Novel polypeptide

Abstract

The present invention provides a CD16a-binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide comprises the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the CD16a-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion. The present invention further provides a pharmaceutical composition comprising the CD16a-binding polypeptide, and the use of the CD16a-binding polypeptide or the pharmaceutical composition as a medicament for use in the treatment or prevention of cancer, particularly multiple myeloma.

Description

Technical Field

[0001] The present invention relates to an immune cell-engaging polypeptide comprising at least one CD16a-binding polypeptide. The present invention also relates to pharmaceutical compositions comprising the immune cell-engaging polypeptides, and their use in the treatment and / or prevention of cancer.

Background Art

[0002] Immunotherapy has proven to be an effective treatment for some cancers by approved therapies that constitute monoclonal antibodies and bispecific antibodies, immunomodulatory drugs, and CAR-T treatments. However, despite the activity of these treatments, some patients exhibit very short responses or do not respond to the treatment. Side effects from some immunotherapies, particularly those related to exacerbated cytokine release, can be severe. In fact, cytokine release syndrome is one of the most common serious side effects of T cell-engaging immunotherapeutic 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, further treatment options in cancer immunotherapy are still needed.

[0003] One obvious drawback of current treatment modalities is 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), for example, cell-mediated immunosuppression via tumor-derived regulatory T cells, regulation of antigen presentation and MHC I expression, or changes in the expression of other ligands that reduce tumor cell killing by CD8+ T cells and natural killer (NK) cells, for example, increased expression of inhibitory checkpoint ligands (such as programmed cell death ligand 1 (PD-L1) and HLA-E) (Vinay D et al, Immune evasion in cancer: Mechanistic basis and therapeutic strategies, Seminars in Cancer Biology. 2015:35 (suppl):S185, https: / / doi.org / 10.1016 / j.semcancer.2015.03.004, Ben-Shmuel A et al, Unleashing Natural Killer Cells in the Tumor Microenvironment - The Next Generation of Immunotherapy? Front Immunol. 2020;11:275, doi:10.3389 / fimmu.2020.00275).

[0004] NK cells are components of the innate immune system, and their functions include cytokine secretion and cell death by the secretion of perforin- and granzyme-containing cytotoxic granules. They are capable of antibody-dependent cell-mediated cytotoxicity (ADCC) when target cells such as tumor cells are bound by IgG antibodies. Binding of the Fc antibody region to the CD16 receptor (FCγRIII) on NK cells abrogates inhibitory signals and induces cytokine secretion and lysis of target cells (Vivier E et al, Functions of natural killer cells, Nat Immunol. 2008;9:503, https: / / doi.org / 10.1038 / ni1582, Pallmer K and Oxenius A, Recognition and regulation of T cells by NK cells, Front Immunol 2016, 7:251, https: / / doi.org / 10.3389 / fimmu.2016.00251).

[0005] CD16 is expressed in two forms, CD16a and CD16b, which differ in their expression patterns and affinity for IgG Fc. CD16a is the form mainly expressed on NK cells and is found on monocytes. In contrast, CD16b is mostly found on neutrophils, but its expression can also be induced on eosinophils. Despite a high degree of sequence similarity (about 96%) between CD16a and CD16b, CD16a has a much higher binding affinity for IgG. (Roberts JT and Barb AW, A single amino acid distorts the Fcγreceptor IIIb / CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a, J Biol Chem. 2018;293(51):19899-19908, doi:10.1074 / jbc.RA118.005273). Genotypic variations of the CD16a (FcγRIIIa) receptor itself can also change its binding affinity: the FcγRIIIa-176V / F polymorphism (rs396991) (in some publications where the leader sequence is excluded, position 176 is reported as position 158, and this numbering is also used in the examples in this specification) results in either valine (V) or phenylalanine (F) at position 176, giving rise to variable binding phenotypes (F / F: low affinity; V / V or V / F: high affinity and thus higher NK cell-mediated ADCC). (Chong KT et al., Distribution of the FcγRIIIa 176 F / V polymorphism between healthy Chinese, Malays and Asian Indiana in Singapore, Br J Clin Pharmacol 2006;63(3):328-332, doi:10.1111 / j.1365-2125.2006.02771.x).

[0006] Recently, there has been growing interest in harnessing NK cell responses for cancer immunotherapy. These include the use of checkpoint inhibitor blocking agents, ex vivo expansion and administration of NK cells, generation of CAR-NK cells (similar to CAR-T cell therapy), and the use of bivalent or multivalent NK cell engagers that bridge NK cells to cancer cells expressing specific antigens (Hofer E and Koehl U, Natural Killer Cell-Based Cancer Immunotherapies: From Immune Evasion to Promising Targeted Cellular Therapies, Front Immunol. 2017;8:745, https: / / doi.org / 10.3389 / fimmu.2017.00745).

[0007] Among the antibody-based NK engagers under development are Affimed's AFM13 (anti-CD16a / CD30 tetravalent bispecific antibody) and AFM24 (anti-CD16a / EGFR IgG1-scFv fusion antibody). Both AFM13 and AFM24 are being further tested for their ability to induce tumor cell killing by macrophages via antibody-dependent cellular phagocytosis (ADCP) (Wingert S et al., Preclinical evaluation of AFM24, a novel CD16A-specific innate immune cell engager targeting EGFR-positive tumors. MAbs. 2021, 13(1):1950264.doi:10.1080 / 19420862.2021.1950264, Wingert S et al, CD16A-Specific Quaternary Bispecific Immune Cell Engager Potently Induce Antibody-Dependent Cellular Phagocytosis(ADCP)on Macrophages, Blood 2018;132(Suppl 1):1111,https: / / doi.org / 10.1182 / blood-2018-99-118427). Other bivalent and multivalent NK engagers include camelid VHH antibody-derived BiKEs and TriKEs such as GT Biopharma's GTB-3650, which contains CD33 and CD16a targeting regions conjugated to the costimulatory molecule IL15.

[0008] A further bispecific cell engager under development is RO7297089. RO7297089 is a bispecific tetravalent antibody targeting CD16a and BCMA (B-cell maturation antigen). BCMA is highly expressed on MM cells. Its properties are described in Kakiuchi-Kiyota et al. (Leukemia (2022) 36:1006-1014; https: / doi.org / 10.1038 / s41375-021-01478-w), and the findings from the Phase I dose escalation trial of RO7297089 in patients with relapsed / refractory multiple myeloma (Study registration NCT04434469) were reported by Plesner et al. (Poster Abtract 2755, Session 653, American Society of Hematology (ASH) Conference, 12 December 2021).

[0009] Despite this progress, antibody-derived NK engagers still share the inherent difficulties associated with existing antibody cancer therapeutics, particularly with regard to immunogenicity and tissue penetration. Therefore, there is still a need for improved cancer immunotherapeutics that can retain the target specificity of antibodies and antibody-based drugs and be delivered more efficiently to tumors while avoiding potential side effects.

[0010] The present invention seeks to address the above-mentioned need.

Summary of the Invention

[0011] The present invention provides a CD16a-binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the CD16a-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion.

[0012] In particular, the present invention Helix 1 contains the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and Helix 2 contains the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein a) X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, X 13 is A, Q or V, X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, M, R or S, or b) X9 is V, X 10 is Q, X 11is M, and X 13 is Q, and X 14 is F, and X 17 is R, and X 18 is K, and X 24 is H, and X 25 is H, and X 27 is S, and X 28 is F, and X 31 is I, and X 32 is K, and X 33 is S, and X 35 is M, and optionally, 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 as such are replaced by alternative residues, or c) X9 is Q, and X 10 is F, and X 11 is Y, and X 13 is R, and X 14 is D, and X 17 is D, and X 18 is L, and X 24 is E, and X 25 is D, and X 27 is K, and X 28 is W, and X 31 is Y, and X 32 is M, and X 33 is S, and X 35 is I, and optionally, 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 as such are replaced by alternative residues, or d) X9 is F, and X10 is W, and X 11 is I, and X 13 is E, and X 14 is S, and X 17 is E, and X 18 is S, and X 24 is I, and X 25 is Y, and X 27 is K, and X 28 is W, and X 31 is K, and X 32 is Y, and X 33 is S, and X 35 is A, and optionally, within helix 1 and helix 2, X n at least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues, and / or X n at least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues not labeled as such are replaced by alternative residues, provides a CD16a binding polypeptide.

[0013] The present invention also relates to i) helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 has the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 SLX 35 wherein, a * ) X9 is V, and X 10 is Q, and X 11 is M, and X 13 is Q, and X 14 is F, and X 17 is R, and X 18 is K, and X 24 is H, and X 25 is H, and X 27is S, X 28 is F, X 31 is I, X 32 is K, X 35 is M, b * )X9 is Q, X 10 is F, X 11 is Y, X 13 is R, X 14 is D, X 17 is D, X 18 is L, X 24 is E, X 25 is D, X 27 is K, X 28 is W, X 31 is Y, X 32 is M, X 35 is I or c * )X9 is F, X 10 is W, X 11 is I, X 13 is E, X 14 is S, X 17 is E, X 18 is S, X 24 is I, X 25 is Y, X 27 is K, X 28 is W, X 31 is K, X 32 is Y, X 35 is A or or ii) Helix 1 and Helix 2 are defined as in i), and within Helix 1 and Helix 2, X n at least 1 and 3 or fewer of the residues are replaced by alternative residues, and / or X n at least 1 and 3 or fewer of the residues not labeled as such are replaced by alternative residues, to provide a CD16a-binding polypeptide.

[0014] The inventors have surprisingly found that the CD16a-binding polypeptide of the present invention based on a non-antibody scaffold is effective in engaging NK cells and inducing ADCC-mediated cancer cell death.

[0015] Accordingly, the present invention provides a novel CD16a engager that is promising as an anti-cancer immunotherapeutic agent.

[0016] The present invention further provides a CD16a-binding polypeptide consisting of the CD16a-binding polypeptide of the present invention and optionally comprising additional binding moieties (e.g., one, two, three or more additional binding moieties).

[0017] The present invention further provides a CD16a-binding oligomer comprising at least two CD16a-binding polypeptides of the present invention.

[0018] The present invention provides a CD16a-binding polypeptide or a CD16a-binding oligomer as defined herein, further comprising additional functional moieties (e.g., at least one, at least two, or at least three, e.g., one, two, three, four or five additional functional moieties).

[0019] Accordingly, the present invention further provides a bispecific or multispecific engager comprising an additional binding moiety in embodiments where the additional functional moiety is an additional binding moiety.

[0020] The present invention further provides the following: - A nucleic acid molecule encoding the CD16a-binding polypeptide or CD16a-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.

[0021] The present invention further provides a method for producing the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention.

[0022] The present invention also provides a pharmaceutical composition comprising the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, nucleic acid molecule or expression vector of the present invention.

[0023] The present invention further provides the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition of the present invention for use in medicine, particularly for the treatment of cancer.

[0024] The present invention also provides the use of the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of cancer.

[0025] The present invention also provides a method for treating cancer, comprising administering to a patient in need thereof the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition of the present invention.

[0026] The present invention further provides a kit comprising the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, nucleic acid molecule, expression vector, pharmaceutical composition of the present invention, 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

[0027]

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Mode for Carrying Out the Invention

[0028] The inventors have found that the polypeptides disclosed herein are effective binders of CD16a and are effectively involved in immune cells. The polypeptides have been found to induce a strong ADCC response against cancer cells in in vitro models. Notably, the inventors have found that such an anti-cancer response favorably compares to the response obtained using the monoclonal antibody elotuzumab, which is approved for the treatment of multiple myeloma in the model. The inventors have further found that such an anti-cancer response favorably compares to the response obtained using a biosimilar of belantamab mafodotin, which is an antibody-drug conjugate for the treatment of multiple myeloma in the model, and the response obtained using daratumumab, which is a monoclonal antibody approved for the treatment of multiple myeloma.

[0029] In its broadest aspect, the invention is a CD16a-binding polypeptide comprising at least one motif that binds to CD16a, the polypeptide having the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and providing a CD16a-binding polypeptide, wherein the CD16a-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion.

[0030] Here, the various elements of the polypeptides of the invention are described in more detail.

[0031] Overall structure In one embodiment, the polypeptide of the present invention may be based on a three-helix backbone, sometimes referred to as an "affibody". An affibody is a small (about 6.5 kDa) engineered affinity ligand based on the Z domain polypeptide, which is a mutated version 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 α-helical bacterial receptor domain, Nature Biotech, 1997:15:772, doi:10.1038 / nbt0897-772). In the full-length affibody, the C-terminal portion contains [second separated portion]-[helix 3]-[C-terminal sequence]. The general structure of an affibody is shown in FIG. 1a.

[0032] 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 can be found that the structure established by the sequence with specific residues may not be strictly helical. Such compounds should be considered within the broadest aspects of the present invention. More preferably, due to the residues in the helix 1 and helix 2 portions, their structures are helical in the sense that they are alpha-helices.

[0033] The sequence of the polypeptide of the present invention The sequences of the CD16a-binding polypeptides disclosed herein can be represented with respect to their constituent amino acids or with respect to the nucleic acid sequences encoding the polypeptides having those amino acid sequences. In the context of this disclosure, the term "amino acid" encompasses any naturally occurring amino acid or non-natural amino acid. The term "non-natural amino acid" as used herein refers to non-proteinaceous (i.e., not encoded) amino acids that may be found in nature or chemically synthesized (e.g., citrulline, hydroxyproline, beta-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 configuration. The amino acid may in particular be a naturally occurring α-amino acid. The amino acid may in particular be a naturally occurring L-amino acid. The amino acid may in particular be a naturally occurring L-α-amino acid.

[0034] Within a polypeptide chain (e.g., the CD16a-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 the polypeptide chain, are referred to as "residues" or "amino acid residues."

[0035] Amino acid sequences herein 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.

[0036] The amino acid sequences recited in this application are shown using standard abbreviations for amino acids.

[0037] The specific sequences shown herein relate to specific embodiments of the invention.

[0038] The present disclosure also includes derivatives of all the sequences described herein (e.g., derivatives of each of the CD16a-binding polypeptides and CD16a-binding oligomeric sequences described herein). Derivatives of the sequences described herein are preferably derivatives in which 1 to 5 (e.g., 1, 2, or 3 amino acid residues) are replaced by alternative residues, such as different naturally occurring amino acids or different non-natural amino acids, or different naturally occurring amino acids or different non-natural amino acids excluding methionine. Preferably, the non-natural amino acids according to the present invention are isoelectronic with naturally occurring amino acids, such as norleucine.

[0039] For example, in one embodiment, one or more (e.g., each) methionine residues of the sequences described herein may be replaced by different naturally occurring amino acids or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine. For example, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues, or 1 methionine residue may be replaced by different naturally occurring amino acids, or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine.

[0040] For example, X 11 may be methionine, or in embodiments where X 11 is methionine, the residue of X 35 may be replaced by different naturally occurring amino acids, or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine. For example, in embodiments where X 35The residue may be replaced by a different naturally occurring amino acid, or a non-natural amino acid, for example, an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine. For example, X 11 and X 35 may be methionine, or in embodiments where it is methionine, X 11 and X 35 The residue may be replaced by a different naturally occurring amino acid, or a non-natural amino acid, for example, an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine. For example, X 32 may be methionine, or in embodiments where it is methionine, X 32 The residue may be replaced by a different naturally occurring amino acid, or a non-natural amino acid, for example, an amino acid selected from isoleucine, leucine, glutamine, and norleucine, particularly isoleucine and norleucine.

