] The results are shown in Figure 19. As can be seen, the hBCMA×hCD16a dual conjugates (Examples 166 and 144) demonstrated CD16a activation in the Lucia luciferase reporter assay in the presence of hBCMA+MM.1s cells, while no detectable activation was observed in the absence of target cells ( Fig.19a In addition, the conjugate induced NK cell-mediated killing of MM.1s cells ( Fig.19b ). Thus, for the conjugates investigated, a robust and target-specific activation leading to NK-mediated cell killing was observed.

[0809] While the present invention has been described and illustrated with reference to specific embodiments, it will be appreciated by those skilled in the art that the present invention is susceptible to many different variations not specifically described herein.

[0810] In the foregoing description, when reference is made to integers or elements with known, obvious or foreseeable equivalents, such equivalents are incorporated herein as if set forth separately. Reference should be made to the claims to determine the true scope of the invention, which should be interpreted as encompass...

Claims

1. A hBCMA-binding polypeptide, comprising at least one motif that binds to hBCMA, wherein the polypeptide comprises the following structure: [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part] The hBCMA binding motif is part [helix 1]-[isolated part]-[helix 2].

2. The hBCMA-binding polypeptide of claim 1, wherein: i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other, X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V; or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

3. The hBCMA-binding polypeptide of claim 2, wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

2.

4. The hBCMA-binding polypeptide of claim 1, wherein: i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO.279], wherein, independently of each other, X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V; or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; And wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

2.

5. The hBCMA-binding polypeptide of claim 2, wherein: i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO.278], and helix 2 contains the sequence FYQKWAFIRX 33 LM, where, independently of each other, X9 is D, E, H, K, N, Q, S or V; X 10 is A, E, F, I, K, M, N, Q, R, S, T, Y, or V; X 11 is E, F or H; X 14 is A, E, H, I, K, L, Q, R, T or Y; X 17 is A, E, S, T or V; X 18 is A, F, H, K, L, M, N, T or S; X 33 is K or S; or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least one and no more than three (e.g., one, two, or three) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 3 (e.g., 1, 2, or 3) of the residues in the protein are replaced by alternative residues (e.g., by alternative residues that are conservative substitutions).

6. The hBCMA-binding polypeptide of claim 5, wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

2.

7. The hBCMA-binding polypeptide of any preceding claim, wherein: Helix 1 contains the sequence X6X7X8 X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 , and / or helix 2 contains the sequence, wherein X6 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or is absent; X7 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or absent; X8 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably E) or absent; X 19 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably L) or is absent; X 23 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or is absent; X 36 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or is absent; and X 37 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or is absent; and preferably wherein X6 is N; X7 is K; and X8 is E; and / or wherein X 36 is D; and X 37 It is D; For example, where X6 is N; X7 is K; X8 is E; X 19 Yes L; X 23 Yes N; X 36 is D; and X 37 It is D.

8. The hBCMA-binding polypeptide of claim 7, wherein: i) helix 1 comprises the sequence NKEETFADLEISNL and helix 2 comprises the sequence NFYQKWAFIRSLMDD, or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, at least 1 and not more than 2 (e.g. 1 or 2) residues are replaced by alternative residues, or (iii) at least 1 and not more than 3 (e.g. 1, 2 or 3) residues in the sequence of helix 1 and / or helix 2 are replaced by alternative residues (e.g. by alternative residues that are conservative substitutions).

9. The hBCMA-binding polypeptide of claim 8, wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

1.

10. The hBCMA-binding polypeptide of claim 7, wherein: i) helix 1 comprises the sequence NKENQFADEEIAAL and helix 2 comprises the sequence NFYQKWAFIRKLMDD, or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, at least 1 and not more than 2 (e.g. 1 or 2) residues are replaced by alternative residues, or (iii) at least one and no more than three (eg, one, two or three) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (eg, by an alternative residue that is a conservative substitution).

11. The hBCMA-binding polypeptide of claim 10, wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

2.

