Novel polypeptide

Abstract

The present invention provides an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the peptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the hBCMA-binding motif is the portion [Helix 1]-[Spacer portion]-[Helix 2]. The present invention further provides a pharmaceutical composition comprising the hBCMA-binding polypeptide, and the use of the hBCMA-binding polypeptide or the pharmaceutical composition for use as a medicament, particularly for the treatment or prevention of cancer.

Description

Technical Field

[0001] The present invention relates to a human BCMA (human BCMA, hBCMA) binding polypeptide. The present invention also relates to a pharmaceutical composition comprising the above-described hBCMA binding polypeptide, and their use in the treatment and / or prevention of cancer.

Background Art

[0002] Immunotherapy has been shown to be an effective treatment for some cancers with approved therapies that include monoclonal antibodies and bispecific antibodies, immunomodulatory drugs, CAR-T therapy, and antibody-drug conjugates. However, despite the activity of these treatments, some patients exhibit a very short response or are unable to respond to the treatment. Side effects from some immunotherapies, particularly those associated with exacerbated cytokine release, can be severe. In fact, cytokine release syndrome is one of the most common serious side effects of T cell-induced immunotherapy agents (Shimabukuro-Vornhagen A et al. Cytokine release syndrome. J Immunother Cancer. 2018;6(1):56. doi:10.1186 / s40425-018-0343-9). Many patients also ultimately become resistant to the available treatments. Thus, despite recent advances, there remains a need for additional treatment options in cancer immunotherapy.

[0003] One obvious obstacle of current treatment modalities is the suboptimal distribution to tumor tissue. The rate of tissue distribution is negatively correlated with molecular size, and thus 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 the production of immunosuppressive cytokines and other molecules (e.g., TGFβ or VEGF), cell-mediated immunosuppression via tumor-derived regulatory T cells, regulation of antigen presentation and MHC I expression, or alteration of the expression of other ligands, e.g., increased expression of inhibitory checkpoint ligands (such as Programmed death-ligand 1 (PD-L1) and HLA-E) that reduce 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(Supplement):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 hematological malignancy that remains incurable despite recent advances in immunotherapy. Several surface proteins that are concentrated on myeloma cells have been identified and function as targets for therapeutic intervention. One such target is BCMA (TNFRSF17), which is highly expressed on myeloma cells but also on plasmacytoid dendritic cells. Two B cell-stimulating ligands, proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF), bind to BCMA and are involved in 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] CAR-T cell therapy and antibody-based therapies targeting BCMA have been proven effective in clinical practice in extending the overall survival of patients. Still, not all patients show a satisfactory response to the treatment, and patients will ultimately relapse from the disease (for example, Teoh, P. J., Chng, W. J. CAR T-cell therapy in multiple myeloma: more room for improvement. Blood Cancer J. 11, 84 (2021). See https: / / doi.org / 10.1038 / s41408-021-00469-5).

[0006] Therefore, there remains a need for improved cancer immunotherapy drugs that can retain the target specificity of antibodies and antibody-based drugs and deliver them more efficiently to tumors while avoiding potential side effects such as immunogenicity.

[0007] The present invention aims to address the above-mentioned need.

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 has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the hBCMA-binding motif is the portion [Helix 1]-[Spacer portion]-[Helix 2].

[0009] In particular, the present invention provides an hBCMA-binding polypeptide, i) Helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], and Helix 2 comprises the sequence FX 25 QKWAFX 31 RX33 LX 35 including [SEQ ID NO: 279], and independently of each other, a) X9 and X 10 are any naturally occurring amino acids, and X 11 is E, F, H, Q, T or Y, and X 14 is any naturally occurring amino acid, and X 17 is A, E, Q, S, T or V, and X 18 is any naturally occurring amino acid, and X 25 is F or Y, and X 31 is I, M, or V, and X 33 is K or S, and X 35 is I, L, M, or V, or alternatively ii) helix 1 and helix 2 are defined as in i), and within helix 1 and helix 2, X n at least 1 and 5 or fewer (e.g., at least 1 and 3 or fewer) of the residues are replaced by alternative residues, and / or X n at least 1 and 5 or fewer (e.g., at least 1 and 3 or fewer) of the residues not labeled with X are replaced by alternative residues, to provide an hBCMA-binding polypeptide.

[0010] The present invention also provides an hBCMA-binding polypeptide, i) helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 including [SEQ ID NO: 278], and helix 2 comprises the sequence FX 25 QKWAFX 31 RX 33 LX 35 including [SEQ ID NO: 279], and independently of each other, a) X9 and X 10 are any naturally occurring amino acids, and X 11 is E, F, H, Q, T or Y, and X 14 is any naturally occurring amino acid, and X 17 is A, E, Q, S, T or V, and X18 is any naturally occurring amino acid, and X 25 is F or Y, and X 31 is I, M, or V, and X 33 is K or S, and X 35 is I, L, M, or V, or alternatively ii) helices 1 and 2 are defined as in i), and within helices 1 and 2, X n at least 1 and up to 5 (e.g., at least 1 and up to 3) of the residues are replaced by alternative residues, and / or X n at least 1 and up to 5 (e.g., at least 1 and up to 3) of the residues not labeled with X are replaced by alternative residues, provides an hBCMA-binding polypeptide having an hBCMA-binding efficacy of at least 1% of SEQ ID NO: 2.

[0011] The inventors of the present application have surprisingly found that such hBCMA-binding polypeptides based on non-antibody scaffolds are effective in binding to cancer cells and inducing cytotoxic drug-mediated or antibody-dependent cellular cytotoxicity-mediated (ADCC-mediated) cancer cell death.

[0012] Accordingly, the present invention provides novel hBCMA-binding polypeptides (also referred to as "engagers") effective in binding to cancer cells. They have found specific uses as cancer cell-binding units in conjugates and fusion proteins capable of inducing cytotoxic drug-mediated or ADCC-mediated cancer cell death, and thus they show promise in anti-cancer immunotherapy.

[0013] The present invention further provides an hBCMA-binding oligomer comprising at least two hBCMA-binding polypeptides of the present invention.

[0014] The present invention further provides the hBCMA-binding polypeptide disclosed herein, or the hBCMA-binding oligomer disclosed herein, which further comprises additional functional moieties (e.g., at least 1, at least 2, or at least 3 additional functional moieties, such as 1, 2, 3, 4, or 5 additional functional moieties).

[0015] Accordingly, in embodiments where the additional functional moiety is an additional binding moiety, the present invention further provides a bispecific or multispecific engager comprising the additional binding moiety.

[0016] The present invention further provides an hBCMA-binding agent-drug conjugate comprising the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention and an additional therapeutic agent.

[0017] The present invention further provides the following. - A nucleic acid molecule encoding the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention, - An expression vector comprising such a nucleic acid molecule, - A host cell comprising such a nucleic acid molecule or vector.

[0018] The present invention further provides a method for producing the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention.

[0019] 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.

[0020] The present invention further provides the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, nucleic acid molecule, expression vector, or pharmaceutical composition disclosed herein for use in medicine, particularly for the treatment of cancer.

[0021] The present invention also provides the use of an 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 the treatment of cancer.

[0022] The present invention also provides a method of treating cancer, the method comprising administering to a patient in need thereof an active ingredient comprising an hBCMA-binding polypeptide or hBCMA-binding oligomer disclosed herein. For example, the hBCMA-binding polypeptide or hBCMA-binding oligomer may be present as a cancer cell-binding unit in a conjugate or fusion protein, such as one capable of inducing cytotoxic drug-mediated or ADCC-mediated cancer cell death. The present invention also provides a method of treating cancer, the method comprising administering to a patient in need thereof a nucleic acid molecule, expression vector or pharmaceutical composition disclosed herein.

[0023] The present invention further provides a kit comprising an hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition disclosed herein, and optionally one or more additional therapeutic agents. Such kits find particular use in the treatment and / or prevention of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

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Mode for Carrying Out the Invention

[0025] The inventors of the present application have found that the hBCMA-binding polypeptides disclosed herein are surprisingly effective in binding to cancer cells. Thus, they find particular uses in the treatment and / or prevention of cancer, for example, as cancer cell-binding units in conjugates and fusion proteins.

[0026] As will be discussed in more detail below, the inventors of the present application have surprisingly found that hBCMA-binding polypeptides based on a three-helix affibody scaffold are effective in binding to BCMA on myeloma cells and induce a strong ADCC-mediated or cytotoxic drug-mediated response against cancer cells. Such ADCC-mediated anti-cancer responses are beneficial in the treatment of multiple myeloma.

[0027] Notably, the inventors of the present application have found that such anti-cancer responses are advantageous compared to the responses obtained using the monoclonal antibody elotuzumab, which is approved for the treatment of multiple myeloma. The inventors of the present application have further found that such anti-cancer responses favorably match the responses obtained using a biosimilar of belantamab mafodotin, which is an antibody-drug conjugate for the treatment of multiple myeloma, and the responses obtained using daratumumab, which is a monoclonal antibody approved for the treatment of multiple myeloma.

[0028] In its broadest aspect, the invention is an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], Provided is an hBCMA-binding polypeptide, wherein the hBCMA-binding motif is the moiety [Helix 1]-[Spacer]-[Helix 2].

[0029] Herein, various elements of the polypeptide of the present invention are described in further detail.

[0030] Overall structure In one embodiment, the polypeptide of the present invention may be based on a three-helix scaffold, sometimes referred to as an “affibody”. An affibody is a small (about 6.5 kDa) engineered affinity ligand based on the Z-domain polypeptide, a variant 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 contains [Second Spacer]-[Helix 3]-[C-terminal sequence]. The general structure of an affibody is shown in FIG. 1.

[0031] The portions of the molecule of the present invention referred to as Helix 1 and Helix 2 (and Helix 3 if present) generally have a helical structure. In some rare embodiments, it may be found that the structure established by a sequence with specific residues is not strictly helical. Such compounds should be considered within the broadest aspects of the present invention. More preferably, the residues in the Helix 1 and Helix 2 portions result in a helical structure in terms of being alpha helices.

[0032] Sequence of the polypeptide of the present invention The sequences of the hBCMA-binding polypeptides disclosed herein can be represented in terms of their constituent amino acids or in terms of the nucleic acid sequences encoding the polypeptides having those amino acid sequences. In the context of this 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 an amino acid that is not found in nature or that is chemically synthesized and does not constitute (i.e., is not encoded by) a protein (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 in any chiral conformation. The amino acid may particularly be a naturally occurring α-amino acid. The amino acid may particularly be a naturally occurring L-amino acid. The amino acid may particularly be a naturally occurring L-α-amino acid.

[0033] Within a polypeptide chain (e.g., the hBCMA-binding polypeptides disclosed herein), amino acids are linked by peptide bonds between the carboxyl group of one amino acid in the chain and the amine group of the next amino acid. Individual amino acids, when linked to a polypeptide chain, are called "residues" or "amino acid residues."

[0034] Amino acid sequences in this specification are shown with the N-terminus to the left, and when the sequence is arranged over multiple lines, the N-terminus is at the upper left. Unless otherwise specified, amino acid residues in the sequence are L-amino acids.

[0035] The amino acid sequences recited in this application are shown using standard abbreviations for amino acids.

[0036] The specific sequences shown in this specification relate to specific embodiments of the invention.

[0037] The present disclosure also includes derivatives of all sequences described herein (e.g., derivatives of each of the hBCMA-binding polypeptides and hBCMA-binding oligomeric sequences described herein). Derivatives of the sequences described herein preferably have 1 to 5 (e.g., 1, 2, or 3) amino acid residues replaced by alternative residues, e.g., different naturally occurring amino acids or different non-natural amino acids, or different naturally occurring amino acids excluding methionine, or different non-natural amino acids. Preferably, the non-natural amino acids according to the present invention are those that are isoelectronic with naturally occurring amino acids, e.g., norleucine.

[0038] The present disclosure also includes derivatives of the sequences described herein in which 1 to 5 (e.g., 1, 2, or 3) amino acid residues can be replaced by alternative residues that are conservative replacements, i.e., the residues are replaced by, e.g., another residue within the same class.

[0039] Aliphatic residues (glycine (G), alanine (A), valine (V), leucine (L), or isoleucine (I)) may be replaced by another aliphatic residue. Hydroxyl-containing residues, sulfur-containing residues, or selenium-containing residues (serine (S), cysteine (C), selenocysteine (U), threonine (T), or methionine (M)) may be replaced by another hydroxyl-containing residue, sulfur-containing residue, or selenium-containing residue. Aromatic residues (phenylalanine (F), tyrosine (Y), or tryptophan (W)) may be replaced by another aromatic residue. Basic residues (histidine (H), lysine (K), arginine (R)) may be replaced by another basic residue. Acidic residues or amides (aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)) may be replaced by another acidic residue or amide.

[0040] In certain embodiments, the sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) are methionine-free. In certain embodiments, in the sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein), at positions where methionine residues are listed, the polypeptide independently has a sequence in which the methionine residue is replaced with a different residue, e.g., a different naturally occurring amino acid or a non-natural amino acid. In certain embodiments, in the sequences described herein, at positions where methionine residues are listed, the polypeptide has a sequence in which the methionine residue is independently replaced with an amino acid selected from isoleucine (I), leucine (L), glutamine (Q), and norleucine, e.g., isoleucine and norleucine.

[0041] In another embodiment, if one or more methionines 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 with alternative amino acids, e.g., different naturally occurring amino acids or non-natural amino acids such as isoleucine (I), leucine (L), glutamine (Q), or norleucine, particularly amino acids selected from isoleucine (I) and norleucine.

[0042] For example, in one embodiment, one or more (e.g., each) methionine residues of the sequences described herein may be replaced with different naturally occurring amino acids or non-natural amino acids such as isoleucine, leucine, glutamine, and norleucine, particularly amino acids selected from isoleucine and norleucine. For example, 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, if present, may be replaced with different naturally occurring amino acids or non-natural amino acids such as isoleucine, leucine, glutamine, and norleucine, particularly amino acids selected from isoleucine and norleucine.

[0043] For example, in embodiments where X9 can be or is methionine, the residue at X9 can be replaced by an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine. For example, X 10 can be or is methionine, in embodiments where X 10 the residue at can be replaced by an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine. For example, X 14 can be or is methionine, in embodiments where X 14 the residue at can be replaced by an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine. For example, X 18 can be or is methionine, in embodiments where X 18 the residue at can be replaced by an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine. For example, X 31 can be or is methionine, in embodiments where X 31 the residue at can be replaced by an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine. For example, X 35 can be or is methionine, in embodiments where X 35The residues at 31 and 35 can be methionine, or in embodiments where they are methionine, the residues at 31 and 35 can be replaced by amino acids selected from isoleucine, leucine, glutamine, and norleucine, particularly different naturally occurring or non-natural amino acids such as isoleucine and norleucine.

[0044] Alternatively, or in addition, in certain embodiments, the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) can contain amino acid substitutions in which one or more residues are replaced by non-natural amino acids.

[0045] For example, in one embodiment, one or more residues of the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may be replaced by non-natural amino acids such as norleucine. For example, 1 to 15 residues may be replaced by non-natural amino acids, for example, 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 residues), 1 to 3 residues (e.g., 1, 2, or 3 residues), or 1 residue may be replaced by a non-natural amino acid (e.g., norleucine).

[0046] For example, in one embodiment, one or more leucine residues of the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may be replaced by non-natural amino acids, preferably norleucine. For example, 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, if present, may be replaced by non-natural amino acids (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein), at the positions where leucine residues are listed, the polypeptide independently has a sequence in which the leucine residue is replaced by a non-natural amino acid, preferably norleucine.

[0047] Alternatively, or in addition, in one embodiment, one or more methionine residues of the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may be replaced by non-natural amino acids, preferably norleucine. For example, 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, if present, may be replaced by non-natural amino acids (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein), at the positions where methionine residues are listed, the polypeptide independently has a sequence in which the methionine residue is replaced by a non-natural amino acid, preferably norleucine.

[0048] For example, in embodiments where X9 can be or is methionine, the residue at X9 may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 10 can be or is methionine, in embodiments where X 10The residue at may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 14 may be methionine, or in embodiments where it is methionine, the residue at X 14 may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 18 may be methionine, or in embodiments where it is methionine, the residue at X 18 may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 31 may be methionine, or in embodiments where it is methionine, the residue at X 31 may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 35 may be methionine, or in embodiments where it is methionine, the residue at X 35 may be replaced by a non-natural amino acid (e.g., norleucine). In particular, in embodiments where X 31 and X 35 may be methionine, or are methionine, the residues at X 31 and X 35 may be replaced by a non-natural amino acid (e.g., norleucine).

[0049] In certain embodiments, one or more methionine residues of the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may be oxidized, i.e., may be in the form of methionine sulfoxide ("Met(O)"). For example, if 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 oxidized (e.g., may be Met(O)). For example, if present, in embodiments where X9 may be methionine, or is methionine, the methionine at X9 may be oxidized (e.g., Met(O)). For example, if present, X 10can be methionine, or in embodiments where it is methionine, X 10 the methionine at may be oxidized (e.g., Met(O)). For example, if present, X 14 can be methionine, or in embodiments where it is methionine, X 14 the methionine at may be oxidized (e.g., Met(O)). For example, if present, X 18 can be methionine, or in embodiments where it is methionine, X 18 the methionine at may be oxidized (e.g., Met(O)). For example, if present, X 31 can be methionine, or in embodiments where it is methionine, X 31 the methionine at may be oxidized (e.g., Met(O)). For example, if present, X 35 can be methionine, or in embodiments where it is methionine, X 35 the methionine at may be oxidized (e.g., Met(O)). In particular, if present, X 31 and X 35 can be methionine, or in embodiments where they are methionine, X 31 and X 35 the methionine at may be oxidized (e.g., Met(O)).

[0050] Alternatively, or in addition, in certain embodiments, the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) 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., one selected from the single peptides of OmpA, DsbA, PhoA, and PelB), a fluorophore tag (e.g., Alexa448), or a tag or moiety for assisting 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 polypeptides and BCMA-binding oligomer sequences described herein.

[0051] Accordingly, the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may further comprise an additional sequence of at least one histidine residue (and optionally at least one tyrosine residue) and / or at least one methionine residue, e.g., at least 4, at least 5, or at least 6 histidine residues (and optionally at least one tyrosine residue, e.g., 1, 2, or 3 tyrosine residues) and / or at least one methionine residue. In one embodiment, the sequences described herein (e.g., the hBCMA-binding polypeptide and hBCMA-binding oligomer sequences described herein) may further comprise an additional sequence of at least 6 histidine residues and optionally at least one tyrosine residue (e.g., 6 histidine residues (e.g., HHHHHH) or 6 histidine residues and 2 tyrosine residues (e.g., YYHHHHHH)) and / or at least one methionine residue (e.g., 1 or 2 methionine residues). Peptide purification tags or moieties, such as the histidine tags or methionine tags described above, may preferably be present at the N-terminus and / or C-terminus of the hBCMA-binding polypeptides 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 2 tyrosine residues) and / or at least one methionine residue (e.g., 1 or 2 methionine residues) may be present at the N-terminus and / or C-terminus of the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein.

[0052] The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may further comprise 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 further comprise a fluorophore tag (e.g., 448Alexa 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 further comprise a signal peptide selected from, for example, OmpA, DsbA, PhoA, and PelB, preferably at the N-terminus. The sequences described herein (e.g., the hBCMA-binding polypeptides and hBCMA-binding oligomer sequences described herein) may further comprise an additional sequence of at least one glycine (e.g., one glycine) at the N-terminus or C-terminus.

[0053] In certain embodiments, the hBCMA-binding polypeptide has an hBCMA-binding efficacy that 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 an hBCMA-binding polypeptide is described herein as having an hBCMA-binding efficacy that is at least X% of a particular peptide (e.g., SEQ ID NO: 2), the IC 50 concentration of the polypeptide for binding to the hBCMA receptor, when measured under the same conditions, is understood to be 100 / X times or less the IC 50 concentration of the particular peptide (e.g., SEQ ID NO: 2) for the hBCMA receptor.

[0054] For example, when the binding efficacy of the hBCMA-binding polypeptide is at least 5%, more preferably at least 10%, 20%, 25%, or 50% of the hBCMA-binding efficacy of a particular peptide (e.g., SEQ ID NO: 2), i.e., the IC 50When measured under the same conditions, the IC of a specific peptide (e.g., SEQ ID NO: 2) against the hBCMA receptor 50 is 20-fold or less, more preferably 10-fold or less, 5-fold or less, 4-fold or less, or 2-fold or less of each of their concentrations.

[0055] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with the peptide of SEQ ID NO: 2 for binding to hBCMA.

[0056] The present invention provides an hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the hBCMA-binding motif is the portion [Helix 1]-[Spacer portion]-[Helix 2].

[0057] Preferably, the hBCMA-binding polypeptide i) Helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], and Helix 2 comprises the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO: 279], and independently of each other, X9 and X 10 are 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 35is I, L, M, or V, or alternatively ii) helices 1 and 2 are defined as in i), and within helices 1 and 2, X n at least 1 and up to 5 (e.g., at least 1 and up to 3) of the residues are replaced by alternative residues, and / or X n at least 1 and up to 5 (e.g., at least 1 and up to 3) of the residues labeled with X and not labeled with X are replaced by alternative residues, and is an hBCMA-binding polypeptide.

[0058] In one embodiment, helix 1 comprises the sequence KEX9X 10 X 11 ADX 14 EIX 17 X 18 and helix 2 comprises the sequence FX 25 QKWAFX 31 RX 33 LX 35 D, for example, helix 1 comprises the sequence NKEX9X 10 X 11 ADX 14 EIX 17 X 18 L and helix 2 comprises the sequence NFX 25 QKWAFX 31 RX 33 LX 35 DD, and X9, X 10 , X 11 , X 14 , X 17 , X 18 , X 25 , X 31 , X 33 , and X 35 are as defined above.

[0059] Preferably, such an hBCMA-binding polypeptide has an hBCMA-binding efficacy that is at least 1% of SEQ ID NO: 2. Preferably, the binding efficacy is at least 5%, e.g., at least 10%, 20%, 25%, or 50% of the hBCMA-binding efficacy of a specific peptide (e.g., SEQ ID NO: 2).

[0060] In certain embodiments, alternatively or additionally, the hBCMA-binding polypeptide competes with SEQ ID NO: 2 for binding to hBCMA.

[0061] In one embodiment, the hBCMA-binding polypeptide is such that i) helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], helix 2 comprises the sequence FX 25 QKWAFX 31 RX 33 LX 35 [SEQ ID NO: 279], and, independently of each other, X9 and X 10 are 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 alternatively ii) helix 1 and helix 2 are as defined in i), and within helix 1 and helix 2, at least 1 and up to 5 (e.g., at least 1 and up to 3) of the X n residues are replaced by alternative residues and / or at least 1 and up to 5 (e.g., at least 1 and up to 3) of the residues not labeled as X n are replaced by alternative residues, An hBCMA-binding polypeptide, wherein the hBCMA-binding efficacy is at least 1% of SEQ ID NO: 2.

[0062] Preferably, 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).

[0063] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 2 of hBCMA.

[0064] As described above, complete identity with the sequences shown for (i) is not required (although in one preferred embodiment of the invention, the peptide has the actually recited sequences). X n Of the residues indicated by the label, at least 1 and up to 8 of the residues can be replaced by alternative residues. The replaced residues may all be within helix 1 or helix 2, or there may be 1 replaced residue in one of them and 1, 2, 3, 4, 5, 6 or 7 replaced residues in the other, or there may be 2 replaced residues in one of them and 1, 2, 3, 4, 5 or 6 replaced residues in the other, or there may be 3 replaced residues in one of them and 1, 2, 3, 4 or 5 replaced residues in the other, or there may be 4 replaced residues in one of them and 1, 2, 3 or 4 replaced residues in the other, or there may be 5 replaced residues in one of them and 1, 2 or 3 replaced residues in the other, or there may be 6 replaced residues in one of them and 1 or 2 replaced residues in the other, or there may be 7 replaced residues in one of them and 1 replaced residue in the other. For example, X nThe residues indicated by the label may have 1, 2, 3, 4, or 5, for example, 1, 2, or 3, for example, 1 replaced residue. X n Since there are 10 residues with labels, a peptide having 5 replaced residues has 50% sequence identity with the listed sequence. For the replacement of 2 residues, it is 80% sequence identity, and for the replacement of 1 residue, it is 90% sequence identity.

[0065] In another embodiment, X n Among the residues not labeled with a residue, at least 1 and up to 5 of the residues may be replaced by alternative residues. All of the replaced residues may be within helix 1 or helix 2, or there may be 1 replaced residue in one of them and 1, 2, 3, 4, or 5 replaced residues in the other, or there may be 2 replaced residues in one of them and 1, 2, or 3 replaced residues in the other, or there may be 3 replaced residues in one of them and 1 or 2 replaced residues in the other, or there may be 4 replaced residues in one of them and 1 replaced residue in the other. For example, X n The residues not indicated by the label may have 1, 2, 3, 4, or 5, for example, 1, 2, or 3, for example, 1 replaced residue. X n Since there are 12 residues without labels, a peptide having 5 replaced residues has 58% sequence identity with the listed sequence. For the replacement of 2 residues, it is 83% sequence identity, and for the replacement of 1 residue, it is 92% sequence identity.

[0066] In another embodiment, X nOf the residues that are not labeled with residues, at least 1 and at most 3 residues can be replaced by alternative residues. All of the replaced residues may be within helix 1 or helix 2, or there may be 1 replaced residue in one of them, 1 or 2 replaced residues in the other, or 2 replaced residues in one of them and 1 replaced residue in the other. For example, X n Among the residues not indicated by the label, there may be 1, 2, or 3, for example, 1 or 2, for example, 1 replaced residue. X n Since there are 12 residues without labels, a peptide having 3 replaced residues has 75% sequence identity with the listed sequence. For replacement of 2 residues, it is 83% sequence identity, and for replacement of 1 residue, it is 92% sequence identity.

[0067] In one embodiment, the total number of residues in the helix 1 and helix 2 portions to be replaced is at least 1 and at most 13, at least 1 and at most 12, at least 1 and at most 11, at least 1 and at most 10, for example, at least 1 and at most 9, at least 1 and at most 8, for example, at least 1 and at most 7, for example, at least 1 and at most 6, for example, at least 1 and at most 5, for example, at least 1 and at most 4, for example, at least 1 and at most 3, for example, at least 1 and at most 2. In particular, there may be a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 replaced residues in these portions.

[0068] X n residues shown as, or X n Amino acid residue replacements for residues not shown as may be conservative replacements. That is, one residue is replaced by another residue within the same class, for example, An aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by another aliphatic residue. A hydroxyl-containing residue, sulfur-containing residue or selenium-containing residue (serine (S), cysteine (C), selenocysteine (U), threonine (T) or methionine (M)) may be replaced by another hydroxyl-containing residue, sulfur-containing residue 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.