[0041] Alternatively, or additionally, in certain embodiments, the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) may contain amino acid substitutions in which one or more residues are replaced by non-natural amino acids.

[0042] For example, in one embodiment, one or more residues of the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) may be replaced by a non-natural amino acid, such as norleucine. For example, 1 to 15 residues may be replaced by a non-natural amino acid, such as 1 to 10 residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues), 1 to 5 residues (e.g., 1, 2, 3, 4, or 5 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).

[0043] For example, in one embodiment, one or more leucine residues of the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-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 CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein), the polypeptide has a sequence in which leucine residues are independently replaced by non-natural amino acids, preferably norleucine, at positions where leucine residues are described.

[0044] Alternatively or additionally, in one embodiment, one or more methionine residues of the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-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 may be replaced by non-natural amino acids (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein), the polypeptide has a sequence in which methionine residues are independently replaced by non-natural amino acids, preferably norleucine, at positions where methionine residues are described.

[0045] For example, X 11 may be methionine, or in embodiments where X is methionine, X 11The residue at 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). For example, X 11 and X 35 may be methionine, or in embodiments where they are methionine, the residues at X 11 and X 35 may be replaced by a non-natural amino acid (e.g., norleucine). For example, X 32 may be methionine, or in embodiments where it is methionine, the residue at X 32 may be replaced by a non-natural amino acid (e.g., norleucine).

[0046] In certain embodiments, one or more methionine residues of the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) may be oxidized, for example, 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, X 11 may be methionine, or in embodiments where it is methionine, if present, the methionine at X 11 may be oxidized (e.g., Met(O)). For example, X 35 may be methionine, or in embodiments where it is methionine, if present, the methionine may be oxidized (e.g., Met(O)). For example, X 11 and X 35 may be methionine, or in embodiments where they are methionine, if present, X 11 and X 35The methionine in 32 may be oxidized (e.g., Met(O)). For example, in embodiments where X

[0047] can be methionine or is methionine, if present, the methionine may be oxidized (e.g., Met(O)).

[0048] Alternatively or additionally, in certain embodiments, the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) include a peptide purification tag or moiety (e.g., a histidine tag (e.g., a polyhistidine tag optionally including tyrosine) 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 selected from the signal peptides of, for example, OmpA, DsbA, PhoA, and PelB), a fluorophore tag (e.g., Alexa448), or a tag or moiety that aids conjugation (a cysteine tag (e.g., a single cysteine at the C or N terminus)). Such tags and / or moieties may preferably be present at the N-terminus and / or C-terminus of the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein.Accordingly, the sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomeric sequences described herein) may further include 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 CD16a-binding polypeptide and CD16a-binding oligomeric sequences described herein) may further include 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 tag or methionine tag described above, may preferably be present at the N-terminus and / or C-terminus of the CD16a-binding polypeptide and CD16a-binding oligomeric 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) 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 CD16a-binding polypeptide and CD16a-binding oligomeric sequences described herein.

[0049] The sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-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 CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) may further comprise a fluorophore tag (e.g., 448 Alexa tag) at the N-terminus or C-terminus. The sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-binding oligomer sequences described herein) may further comprise a signal peptide selected from, for example, OmpA, DsbA, PhoA, and PelB at the N-terminus or C-terminus, preferably at the N-terminus. The sequences described herein (e.g., the CD16a-binding polypeptide and CD16a-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.

[0050] In certain embodiments, the CD16a-binding polypeptide has a CD16a-binding potency 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: 1, 74 or 75. When it is described herein that a CD16a-binding polypeptide has a CD16a-binding potency that is at least X% of a particular peptide (e.g., SEQ ID NO: 1, 74 or 75), the IC 50 concentration of the polypeptide for binding to the CD16a receptor, when measured under the same conditions, is understood to be 100 / X times or less of the IC 50 concentration of the particular peptide (SEQ ID NO: 1, 74 or 75) for the CD16a receptor.

[0051] For example, when the binding potency of a CD16a-binding polypeptide is at least 5%, and more preferably at least 10%, 20%, 25% or 50% of the CD16a-binding potency of a specific peptide (e.g., SEQ ID NO: 1, 74 or 75), i.e., the IC 50 concentration of the alternative polypeptide for binding to the CD16a receptor is 20-fold or less, and more preferably 10-fold, 5-fold, 4-fold or 2-fold of the IC 50 concentration of the specific peptide (e.g., SEQ ID NO: 1, 74 or 75) for the CD16a receptor, when measured under the same conditions.

[0052] In certain embodiments, alternatively or additionally, the CD16a-binding polypeptide competes with SEQ ID NO: 1, 74 and / or 75.

[0053] The CD16a-binding polypeptides of the invention have binding affinity for the CD16a receptor and optionally may have binding affinity for the CD16b receptor. For example, in certain embodiments, the CD16a-binding polypeptides of the invention have similar binding affinity for the CD16a receptor and the CD16b receptor. In certain embodiments, the CD16a-binding polypeptides of the invention have stronger binding affinity for the CD16a receptor than for the CD16b receptor, and in certain embodiments, the CD16a-binding polypeptides of the invention have stronger binding affinity for the CD16b receptor than for the CD16a receptor.

[0054] The CD16a-binding polypeptide of the present invention may have a binding affinity for one or both genotype variations of the CD16a (FcγRIIIa) receptor: the FcγRIIIa-176V / F polymorphism (rs396991) (in some publications where the leader sequence is excluded, position 176 is reported as position 158, and this numbering is also used in the examples herein). Preferably, the CD16a-binding polypeptide of the present invention has a binding affinity for both genotype variations of the CD16a (FcγRIIIa) receptor: the FcγRIIIa-176V / F polymorphism (rs396991). In certain embodiments, the CD16a-binding polypeptide of the present invention has a stronger binding affinity for the valine (V) 176 phenotype, or a stronger binding affinity for the phenylalanine (F) 176 phenotype, or similar binding for both the F and V 176 phenotypes.

[0055] The present invention provides a CD16a-binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and the CD16a-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion.

[0056] Preferably, Helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and Helix 2 comprises the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein, a) X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, X 13 is A, Q or V, X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, M, R or S, or b) X9 is V, X 10 is Q, X 11 is M, X 13 is Q, X 14 is F, X 17 is R, X 18 is K, X 24 is H, X 25 is H, X 27 is S, X 28 is F, X 31 is I, X 32 is K, X 33 is S, X 35 is M, optionally, within helix 1 and helix 2, X nAt 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 as X are replaced by alternative residues or c) X9 is Q, X 10 is F, X 11 is Y, X 13 is R, X 14 is D, X 17 is D, X 18 is L, X 24 is E, X 25 is D, X 27 is K, X 28 is W, X 31 is Y, X 32 is M, X 33 is S, X 35 is I, and optionally, within helices 1 and 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 as X are replaced by alternative residues or d) X9 is F, X 10 is W, X 11 is I, X 13 is E, X 14 is S, X 17 is E, X 18 is S, X 24 is I, X 25 is Y, X 27 is K, X 28 is W, X 31 is K, X 32 is Y, X 33 is S, X 35is A, and optionally, within helix 1 and helix 2, X n At least one and no more than five (e.g., at least one and no more than three) of the residues are replaced by alternative residues and / or X n At least one and no more than five (eg, at least one and no more than three) of the residues not labeled as are replaced by alternative residues.

[0057] In certain embodiments, the CD16a binding polypeptide has helix 1 having the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprises the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 is Q and X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is Q or R, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H and X 25 is A or H, and X 27 is A, I, K, Q, R, S, T or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, M, R or S.

[0058] In a further embodiment, the CD16a binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, X 13 is A, Q or V, X 14 is F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is R or Q, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is H or A, X 27 is A, I, K, Q, R, T, S or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 , is A, H, I, L, M, R or S.

[0059] In a further embodiment, the CD16a binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X28 AFX 31 X 32 X 33 LX 35 comprises, wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X 14 is F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R, and X 18 is A, D, E, F, H, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H, and X 27 is A, I, K, Q, R, T, S or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, M, R or S.

[0060] In a further embodiment, the CD16a binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 comprises, wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X14 is H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R or Q, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H or A, and X 27 is A, I, K, Q, R, T or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, R or S.

[0061] In a further embodiment, the CD16a binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X 14 is H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R, and X 18 is A, D, E, F, H, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H, and X 27 is A, I, K, Q, R, T or V, and X 28 is F or Y, and X31 is I or L, and X 32 is A, E, H, K, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, R or S.

[0062] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a fast binding rate or "on" rate to the CD16a receptor. For example, the CD16a-binding polypeptide has a helix 1 that comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and a helix 2 that comprises the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, N, Q, R, S, T, V, W or Y, X 13 is A, Q or V, X 14 is H, I, K, L, N, Q, R, S, V, W or Y, X 17 is R, X 18 is A, D, E, F, H, K, N, Q, R, S or T, X 24 is H, X 25 is H, X 27 is A, I, K, Q, R, T or V, X 28 is F, X 31 is I or L, X 32 is A, E, H, K, N, Q or R, X 33 is K or S, X 35 is H, I, L, R or S.

[0063] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a slow dissociation rate or "off" rate for the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 having the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 having the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is D, F, H, I, K, L, Q, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 13 is A or Q, X 14 is H, I, K, L, Q, R, S, T, V, W or Y, X 17 is R, X 18 is A, D, E, F, H, K, N, Q, R, S, T or V, X 24 is H, X 25 is H, X 27 is A, I, K, Q, R, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, N, Q or R, X 33 is K or S, X 35 is A, H, I, L or R.

[0064] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a high binding affinity for the CD16a receptor, e.g., the KD D for the CD16a receptor is less than 250 nM. For example, the CD16a-binding polypeptide has helix 1 having the sequence X9X 10 X 11 AX 13 X14 EIX 17 X 18 comprises, wherein helix 2 is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 comprises, wherein X9 is D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, N, Q, R, S, T, V, W or Y, X 13 is A or Q, X 14 is H, I, K, L, Q, R, S, V, W or Y, X 17 is R, X 18 is A, F, H, K, N, Q, R, S or T, X 24 is H, X 25 is H, X 27 is A, I, K, Q, R, T or V, X 28 is F or Y, X 31 is I or L, X 32 is E, H, K, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, R or S.

[0065] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a particularly fast binding rate or "on" rate to the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 being the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 comprises, wherein helix 2 is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35which includes, wherein X9 is F, L, Q, T or Y, X 10 is Q, X 11 is A, F, H, I, L, N, Q, S, or Y, X 13 is A or Q, X 14 is I, K, Q, R or V, X 17 is R, X 18 is A, E, H, K, Q, R, T or V, X 24 is H, X 25 is H, X 27 is A, I, K, Q, R or V, X 28 is F, X 31 is I, X 32 is A, H, K, N, Q or R, X 33 is K or S, X 35 is H, I or L.

[0066] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a particularly slow dissociation rate or "off" rate for the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 that is the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 that is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 and includes, wherein X9 is I, L, Q, T or V, X 10 is Q, X 11 is A, E, F, H, I, S, V or Y, X 13 is Q, X 14 is K, L, R, V, W or Y, X 17 is R, X 18 is A, H, K, Q, R, S or T, X 24 is H, X 25is H, and X 27 is I, K, Q, R, T or V, and X 28 is F, and X 31 is I or L, and X 32 is K, N or R, and X 33 is K or S, and X 35 is I or L.

[0067] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a particularly high binding affinity for the CD16a receptor, e.g., a KD of less than 100 nM for the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is I, L, Q or V, X 10 is Q, X 11 is A, E, H, I, S, W or Y, X 13 is Q, X 14 is K, L, R, V, W or Y, X 17 is R, X 18 is A, H, K, Q, R, S or T, X 24 is H, X 25 is H, X 27 is K, Q, R, T or V, X 28 is F, X 31 is I, X 32 is K, N or R, X 33 is K or S, X 35 is I or L.

[0068] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has at least two of the following: a fast binding rate or "on" rate to the CD16a receptor, and / or a slow dissociation rate or "off" rate to the CD16a receptor, and / or a high binding affinity to the CD16a receptor, e.g., a KD of less than 100 nM for the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 comprising the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprising the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is I, L, Q or V, X 10 is Q, X 11 is A, E, H, I, S or Y, X 13 is Q, X 14 is K, L, R, V, W or Y, X 17 is R, X 18 is H, K, Q, R, S or T, X 24 is H, X 25 is H, X 27 is K, Q, R, T or V, X 28 is F, X 31 is I, X 32 is N or K, X 33 is K or S, X 35 is I or L.

[0069] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has a particularly slow dissociation rate or "off" rate to the CD16a receptor and a high binding affinity to the CD16a receptor, e.g., a K DIt has. For example, the CD16a-binding polypeptide has helix 1 with the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 with the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is L or V, X 10 is Q, X 11 is A, I, S or Y, X 13 is Q, X 14 is K, R or V, X 17 is R, X 18 is K, Q, R, S or T, X 24 is H, X 25 is H, X 27 is K, R or V, X 28 is F, X 31 is I, X 32 is N or K, X 33 is K or S, X 35 is I or L.

[0070] In certain preferred embodiments, the CD16a-binding polypeptide of the invention binds to the CD16a and CD16b receptors. For example, the CD16a-binding polypeptide has helix 1 with the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 with the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is D, H, I, K, L, Q, T or V, X10 is Q, X 11 is A, D, E, F, H, I, K, N, R, S, V, W or Y, X 13 is Q, X 14 is K, L, Q, R, S, V, W or Y, X 17 is R, X 18 is A, H, K, N, Q, R, S or T, X 24 is H, X 25 is H, X 27 is A, I, K, Q, R, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, N, Q or R, X 33 is K or S, X 35 is A, I, L, or R.

[0071] In certain preferred embodiments, the CD16a-binding polypeptide of the invention has binding preference for the CD16a receptor as compared to the CD16b receptor. For example, the CD16a-binding polypeptide has helix 1 that is the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 that is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is K, Q or Y, X 10 is Q, X 11 is I or Q, X 13 is Q, X 14 is W or Y, X 17 is R, X 18 is H, K or R, X 24 is H, X 25 is H, X 27 is A, K or T, X28 is F, X 31 is I, X 32 is A, K or Q, X 33 is K or S, X 35 is I or L.

[0072] In certain preferred embodiments, the CD16a-binding polypeptide of the present invention has binding preference for the CD16b receptor as compared to the CD16a receptor. For example, the CD16a-binding polypeptide has helix 1 that is the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 including, and helix 2 that is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 including, wherein X9 is L, V or Y, X 10 is Q, X 11 is I, N or Q, X 13 is Q, X 14 is K, R or Q, X 17 is R, X 18 is E, A or V, X 24 is H, X 25 is H, X 27 is K or Q, X 28 is F, X 31 is I, X 32 is H, K or Q, X 33 is K or S, X 35 is I or L.

[0073] In certain very preferred embodiments, the CD16a-binding polypeptide of the present invention is particularly active in functional assays, such as CD16 reporter assays and / or cell killing assays. For example, the CD16a-binding polypeptide has helix 1 that is the sequence X9X 10X 11 AX 13 X 14 EIX 17 X 18 comprising, helix 2 being the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is L, Q, T or V, X 10 is Q, X 11 is I, V or Y, X 13 is Q, X 14 is K, R or Y, X 17 is R, X 18 is K, R, S or T, X 24 is H, X 25 is H, X 27 is I, T or V, X 28 is F, X 31 is I or L, X 32 is K or N, X 33 is K or S, X 35 is I or L.