12. The hBCMA-binding polypeptide of any preceding claim, wherein the isolated portion is a sequence of 1 to 5 (preferably 2 to 5, such as 2, 3, 4 or 5; or such as 3 to 5, such as 3, 4 or 5, and more preferably 3) naturally occurring amino acids.

13. The hBCMA-binding polypeptide of any preceding claim, wherein the isolated portion has sequence X 20 X 21 X 22 ,in X 20 is any naturally occurring amino acid (preferably S, T, M, P, F, Y or W; more preferably P); X 21 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N); X 22 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably L); and wherein optionally X 20 , X 21 or X 22 One or both (e.g., optionally 1) of are absent; and preferably wherein X 20 It is P; X 21 is N; and X 22 is L (eg, the isolated portion has the sequence PNL).

14. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion is absent or It is a sequence of 1 to 15 (preferably 1 to 10 or 1 to 8, more preferably 1 to 5, such as 1, 2, 3, 4 or 5) naturally occurring amino acids.

15. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a It is M or not present; X b It is M or not present; X1 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably V or G; most preferably V) or absent; X2 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or absent; X3 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or absent; X4 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or is absent; and X5 is any naturally occurring amino acid (preferably F, Y or W; more preferably F) or absent; Preferably, X a M or not present, X b is M or absent, X1 is V, G or absent (preferably V or absent), X2 is D or absent, X3 is N or absent, X4 is K or absent, and X5 is F or absent.

16. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion is absent, or the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a It is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b not present, X1 is not present, X2 is D, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 is N, X4 is K, and X5 is F; or X a Does not exist, X b b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; or X a Does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F.

17. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is any naturally occurring amino acid (preferably G, A, V, L, or I; more preferably V or G; and most preferably V) or absent; X2 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or absent; X3 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or absent; X4 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or is absent; and X5 is any naturally occurring amino acid (preferably F, Y or W; more preferably F) or absent; And preferably wherein X1 is V, G or absent, X2 is D or absent, X3 is N or absent, X4 is K or absent, and X5 is F or absent.

18. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion is absent, or the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F.

19. The hBCMA-binding polypeptide of any preceding claim, wherein the C-terminal portion is absent or is a sequence of 1 to 50 (eg, 1 to 35, 1 to 30, 15 to 25, or 18 to 22) naturally occurring amino acids.

20. The hBCMA-binding polypeptide of claim 19, wherein the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 ,in X 38 is a sequence of 1 to 14 (preferably 1 to 9 or 1 to 7, more preferably 1 to 4, such as 1, 2, 3 or 4) naturally occurring amino acids (and preferably X 38 is any naturally occurring amino acid (most preferably X 38 Yes P)), X 39 is any naturally occurring amino acid (preferably S, T, M, P, F, Y or W; more preferably S), X 56 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably A) or is absent, X 57 is any naturally occurring amino acid (preferably P) or is absent; and X 58 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or is absent; (For example, X 38 It is P; X 39 Yes S; X 56 A or not; X 57 Is P or does not exist; X 58 is K or does not exist) and optionally wherein, At least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5) (preferably at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues; and preferably optionally wherein, At least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5) (preferably at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues as conservative substitutions.

21. The hBCMA-binding polypeptide of claim 20, wherein the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 ,in X 56 It's A, X 57 is P, and X 58 It is K; X 56 A; or X 57 is P, and X 58 does not exist; X 56 It's A, X 57 does not exist; and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 Does not exist. and optionally wherein, At least 1 and no more than 3 (e.g. 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues; and preferably optionally wherein at least 1 and no more than 3 (e.g. 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues as conservative substitutions; and Preferably, X 38 is P, and X 39 It’s S.