[0069] Alternatively, X n The residue shown as or X n Amino acid residue substitutions for residues not shown as may be non-conservative substitutions, i.e., the residue is replaced by another residue of a different class, e.g., 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 e.g., may be replaced by an amino acid having the opposite characteristics, e.g., a lysine (K) residue may be replaced by an aspartic acid (D) residue.

[0070] Alternatively, X n The residue shown as or X nFor amino acid residue substitutions for residues not shown as, non-standard (also known as non-natural) amino acids may be used, i.e., amino acids not found in natural polypeptide chains. As described above, the term "non-natural amino acid" as used herein refers to an amino acid that may be found in nature or chemically synthesized and does not constitute (i.e., is not encoded) a protein (e.g., citrulline, hydroxyproline, β-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid, or nitroarginine). These may be produced as secondary metabolites or chemically synthesized. Preferably, the non-natural amino acids according to the present invention are isoelectronic with naturally occurring amino acids, for example, norleucine. Thus, X n the residue shown as or X n For amino acid residue substitutions for residues not shown as, the amino acid residue substitutions may be non-natural amino acids according to the present invention, those that are isoelectronic with naturally occurring amino acids, preferably norleucine.

[0071] In another embodiment, the hBCMA-binding polypeptide is i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], helix 2 contains the sequence FYQKWAFIRX 33 LM, and 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, Alternatively, ii) Helix 1 and Helix 2 are defined as in i), and within Helix 1 and Helix 2, X n at least 1 and no more than 3 (e.g., 1, 2, or 3) of the residues are replaced by alternative residues, and / or X n at least 1 and no more than 3 (e.g., 1, 2, or 3) of the residues that are not labeled with X are replaced by alternative residues, and is an hBCMA-binding polypeptide.

[0072] Preferably, such an hBCMA-binding polypeptide has an hBCMA-binding efficacy that is at least 1% 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).

[0073] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 2 for binding to hBCMA.

[0074] As noted above, complete identity with the sequences shown for (i) is not required (although in one preferred embodiment of the invention, the peptide has the actually recited sequences). X n Of the residues shown with labels, at least 1 and no more than 3 (e.g., 1, 2, or 3) of the residues can be replaced by alternative residues. The replaced residues can all be within Helix 1 or Helix 2, or there can be 1 replaced residue in one of them and 1 or 2 replaced residues in the other. For example, X n There may be 1, 2, or 3, such as 1 or 2, such as 1 replaced residue in the residues shown with labels. X n Since there are 7 residues with labels, a peptide with 3 replaced residues has 57% sequence identity with the recited sequence. For 2 residue replacements, it is 71% sequence identity, and for 1 residue replacement, it is 86% sequence identity.

[0075] X n Of the residues and unlabeled residues, at least 1 and up to 3 (e.g., 1, 2, or 3) of the residues can be replaced by alternative residues. All of the replaced residues may be within helix 1 or helix 2, or there may be 1 replaced residue in one of them and 1 or 2 replaced residues in the other. For example, X n There may be 1, 2, or 3, e.g., 1 or 2, e.g., 1 replaced residue in the unlabeled residues. X n Since there are 15 residues without labels, a peptide having 3 replaced residues has 80% sequence identity with the recited sequence. For replacement of 2 residues, it is 87% sequence identity, and for replacement of 1 residue, it is 93% sequence identity.

[0076] In one embodiment, the total number of residues in the helices 1 and 2 portions to be substituted is at least 1 and up to 6, e.g., at least 1 and up to 5, at least 1 and up to 4, e.g., at least 1 and up to 3, e.g., at least 1 and up to 3. In particular, there may be a total of 1, 2, 3, 4, 5, or 6 replacement residues in these portions.

[0077] X n The residue shown as or X n Amino acid residue replacements for residues not shown as can be conservative replacements, non-conservative replacements, or non-standard (also known as non-natural) amino acids, as described above herein.

[0078] In a further embodiment, the hBCMA-binding polypeptide does not have a methionine residue at position X9, X 10 , X 14 , X 18 , X 31 or X 35 For example, helix 1 has the sequence X9X10 X 11 ADX 14 EIX 17 X 18 including [Array No. 278], wherein Helix 2 is the array FX 25 QKWAFX 31 RX 33 LX 35 an hBCMA-binding polypeptide comprising [Array No. 279], and independently of one another, X9 and X 10 is a non-natural amino acid (e.g., norleucine) or any naturally occurring amino acid other than methionine, X 11 is E, F, H, Q, T or Y, X 14 is a non-natural amino acid (e.g., norleucine) or any naturally occurring amino acid other than methionine, X 17 is A, E, Q, S, T or V, X 18 is a non-natural amino acid (e.g., norleucine) or any naturally occurring amino acid other than methionine, X 25 is F or Y, X 31 is I, V, L, glutamine, or a non-natural amino acid (e.g., norleucine) (preferably I, L, V, glutamine, or norleucine), X 33 is K or S, X 35 is I, L, V, glutamine, or a non-natural amino acid (e.g., norleucine) (preferably I, L, V, glutamine, or norleucine), an hBCMA-binding polypeptide, or In a further embodiment, the hBCMA-binding polypeptide has no methionine residue at position X9, X 10 , X 14 , X 18 , X 31 or X 35 , for example, Helix 1 has the sequence X9X 10 X 11 ADX 14 EIX17 X 18 [SEQ ID NO: 278], wherein helix 2 has the sequence FX 25 QKWAFX 31 RX 33 LX 35 An hBCMA-binding polypeptide comprising [SEQ ID NO: 279], wherein, independently of each other, X9 and X 10 is any naturally occurring amino acid except norleucine or methionine, X 11 is E, F, H, Q, T or Y, X 14 is any naturally occurring amino acid except norleucine or methionine, X 17 is A, E, Q, S, T or V, X 18 is any naturally occurring amino acid except norleucine or methionine, X 25 is F or Y, X 31 is I, V, L, glutamine or norleucine (e.g., I, V or norleucine, preferably I or norleucine), X 33 is K or S, X 35 is I, L, V glutamine or norleucine (e.g., I, V or norleucine, preferably I or norleucine), an hBCMA-binding polypeptide, or In a further embodiment, the hBCMA-binding polypeptide has no methionine residues at positions X9, X 10 , X 14 , X 18 , X 31 or X 35 , for example, helix 1 has the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], wherein helix 2 has the sequence FX 25 QKWAFX 31 RX 33 LX 35An hBCMA-binding polypeptide comprising [SEQ ID NO: 279], independently of each other, X9 and X 10 is any naturally occurring amino acid except methionine, X 11 is E, F, H, Q, T or Y, X 14 is any naturally occurring amino acid except methionine, X 17 is A, E, Q, S, T or V, X 18 is any naturally occurring amino acid except methionine, X 25 is F or Y, X 31 is I or V, X 33 is K or S, X 35 is I, L or V, an hBCMA-binding polypeptide.

[0079] In a further embodiment, the hBCMA-binding polypeptide has no methionine residue at position X9, X 10 , X 14 , X 18 , X 31 or X 35 , for example, helix 1 comprises the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 comprises [SEQ ID NO: 278], and helix 2 comprises the sequence FYQKWAFIRX 33 LX 35 , independently of each other, X9 is D, E, H, K, N, Q, S, or V, X 10 is A, E, F, I, K, N, Q, R, S, T, Y, V, L, or a non-natural amino acid (e.g., norleucine), X 11 is E, F, or H, X 14is 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, N, T, S, I, Q, or a non-natural amino acid (e.g., norleucine), X 33 is K or S, X 35 is any naturally occurring amino acid other than methionine (e.g., a naturally occurring amino acid isoelectronic with methionine, e.g., isoleucine), or a non-natural amino acid (preferably a naturally occurring amino acid isoelectronic with methionine, e.g., norleucine), an hBCMA-binding polypeptide, or In a further embodiment, the hBCMA-binding polypeptide has no methionine residue at position X9, X 10 、X 14 、X 18 、X 31 or X 35 For example, 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 LX 35 is an hBCMA-binding polypeptide, and independently of each other, X9 is D, E, H, K, N, Q, S, or V, X 10 is A, E, F, I, K, N, Q, R, S, T, Y, V, L or norleucine, 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, N, T, S, I, Q, or norleucine, X 33 is K or S, X 35 is a hBCMA-binding polypeptide that is a naturally occurring amino acid isosteric with methionine (e.g., isoleucine), or norleucine, or In a further embodiment, the hBCMA-binding polypeptide has no methionine residue at position X9, X10, X14, X18, X31 or X35; for example, helix 1 has the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 contains [SEQ ID NO: 278], and helix 2 contains the sequence FYQKWAFIRX 33 LX 35 is a hBCMA-binding polypeptide, and independently of each other, X9 is D, E, H, K, N, Q, S, or V, X 10 is A, E, F, I, K, 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, N, T, or S, X 33 is K or S, X 35 is isoleucine or norleucine, a hBCMA-binding polypeptide, Preferably, in the above-described 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).

[0080] Preferably, in the above-described embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 2.

[0081] In an advantageous embodiment of the hBCMA-binding polypeptide of the present invention, proline (P) and cysteine (C) are not present in helix 1 or helix 2 of the hBCMA-binding polypeptide of the present invention. Advantageously, glycine (G) is also not present in helix 1 or helix 2 of the hBCMA-binding polypeptide of the present invention.

[0082] In certain embodiments of the present invention, such as the embodiments described above, the hBCMA-binding polypeptide wherein helix 1 comprises the sequence X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 and / or helix 2 comprises the sequence X 23 FX 25 QKWAFX 31 RX 33 LX 35 X 36 X 37 is an hBCMA-binding polypeptide.

[0083] In such embodiments, X6 can be any naturally occurring amino acid or can be absent, X7 can be any naturally occurring amino acid or can be absent, X8 can be any naturally occurring amino acid or can be absent, X 19 can be any naturally occurring amino acid or can be absent, X 23 can be any naturally occurring amino acid or can be absent, X 36 can be any naturally occurring amino acid or can be absent, X 37 can be any naturally occurring amino acid or can be absent.

[0084] 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, X23 may be any naturally occurring amino acid, and X 36 may be any naturally occurring amino acid, and X 37 may be any naturally occurring amino acid.

[0085] In another preferred embodiment, X6 may be D, E, N or Q, or may be absent, X7 may be H, K or R, or may be absent, X8 may be D, E, N or Q, or may be absent, and X 19 may be G, A, V, L or I, or may be absent, and X 23 may be D, E, N or Q, or may be absent, and X 36 may be D, E, N or Q, or may be absent, and X 37 may be D, E, N or Q, or may be absent, In another preferred embodiment, X6 may be D, E, N or Q, X7 may be H, K or R, X8 may be D, E, N or Q, and X 19 may be G, A, V, L or I, and X 23 may be D, E, N or Q, and X 36 may be D, E, N or Q, and X 37 may be D, E, N or Q.

[0086] In another preferred embodiment, X6 may be N, or may be absent, X7 may be K, or may be absent, X8 may be E, or may be absent, and X 19 may be L, or may be absent, and X 23 may be N, or may be absent, and X 36 may be D, or may be absent, and X 37 may be D, or may be absent.

[0087] 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 37is D.

[0088] In a highly particularly preferred embodiment, X6 is N, X7 is K, X8 is E, X 19 is L, X 23 is N, X 36 is D, X 37 is D. In such an embodiment, 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, In an advantageous embodiment of the hBCMA-binding polypeptide 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.

[0089] Preferably, such an hBCMA-binding polypeptide has hBCMA-binding efficacy that is at least 1% of SEQ ID NO: 2.

[0090] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 2 for being of hBCMA.

[0091] In another preferred embodiment, the hBCMA-binding polypeptide i) Helix 1 contains the sequence NKEETFADLEISNL and Helix 2 contains the sequence NFYQKWAFIRSLMDD, or ii) Helix 1 and Helix 2 are defined as in i), and within Helix 1 and Helix 2, at least 1 and no more than 2 (e.g., 1 or 2) residues are replaced by alternative residues, or or (iii) an hBCMA-binding polypeptide in which at least one and no more than three (e.g., 1, 2, or 3) residues within the sequence of helix 1 and / or helix 2 are replaced by alternative residues (e.g., replaced by alternative residues that are conservative replacements).

[0092] Preferably, such an hBCMA-binding polypeptide has an hBCMA-binding efficacy that is at least 1% of SEQ ID NO: 1.

[0093] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 1.

[0094] In another preferred embodiment, the hBCMA-derived polypeptide is i) helix 1 comprises the sequence NKENQFADEEIAAL and helix 2 comprises the sequence NFYQKWAFIRKLMDD, or or ii) helix 1 and helix 2 are as defined in i), and within helix 1 and helix 2, at least one and no more than two (e.g., 1 or 2) residues are replaced by alternative residues, or or (iii) an hBCMA-derived polypeptide in which at least one and no more than three (e.g., 1, 2, or 3) residues within the sequence of helix 1 and / or helix 2 are replaced by alternative residues (e.g., replaced by alternative residues that are conservative replacements).

[0095] Preferably, such an hBCMA-binding polypeptide has an hBCMA-binding efficacy that is at least 1% of SEQ ID NO: 2.

[0096] In certain embodiments, alternatively or in addition, the hBCMA-binding polypeptide competes with SEQ ID NO: 2.

[0097] In one embodiment, the hBCMA-binding polypeptide is a) Helix 1 contains the sequence ETFADLEISN, and Helix 2 contains the sequence FYQKWAFIRSLM. For example, Helix 1 contains the sequence NKEETFADLEISNL, and Helix 2 contains the sequence NFYQKWAFIRSLMDD.

[0098] In a further embodiment, the hBCMA binding polypeptide is b) Helix 1 contains the sequence NQFADEEIAA, and Helix 2 contains the sequence FYQKWAFIRKLM. For example, Helix 1 contains the sequence NKENQFADEEIAAL, and Helix 2 contains the sequence NFYQKWAFIRKLMDD.

[0099] As described above herein, some residues may each be replaced by alternative residues. For example, at least 1 and 3 or less, at least 1 and 2 or less, or 1 residue may be replaced by an alternative residue, e.g., 3, 2, or 1 residue may be replaced by an alternative residue. Amino acid residue replacements can be conservative replacements or non-conservative replacements, or non-standard (also known as non-natural) amino acids, as described above herein.

[0100] In an advantageous embodiment of the hBCMA binding polypeptide 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.

[0101] In one embodiment, the hBCMA binding motif, which is the portion [Helix 1]-[spacer portion]-[Helix 2], is X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 FX 25QKWAFX 31 RX 33 LX 35 X 36 X 37 and has (i.e., has this sequence), X 20 where X is any naturally occurring amino acid, 21 where X is any naturally occurring amino acid, 22 where X is any naturally occurring amino acid, and optionally, 20 X, 21 or X, 22 is / are absent, and the other residues are as defined above.

[0102] More preferably, X6 may be D, E, N or Q, or may be absent, X7 may be H, K or R, or may be absent, X8 may be D, E, N or Q, or may be absent, X9, X 10 X, 11 X, 14 X, 17 and X 18 are as defined above, X 19 may be G, A, V, L or I, or may be absent, X 20 may be S, T, M, P, F, Y or W (e.g., P), X 21 may be D, E, N or Q, X 22 may be G, A, V, L, I, or may be absent, X 23 may be D, E, N or Q, or may be absent, X 25 X, 31 X, 33 and X 35 are as defined above, X 36 may be D, E, N or Q, or may be absent, X 37 may be D, E, N or Q, or may be absent.

[0103] For example, X6 may be D, E, N or Q, X7 may be H, K or R, X8 may be D, E, N or Q, X9, X10 , X 11 , X 14 , X 17 and X 18 is as defined above, X 19 may be G, A, V, L or I, X 20 may be S, T, M, P, F, Y or W (e.g., P), X 21 may be D, E, N or Q, X 22 may be G, A, V, L, I, X 23 may be D, E, N or Q, X 25 , X 31 , X 33 and X 35 is as defined above, X 36 may be D, E, N or Q, X 37 may be D, E, N or Q.

[0104] In a particularly preferred embodiment, X6 is N, X7 is K, X8 is E, X9, X 10 , X 11 , X 14 , X 17 and X 18 is as defined above, X 19 is 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 22 is G, A, V, L or I (e.g., L), X 23 is N, X 25 , X 31 , X 33 and X 35 is as defined above, X 36 is D, X 37 is D.

[0105] For example, the X9-X 35 portion of the hBCMA binding motif is selected from the group consisting of the following.

[0106]

Table 1-1

[0107] [Table 1-2]

[0108] [Table 1-3]

[0109] [Table 1-4]

[0110] [Table 1-5]

[0111] In the polypeptides in the above table, optionally 1 to 5 residues (for example, 1, 2, 3, 4 or 5 residues), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues. Preferably, in embodiments where optionally 1 to 5 residues (for example, 1, 2, 3, 4 or 5 residues), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues, the alternative residues are conservative replacements. In a preferred embodiment, the sequence is one of SEQ ID NOs: 170 to 275. In a preferred embodiment, the hBCMA binding motif is selected from the group consisting of the following.

[0112] [Table 2]

[0113] In the polypeptides in the table above, optionally 1 to 5 residues (e.g., 1, 2, 3, 4, or 5 residues), preferably 1, 2, or 3 residues in the sequence are replaced by alternative residues. Preferably, in embodiments where optionally 1 to 5 residues (e.g., 1, 2, 3, 4, or 5 residues), preferably 1, 2, or 3 residues in the sequence are replaced by alternative residues, the alternative residues are conservative replacements.

[0114] In a preferred embodiment, the hBCMA binding motif sequence further has the residues NKE at positions X6X7X8 (i.e., X6 is N, X7 is K, and X8 is E). In a preferred embodiment, the motif sequence further has 36 X 37 the residues DD at position X (i.e., X 36 is D and X 37 is D). For example, the motif sequence has NKE at positions X6X7X8 and DD at positions X 36 X 37 (i.e., X6 is N, X7 is K, X8 is E, X 36 is D, and X 37 is D).

[0115] Thus, in a preferred embodiment, the hBCMA binding motif sequence is 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 , X6X7X8X9X 10 X 11 ADX 14 EIX 17 X 18 X 19 X20 X 21 X 22 X 23 FX 25 QKWAFX 31 RX 33 LX 35 DD, and 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 may be selected from DD.

[0116] As described above, in embodiments of the present invention, as described herein, some residues may each be replaced by alternative residues. For example, in the above-described embodiments, even if not explicitly mentioned, in the sequences of helix 1 and / or helix 2 defined above, optionally 1 to 5 (e.g., 1, 2, 3, 4, or 5), preferably optionally 1 to 3 (e.g., 1, 2, or 3) residues may be replaced by alternative residues (e.g., replaced by alternative residues that are conservative replacements). In any such alternative polypeptide of the present invention where the alternative residues are in place, 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., the binding efficacy of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions (e.g., as described above)). Alternatively, or in addition, for example, in any such alternative polypeptide of the present invention where the alternative residues are in place, the hBCMA-binding polypeptide competes with SEQ ID NO: 2 for hBCMA binding.

[0117] More preferably, the binding potency is at least 5%, more preferably at least 10%, 20%, 25% or 50% of the binding potency of the peptide of SEQ ID NO: 2 (i.e., the binding potency of the peptide of SEQ ID NO: 2 to hBCMA when measured under the same conditions).

[0118] In an advantageous embodiment of the hBCMA-binding polypeptide of the invention, proline (P) and cysteine (C) are not present in the hBCMA-binding motif.

[0119] Advantageously, glycine (G) is also not present in the hBCMA-binding motif.

[0120] As described above herein, the hBCMA-binding polypeptide of the invention has the overall structure [N-terminal portion]-[helix 1]-[spacer portion]-[helix 2]-[C-terminal portion].

[0121] The spacer portion can be a sequence of 1 to 5 (preferably 1, 2, 3, 4 or 5) naturally occurring amino acids. Preferably, the spacer portion is a sequence of 2 to 5 (e.g., 2, 3, 4 or 5) naturally occurring amino acids. For example, the spacer portion is a sequence of 3 to 5 (e.g., 3, 4 or 5) naturally occurring amino acids. Preferably, the spacer portion is a sequence of 3 amino acids.

[0122] In a particular embodiment, the spacer 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, X 22 is any naturally occurring amino acid, and optionally, one or two of X 20 X 21 or X 22 are absent. For example, none of X 20 X 21 or X 22 are present, or one of X 20 X 21 or X22 One of them does not exist, or X 20 , X 21 or X 22 Two of them do not exist. More preferably, X 20 , X 21 or X 22 None of them exist, or X 20 , X 21 or X 22 One of them does not exist. Most preferably, X 20 , X 21 or X 22 None of them exist, that is, the separated part has the sequence X 20 X 21 X 22 has X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid, X 22 is any naturally occurring amino acid.

[0123] Preferably, X 20 is S, T, M, P, F, Y or W (e.g., P or T), X 21 is D, E, N, Q, X 22 is G, A, V, L, I, and optionally, X 20 , X 21 or X 22 One or two (e.g., one) of them do not exist. In one embodiment, X 20 is P, S, C, U, T, or M, X 21 is D, E, N or Q, X 22 is G, A, V, L, I. More preferably, X 20 is P or T, X 21 is N, X 22 is L. For example, the separated part has the sequence PNL or TNL.

[0124] 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, such as 1, 2, 3, 4, or 5 naturally occurring amino acids.

[0125] In certain embodiments, the N-terminal portion has the sequence X a X b having X1X2X3X4X5, where X a is any naturally occurring amino acid (such as M) or is absent, and X b is any naturally occurring amino acid (such as 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.

[0126] Preferably, the N-terminal portion has the sequence X a X b having X1X2X3X4X5, where X a is M or is absent, and X b is M or is absent, X1 is G, A, V, L, or I or is absent, X2 is D, E, N, or Q or is absent, X3 is D, E, N, or Q or is absent, X4 is H, K, or R or is absent, and X5 is F, Y, or W or is absent. More preferably, X a is M or is absent, and X b is M or is absent, X1 is V, G or is absent (for example, V or is absent), X2 is D or is absent, X3 is N or is absent, X4 is K or is absent, and X5 is F or is absent. More preferably, X a is M or is absent, and X bis M or does not exist, X1 is V or does not exist, X2 is D or does not exist, X3 is N or does not exist, X4 is K or does not exist, X5 is F or does not exist.

[0127] In certain embodiments, preferably, none of X a , X b , X1, X2, X3, X4, and X5 exist, or X a , and / or X b does not exist, and the other residues do not all not exist, or X a , X b , and X1 do not exist, and the other residues do not all not exist, or X a , X b , X1, and X2 do not exist, and the other residues do not all not exist, X a , X b , X1, X2, and X3 do not exist, and the other residues do not all not exist, X a , X b , X1, X2, X3, and X4 do not exist, and the other residues do not all not exist, or X a , X b , X1, X2, X3, X4, and X5 all do not exist.

[0128] In certain embodiments, preferably, none of X1, X2, X3, X4, and X5 exist, X1 does not exist, and the other residues do not all not exist, X1 and X2 do not exist, and the other residues do not all not exist, X1, X2, and X3 do not exist, and the other residues do not all not exist, X1, X2, X3, and X4 do not exist, and the other residues do not all not exist, or X1, X2, X3, X4, and X5 all do not exist. In such embodiments, optionally, X a , and / or X b does not exist.

[0129] 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, X5 is F, or X a , and X b is absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X a , and X b is absent, X1 is absent, X2 is D, X3 is N, X4 is K, X5 is F, or X a , and X b is absent, X1 is absent, X2 is absent, X3 is N, X4 is K, X5 is F, or X a , and X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is K, X5 is F, or X a , and X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is F, or X a , and X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent.

[0130] In certain embodiments, the N-terminal portion has the sequence X1X2X3X4X5, where X1 is any naturally occurring amino acid (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.

[0131] In certain embodiments, preferably, none of X1, X2, X3, X4, and X5 are present, or X a , and / or X b is not present, and the other residues are not absent, or X a , X b , and X1 are not present, and the other residues are not absent, or X a , X b , X1, and X2 are not present, and the other residues are not absent, X a , X b , X1, X2, and X3 are not present, and the other residues are not absent, X a , X b , X1, X2, X3, and X4 are not present, and the other residues are not absent, or X a , X b , X1, X2, X3, X4, and X5 are all not present.

[0132] For example, the N-terminal portion has the sequence X1X2X3X4X5, where X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X1 is not present, X2 is D, X3 is N, X4 is K, X5 is F, or X1 is not present, X2 is not present, X3 is N, X4 is K, X5 is F, or X1 is not present, X2 is not present, X3 is not present, X4 is K, X5 is F, or X1 is not present, X2 is not present, X3 is not present, X4 is not present, X5 is F, or X1 is not present, X2 is not present, X3 is not present, X4 is not present, X5 is not present. For example, X1 is V or G (preferably, V), X2 is D, X3 is N, X4 is K, X5 is F, or X1 is not present, X2 is not present, X3 is not present, X4 is not present, X5 is not present, i.e., the N-terminal portion has the sequence VDNKF, GDNKF or is not present, more preferably, has the sequence VDNKF or is not present.

[0133] In one preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, that is, the N-terminal portion has the sequence VDNKF or GDNKF, and more preferably, the N-terminal portion has the sequence VDNKF.

[0134] In one preferred embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent, that is, the N-terminal portion is absent.

[0135] 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, such as 18, 19, 20, 21, or 22 naturally occurring amino acids. Preferably, the C-terminal portion has a sequence that enhances target binding by the hBCMA binding motif of helix 1 and helix 2.

[0136] In certain embodiments, the C-terminal portion is absent or the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 and 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, X 58is any naturally occurring amino acid or is absent, and optionally 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0137] 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, e.g., 1, 2, 3, or 4. In a particularly preferred embodiment, X 38 is any naturally occurring amino acid (i.e., any single (1) naturally occurring amino acid residue).

[0138] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 where X 38 is P, X 39 is S, T, M, P, F, Y, or W, X 56 is G, A, V, L, I or is absent, X 57 is P or is absent, X 58 is H, K, R or is absent, and optionally 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements. In another preferred embodiment, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 where X 56 is G, A, V, L, I or is absent, X 57 is P or is absent, X 58is H, K, R, or absent, and 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, e.g., by alternative residues that are conservative replacements.

[0139] In a particularly preferred embodiment, the C-terminal portion is X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 having, where X 38 is P, X 39 is S, X 56 is A or absent, X 57 is P or absent, X 58 is K or absent, and 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, e.g., by alternative residues that are conservative replacements. In another particularly preferred embodiment, the C-terminal portion is PSQSANLLAEAKKLNDAQX 56 X 57 X 58 having, where X 56 is A or absent, X 57 is P or absent, X 58 is K or absent, and 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, e.g., by alternative residues that are conservative replacements.