[0074] In certain particularly preferred embodiments, the CD16a-binding polypeptide of the invention is highly particularly active in functional assays, such as CD16 reporter assays and / or cell killing assays. For example. For example, the CD16a-binding polypeptide has helix 1 being the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 comprising, helix 2 being the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein X9 is L, T or V, X 10 is Q, X11 is I, V or Y, and X 13 is Q, and X 14 is K or R, and X 17 is R, and X 18 is R, S or T, and X 24 is H, and X 25 is H, and X 27 is I or V, and X 28 is F, and X 31 is I or L, and X 32 is K or N, and X 33 is K, and X 35 is I or L.

[0075] In certain embodiments of the present invention, the CD16a-binding polypeptide is X 11 position or X 35 position without a methionine residue, for example, a CD16a-binding polypeptide, Helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and Helix 2 comprises the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein, X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y or a non-natural amino acid (e.g., norleucine), and X 13 is A, Q or V, and X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is Q or R, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, R, S or a non-natural amino acid (e.g., norleucine), or X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine, leucine, glutamine or norleucine (e.g., A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine or norleucine), X 13 is A, Q or V, X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, R, S, isoleucine, leucine, glutamine or norleucine (e.g., A, H, I, L, R, S, isoleucine or norleucine), or X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 13 is A, Q or V, X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, R or S.

[0076] X 11 position or X 35 In a further embodiment having no methionine residue at the X or X position, the CD16a-binding polypeptide, the CD16a-binding polypeptide, helix 1 is the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 contains, and helix 2 is the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 contains, wherein, X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y or a non-natural amino acid (e.g., norleucine), X 13 is A, Q or V, X 14is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S, T or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35 is A, H, I, L, R, S or a non-natural amino acid (e.g., norleucine), or X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, X 10 is Q, X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine, leucine, glutamine or norleucine (e.g., A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine or norleucine), X 13 is A, Q or V, X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, X 17 is Q or R, X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, X 24 is H, X 25 is A or H, X 27 is A, I, K, Q, R, S or V, X 28 is F or Y, X 31 is I or L, X 32 is A, E, H, K, L, N, Q or R, X 33 is K or S, X 35is A, H, I, L, R, S, isoleucine, leucine, glutamine or norleucine (e.g., A, H, I, L, R, S, isoleucine or norleucine), or X9 is A, D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is Q or R, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is A or H, and X 27 is A, I, K, Q, R, S, T or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, R or S.

[0077] X 11 position or X 35 In a further embodiment having no methionine residue at the position, the CD16a-binding polypeptide, the CD16a-binding polypeptide, Helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 comprises the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 wherein, X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y or a non-natural amino acid (e.g., norleucine), and X 13 is A, Q or V, and X 14 is F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R or Q, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H or A, and X 27 is A, I, K, Q, R, T, S or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, R, S or a non-natural amino acid (e.g., norleucine), or X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine, leucine, glutamine or norleucine (e.g., A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, isoleucine or norleucine), and X 13 is A, Q or V, and X 14 is F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R or Q, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H or A, and X 27 is A, I, K, Q, R, T, S or V, and X 28 is F or Y, and X31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is A, H, I, L, R, S, isoleucine, leucine, glutamine or norleucine (e.g., A, H, I, L, R, S, isoleucine or norleucine), or X9 is D, F, H, I, K, L, Q, R, T, V or Y, and X 10 is Q, and X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 13 is A, Q or V, and X 14 is F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X 17 is R or Q, and X 18 is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X 24 is H, and X 25 is H or A, and X 27 is A, I, K, Q, R, T, S or V, and X 28 is F or Y, and X 31 is I or L, and X 32 is A, E, H, K, L, N, Q or R, and X 33 is K or S, and X 35 is one that is A, H, I, L, R or S.

[0078] In certain embodiments, the CD16a-binding polypeptide has a CD16a-binding potency 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: 1 (i.e., the binding potency of the peptide of SEQ ID NO: 1 to the CD16a receptor as measured under the same conditions). Preferably, the CD16a-binding polypeptide has a CD16a-binding potency that is at least 10% of SEQ ID NO: 1.

[0079] In certain embodiments, alternatively or additionally, the CD16a binding polypeptide competes with SEQ ID NO:1.

[0080] In an advantageous embodiment of the CD16a binding polypeptide of the invention, proline (P) and cysteine ​​(C) are not present in either helix 1 or helix 2 of the CD16a binding polypeptide of the invention. Advantageously, glycine (G) is also not present in either helix 1 or helix 2 of the CD16a binding polypeptide of the invention.

[0081] In certain embodiments, the CD16a binding polypeptide of the invention comprises: Helix 1 has the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 and / or helix 2 comprises the sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 Including, wherein X6 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably N) or absent (e.g., X6 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably N)), X7 is any naturally occurring amino acid (preferably H, K or R, more preferably K) or absent (e.g., X7 is any naturally occurring amino acid (preferably H, K or R, more preferably K)), X8 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably E) or absent (e.g., x8 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably E)), and X 19is any naturally occurring amino acid (preferably G, A, V, L or I, more preferably L), or is absent (e.g., X 19 is any naturally occurring amino acid (preferably G, A, V, L or I, more preferably L), and X 23 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably N), or is absent (e.g., X 23 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably N), and X 36 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably D), or is absent (e.g., X 36 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably D), and X 37 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably D), or is absent (e.g., X 37 is any naturally occurring amino acid (preferably D, E, N or Q, more preferably D)).

[0082] In certain embodiments, the CD16a-binding polypeptide of the invention is Helix 1 is the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 and / or Helix 2 is the sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 and In the formula, X6 is D, E, N, or Q (more preferably N), or does not exist (for example, X6 is D, E, N, or Q, and more preferably N), X7 is H, K, or R (preferably K), or does not exist (for example, X7 is H, K, or R, and more preferably K), X8 is D, E, N, or Q (preferably E), or does not exist (for example, X8 is D, E, N, or Q, and more preferably E), X 19 is G, A, V, L, or I (preferably L), or does not exist (for example, X 19 is G, A, V, L, or I, and more preferably L), X 23 is D, E, N, or Q (preferably N), or does not exist (for example, X 23 is D, E, N, or Q, and more preferably N), X 36 is D, E, N, or Q, more preferably D), or does not exist (for example, X 36 is D, E, N, or Q, and more preferably D), X 37 is D, E, N, or Q (preferably D), or does not exist (for example, X 37 is D, E, N, or Q, and more preferably D).

[0083] In certain specific embodiments of the present invention such as the above embodiments, Helix 1 includes the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 and / or Helix 2 includes the sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 .

[0084] In such an embodiment, X6 can be any naturally occurring amino acid or may be absent, X7 can be any naturally occurring amino acid or may be absent, X8 can be any naturally occurring amino acid or may be absent, X 19 can be any naturally occurring amino acid or may be absent, X 23 can be any naturally occurring amino acid or may be absent, X 36 can be any naturally occurring amino acid or may be absent, X 37 can be any naturally occurring amino or may be absent.

[0085] More preferably, X6 may be any naturally occurring amino acid, X7 may be any naturally occurring amino acid, X8 may be any naturally occurring amino acid, X 19 may be any naturally occurring amino acid, X 23 may be any naturally occurring amino acid, X 36 may be any naturally occurring amino acid, X 37 may be any natural amino acid.

[0086] In another preferred embodiment, X6 can be D, E, N, Q or may be absent, X7 can be H, K, R or may be absent, X8 can be any D, E, N, Q or may be absent, X 19 can be G, A, V, L, I or may be absent, X 23 can be G, A, V, L, I or may be absent, X 36 can be G, A, V, L, I or may be absent, X 37 can be G, A, V, L, I or may be absent. More preferably, X8 can be E or may be absent, X 19 can be L or may be absent, X 23 can be D or may be absent, X 36 can be D or may be absent, X37 may be D or may not exist.

[0087] In an even more preferred embodiment, X6 may be D, E, N or Q, X7 may be H, K or R, X8 may be any D, E, N or Q, X 19 may be G, A, V, L or I, X 23 may be G, A, V, L, or I, X 36 may be G, A, V, L or I, X 37 may be G, A, V, L or I.

[0088] In another preferred embodiment, X6 may be N or may not exist, X7 may be K or may not exist, X8 may be E or may not exist, X 19 may be L or may not exist, X 23 may be D or may not exist, X 36 may be D or may not exist, X 37 may be D or may not exist.

[0089] In a particularly preferred embodiment, X6 is N, X7 is K, and X8 is E. In another particularly preferred embodiment, X 36 is D, X 37 is D.

[0090] In a very particularly preferred embodiment, X6 is N, X7 is K, X8 is E, X 19 is L, X 23 is D, X 36 is D, X 37 is D. In such an embodiment, Helix 1 comprises the sequence NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 L and / or Helix 2 is the sequence DX 24 X25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 contains DD.

[0091] In one embodiment, the CD16a-binding polypeptide Helix 1 contains the sequence NKEVQMAQFEIRKL, and Helix 2 contains the sequence NHHQSFAFIKSLMDD, Optionally, at least 1 and up to 5 (e.g., 1, 2, 3, 4, or 5, or, e.g., at least 1 and up to 3 (e.g., 1, 2, or 3)) residues in the sequences of Helix 1 and / or Helix 2 are replaced by alternative residues (e.g., replaced by alternative residues that are conservative replacements).

[0092] In certain embodiments, such a CD16a-binding polypeptide has at least 1%, at least 5%, or at least 10% (e.g., at least 15%, 20%, 25%, or 50%) of the CD16a-binding efficacy of SEQ ID NO: 1. Preferably, such a CD16a-binding polypeptide has at least 10% of the CD16a-binding efficacy of SEQ ID NO: 1. In certain embodiments, alternatively or additionally, such a CD16a-binding polypeptide competes with SEQ ID NO: 1.

[0093] In one embodiment, the CD16a-binding polypeptide Helix 1 contains the sequence NKEQFYARDEIDLL, and Helix 2 contains the sequence NEDQKWAFYMSLIDD, Optionally, at least 1 and up to 5 (e.g., 1, 2, 3, 4, or 5, or, e.g., at least 1 and up to 3 (e.g., 1, 2, or 3)) residues in the sequences of Helix 1 and / or Helix 2 are replaced by alternative residues (e.g., replaced by alternative residues that are conservative replacements).

[0094] In certain embodiments, such a CD16a-binding polypeptide has at least 1%, at least 5%, or at least 10% (e.g., at least 15%, 20%, 25%, or 50%) of the CD16a-binding potency of SEQ ID NO: 74. Preferably, such a CD16a-binding polypeptide has a CD16a-binding potency that is at least 10% of SEQ ID NO: 74. In certain embodiments, alternatively or additionally, such a CD16a-binding polypeptide competes with SEQ ID NO: 74.

[0095] In one embodiment, the CD16a-binding polypeptide is where helix 1 comprises the sequence NKEFWIAESEIESL and helix 2 comprises the sequence NIYQKWAFKYSLADD, Optionally, at least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5, or, e.g., at least 1 and no more than 3 (e.g., 1, 2, or 3)) residues in the sequences of helix 1 and / or helix 2 are replaced by alternative residues (e.g., alternative residues that are conservative replacements).

[0096] In certain embodiments, such a CD16a-binding polypeptide has at least 1%, at least 5%, or at least 10% (e.g., at least 15%, 20%, 25%, or 50%) of the CD16a-binding potency of SEQ ID NO: 75. Preferably, such a CD16a-binding polypeptide has a CD16a-binding potency that is at least 10% of SEQ ID NO: 75. In certain embodiments, alternatively or additionally, such a CD16a-binding polypeptide competes with SEQ ID NO: 75.

[0097] In one embodiment, the CD16a-binding motif that is a [helix 1]-[spacer]-[helix 2] moiety is as follows (i.e., has the following sequence): X6X7X8X9X 10 X 11 AX13 X 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 In the formula, X 20 is any naturally occurring amino acid, and X 21 is any naturally occurring amino acid, and X 22 is any naturally occurring amino acid, and optionally, X 20 , X 21 or X 22 of which one or two (e.g., optionally one) do not exist, and the other residues are as defined above.

[0098] More preferably, X6 can be D, E, N, Q or does not exist, X7 can be H, K, R or does not exist, X8 can be any D, E, N, Q or does not exist, X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 are as defined above, and X 19 can be G, A, V, L, I or does not exist, X 20 can be S, T, M, P, F, Y or W (e.g., P or T), X 21 can be D, E, N or Q, X 22 can be G, A, V, L, I or does not exist, X 23 can be G, A, V, L, I or does not exist, X 36 can be G, A, V, L, I or does not exist, X 37It can be G, A, V, L, I, or it may not exist. Optionally, X 20 X 21 X 22 One or two of them (e.g., optionally one) do not exist.

[0099] Preferably, X6 may be D, E, N, or Q, X7 may be H, K, or R, X8 may be any D, E, N, or Q, X9, X 10 X 11 X 13 X 14 X 17 and X 18 are 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 or T), X 21 may be D, E, N, or Q, X 22 may be G, A, V, L, or I, X 23 may be G, A, V, L, or I, X 36 may be G, A, V, L, or I, X 37 may be G, A, V, L, or I. Optionally, X 20 X 21 X 22 One or two of them (e.g., optionally one) do not exist.

[0100] For example, X6 may be D, E, N, or Q, X7 may be H, K, or R, X8 may be any D, E, N, or Q, X9, X 10 X 11 X 13 X 14 X 17 and X 18 are 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 or T), X 21may be D, E, N or Q, and X 22 may be G, A, V, L or I; X 23 may be G, A, V, L or I; X 36 may be G, A, V, L or I; X 37 may be G, A, V, L or I.

[0101] More preferably, X8 can be E or is absent, and X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 is as defined above, and X 19 may be L or absent, and X 23 can be D or absent, and X 24 , X 25 , X 27 , X 28 , X 31 , X 32 , X 33 and X 35 is as defined above, and X 36 can be D or absent, and X 37 can be D or absent.

[0102] In a particularly preferred embodiment, X6 is N, X7 is K, X8 is E, and X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 is as defined above, and X 19 is L and X 20 is S, T, M, P, F, Y or W (e.g., P or T), and X 21 is D, E, N or Q (e.g., N), and X 22 is G, A, V, L or I (e.g., L), and X 23 is D and X 36 is D and X 37is D. Even more preferably, X6 is N, X7 is K, X8 is E, X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 are as defined above, and X 19 is L, X 20 is P or T, X 21 is N, X 22 is L, X 23 is D, X 36 is D, X 37 is D.

[0103] For example, the CD16a binding motif is selected from the group consisting of SEQ ID NOs: 150 to 221, as shown in FIG. 24.

[0104] For example, the CD16a binding motif is selected from the group consisting of:

[0105] [Table 1]

[0106] In the polypeptides of FIG. 24 and the above table, the N-terminus of helix 1 may further comprise the sequence X6X7X8, where X6, X7, and X8 are as defined above, preferably NKE (i.e., X6 is N, X7 is K, and X8 is E). In the polypeptides of FIG. 24 and the above table, the C-terminus of helix 2 may further comprise the sequence X 36 X 37 , where X 36 and X 37 are as defined above, preferably DD, i.e., X 36 is D and X 37 is D).

[0107] In the polypeptide of FIG. 24 and the above table, 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 replacement residues are conservative replacements.