22. The hBCMA-binding polypeptide of any preceding claim, wherein: (i) the isolated portion has a sequence X 20 X 21 X 22 ; and / or The N-terminal portion has the sequence X a X b X1X2X3X4X5; and / or The C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 ; Wherein, in the separation part, X 20 It is P; X 21 Yes N; X 22 It is L; wherein in the N-terminal portion, X a M or not present; X b is M or absent; X1 is V or G (preferably V), or absent; X2 is D or G, or absent; X3 is G or N, or absent; X4 is K or absent; X5 is F or absent; and wherein in the C-terminal portion, X 56 A or not; X 57 Is P or does not exist; X 58 It is K or not present; or (ii) the separation portion, the N-terminal portion and the C-terminal portion are as defined in (i), wherein optionally (a) within each portion, 1, 2 or 3 residues are replaced by alternative residues; or (b) within those portions, at least 1 and no more than 10 (eg no more than 5, such as 1, 2, 3, 4 or 5) residues are collectively replaced by the alternative residues.

23. The hBCMA-binding polypeptide of any preceding claim, wherein the isolated portion has the sequence PNL.

24. The hBCMA-binding polypeptide of any preceding claim, wherein: (i) the isolated portion has the sequence PNL and the hBCMA binding motif is flanked by an N-terminal portion X1X2X3X4X5 and a C-terminal portion PSQSANLLAEAKKLNDAQAPK, wherein in the N-terminal portion, X1 is G, V or missing; X2 is D or missing; X3 is N or missing; X4 is K or missing; X5 is F or missing; or (ii) the isolated portion, the N-terminal portion and the C-terminal portion are as defined in (i), wherein within those portions at least 1 and no more than 5 residues are replaced together by alternative residues.

25. The hBCMA-binding polypeptide of any preceding claim, wherein the N-terminal portion has the sequence VDNKF.

26. The hBCMA-binding polypeptide of any one of claims 12 to 25, wherein the hBCMA binding efficacy is at least 1% of SEQ ID NO:

2.

27. The hBCMA-binding polypeptide according to any preceding claim, wherein the hBCMA-binding motif sequence is selected from SEQ ID NOs. 170 to 275; Preferably selected from SEQ ID NO.170-188; And wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions.

28. The hBCMA-binding polypeptide of claim 1 or claim 2, comprising a sequence selected from SEQ ID NO. 1, 2, 23-39, 41-119 or 121-128, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions; or The hBCMA-binding polypeptide according to claim 1 or claim 2, comprising a sequence selected from SEQ ID NO. 2 and 23-39, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions.

29. The hBCMA-binding polypeptide of claim 1 or claim 2, wherein the sequence of the hBCMA-binding polypeptide is selected from the group consisting of: VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEA KKLNDAQAPK[SEQ ID NO:1]; or VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAE AKKLNDAQAPK[SEQ ID NO:2]; or VDNKFNKEEIFADREIAFLPNLNFYQKWAFIRKLMDDPSQSANLLAEA KKLNDAQAPK[SEQ ID NO:23]; or VDNKFNKEHQFADYEIAMLPNLNFYQKWAFIRSLMDDPSQSANLLAE AKKLNDAQAPK[SEQ ID NO:33].

30. The hBCMA-binding polypeptide of claim 1 or claim 2, wherein the sequence of the hBCMA-binding polypeptide is selected from SEQ ID NOs. 23-39.

31. The hBCMA-binding polypeptide of any preceding claim, wherein the hBCMA-binding polypeptide does not comprise methionine.

32. An hBCMA-binding polypeptide having a sequence according to any preceding claim, wherein: At the positions where methionine residues are listed, the polypeptide has a sequence in which the methionine residues are independently substituted with a different naturally occurring amino acid or a non-natural amino acid (e.g., a different naturally occurring amino acid or norleucine); and preferably, each methionine residue is independently substituted with an amino acid selected from isoleucine (I), leucine (L), glutamine (Q) and norleucine; and more preferably, each methionine residue is independently substituted with norleucine or isoleucine.