[0140] For example, in certain embodiments, the C-terminal portion is the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 having, where X 38 is P, X39 is S, X 56 is A, X 57 is P, X 58 is K, or X 56 is A, or X 57 is P, X 58 does not exist, or X 56 is A, X 57 does not exist, X 58 does not exist, or X 56 does not exist, X 57 does not exist, X 58 does not exist. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements. Or, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 56 is A, X 57 is P, X 58 is K, or X 56 is A, or X 57 is P, X 58 does not exist, or X 56 is A, X 57 does not exist, X 58 does not exist, or X 56 does not exist, X 57 does not exist, X 58 does not exist. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0141] One preferably, the C-terminal portion is the sequence X 38 X 39QSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 38 is P, X 39 is S, X 56 is A, X 57 is P, X 58 is K. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements. Or, for example, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 56 is A, X 57 is P, X 58 is K. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0142] Alternatively preferably, the C-terminal portion is the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 38 is P, X 39 is S, X 56 is absent, X 57 is absent, X 58does not exist. In such an embodiment, 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, e.g., by alternative residues that are conservative replacements. Or, for example, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 and X 56 does not exist, and X 57 does not exist, and X 58 does not exist. In such an embodiment, 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, e.g., by alternative residues that are conservative replacements.

[0143] In one embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5, where X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, and 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, X5 is F, or X a does not exist, X b does not exist, X1 is V or G (preferably, V), X2 is D, X3 is N, X4 is K, X5 is F, or X a does not exist, X b does not exist, X1 does not exist, X2 is D, X3 is N, X4 is K, 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, X5 is F, or Xa does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, X4 is K, 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, 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, X5 does not exist (e.g., X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X a does not exist, X b does not exist, X1 is V or G (preferably, V), X2 is D, X3 is N, X4 is K, 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, X5 does not exist), the C-terminal part is the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 38 is P, X 39 is S, T, M, P, F, Y or W, X 56 is G, A, V, L, I or does not exist, X 57 is P, X 58 is H, K, or R, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements, more preferably, X 38 is P, X 39 is S, X 56 is A, X 57is P and X 58 is K and optionally 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0144] In another embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5X a X b X1X2X3X4X5, where X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, and X a is absent, X b is absent, X1 is absent, X2 is D, X3 is N, X4 is K, X5 is F, and X a is absent, X b is absent, X1 is absent, X2 is absent, X3 is N, X4 is K, X5 is F, and X a is absent, X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is K, X5 is F, and X a is absent, X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is F, or X a is absent, X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent (e.g., X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X a is absent, X bis absent, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X a is absent, X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent), the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 56 is G, A, V, L, or I, or is absent, X 57 is P, X 58 is H, K, or R, and optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements, and more preferably, X 56 is A, X 57 is P, X 58 is K, and optionally, 1 to 5 (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0145] In one embodiment, the N-terminal portion is the sequence X a X b has X1X2X3X4X5, X a 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, X5 is F, Y, or W (e.g., X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F), the C-terminal portion is the sequence X 38 X 39QSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 38 is P, X 39 is S, T, M, P, F, Y or W (preferably S), X 56 is G, A, V, L or I (preferably A), X 57 is P, X 58 is H, K, or R (preferably K), X 56 is G, A, V, L or I (preferably A), X 57 is P, X 58 does not exist, X 56 is G, A, V, L or I (preferably A), X 57 does not exist, X 58 does not exist, or X 56 does not exist, X 57 does not exist, X 58 does not exist. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0146] In one embodiment, the N-terminal portion has the sequence X a X b X1X2X3X4X5, X a 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, X5 is F, Y, or W (e.g., X a is M, X b is M, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F), and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, X 56is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is H, K, or R (preferably K), and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is absent, and X 56 is G, A, V, L or I (preferably A), and X 57 is absent, and X 58 is absent or 56 is absent, and X 57 is absent, and X 58 is absent. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0147] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, where X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, X1 is absent, X2 is D, X3 is N, X4 is K, X5 is F, X1 is absent, X2 is absent, X3 is N, X4 is K, X5 is F, X1 is absent, X2 is absent, X3 is absent, X4 is K, X5 is F, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is F or a is absent, and X b is absent, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F or X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent), and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX56 X 57 X 58 has X, 38 is P, and X 39 is S, T, M, P, F, Y or W, and X 56 is G, A, V, L, I or does not exist, and X 57 is P, and X 58 is H, K or R, and optionally 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements, and more preferably, X 38 is P, and X 39 is S, and X 56 is A, and X 57 is P, and X 58 is K, and optionally 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0148] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, where X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, X1 does not exist, X2 is D, X3 is N, X4 is K, X5 is F, X1 does not exist, X2 does not exist, X3 is N, X4 is K, X5 is F, X1 does not exist, X2 does not exist, X3 does not exist, X4 is K, X5 is F, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, X5 is F, or X a does not exist, and X bdoes not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist (for example, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist), and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, and X 56 is G, A, V, L, or I, or does not exist, and X 57 is P, and X 58 is H, K, or R, and optionally, 1 to 5 (for example, 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, replaced by alternative residues that are conservative replacements, and more preferably, X 56 is A, and X 57 is P, and X 58 is K, and optionally, 1 to 5 (for example, 1, 2, 3, 4, or 5, preferably 1, 2, or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, replaced by alternative residues that are conservative replacements.

[0149] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, where 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, X5 is F, Y, or W (for example, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F), and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 has, and X 38 is P, and X 39is S, T, M, P, F, Y or W (preferably S), and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is H, K, or R (preferably K), and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is absent, and X 56 is G, A, V, L or I (preferably A), and X 57 is absent, and X 58 is absent or 56 is absent, and X 57 is absent, and X 58 is absent. More preferably, X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is H, K, or R (preferably K), or 56 is absent, and X 57 is absent, and X 58 is absent. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0150] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, where 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 has, and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X58 is H, K, or R (preferably K), and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is absent, and X 56 is G, A, V, L or I (preferably A), and X 57 is absent, and X 58 is absent or X 56 is absent, and X 57 is absent, and X 58 is absent. More preferably, X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is H, K, or R (preferably K), or X 56 is absent, and X 57 is absent, and X 58 is absent. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3) of the residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0151] In a highly preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, and X 56 is absent, and X 57 is absent, and X 58 is absent.

[0152] In an alternative highly preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, X5 is F, and X 56 is G, A, V, L or I (preferably A), and X 57 is P, and X 58 is H, K, or R (preferably K).

[0153] In an alternative highly preferred embodiment, Xa does not exist, X b does not exist, X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 56 is G, A, V, L or I (preferably A), X 57 is P, X 58 is H, K, or R (preferably K).

[0154] In certain embodiments, (i) the separated portion has the 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 is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, In the above-mentioned separated portion, X 20 is P, X 21 is N, X 22 is L, In the above-mentioned N-terminal portion, X a is M or does not exist, X b is M or does not exist, X1 is V or G (preferably V) or does not exist, X2 is D or does not exist, X3 is N or does not exist, X4 is K or does not exist, X5 is F or does not exist, In the above-mentioned C-terminal portion, X 56 is A or does not exist, X 57 is P or does not exist, X 58 is K or does not exist, or (ii) the separated portion, the N-terminal portion, and the C-terminal portion are as defined in (i), and optionally, (a) within each portion, 1, 2 or 3 residues are replaced by alternative residues, or (b) Collectively within those portions, at least one and up to 10 (e.g., up to 5, e.g., 1, 2, 3, 4, or 5) residues are replaced by alternative residues.

[0155] In such embodiments, preferably, the separated portion has the sequence PNL.

[0156] Alternatively, or in addition, in such embodiments, the N-terminal portion has the sequence MMVDNKF or VDNKF.

[0157] In another embodiment, the N-terminal portion may comprise the sequence X1X2X3X4X5, where X1 is 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. For example, the N-terminal portion may comprise the sequence VDNKF, the sequence DNKF, the sequence NKF, the sequence KF, or the sequence F.

[0158] The separated portion may comprise the sequence PNL.

[0159] The C-terminal portion may have the structure [second separated portion]-[helix 3]-[C-terminal sequence]. For example, - the second separated portion comprises the sequence PS, - the helix 3 portion comprises the sequence QSANLLAEAKKLNDAQ, - the C-terminal sequence comprises the sequence APK.

[0160] For example, the C-terminal portion may comprise the sequence PSQSANLLAEAKKLNDAQAPK.

[0161] Among these five parts (N-terminal part, separated part, second separated part, helix 3, and C-terminal sequence), at least one and up to five of these residues can be replaced by alternative residues. For example, the number of residues to be replaced is at least one and up to four, for example, at least one and up to three, for example, at least one and up to two. For example, there can be a total of 1, 2, 3, 4, or 5 replacement residues in these parts.

[0162] In an advantageous embodiment of the hBCMA-binding polypeptide of the invention, when the C-terminal part comprises a helical or substantially helical region (such as helix 3 described immediately above), proline (P) and cysteine (C) are not present in that helical or substantially helical region. In another embodiment, furthermore glycine (G) is also not present in that helical or substantially helical region.

[0163] Thus, across the entire hBCMA-binding polypeptide of the present invention (i.e., across the entire structure [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]), at least 1 and up to 18 residues can be replaced by alternative residues. For example, the number of residues to be replaced can be at least 1 and up to 18, at least 1 and up to 17, at least 1 and up to 16, at least 1 and up to 15, at least 1 and up to 14, at least 1 and up to 13, at least 1 and up to 12, at least 1 and up to 11, at least 1 and up to 10, at least 1 and up to 9, at least 1 and up to 8, at least 1 and up to 7, or at least 1 and up to 6. In particular, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 replacement residues can be present across the entire structure [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]. Amino acid residue replacements can be conservative replacements, non-conservative replacements, or non-standard (also known as non-natural) amino acids, as described above herein.

[0164] Preferably, across the entire hBCMA-binding polypeptide of the present invention (i.e., across the entire structure [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]), at least 1 and up to 15 residues can be replaced by alternative residues. For example, the number of residues to be replaced can be at least 1 and up to 13, at least 1 and up to 12, at least 1 and up to 11, at least 1 and up to 10, at least 1 and up to 9, at least 1 and up to 8, at least 1 and up to 7, or at least 1 and up to 6.

[0165] In particular, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 replacement residues may be present throughout the overall structure [N-terminal portion]-[Helix 1]-[Separation portion]-[Helix 2]-[C-terminal portion]. Amino acid residue replacements can be conservative replacements, non-conservative replacements, or non-standard (also known as non-natural) amino acids, as described above herein.

[0166] In one aspect of the invention, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1, 2, 23-39, 41-119, or 121-128 shown in Table 1. In such sequences, optionally, 1 to 5 (preferably 1, 2 or 3) residues within the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements. In one embodiment, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NOs: 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 NOs: 1, 2, 23-39, 41-119, or 121-128.

[0167] In one aspect of the invention, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NOs: 2 and 23-39 as shown in Table 1. In such sequences, optionally, 1 to 5 (preferably 1, 2 or 3) residues within the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements. In one embodiment, the hBCMA-binding polypeptide comprises a sequence selected from SEQ ID NOs: 2 and 23-39. In one embodiment, the hBCMA-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 2 and 23-39.

[0168] In one aspect of the invention, the hBCMA-binding polypeptide has the sequence a) comprising VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1], In a further aspect of the invention, the hBCMA-binding polypeptide has the sequence b) VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 2], and In a further aspect of the invention, the hBCMA-binding polypeptide has the sequence c) VDNKFNKEEIFADREIAFLPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 23], and In a further aspect of the invention, the hBCMA-binding polypeptide has the sequence c) VDNKFNKEHQFADYEIAMLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 33].

[0169] As described above herein, some of the residues in the polypeptides of the invention may be replaced by alternative residues. For example, in the polypeptides shown in a), b), c) or d) immediately above, at least 1 and up to 5 of the residues may be replaced by alternative residues. For example, the number of residues to be replaced may be at least 1 and up to 4, for example at least 1 and up to 3, for example at least 1 and up to 2. In particular, there may be a total of 1, 2, 3, 4 or 5 replacement residues in these portions. Since there are 58 residues in the polypeptide of this aspect of the invention, a peptide having 5 replaced residues has 91% sequence identity (91.4%) with the recited sequence. For 4 residue replacements it is 93% (93.1%), for 3 residue replacements it is 95% (94.8%), for 2 residue replacements it is 97% (96.6%), and for 1 residue replacement it is 98% sequence identity (98.3%).

[0170] As disclosed above herein, in one embodiment, helix 1 has the sequence X9X 10 X 11 ADX 14 EIX 17 X 18comprising [SEQ ID NO: 278], wherein helix 2 is the sequence FX 25 QKWAFX 31 RX 33 LX 35 comprising [SEQ ID NO: 279], wherein, independently of one another, X9 and X 10 are 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, as described herein, some residues may each be substituted by an alternative residue. In any such alternative polypeptide of the invention where the alternative residue is in place, 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 when measured under the same conditions.

[0171] At a binding efficacy of 0.1%, the EC50 concentration of the alternative polypeptide for binding to hBCMA is understood to be 1000-fold or less of the EC 50 concentration of the peptide of SEQ ID NO: 2 for binding to hBCMA when measured under the same conditions.

[0172] 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 for binding to hBCMA when measured under the same conditions. That is, the EC 50 concentration of the alternative polypeptide for binding to hBCMA is 50 500-fold or less, 100-fold or less, 50-fold or less, 25-fold or less, 20-fold or less, 10-fold or less, 5-fold or less, 4-fold or less or 2-fold or less of the EC concentration of the peptide of SEQ ID NO: 2 for binding to hBCMA when measured under the same conditions.

[0173] When disclosed above in this specification, in another embodiment, helix 1 is the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 [SEQ ID NO: 278], and helix 2 is the sequence FYQKWAFIRX 33 LM, and independently of each other, X9 is D, E, H, K, N, Q, S, or V, and X 10 is A, E, F, I, K, M, N, Q, R, S, T, Y, or V, and X 11 is E, F, or H, and X 14 is A, E, H, I, K, L, Q, R, T, or Y, and X 17 is A, E S, T, or V, and X 18 is A, F, H, K, L, M, N, T, or S, and X 33 is K or S. In this embodiment, as in other embodiments, as described herein, some residues may each be replaced by alternative residues. In any such alternative polypeptide of the invention where the alternative residues are in place, 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 when measured under the same conditions.

[0174] At a binding efficacy of 0.1%, the EC 50 concentration of the alternative polypeptide for binding to hBCMA is understood to be 1000-fold or less of the EC 50 concentration of the peptide of SEQ ID NO: 2 for binding to hBCMA when measured under the same conditions.

[0175] 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 for binding to hBCMA when measured under the same conditions. That is, the EC 50 concentration of the alternative polypeptide for binding to hBCMA is the EC of the peptide of SEQ ID NO: 2 for binding to hBCMA when measured under the same conditions.50 The concentration is 500 times or less, 100 times or less, 50 times or less, 25 times or less, 20 times or less, 10 times or less, 5 times or less, 4 times or less, or 2 times or less.

[0176] As disclosed herein above, in another embodiment, helix 1 comprises the sequence ETFADLEISN and helix 2 comprises the sequence FYQKWAFIRSLM. In this embodiment, as in other embodiments, some residues may each be replaced by alternative residues as described herein. In any such alternative polypeptides of the invention with alternative residues in place, binding to hBCMA is maintained.

[0177] 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.

[0178] At 0.1% binding efficacy, the EC of the alternative polypeptide for binding to hBCMA 50 The EC50 concentration of the peptide of SEQ ID NO:2 to hBCMA when measured under the same conditions. 50 It is understood that the concentration is 1000 times or less.

[0179] 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, i.e., the EC of the alternative polypeptide for binding to hBCMA. 50 The EC concentration of peptide of SEQ ID NO:2 to hBCMA when measured under the same conditions. 50 The concentration is 500 times or less, 100 times or less, 50 times or less, 25 times or less, 20 times or less, 10 times or less, 5 times or less, 4 times or less, or 2 times or less.

[0180] Also, as disclosed above in this specification, in another embodiment, helix 1 comprises the sequence NQFADEEIAA and helix 2 comprises the sequence FYQKWAFIRKLM. In this embodiment, as in other embodiments, some residues may each be substituted by alternative residues as described herein. In any such alternative polypeptide of the invention where the alternative residues are in place, 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.

[0181] At a binding efficacy of 0.1%, the EC of the alternative polypeptide for binding to hBCMA 50 concentration, when measured under the same conditions, is understood to be 1000-fold or less of the EC of the peptide of SEQ ID NO: 2 for hBCMA 50 concentration.

[0182] 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 for hBCMA when measured under the same conditions. That is, the EC of the alternative polypeptide for binding to hBCMA 50 concentration, when measured under the same conditions, is 500-fold or less, 100-fold or less, 50-fold or less, 25-fold or less, 20-fold or less, 10-fold or less, 5-fold or less, 4-fold or less, or 2-fold or less of the EC of the peptide of SEQ ID NO: 2 for hBCMA 50 concentration.

[0183] The present invention relates to an hBCMA-binding polypeptide, wherein the hBCMA-binding polypeptide consists of one motif that binds to hBCMA, and the above-mentioned polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], The hBCMA binding motif is the portion [Helix 1]-[Spacer]-[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., 1, 2, or 3 additional functional portions), and further provides an hBCMA binding polypeptide.

[0184] The present invention further provides an hBCMA binding polypeptide comprising the CD16a binding polypeptide of the present invention and optionally an additional binding portion. The present invention further provides an hBCMA binding polypeptide comprising the hBCMA binding polypeptide of the present invention, wherein the hBCMA binding polypeptide further comprises an hBCMA binding polypeptide (and optionally one or more additional functional portions as described below (e.g., 1, 2, 3, 4, 5, 6 or more), e.g., 1, 2, or 3 additional functional portions).

[0185] In one embodiment, the hBCMA binding polypeptide (e.g., the hBCMA binding polypeptide of the present invention described above or below herein) consists of one motif that binds to hBCMA, and the polypeptide has the following structure: consisting of [N-terminal portion]-[Helix 1]-[Spacer]-[Helix 2]-[C-terminal portion], wherein the hBCMA binding motif is the portion [Helix 1]-[Spacer]-[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), e.g., 1, 2, or 3 additional functional portions).

[0186] In one embodiment, the hBCMA binding polypeptide consists of the hBCMA binding polypeptide of the present invention (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., 1, 2, or 3 additional functional portions).

[0187] To avoid misunderstanding, in embodiments where 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 connected to a further hBCMA binding polypeptide, i.e., the hBCMA binding polypeptide is not part of an hBCMA binding oligomer of the present invention.

[0188] In another embodiment, the hBCMA binding polypeptide (e.g., the hBCMA binding polypeptide of the present invention described hereinabove or hereinbelow) consists of one motif that binds to hBCMA, and the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], the hBCMA binding motif being the portion [Helix 1]-[Spacer portion]-[Helix 2], and the polypeptide further comprising one or more additional functional portions (e.g., 1, 2, 3, 4, 5, 6 or more), e.g., 1, 2, or 3 additional functional portions, as described hereinbelow. In another highly preferred embodiment, the hBCMA binding polypeptide consists of the hBCMA binding polypeptide of the present invention and further comprises one or more additional functional portions (e.g., 1, 2, 3, 4, 5, 6 or more), e.g., 1, 2, or 3 additional functional portions (preferably 1 or 2 additional functional portions), as described hereinbelow.

[0189] Multimeric hBCMA binding polypeptide: hBCMA binding oligomer The present invention further provides an hBCMA binding oligomer comprising at least 2 (i.e., 2 or more than 2, e.g., 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 2 hBCMA binding polypeptides of the present invention. In another embodiment, the hBCMA binding oligomer of the present invention comprises at least 3, at least 4, at least 5 or at least 6 hBCMA binding polypeptides of the present invention, e.g., 3, 4, 5 or 6 or more hBCMA binding polypeptides of the present invention.

[0190] This aspect of the invention may also be defined such that the hBCMA-binding polypeptide comprises two or more hBCMA-binding moieties or hBCMA-binding peptides optionally connected via one or more linkers, e.g., two, three, four, five, six or more hBCMA-binding moieties or hBCMA-binding peptides optionally connected via one or more linkers, e.g., two, three, or four hBCMA-binding moieties or hBCMA-binding peptides optionally connected via one or more linkers, e.g., two hBCMA-binding moieties or hBCMA-binding peptides optionally connected via one or more linkers. This definition is used in the following numbered embodiments of the invention to refer to the oligomeric aspect of the hBCMA-binding polypeptides of the invention.

[0191] The hBCMA-binding oligomers of the invention comprise at least a first hBCMA-binding polypeptide which is an hBCMA-binding polypeptide of the invention and a second hBCMA-binding polypeptide which is an hBCMA-binding polypeptide of the invention. The first and second hBCMA-binding polypeptides may have the same sequence. Alternatively, the first and second hBCMA-binding polypeptides may have different sequences. The hBCMA-binding oligomers of the invention may optionally further comprise a third hBCMA-binding polypeptide which is an hBCMA-binding polypeptide of the invention. The third hBCMA-binding polypeptide may have the same sequence as the first and / or second hBCMA-binding polypeptide sequences. Alternatively, the third hBCMA-binding polypeptide may have a sequence different from the first and second hBCMA-binding polypeptides. The hBCMA-binding oligomers of the invention may optionally further comprise a fourth hBCMA-binding polypeptide which is an hBCMA-binding polypeptide of the invention. The fourth hBCMA-binding polypeptide may have the same sequence as the first and / or second and / or third hBCMA-binding polypeptide sequences. Alternatively, the fourth hBCMA-binding polypeptide may have a sequence different from the first, second and third hBCMA-binding polypeptides.

[0192] 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 SEQ ID NOs: 170 to 275 (preferably SEQ ID NOs: 170 to 188), optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements), and a second hBCMA-binding polypeptide comprising a second binding motif selected from SEQ ID NOs: 170 to 275 (preferably SEQ ID NOs: 170 to 188), optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements). The first and second hBCMA-binding motifs may have the same or different sequences.

[0193] In a preferred embodiment, the hBCMA-binding oligomer of the present invention comprises a first hBCMA-binding polypeptide having a sequence selected from SEQ ID NOs: 1, 2, 23 to 39, 41 to 119, or 121 to 128 (preferably SEQ ID NOs: 2 and 23 to 39), optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements), and a second hBCMA-binding polypeptide having a sequence selected from SEQ ID NOs: 1, 2, 23 to 39, 41 to 119, or 121 to 128 (preferably SEQ ID NOs: 2 and 23 to 39), optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably alternative residues that are conservative replacements). The first and second hBCMA-binding polypeptides may have the same or different sequences.

[0194] The hBCMA-binding polypeptides within the hBCMA-binding oligomers of the present invention may be separated by linkers. For example, each hBCMA-binding polypeptide within the hBCMA-binding oligomers of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein, for example, a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, or a non-amino acid linker. When the hBCMA-binding oligomers of the present invention contain more than one linker, the linkers may be the same or different. Preferably, the linker for the hBCMA-binding oligomers of the present invention comprises or has a sequence selected from the group consisting of 1 to 50 (for example, 1 to 25, for example, 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, preferably 1 to 20 (for example, 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, for example, a sequence consisting of G, S, and T (preferably G and S).

[0195] In a preferred embodiment, the linker for the hBCMA-binding oligomers of the present invention is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. For example, the linker is G or comprises or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0196] In a preferred embodiment, the linker for the hBCMA-binding oligomer of the present invention is G, or contains or has the sequence GGGSG. For example, the linker contains or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the hBCMA-binding oligomer of the present invention contains or has the sequence GGGGS. For example, the linker contains or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0197] Alternatively, the hBCMA-binding polypeptides in the hBCMA-binding oligomer of the present invention may not be separated by a linker (i.e., they may be directly connected to each other).

[0198] In a preferred embodiment, the hBCMA-binding oligomer does not contain a linker.

[0199] In one embodiment, the hBCMA-binding oligomer of the present invention contains at least 2 (e.g., 2) hBCMA-binding polypeptides, and the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], Each N-terminal portion within the oligomer may have the same sequence or different sequences, each C-terminal portion within the oligomer may have the same sequence or different sequences, each spacer portion within the oligomer may have the same sequence or different sequences, each helix 1 portion within the oligomer may have the same sequence or different sequences, and each helix 2 portion within the oligomer may have the same sequence or different sequences.

[0200] In such an embodiment, the linker preferably comprises or has a sequence selected from the group consisting of 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, such as G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker is G or comprises or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0201] Alternatively, the linker may be absent, i.e., the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] and comprises 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 has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] and each linker portion within the oligomer may have the same sequence or different sequences, each N-terminal portion within the oligomer may have the same sequence or different sequences, or two of them may have the same sequence and one may have a different sequence, each C-terminal portion may have the same sequence or different sequences, or two of them may have the same sequence and one may have a different sequence, each spacer portion within the oligomer may have the same sequence or different sequences, or two of them may have the same sequence and one may have a different sequence, each Helix 1 portion within the oligomer may have the same sequence or different sequences, or two of them may have the same sequence and one may have a different sequence, each Helix 2 portion within the oligomer may have the same sequence or different sequences, or two of them may have the same sequence and one may have a different sequence.

[0202] In such embodiments, the linker preferably comprises or has a sequence selected from the group consisting of 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, such as G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. Also, for example, the linker is G or comprises or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0203] Alternatively, one or both linkers may be absent, i.e., the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] is included.

[0204] In some embodiments of the present invention, the hBCMA-binding oligomer is two or more hBCMA-binding polypeptides optionally connected via one or more linkers, for example, 2, 3, 4, 5, 6 or more hBCMA-binding polypeptides optionally connected via one or more linkers, for example, 2, 3, or 4 hBCMA-binding polypeptides optionally connected via one or more linkers, for example, 2 hBCMA-binding polypeptides optionally connected via one or more linkers.

[0205] For example, such an oligomer may comprise two hBCMA-binding polypeptides, each having a sequence in which Helix 1 comprises the sequence NKEETFADLEISNL and Helix 2 comprises the sequence NFYQKWAFIRSLMDD. As described elsewhere, such a portion may have some residues substituted by alternative residues. For example, each of the two polypeptides may have the sequence VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO:1].

[0206] In an alternative example, such an oligomer may comprise two hBCMA-binding polypeptides, each having a sequence in which Helix 1 comprises the sequence NKENQFADEEIAAL and Helix 2 comprises the sequence NFYQKWAFIRKLMDD. As described elsewhere, such a portion may have some residues substituted by alternative residues. For example, each of the two polypeptides may have the sequence It may have [[SEQ ID NO:2]].