[0108] In a preferred embodiment, the sequence is one of SEQ ID NOs: 166, 168, 178, 182, or 202 (i.e., as found in one of Example Compounds 17, 19, 29, 33, and 53) (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, and the replacement residues are conservative replacements).

[0109] In another preferred embodiment, the sequence is one of SEQ ID NOs: 164, 166, 168, or 200, or one of SEQ ID NOs: 164, 166, or 168 (i.e., as found in one of Example Compounds 15, 17, 19, 51 or as found in one of Example Compounds 15, 17, 19) (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, and the replacement residues are conservative replacements).

[0110] In a preferred embodiment, the 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 the residues DD at its X 36 X 37 position (i.e., X 36 is D and X 37 is D). For example, the motif sequence has NKE at positions X6X7X8 and X 36 X 37having DD at the position (i.e., X6 is N, X7 is K, X8 is E, X 36 is D, X 37 is D). Thus, in a preferred embodiment, the motif sequence is NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 , X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 DD, and NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33LX 35 can be selected from DD.

[0111] 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 embodiments, even if not explicitly mentioned, optionally 1 to 5 (e.g., 1, 2, 3, 4 or 5), preferably optionally 1 to 3 (e.g., 1, 2 or 3) residues in the sequences of helix 1 and / or helix 2 defined above are 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 the CD16a receptor is maintained. For example, the CD16a binding potency is at least 1% of the binding potency of the peptide of SEQ ID NO: 1 (i.e., the binding potency of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions (above)). Alternatively or additionally, for example, in any such alternative polypeptide of the present invention where the alternative residues are in place, the CD16a binding polypeptide competes with SEQ ID NO: 1.

[0112] In certain preferred embodiments, the binding potency is at least 5%, and more preferably at least 10%, 20%, 25% or 50% of the binding potency of the peptide of SEQ ID NO: 1 (i.e., the binding potency of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions).

[0113] In advantageous embodiments of the CD16a binding polypeptide of the present invention, proline (P) and cysteine (C) are not present in the CD16a binding motif of the CD16a binding polypeptide of the present invention. Advantageously, glycine (G) is also not present in the CD16a binding motif of the CD16a binding polypeptide of the present invention.

[0114] In certain embodiments of the present invention, the CD16a binding polypeptide is i) helix 1 is sequence X9X 10 X11 AX 13 X 14 EIX 17 X 18 comprising, helix 2 having the array X 24 X 25 QX 27 X 28 AFX 31 X 32 SLX 35 having, wherein a * ) X9 is V, X 10 is Q, X 11 is M, X 13 is Q, X 14 is F, X 17 is R, X 18 is K, X 24 is H, X 25 is H, X 27 is S, X 28 is F, X 31 is I, X 32 is K, X 35 is M, b * ) X9 is Q, X 10 is F, X 11 is Y, X 13 is R, X 14 is D, X 17 is D, X 18 is L, X 24 is E, X 25 is D, X 27 is K, X 28 is W, X 31 is Y, X 32 is M, X 35 is I, or c * ) X9 is F, X 10 is W, X 11 is I, X 13 is E, X 14 is S, X 17 is E, X 18 is S, X 24is I, X 25 is Y, X 27 is K, X 28 is W, X 31 is K, X 32 is Y, X 35 is A, or or 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 5 (e.g., 1, 2, 3, 4, or 5, or, e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues, and / or, X n at least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5, or, e.g., at least 1 and no more than 3) of the residues not labeled as X are replaced by alternative residues.

[0115] In another embodiment, a * ) X9 is V, X 10 is Q, X 11 is I or norleucine, X 13 is Q, X 14 is F, X 17 is R, X 18 is K, X 24 is H, X 25 is H, X 27 is S, X 28 is F, X 31 is I, X 32 is K, X 35 is I or norleucine.

[0116] In one embodiment, Helix 1 comprises the sequence KEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 and Helix 2 comprises the sequence X 24 X 25 QX 27 X 28 AFX31 X 32 SLX 35 including D, for example, helix 1 has the sequence NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 including L, helix 2 has the sequence NX 24 X 25 QX 27 X 28 AFX 31 X 32 SLX 35 including DD.

[0117] As described above, complete identity with the sequences shown for (i) is not required (however, in one preferred embodiment of the invention, this peptide has the exactly described sequence). X n Among the residues indicated by the label, in certain embodiments, at least 1 and up to 8 of the residues may be replaced with alternative residues. The replaced residues may be within helix 1 or helix 2, or there may be one or more replaced residues within each of helix 1 and helix 2 (for example, there may be a replaced residue in one of them and one or two replaced residues in the other). For example, X n In the residues indicated by the label, 1, 2, 3, 4, 5, 6, 7, or 8 (for example, 1, 2, 3, 4, or 5, or 1, 2, or 3), for example 1, 2, 3, 4, or 5, for example 1 or 2, or 1 replaced residue may be present. X n Since there are 15 residues with the label, a peptide with 5 residues replaced has 67% sequence identity with the described sequence. For 3 residue replacements it is 80%, and for 1 residue replacement it is 93% sequence identity. For 2 residue replacements it is 87%, and for 1 residue replacement it is 93% sequence identity. X nIn embodiments where there are 14 residues with a label, the peptide with 5 residues replaced has 64% sequence identity with the described sequence. For 3 residues replaced, it is 79%, and for 1 residue replaced, it is 93% sequence identity. For 2 residues replaced, it is 86%, and for 1 residue replaced, it is 93% sequence identity.

[0118] X n Among the residues not labeled as residues, at least 1 and up to 5 residues may be replaced with alternative residues. Preferably, at least 1 and up to 3 of the residues may be replaced with alternative residues. The replaced residues may be within either helix 1 or helix 2, or there may be one or more replaced residues within each of helix 1 and helix 2 (e.g., there may be a replaced residue in one of them and one or two replaced residues in the other). For example, X n There may be 1, 2, or 3, particularly 1 or 2, for example, 1 replaced residue among the residues not indicated by the label. X n Since there are 8 residues without a label, the peptide with 3 residues replaced has 63% sequence identity with the described sequence. For 2 residues replaced, it is 75%, and for 1 residue replaced, it is 88% sequence identity.

[0119] In one embodiment, the total number of residues in the replaced helix 1 and helix 2 portions is at least 1 and up to 11 or less (e.g., 10 or less, e.g., 9 or less, e.g., 8 or less, e.g., 7 or less), for example at least 1 and up to 6 or less, for example at least 1 and up to 5 or less, at least 1 and up to 4 or less, for example at least 1 and up to 3 or less, for example at least 1 and up to 2 or less. In particular, there may be a total of 1, 2, 3, 4, 5, or 6 replaced residues in those portions.

[0120] For example, X n the residue shown as or X nAmino acid residue replacements of residues not shown as such can be conservative replacements. That is, a residue is replaced with another residue of the same class, for example, an aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced with another aliphatic residue. A hydroxyl, sulfur or selenium-containing residue (serine (S), cysteine (C), selenocysteine (U), threonine (T) or methionine (M)) may be replaced with another hydroxyl, sulfur or selenium-containing group. An aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)) may be replaced with another aromatic residue. A basic residue (histidine (H), lysine (K), arginine (R)) may be replaced with another basic residue. An acidic residue or amide (aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)) may be replaced with another acidic residue or amide.

[0121] Alternatively, X n a residue shown as such or an amino acid residue replacement of a residue not shown as X n as such may be a non-conservative replacement, that is, the residue is replaced with an alternative residue of a different class, for example, an aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced with an aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)), or for example, it may be replaced with an amino acid having the opposite characteristic, for example, replacement of a lysine (K) residue with an aspartic acid (D) residue.

[0122] In one embodiment, the CD16a binding polypeptide is a *)Helix 1 contains the sequence VQMAQFEIRK (SEQ ID NO: 230), and Helix 2 contains the sequence HHQSFAFIKSLM (SEQ ID NO: 231). For example, Helix 1 contains the sequence NKEVQMAQFEIRKL (SEQ ID NO: 232), and Helix 2 contains the sequence NHHQSFAFIKSLMDD (SEQ ID NO: 233).

[0123] In such embodiments, optionally, at least 1 and up to 5 (e.g., 1, 2, 3, 4, or 5, or e.g., at least 1 and up to 3 (e.g., 1, 2, or 3)) residues in the sequences of Helix 1 and / or Helix 2 are replaced by alternative residues (e.g., alternative residues that are conservative replacements). In a further embodiment, the CD16a-binding polypeptide is b * )Helix 1 contains the sequence QFYARDEIDL (SEQ ID NO: 234), and Helix 2 contains the sequence EDQKWAFYMSLI (SEQ ID NO: 235). For example, Helix 1 contains the sequence NKEQFYARDEIDLL (SEQ ID NO: 236), and Helix 2 contains the sequence NEDQKWAFYMSLIDD (SEQ ID NO: 237).

[0124] In such embodiments, optionally, at least 1 and up to 5 (e.g., 1, 2, 3, 4, or 5, or e.g., at least 1 and up to 3 (e.g., 1, 2, or 3)) residues in the sequences of Helix 1 and / or Helix 2 are replaced by alternative residues (e.g., alternative residues that are conservative replacements). In a further embodiment, the CD16a-binding polypeptide is c * )Helix 1 contains the sequence FWIAESEIES (SEQ ID NO: 238), and Helix 2 contains the sequence IYQKWAFKYSLA (SEQ ID NO: 239). For example, Helix 1 contains the sequence NKEFWIAESEIESL (SEQ ID NO: 240), and Helix 2 contains the sequence NIYQKWAFKYSLADD (SEQ ID NO: 241).

[0125] In such embodiments, optionally, at least 1 and up to 5 (e.g., 1, 2, 3, 4 or 5, or e.g., at least 1 and up to 3 (e.g., 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by alternative residues (e.g., replaced by alternative residues that are conservative substitutions).

[0126] As discussed above, some residues may each be replaced by an alternative residue.

[0127] The CD16a-binding polypeptide of the present invention has the overall structure [N-terminal portion]-[helix 1]-[spacer portion]-[helix 2]-[C-terminal portion].

[0128] The spacer portion can be a sequence of 1 to 5 (e.g., 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.

[0129] In certain embodiments, the spacer portion has the sequence X 20 X 21 X 22 wherein 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 X 22One 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, i.e., the separated portion has the sequence X 20 X 21 X 22 wherein X 20 is any naturally occurring amino acid, and X 21 is any naturally occurring amino acid, and X 22 is any naturally occurring amino acid.

[0130] Preferably, X 20 is S, T, M, P, F, Y or W (e.g., P or T). X 21 is D, E, N or Q, and X 22 is G, A, V, L or I, wherein X 20 , X 21 or X 22 One or two (e.g., one) of them do not exist.

[0131] In a preferred embodiment, X 20 is S, T, M, P, F, Y or W (e.g., P or T), X 21 is D, E, N or Q, and X 22 is G, A, V, L, I. More preferably, X 20 is P or T, X 21 is N, and X 22 is L. For example, the separated portion has the sequence PNL or TNL.

[0132] 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.

[0133] In certain embodiments, the N-terminal portion has the sequence X1X2X3X4X5, where 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.

[0134] Preferably, the N-terminal portion has the sequence X1X2X3X4X5, where X1 is G, A, V, L, I or is absent, X2 is D, E, N, Q or is absent, X3 is D, E, N, Q or is absent, X4 is H, K, R or is absent, and X5 is F, Y, W or is absent. More preferably, X1 is V, G or is absent (e.g., 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.

[0135] In certain embodiments, preferably none of X1, X2, X3, X4, and X5 are present, or X1 is absent and the other residues are absent, or X1 and X2 are absent and the other residues are absent, or X1, X2, and X3 are absent and the other residues are absent, or X1, X2, X3, and X4 are absent and the other residues are absent, or all of X1, X2, X3, X4, and X5 are absent.

[0136] In a particular particularly preferred 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, and X5 is F, or X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent. For example, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, i.e., the N-terminal portion has the sequence VDNKF, GDNKF or is absent, and more preferably has the sequence VDNKF or is absent.

[0137] In one preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, i.e., the N-terminal portion has the sequence VDNKF or GDNKF, and more preferably the N-terminal portion has the sequence VDNKF.

[0138] In one preferred embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, i.e., the N-terminal portion is absent.

[0139] 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, for example, a sequence of 18, 19, 20, 21, or 22 naturally occurring amino acids. Preferably, the C-terminal portion has a sequence that enhances target binding by the CD16a binding motifs of helix 1 and helix 2.

[0140] In certain embodiments, the C-terminal portion is absent or has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 wherein X 38 is a sequence of 1 to 14 naturally occurring amino acids, X 39 is any naturally occurring amino acid, X 56 is either any naturally occurring amino acid or is absent, X 57 is either any naturally occurring amino acid or is absent, X 58 is either 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 amino acid residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative replacements.

[0141] In certain embodiments, X 38 is a sequence of 1 to 9, 1 to 7, or 1 to 5 naturally occurring amino acids. More preferably, X 38 is a sequence of 1 to 4, for example, 1, 2, 3, or 4 amino acids. In a particularly preferred embodiment, X 38is any naturally occurring amino acid (i.e., any single (one) naturally occurring amino acid residue).

[0142] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 wherein 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., by alternative residues that are conservative replacements. In another preferred embodiment, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 wherein 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 amino acid residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0143] In a particularly preferred embodiment, the C-terminal portion has X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 wherein X 38 is P, X 39 is S, X 56is A or does not exist, X 57 is P or does not exist, X 58 is K or does not exist, 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. In another particularly preferred embodiment, the C-terminal portion is PSQSANLLAEAKKLNDAQX 56 X 57 X 58 has, wherein X 56 is A or does not exist, X 57 is P or does not exist, X 58 is K or does not exist, 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.

[0144] For example, in a particular embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 has, wherein X 38 is P, X 39 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 58does 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., alternative residues that are conservative replacements. Alternatively, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 having, wherein 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., alternative residues that are conservative replacements.

[0145] In one preferred embodiment, the C-terminal portion is the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 having, wherein X 38 is P, X 39 is S, X 56 is A, X 57 is P, X 58is 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., by alternative residues that are conservative replacements. Alternatively, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 wherein 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., by alternative residues that are conservative replacements.

[0146] In another preferred alternative embodiment, the C-terminal portion is the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 wherein X 38 is P, X 39 is S, X 56 is absent, X 57 is absent, 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. Alternatively, for example, the C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 wherein X 56 is absent, X 57 is absent, X 58does 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., replaced by alternative residues that are conservative replacements.

[0147] In one 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, and X5 is F, or X1 does not exist, X2 is D, X3 is N, X4 is K, and X5 is F, or X1 does not exist, X2 does not exist, X3 is N, X4 is K, and X5 is F, or X1 does not exist, X2 does not exist, X3 does not exist, X4 is K, and X5 is F, or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F, or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 does not exist), and 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 does not exist, 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 QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements, and more preferably, X 38 is P, X 39 is S, X 56is 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 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, or X1 is absent, X2 is D, X3 is N, X4 is K, X5 is F, or X1 is absent, X2 is absent, X3 is N, X4 is K, X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is K, X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is F, or 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 PSQSANLLAEAKKLNDAQX 56 X 57 X 58 having the formula, where X 56 is G, A, V, L, I, or 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 58is 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.

[0149] In one 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).

[0150] In a highly preferred alternative embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, X5 is absent, 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).