33. The hBCMA-binding polypeptide of any preceding claim, wherein one or more residues (e.g., 1 to 5 residues, such as 1, 2, 3, 4, or 5) of the hBCMA-binding polypeptide are substituted with a non-natural amino acid, such as norleucine; and / or wherein one or more methionine residues when present (e.g., 1 or 2 methionine residues when present) are substituted with an unnatural amino acid, such as norleucine; and / or wherein one or more leucine residues when present (e.g., 1 to 5 leucine residues when present) are substituted with norleucine; and / or wherein one or more methionine residues when present (eg 1 or 2 methionine residues when present) are oxidized (eg are Met(O)).

34. An hBCMA-binding oligomer comprising at least two hBCMA-binding polypeptides as defined in any one of claims 1 to 33, such as 2, 3, 4 or 5 hBCMA-binding polypeptides as defined in any one of claims 1 to 33.

35. The hBCMA-binding oligomer of claim 34, comprising at least two hBCMA-binding polypeptides, wherein the first hBCMA-binding polypeptide is as defined in any one of claims 2 to 33, and the second hBCMA-binding polypeptide is as defined in any one of claims 2 to 33.

36. The hBCMA-binding oligomer of claim 35, comprising at least two hBCMA-binding polypeptides, wherein the first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide have the same sequence.

37. The hBCMA-binding oligomer of claim 35, comprising at least two hBCMA-binding polypeptides, wherein the first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide have different sequences.

38. The hBCMA-binding oligomer of claim 34, comprising at least two hBCMA-binding polypeptides, wherein: The first hBCMA-binding polypeptide comprises a first binding motif selected from SEQ ID NOs. 170 to 275 (preferably selected from SEQ ID NOs. 170-188) or comprises a first sequence selected from SEQ ID NOs. 1, 2, 23-39, 41-119 or 121-128; And the second hBCMA-binding polypeptide comprises a second binding motif selected from SEQ ID NOs. 170 to 275 (preferably selected from SEQ ID NOs. 170-188) or comprises a second sequence selected from SEQ ID NOs. 1, 2, 23-39, 41-119 or 121-128.

39. The hBCMA-binding oligomer of claim 38, comprising at least two hBCMA-binding polypeptides, wherein the first hBCMA-binding polypeptide and the second hBCMA-binding polypeptide have the same sequence, or the selected first binding motif and the second binding motif have the same sequence.

40. The hBCMA-binding oligomer of claim 38, comprising at least two hBCMA-binding polypeptides, wherein the selected first and second binding motifs or the first and second hBCMA-binding polypeptides have different sequences.

41. The hBCMA-binding oligomer of any one of claims 34 to 40, wherein the hBCMA-binding polypeptides are each separated by a linker.

42. The hBCMA-binding oligomer of claim 41, wherein the linker is a sequence of 1 to 50 (e.g., 1 to 25) naturally occurring amino acids; preferably a sequence of 1 to 25 (e.g., 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S).

43. The hBCMA-binding oligomer of claim 41 or 42, wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGS GGGGS GGGGS G, GGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGG, GGS GGGGS GGGGS GG, GGGGS, GGGGSGGGGS GGGGS, GGGGS, GGGGSGGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS.

44. The hBCMA-binding oligomer according to any one of claims 41 to 43, wherein the hBCMA-binding oligomer comprises at least 2 (e.g., 2) hBCMA-binding polypeptides, and the hBCMA-binding oligomer comprises the following structure: [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]; wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (e.g. 1-20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S) (e.g., wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGSGGGGSGGGGSG, GGSGGGGSGGGGSG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG, GGGGS, GGGGSGGGGSGGGGSGG, GGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGS GGGGS; wherein each N-terminal portion in the oligomer can have the same sequence or have a different sequence; Each C-terminal portion in the oligomer can have the same sequence or a different sequence; Each separate portion of the oligomer can have the same sequence or a different sequence; Each helix 1 portion in the oligomer can have the same sequence or a different sequence; and Each helix 2 portion in the oligomer can have the same sequence or a different sequence.