[0207] 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 moieties may have some residues substituted by alternative residues. For example, one of the two polypeptides may have the sequence VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLAEAKKLNDAQAPK [[SEQ ID NO:]] and the other may have the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [[SEQ ID NO:2]] and

[0208] Linker When present, the linker may join together and connect two or more functional moieties (further defined hereinbelow) of the hBCMA-binding polypeptide of the invention (e.g., within the hBCMA-binding oligomer of the invention), or the linker may join together and connect the hBCMA-binding polypeptide of the invention and an additional functional moiety of the invention, or the linker may join together and connect two or more functional moieties of the hBCMA-binding agent-drug conjugate of the invention. The linker may also join together and connect the additional functionality of the invention and additional functional moieties of the invention in embodiments where more than one additional functional moiety is present. Those skilled in the art will be aware that different types of linkers with various properties are known in the art and can select an appropriate linker depending 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, the linker may be selected to increase stability, or improve folding, increase expression, improve biological activity, enable targeting, or alter pharmacokinetics. Non-amino acid linkers may also be referred to as synthetic linkers.

[0209] To avoid misunderstanding, in certain embodiments, the linker may not join together and connect two hBCMA-binding polypeptides of the invention (e.g., within the hBCMA-binding oligomer of the invention), and / or the linker may not join together and connect the hBCMA-binding polypeptide of the invention and an additional functional moiety of the invention, and / or in embodiments where more than one additional functional moiety is present, the linker may not join together and connect the additional functionality of the invention and additional functional moieties of the invention. For example, in certain embodiments, two hBCMA-binding polypeptides of the invention (e.g., within the hBCMA-binding oligomer of the invention) may be directly connected, and / or the hBCMA-binding polypeptide or oligomer of the invention may be directly connected to an additional functional moiety of the invention, and / or additional functional moieties of the invention may be directly connected to additional functional moieties of the invention.

[0210] In one embodiment, an hBCMA-binding polypeptide, hBCMA-binding oligomer, or hBCMA-binding agent-drug conjugate according to any aspect disclosed herein further comprises at least one linker selected from, for example, a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, and a synthetic linker. In one embodiment, the linker described above is between two or more hBCMA-binding polypeptides (e.g., within the hBCMA-binding oligomers of the present invention), or between an hBCMA-binding polypeptide or hBCMA-binding oligomer and an additional functional moiety (e.g., an immune signaling molecule or an additional binding moiety) (e.g., described in more detail below). In another embodiment, the linker described above is between an hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention and a therapeutic agent (e.g., described in more detail below).

[0211] In one embodiment, an hBCMA-binding oligomer according to any aspect disclosed herein comprises at least one linker selected from, for example, 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. Additional linkers between an hBCMA-binding polypeptide or hBCMA-binding oligomer and an additional functional moiety, e.g., an immune signaling molecule or an additional binding moiety (such as described in more detail below), may also be present in an hBCMA-binding oligomer according to any aspect disclosed herein. Additional linkers between additional functional moieties (e.g., an immune signaling molecule or an additional binding moiety (such as described in more detail below)) and additional functional moieties (e.g., an immune signaling molecule or an additional binding moiety (such as described in more detail below)) may also be present in an hBCMA-binding oligomer according to any aspect disclosed herein.

[0212] In embodiments and aspects of the invention that include more than one linker, each linker may be the same, or different, or some linkers may be the same and some may be different (e.g., in embodiments having three or more linkers (e.g., 3, 4, 5, 6, 7, 8 or more linkers)).

[0213] Flexible amino acid linkers may be used when the linked domains require some degree of distance and conformational freedom, and may be advantageous in some embodiments of the invention. Such linkers generally consist of small non-polar (e.g., G) or polar (e.g., S or T) amino acids. Some flexible linkers consist mainly of stretches of G and S residues, e.g., (GGGGS)p or (GGGSG)p. Other examples include (GGSGG)p, (GSGGG)p or (SGGGG)p.

[0214] By adjusting the copy number "p", linker optimization is possible to achieve appropriate separation between functional moieties or to maintain the necessary interactions between moieties. In one embodiment, the linker is (GGGSG)p, e.g., (GGGSG)1, (GGGSG)2 or (GGGSG)3, e.g., (GGGSG)3. In one embodiment, the linker is (GGGGS)p, e.g., (GGGSG)1, (GGGGS)2 or (GGGGS)3, e.g., (GGGGS)3. In one embodiment, the linker is (GGSGG)p, e.g., (GGGSG)1, (GGSGG)2 or (GGSGG)3, e.g., (GGSGG)3. In one embodiment, the linker is (GSGGG)p, e.g., (GGGSG)1, (GSGGG)2 or (GSGGG)3, e.g., (GSGGG)3. In one embodiment, the linker is (SGGGG)p, e.g., (GGGSG)1, (SGGGG)2 or (SGGGG)3, e.g., (SGGGG)3. In another embodiment, the linker is G.

[0215] In one embodiment, the linker of the present invention comprises or has a sequence selected from the group consisting of 1 to 50 (for example, 1 to 40, 1 to 30, or 1 to 25, for example, 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, preferably 1 to 20 (for example, 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, for example, a sequence selected from the group consisting of G, S and T. In one embodiment, the linker of the present invention comprises or has a sequence selected from the group consisting of 1 to 50 (for example, 1 to 25, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, or 1 to 20 (for example, 1, 5, 10, 15 or 20, or 1 to 15, for example, 1, 5, 10 or 15) naturally occurring amino acids, for example, a sequence selected from the group consisting of G and S. For example, the linker 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.

[0216] Also, 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 comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0217] In a preferred embodiment, the linker of the present invention is G, or contains or has the sequence GGGSG. For example, the linker contains or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the hBCMA-binding oligomer of the present invention contains or has the sequence GGGGS. For example, the linker contains or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGSG.

[0218] Apart from the G and S linkers, other flexible linkers are known in the art, such as additional amino acid residues such as T and A to maintain flexibility, and G and S linkers containing polar amino acid residues to improve solubility. In general, it is known in the art that the linker sequence and length can affect the characteristics of the linked moieties, and thus, one of ordinary skill in the art can select an appropriate linker for use in the binding polypeptides described herein.

[0219] Other types of linkers, such as rigid linkers and / or cleavable linkers, can also be used to connect domains in a multi-domain construct to improve or control their biological activities. 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).

[0220] Alternatively, different portions of the conjugate polypeptides or oligomers described herein (e.g., two or more hBCMA-binding polypeptides, an hBCMA-binding polypeptide and an additional functional portion, such as an immune signaling molecule or an additional binding portion, 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 may be cleavable (e.g., MC-Val-Ala-PAB linker) or non-cleavable (e.g., MPB linker). Those skilled in the art will recognize other linkers suitable for use in the conjugate polypeptides described herein.

[0221] Regarding the above description of conjugate polypeptides comprising hBCMA-binding polypeptides according to the present disclosure, it should be noted that the designation of the first, second, and further portions or functional portions is made to clearly distinguish, on the one hand, one or more hBCMA-binding polypeptides according to the invention and, on the other hand, a binding portion or functional portion exhibiting other functions. These designations are not intended to refer to the actual order of the different regions of the conjugate polypeptide. Similarly, the designation of the first and second portions or functional portions (or monomer units) is made to clearly distinguish the units described above. Thus, for example, the first portion or functional portion (or monomer unit) described above may appear, but is not limited to, at the N-terminus, central, or C-terminus of the conjugate polypeptide.

[0222] The hBCMA-binding polypeptides within the hBCMA-binding oligomers of the present invention may be separated by linkers. For example, each hBCMA-binding polypeptide within the hBCMA-binding oligomers of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein. If the hBCMA-binding oligomers of the present invention contain more than one linker, the linkers may be the same or different.

[0223] Preferably, the linker for the hBCMA-binding oligomer of the present invention comprises, or has, a sequence selected from the group consisting of 1 to 50 (e.g., 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, such as G, S, and T (preferably G and S).

[0224] In a preferred embodiment, the linker for the hBCMA-binding oligomer of the present invention is G or comprises, or has, the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker comprises, or has, the sequences 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 sequences GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0225] In embodiments of the present invention that include additional functional moieties, the hBCMA-binding polypeptide or the hBCMA-binding polypeptide and the additional functional moieties within the hBCMA-binding oligomer of the present invention may be separated by a linker. For example, each additional functional moiety and the hBCMA-binding polypeptide within the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein. In embodiments of the present invention that include two or more additional functional moieties, the additional functional moieties may be separated by a linker. Preferably, the linker is a linker as defined herein.

[0226] When the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention contains more than one linker (i.e., when at least two hBCMA-binding polypeptides and at least one additional functional moiety are present, or when at least one hBCMA-binding polypeptide and at least two additional functional moieties are present), the linkers may be the same or different. Preferably, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer containing an additional functional moiety is 1 to 50 (e.g., 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, for example, a sequence selected from the group consisting of G, S, and T (preferably G and S), or has such a sequence.

[0227] In a preferred embodiment, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer containing an additional functional moiety is G or contains or has the sequences GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG, for example, the linker contains or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the hBCMA-binding oligomer of the present invention contains or has the sequence GGGGS, for example, the linker contains or has the sequences GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS.

[0228] Additional functional moiety The hBCMA-binding polypeptides disclosed herein can be connected to one or more additional functional moieties, for example, binding partners that recognize immune cell surface molecules. The hBCMA-binding oligomers disclosed herein can be connected to one or more functional moieties, for example, binding partners that recognize immune cell surface molecules. Thus, in one embodiment, at least one hBCMA-binding polypeptide (or hBCMA-binding oligomer) is optionally connected to one or more additional functional moieties via the linker described above.

[0229] "Functional moiety", as used herein, refers to a component or "moiety" having a particular desired biological activity.

[0230] One or more additional components (i.e., one or more additional functional moieties) may be, for example, signaling molecules or derivatives thereof. Examples of suitable signaling molecules include immune signaling molecules or derivatives thereof, such as cytokines or derivatives thereof, such as IL-15 and derivatives thereof.

[0231] One or more additional components (i.e., one or more additional functional moieties) may be one or more additional binding moieties, for example, one or more binding partners that recognize cell surface proteins or antigens, such as immune cell surface proteins or cell surface tumor antigens (also referred to as cancer cell surface antigens or cancer cell surface targets). The cell surface tumor antigen may be, for example, a tumor-associated antigen or a tumor-specific antigen.

[0232] Such additional binding moieties may be specific for, for example, immune cell surface proteins, such as NK cell activating receptors, such as CD16a. In particular, such CD16a binding moieties may be, for example, immunocyte-inducing polypeptides as disclosed in UK Patent Application No. 2208027.9 (which may be obtained from publicly available files at the time of publication or at the time of publication of other patent applications, such as international patent applications claiming priority therefrom). Such "dual-engager" binding polypeptides may include, for example, at least one hBCMA binding polypeptide as disclosed herein, such as 1, 2, 3 or more hBCMA binding polypeptides as disclosed herein, or at least one hBCMA binding oligomer as disclosed herein. At least one (e.g., 1, 2, 3 or more) hBCMA binding polypeptide as disclosed herein, or at least one hBCMA binding oligomer as disclosed herein, may be connected to, for example, at least one CD16a binding polypeptide as described above, such as 1, 2, 3 or more CD16a binding polypeptides. In such "dual-engager" binding polypeptides, the additional binding moiety (e.g., a binding moiety specific for an immune cell surface molecule such as CD16a, such as at least one CD16a binding polypeptide as described above) may be connected to the N-terminus or C-terminus of the 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 of the linkers described above. Alternatively, the additional binding moiety (e.g., a binding moiety specific for an immune cell surface molecule such as CD16a, such as at least one CD16a binding polypeptide as described above) may be connected between two hBCMA binding moieties and optionally separated by one or more of the linkers described above. Non-limiting examples of such "dual-engager" binding polypeptides comprising one hBCMA binding polypeptide and one or more CD16a binding polypeptides are given in Table 1.The sequences of these bispecific engagers containing a C-terminal His6 tag are provided as [SEQ ID NOs: 130 - 133], the sequences without a C-terminal His6 tag are provided as [SEQ ID NOs: 139 - 142], additional non-limiting examples of "bispecific engager" polypeptides containing two hBCMA-binding polypeptides and a portion targeting CD16a are provided in Table 1, the sequences of these bispecific engagers containing a C-terminal His6 tag are provided as [SEQ ID NOs: 134 - 138], the sequences without a C-terminal His6 tag are provided as [SEQ ID NOs: 143 - 147], non-limiting examples of such "bispecific engager" binding polypeptides containing one hBCMA-binding polypeptide and one or more CD16a-binding polypeptides are also provided, the sequences of these bispecific engagers containing a C-terminal His6 tag are provided as SEQ ID NOs: 154 - 163, and the sequence without a C-terminal His6 tag is provided as SEQ ID NO: 166. Further non-limiting examples of "bispecific engager" polypeptides containing two hBCMA-binding polypeptides and a portion targeting CD16a are also provided, examples of the sequences of such bispecific engagers containing a C-terminal His6 tag include SEQ ID NOs: 149, 150, 151, 152, 153, and examples of the sequences without a C-terminal His6 tag include SEQ ID NO: 167.

[0233] In certain embodiments, the hBCMA-binding polypeptides of the invention (e.g., "bispecific engager" polypeptides, e.g., hBCMA-binding polypeptides incorporated into a bispecific engager polypeptide further comprising at least one portion targeting an NK cell surface target, e.g., CD16a) are particularly active in functional assays, such as CD16 reporter assays and / or cell killing assays. Exemplary sequences of such bispecific engager polypeptides include SEQ ID NOs: 149, 150, 151, 158, 160, and 161 (also disclosed in the examples hereinbelow).

[0234] One of ordinary skill in the art will understand that such non-limiting examples of dual-engagers (as well as other dual-engagers comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer disclosed herein connected to a CD16a-binding moiety, and other molecules comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer connected to different functional moieties (e.g., additional binding moieties described below)) may optionally include one or more additional N-termini, C-termini, or other modifications, such as a C-terminal His6 tag, depending on the intended use or expression, purification, etc. requirements of the engager. Non-limiting examples of such N-terminal, C-terminal, or other modifications include peptide purification tags or moieties (e.g., histidine tags (e.g., polyhistidine tags) or methionine tags (e.g., single methionine tags or polymethionine tags)), signaling tags or moieties (e.g., glycine residues, or signal peptides selected from, e.g., OmpA, DsbA, PhoA, and PelB), fluorophore tags (e.g., Alexa448), or tags or moieties to assist conjugation (cysteine tags (e.g., 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 dual-engager comprising at least one hBCMA-binding polypeptide or at least one hBCMA-binding oligomer described herein.

[0235] Additional examples of additional components (i.e., additional functional moieties) include B-cell maturation antigen (BCMA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Programmed cell death protein 1 (PD-1), A Disintegrin And Metalloprotease 17 (ADAM17), 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), MUC16, MUC17, MUC1, trophoblast glycoprotein (TPBG / 5T4 / WAIF1), V-set domain-containing T cell activation inhibitor 1 (B7-H4 / VTCN1 / B7x / B7S1), cluster of differentiate 20 (CD20), B-lymphocyte surface antigen B4 (CD19), sialic acid-binding Ig-like lectin 2 (CD22), TNF receptor superfamily member 8 (CD30), natural cytotoxicity triggering receptor 1 (NKp46), and additional binding moieties that are binding partners that recognize NKG2D.

[0236] 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 selected from the group consisting of BCMA, ADAM17, 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.

[0237] 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, such as BCMA, CD20, CD19, CD22, or CD30.

[0238] Alternatively, or in addition, such additional binding moieties may be specific for different targets on multiple myeloma cells other than, for example, hBCMA. For example, the additional binding moiety may bind to different targets on the same multiple myeloma cells.

[0239] To avoid misunderstanding, such examples of additional binding moieties, such as binding partners that recognize cell surface proteins or antigens, are non-limiting and are set forth herein for illustrative purposes. The additional binding moieties referred to in this context are not the hBCMA-binding polypeptides of the present invention.

[0240] In one embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention is optionally connected to one or more additional functional moieties (e.g., one or more additional binding moieties and / or signaling molecules) via the linker described above. For example, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is connected to 1, 2, 3, 4 or more additional functional moieties (e.g., 1, 2, 3, 4 or more additional binding moieties and / or signaling molecules). In certain embodiments, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is connected to 1 or 2 additional functional moieties (e.g., 1 or 2 additional binding moieties and / or signaling molecules). In certain embodiments, at least one hBCMA-binding polypeptide or hBCMA-binding oligomer is connected to 1 additional functional moiety (e.g., 1 additional binding moiety or signaling molecule).

[0241] The hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention containing one or more (e.g., 1, 2, 3, 4, 5 or 6 or more) additional functional moieties may be referred to as a heteromultimer. The hBCMA-binding polypeptide of the present invention consisting of only one hBCMA-binding polypeptide and one additional functional moiety and containing no other functional moieties (i.e., no further hBCMA-binding polypeptide functional moieties or additional functional moieties) may be referred to as a heterodimer. The hBCMA-binding polypeptide of the present invention consisting of only one hBCMA-binding polypeptide and two additional functional moieties and containing no other functional moieties may be referred to as a heterotrimer. The hBCMA-binding oligomer of the present invention consisting of only two hBCMA-binding polypeptide functional moieties and one additional functional moiety and containing no other functional moieties may be referred to as a heterotrimer.

[0242] In certain preferred embodiments, the additional functional moiety is a signaling molecule. The signaling molecule, for example, an immune signaling molecule such as a cytokine, for example, IL-15 or a derivative thereof, may be connected to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above. Alternatively, one or more signaling molecules may be connected between two hBCMA-binding polypeptides within the hBCMA-binding oligomer and may be optionally separated by one or more of the linkers or linking sequences described above. Preferably, the signaling molecule may be connected to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above. Preferably, the signaling molecule may be connected to the C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above.

[0243] In certain preferred embodiments, the additional functional moiety is an additional binding moiety. For example, the additional functional moiety is an additional binding moiety that is a binding partner that recognizes one of CTLA-4, PD-1, BCMA, ADAMI7, 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. For example, the additional functional moiety is an additional binding moiety that is specific for one of CTLA-4, PD-1, BCMA, ADAMI7, 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.

[0244] In certain preferred embodiments, the additional binding moiety is specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA). In certain preferred embodiments, the additional binding moiety is specific for an immune cell surface protein (e.g., an NK cell surface protein, e.g., CD16a, NKp46, NKG2D, PD-1, particularly CD16a).

[0245] As described above, a binding partner that recognizes an additional binding moiety, e.g., the NK cell surface protein CD16a, may be connected to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above. Alternatively, one or more additional binding moieties may be connected between two hBCMA-binding polypeptides within the hBCMA-binding oligomer and may optionally be separated by one or more of the linking sequences described above. Preferably, a binding partner that recognizes an additional binding moiety, e.g., the NK cell surface protein CD16a, may be connected to the N-terminus or C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above. More preferably, a binding partner that recognizes an additional binding moiety, e.g., the NK cell surface protein CD16a, may be connected to the C-terminus of the hBCMA-binding polypeptide or hBCMA-binding oligomer, optionally via the linker described above.

[0246] The inventors of the present application have surprisingly found that a "dual-engager" polypeptide comprising an hBCMA-binding polypeptide or hBCMA-binding oligomer disclosed herein can retain its hBCMA-binding ability when fused to an additional binding moiety that targets the NK cell surface protein CD16a. This "dual-engager" polypeptide comprising an hBCMA-binding polypeptide disclosed herein fused to a CD16a-binding moiety can also surprisingly activate NK cells in the presence of BCMA-expressing tumor cells.

[0247] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention further comprises one or more additional functional moieties, for example, at least 1, at least 2, or at least 3 or at least 4 functional moieties. For example, in certain embodiments, the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention further comprises 1, 2, 3, 4, or 5 additional functional moieties.

[0248] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 2, at least 3 or at least 4 additional functional moieties (e.g., 2, 3, 4, or 5 additional functional moieties), each additional functional moiety may be the same, or different, or some of the additional functional moieties may be the same and some may be 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)).

[0249] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 2, at least 3 or at least 4 additional functional moieties (e.g., 2, 3, 4, or 5 additional functional moieties), each additional functional moiety may have the same function, or different functions, or some of the 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)).

[0250] In embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 2, at least 3, or at least 4 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 other than hBCMA, or an immune cell surface target, such as an NK cell surface protein, such as an additional binding moiety specific for 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 one 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 other than 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 other than BCMA, and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof. Alternatively, for example, in one preferred embodiment, the first additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, such as CD16a), 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 an NK cell target (e.g., CD16a), and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof.

[0251] In another preferred embodiment, the first additional functional part may include an additional binding part (e.g., an additional binding part specific to a cancer cell surface target, e.g., a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target specific to CD16a), and the second additional functional part may include an additional binding part (e.g., an additional binding part specific to a cancer cell surface target, e.g., a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target specific to CD16a). For example, the first additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a myeloma cell surface antigen other than BCMA), and the second additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a myeloma cell surface antigen other than BCMA). In particular, the first additional functional part may include an additional binding part specific to a myeloma cell surface antigen other than BCMA, and the second additional functional part may include an additional binding part specific to a myeloma cell surface antigen other than BCMA.

[0252] Alternatively, for example, the first additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a myeloma cell surface antigen other than BCMA), and the second additional functional part may include an additional binding part specific to an immune cell surface target (e.g., an NK cell target, e.g., CD16a). In particular, the first additional functional part may include an additional binding part specific to a myeloma cell surface antigen other than BCMA, and the second additional functional part may include an additional binding part specific to an NK cell target (e.g., CD16a).

[0253] Alternatively, in another embodiment, the first additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof, and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0254] If present (e.g., in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 3 or at least 4 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 specific for a cancer cell surface target, such as a myeloma cell surface antigen other than BCMA, or an immune cell target, such as an NK cell target, such as CD16a), or may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0255] If present (e.g., in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 4 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 specific for a cancer cell surface target, such as a myeloma cell surface antigen other than BCMA, or an immune cell target, such as an NK cell target, such as CD16a), or may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0256] When present (for example, in embodiments where the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 5 additional functional moieties (e.g., 5 or 6), the fifth additional functional moiety may comprise an additional binding moiety (e.g., a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., an additional binding moiety specific for a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target, e.g., specific for CD16a), or may comprise an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof).

[0257] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least 3 (e.g., 3, 4, or 5) additional functional moieties, the first additional functional moiety may comprise an additional binding moiety (e.g., a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., an additional binding moiety specific for a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target, e.g., specific for CD16a), the second additional functional moiety may comprise an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof, and the third additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target, e.g., specific for CD16a).

[0258] For example, in one embodiment, the first additional functional portion may include an additional binding portion specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA), the second additional functional portion may include a cytokine, e.g., IL-15 or a derivative thereof, and the third additional functional portion may include an additional binding portion specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., 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 specific for BCMA, the second additional functional portion may include a cytokine, e.g., IL-15 or a derivative thereof, and the third additional functional portion may include an additional binding portion specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA). In another embodiment, the first additional functional portion may include an additional binding portion specific for BCMA, the second additional functional portion may include a cytokine, e.g., IL-15 or a derivative thereof, and the third additional functional portion may include an additional binding portion specific for an immune cell target (e.g., an NK cell target).

[0259] For example, the first additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., a multiple myeloma cell surface antigen other than BCMA), the second additional functional part may be an additional binding part specific to a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., a multiple myeloma cell surface antigen other than BCMA), and the third additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., a multiple myeloma cell surface antigen other than BCMA) or an immune cell target (e.g., an NK cell target, e.g., CD16a). In particular, the first additional functional part may include an additional binding part specific to a multiple myeloma cell surface antigen other than BCMA, the second additional functional part may include an additional binding part specific to a multiple myeloma cell surface antigen other than BCMA, and the third additional functional part may include an additional binding part specific to a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., a multiple myeloma cell surface antigen other than BCMA). In another embodiment, the first additional functional part may include an additional binding part specific to a multiple myeloma cell surface antigen other than BCMA, the second additional functional part may include an additional binding part specific to a multiple myeloma cell surface antigen other than BCMA, and the third additional functional part may include an additional binding part specific to an immune cell target (e.g., an NK cell target, e.g., CD16a).

[0260] Alternatively, the first additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), the second additional functional moiety may be an additional binding moiety specific for an immune cell surface target (e.g., an NK cell target, e.g., CD16a), and the third additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., a multiple myeloma cell surface antigen other than BCMA) or an immune cell target (e.g., an NK cell target, e.g., CD16a). In particular, the first additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), the second additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), and the third additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., an immune cell target (e.g., an NK cell target, e.g., CD16a)). In another embodiment, the first additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), the second additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), and the third additional functional moiety may comprise an additional binding moiety specific for a multiple myeloma cell surface antigen other than BCMA.

[0261] In one embodiment, the hBCMA-binding polypeptide, or hBCMA-binding oligomer, comprising the additional binding moiety of the invention has an additional binding moiety separated from the hBCMA-binding polypeptide or hBCMA-binding oligomer by a linker. The linker may be any linker defined hereinabove. For example, a linker selected from a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, and a non-amino acid linker.

[0262] Preferably, the linker for the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding moiety comprises, or has, a sequence selected from the group consisting of 1 to 50 (e.g., 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, for example, G, S and T (preferably G and S).

[0263] 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 GGGSGGGGSGGGGSGGGGSGGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0264] In a preferred embodiment, the linker for the hBCMA-binding oligomer of the present invention is G, or contains or has the sequence GGGSG. For example, the linker contains or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the hBCMA-binding oligomer of the present invention contains or has the sequence GGGGS. For example, the linker contains or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

[0265] Alternatively, in one embodiment, the hBCMA-binding polypeptide, or hBCMA-binding oligomer, comprising an additional binding portion of the present invention has an additional binding portion that is 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 connected to the additional binding portion).

[0266] In a preferred embodiment, the hBCMA-binding polypeptide or hBCMA-binding oligomer comprising an additional binding portion of the present invention does not contain a linker. In a preferred embodiment, the hBCMA-binding polypeptide or CD16a-binding oligomer is directly connected to the additional binding portion.

[0267] In certain embodiments, the hBCMA-binding polypeptide of the present invention comprising an additional functional portion has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion], [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], or include [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part], If there are more than one additional functional parts, each additional functionality may be the same or different.

[0268] In such embodiments, the linker preferably comprises or has a sequence selected from the group consisting of 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, such as G, S and T (preferably G and S). For example, the linker is G or includes or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker is G or includes or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0269] Alternatively, the linker may be absent, i.e., the CD16a-binding polypeptide or CD16a-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion], [Additional functional portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion]-[Additional functional portion], [Additional functional portion]-[Additional functional portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], or [Additional functional portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion].

[0270] In such embodiments, more preferably, the hBCMA-binding polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion] (or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion]-[Additional functional portion]).