[0151] In another preferred embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is G, A, V, L, or I, X2 is D, E, N, or Q, X3 is D, E, N, or Q, X4 is H, K, or R, and X5 is F, Y, or W (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F), and the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 and wherein X 38 is P, and X 39 is S, T, M, P, F, Y, or W (preferably S), or 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 X56 is G, A, V, L or I (preferably, A), and X 57 is P, and X 58 is absent or 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 QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., by alternative residues that are conservative replacements.

[0152] In a preferred embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is G, A, V, L, or I; X2 is D, E, N or Q; X3 is D, E, N or Q; X4 is H, K, or R; and X5 is F, Y, or W (e.g., X1 is V or G (preferably, V), X2 is D, X3 is N, X4 is K, and X5 is F), and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 having, wherein 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 G, A, V, L or I (preferably, A), and X 57 is P, and X 58 is absent or X56 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 QSANLLAEAKKLNDAQ are replaced by alternative residues, e.g., replaced by alternative residues that are conservative replacements.

[0153] In one 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.

[0154] In a highly preferred alternative embodiment, X1 is V or G, 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).

[0155] In certain embodiments, (i) the isolated moiety has the sequence X 20 X 21 X 22 and / or the N-terminal moiety has the sequence X1X2X3X4X5, and / or the C-terminal moiety has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X58 having, in the separating part, X 20 is P or T, and X 21 is N, and X 22 is L, in the N-terminal part, X1 is V or G (preferably V), or absent, X2 is D or absent, X3 is N or absent, X4 is K or absent, X5 is F or absent, in the C-terminal part, X 56 is A or absent, and X 57 is P or absent, and X 58 is K or absent.

[0156] Alternatively, (ii) the separating part, the N-terminal part, and the C-terminal part are as defined in (i) above, and optionally, (a) within each part, 1, 2, or 3 residues are replaced by alternative residues, or (b) within those parts taken together, at least 1 and up to 10 (e.g., up to 5, e.g., 1, 2, 3, 4, or 5) residues are replaced by alternative residues.

[0157] In such embodiments, preferably, the separating part has the sequence PNL or TNL. Alternatively or additionally, in such embodiments, the N-terminal part has the sequence VDNKF.

[0158] In one embodiment, the N-terminal part may comprise the sequence X1X2X3X4X5, wherein X1 is V or absent, X2 is D or absent, X3 is N or absent, X4 is K or absent, X5 is F or absent.

[0159] For example, the N-terminal part may comprise the sequence VDNKF.

[0160] The separating part includes the array X 20 X 21 X 22 may be included, where in the formula, X 20 is P or T, X 21 is N, X 22 is L.

[0161] For example, the separating part may include the array PNL.

[0162] The C-terminal part may include the array PSQSANLLAEAKKLNDAQX 56 X 57 X 58 where in the formula, X 56 is A or does not exist, X 57 is P or does not exist, X 58 is K or does not exist.

[0163] For example, the C-terminal part may include the array PSQSANLLAEAKKLNDAQAPK or PSQSANLLAEAKKLNDAQ. The C-terminal part may have the structure [second separating part]-[helix 3]-[C-terminal sequence]. For example, - The second separating part includes the array PS, - The helix 3 part includes the array QSANLLAEAKKLNDAQ, - The C-terminal sequence includes the array APK or does not exist.

[0164] In such embodiments, within those five parts taken together (N-terminal part, separating part, second separating part, helix 3, and C-terminal sequence), at least 1 and 8 or fewer (e.g., 5) residues may be replaced by alternative residues. For example, the number of replaced residues may be at least 1 and 7 or fewer, at least 1 and 6 or fewer, at least 1 and 5 or fewer, at least 1 and 4 or fewer, e.g., at least 1 and 3 or fewer, e.g., at least 1 and 2 or fewer. In particular, there may be a total of 1, 2, 3, 4, or 5 replaced residues in those parts.

[0165] Thus, in such embodiments, across the entire CD16a-binding polypeptide of the 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 11 residues may be replaced by alternative residues. For example, the number of replaced residues may be at least 1 and up to 10, such as at least 1 and up to 9, such as at least 1 and up to 8, such as at least 1 and up to 7, such as at least 1 and up to 6. In particular, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 replacement residues may be present across the entire structure [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]. In an advantageous embodiment of the CD16a-binding polypeptide of the invention, the C-terminal portion is a helical or substantially helical region (e.g., Helix 3 described immediately above, e.g., the sequence QSANLLAEAKKLNDAQ), and 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, e.g., Helix 3 described immediately above, e.g., the sequence QSANLLAEAKKLNDAQ).

[0166] In one aspect of the invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1-75 shown in Table 23. In such sequences, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1-75. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 1-75.

[0167] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 73 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 73. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 1 to 73.

[0168] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 11 to 73 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 11 to 73. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 11 to 73.

[0169] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 67 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2 or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 11 to 67. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 1 to 67.

[0170] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 19, 33, 17, 29, 53, 16, 25, 15, 51, 36, 49, 55, 43, 24, 56, 12, 28, 21, 59, 52, 32, 18, 27, 35, and 11 shown in the table of FIG. 23. In such sequences, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 19, 33, 17, 29, 53, 16, 25, 15, 51, 36, 49, 55, 43, 24, 56, 12, 28, 21, 59, 52, 32, 18, 27, 35, and 11. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 19, 33, 17, 29, 53, 16, 25, 15, 51, 36, 49, 55, 43, 24, 56, 12, 28, 21, 59, 52, 32, 18, 27, 35, and 11.

[0171] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 34, 45, and 51 shown in Table FIG. 23. In such sequences, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 34, 45, and 51. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 34, 45, and 51.

[0172] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 26, 35, and 47 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 26, 35, and 47. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 26, 35, and 47.

[0173] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 12, 17, 19, 29, 33, 49, 51, and 53 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 12, 17, 19, 29, 33, 49, 51, and 53. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 12, 17, 19, 29, 33, 49, 51, and 53.

[0174] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 17, 19, 29, 33, and 53 shown in Table 23. In such a sequence, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 17, 19, 29, 33, and 53. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 29, 33, and 53.

[0175] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 15, 17, 19, and 51 shown in Table 23. In such sequences, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 15, 17, 19, and 51. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, and 51.

[0176] In one aspect of the present invention, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 15, 17, and 19 shown in Table 23. In such sequences, optionally, 1 to 5 (preferably, optionally 1, 2, or 3) residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements. In one embodiment, the CD16a-binding polypeptide comprises a sequence selected from SEQ ID NOs: 15, 17, and 19. In one embodiment, the CD16a-binding polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 15, 17, and 19.

[0177] In one aspect of the present invention, the CD16a-binding polypeptide has the sequence: a * )VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLA EAKKLNDAQAPK [SEQ ID NO: 1]. In a further aspect of the present invention, the CD16a-binding polypeptide has the sequence: b * )VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLL AEAKKLNDAQAPK [SEQ ID NO: 2]. In a further aspect of the present invention, the CD16a-binding polypeptide has the sequence: c *)It contains VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLA EAKKLNDAQAPK [SEQ ID NO: 3].

[0178] As discussed above, some residues in the polypeptide of the present invention may be replaced by alternative residues. For example, a * ) immediately above, b * ) or c * ) In the polypeptides shown, at least 1 and up to 5 of the residues may be replaced by alternative residues. For example, the number of replaced residues 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 those portions. Since the polypeptides of this aspect of the present invention have 58 residues, a peptide with 5 residues replaced has 91% sequence identity (91.4%) with the described 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%).

[0179] As described above, in one embodiment, a * ) helix 1 contains the sequence VQMAQFEIRK and helix 2 contains the sequence HHQSFAFIKSLM. In that embodiment, as in other embodiments, as described herein, some residues may each be replaced by alternative residues. In any such alternative polypeptide of the present invention where the alternative residues are in place, binding to the CD16a receptor is maintained. For example, the CD16a binding potency is at least 1% of the binding potency of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions.

[0180] At 1% binding potency, the IC of the alternative polypeptide for binding to the CD16a receptor 50The concentration, when measured under the same conditions, is understood to be 100 times or less the IC of the peptide of SEQ ID NO:1 to the CD16a receptor. 50 It is understood to be 100 times or less the IC of the peptide of SEQ ID NO:1 to the CD16a receptor.

[0181] More preferably, the binding potency, when measured under the same conditions, is at least 5%, 10%, 20%, 25% or 50% of the binding potency of the peptide of SEQ ID NO:1 to the CD16a receptor. That is, the IC of the alternative polypeptide for binding to the CD16a receptor 50 The concentration, when measured under the same conditions, is the IC of the peptide of SEQ ID NO:1 to the CD16a receptor 50 It is 20 times, 10 times, 5 times, 4 times or 2 times or less the IC of the peptide of SEQ ID NO:1 to the CD16a receptor.

[0182] Also, as described above, in one embodiment, b * ) Helix 1 contains the sequence QFYARDEIDL and helix 2 contains the sequence EDQKWAFYMSLI. In that 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 the CD16a receptor is maintained. For example, the CD16a binding potency, when measured under the same conditions, is at least 1% of the binding potency of the peptide of SEQ ID NO:74 to the CD16a receptor.

[0183] At a binding potency of 1%, the IC of the alternative polypeptide for binding to the CD16a receptor 50 The concentration, when measured under the same conditions, is the IC of the peptide of SEQ ID NO:74 to the CD16a receptor 50 It is understood to be 100 times or less the IC of the peptide of SEQ ID NO:74 to the CD16a receptor.

[0184] More preferably, the binding potency, when measured under the same conditions, is at least 5%, 10%, 20%, 25% or 50% of the binding potency of the peptide of SEQ ID NO:74 to the CD16a receptor. That is, the IC of the alternative polypeptide for binding to the CD16a receptor 50 The concentration, when measured under the same conditions, is the IC of the peptide of SEQ ID NO:74 to the CD16a receptor50 It is 20 times, 10 times, 5 times, 4 times or 2 times or less of the concentration.

[0185] Also, as described above, in one embodiment, c * ) Helix 1 contains the sequence FWIAESEIES, and helix 2 contains the sequence IYQKWAFKYSLA. In that embodiment, as in other embodiments and as described herein, some residues may each be substituted by alternative residues. In any such alternative polypeptide of the invention where the alternative residues are in place, binding to the CD16a receptor is maintained. For example, the CD16a binding potency is at least 1% of the binding potency of the peptide of SEQ ID NO: 75 to the CD16a receptor when measured under the same conditions.

[0186] At 1% binding potency, the IC of the alternative polypeptide for binding to the CD16a receptor 50 concentration, when measured under the same conditions, is 100 times or less of the IC of the peptide of SEQ ID NO: 75 to the CD16a receptor 50 concentration. It is understood that it is 100 times or less.

[0187] More preferably, the binding potency is at least 5%, 10%, 20%, 25% or 50% of the binding potency of the peptide of SEQ ID NO: 75 to the CD16a receptor when measured under the same conditions. That is, the IC of the alternative polypeptide for binding to the CD16a receptor 50 concentration, when measured under the same conditions, is 20 times, 10 times, 5 times, 4 times or 2 times or less of the IC of the peptide of SEQ ID NO: 75 to the CD16a receptor 50 concentration.

[0188] The present invention further provides a CD16a-binding polypeptide, which consists of one motif that binds to CD16a, and the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], and The CD16a-binding motif is the [Helix 1]-[Spacer]-[Helix 2] portion (and optionally, the peptide further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions described below, e.g., 1, 2 or 3 additional functional portions), and provides a CD16a-binding polypeptide.

[0189] The present invention further provides a CD16a-binding polypeptide of the present invention, which consists of the CD16a-binding polypeptide (and optionally further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions described below, e.g., 1, 2 or 3 additional functional portions).

[0190] In a highly preferred embodiment, the CD16a-binding polypeptide (e.g., the CD16a-binding polypeptide of the present invention described above or below) consists of one motif that binds to CD16a, and the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer]-[Helix 2]-[C-terminal portion], The CD16a-binding motif is the [Helix 1]-[Spacer]-[Helix 2] portion (and optionally, the peptide further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions described below, e.g., 1, 2 or 3 additional functional portions), and provides a CD16a-binding polypeptide.

[0191] In a highly preferred embodiment, the CD16a-binding polypeptide consists of the CD16a-binding polypeptide of the present invention (and optionally further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions described below, e.g., 1, 2 or 3 additional functional portions), and provides a CD16a-binding polypeptide.

[0192] To avoid misunderstanding, in embodiments where the CD16a-binding polypeptide consists of a CD16a-binding motif and / or a CD16a-binding polypeptide, the CD16a-binding polypeptide is not connected to a further CD16a-binding polypeptide, i.e., the CD16a-binding polypeptide is not part of the CD16a-binding oligomers of the invention.

[0193] In another highly preferred embodiment, the CD16a-binding polypeptide (e.g., the CD16a-binding polypeptide of the invention as described above or below) consists of one motif that binds to CD16a, and the polypeptide has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], The CD16a-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion, and optionally, the polypeptide further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions as described below, e.g., 1, 2 or 3 additional functional portions), providing a CD16a-binding polypeptide. In another highly preferred embodiment, the CD16a-binding consists of the CD16a-binding polypeptide of the invention and further comprises one or more (e.g., 1, 2, 3, 4, 5, 6 or more) additional functional portions as described below, e.g., 1, 2 or 3 additional functional portions (preferably 1 or 2 additional functional portions).

[0194] Multimeric CD16a-binding polypeptide: CD16a-binding oligomer The present invention further provides a CD16a-binding oligomer comprising at least two (i.e., two or more, for example, two, three, four, five, six or more, preferably two, three or four)) CD16a-binding polypeptides of the present invention. In a preferred embodiment, the CD16a-binding oligomer of the present invention comprises two CD16a-binding polypeptides of the present invention. In another embodiment, the CD16a-binding oligomer of the present invention comprises at least three, at least four, at least five or at least six CD16a-binding polypeptides of the present invention, for example, three, four, five or six or more CD16a-binding polypeptides of the present invention.

[0195] The CD16a-binding polypeptides of the CD16a-binding oligomer of the present invention may optionally be connected via one or more linkers. The CD16a-binding oligomer of the present invention has multiple CD16a-binding motifs, that is, each CD16a-binding polypeptide it contains has a CD16a-binding motif ([helix 1]-[separating part]-[helix 2]), so it is a multimeric binder.

[0196] This aspect of the present invention can also be defined such that the CD16a-binding polypeptide comprises two or more CD16a-binding moieties or CD16a-binding peptides optionally connected via one or more linkers, for example, two, three, four, five, six or more CD16a-binding moieties or CD16a-binding peptides optionally connected via one or more linkers, for example, two, three or four CD16a-binding moieties optionally connected via one or more linkers. This definition is used to refer to the aspect of the CD16a-binding oligomer of the present invention in the following numbered embodiments of the present invention.

[0197] The CD16a-binding oligomer of the present invention comprises at least a first CD16a-binding polypeptide which is a CD16a-binding polypeptide of the present invention and a second CD16a-binding polypeptide which is a CD16a-binding polypeptide of the present invention. The first and second CD16a-binding polypeptides may have the same sequence. Alternatively, the first and second CD16a-binding polypeptides may have different sequences. The CD16a-binding oligomer of the present invention may optionally further comprise a third CD16a-binding polypeptide which is a CD16a-binding polypeptide of the present invention. The third CD16a-binding polypeptide may have the same sequence as the first and / or second CD16a-binding polypeptide sequences. Alternatively, the third CD16a-binding polypeptide may have a sequence different from the first and second CD16a-binding polypeptides. The CD16a-binding oligomer of the present invention may optionally further comprise a fourth CD16a-binding polypeptide which is a CD16a-binding polypeptide of the present invention. The fourth CD16a-binding polypeptide may have the same sequence as the first and / or second and / or third CD16a-binding polypeptide sequences. Alternatively, the fourth CD16a-binding polypeptide may have a sequence different from the first, second and third CD16a-binding polypeptides.