45. The hBCMA-binding oligomer of claim 44, wherein the hBCMA-binding oligomer comprises at least 3 (e.g., 3) hBCMA-binding polypeptides, and the hBCMA-binding oligomer comprises the following structure: [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]; wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (e.g. 1-20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S) (e.g., wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGGGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS; and wherein each linker moiety in the oligomer can have the same sequence or have a different sequence; Each N-terminal portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each C-terminal portion can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each separate portion of the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each helix 1 portion in the oligomer can have the same sequence, can have a different sequence, or two can have the same sequence and one can have a different sequence; and Each helix 2 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence.

46. ​​The hBCMA-binding polypeptide of one of claims 2 to 33, or the hBCMA-binding oligomer of any one of claims 34 to 45, further comprising additional functional moieties (e.g., at least one, at least two, or at least three; such as 1, 2, 3, 4, or 5 additional functional moieties).

47. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 46, wherein the additional functional moiety comprises an immune signaling molecule or a derivative thereof, such as a cytokine or a derivative thereof, such as IL-15 or a derivative thereof.

48. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 46, wherein the additional functional moiety comprises an additional binding moiety.

49. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 48, wherein the additional binding moiety is specific for an immune cell surface target, such as a NK cell activating receptor, such as CD16a.

50. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 46, wherein the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two (e.g., 2, 3, 4, or 5) additional functional moieties, wherein each additional functional moiety can be the same or can be different.

51. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 50, wherein the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two (eg, 2, 3, 4, or 5) additional functional moieties, wherein the first additional functional moiety comprises an additional binding moiety (e.g., an additional binding moiety specific for an immune cell surface target, such as an NK cell activation receptor, such as CD16a); and wherein the second additional functional moiety comprises an immune signaling molecule or a derivative thereof, such as a cytokine or a derivative thereof, such as IL-15 or a derivative thereof; or wherein the first additional functional portion comprises an additional binding portion (e.g., an additional binding portion that is specific for an immune cell surface target such as an NK cell activation receptor such as CD16a); and wherein the second additional functional portion comprises an additional binding portion (e.g., an additional binding portion that is specific for an immune cell surface target such as an NK cell activation receptor such as CD16a).

52. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 51, comprising additional functional moieties, wherein the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 3 (e.g., 3, 4, or 5) additional functional moieties, wherein the third additional functional moiety comprises an additional binding moiety that is specific for an immune cell surface target, such as an NK cell activation receptor, such as CD16a, or comprises an immune signaling molecule or a derivative thereof, such as a cytokine or a derivative thereof, such as IL-15 or a derivative thereof.

53. The hBCMA-binding polypeptide or hBCMA-binding oligomer according to any one of claims 46 to 52, wherein the additional functional moiety is separated from the hBCMA-binding polypeptide or the hBCMA-binding oligomer by a linker, For example, wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (eg 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S).

54. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 65, wherein the linker wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSG, GGS GGGGS GGGGS GG, GGGSGGGGSGGGGSGGGGSGG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS or GGGGS GGGGS GGGGS GGGGS.

55. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claim 53 or 54, comprising an additional functional moiety, wherein the hBCMA-binding polypeptide comprises the following structure: [N-terminal part] - [helix 1] - [isolation part] - [helix 2] - [C-terminal part] - [linker] - [additional functional part]; or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]; or [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part]; [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]; or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Additional functional part]; wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (e.g. 1-20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S) (e.g., wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS or GGGGS GGGGS GGGGS GGGGS). and in, When there is more than one additional functional part, each additional functional part can be the same or different.

56. The hBCMA-binding polypeptide or hBCMA-binding oligomer of claims 53 to 55, comprising the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLP NLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK[SEQ ID NO.139]; VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQS ANLLAEAKKLNDAQAPK[SEQ ID NO.140]; <h2 style=";text-align:left;direction:ltr">VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQS ANLLAEAKKLNDAQAPK[SEQ ID NO.141]; <h2 style=";text-align:left;direction:ltr"> VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGGSGGGGVDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQS ANLLAEAKKLNDAQAPK[SEQ ID NO.142]; <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.143]; <h2 style=";text-align:left;direction:ltr"> VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGVDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKL NDAQAPK[SEQ ID NO.144]; <h2 style=";text-align:left;direction:ltr"> VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQS <h2 style=";text-align:left;direction:ltr"> ANLLAEAKKLNDAQAPKGGGSGGGGGSGGGGSGVDNKFNKENQF <h2 style=";text-align:left;direction:ltr"> ADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQ APKGGGSGGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHH QSFAFIKSLMDDPSQS ANLLAEAKKLNDAQAPK [SEQ ID NO. 145].