[0271] In such embodiments, preferably, each additional functional portion is an additional binding portion specific for an NK cell target, such as CD16a. Most preferably, each additional functional portion is an additional binding portion specific for CD16a, and In certain embodiments, the hBCMA-binding oligomer of the invention comprising an additional functional portion has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion], [Additional functional portion]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[-Linker]-[Additional functional portion], [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], comprising Each linker portion within the oligomer may have the same sequence or different sequences, each N-terminal portion within the oligomer may have the same sequence or different sequences, each C-terminal portion within the oligomer may have the same sequence or different sequences, each spacer portion within the oligomer may have the same sequence or different sequences, each Helix 1 portion within the oligomer may have the same sequence or different sequences, each Helix 2 portion within the oligomer may have the same sequence or different sequences.

[0272] In such embodiments, preferably, each additional functional portion is an additional binding portion specific for an NK cell target, such as CD16a. Most preferably, each additional functional portion is an additional binding portion specific for CD16a, In such embodiments, the linker preferably comprises or has a sequence selected from the group consisting of 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, preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20, more preferably 1 to 15) naturally occurring amino acids, such as G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. Also, for example, the linker is G or comprises or has the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0273] In such embodiments, one or more linkers may be absent.

[0274] In such embodiments, more preferably, the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion] and more preferably, [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion] and

[0275] In such embodiments, preferably, each additional functional portion is an additional binding portion specific for an NK cell target, such as CD16a. Most preferably, each additional functional portion is an additional binding portion specific for CD16a.

[0276] In such embodiments, one or more linkers may be absent.

[0277] In a particularly preferred embodiment, the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion].

[0278] In such embodiments, preferably, each additional functional portion is an additional binding portion specific for an NK cell target, such as CD16a. Most preferably, each additional functional portion is an additional binding portion specific for CD16a.

[0279] In certain embodiments, the hBCMA-binding oligomers of the invention comprising an additional functional portion comprise at least 3 hBCMA-binding polypeptides and have the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 2]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion], [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], and each linker part within the oligomer may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence; each N-terminal part within the oligomer may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence; each C-terminal part may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence; each spacer part within the oligomer may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence; each helix 1 part within the oligomer may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence; and each helix 2 part within the oligomer may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence.

[0280] In such embodiments, preferably, each additional functional part is an additional binding part specific for an NK cell target, such as CD16a. Most preferably, each additional functional part is an additional binding part specific for CD16a.

[0281] In such embodiments, the linker is preferably as defined above. In such embodiments, one or more linkers may be absent.

[0282] In such embodiments, more preferably, the hBCMA-binding oligomer has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion], or [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion]-[Additional functional portion].

[0283] In such embodiments, more preferably, each additional functional portion is an additional binding portion specific for an NK cell target, such as CD16a. Most preferably, each additional functional portion is an additional binding portion specific for CD16a.

[0284] In the above embodiments defining the structure of a CD16a-binding polypeptide or CD16a-binding oligomer comprising an additional functional portion, each additional functional portion can be any one described herein. For example, each additional functional portion, independently, an additional binding portion (e.g., an additional binding portion specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen other than BCMA, or an immune cell target, e.g., an NK cell target, e.g., CD16a), and an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof, may be selected.

[0285] For example, each additional functional portion, independently, an additional binding portion specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, e.g., a myeloma cell surface antigen other than BCMA), An additional binding moiety specific for an immune cell target (e.g., an NK cell target, e.g., CD16a), and It may be selected from immune signaling molecules, such as cytokines, such as IL-15 or a derivative thereof.

[0286] For example, each additional functional moiety is independently An additional binding moiety specific for a myeloma cell surface antigen other than BCMA, An additional binding moiety specific for an NK cell target, e.g., CD16a, and It may be selected from immune signaling molecules, such as cytokines, such as IL-15 or a derivative thereof.

[0287] For example, each additional functional moiety is independently An additional binding moiety specific for a myeloma cell surface antigen other than BCMA, and An additional binding moiety specific for an NK cell target, e.g., CD16a, and It may be selected from cytokines, such as IL-15 or a derivative thereof.

[0288] For example, each additional functional moiety is independently An additional binding moiety specific for a myeloma cell surface antigen other than BCMA, and An additional binding moiety specific for an NK cell target, e.g., CD16a, and It may be selected from IL-15 or a derivative thereof.

[0289] In a preferred embodiment, each additional functional moiety is independently An additional binding moiety specific for a myeloma cell surface antigen other than BCMA, and An additional binding moiety specific for CD16a, and It may be selected from IL-15 or a derivative thereof.

[0290] In a preferred embodiment, each additional functional moiety is independently An additional binding moiety specific for CD16a, and It may be selected from cytokines, such as IL-15 or derivatives thereof.

[0291] In a preferred embodiment, each additional functional moiety is independently an additional binding moiety specific for a myeloma cell surface antigen other than BCMA, and an additional binding moiety specific for CD16a, and may be selected therefrom.

[0292] In a preferred embodiment, each additional functional moiety is an additional binding moiety specific for CD16a.

[0293] 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 may be an additional binding moiety (e.g., an additional binding moiety specific for an immune cell surface molecule, e.g., an NK cell target, e.g., CD16a). For example, each additional functional moiety can be an additional binding moiety for CD16a.

[0294] Further non-limiting examples of additional binding moieties include aptamers, monobodies, nanobodies, small molecules, and antibodies. In particular, the additional binding moiety may be an antibody, e.g., a monoclonal antibody, e.g., a monoclonal antibody specific for hBCMA, e.g., a monoclonal antibody specific for CD3 (e.g., foralumab, otelixizumab, teprotumumab, bisilizumab, OKT3, UCHT1, SP34 or F2B), a monoclonal antibody specific for CD38 (e.g., daratumumab or isatuximab), or e.g., a monoclonal antibody specific for SLAMF7 (e.g., elotuzumab). The additional binding moiety may also be a T cell engager.

[0295] The additional binding moiety may be connected to the N-terminus or C-terminus of at least one hBCMA-binding polypeptide, optionally via the linker described above. Alternatively, the additional binding moiety may be connected between two hBCMA-binding polypeptides and optionally separated by the linker described above.

[0296] hBCMA-binding agent-drug conjugate Alternatively, or in addition, an hBCMA-binding polypeptide or hBCMA-binding oligomer disclosed herein may be connected 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 typically covalently binds to one or more therapeutic agents, optionally to one or more of the linkers described above. Non-limiting examples of such therapeutic agents include cytotoxic drugs such as mitomycin C, desmethyltopotecan, SN-38, MMAE, MMAF, doxorubicin, pyrrolobenzodiazepine, amanitin, maytansinoid (e.g., maytansinoid DM1 or maytansinoid DM4), or duocarmycin (e.g., duocarmycin 5.2). Further non-limiting examples of therapeutic agents include radioisotopes or radiolabeled compounds, which may be connected to the hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention (e.g., to form a radioligand). The hBCMA-binding polypeptide or hBCMA oligomer of the present invention connected to a radioisotope or radiolabeled compound finds use in the diagnosis (e.g., by imaging) or treatment (e.g., by radiotherapy) of cancer.

[0297] To avoid misunderstanding, during the generation of an hBCMA-binding agent-drug conjugate according to the present invention, one or more therapeutic agents (e.g., MMAF) are necessarily modified by reaction of a functional group (e.g., -NH, -OH or -SH (e.g., within Cys)) at the attachment point with any linking group or linker used. Thus, one of ordinary skill in the art will understand that the therapeutic agent in an hBCMA-binding agent-drug conjugate (e.g., an hBCMA-binding agent-MMAF conjugate) contains such modifications.

[0298] Generation of polypeptides The hBCMA-binding polypeptides of the present invention can be produced 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, insects, 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 either chemical synthesis or recombinant protein production methods.

[0299] The hBCMA-binding polypeptides of the present invention can also be fused to different molecules with therapeutic potential, such as immunoglobulins or polypeptides with therapeutic potential, via the recombinant or chemical synthesis techniques described above.

[0300] Modulation of polypeptide properties The pharmacokinetic properties of the hBCMA-binding polypeptides of the present 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 (e.g., XTEN or PAS), FcRn-binding ligands, such as the Fc domain of serum albumin or immunoglobulins, or polypeptides that can bind to serum proteins with long in vivo half-lives, such as serum albumin or immunoglobulins.

[0301] Formulations The hBCMA-binding polypeptides or hBCMA-binding oligomers (particularly, hBCMA-binding agent-drug conjugates) according to the present invention can be present in formulations, particularly pharmaceutical formulations.

[0302] In certain embodiments, the invention provides a nucleic acid molecule encoding an hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention. The nucleic acid molecule encoding an hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention finds use, for example, as a medicament for use in the treatment of cancer. The nucleic acid molecule may be, for example, a DNA or RNA molecule, such as an mRNA molecule. The nucleic acid molecule according to the invention may be present in a formulation, in particular a pharmaceutical formulation. Accordingly, the invention further provides a formulation, in particular a pharmaceutical formulation, of a nucleic acid molecule (such as DNA or RNA, in particular an mRNA molecule) encoding an hBCMA-binding polypeptide or hBCMA-binding oligomer of the invention.

[0303] Suitable pharmaceutical formulations include, for example, those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), and intrathecal), or intraperitoneal administration, although the most suitable route may depend, for example, on the condition and disorder of the subject being treated.

[0304] In one embodiment of the invention, an hBCMA-binding polypeptide or hBCMA-binding oligomer (in particular, an hBCMA-binding agent-drug conjugate) or a nucleic acid (such as a DNA or RNA molecule of the invention, such as an mRNA molecule) according to the invention is administered as a pharmaceutical formulation suitable for oral or parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), and intrathecal) administration.

[0305] Pharmaceutical formulations suitable for oral administration can be presented as individual units, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient, as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions. The peptides of the present invention may also be presented as a bolus, lozenge, or paste. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical treatises, such as Remington’s Pharmaceutical Sciences by E.W. Martin. Also, see Wang, Y.J. and Hanson, M.A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0306] Formulations for parenteral administration may include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending and thickening agents. Preferably, the formulations may be presented in unit dosage or divided dosage containers, such as sealed ampoules and vials. The formulations may be stored in a freeze-dried state that requires only the addition of a sterile liquid carrier, such as physiological saline, a physiologically acceptable solution, or water for injection, immediately prior to use.

[0307] Immediate injection and infusion solutions and suspensions can be prepared from sterile powders, granules, or other dry compositions. Exemplary compositions for parenteral administration include, for example, injectable solutions or suspensions that may contain suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents, and suspending agents that include synthetic monoglycerides or diglycerides, and fatty acids that include oleic acid or Cremaphor.

[0308] The hBCMA-binding polypeptide or hBCMA-binding oligomer of the present invention (particularly, an hBCMA-binding agent-drug conjugate), and a pharmaceutical preparation containing such a polypeptide, oligomer or binding agent-drug conjugate, or the nucleic acid of the present invention (for example, the DNA or RNA molecule of the present invention, for example, the mRNA molecule of the present invention), and a pharmaceutical preparation containing these nucleic acid molecules find use in the treatment and / or prevention of cancer, for example, multiple myeloma.

[0309] The amount of the hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly, an hBCMA-binding agent-drug conjugate), or nucleic acid molecule required to achieve a therapeutic effect will vary depending on the specific route of administration and the characteristics of the subject under treatment, such as species, age, weight, gender, medical condition, specific disease and its severity, as well as other relevant medical and physical factors. A person skilled in the art can readily determine and administer an effective amount of the binding polypeptide, oligomer, binding agent-drug conjugate, and / or a composition containing them, or nucleic acid molecule and / or a composition containing it, required for the treatment and / or prevention of cancer.

[0310] The hBCMA-binding polypeptide, hBCMA-binding oligomer (particularly, an hBCMA-binding agent-drug conjugate), or nucleic acid molecule of the present invention, or a pharmaceutical preparation thereof may be administered, for example, daily, weekly, every two weeks, every three weeks or every four weeks, or as a high single dose, depending on the subject to be treated and the severity of the cancer.

[0311] The hBCMA-binding polypeptide, hBCMA-binding oligomer (in particular, hBCMA-binding agent-drug conjugate), or nucleic acid molecule of the present invention, or a pharmaceutical formulation thereof, may be administered, for example, in parenteral or oral dosage forms. Parenteral administration includes intravenous (into a vein, e.g., a central vein or a peripheral vein, as a bolus or infusion), intraarterial (into an artery, e.g., a central artery or a peripheral artery), intraosseous injection (into the bone marrow), intramuscular (into a muscle), intradermal (into the dermis), and subcutaneous (under the skin) administration. In a preferred embodiment, the dosage of the present invention is administered intravenously or intraarterially, more preferably by intravenous infusion (e.g., central venous infusion or peripheral intravenous infusion). In another preferred embodiment, the dosage of the present invention is administered by subcutaneous injection. Thus, pharmaceutical formulations particularly useful in the present invention are suitable for intravenous administration, more specifically intravenous infusion, or subcutaneous administration.

[0312] The hBCMA-binding polypeptide, hBCMA-binding oligomer (in particular, hBCMA-binding agent-drug conjugate) or nucleic acid molecule of the present invention, or a pharmaceutical formulation thereof, may be administered as part of a treatment cycle. In a treatment cycle, the polypeptide described above may be administered on day 1 of the cycle, the cycle lasts for X days, and for the next X - 1 days, there is no further administration of the polypeptide of the present invention. X may be, for example, from 1 to 42 days, for example, from 2 to 28 days, for example, from 2 to 21 days, for example, from 2 to 14 days. Alternatively, the binding polypeptide may be administered, for example, as a divided dose on days 1 and 2 of the cycle.

[0313] This cycle may be repeated one or more times depending on the category, class, or stage of the cancer to be treated. For example, the cycle may 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) may be administered by repeating the 7-day cycle 8 times, followed by repeating the 14-day cycle 16 times, and optionally, followed by further repeating the 28-day cycle. For example, it may be administered in 21-day cycles (i.e., once every three weeks) until disease progression or unacceptable toxicity. A physician or clinician of ordinary skill can readily determine the number of cycles of the hBCMA-binding polypeptide (e.g., hBCMA-binding agent-drug conjugate) necessary to prevent, counteract, or halt cancer progression.

[0314] Combination therapy The hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly, an hBCMA-binding agent-drug conjugate) or nucleic acid molecule disclosed herein may be used as the sole active ingredient in the present invention, but it is also possible to use it in combination with one or more additional therapeutic agents, and the use of such a combination provides one embodiment of the present invention. Such additional therapeutic agents may be agents useful for the treatment and / or prevention of cancer, or other pharmaceutically active materials. Such agents are known in the art.

[0315] Non-limiting examples of additional therapeutic agents for use in the present invention include proteasome inhibitors (Pl) (e.g., carfilzomib, bortezomib or ixazomib), immunomodulatory agents (IMiD) (e.g., lenalidomide, thalidomide or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, melphufen or bendamustine), anthracyclines (e.g., doxorubicin), steroids (e.g., dexamethasone, prednisone or prednisolone), BCL-2 inhibitors (e.g., venetoclax), histone deacetylase (HDAC) inhibitors (e.g., panobinostat), anti-CD38 agents (e.g., daratumumab or isatuximab), anti-SLAMF7 agents (e.g., elotuzumab), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), or ADAM17 inhibitors. For example, the additional therapeutic agent may be selected from proteasome inhibitors (e.g., carfilzomib or bortezomib), immunomodulatory agents (e.g., lenalidomide or thalidomide), alkylating agents (e.g., melphalan or melphufen), steroids (e.g., dexamethasone or prednisone), anti-CD38 agents (e.g., daratumumab), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), and ADAM17 inhibitors.

[0316] One or more additional therapeutic agents can be used simultaneously, continuously, or separately from the administration of the dosage of the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, or nucleic acid of the present invention. The individual components of such combinations can be administered separately at different times during the course of treatment, or can be administered simultaneously in a divided or single combination form.

[0317] In one embodiment of the present invention, the one or more additional therapeutic agents are selected from PIs, IMiDs, and steroids. For example, the one or more additional therapeutic agents are PIs (e.g., bortezomib or carfilzomib), IMiDs (e.g., lenalidomide, thalidomide, and pomalidomide), and steroids (e.g., prednisone, prednisolone, and dexamethasone). For example, the one or more therapeutic agents are bortezomib, thalidomide, and dexamethasone.

[0318] In one embodiment of the present invention, the one or more additional therapeutic agents are selected from PIs, alkylating agents, and steroids. For example, the one or more additional therapeutic agents are PIs (e.g., bortezomib or carfilzomib), alkylating agents (e.g., cyclophosphamide, melphalan, melphlufen, or bendamustine), and steroids (e.g., prednisone, prednisolone, and dexamethasone). For example, the one or more therapeutic agents are bortezomib, melphalan or melphlufen, and prednisone.

[0319] In one embodiment of the present invention, the one or more additional therapeutic agents are selected from PIs and steroids. For example, the one or more therapeutic agents are PIs (e.g., bortezomib or carfilzomib), and steroids (e.g., prednisone, prednisolone, and dexamethasone). For example, the PI is bortezomib and the steroid is dexamethasone.

[0320] In one embodiment of the present invention, the one or more additional therapeutic agents are selected from IMiDs and steroids. For example, the 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.

[0321] In another embodiment of the present invention, one or more therapeutic agents can be selected from NK cell-based or T cell-based therapies. For example, when the hBCMA-binding polypeptide of the present invention is connected to an additional binding moiety specific for an NK cell surface protein (e.g., an NK cell-binding polypeptide), allogeneic NK cells can be administered. For example, these NK cells can be co-infused with the polypeptide.

[0322] Alternatively, the hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly, an hBCMA-binding agent-drug conjugate) or nucleic acid molecule according to the present invention can be combined with a stem cell transplantation procedure, such as a therapeutic technique like autologous stem cell transplantation or allogeneic stem cell transplantation. Thus, in one embodiment of the present invention, the hBCMA-binding polypeptide disclosed herein can be combined with an autologous stem cell transplantation technique. In another embodiment of the present invention, the hBCMA-binding polypeptide disclosed herein can be combined with an allogeneic stem cell transplantation technique.

[0323] The simultaneous, sequential, or separate administration of one or more additional therapeutic agents or therapeutic techniques using the hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate, or nucleic acid molecule of the present invention further enhances their effectiveness in the treatment and / or prevention of cancer.

[0324] Kit The present invention provides a kit comprising the hBCMA-binding polypeptide, hBCMA-binding oligomer (particularly an hBCMA-binding agent-drug conjugate) or nucleic acid molecule disclosed herein, and one or more additional therapeutic agents useful in the treatment and / or prevention of cancer.

[0325] Non-limiting examples of additional therapeutic agents for use in the kit of the present invention include proteasome inhibitors (Pl) (e.g., carfilzomib, bortezomib or ixazomib), immunomodulatory agents (IMiD) (e.g., lenalidomide, thalidomide or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, melphlufen or bendamustine), anthracyclines (e.g., doxorubicin), steroids (e.g., dexamethasone, prednisone or prednisolone), BCL-2 inhibitors (e.g., venetoclax), histone deacetylase (HDAC) inhibitors (e.g., panobinostat), anti-CD 38 agents (e.g., daratumumab or isatuximab), anti-SLAMF7 agents (e.g., elotuzumab), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), or ADAM17 inhibitors.

[0326] For example, one or more additional therapeutic agents may be selected from proteasome inhibitors (e.g., carfilzomib or bortezomib), immunomodulatory agents (e.g., lenalidomide or thalidomide), alkylating agents (e.g., melphalan or melphlufen), steroids (e.g., dexamethasone or prednisone), anti-CD38 agents (e.g., daratumumab), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), and ADAM17 inhibitors.

[0327] One or more additional therapeutic agents may be selected from, for example, proteasome inhibitors (e.g., carfilzomib or bortezomib), immunomodulatory agents (e.g., lenalidomide or thalidomide), alkylating agents (e.g., melphalan or melphlufen), and steroids (e.g., dexamethasone or prednisone).

[0328] In one embodiment of the present invention, the kit of the present invention finds use in the treatment and / or prevention of cancer.

[0329] To avoid misunderstanding, the hBCMA-binding polypeptides, hBCMA-binding oligomers (especially hBCMA-binding agent-drug conjugates), or nucleic acid molecules disclosed herein are present in the kits according to the invention in forms and amounts suitable for use according to the invention. Suitable pharmaceutical formulations are described herein. A person skilled in the art can readily determine the amount of the hBCMA-binding polypeptide or oligomer (e.g., in the form of an hBCMA-binding agent-drug conjugate) or nucleic acid molecule disclosed herein that is suitable for use according to the invention.

[0330] Use of the polypeptides of the invention The hBCMA-binding polypeptides, hBCMA-binding oligomers (especially hBCMA-binding agent-drug conjugates), or nucleic acid molecules of the invention, and pharmaceutical formulations or kits of the invention comprising the above-described hBCMA-binding polypeptides, hBCMA-binding oligomers, hBCMA-binding agent-drug conjugates, or nucleic acid molecules find use in medicine, for example, in the treatment and / or prevention of 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 can include leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, small lymphocytic lymphoma, and other high-grade B-cell lymphomas), or plasma cell neoplasms and myelomas (e.g., MGUS, plasmacytoma, smoldering myeloma, multiple myeloma, light chain myeloma, or non-secretory myeloma).

[0331] Preferably, the hBCMA-binding polypeptide or hBCMA-binding oligomer (particularly, the 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 containing the above-described hBCMA-binding polypeptide, oligomer, binding agent-drug conjugate or nucleic acid molecule find use in the treatment and / or prevention of myeloma (e.g., MGUS, plasmacytoma, smoldering 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 containing the above-described hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate or nucleic acid molecule find use in the treatment and / or prevention of multiple myeloma.

[0332] Alternatively, or in addition, the hBCMA-binding polypeptide, hBCMA-binding oligomer (particularly, the hBCMA-binding agent-drug conjugate) or nucleic acid molecule of the present invention, and the pharmaceutical preparation or kit of the present invention containing the above-described hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate or nucleic acid molecule may also find use in the treatment and / or prevention of 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, Sjögren's syndrome, systemic lupus erythematosus, or type 1 diabetes, particularly autoimmune disorders involving hBCMA signaling, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and optic neuritis, and Sjögren's syndrome.

[0333] The hBCMA-binding polypeptide, hBCMA-binding oligomer (particularly, hBCMA-binding agent-drug conjugate) or nucleic acid molecule of the present invention also finds use in biotechnology and research applications such as, for example, the detection of hBCMA in a biological sample. The hBCMA-binding polypeptide, hBCMA-binding oligomer, hBCMA-binding agent-drug conjugate or nucleic acid molecule of the present invention may be used, for example, in an enzyme-linked immunosorbent assay (ELISA), or may be conjugated to a reporter such as a fluorophore for use, for example, in flow cytometry or immunohistochemistry.

Example

[0334] The following examples illustrate the present invention.

[0335] Preparation Example 1: Selection of Binding Agents to hBCMA Using Phage Display In this example, human BCMA (hBCMA) was used as a target in phage display selection using a phage-based affibody library. Individual clones obtained after four selection cycles were assayed for binding to hBCMA in monoclonal phage-ELISA (enzyme-linked immunosorbent assay), and ELISA-positive clones were DNA sequenced.

[0336] Materials and Methods Affibody Library. 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 modified with an albumin binding domain (ABD WT) It is designed for phage display of the encoded affibody library members as an in-frame fusion to an amber stop codon and a truncated form of the M13 phage coat protein 3 (residues 249 - 406). A synthetic 121-base-long oligonucleotide (5’-GCGCTTTGGCTTGGGTCATCXXXTAAACTXXXYYYGAAGGCXXXXXXTTGXXXXXXGTTCAGGTTCGGCAGXXXXXXGATCTCXXXXXXCGCXXXXXXXXXTTCTTTGTTGAATTTGTTGT-3’) [SEQ ID NO: 280] encoding amino acids 3 - 41 (reverse complementary strand) of the Z domain (Nilsson et al. (1987) Protein Eng. 1:107 - 113), in which the wild-type codons for 14 positions in the domain were randomized using a mixture of trinucleotide codon building blocks (XXX = equal codon mixture for all 20 amino acids except Cys and Pro, YYY = 60% Ile and 10% each of His, Tyr, Lys, and Asp), was used as the forward primer (5-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3’) [SEQ ID NO: 281] and the reverse (5-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3’) [SEQ ID NO: 282] were used as templates for PCR amplification. Approximately 6.4 μg of the PCR product double-digested with XhoI and NheI and gel-purified (Qiagen, Germany) was ligated to 35 μg of the pAffi-1 phagemid vector double-digested with XhoI and NheI and gel-purified using T4 DNA ligase. The resulting ligation mixture was desalted using column purification (Qiagen, Germany), divided into 24 aliquots, and used to electroporate 25 μl of electrocompetent ER2738 E. coli cells (F’, glnV amber suppressor) (Lucigen, USA) (0.1 cm BioRad cuvette).

[0337] 970 μl of 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, then pooled (6 electroporations per pool), incubated at 37 °C for 1 hour with shaking, and then the cell pool was titrated by spreading a dilution series onto Amp (ampicillin) plates. The cell pool was transferred to four 5-liter shaking flasks containing 1 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. The overnight cell culture was pelleted by centrifugation, resuspended in 50 ml of cold 40% glycerol, and subsequently distributed into 29 tubes of approximately 3.5 ml of cell / glycerol solution per tube. Titration after electroporation and OD after overnight culture 600 From the measurement, the library size (diversity) was calculated to be approximately 3×10 10 and it was calculated that each 3.5 ml aliquot of cells contained a cell number corresponding to approximately 10,9-fold of the library size. The tubes containing the cells were stored at -80 °C until used for phage stock preparation using M13KO7 helper phage.

[0338] Generation of naive phage library stock. For the generation of the afibody-displaying phage stock, 2 ml of the library glycerol stock was dispensed and inoculated into three E flasks containing 750 ml of TSB+Y, 1% (w / v) glucose, 10 μg / ml Tet (tetracycline), and 100 μg / ml Carb (carbenicillin). When the culture reached OD 600It was grown at 37°C while shaking at 150 rpm until it reached =1. The cells were infected with M13KO7 helper phage (New England Biolabs) at 600 μl per E flask (multiplicity of infection (MOI) of 5), gently rotated, incubated at 37°C for 15 minutes without shaking, and then incubated at 70 rpm and 37°C for 15 minutes. The cells of each culture were centrifuged and resuspended in 670 ml of TSB+Y, 100 μg / ml of Carb, and 1 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside). 25 μg / ml of Kan (kanamycin) was added 2 hours after inoculation. The cultures were incubated at 37°C for 16 - 18 hours while shaking at 200 rpm. The phage library stock was recovered by precipitating it twice with 20% (w / v) PEG6000 / 2.5 M NaCl. The titer of the stock was measured by spot titration, and polymerase chain reaction screening was used to analyze the proportion of phage particles carrying phagemids with affibody inserts.