[0198] In a preferred embodiment, the CD16a-binding oligomer of the present invention comprises a first CD16a-binding polypeptide comprising a first binding motif selected from SEQ ID NOs: 150 to 221 (and optionally 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably conservative replacements) and a second CD16a-binding polypeptide comprising a second binding motif selected from SEQ ID NOs: 150 to 221 (and optionally 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably conservative replacements). The first and second CD16a-binding motifs may have the same or different sequences.

[0199] In a preferred embodiment, the CD16a-binding oligomer of the present invention comprises a first CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative substitutions), and a second CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative substitutions). The first and second CD16a-binding polypeptides may have the same or different sequences.

[0200] In another embodiment, the CD16a-binding oligomer of the present invention comprises a first CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative replacements), a second CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative replacements), and a third CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative replacements). The first, second and third CD16a-binding polypeptides may have the same sequence, or each may have a different sequence, or two of the sequences may be the same and one may be different). Optionally, a fourth CD16a-binding polypeptide having a sequence selected from SEQ ID NOs: 1 to 75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably residues that are conservative replacements) may be present, which may be the same as or different from any one of the first, second or third sequences.

[0201] In a preferred embodiment, the CD16a-binding oligomer of the present invention comprises a first CD16a-binding polypeptide having a sequence selected from SEQ ID NO: 1 and 74-75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably conservative replacements), and a second CD16a-binding polypeptide having a sequence selected from SEQ ID NO: 1 and 74-75 (and optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence may be replaced by alternative residues and preferably conservative replacements). The first and second CD16a-binding polypeptides may have the same or different sequences (for example, the first CD16a-binding polypeptide may have SEQ ID NO: 1, the second CD16a-binding polypeptide may have SEQ ID NO: 1, or the first CD16a-binding polypeptide may have SEQ ID NO: 74, the second CD16a-binding polypeptide may have SEQ ID NO: 1, the first CD16a-binding polypeptide may have SEQ ID NO: 75, the second CD16a-binding polypeptide may have SEQ ID NO: 1).

[0202] The CD16a-binding polypeptides in the CD16a-binding oligomers of the present invention may be separated by linkers. For example, each CD16a-binding polypeptide in the CD16a-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, or a cleavable amino acid linker or a non-amino acid linker. When the CD16a-binding oligomers of the present invention contain two or more linkers, the linkers may be the same or different. Preferably, the linker for the CD16a-binding oligomers of the present invention is selected from the group consisting of, for example, G, S, and T (preferably G and S), and contains or has 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, and preferably 1 to 20 (for example, 1, 5, 10, 15, or 20, and more preferably 1 to 15) naturally occurring amino acids in sequence.

[0203] In a preferred embodiment, the linker for the CD16a-binding oligomers of the present invention is G or contains or has the sequences GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. For example, the linker is G or contains 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.

[0204] In a preferred embodiment, the linker for the CD16a-binding oligomer of the present invention is G, or comprises or has the sequence GGGSG. For example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the CD16a-binding oligomer of the present invention comprises or has the sequence GGGGS. For example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0205] Alternatively, the CD16a-binding polypeptides in the CD16a-binding oligomer of the present invention may not be separated by a linker (i.e., they may be directly attached to each other).

[0206] In a preferred embodiment, the CD16a-binding oligomer does not contain a linker.

[0207] In one embodiment, the CD16a-binding oligomer of the present invention comprises at least two (e.g., two) CD16a-binding polypeptides, and the CD16a-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 in the oligomer may have the same sequence or different sequences, each C-terminal portion in the oligomer may have the same sequence or different sequences, each spacer portion in the oligomer may have the same sequence or different sequences, each helix 1 portion in the oligomer may have the same sequence or different sequences, and each helix 2 portion in the oligomer may have the same sequence or different sequences.

[0208] In such an embodiment, the linker preferably comprises or has from 1 to 25 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids selected from the group consisting of, for example, G, S and T (preferably G and S), and preferably from 1 to 20 (such as 1, 5, 10, 15 or 20, and more preferably from 1 to 15) sequences of naturally occurring amino acids. For example, the linker may be G or may comprise or have the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker may be G or may comprise or have the sequences GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG or SGGGGSGGGGSGGGGSGGGG.

[0209] Alternatively, the linker may be absent, i.e., the CD16a-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].

[0210] In one embodiment, the CD16a-binding oligomer of the present invention comprises at least three (such as 3) CD16a-binding polypeptides, and the CD16a-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], comprising Each linker portion in the oligomer may have the same sequence or different sequences, each N-terminal portion in 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 in 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 in 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, and each Helix 2 portion in 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.

[0211] In such embodiments, the linker preferably comprises or has from 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 selected from the group consisting of, for example, G, S and T (preferably G and S), and preferably from 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably from 1 to 15) sequences of naturally occurring amino acids. For example, the linker is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG, and, 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.

[0212] Alternatively, one or both linkers may be absent, i.e., the CD16a-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 It includes [N-terminal portion]-[Helix 1]-[Separation portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Separation portion]-[Helix 2]-[C-terminal portion]-[N-terminal portion]-[Helix 1]-[Separation portion]-[Helix 2]-[C-terminal portion].

[0213] In one embodiment, the CD16a-binding oligomer has the sequence VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAF IKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 242], and 1, 2, or 3 residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements.

[0214] In another embodiment, the CD16a-binding oligomer has the sequence VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 243], and optionally, 1, 2, or 3 residues in the sequence are replaced by alternative residues, preferably residues that are conservative replacements.

[0215] In another embodiment, the CD16a-binding oligomer has the sequence VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 244], and 1, 2, or 3 residue moieties in the sequence are replaced by alternative residues, preferably residues that are conservative replacements.

[0216] In a further embodiment, the CD16a-binding oligomer of the present invention may comprise two CD16a-binding polypeptides, each having a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, each of the two CD16-α-binding polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0217] In an alternative, the CD16a-binding oligomer of the present invention may comprise two CD16a-binding polypeptides, one having a sequence in which helix 1 comprises the sequence QFYARDEIDL and helix 2 comprises the sequence EDQKWAFYMSLI, and the other having a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, one of the two CD16a-binding polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 2], and the other may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0218] In the alternative example, the CD16a-binding oligomer of the present invention may comprise two CD16a-binding polypeptides, one of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM, and the other of which has a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, one of the two polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1], and the other may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:75].

[0219] For example, such a C16a-binding oligomer may comprise three CD16a-binding polypeptides, each of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, each of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1].

[0220] In an alternative example, the CD16a-binding oligomer of the present invention may comprise three CD16a-binding polypeptides, two of which have a sequence in which helix 1 comprises the sequence QFYARDEIDL and helix 2 comprises the sequence EDQKWAFYMSLI, and the other one has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, two of the three polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 74], and the other one may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0221] In an alternative example, the CD16a-binding oligomer of the present invention may comprise three CD16a-binding polypeptides, one of which has a sequence in which helix 1 comprises the sequence QFYARDEIDL and helix 2 comprises the sequence EDQKWAFYMSLI, and the other two have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, one of the three polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 74], and the other two may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0222] In an alternative example, the CD16a-binding oligomer of the present invention may comprise three CD16a-binding polypeptides, one of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM, and the other two have a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, one of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1], and the other two may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:75].

[0223] In an alternative example, the CD16a-binding oligomer of the present invention may comprise three CD16a-binding polypeptides, two of which have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM, and the other one has a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, two of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1], and the other one may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:75].

[0224] For example, such a CD16a-binding oligomer of the present invention may comprise four CD16a-binding polypeptides, each of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, each of the four polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1].

[0225] In an alternative example, the CD16a-binding oligomer of the present invention may comprise four CD16a-binding polypeptides, two of which have a sequence in which helix 1 comprises the sequence QFYARDEIDL and helix 2 comprises the sequence EDQKWAFYMSLI, and the other two have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, two of the four polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:74], and the other two may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1].

[0226] In an alternative example, the CD16a-binding oligomer of the present invention may comprise four CD16a-binding polypeptides, two of which have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM, and the other two have a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As shown elsewhere, such sequences may have some residues substituted by alternative residues. For example, two of the four polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:1], and the other two may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO:75].

[0227] In the CD16a-binding oligomer of the present invention, which may also be referred to as a multimeric binder, two or more CD16a-binding moieties are optionally connected via a linker as defined herein. When two or more linkers are present in the CD16a-binding oligomer of the present invention, the linkers may be the same or different. For example, the linker may be an amino acid sequence, and they may have the same or different amino acid sequences.

[0228] Linker When present, the linker connects together two or more functional portions of the polypeptides of the invention (further defined hereinbelow). For example, the linker may connect together two CD16a-binding polypeptides of the invention (e.g., in the CD16a-binding oligomers of the invention), or the linker may connect together a CD16a-binding polypeptide of the invention and an additional functional portion of the invention. The linker may also, in embodiments where two or more additional functional portions are present, connect together additional functional portions of the invention. Those skilled in the art are 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. Examples of linker types include, for example, flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers, and non-amino acid linkers may also be used. For example, the linker may be selected to increase stability or improve folding, to increase expression, to improve biological activity, to enable targeting, or to alter pharmacokinetics. Non-amino acid linkers may be referred to as synthetic linkers.

[0229] To avoid misunderstanding, in certain embodiments, the linker may not connect together two CD16a-binding polypeptides of the invention (e.g., in the CD16a-binding oligomers of the invention), and / or the linker may not connect together a CD16a-binding polypeptide of the invention and an additional functional portion of the invention, and / or the linker may not connect together additional functional portions of the invention in embodiments where two or more additional functional portions are present. For example, in certain embodiments, two CD16a-binding polypeptides of the invention (e.g., in the CD16a-binding oligomers of the invention) may be directly connected to an additional functional portion of the invention, and / or a CD16a-binding polypeptide or oligomer of the invention may be directly connected to an additional functional portion of the invention, and / or an additional functional portion of the invention may be directly connected to an additional functional portion of the invention.

[0230] In one embodiment, a CD16a-binding polypeptide according to any aspect disclosed herein, or a CD16a-binding oligomer according to any aspect disclosed herein, further comprises at least one linker selected from at least one of a flexible amino acid linker, a rigid amino acid linker, and a cleavable amino acid linker. In one embodiment, the linker is, for example, between two or more CD16a-binding polypeptides in the CD16a-binding oligomer of the invention, or between a CD16a-binding polypeptide or CD16a-binding oligomer and an additional functional moiety, such as an immune signaling molecule or an additional binding moiety (e.g., described in more detail below).

[0231] In one embodiment, a CD16a-binding oligomer according to any aspect disclosed herein comprises at least one linker selected from at least one of a flexible amino acid linker, a rigid amino acid linker, and a cleavable amino acid linker. The linker is between two or more CD16a-binding polypeptides. A further linker between a CD16a-binding polypeptide or CD16a-binding oligomer and an additional functional moiety, such as an immune signaling molecule or an additional binding moiety (e.g., described in more detail below) may also be present in a CD16a-binding oligomer according to any aspect disclosed herein. A further linker between an additional functional moiety (e.g., an immune signaling molecule or an additional binding moiety (e.g., described in more detail below)) and an additional functional moiety (e.g., an immune signaling molecule or an additional binding moiety (e.g., described in more detail below)) may also be present in a CD16a-binding oligomer according to any aspect disclosed herein.

[0232] In embodiments and aspects of the invention that include two or more linkers, 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)).

[0233] Flexible linkers can 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, such as, for example, (GGGGS)p or (GGGSG)p. Other examples include (GGSGG)p, (GSGGG)p or (SGGGG)p. Adjusting the copy number "p" allows optimization of the linker to achieve an appropriate separation between functional moieties or to maintain the necessary intermolecular interactions. In a preferred embodiment, the linker is (GGGSG)p, for example, (GGGSG)1, (GGGSG)2 or (GGGSG)3, for example, (GGGSG)3. In one embodiment, the linker is (GGGGS)p, for example, (GGGGS)1, (GGGGS)2 or (GGGGS)3, for example, (GGGGS)3. In one embodiment, the linker is (GGSGG)p, for example, (GGSGG)1, (GGSGG)2 or (GGSGG)3, for example, (GGSGG)3. In one embodiment, the linker is (GSGGG)p, for example, (GSGGG)1, (GSGGG)2 or (GSGGG)3, for example, (GSGGG)3. In one embodiment, the linker is (SGGGG)p, for example, (SGGGG)1, (SGGGG)2 or (SGGGG)3, for example, (SGGGG)3. In another embodiment, the linker is G.

[0234] In one embodiment, the linker of the present invention comprises or has a sequence of 1 to 50 (e.g., 1 to 40, 1 to 30, or 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 selected from the group consisting of, for example, G, S and T, preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably 1 to 15) naturally occurring amino acids. In one embodiment, the linker of the present invention comprises or has a sequence of 1 to 50 (e.g., 1 to 25, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, or 1 to 20, e.g., 1, 5, 10, 15 or 20, or 1 to 15, e.g., 1, 5, 10 or 15) naturally occurring amino acids selected from the group consisting of, for example, G and S. For example, the linker of the present invention is G or comprises or has the sequence GGGSG, e.g., the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG.

[0235] Also, for example, the linker 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.

[0236] 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 CD16a-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.

[0237] Apart from G and S linkers, other flexible linkers are known in the art, such as additional amino acid residues like 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.

[0238] Other types of linkers, such as rigid 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 2012.09.039).

[0239] Alternatively, different binding portions of the binding polypeptides described herein (e.g., two or more CD16a binding polypeptides, or a CD16a binding polypeptide and an additional functional portion, e.g., an immune signaling molecule or an additional binding portion) can be covalently linked by a chemical linker. Such chemical linkers can be generated, for example, by maleimide or "click" chemistry. Those skilled in the art will recognize other linkers suitable for use in the binding polypeptides described herein.

[0240] Regarding the above description of the binding polypeptide comprising a CD16a binding polypeptide according to the present disclosure, it should be noted that the designation of the first portion, the second portion and the further portion is made for reasons of clarity to distinguish, on the one hand, the CD16a binding polypeptide according to the invention and, on the other hand, the binding portion exhibiting other functions. These designations are not intended to refer to the actual order of the different regions of the binding polypeptide. Similarly, the designation of the first and second portions (or monomer units) is made for reasons of clarity to distinguish the units. Thus, for example, the first portion (or monomer unit) can occur at the N-terminus, the middle, or the C-terminus of the binding polypeptide without limitation.

[0241] The CD16a binding polypeptides in the CD16a binding oligomers of the present invention may be separated by a linker. For example, each CD16a binding polypeptide in the CD16a binding oligomers of the present invention may be separated by a linker. Preferably, the linker is a linker as defined herein. If the CD16a binding oligomers of the present invention contain two or more linkers, the linkers may be the same or different.

[0242] Preferably, the linker for the CD16a-binding oligomer of the present invention comprises or has a sequence 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 selected from the group consisting of, for example, G, S and T (preferably G and S), and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably 1 to 15) naturally occurring amino acids.