57. An hBCMA-binding agent-drug conjugate comprising the hBCMA-binding polypeptide or hBCMA-binding oligomer according to any one of claims 1 to 56 and an additional therapeutic agent.

58. The hBCMA binding agent-drug conjugate of claim 57, wherein the additional therapeutic agent is a cytotoxic drug, such as MMAF, MMAE, doxorubicin, pyrrolobenzodiazepine, Amanitine, maytansinoids, dolastatin, mitomycin C, desmethyltopotecan, or SN-38.

59. The hBCMA-binding agent-drug conjugate of claim 57 or claim 58, wherein the hBCMA-binding polypeptide is linked to the additional therapeutic agent via a linker.

60. A nucleic acid molecule encoding the hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56; or encoding the hBCMA-binding oligomer of any one of claims 34 to 56.

61. An expression vector comprising the nucleic acid molecule according to claim 60.

62. A host cell comprising the nucleic acid molecule according to claim 60 or the expression vector according to claim 61.

63. A method for preparing the hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the method comprising maintaining the host cell of claim 62 under optimal conditions for expression of nucleic acid, and isolating the hBCMA-binding polypeptide or hBCMA-binding oligomer.

64. A pharmaceutical composition comprising the hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the hBCMA-binding agent-drug conjugate of any one of claims 57 to 59, the nucleic acid molecule of claim 60, or the expression vector of claim 61.

65. The hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the hBCMA-binding agent-drug conjugate of claims 57 to 59, the nucleic acid molecule of claim 60, the expression vector of claim 61 and / or the pharmaceutical composition of claim 64 for use in medicine.

66. The hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the hBCMA-binding agent-drug conjugate of claims 57 to 59, the nucleic acid molecule of claim 60, the expression vector of claim 61 and / or the pharmaceutical composition of claim 64 for use in treating cancer.

67. The hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the hBCMA-binding agent-drug conjugate of claims 57 to 59, the nucleic acid molecule of claim 60, the expression vector of claim 61 and / or the pharmaceutical composition of claim 64 for use according to claim 66, wherein the cancer is multiple myeloma.

68. Use of the hBCMA-binding polypeptide of any one of claims 1 to 33 or 46 to 56, the hBCMA-binding oligomer of any one of claims 34 to 56, the hBCMA-binding agent-drug conjugate of claims 57 to 59, the nucleic acid molecule of claim 60, the expression vector of claim 61 and / or the pharmaceutical composition of claim 64 for the manufacture of a medicament for treating cancer.

69. A method of treating cancer, the method comprising administering to a patient in need thereof an hBCMA-binding polypeptide according to any one of claims 1 to 33 or 46 to 56, an hBCMA-binding oligomer according to any one of claims 34 to 56, an hBCMA-binding agent-drug conjugate according to claims 57 to 59, a nucleic acid molecule according to claim 60, an expression vector according to claim 61, and / or a pharmaceutical composition according to claim 64.

70. A kit comprising an hBCMA-binding polypeptide according to any one of claims 1 to 33 or 46 to 56, an hBCMA-binding oligomer according to any one of claims 34 to 56, an hBCMA-binding agent-drug conjugate according to claims 57 to 59, or a pharmaceutical composition according to claim 64, and optionally one or more additional therapeutic agents.

71. The kit of claim 70, wherein the one or more additional therapeutic agents are selected from a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflufenamic acid), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

72. A kit according to claim 70 or claim 71 for use in treating cancer, such as multiple myeloma.

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