[0339] Phage display selection of hBCMA-binding candidates. Biotinylated hBCMA-Fc (human TNFRSF17 / BCMA / CD269 protein (His and human IgG1 Fc tag)), biotinylated (Sino Biological Inc., Eschborn, Germany, catalog number 10620-H03H-B, corresponding to residues 1 - 54 of Uniprot entry Q02223) was used at concentrations of 150 nM (cycle 1), 100 nM (cycle 2) and 50 nM (cycles 3 - 4) for 4 cycles of panning. 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 non-specific 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). Furthermore, in cycles 2 - 4, the phage stock in PBS-T was preincubated for 30 min at room temperature (RT) with SA beads (Herceptin, Roche, Basel, Germany) containing 0.1% (w / v) BSA and biotinylated trastuzumab mAb (containing the Fc region) under constant end-over-end (eoe) rotation to perform negative selection by removing phages carrying binders to SA and Fc. The amount of phage stock used was approximately 8×10 in cycle 1 and subsequent cycles 11It was a colony forming unit (cfu). The biotinylated target protein was immobilized on 1.5 mg of beads at room temperature and eoe for 1 hour. The target-containing beads were incubated with 1% (w / v) BSA in PBS-T at room temperature and eoe for 30 minutes, then washed with PBS-T, and then the pre-incubated phage stock was added for selection. The selection process was carried out at room temperature, and the incubation time for selection was 3 hours (cycle 1). In subsequent cycles, it was washed 1, 3, 6, or 10 times at room temperature using PBS-T eoe. The final wash volume was transferred to a new 1% (w / v) BSA-pretreated tube to remove the sticky binder adhering to the tube wall. The antigen-binding phage was eluted by incubating with 0.3 M acetic acid (pH 2.8) at room temperature and eoe for 15 minutes. Subsequently, the eluate was transferred to a new pretreated tube and neutralized with an equal volume of 1 M Tris-HCl (pH 8).

[0340] Phage stock amplification. A new phage stock was generated by infecting Escherichia coli XL-1 Blue cells (Agilent) grown in TSB+Y containing 10 μg / ml of Tet with the phage eluate (total volume after cycle 1, half volume after cycles 2-3) while shaking at 150 rpm until the OD 600 reached 0.5 - 0.8 at 37°C. 15 ml of the bacterial culture was used for cycles 1 - 2, and 7.5 ml was used for cycles 3 - 4. The infected culture was gently rotated and incubated at 37°C for 25 minutes without shaking, and then incubated for 15 minutes while shaking at 70 rpm and 37°C. Then, the culture was centrifuged, resuspended in TSB+Y, and plated on a blood agar plate (40 g / l blood agar) containing 100 μg / ml of Carb and 1% (w / v) glucose, and incubated at 37°C for 16 - 18 hours. The bacterial colonies were scraped and collected in TSB+Y, inoculated into 200 ml of TSB+Y containing 100 μg / ml of Carb, and the OD 600It was grown at 37 °C while shaking at 150 rpm until it reached 0.5 - 0.8. 30 ml after cycle 1, 20 ml after cycle 2, or 10 ml after the cycle was super-infected with M13KO7 helper phage (MOI of about 10), gently rotated, incubated at 37 °C for 25 minutes without shaking, and then incubated at 70 rpm and 37 °C for 15 minutes. The culture was centrifuged and resuspended in 150 ml of TSB+Y containing 100 μg / ml of Carb and 1 mM of IPTG. 25 μg / ml of Kan was added 2 hours after inoculation. The culture was incubated at 37 °C while shaking at 150 rpm for 16 - 18 hours. The phage library stock was recovered by precipitating twice with 20% (w / v) PEG6000 / 2.5 M NaCl. The titer of the stock was measured by spot titration on Carb plates, and the proportion of colonies carrying phagemids with the correct-sized affibody insert was analyzed using polymerase chain reaction screening.

[0341] Preparation of monoclonal phage supernatant. Following cycle 4, 48 bacterial colonies that generated PCR products of the correct size were individually grown in 500 μl of TSB+Y / Carb in a 96-well deep-well plate at 30 °C for 16 - 18 hours while shaking at 250 rpm. 30 μl of the overnight culture was inoculated into 720 μl of TSB+Y / Carb in a new 96-well deep-well plate, incubated at 37 °C and 250 rpm for 2 hours, and then super-infected by adding an excess of M13K07 helper phage in 100 ul of TSB+Y / Carb per well. The plate was incubated at 37 °C for 30 minutes without shaking. Finally, 150 μl of TSB+Y / Carb / IPTG / Kan per well was added. The final concentrations were 100 μg / ml of Carb, 1 mM of IPTG, and 25 μg / ml of Kan. The deep-well plate was incubated at 37 °C and 250 rpm for 16 - 18 hours. The next day, the phage supernatant was recovered by centrifugation.

[0342] Candidate monoclonal phage-ELISA screening. MaxiSorp ELISA plates (Clear Flat-Bottom Immuno Nonsterile 384-Well Plates, catalog number 464718, Thermo Fisher Scientific) were coated with 30 μl of 5 μg / ml hBCMA-Fc (corresponding to residues 1 - 54 of NCBI entry NP_001183.2 fused to rabbit Fc, Sino Biological, catalog number 10620-H15H), 15 μg / ml human serum albumin (HSA, Sigma product number SRP6182) (for evaluation of proper presentation of an expression cassette containing an affibody, an affibody albumin-binding domain, and a cleavable protein 3), 10 μg / ml trastuzumab (as a control for binding to the Fc tag), or 5 μg / ml of an irrelevant control protein (human CD38, His Tag, Aero Biosystems, catalog number CD8-H5224, corresponding to residues 43 - 300 of NCBI entry NP_001766.2) in 100 mM sodium carbonate buffer pH 8.5 / well (1 / 4 of the well with each coated protein). The coating solution was removed, the plates were washed twice with PBS-T, and blocked with PBS supplemented with 1% (w / v) BSA for 1 hour at room temperature with gentle shaking. The blocking solution was discarded, and the wells were incubated with 10 μl of phage supernatant diluted 1:3 with 20 μl of PBS-T for 1 hour at room temperature with gentle shaking. The supernatant was removed, and the plates were washed three times with PBS-T. 30 μl of 1:5000 α-M13-HRP (Sigma-Aldrich, Stockholm, Sweden) in PBS-T was added and incubated for 30 minutes at room temperature with gentle shaking, after which the plates were 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 stopped by adding 30 μl of 2 M H2SO4 after 15 - 25 minutes.The absorbance at 450 nm was measured using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). Candidates considered to be ELISA positive clones had high HSA signals and relatively high signals against hBCMA-Fc compared to the signals observed against trastuzumab (Fc control) and other irrelevant target controls.

[0343] 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).

[0344] Results To identify polypeptides that bind to hBCMA, an affibody phage library was constructed and used as input in the first cycle of a 4-cycle phage display selection campaign as described in Materials and Methods (above). After the fourth selection cycle, E. coli cells were infected with phage eluates from the acid and trypsin selection tracks to obtain individual colonies on carbenicillin plates. Phage-ELISA experiments using microtiter plates coated with four test antigens (HSA, hBCMA-Fc, trastuzumab (Fc), and CD38) were used to perform binding analysis on 48 randomly selected colonies (clones) with an affibody gene insert of the correct size from each track. DNA sequencing was subsequently performed on one clone that showed a strong ELISA signal against HSA and hBCMA-Fc and a low signal against the trastuzumab (Fc) and CD38 controls. This clone was named Fa-G6, and its deduced amino acid sequence is described in Table 1 and the Sequence Listing as SEQ ID NO:1. Table 1 also lists the remaining sequences of the present disclosure along with references to the corresponding SEQ ID NOs. Throughout the chapter of this example, an example compound having the number X is a polypeptide having the amino sequence of the same number, i.e., SEQ ID NO:X.

[0345] Biological Example 2: Initial Biosensor Characterization of Candidate hBCMA-Binding Clone Fa-G6 In this example, the hBCMA-binding polypeptide Fa-G6 corresponding to SEQ ID NO:1 was subcloned, expressed, and purified as a His6-affibody-ABD WT fusion protein [SEQ ID NO:3] and first analyzed for binding to hBCMA-Fc by surface plasmon resonance.

[0346] Materials and Methods Subcloning of Fa-G6 polypeptide variants. The DNA fragment encoding the Fa-G6 binding polypeptide candidate variant [SEQ ID NO: 1] was amplified by PCR from the library phagemid vector pAffi-1 using specific primers that introduced restriction sites for XhoI and AscI. The fragment was cleaved and ligated into a T7 promoter-based E. coli expression vector prepared using the same restriction enzymes. This generated a monomeric polypeptide construct with an N-terminal His6 tag and a C-terminal ABD WT The DNA construct was sequence-verified using Sanger sequencing (Microsynth). The amino acid sequence of this affibody variant is listed in Table 1 and the Sequence Listing as SEQ ID NO: 3.

[0347] Expression and purification. E. coli BL21(DE3) cells were transformed with the plasmid containing the expression DNA construct and cultured at 37 °C for 16 - 18 h with shaking at 150 rpm in 10 ml of TSB+Y containing 25 μg / ml of Kan. The culture was inoculated 1:100 into 200 ml of TSB+Y containing 25 μg / ml of Kan and grown at 37 °C with shaking at 150 rpm until OD 600 = 0.6 - 1.

[0348] IPTG was added until a final concentration of 1 mM was reached to induce protein expression. The culture was incubated at 25 °C for 16 - 18 h with shaking at 150 rpm, and the cells were harvested by centrifugation. The cell pellet was resuspended in denaturing lysis buffer (7 M guanidinium chloride, 47 mM Na2HPO4, 2.65 mM NaH2PO4, 10 mM Tris-HCl, 100 mM NaCl, pH 8). After incubation at 37 °C for 2 h with shaking at 150 rpm, 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 for 30 min at room temperature with end-over-end rotation. 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), followed by hBCMA-binding affibody Fa-G6-ABD WT The fusion protein was 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 incubating for 10 min at room temperature with end-over-end rotation. After IMAC purification, the protein buffer was exchanged to PBS using a PD-10 desalting column (Catalog No. 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm purity and concentration, and the absorbance was measured at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA). The low molecular weight marker was from Cytiva (Product No. 17044601).

[0349] N-terminal His6 tag and C-terminal ABD WT Biosensor analysis of the hBCMA-binding variant Fa-G6 having Using a Biacore T200 instrument (Cytiva), the real-time interaction between the His6-Fa-G6-ABD WT fusion protein [SEQ ID NO: 3] and hBCMA was analyzed. The 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 and used as a reference cell. His6-Fa-G6-ABD WT The fusion protein [SEQ ID NO: 3] was diluted with electrophoresis buffer PBS-T and then the binding analysis was performed 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 (Fc-containing) control surface.

[0350] Results To examine whether the anti-hBCMA binding polypeptide candidate clone Fa-G6 [SEQ ID NO: 1] identified via phage-ELISA and DNA sequencing showed target binding when expressed as a soluble protein, this was subcloned and expressed in E. coli cytoplasm as His6-polypeptide-ABD WT fusion protein [SEQ ID NO: 3] and purified. ABD WTThe moiety is a small serum albumin-binding protein and can be used for affinity chromatography purification and / or immobilization using human serum albumin as a ligand if desired. Analysis of the fusion protein purified by immobilised metal ion affinity chromatography (IMAC) using a hexahistidyl (His6) gene fusion partner in the protein by SDS-PAGE showed that the protein was of high purity and had an approximate molecular weight according to its amino acid sequence. Injection of the fusion protein at a concentration of 200 nM onto the surface of a sensor chip containing immobilized hBCMA-Fc showed that the fusion protein bound to hBCMA-Fc. Figure 2a shows, in schematic form, a typical binding experiment. The resulting binding experiment trace is shown in Figure 2b. No binding to the trastuzumab (Fc) control was observed (data not shown).

[0351] Biological Example 3: Biosensor evaluation of N-terminal truncated variants of the hBCMA-binding clone Fa-G6 In this example, residues 1 - 5 at the N-terminus of the hBCMA-binding clone Fa-G6 [SEQ ID NO: 1] were sequentially cleaved to investigate the effect on the binding interaction between Fa-G6 and hBCMA.

[0352] Materials and methods Cloning of the hBCMA-binding clone for construction of the cleavage variants. A DNA fragment encoding the hBCMA-binding polypeptide candidate [SEQ ID NO: 1] was amplified from the library vector pAffi-1 using specific primers designed to introduce overhangs into the full coding sequence, or a coding sequence having a deletion extending to the first 5 residues at the N-terminus and having ends complementary to the ends of the linearized expression vector (one for each cleavage variant). Five monomeric affibody constructs with a C-terminal His6 tag (a wild-type Fa-G6 variant and four cleavage mutants with deletion cleavages corresponding to the -2, -3, -4, and -5 residues at the affibody N-terminus, respectively) were cloned using the In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden). 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.

[0353] Expression and purification of cleavage variants. E. coli BL21(DE3) cells were transformed with plasmids containing the five constructs with a C-terminal His6 tag and cultured at 37 °C for 16-18 h with shaking at 150 rpm in 10 ml of TSB+Y containing 25 μg / ml of Kan. The culture was inoculated 1:100 into 200 ml of TSB+Y containing 25 μg / ml of Kan, and the OD 600It was grown at 37 °C while shaking at 150 rpm until it reached 0.6 - 1. IPTG was added until the final concentration reached 1 mM to induce protein expression. The culture was incubated at 25 °C for 16 - 18 hours while shaking at 150 rpm, and the cells were collected by centrifugation. The cell pellet was resuspended in denaturing lysis buffer. After incubating at 37 °C for 2 hours while shaking at 150 rpm, 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 number 89966, Thermo Scientific). The supernatant was incubated with the resin at room temperature for 30 minutes while end - over - end rotating. Contaminants were removed by washing three times with wash buffer, and then the Fa - G6 affibody variant was eluted with elution buffer by incubating at room temperature for 10 minutes while end - over - end rotating.

[0354] After IMAC purification, the protein buffer was exchanged to PBS using a PD - 10 desalting column (catalog number 17085101, Cytiva). SDS - PAGE analysis was performed to confirm the purity and concentration, and the absorbance was measured at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen). The low - molecular - weight marker was from Cytiva (product number 17044601).

[0355] Biosensor analysis of the cleavage mutant. Using a Biacore T200 instrument (Cytiva), the interaction between four cleavage mutants [Example Compounds 5 - 8 having SEQ ID NOs: 5 - 8] and hBCMA was analyzed in real time and compared with the wild-type hBCMA binder Fa-G6 [SEQ ID NO: 4]. The protein ligand hBCMA-Fc (Sino Biological Inc., Catalog No. 10620-H15H) was immobilized (1000 RU) on a Series S CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated for use as a reference cell. The analyte was diluted with electrophoresis buffer PBS-T, and the binding assay was performed 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. The wild-type Fa-G6-His6 construct and the four cleavage mutants described above were injected onto the hBCMA-Fc surface at a concentration of 20 nM.

[0356] Results To investigate whether cleavage of the N-terminal residue affects the binding of the Fa-G6 clone to hBCMA, cleavage corresponding to the -2, -3, -4, and -5 residues at the N-terminus of the polypeptide was performed to obtain four cleavage mutants, which together with the wild-type Fa-G6 were expressed in the E. coli cytoplasm and purified as His6 fusion proteins [SEQ ID NOs: 4 - 8]. Analysis of the IMAC-purified fusion proteins using His6 fusion proteins in the protein by SDS-PAGE showed that all five proteins were of high purity and had an approximate molecular weight according to their amino acid sequences. Injection of four cleavage mutants and the wild-type binder with successively smaller molecular weights onto the sensor chip surface containing immobilized hBCMA-Fc showed that cleavage up to residue 5 at the N-terminus did not impair the binding response profile to hBCMA-Fc compared to the parental full-length Fa-G6 (Figure 3).

[0357] Biological Example 4: Alanine Scan of the hBCMA Binding Clone Fa-G6 The hBCMA-binding Fa-G6 clone promoted the development of a second-generation hBCMA binder based on the parental Fa-G6 binder, which exhibited relatively low solubility and / or thermal stability characteristics and potentially different biophysical properties compared to Fa-G6. In this example, alanine scanning of Fa-G6 was performed to investigate which positions were important for the binding interaction between Fa-G6 and hBCMA and used as the basis for the design of a second-generation library based on Fa-G6.

[0358] Materials and Methods Site-directed mutagenesis of the hBCMA-binding clone Fa-G6. The DNA fragment encoding the Fa-G6-binding polypeptide candidate variant [SEQ ID NO: 1] was amplified by PCR from the library phagemid vector pAffi-1 using specific primers that introduced restriction sites. The fragment was cleaved and ligated into a T7 promoter-based E. coli expression vector prepared using the same restriction enzyme. This generated 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 the DNA template in individual mutagenesis PCR reactions to substitute the original codons at 14 various positions with alanine codons using primers designed for each position. The 14 alanine substitution variants were sequence-verified using Sanger sequencing (Microsynth), and their amino acid sequences are listed in Table 1 and the Sequence Listing as SEQ ID NOs: 9-22.

[0359] Expression and purification of alanine variants. E. coli BL21(DE3) cells were separately transformed with individual plasmids encoding 14 alanine variants [SEQ ID NOs: 9 - 22], each with an N-terminal His6, and the wild-type His6-Fa-G6 construct [SEQ ID NO: 129], and cultured at 37°C for 16 - 18 hours with shaking at 150 rpm in 10 ml of TSB+Y containing 25 μg / ml of Kan. The culture was inoculated 1:100 into 200 ml of TSB+Y containing 25 μg / ml of 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 incubated at 25°C for 16 - 18 hours with shaking at 150 rpm, and the cells were harvested by centrifugation. The cell pellet was resuspended in denaturing lysis buffer. After incubation at 37°C for 2 hours with shaking at 150 rpm, 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 for 30 minutes at room temperature while end-over-end rotating. Contaminants were removed by washing three times with wash buffer, and then the hFa-G6-His6 variant was eluted with elution buffer by incubating for 10 minutes at room temperature while end-over-end rotating. After IMAC purification, the protein buffer was exchanged to PBS using a PD-10 desalting column (Catalog No. 17085101, Cytiva). SDS-PAGE analysis was performed to confirm purity and concentration, and the absorbance was measured at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen). The low molecular weight marker was from Cytiva (Product No. 17044601).

[0360] Biosensor analysis of alanine variants. The interaction between His6-tagged wild-type Fa-G6 polypeptide [SEQ ID NO: 129] and 14 alanine variants [SEQ ID NOs: 9-22] with hBCMA was analyzed in real time using a Biacore T200 instrument (Cytiva). The protein ligand hBCMA-Fc (Sino Biological Inc., Catalog No. 10620-H15H) was immobilized (3500 RU) onto a Series S CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated for use as a reference cell. The analyte was diluted with running buffer PBS-T and then the binding assay was performed 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. The wild-type His6-Fa-G6 construct and the 14 alanine variants described above were injected onto the hBCMA-Fc surface at a common concentration of 200 nM.

[0361] Results To investigate which of the amino acids occupying 14 various positions in the Fa-G6 clone are most important for the binding interaction to hBCMA, site-directed alanine scanning mutagenesis was performed to obtain 14 alanine variants for subsequent design of a second-generation library based on Fa-G6. These were expressed in the E. coli cytoplasm together with wild-type Fa-G6 (His6-Fa-G6, [SEQ ID NO: 129]) and purified as His6-affibody fusion proteins [SEQ ID NOs: 9-22]. Analysis of the IMAC-purified fusion proteins using SDS-PAGE with 6 gene fusion partners in the protein showed that all 15 proteins were of high purity and had an approximate molecular weight according to their amino acid sequences.

[0362] Figure 4 shows the injection of 14 alanine variants and wild-type Fa-G6 binder onto the surface of a sensor chip containing immobilized hBCMA-Fc. The resulting sensorgrams showed that substitution of alanine at positions 9, 10, 11, 14, and 18 decreased the binding of these variants to hBCMA, whereas substitution of alanine at positions 13, 24, 25, 27, 28, 31, 32, and 35 impaired the binding. Substitution of alanine at position 17 suggested an improvement in hBCMA-Fc binding.

[0363] Preparation Example 5: Construction, Selection, and Phage ELISA of the Second Generation Library In this example, two different polypeptide libraries were constructed based on the randomization of specific variable positions of the Fa-G6 clone and randomization to various extents. These two libraries were used for the selection of new hBCMA binders that have diversified biophysical properties compared to the existing Fa-G6 and the potential to be used for different application purposes. Individual clones obtained after three rounds of phage display selection cycles were assayed for binding to hBCMA in monoclonal phage-ELISA, and ELISA-positive clones were DNA sequenced.

[0364] Materials and Methods Second generation library of hBCMA affibody Fa-G6. Two M13 phage display second-generation selection libraries were prepared based on the Fa-G6 clone. The phagemid vectors used were pAffi-1 (Grbnwall et al. (2007) J. Biotechnol. 128:162-183), and its variant designated pAffi-100. The pAffi-1 phagemid containing the lac promoter and the OmpA signal peptide was designed for phage display of the affibody library members encoded as an in-frame fusion to 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 affibody library members as an in-frame fusion to the lac promoter, the OmpA signal peptide, and ABD, but further contains a trypsin protease cleavage site before the amber stop codon, followed by the full-length form of the M13 phage coat protein 3.

[0365] Oligo A (5’-ACAACAAATTCAACAAAGAA X01 X01 X03 GCG X04 X01 GAGATC X06 X06 CTGCCGAACCTGAAC X03 X09 CAA X10 XI 1 GCCTTC X12 X13 X14 TTA X15 GATGACCCAAGCCAAAGCGC-3’)[SEQ ID NO: 283] and oligo B (5’-ACAACAAATTCAACAAAGAA X01 X01 Y03 GCG Y04 X01 GAGATC X06 X06 CTGCCGAACCTGAAC Y03 Y09 CAA Y10 Y11 GCCTTC Y12 Y13 X14 TTA Y15 GATGACCCAAGCCAAAGCGC-3’)[SEQ ID NO: 284], which are two synthetic 121-base oligonucleotides based on the Fa-G6 sequence, encode amino acid positions 3 to 41 (reverse complementary strand) corresponding to the Z domain numbering (Nilsson et al. (1987) Protein Eng.1:107-113), the wild-type codons at 14 positions in the domain were randomized using a mixture of trinucleotide codon building blocks (excluding the amino acid codons for Cys, Pro, and Gly, X01 = an equal mixture of codons for all 17 amino acids, X03 = 90% is Phe, and the remaining 10% is evenly distributed among the remaining amino acids, X04 = 90% is Asp, and the remaining 10% is evenly distributed among the remaining amino acids, X06 = 20% is Ala, and the remaining 80% is evenly distributed among the remaining amino acids, X09 = 90% is Tyr, and the remaining 10% is evenly distributed among the remaining amino acids, X10 = 90% is Lys, and the remaining 10% is evenly distributed among the remaining amino acids, X11 = 90% is Trp, and the remaining 10% is evenly distributed among the remaining amino acids, X12 = 90% is Ile, and the remaining 10% is evenly distributed among the remaining amino acids, X13 = 90% is Arg, and the remaining 10% is evenly distributed among the remaining amino acids, X14 = 90% is Ser and 10% is Lys, X15 = 90% is Met, and the remaining 10% is evenly distributed among the remaining amino acids, Y03 = 60% is Phe, and the remaining 40% is evenly distributed among the remaining amino acids, Y04 = 60% is Asp, and the remaining 40% is evenly distributed among the remaining amino acids, Y09 = 60% is Tyr, and the remaining 40% is evenly distributed among the remaining amino acids, Y10 = 60% is Lys, and the remaining 40% is evenly distributed among the remaining amino acids, Y11 = 60% is Trp, and the remaining 40% is evenly distributed among the remaining amino acids, Y12 = 60% is Ile, and the remaining 40% is evenly distributed among the remaining amino acids, Y13 = 60% is Arg, and the remaining 40% is evenly distributed among the remaining amino acids, Y15 = 60% is Met, and the remaining 40% is evenly distributed among the remaining amino acids), during synthesis, the forward primer... (5’-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3’)[SEQ ID NO: 285] and the reverse primer [5’-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3’)(SEQ ID NO: 286] were used as templates for PCR amplification.

[0366] Double-digest with approximately 5.4 μg of XhoI and NheI, and ligate the gel-purified (Qiagen, Germany) PCR products of oligo A and oligo B, respectively, to the gel-purified pAffi-1 and pAffi-100 phagemid vectors double-digested with 30 μg of XhoI and NheI using T4 DNA ligase, respectively.

[0367] Desalt each resulting ligation mixture using column purification (Qiagen, Germany), divide it into 24 portions, and use this to electroporate 25 μl of electrocompetent ER2738 E. coli cells (F’, glnV amber suppressor) (Lucigen, USA) (0.1 cm BioRad cuvette). Add 970 μl of 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) to the electroporated cells, then pool (6 electroporations per pool), incubate at 37 °C for 1 hour with shaking, and then titrate the pool of cells by spreading a dilution series onto ampicillin plates. Transfer to four 5-liter shake flasks 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 each. Pellet the overnight cell culture by centrifugation, resuspend in 50 ml of cold 40% glycerol, and subsequently distribute into approximately 29 tubes of about 2 ml of cell / glycerol solution per tube. Titration after electroporation and OD measurement after overnight culture 600 From the measurement, the library size (diversity) of oligo A in pAffi-1 was calculated to be approximately 3.4×10 7 and it was calculated that each 2 ml aliquot of cells contains the number of cells corresponding to a library size of approximately 147-fold. For oligo B in pAffi-100, the calculated library size (diversity) was approximately 5.9×10 7and each 2 ml aliquot of cells contained a cell number corresponding to a library size of approximately 84 - fold. Tubes containing the cells were stored at - 80 °C until used for phage stock preparation using M13KO7 helper phage.

[0368] Phage stock preparation. For the generation of the afibody - displaying phage stock, 0.5 ml of each library glycerol stock was aliquoted and inoculated into one baffled - free Erlenmeyer flask containing 500 ml of 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 OD 600 = 1. 25 ml of cells per culture were infected with M13KO7 helper phage (New England Biolabs) at a multiplicity of infection (MOI) of 5, gently rotated, incubated at 37 °C for 15 minutes without shaking, and then incubated at 70 rpm, 37 °C for 15 minutes. The cells of each culture were centrifuged and resuspended in two baffled Erlenmeyer flasks containing 700 ml of TSB + Y, 100 μg / ml Carb, and 1 mM IPTG (isopropyl β - D - 1 - thiogalactopyranoside) for each library. 25 μg / ml Kan (kanamycin) was added 2 hours after inoculation. The cultures were incubated at 37 °C with shaking at 200 rpm for 16 - 18 hours. The phage library stock was recovered by precipitation twice with 20% (w / v) PEG6000 / 2.5 M NaCl. The stock titer was measured by spot titration, and the proportion of colonies carrying phagemids with afibody inserts of the correct size was analyzed using polymerase chain reaction screening.