[0243] In a preferred embodiment, the linker for the CD16a-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 CD16a-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 GGGGSGGGGSGGGGSGGGGSG.

[0244] In embodiments of the present invention that include additional functional moieties, the CD16a-binding polypeptide or the CD16a-binding polypeptide and the additional functional moieties in the CD16a-binding oligomer of the present invention may be separated by a linker. For example, each additional functional moiety and the CD16a-binding polypeptide in the CD16a-binding polypeptide or CD16a-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.

[0245] When the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention contains two or more linkers (i.e., when there are at least two CD16a-binding polypeptides and at least one additional functional moiety, or when there is at least one CD16a-binding polypeptide and at least two additional functional moieties), the linkers may be the same or different. Preferably, the linker for the CD16a-binding polypeptide or CD16a-binding oligomer containing an additional functional moiety is selected from the group consisting of, for example, G, S, and T (preferably G and S), and 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, and preferably contains or has a sequence of 1 to 20 (e.g., 1, 5, 10, 15, or 20, and more preferably 1 to 15) naturally occurring amino acids.

[0246] In a preferred embodiment, the linker for the CD16a-binding polypeptide or CD16a-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 CD16a-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.

[0247] Additional functional moiety The CD16a-binding polypeptides disclosed herein may be linked to one or more additional functional moieties, for example via the linkers described above. The CD16a-binding oligomers disclosed herein may be linked to one or more additional functional moieties, for example via the linkers described above. Thus, in one embodiment, at least one CD16a-binding polypeptide (or CD16a-binding oligomer) is optionally attached to one or more additional functional moieties via a linker described herein.

[0248] To avoid misunderstanding, the CD16a-binding polypeptides or CD16a-binding oligomers of the invention may be directly attached to one or more additional functional moieties (i.e., not attached via a linker).

[0249] As used herein, "functional moiety" refers to a component or "moiety" having a particular desired biological activity.

[0250] One or more additional components (i.e., one or more additional functional moieties) may be, for example, signaling molecules. Examples of suitable signaling molecules include cytokines, such as immune signaling molecules such as IL-15 and its derivatives.

[0251] One or more additional components (i.e., one or more additional functional moieties) can 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 called cancer cell surface antigens). Cell surface tumor antigens can be, for example, tumor-associated antigens or tumor-specific antigens.

[0252] 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), disintegrin and metalloprotease 17 (ADAM17), programmed cell 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), claudin 8.2 (CLDN18.2), delta-like protein 3 (DLL3), mucin 16 (MUC16), mucin 17 (MUC17), mucin 1 (MUC1), trophoblast glycoprotein (TPBG / 5T4 / WAIF1), V-set domain-containing T cell activation inhibitor 1 (B7-H4 / VTCN1 / B7x / B7S1), cluster of differentiation 20 (CD20), B lymphocyte surface antigen B4 (CD19), sialic acid-binding Ig-like lectin 2 (CD22), TNF receptor superfamily member 8 (CD30), natural cytotoxicity triggering receptor 1 (NKp46), or an additional binding moiety that is a binding partner that recognizes NKG2D. Examples of additional components (i.e., additional functional moieties) include an additional binding moiety that is a binding partner that recognizes a cell surface tumor antigen or cancer cell surface target 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. Examples of additional components (i.e., additional functional moieties) include an additional binding moiety that is a binding partner that recognizes an immune cell surface protein or immune cell surface target selected from the group consisting of CTLA-4, PD-1, NKp46, NKG2D, 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. (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 identified herein by way of illustration). The additional binding moieties referred to in this context are not the CD16a binding polypeptides of the present invention.

[0253] In one embodiment, the CD16a binding polypeptide or CD16a binding oligomer of the present invention is optionally attached, via the linker described above, to one or more additional functional moieties (e.g., one or more additional binding moieties and / or signaling molecules). For example, at least one CD16a binding polypeptide or CD16a binding oligomer is attached to one, two, three, four or more additional functional moieties (e.g., one, two, three, four or more additional binding moieties and / or signaling molecules). In certain embodiments, at least one CD16a binding polypeptide or CD16a binding oligomer is attached to one or two additional functional moieties (e.g., one or two additional binding moieties or signaling molecules). In certain embodiments, at least one CD16a binding polypeptide or CD16a binding oligomer is attached to one functional moiety (e.g., one additional binding moiety or signaling molecule).

[0254] In certain preferred embodiments, the additional functional moiety is a signaling molecule. A signaling molecule, such as an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof, may be attached to the N-terminus or C-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above. Alternatively, one or more signaling molecules may be attached between two CD16a-binding polypeptides in a CD16a-binding oligomer, optionally separated by one or more linking sequences as described above. Preferably, the signaling molecule may be attached to the N-terminus or C-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above. Preferably, the signaling molecule may be attached to the N-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above.

[0255] 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 the following: CTLA-4, PD-1, BCMA, ADAM17, 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 a binding partner specific for one of the following: CTLA-4, PD-1, BCMA, ADAM17, 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.

[0256] In certain preferred embodiments, the additional binding moiety is specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen such as BCMA). In certain preferred embodiments, the additional binding moiety is specific for an immune cell surface target (e.g., an NK cell target such as NKp46, NKG2D, PD-1, etc.).

[0257] An additional binding moiety, such as a binding partner that recognizes the cell surface tumor antigen BCMA, may be attached to the N-terminus or C-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above. Alternatively, one or more additional binding moieties may be attached between two CD16a-binding polypeptides in a CD16a-binding oligomer, optionally separated by one or more of the linking sequences described above. Preferably, an additional binding moiety, such as a binding partner that recognizes the cell surface tumor antigen BCMA, may be attached to the N-terminus or C-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above. More preferably, an additional binding moiety, such as a binding partner that recognizes the cell surface tumor antigen BCMA, may be attached to the N-terminus of the CD16a-binding polypeptide or CD16a-binding oligomer, optionally via the linker described above.

[0258] The inventors have surprisingly found that a "dual-engager" polypeptide comprising a CD16a-binding polypeptide or CD16a-binding oligomer disclosed herein can retain its CD16a-binding ability when fused to an additional binding moiety that targets the multiple myeloma antigen BCMA. This "dual-engager" polypeptide comprising a CD16a-binding polypeptide disclosed herein fused to a BCMA-binding moiety can also surprisingly activate NK cells in the presence of BCMA-expressing tumor cells.

[0259] In a preferred embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention further comprises one or more additional functional moieties, for example, at least one, at least two, or at least three, or at least four additional functional moieties. For example, in certain embodiments, the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention further comprises one, two, three, four or five additional functional moieties. In a particularly preferred embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention further comprises one, two or three additional functional moieties, and most preferably, further comprises two additional functional moieties.

[0260] In embodiments where the CD16a-binding polypeptide or CD16a-binding oligomer comprises at least two, at least three, or at least four additional functional moieties (e.g., two, three, four, or five 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 three or at least four additional functional moieties (e.g., three, four, five, six or more additional functional moieties)).

[0261] In embodiments where the CD16a-binding polypeptide or CD16a-binding oligomer comprises at least two, at least three or at least four additional functional moieties (e.g., two, three, four or five 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 three or at least four additional functional moieties (e.g., three, four, five, six or more additional functional moieties)).

[0262] In embodiments where the CD16a-binding polypeptide or CD16a-binding oligomer comprises at least two, at least three, or at least four additional functional moieties (e.g., two, three, four, or five), the first additional functional moiety may comprise an additional binding moiety (e.g., an additional binding specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA, or an immune cell target, e.g., an additional binding specific for an NK cell target), and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, e.g., 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, e.g., BCMA), and the second additional functional moiety may comprise a cytokine, e.g., IL-15 or a derivative thereof. In particular, the first additional functional moiety may comprise an additional binding moiety specific for BCMA, and the second additional functional moiety may comprise a cytokine, e.g., 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), and the second additional functional moiety may comprise a cytokine, e.g., 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, and the second additional functional moiety may comprise a cytokine, e.g., IL-15 or a derivative thereof.

[0263] In a highly preferred embodiment, the first additional functional moiety may include an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a multiple myeloma cell surface antigen, such as an additional binding moiety specific for BCMA, or an immune cell target, such as an additional binding moiety specific for an NK cell target), and the second additional functional moiety may include an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a multiple myeloma cell surface antigen, such as an additional binding moiety specific for BCMA, or an immune cell target, such as an additional binding moiety specific for an NK cell target). For example, the first additional functional moiety may include an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, such as BCMA), and the second additional functional moiety may include an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, such as BCMA). In particular, the first additional functional moiety may include an additional binding moiety specific for BCMA, and the second additional functional moiety may include an additional binding moiety specific for BCMA.

[0264] Alternatively, for example, the first additional functional moiety may include an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, such as BCMA), and the second additional functional moiety may include an additional binding moiety specific for an immune cell surface target (e.g., an NK cell target). In particular, the first additional functional moiety may include an additional binding moiety specific for BCMA, and the second additional functional moiety may include an additional binding moiety specific for an NK cell target.

[0265] In another embodiment, the first additional functional moiety may include an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof, and the second additional functional moiety may include an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0266] When present (e.g., in embodiments where the CD16a-binding polypeptide or CD16a-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, e.g., a myeloma cell surface antigen, e.g., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target), or an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof. Preferably, when present, the third additional functional moiety comprises 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., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target, particularly an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA).

[0267] When present (e.g., in embodiments where the CD16a-binding polypeptide or CD16a-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, e.g., a myeloma cell surface antigen, e.g., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target), or an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof. Preferably, when present, the fourth additional functional moiety comprises 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., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target, particularly an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA).

[0268] If present (e.g., in embodiments where the CD16a-binding polypeptide or CD16a-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., an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target), or an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0269] In a preferred embodiment, the CD16a-binding polypeptide or CD16a-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., an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target), 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., BCMA, or an immune cell target, e.g., an additional binding moiety specific for an NK cell target).

[0270] In one embodiment, the first 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., BCMA), the second additional functional moiety may comprise a cytokine, e.g., IL-15 or a derivative thereof, 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., BCMA) or an immune cell target (e.g., an NK cell target). In particular, the first additional functional moiety may comprise an additional binding moiety specific for BCMA, the second additional functional moiety may comprise a cytokine, e.g., IL-15 or a derivative thereof, 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., BCMA). In another embodiment, the first additional functional moiety may comprise an additional binding moiety specific for BCMA, the second additional functional moiety may comprise a cytokine, e.g., IL-15 or a derivative thereof, and the third additional functional moiety may comprise an additional binding moiety specific for an immune cell target (e.g., an NK cell target).

[0271] For example, the first additional functional moiety may include an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA), the second additional functional moiety may be an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA), and the third additional functional moiety may include an additional binding moiety 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 moiety may include an additional binding moiety specific for BCMA, the second additional functional moiety may include an additional binding moiety specific for BCMA, and the third additional functional moiety may include an additional binding moiety 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 moiety may include an additional binding moiety specific for BCMA, the second additional functional moiety may include an additional binding moiety specific for BCMA, and the third additional functional moiety may include an additional binding moiety specific for an immune cell target (e.g., an NK cell target).

[0272] In one embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer comprising the additional binding moiety of the present invention has an additional binding moiety separated from the CD16a-binding polypeptide or CD16a-binding oligomer by a linker. The linker can be any linker as defined above. For example, the linker is selected from a flexible amino acid linker, a rigid amino acid linker, a cleavable amino acid linker, and a non-amino acid linker.

[0273] Preferably, the linker for the CD16a-binding polypeptide or CD16a-binding oligomer comprising an additional binding moiety comprises or has a sequence 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 selected from the group consisting of, for example, G, S and T (preferably, G and S), and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably 1 to 15) naturally occurring amino acids.

[0274] In a preferred embodiment, the linker for the CD16a-binding polypeptide or CD16a-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.

[0275] In a preferred embodiment, the linker for the CD16a-binding oligomer of the present invention is G, or comprises or has the sequence GGGSG. For example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the CD16a-binding oligomer of the present invention comprises or has the sequence GGGGS. For example, the linker comprises or has the sequence GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

[0276] Alternatively, in one embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer comprising an additional binding moiety of the present invention has an additional binding moiety that is not separated from the CD16a-binding polypeptide or CD16a-binding oligomer by a linker (i.e., the CD16a-binding polypeptide or CD16a-binding oligomer is directly attached to the additional binding moiety).

[0277] In a preferred embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer comprising an additional binding moiety of the present invention does not comprise a linker. In one preferred embodiment, the CD16a-binding polypeptide or CD16a-binding oligomer is directly attached to the additional binding moiety.

[0278] In certain embodiments, the CD16a-binding polypeptide of the present invention comprising an additional functional moiety has the following structure: [N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional moiety], [Additional functional moiety]-[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 moiety]-[Additional functional moiety], [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part], and if there are two or more additional functional parts, each additional functional part may be the same or different.

[0279] In such embodiments, the linker preferably comprises or has a sequence 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 selected from the group consisting of, for example, G, S and T (preferably G and S), and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably 1 to 15) naturally occurring amino acids. 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.

[0280] 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].

[0281] In such embodiments, more preferably, the CD16a-binding polypeptide has the following structure: [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], [Additional functional portion]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion], or [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Linker]-[Additional functional portion] (or, the following structure [Additional functional portion]-[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], or [Additional functional portion]-[N-terminal portion]-[Helix 1]-[Spacer portion]-[Helix 2]-[C-terminal portion]-[Additional functional portion]).

[0282] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA). Most preferably, each additional functional moiety is an additional binding moiety specific for BCMA.

[0283] Even more preferably, the CD16a binding polypeptide has the following structure: [Additional functional moiety]-[Linker]-[N-terminal moiety]-[Helix 1]-[Spacer moiety]-[Helix 2]-[C-terminal moiety], or [Additional functional moiety]-[Additional functional moiety]-[Linker]-[N-terminal moiety]-[Helix 1]-[Spacer moiety]-[Helix 2]-[C-terminal moiety].

[0284] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA). Most preferably, each additional functional moiety is an additional binding moiety specific for BCMA.

[0285] In a very particularly preferred embodiment of the invention, the CD16a binding polypeptide has the following structure: [Additional functional moiety]-[Additional functional moiety]-[Linker]-[N-terminal moiety]-[Helix 1]-[Spacer moiety]-[Helix 2]-[C-terminal moiety].

[0286] In such embodiments, preferably, each additional functional moiety is an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA). Most preferably, each additional functional moiety is an additional binding moiety specific for BCMA. Optionally, in such embodiments, the linker may be absent.

[0287] In another very particularly preferred embodiment of the invention, the CD16a binding polypeptide has the following structure: It includes an [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part].

[0288] In such embodiments, preferably, each additional functional part is an additional binding part specific to a cancer cell surface target (e.g., a myeloma cell surface antigen, e.g., BCMA). Most preferably, each additional functional part is an additional binding part specific to BCMA. Optionally, in such embodiments, the linker may be absent.

[0289] In certain embodiments, the CD16a-binding oligomer of the present invention comprising an additional functional part has the following structure: [N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part], [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], [N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], [N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Additional functional part], [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[-Linker]-[Additional functional part], [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], [N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]. Or [N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part] and consists of Each linker part in the oligomer may have the same sequence or different sequences, and each N-terminal part in the oligomer may have the same sequence or different sequences. Each C-terminal part in the oligomer may have the same sequence or different sequences, each spacer part in the oligomer may have the same sequence or different sequences, each helix 1 part in the oligomer may have the same sequence or different sequences, and each helix 2 part in the oligomer may have the same sequence or different sequences.