[0369] Selection from the secondary library of the BCMA - binding clone Fa - G6. Recombinant biotinylated hBCMA-Fc was used at concentrations of 140 nM in cycle 1, 40 nM in cycle 2, and 25 nM in cycle 3 to perform 3 rounds of panning in 6 separate selection tracks using 2 optimized selection library stocks. In 3 additional selection tracks that 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. Trastuzumab containing human IgG1 Fc was used at 300 nM in all selection cycles for simultaneous negative selection. 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 non-specific 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). Furthermore, phage stocks in PBS-T were pre-incubated with 0.1% (w / v) BSA and beads for 30 minutes at room temperature (RT) under constant end-over-end (eoe) rotation to remove phages that retained binders to SA. The amount of phage stock used was 10 12 colony forming units (cfu) in cycle 1, 10 13 cfu in cycle 2, and 10 11~13 cfu in cycle 3.

[0370] For three tracks using solid-phase selection, biotinylated target protein was immobilized on 0.5 mg of beads at RT for 1 hour end-over-end (eoe). Target-containing beads were incubated with 1% (w / v) BSA in PBS-T at room temperature and eoe for 30 minutes, then washed with PBS-T and pre-incubated phage stock and trastuzumab were added, and selection was performed at room temperature and eoe for 2 hours. For six tracks by liquid-phase selection, pre-incubated phage stock and trastuzumab were added to biotinylated target protein and selection was performed in solution. The selection step was carried out at room temperature and the incubation time for selection was 2 hours for cycles 1 and 3 or 1.5 hours for cycle 2. Three of these tracks included competition with non-biotinylated target (off-rate selection), which was added after the aforementioned incubation and incubated for an additional 1 hour. The phage-antigen complex was captured by incubating with 0.5 mg of SA beads at room temperature and eoe for 30 minutes.

[0371] The SA beads-captured phage-antigen complexes were washed with PBS-T for a total of 5 minutes, 10 minutes, or 20 minutes at room temperature, eoe, for each subsequent cycle. For three tracks including competition with non-biotinylated targets, the washes in cycles 2-3 were performed for 4 minutes at room temperature and eoe using 100 nM non-biotinylated hBCMA in PBS-T. The final wash volume was transferred to a new 1% (w / v) BSA-pretreated tube to remove the sticky binder adhering to the tube wall. For six of the selected tracks, the antigen-bound phages were eluted by incubating with 0.5 M acetic acid (pH 2.8) at room temperature, eoe for 15 minutes, and then the eluate was transferred to a new pretreated tube and neutralized with an equal volume of 1 M Tris-HCl (pH 8). For the remaining three selected tracks, the antigen-bound phages were eluted by incubating 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 at room temperature, eoe for 30 minutes, and then the eluate was transferred to a new pretreated tube. After cycles 1-2, a new phage stock was generated by growing Escherichia coli XL-1 Blue cells (Agilent) in TSB+Y containing 10 μg / ml Tet at 37 °C with shaking at 150 rpm until OD 600 reached 0.5 - 0.8, and then infecting with the phage eluate (total volume after cycle 1, half the volume after cycle 2) (100 ml after cycle 1, 50 ml after cycle 2). The infected culture was gently rotated and incubated at 37 °C for 25 minutes without shaking, and then incubated with shaking at 70 rpm, 37 °C for 15 minutes. The culture was then centrifuged, resuspended in TSB+Y, and plated on blood agar plates (40 g / l blood agar) containing 100 μg / ml Carb and 1% (w / v) glucose, and incubated at 37 °C for 16 - 18 hours. Bacterial colonies were scraped into TSB+Y, inoculated into 200 ml of TSB+Y containing 100 μg / ml Carb, and OD 600It was grown at 37 °C while shaking at 150 rpm until it reached 0.5. 30 ml was super-infected with M13KO7 helper phage (MOI of 8) or KM13 helper phage (MOI of 33 in cycle 1 and MOI of 5 in cycle 2), gently rotated, incubated at 37 °C for 25 minutes without shaking, and then incubated at 37 °C for 15 minutes while shaking at 70 rpm. The culture was centrifuged and resuspended in 150 ml of TSB+Y containing 100 μg / ml of Carb and 1 mM of IPTG.

[0372] 25 μg / ml of Kan was added 2 hours after inoculation. The culture was incubated at 37 °C for 16 - 18 hours while shaking at 150 rpm. The phage library stock was recovered by precipitating twice with 20% (w / v) PEG6000 / 2.5 M NaCl. The titer of the stock was measured by spot titration, and the proportion of phage particles carrying phagemids with affibody inserts was analyzed using polymerase chain reaction screening.

[0373] Monoclonal phage ELISA. Following cycle 3, 20 - 21 bacterial colonies representing 9 selected tracks equally and generating PCR products of the correct size were individually grown in 500 μl of TSB+Y / Carb in a 96 - well deep - well plate at 30 °C for 16 - 18 hours with shaking at 250 rpm. 30 μl of the overnight culture was inoculated into 720 μl of TSB+Y / Carb in a new 96 - well deep - well plate, incubated at 37 °C, 250 rpm for 2 hours, and then superinfected by adding M13K07 helper phage (MOI of 8) in 100 ul of TSB+Y / Carb per well. The plate was incubated at 37 °C for 30 minutes without shaking. Finally, 150 μl of TSB+Y / Carb / IPTG / Kan was added per well. The final concentrations were 100 μg / ml of Carb, 1 mM of IPTG, and 25 μg / ml of Kan. The deep - well plate was incubated at 37 °C, 250 rpm for 16 - 18 hours. The next day, the phage supernatant was recovered by centrifugation.

[0374] Optimized candidate monoclonal phage - ELISA screening. MaxiSorp ELISA plates Clear Flat - Bottom Immuno Nonsterile 384 - Well Plates, catalog number 464718, Thermo Fisher Scientific) were coated with 30 μl of 1 μg / ml biotinylated hBCMA - Fc, 20 μg / ml HSA (for evaluation of proper presentation of an expression cassette containing a three - component fusion protein comprising an albumin - binding domain and truncated (pFaffi - 1) or full - length (pFaffi - 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) per well at 4°C for 16 - 18 hours with gentle shaking (1 / 4 of the wells with each coated protein). The coating solution was removed, the plates were washed twice with PBS - T, and blocked with PBS supplemented with 1% (w / v) BSA for 1 hour at room temperature with gentle shaking. The blocking solution was discarded, and the wells were incubated with 10 μl of phage supernatant diluted 1:3 in 20 μl of PBS - T for 1 hour at room temperature with gentle shaking. The supernatant was removed, and the plates were washed three times with PBS - T. 30 μl of 1:5000 α - M13 - HRP (Sigma - Aldrich, Stockholm, Sweden) in PBS - T was added and incubated for 30 minutes at room temperature with gentle shaking, then the plates were 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 stopped by adding 30 μl of 2 M H2SO4 after 15 - 25 minutes. Absorbance at 450 nm was measured using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). Candidates considered ELISA - positive clones had high HSA signals and relatively high signals against hBCMA compared to the SA and trastuzumab controls.

[0375] DNA sequencing. After ELISA screening, 120 ELISA-positive clones were sent for sequencing by Sanger sequencing (Microsynth).

[0376] Results To isolate second-generation polypeptides that bind to hBCMA, two affibody phage libraries were constructed mainly based on the results from the hBCMA-binding clone Fa-G6 and alanine scanning. These two libraries were used as input in the first cycle of a 3-cycle selection campaign as described in Materials and Methods (above). After the third selection cycle, E. coli cells were infected with eluates from 9 selected tracks to obtain individual colonies on Carb plates. For binding analysis using phage-ELISA experiments, 20 - 21 clones were randomly picked from each track. Most of the clones analyzed showed strong ELISA signals against HSA and hBCMA and low signals against the controls used, for which DNA sequencing was performed. 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 the Sequence Listing as SEQ ID NO: 2 (clone 1-E6) and 23 - 128.

[0377] Biological Example 6: Characterization of Second-Generation Candidate hBCMA-Binding Polypeptides In this example, a subset of 18 second-generation polypeptide variants corresponding to SEQ ID NOs: 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) was subcloned, expressed, purified as an Affibody-His6 fusion protein, and initially evaluated for their binding to hBCMA-Fc by surface plasmon resonance. Circular dichroism (CD) spectroscopy was used to evaluate the secondary structure content and their thermal denaturation profiles.

[0378] Materials and Methods Cloning of second-generation hBCMA-binding clones. DNA fragments encoding a subset of 18 new candidate hBCMA binders [SEQ ID NOs: 2 and 23-39] equally representing 9 selection tracks were amplified from the library vector pAffi-1 using specific primers designed to introduce complementary overhangs at the ends of the linearized expression vector. Eighteen monomeric polypeptide constructs with a C-terminal His6 tag were cloned using the In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden). Eighteen constructs containing SEQ ID NOs: 2 and 23-39 in the form of [polypeptide sequence]-YYHHHHHH were sequence-verified using Sanger sequencing (Microsynth).

[0379] Expression and purification. E. coli BL21(DE3) cells were transformed with plasmids containing DNA constructs encoding 18 polypeptide variants each with a C-terminal His6 tag and the parental Fa-G6-His6 construct, and cultured at 37 °C for 16-18 h with shaking at 150 rpm in 10 ml of TSB+Y containing 25 μg / ml of Kan. The culture was inoculated 1:100 into 200 ml of TSB+Y containing 25 μg / ml of Kan, and OD 600It was grown at 37°C while shaking at 150 rpm until it reached 0.6 - 1. IPTG was added until the final concentration reached 1 mM to induce protein expression. The culture was incubated at 25°C for 16 - 18 hours while shaking at 150 rpm, and the cells were collected by centrifugation. The cell pellet was resuspended in denaturing lysis buffer. After incubating at 37°C for 2 hours while shaking at 150 rpm, 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 room temperature for 30 minutes while end - over - end rotating. Contaminants were removed by washing three times with wash buffer, and then the hBCMA - binding polypeptide was eluted with elution buffer by incubating at room temperature for 10 minutes while end - over - end rotating.

[0380] After IMAC purification, the protein buffer was exchanged to PBS using a PD - 10 desalting column (Catalog No. 17085101, Cytiva). SDS - PAGE analysis was performed to confirm the purity and concentration, and the absorbance was measured at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen). The low - molecular - weight marker was from Cytiva (Product No. 17044601).

[0381] Biosensor analysis of the second-generation Fa-G6 variants. Using a Biacore T200 instrument (Cytiva), the real-time interactions between 18 second-generation variants expressed and purified as polypeptide-His6 (SEQ ID NOs: 2 and 23-39 in the form of [polypeptide sequence]-YYHHHHHH) and hBCMA were analyzed. The protein ligand hBCMA-Fc (Sino Biological Inc., catalog number 10620-H15H) was immobilized (3500 RU) onto a Series S CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. One flow cell was activated and deactivated for use as a reference cell. Analytes were diluted in running buffer PBS-T and then binding assays were performed 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. The 18 second-generation variants and the parental Fa-G6 binder were injected at 100 nM onto the hBCMA-Fc surface.

[0382] CD analysis. Using a Chirascan CD Spectrometer (Applied Photophysics, Leatherhead, United Kingdom), the secondary structure content of the Fa-G6 binder and the second-generation binders was determined by recording CD spectra in the range of 195-260 nm at 20 °C, and their thermal denaturation profiles were determined by recording the CD ellipticity at 221 nm during heating from 20 °C to 92 °C.

[0383] Results To examine whether a subset of the new candidate hBCMA-binding variants that emerged from selections from the second-generation library and were identified by phage ELISA and DNA sequencing showed hBCMA target binding when expressed as soluble proteins, 18 new variants [SEQ ID NO: 2 and 23-39] were selected to equally represent 9 selection tracks, subcloned, cytoplasmically expressed in E. coli, and purified as Affibody His6 fusion proteins. When the fusion proteins purified by IMAC using the His6 gene fusion partner in the proteins were analyzed by SDS-PAGE, all 18 proteins were shown to be of high purity and to have an approximate molecular weight according to their amino acid sequences. The 18 candidate optimized variants were separately injected onto a sensor chip immobilized with hBCMA-Fc at a single concentration of 100 nM. The resulting sensorgrams (Figure 5) showed that all 18 new variants bound to hBCMA. The CD spectra obtained showed that all the binders analyzed had a high α-helix secondary structure content at 20 °C, and from the thermal denaturation profiles of the second-generation hBCMA binders (Figure 6a), for most of these binders, an improved resistance to thermal denaturation could also be observed compared to the parental Fa-G6 clone included in the analysis. One of the new variants, clone 1-E6, showed a melting temperature (Tm) of 63 °C determined by variable temperature measurement, which is 11 °C higher than that observed for the parental Fa-G6 clone (Tm of 52 °C). This is shown in Figure 6b.

[0384] Collectively, the results showed that a pool of new hBCMA-binding polypeptides based on the parental Fa-G6 binder with diverse biophysical properties compared to Fa-G6 was generated.

[0385] Biological Example 7: Anti-hBCMA drug conjugates are cytotoxic to BCMA + multiple myeloma cells In this example, an hBCMA binder-drug conjugate was synthesized and its cell-binding and cancer cell killing abilities were tested using multiple myeloma cells.

[0386] MMAF conjugation for generating hBCMA binder-drug conjugate The hBCMA binder, SEQ ID NO: 2 (6756.5 Da), was synthetically generated by Almac Science Limited (Scotland). This protein was composed of 59 natural amino acids containing a terminal cysteine residue with a free thiol. The selected toxin was MMAF with a non-cleavable MPB linker. The unbound cysteine residue thiol functional groups were utilized by a thiol-Michael addition reaction to the electrophilic maleimide moiety of the linker. Buffer exchange and purification were performed using an Amicon Ultra centrifugal filter (3 kDa cut-off). HPLC-MS during the reaction progress was performed using an Agilent 1100 series liquid chromatograph / mass selective detector (MSD, single quadrupole) equipped with an electrospray interface and a UV diode array detector. The analysis was performed using an ACE 3 C8 (3.0 × 50 mm) column with a gradient of acetonitrile in 0.1% TFA aqueous solution for 3 minutes at a flow rate of 1 mL / min. Purity and ID analysis were performed using an Agilent 1290 Ultra Performance Liquid Chromatograph / Quadrople Time of Flight (Q-ToF) mass spectrometry detector. The analysis was performed using an ACE 3 C8 5μ (3.0 × 50 mm) with UV detection at 280 nm with a gradient of acetonitrile in 0.1% formic acid aqueous solution and a flow rate of 0.8 mL / min. The Fa-G6 null mutant of the polypeptide lacking binding ability was also synthetically synthesized using the same method as above.

[0387] Tris(2-carboxyethyl)phosphine HCl (4.24 mg, 14.8 μM) of TCEP HCl (8.47 mM) was dissolved in PBS (pH 7.4) (2 mL, 10 mM) and gently vortexed. MPB-MMAF solution (2.06 mM) MPB-MMAF (4.00 mg, 4.11 μmol) was dissolved in dry DMF (2.0 mL) and gently vortexed. Both solutions were freshly prepared before use. hBCMA binder 1-E6 (10 mg, 1.48 μmol) was weighed into a glass vial, dissolved in PBS buffer (4 mL, pH 7.4, 10 mM), and TCEP (2.0 mL, 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 5000 rpm (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 filter unit to a 10 mL glass vial, and then PBS pH 7.4 (4 mL) and MPB-MMAF in dry DMF (2 mL, 2.78 molar equivalents) were added. The reaction mixture was gently stirred at room temperature for 2 hours. The reaction mixture was diluted with 10% DMF in PBS (2.0 mL, pH 7.4), and the resulting solution was centrifuged at 5000 rpm for 25 minutes in a filter unit. When the washing by this filtration method was performed 8 cycles, no trace of unconjugated toxin was detected by LCMS. Next, the buffer was exchanged with an arginine-containing buffer pH 7.8 (50 mM Arg, 75 mM NaCl, 2% (w / w) sucrose, 0.01% (w / w) polysorbate 20). The buffer exchange was performed in a centrifugal filter unit at 5000 rpm (6 °C) for 25 minutes. The buffer exchange step was repeated 7 times to obtain a substance having a UPLC UV purity of 87% and HRMS (deconvoluted): 7729.

[0388] Protection of 1-E6 and Fa-G6 null variant polypeptides The protocol for generating the protected non-toxic form of the polypeptide had only one major difference, namely that the terminal cysteine residue with a free thiol was reacted with 1-ethylpyrrole-2,5-dione at a molar equivalent of 1.50 compared to the linker drug, and was essentially the same as MMAF conjugation. This procedure yielded a protected form with a UPLC UV purity of 99% and HRMS (deconvoluted): 6681 for 1-E6 and 6637 for Fa-G6.

[0389] BCMA-binding ELISA A 96-well assay plate (3690, Costar) was coated with 1 μg / ml of BCMA (193-BC-050, R&D) and incubated overnight at 4°C. After blocking, serial dilutions of the protected 1-E6 and 1-E6-MPB-MMAF in blocking buffer (37528, Thermo Scientific) were added and incubated. Then, an anti-Affibody antibody (20.1000.01.0005, Affibody AB) and an anti-goat antibody-HRP (PAI-28664, Invitrogen) in blocking buffer were used. The reaction was deconvoluted using 1-Step™ Ultra TMB-ELISA Substrate Solution (34028, Thermo Scientific)) and stopped by adding 2M sulfuric acid. Absorbance was read at 450 nm using a SpectraMax plate reader. All incubation steps were performed for 1 hour at room temperature, and the assay plate was washed 4 times with PBST after each incubation.

[0390] MMAF conjugation decreased the affinity of 1E6 for BCMA to some extent (Figure 7). Nevertheless, it was demonstrated that 1-E6-MPB-MMAF exhibited binding affinity for BCMA.

[0391] Cell viability of BCMA + MM.1S cells 4×10 5Individual MM.1s cells (CRL-2974, ATCC) were treated with various concentrations of 1-E6-MPB-MMAF for 72 hours in the presence of 5 μM of 1-E6 or Fa-G6_W24A, the protected F28A. DMSO-treated wells and 200 μM chlorpromazine hydrochloride (C8138, Sigma-Aldrich)-treated wells served as controls for viable and dead cells, respectively. After incubation, CellTiter-Glo (G924B, Promega) was dispensed into the cells and luminescence was read using a PerkinElmer EnVision Multilabel Plate Reader.

[0392] 1-E6-MPB-MMAF was cytotoxic to BCMA+ cells in a dose-dependent manner (Figure 8). The potency was lower in the presence of competing 1-E6 compared to the non-competing Fa-G6 null variant of the polypeptide. Thus, it could be demonstrated that the drug conjugate of 1-E6 could be useful for targeting the growth of BCMA+ myeloma cells.

[0393] Biological Example 8: The hBCMA engager of the present invention conjugated to an hCD16a binder induces immune cell activation in the presence of tumor cells Experiments were conducted to evaluate the tendency of the anti-hBCMA engager of the present invention connected to an hCD16a binder to elicit an IFNγ response in co-cultures of human PBMC and BCMA-positive multiple myeloma cells. PBMCs (4W-270, Lonza) thawed and rested overnight were co-cultured with MM.1s cells (CRL-2974, ATCC) at an effector-to-target ratio (E:T) of 25:1 in a V-bottom 96-well plate (249935, Thermo Scientific). A total of 4 × 10 5 cells in 200 μl / well were treated with 200 nM of the hBCMAxhCD16a dual engager or 200 nM of elotuzumab (300 mg of Empliciti, Bristol-Myers Squibb) in a humidified 5% CO2 incubator at 37°C for 4 hours.

[0394] The supernatant was collected and subsequently centrifuged at 600 g for 5 minutes. IFN-γ levels were determined by enzyme-linked immunoassay (ELISA) using the Human IFN-γ ELISA MAX (trademark) Deluxe Set (430104, BioLegend). The data was processed using Excel and plotted using GraphPad Prism9.

[0395] The hBCMA×hCD16a dual-engager constructs containing the hBCMA-binding polypeptide of the present invention (1-E6, SEQ ID NO: 2) genetically fused to an hCD16a-binding arm composed of CD16a-binding polypeptides (referred to herein as A10 (SEQ ID NO: 167), H09 (SEQ ID NO: 276), and A11 (SEQ ID NO: 277)) were examined in either monomeric or dimeric form. The dual-engagers 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]). The sequence information and SEQ ID NO: references for each of these four dual-engager constructs are given in Table 1 below. Corresponding constructs carrying a null non-hBCMA-binding polypeptide (Fa-G6 null) were also evaluated. The anti-SLAMF7 monoclonal antibody elotuzumab was used as a positive control, and single cultures of PBMC and MM.1s served as negative controls. All dual-engagers containing a BCMA-binding polypeptide induced an IFNγ response in co-cultures of PBMC and MM.1S cells, which was greater than the response of the positive control elotuzumab at a concentration of 200 nM for all engagers. (Figure 9). Dual-engager constructs lacking hBCMA binding showed no IFNγ response or a limited IFNγ response in co-cultures of PBMC and MM.1S cells. These experiments illustrate the hBCMA-dependent activation of PBMC against the BCMA-positive MM.1S myeloma cell line induced by the anti-hBCMA×CD16a NK cell engagers of the present invention.

[0396] The CD16a-binding polypeptides referred to herein as A10, H09, and A11 have the following sequences.

[0397]

Table 3

[0398] Biological Example 9: The hBCMA engager of the present invention conjugated to an hCD16a binder induces CD16-mediated activation in the presence of tumor cells In a reporter assay using the Jurkat-Lucia NFAT cell line commercially available from Invivogen, experiments were conducted to evaluate the tendency of the anti-hBCMA engager of the present invention conjugated to the hCD16a binder A10 to elicit a CD16-dependent response. Using 100,000 cells / mL of the Jurkat-Lucia NFAT cell line and 50,000 cells / mL of MMIS., the cells were cultured according to the manufacturer's specifications. The cells were incubated with the engager construct for 24 hours.

[0399] Anti-hBCMA dual engager constructs containing either one or two hBCMA-binding polypeptides (1-E6) of the present invention genetically fused to the hCD16a-binding arm A10 were evaluated. Sequence information and sequence number references for these dual engager constructs are given in Table 1 below [SEQ ID NOs: 130, 134-138]. Constructs carrying a null non-hBCMA-binding polypeptide (Fa-G6 null, designated "null" in FIG. 10) were also evaluated. The anti-SLAMF7 monoclonal antibody elotuzumab was used as a positive control. All dual engagers containing the BCMA-binding polypeptide induced a CD16-mediated response in the presence of MM.1S cells (FIG. 10). Dual engager constructs lacking hBCMA binding did not show a response. These experiments illustrate hBCMA-dependent activation by the anti-hBCMA×CD16a NK cell engager of the present invention.

[0400] [Table 4-1]

[0401] [Table 4-2]

[0402] [Table 4-3]

[0403]

Table 4-4

[0404]

Table 4-5

[0405]

Table 4-6

[0406]

Table 4-7

[0407]

Table 4-8

[0408] Table 1 shows the sequences of the polypeptides disclosed in this application, including some of the dual engagers [SEQ ID NOs: 130 to 147].

[0409]

Table 5

[0410] Table 2 shows the sequences of the primers and synthetic oligonucleotides used in the preparation examples of this application.

[0411] Biological Example 10: Activation of hCD16a by an hBCMA×hCD16a dual engager carrying an IL-15 cytokine polypeptide:

[0412] Materials and Methods: The cDNA encoding the polypeptide of the present invention having a stop codon was synthesized and ligated to the Ndel Xhol restriction sites of the pET29 vector. E. coli BL21(DE3) was transformed with the vector under kanamycin selection. The compound referred to herein as Example Compound 148 (of SEQ ID NO: 148) was expressed by induction with IPTG at an OD of 0.6 and harvested after 16 hours. Example Compound 148 was purified from inclusion bodies by dissolving it in 1×PBS supplemented with 30 mM imidazole, 6 M urea, and 10 mM glutathione (reduced form). Soluble substances were purified by IMAC using 1×PBS supplemented with 500 mM imidazole and 6 M urea as the elution buffer. Subsequently, Example Compound 148 was refolded by dialysis against 1×PBS.

[0413] Lucia Luciferase reporter assay In Jurkat-Lucia™ NFAT-CD16 cells (InvivoGen), the tendency of compounds to stimulate CD16a activation was evaluated and compared to the antibody-dependent cellular cytotoxicity (ADCC) CD16a activation of elotuzumab (Empliciti, Bristol Myers Squibb, clinical grade α-SLAMF7 monoclonal antibody) according to the method of Example 11.

[0414] Results An hBCMA×hCD16a dual engager (Example Compound 148 = SEQ ID NO: 148) carrying the IL-15 cytokine sequence was constructed and expressed as a soluble gene product in E. coli (DE3). The CD16a activation responses of the engager construct in the presence and absence of target MM.1s cells are shown in FIG. 11.

[0415] It can be seen from this figure that the affibody-based IL-15-containing dual engager can activate CD16a in a target-specific manner.

[0416] [Table 6]

[0417] Biological Example 11: CD16a Activation and NK Cell-Mediated Cell Killing of hBCMA×hCD16a Bispecific Engager Constructs with Different Domain Orders and Linkers Compounds with targeting domains of different distances and orders in the bispecific engager constructs were prepared. The tendency of hBCMA×hCD16a bispecific engagers to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated. The hCD16a-binding polypeptide was in each case the polypeptide having SEQ ID NO: 167. The hBCMA-binding polypeptide was in each case the polypeptide having SEQ ID NO: 2.

[0418] [Table 7-1]

[0419] [Table 7-2]

[0420] Design, construction, expression, and protein purification of hBCMA×hCD16a binding polypeptide engager constructs. The hBCMA×hCD16a binding constructs were designed for expression and purification as follows. cDNA encoding each hBCMA×hCD16a binding construct carrying a stop codon was synthesized and ligated into the Ndel Xhol restriction sites of the pET29 vector. E. coli BL21(DE3) was transformed with the vector under kanamycin selection, and the construct was expressed by induction with IPTG at an OD of 0.6 and harvested after 16 hours. The soluble cytosolic product was recovered and resuspended in 1×PBS supplemented with 30 mM imidazole. Purification by IMAC was achieved using 1×PBS supplemented with 500 mM imidazole as the elution buffer. Further purification was achieved by RP-HPLC, and the identity / purity was confirmed by analysis using SDS-PAGE and LC-MS / MS.

[0421] Both the heterodimeric hBCMA×hCD16a dual binding constructs (Example Compounds 130, 154, 155, 156 and 157) and the heterotrimeric hBCMA×hCD16a binding constructs containing two BCMA binding polypeptides (Example Compounds 149, 150, 151, 152 and 153) were evaluated.

[0422] The tendency of hBCMA×hCD16a dual engagers to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated.

[0423] CD16 activation Jurkat-Lucia™ Luciferase reporter assay The tendency of hBCMA×hCD16a dual engagers to stimulate CD16a activation was evaluated in the Lucia Luciferase reporter assay and compared to the antibody-dependent cell cytotoxicity (ADCC) potency of elotuzumab (clinical grade α-SLAMF7 monoclonal antibody) through the evaluation of CD16a activation. Jurkat-Lucia™ NFAT-CD16 cells (Invivogen) were seeded in 96-well flat-bottom plates with MM.1s cells at an effector-to-target ratio (E:T) of 2:1. 3×10 in 200 μl per well 5Cells were treated with 20 or 200 nM of the reagent of interest for 24 hours. The supernatant was collected, and the Lucia luciferase activity reflecting the induced ADCC response was evaluated using QUANTI Luc™ Gold (InvivoGen). The response was normalized against the maximum response of elotuzumab.

[0424] Calcein release-based in vitro cytotoxicity assay The cytotoxic activity of NK cells was evaluated in a calcein release-based cytotoxicity assay previously described by (Gauthier et al, 2023) PMID36635380. Briefly, MM1.S target cells were pre-labeled with calcein-AM (Biolegend) and co-cultured with NK cells at a 1:1 ratio in 96-well v-bottom plates. After the effector and target cells, the engager was added to the co-culture last. 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 the killed MM1.S cells was quantified with a SpectraMax i3x (Molecular Devices). The percentage of specific lysis was calculated in Microsoft Excel (Microsoft) using only target cells for spontaneous release and target cells containing surfactant for maximum release. The cytotoxic activity of NK cells in the presence of the engager was compared with the antibody-dependent cytotoxic activity of belantamab biosimilar (purchased from IchorBio).

[0425] Results An hBCMA×hCD16a dual engager construct containing the polypeptide of SEQ ID NO: 167 and one or two hBCMA-binding polypeptides of SEQ ID NO: 2 genetically fused to the hCD16a-binding arm consisting of a C-terminal His6 tag was constructed and expressed as a soluble gene product in E. coli (DE3). Figures 12a and 12b show the hCD16a activation response of the engager construct in the presence or absence of target MM.1s cells.

[0426] The results show that when the afibody-based heterodimeric hBCMA×hCD16a double-binding construct and the heterotrimeric hBCMA×hCD16a binding construct (here composed of the polypeptides of SEQ ID NO: 2 (for hBCMA) and SEQ ID NO: 167 (for hCD16a)) are genetically fused together in either order, they can activate hCD16a in a target-specific manner. For the engager with a linker length of 0 to 15 amino acids, good activation was observed.

[0427] Biological Example 12: Evaluation of domain boundaries in hBCMA×hCD16a double-engager constructs using the Jurkat CD16a activation reporter assay and the NK-mediated cell killing assay An hBCMA×hCD16a double-binding construct with cleavage at both the N-terminus and C-terminus of both the BCMA-binding polypeptide and the CD16a-binding A10 afibody was generated and evaluated in an hCD16a-mediated activation cell-based reporter assay and cell killing. This enabled the evaluation of the domain boundaries of the targeting domains in the double-engager construct.

[0428]

Table 8

[0429] Design, construction, expression, and protein purification of the hBCMA×hCD16a CD16a-binding polypeptide double-engager construct. The hBCMA×hCD16a double-binding construct was designed for expression and purification as described above in Biological Example 11. The prepared heterodimeric hBCMA×hCD16a double-binding constructs are listed above (SEQ ID NOs: 156 and 158 - 163).

[0430] The tendency of the hBCMA×hCD16a double-engager to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated according to the protocol described above in Biological Example 11.

[0431] Result An hBCMA×hCD16a dual-engager construct containing one BCMA-binding polypeptide of SEQ ID NO: 2 genetically fused to an hCD16a-binding arm consisting of the polypeptide of SEQ ID NO: 167 (with cleavage in some cases) and a C-terminal His6 tag was constructed and expressed as a soluble gene product in E. coli (DE3).

[0432] Figure 13a shows the CD16a activation response of the engager construct in the presence or absence of target MM.1s cells. Furthermore, the engager induced NK cell-mediated killing of MM.1S cells (Figure 13b).

[0433] The results show that an affibody-based heterodimeric hBCMA×hCD16a double-binding construct (here consisting of the polypeptides of SEQ ID NO: 2 (for hBCMA) and SEQ ID NO: 167 (for hCD16a)), when genetically fused together (with cleavage at specific positions), can activate hCD16a in a target-specific manner.

[0434] Biological Example 13: NK cell-mediated multiple myeloma cell killing The ability of an hBCMA×hCD16a dual-engager to promote NK cell-mediated lysis and cell killing was evaluated by flow cytometry assays and Incucyte-based assays. The compounds investigated were as shown in the following table.

[0435] [Table 9]

[0436] The compounds were generated according to the preparation described in Biological Example 11.

[0437] Flow cytometry-based in vitro assay. NK cell function was evaluated in a previously described flow cytometry-based assay (Bryceson, Fauriat et al. 2010). Briefly, NK cells were co-cultured with target cells at a 1:1 E:T ratio for 6 hours, after which they were stained for the degranulation marker CD107a and interferon-gamma. After 1 hour, BD GolgiStop (BD Biosciences) was added at a concentration of 1 / 1500. For the cytotoxicity assay, target cells were pre-stained with CellTrace Violet (Invitrogen) and co-incubated with NK cells at different E:T ratios for 8 - 12 hours based on the kinetics from the live cell imaging assay. Samples were acquired on either a BD LSR II or FACSymphony A5 (BD Biosciences). Data were analyzed with FlowJo version 10 (BD Biosciences).

[0438] Incucyte-based in vitro cytotoxicity assay. Real-time tumor cell killing was measured on an Incucyte S3 platform (Essen Biosciences). NK cells were co-cultured with MM.1s cells labeled with the red fluorescent protein mCherry on 96-well plates pre-coated with 0.01% poly-L-ornithine (Sigma-Aldrich) at various E:T ratios according to the assay setup. In assays with reagents (e.g., afibody constructs), these were added after the effector and target cells. Scans were performed every 60 minutes for at least 24 hours using a 10x objective lens, and images were analyzed using Incucyte Controller v2020A (Essen Biosciences). The same masking was used for repeated analysis. In the experiments conducted, cells were treated with 100 nM of a dual-engager, null variant (does not bind to hBCMA), or daratumumab.

[0439] Results The ability of Example Compound 131, a dual-engager, to induce lysis of MM.1s cells was examined. The results are shown in Figure 14a. The results are from one representative experiment (n = 4). In a 24-hour killing assay, Example Compound 131 induced rapid and excellent lysis of MM.1s cells compared to both daratumumab and the control null variant anti-hBCMA-null-H09-A10-His6. The bulk cytotoxic effect of Example Compound 131 was seen within the first 8 - 12 hours after the start of co-culture.

[0440] Finally, flow cytometry-based killing assays were performed to establish the functional EC 50 and EC 90 values of three constructs of Example Compounds 131, 132, and 130. The frequency of dead target cells was calculated based on staining of a dead cell marker on target cells pre-incubated with cell trace violet (CTV). The results are shown in Figure 14b. The results are from two independent experiments (n = 8), with spontaneous target cell death subtracted. The plateau level of cytotoxicity was highest for Example Compound 131 and lowest for Example Compound 132, which is consistent with the degranulation response and the latter construct showing a higher fratricide effect. Compared to Example Compound 130, the plateau cytotoxicity for Example Compound 131 was achieved at a lower dose. The EC 50 values were 0.4 nM for Example Compound 131 and Example Compound 132, and 1.8 nM for Example Compound 130, while the EC 90 values were 2.0, 3.8, and 14.7 nM, respectively. The data demonstrate that these dual-engagers induce a strong response.

[0441] Biological Example 14: Enhancement of NK cell-mediated lysis of multiple myeloma cells in an hBCMA-selective manner. The ability of an hBCMA×hCD16a dual engager to enhance NK cell-mediated lysis of multiple myeloma cells in a BCMA-selective manner was examined. The compound tested was Example Compound 131.

[0442] Genetic cell manipulation A BCMA knockout variant of the cell line MM.1s was generated using CRISPR-Cas9. A BCMA overexpression variant of the cell line MM.1s and a variant expressing the red fluorescent protein mCherry were performed by lentiviral transfection using vectors designed and ordered from VectorBuilder (VectorBuilder Inc.). MM.1s cells expressing the nuclear red fluorescent protein mCherry were transduced with 0.3 MOI of 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) using a Sony SH800 cell sorter (Sony Biotechnology). MM.1s with overexpressed BCMA gene: On day 1, 1 million MM.1s cells were transduced with 10 MOI of lentiviral particles (designated SK-V93 and shown in Figure S6A) and seeded on a 24-well plate. On day 4, the cells were washed in PBS and seeded at 500,000 cells per milliliter in fresh medium. After sufficient growth, the cells were subjected to FACS. MM.1s with the BCMA gene knocked out: The following sgRNA sequences were used: sgRNA#0, gacgagtttaaaaacac (SEQ ID NO: 287); sgRNA#1, gagcttaataatttctt (SEQ ID NO: 288); and sgRNA#2, gtgaccaattcagtgaa (SEQ ID NO: 289). The sgRNA and Cas9 were assembled at a ratio of 9:1 on a sterile 96-well U-bottom plate.

[0443] 300,000 MM.1s cells were used per nucleofection. Five days later, the efficacy of the knockout was confirmed by PCR and Sanger sequencing and analyzed by Synthego Performance Analysis (ICE) (Synthego). After sufficient growth, the cells were subjected to FACS.

[0444] Flow cytometry-based in vitro assay. NK cell function was evaluated in a previously described flow cytometry-based assay (Bryceson, Fauriat et al. 2010). Briefly, NK cells were co-cultured with target cells at an E:T ratio of 1:1 for 6 hours and then stained for the degranulation marker CD107a and interferon-gamma. One hour later, BD GolgiStop (BD Biosciences) was added at a concentration of 1 / 1500.

[0445] Results A clear correlation between BCMA expression levels and NK cell activation was observed using engineered MM.1s target cells in which BCMA was knocked out or overexpressed (see Figure 15). The data demonstrate that the dual engager Example Compound 131 at a concentration of 100 nM induces a response with specificity for BCMA. In the figure, the results are shown for MM.1s wild type (WT), BCMA knock-out (KO), and BCMA overexpression (OE). The results are from one representative experiment (n = 4).

[0446] Biological Example 15: Additional anti-hBCMA drug conjugates are cytotoxic to + multiple myeloma cells Example compounds having the Fa-G6 sequence (SEQ ID NO: 1) with a c-terminal Cys were generated by conventional solid peptide synthesis. The compound had SEQ ID NO: 164.

[0447]

Table 10

[0448] The MC-MMAF linker toxin was dissolved in DMF organic solvent and conjugated at a molar ratio of 2:1. The product with one mc-MMAF was isolated by RP-HPLC and prepared in 10% IPA in PBS buffer.

[0449] By using the CellTiter-Blue® 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 the concentration range of 1 ng / mL to 30 μg / mL.

[0450] Results The results are shown in Figure 16. The Fa-G6-MMAF affibody drug conjugate showed anticancer activity in all multiple myeloma cell lines investigated, and the EC 50 values were in the range of 0.19 to 3.5 μg / mL. Therefore, Fa-G6-MMAF showed activity in some BCMA+ multiple myeloma cell lines.

[0451] Biological Example 16A: Compound containing unnatural amino acid norleucine Experiment A polypeptide having the sequence of SEQ ID NO: 165 (aBCMA-1-E6-(6-55)M35nL) was synthesized by solid-phase peptide synthesis (SPSS), purified by RP-HPLC, and lyophilized. The exact Mw was confirmed by LC-MS / MS.

[0452]

Table 11

[0453] The binding affinity for the extracellular domain of BCMA was studied by SPR on a Biacore T200. BCMA was immobilized on a CM5 chip (Cytiva) with 1300Ru as the ligand. The example compound with SEQ ID NO: 165 was used as the analyte, and the response was monitored in the concentration range of 1 - 100 nM. The data was fitted to a 1:1 binding mode.

[0454] Results It was observed that the example compound with SEQ ID NO: 165 binds to BCMA with an apparent dissociation constant of 10 nM.

[0455] Biological Example 16B: Evaluation of domain boundaries in BCMA×hCD16a dual - engager constructs using Jurkat CD16a activation reporter assay and NK - mediated cell killing assay An hBCMA×hCD16a dual - binding construct was generated and evaluated in hCD16a - mediated activation cell - based reporter assay and cell killing as described above in Biological Example 11. The activities were compared for an engager with the full - length aBCMA 1E6 domain (SEQ ID NO: 156), an engager with a cleavage at both the N - terminus and C - terminus (SEQ ID NO: 160), and an engager carrying the V1 G - point mutation (SEQ ID NO: 163).

[0456] [Table 12]

[0457] Design, construction, expression, and protein purification of hBCMA×hCD16a CD16a - binding polypeptide dual - engager constructs. The hBCMA×hCD16a dual - binding construct was designed for expression and purification according to Biological Example 11. The prepared heterodimeric hBCMA×hCD16a dual - binding constructs are listed above (SEQ ID NO: 156, 160, and 163).

[0458] The tendency of the hBCMA×hCD16a dual engager to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated according to the protocol described in Biological Example 11.

[0459] Results An hBCMA×hCD16a dual engager 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). Figure 17a shows the CD16a activation response of the engager construct in the presence or absence of target MM.1s cells. Furthermore, the engager induced NK cell-mediated killing of MM.1s cells, as shown in Figure 17b.

[0460] The results show that the hBCMA-binding arm (here composed of the polypeptide of SEQ ID NO: 2 (1E6)) can be shortened and still retain hCD16a activation. Furthermore, valine 1 can be replaced with glycine without loss of activity.

[0461] Biological Example 17: Anti-BCMA engagers induce immune cell activation in the presence of tumor cells Experiments were conducted to evaluate the tendency of an anti-BCMA engager example compound having SEQ ID NO: 130, which elicits an IFNγ response, in co-cultures of human PBMC and the BCMA-positive multiple myeloma cell line MM.IS. PBMC (4W-270, Lonza), thawed and rested overnight, were co-cultured with MM.1s cells (CRL-2974, ATCC) at an effector-to-target ratio of 25:1 in a V-bottom 96-well plate (249935, Thermo Scientific). A total of 4×10 5 cells in 200 μl / well were treated with 200 nM anti-BCMA engager or 200 nM elotuzumab (300 mg of Empliciti, Bristol-Myers Squibb) for 4 hours at 37 °C in a humidified 5% CO2 incubator.

[0462] The supernatant was collected and subsequently centrifuged at 600 g for 5 minutes. IFN-γ levels were determined by enzyme-linked immunoassay (ELISA) using the Human IFN-γ ELISA MAX (trademark) Deluxe Set (430104, BioLegend). The data was processed using Excel and plotted using GraphPad Prism 9.

[0463] An anti-BCMA engager construct having SEQ ID NO: 130 containing a BCMA-binding polypeptide (referred to herein as 1-E6 (SEQ ID NO: 2)) genetically fused to an hCD16a-binding polypeptide (referred to herein as A10 (SEQ ID NO: 167)) was evaluated. The SLAMF7 monoclonal antibody elotuzumab was used as a positive control, and single cultures of PBMC and MM.1s functioned as negative controls. The dual engager containing the BCMA-binding polypeptide induced an IFNγ response in co-cultures of PBMC and MM.1s cells, and the response was greater than that of the positive control elotuzumab (Figure 18). The dual engager construct lacking BCMA binding did not show an IFNγ response in co-cultures of PBMC and MM.1s cells. This experiment illustrates the BCMA-dependent activation of PBMC against BCMA-positive MM.1s myeloma cell lines induced by the anti-BCMA engagers described herein.

[0464] Biological Example 18: CD16a activation and NK cell-mediated cell killing by an hBCMA×hCD16a dual engager having an hCD16a-binding polypeptide hBCMA×hCD16a dual engagers containing hBCMA-binding polypeptides having SEQ ID NOs: 166 and 144 were expressed in E. coli (DE3) and then characterized for their CD16a activation properties.

[0465] cDNA encoding each hBCMA×hCD16a dual engager carrying a stop codon was synthesized and ligated into the Ndel Xhol restriction sites of the pET29 vector. E. coli BL21(DE3) was transformed with the vector under kanamycin selection, and the construct was expressed by induction with IPTG at an OD of 0.6 and harvested after 16 hours. The soluble cytosolic product was recovered and resuspended in 1×PBS. Heat denaturation at 95°C for 7 minutes was applied, and the precipitated protein was pelleted by centrifugation at 20,000×g. Further purification was achieved by RP-HPLC, and the identity / purity was confirmed by SDS-PAGE and LC / MS / MS analysis. The tendency of the hBCMA×hCD16a dual engager to stimulate CD16a activation and enhance NK cell-mediated killing was evaluated according to the protocol described in Biological Example 11.

[0466]

Table 13

[0467] Results The results are shown in Figure 19. The hBCMA×hCD16a dual engagers (Examples 166 and 144) showed CD16a activation in the Lucia Luciferase reporter assay in the presence of hBCMA+MM.1s cells, but no detectable activation was seen in the absence of target cells (Figure 19a). Furthermore, the engagers induced NK cell-mediated killing of MM.1s cells (Figure 19b). Thus, robust target-specific activation leading to NK-mediated cell killing was seen for the engagers investigated.

[0468] Although the present invention has been described and illustrated in connection with specific embodiments, it will be understood by those skilled in the art that the present invention can be useful in many different variations that are not specifically illustrated herein.

[0469] In the foregoing specification, if an integer or element having known, obvious, or foreseeable equivalents is described, such equivalents are incorporated herein as if individually expressly set forth. The claims should be referred to in order to determine the true scope of the...

Claims

1. An hBCMA-binding polypeptide comprising at least one motif that binds to hBCMA, wherein the polypeptide has the following structure: It includes [N-terminal portion] - [Helix 1] - [Separation portion] - [Helix 2] - [C-terminal portion], The hBCMA-binding polypeptide wherein the hBCMA-binding motif is a partial [helix 1] - [separation portion] - [helix 2].

2. i) Helix 1 includes the sequence X ９ X １０ X １１ ADX １４ EIX １７ X １８ [SEQ ID NO: 278], and Helix 2 includes the sequence FX ２５ QKWAFX ３１ RX ３３ LX ３５ [SEQ ID NO: 279], and independently of each other X ９ and X １０ However, it is any naturally occurring amino acid, X １１ However, it is E, F, H, Q, T, or Y. X １４ However, it is any naturally occurring amino acid, X １７ However, it is A, E, Q, S, T or V, X １８ However, it is any naturally occurring amino acid, X ２５ However, it is either F or Y, X ３１ However, it is I, M, or V, X ３３ However, it is K or S, X ３５ However, is it I, L, M, or V? Alternatively, ii) Helix 1 and Helix 2 are defined as in i), and within Helix 1 and Helix 2, X ｎ At least one and no more than five residues are replaced by substitute residues, and / or X ｎ The hBCMA-binding polypeptide according to claim 1, wherein at least one and no more than five unlabeled residues are replaced by substitute residues.

3. Helix 1 is in sequence X ６ X ７ X ８ X ９ X １０ X １１ ADX １４ EIX １７ X １８ X １９ including and / or helix 2 including the sequence X 23 FX 25 QKWAFX 31 RX 33 LX 35 X 36 X 37, X ６ However, it is either D, E, N, or Q, or does not exist. X ７ However, it is either H, K, or R, or does not exist. X ８ However, it is either D, E, N, or Q, or does not exist. X １９ However, it is G, A, V, L, or I, or does not exist. X ２３ However, it is either D, E, N, or Q, or does not exist. X ３６ However, it is either D, E, N, or Q, or does not exist. X ３７ The hBCMA-binding polypeptide according to claim 1, wherein the polypeptide is D, E, N, or Q, or is not present.

4. i) Helix 1 contains the sequence NKENQFADEEIAAL and helix 2 contains the sequence NFYQKWAFIRKLMDD, or ii) helix 1 and helix 2 are defined as in i), and within helix 1 and helix 2, at least one and no more than three residues in the sequence of helix 1 and / or helix 2 are replaced by substitute residues, according to claim 3.

5. The hBCMA-binding polypeptide according to claim 1, wherein the separated portion is a sequence of 1 to 5 naturally occurring amino acids.

6. The aforementioned N-terminal portion It does not exist, or The hBCMA-binding polypeptide according to claim 1, wherein the sequence is of 1 to 15 naturally occurring amino acids.

7. The C-terminal portion is Does not exist, or The hBCMA-binding polypeptide according to claim 1, wherein the sequence is 1 to 50 naturally occurring amino acids.

8. The separation portion is array X ２０ X ２１ X ２２ Having, and / or The aforementioned N-terminal portion is sequence X ａ X ｂ X １ X ２ X ３ X ４ X ５ Having, and / or The C-terminal portion is arranged PSQSANLLAEAKKLNDAQX ５６ X ５７ X ５８ It has, In the aforementioned separation portion, X ２０ However, P is X ２１ However, N is X ２２ However, it is L, In the aforementioned N-terminal portion, X ａ However, it is M, or does not exist, X ｂ However, it is M, or does not exist, X １ However, it is either V or G, or does not exist, X ２ However, it is either D or G, or does not exist, X ３ However, it is either G or N, or does not exist, X ４ However, K is either true or does not exist, X ５ However, it is either F or does not exist. In the C-terminal portion, X ５６ However, A is either true or does not exist, X ５７ However, P is either true or does not exist, X ５８ The hBCMA-binding polypeptide according to claim 1, wherein K is present or absent.

9. The hBCMA-binding polypeptide according to claim 1, wherein the separated portion has a sequence PNL.

10. The separation portion has sequence PNL, and the hBCMA binding motif is adjacent to the N-terminal portion X1X2X3X4X5 and the C-terminal portion PSQSANLLLAEAKKLNDAQAPK, In the aforementioned N-terminal portion, X １ However, it is either G, V, or missing, X ２ However, it is either D or missing, X ３ However, it is either N or missing, X ４ However, it is either K or missing, X ５ The hBCMA-binding polypeptide according to claim 1, wherein F is present or is missing.

11. The hBCMA-binding polypeptide according to claim 1, wherein the N-terminal portion has the sequence VDNKF.

12. The hBCMA-binding polypeptide according to claim 1, wherein the hBCMA-binding efficacy is at least 1% of that of SEQ ID NO:

2.

13. The sequence of the hBCMA-binding polypeptide is, VDNKFNKEETFADLEISNLPNLNFYQKWAFIRSLMDDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 1], or VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence ID 2], or VDNKFNKEEIFADREIAFLLPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 23], or VDNKFNKEHQFADYEIAMLPNLNFYQKWAFIRSLMDDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 33] An hBCMA-binding polypeptide according to claim 1, selected from the above.

14. An hBCMA-binding oligomer comprising at least two hBCMA-binding polypeptides as described in claim 1.

15. The hBCMA-binding oligomer according to claim 14, wherein each of the hBCMA-binding polypeptides is separated by a linker.

16. The hBCMA-binding polypeptide according to claim 1, further comprising an additional functional portion.

17. The hBCMA-conjugated oligomer according to claim 14, further comprising an additional functional portion.

18. The hBCMA-binding polypeptide according to claim 16 or the hBCMA-binding oligomer according to claim 17, wherein the additional functional portion comprises an immune signaling molecule or a derivative thereof.

19. The hBCMA-binding polypeptide according to claim 16 or the hBCMA-binding oligomer according to claim 17, wherein the additional functional portion includes an additional binding portion.

20. The hBCMA-binding polypeptide or hBCMA-binding oligomer according to claim 19, wherein the additional binding portion is specific to an immune cell surface target.

21. The hBCMA-binding polypeptide or hBCMA-binding oligomer according to claim 20, wherein the additional binding portion is specific to an NK cell activating receptor, for example, CD16a.

22. The hBCMA-binding polypeptide or hBCMA-binding oligomer according to claim 16 or claim 17, wherein the hBCMA-binding polypeptide or hBCMA-binding oligomer comprises at least two additional functional moieties, each of which may be the same or different.

23. The hBCMA-binding polypeptide of claim 16 or the hBCMA-binding oligomer of claim 17, wherein the additional functional portion is separated from the hBCMA-binding polypeptide or hBCMA-binding oligomer by a linker.

24. The following array VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence No. 139], VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGG GSGVDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGS GGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence No. 140], VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGGGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGGGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLMNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGGGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLMNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence No. 141], VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGGGVGVDNKFNKEQFYARDEIDLLLPNLNEDQKWAFYMSLIDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence No. 142], VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGSGVDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence ID 143], VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGVGVDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence ID 144], or VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGGGVDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGGGVDNKFNKEVQMAQFEIRKLMPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence No. 145] The hBCMA-binding polypeptide according to claim 1 or the hBCMA-binding oligomer according to claim 14, comprising the above.

25. An hBCMA-binding agent-drug conjugate comprising an hBCMA-binding polypeptide according to claim 1, or an hBCMA-binding oligomer containing at least two hBCMA-binding polypeptides according to claim 1, and an additional therapeutic agent.

26. The hBCMA-binding agent-drug conjugate according to claim 25, wherein the additional therapeutic agent is a cytotoxic drug.

27. The hBCMA-binding polypeptide is linked to the additional therapeutic agent via a linker, according to claim 25.

28. A nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or encoding an hBCMA-binding oligomer according to any one of claims 14, 15 or 17.

29. An expression vector comprising a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17.

30. A host cell comprising an expression vector containing a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, or a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17.

31. A method for producing an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, the method comprising: maintaining a host cell containing an expression vector comprising a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, or a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, under conditions optimal for the expression of the nucleic acid; and isolating the hBCMA-binding polypeptide or hBCMA-binding oligomer.

32. A pharmaceutical composition comprising an expression vector comprising an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, an hBCMA-binding agent-drug conjugate according to any one of claims 25 to 27, a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17, or a nucleic acid molecule encoding an hBCMA-binding polypeptide according to any one of claims 1 to 13 or 16, or an hBCMA-binding oligomer according to any one of claims 14, 15 or 17.

33. The pharmaceutical composition according to claim 32 for use in the treatment of cancer.

34. The pharmaceutical composition according to claim 33, wherein the cancer is multiple myeloma.

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