[0290] In such embodiments, the linker preferably comprises or has from 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 selected from the group consisting of, for example, G, S and T (preferably G and S), and preferably from 1 to 20 (e.g., 1, 5, 10, 15 or 20, and more preferably from 1 to 15) sequences of naturally occurring amino acids. For example, the linker is G or comprises or has the sequences GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG, and, 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.

[0291] In such embodiments, one or more linkers may not be present.

[0292] In such embodiments, more preferably, the CD16a-binding oligomer has the following structure: [Additional functional moiety]-[Linker]-[N-terminal moiety]-[Helix 1]-[Spacer moiety]-[Helix 2]-[C-terminal moiety]-[Linker]-[N-terminal moiety]-[Helix 1]-[Spacer moiety]-[Helix 2]-[C-terminal moiety], [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[-Linker]-[Additional functional part]. Or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part].

[0293] More preferably, the CD16a-binding oligomer has the following structure: [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part], or [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Spacer part]-[Helix 2]-[C-terminal part].

[0294] In certain embodiments, the CD16a-binding oligomer of the invention comprising an additional functional part comprises at least three CD16a-binding polypeptides and 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], [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 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] comprising, Each linker portion in 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 portion in 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 portion may have the same sequence, may have different sequences, or two may have the same sequence and one may have a different sequence. Each separated portion in 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 portion in 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 2 portion in 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.

[0295] In such embodiments, the linker is preferably as defined above. In such embodiments, one or more linkers may not be present.

[0296] In such embodiments, more preferably, the CD16a-binding oligomer has the following structure: [Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Separated portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 2]-[Separated portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Separated portion]-[Helix 2]-[C-terminal portion], or [Additional functional portion]-[Additional functional portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Separated portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 2]-[Separated portion]-[Helix 2]-[C-terminal portion]-[Linker]-[N-terminal portion]-[Helix 1]-[Separated portion]-[Helix 2]-[C-terminal portion].

[0297] In the above embodiments that define the structure of a CD16a-binding polypeptide or CD16a-binding oligomer comprising additional functional moieties, each additional functional moiety can be any one described herein. For example, each additional functional moiety can independently be an additional binding moiety (e.g., an additional binding moiety 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), and an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0298] For example, each additional functional moiety can independently be an additional binding moiety specific for a cancer cell surface target (e.g., a multiple myeloma cell surface antigen, e.g., BCMA), an additional binding moiety specific for an immune cell target (e.g., an NK cell target), and an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0299] For example, each additional functional moiety can independently be an additional binding moiety specific for BCMA, an additional binding moiety specific for an NK cell target, and an immune signaling molecule, e.g., a cytokine, e.g., IL-15 or a derivative thereof.

[0300] For example, each additional functional moiety can independently be an additional binding moiety specific for BCMA, and an additional binding moiety specific for an NK cell target, and a cytokine, e.g., IL-15 or a derivative thereof.

[0301] For example, each additional functional moiety can independently be an additional binding moiety specific for BCMA, and an additional binding moiety specific for an NK cell target, and It may be selected from IL-15 or its derivatives.

[0302] In one preferred embodiment, each additional functional moiety is independently an additional binding moiety specific for BCMA, and an additional binding moiety specific for an NK cell target, and a cytokine, for example, it may be selected from IL-15 or its derivatives.

[0303] In one preferred embodiment, each additional functional moiety is independently an additional binding moiety specific for BCMA, and may be selected from additional binding moieties specific for an NK cell target.

[0304] In one preferred embodiment, each additional functional moiety is an additional binding moiety specific for BCMA.

[0305] In the above embodiments defining the structure of the CD16a-binding polypeptide or CD16a-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 a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA). For example, each additional functional moiety may be an additional binding moiety for BCMA.

[0306] In a preferred embodiment, the additional binding moiety for BCMA is an hBCMA-binding polypeptide comprising at least 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], and the hBCMA-binding motif is the [Helix 1]-[Spacer portion]-[Helix 2] portion.

[0307] For example, it is an hBCMA-binding polypeptide as defined in the PCT application filed on May 31, 2023, under the name Oncopeptides Innovation 1 AB, which claims priority to UK Patent Application Nos. 2208027.9 and 2214718.5. The content of this PCT application is incorporated herein by reference.

[0308] In particular, the hBCMA-binding polypeptide is i) Helix 1 contains the sequence X9X 10 X 11 ADX 14 EIX 17 X 18 and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 and, independently of each other, a) X9 and X 10 are any naturally occurring amino acids, 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 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 5 (e.g., at least 1 and no more than 3) of the X n residues are replaced by alternative residues and / or at least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues not labeled as X n are replaced by alternative residues.

[0309] For example, the hBCMA-binding polypeptide has at least 1% of the hBCMA-binding potency of the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 226].

[0310] In a highly preferred embodiment, the hBCMA-binding polypeptide is the polypeptide of SEQ ID NO: 226.

[0311] CD16a binder-drug conjugate Alternatively or additionally, a CD16a-binding polypeptide or CD16a-binding oligomer disclosed herein may be attached to a therapeutic agent to form a CD16a binder-drug conjugate. Thus, in one embodiment, at least one CD16a-binding polypeptide or CD16a-binding oligomer is typically covalently attached to one or more therapeutic agents, optionally via a linker as 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).

[0312] To avoid misunderstanding, during the generation of a CD16a binder-drug conjugate according to the present invention, one or more therapeutic agents (e.g., MMAF) are necessarily modified by the reaction between a functional group at the attachment point (e.g., -NH, -OH or -SH (e.g., in Cys)) and any attachment group or linker used. Thus, those skilled in the art will understand that the therapeutic agent in a CD16a binder-drug conjugate (e.g., a CD16a binder-MMAF conjugate) contains such modifications.

[0313] Polypeptide production The polypeptide of the present invention can be produced using methods known in the art. For example, the polypeptide can be prepared by chemical synthesis methods or, for example, by recombinant protein production techniques in bacteria, yeast, insects, fungi, plants or mammalian cells.

[0314] The polypeptide of the present invention can also be fused to different molecules with therapeutic potential (e.g., immunoglobulins with therapeutic potential) via the recombinant or chemical synthesis techniques as described above.

[0315] Regulation of polypeptide properties The pharmacokinetic properties of the polypeptide of the present invention can be adjusted by methods known in the art. For example, the polypeptide can be linked to moieties that extend plasma half-life, such as polyethylene glycol polymers, unstructured polypeptides (e.g., XTEN or PAS), or FcRn-binding ligands, such as serum albumin or the Fc domain of immunoglobulins.

[0316] Formulations The polypeptide according to the present invention (e.g., the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention, or the CD16a-binding agent-drug conjugate) may be present in a formulation, and particularly in a pharmaceutical formulation.

[0317] In certain embodiments, the present invention provides a nucleic acid molecule encoding the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention. The nucleic acid molecule encoding the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention may be used as a medicament, for example, for 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 present invention may be present in a formulation, and particularly in a pharmaceutical formulation. Thus, the present invention further provides a formulation, and particularly a pharmaceutical formulation, of a nucleic acid molecule (e.g., DNA or RNA, and particularly an mRNA molecule) encoding the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention.

[0318] Pharmaceutical formulations include, for example, those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), and intramedullary), or intraperitoneal administration, although the most preferred route may depend, for example, on the condition and disorder of the subject under treatment.

[0319] In one embodiment of the invention, the CD16a-binding polypeptide or CD16a-binding oligomer (particularly a CD16a-binding agent-drug conjugate) or nucleic acid (e.g., a DNA or RNA molecule of the invention, e.g., 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 intramedullary) administration.

[0320] 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 a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. The peptides of the invention may also be presented as a bolus, lozenge, or paste. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical texts, such as Remington’s Pharmaceutical Sciences by E.W. Martin. See also Wang, Y.J. and Hanson, M.A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0321] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain an antioxidant, a buffer, a bacteriostat, and a solute that renders the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include a suspending agent and a thickening agent. Preferably, the formulation may be presented in unit dose or multi-dose containers, for example, sealed ampoules and vials. The formulation may be stored in a freeze-dried (lyophilized) state which requires only the addition of a sterile liquid carrier, for example, physiological saline, a physiologically acceptable solution, or water for injection, immediately prior to use.

[0322] Immediate injection and infusion solutions and suspensions may be prepared from sterile powders, granules or other dry compositions. Exemplary compositions for parenteral administration include, for example, injectable solutions or suspensions containing a suitable non-toxic, parenterally acceptable diluent or solvent, for example, mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agent, and a suspending agent including synthetic monoglycerides or diglycerides, and a fatty acid including oleic acid or Cremaphor.

[0323] Dosing schedule The CD16a-binding polypeptide or CD16a-binding oligomer of the present invention (particularly, a CD16a-binding agent-drug conjugate), and a pharmaceutical formulation comprising such a polypeptide, oligomer or binding agent-drug conjugate, or the nucleic acid molecule of the present invention (for example, a DNA or RNA molecule of the present invention, for example, an mRNA molecule of the present invention), and a pharmaceutical formulation comprising those nucleic acid molecules find use in the treatment and / or prevention of cancer, for example, multiple myeloma.

[0324] The amount of the CD16a-binding polypeptide or CD16a oligomer (in particular, a CD16a-binding agent-drug conjugate), or the diffusible molecule required to achieve a therapeutic effect will vary depending on the particular route of administration and the characteristics of the subject under treatment, such as species, age, weight, gender, medical condition, specific IBD and its severity, as well as other relevant medical and physical factors. One of ordinary skill in the art can readily determine and administer an effective amount of the CD16a-binding polypeptide, CD16a oligomer, CD16a-binding agent-drug conjugate and / or composition containing the same, or the nucleic acid molecule and / or composition containing the same for the treatment and / or prevention of cancer.

[0325] The CD16a-binding polypeptide, CD16a-binding oligomer (in particular, a CD16a-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 and the severity of the cancer being treated.

[0326] The CD16a-binding polypeptide, CD16a-binding oligomer (in particular, a CD16a-binding agent-drug conjugate) or nucleic acid molecule of the present invention, or a pharmaceutical preparation thereof, may be administered, for example, in parenteral or oral dosage forms. Parenteral administration includes intravenous (into a vein, e.g., a central or peripheral vein, as a bolus or infusion), intraarterial (into an artery, e.g., a central or peripheral artery), intraosseous injection (into the bone marrow), intramuscular (into a muscle), intradermal (into the dermis), and subcutaneous (under the skin) administration. In one preferred embodiment, the dosage of the present invention is administered intravenously or intraarterially, and 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 preparations particularly useful in the present invention are those suitable for intravenous administration, more particularly intravenous infusion, or subcutaneous administration.

[0327] The CD16a-binding polypeptide, CD16a-binding oligomer (in particular, CD16a-binding agent-drug conjugate) or nucleic acid molecule of the present invention, and pharmaceutical formulations thereof may be administered as part of a treatment cycle. In the treatment cycle, these polypeptides 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 CD16a-binding polypeptide or CD16a-binding oligomer of the present invention. X may be, for example, 1 to 42 days, for example 2 to 14 days. Alternatively, the CD16a-binding polypeptide or CD16a-binding oligomer may be administered, for example, on days 1 and 2 of the cycle, for example, as divided doses.

[0328] This cycle may be repeated one or more times depending on the category, class, or stage of the cancer being treated. For example, the cycle may be repeated 1 to 100 times, for example 2 to 50 times, for example 8 to 40 times, for example 8 or 16 times. For example, the CD16a-binding polypeptide or CD16a-binding oligomer, or nucleic acid molecule of the present invention may be administered by repeating an 8-day cycle 8 times, followed by repeating a 14-day cycle 16 times, and optionally, followed by further repeating a 28-day cycle. A normal skilled physician or clinician can easily determine the number of cycles of the CD16a-binding polypeptide (e.g., CD16a-binding agent-drug conjugate) necessary to prevent, counteract, or halt the progression of cancer.

[0329] Combined therapy The CD16a-binding polypeptide or CD16a-binding oligomer (in particular, CD16a-binding agent-drug conjugate) or nucleic acid molecule disclosed herein may be used as the sole active ingredient in the present invention, but can also be used 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 or prevention of cancer, or other pharmaceutically active materials. Such agents are known in the art.

[0330] Non-limiting examples of additional therapeutic agents for use in the present invention include proteasome inhibitors (PIs) (e.g., carfilzomib, bortezomib, or ixazomib), immunomodulatory drugs (IMiDs) (e.g., lenalidomide, thalidomide, or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, bendamustine, or melphufen), 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), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), or ADAM17 inhibitors. For example, the additional therapeutic agent can be selected from proteasome inhibitors (e.g., carfilzomib or bortezomib), immunomodulatory drugs (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.

[0331] One or more additional therapeutic agents can be used simultaneously, sequentially, or separately from the administration of the dosage of the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, or nucleic acid molecule of the present invention. The individual components of such a combination can be administered separately at different times during the course of treatment, or can be administered simultaneously in a divided or single combination form.

[0332] 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). Preferably, the one or more therapeutic agents are bortezomib, thalidomide, and dexamethasone.

[0333] 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). Preferably, the one or more therapeutic agents are bortezomib, melphalan, melphlufen, and prednisone.

[0334] 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). Preferably, the PI is bortezomib and the steroid is dexamethasone.

[0335] 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). Preferably, the IMiD is lenalidomide and the steroid is dexamethasone.

[0336] In another embodiment of the present invention, the one or more therapeutic agents may be selected from NK cell-based or T cell-based therapies.

[0337] Alternatively, the CD16a-binding polypeptide or CD16a-binding oligomer (particularly, a CD16a-binding agent-drug conjugate) or nucleic acid molecule according to the present invention may be combined with a stem cell transplantation procedure, such as a therapeutic procedure of autologous stem cell transplantation or allogeneic stem cell transplantation. Thus, in one embodiment of the present invention, the CD16a-binding polypeptide disclosed herein may be combined with an autologous stem cell transplantation procedure. In another embodiment of the present invention, the CD16a-binding polypeptide disclosed herein may be combined with an allogeneic stem cell transplantation procedure.

[0338] Simultaneous, sequential or separate administration of one or more additional therapeutic agents or therapeutic procedures with the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate, or nucleic acid molecule of the present invention further enhances their effectiveness in the treatment and / or prevention of cancer.

[0339] Kit The present invention provides a kit comprising the CD16a-binding polypeptide, CD16a-binding oligomer (particularly, a CD16a-binding agent-drug conjugate) or nucleic acid molecule disclosed herein, and one or more additional therapeutic agents useful for the treatment and / or prevention of cancer.

[0340] Non-limiting examples of additional therapeutic agents for use in the kits of the present invention include proteasome inhibitors (PIs) (e.g., carfilzomib, bortezomib, or ixazomib), immunomodulatory drugs (IMiDs) (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-CD38 agents (e.g., daratumumab or isatuximab), immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, or PD-L1 inhibitors), or ADAM17 inhibitors. One or more additional therapeutic agents can be selected, for example, from proteasome inhibitors (e.g., carfilzomib or bortezomib), immunomodulatory drugs (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.

[0341] In one embodiment of the invention, the kits of the invention find use in the treatment and / or prevention of cancer.

[0342] To avoid misunderstanding, the CD16a-binding polypeptides, CD16a-binding oligomers (in particular, CD16a-binding agent-drug conjugates), or nucleic acid molecules disclosed herein are present in the kits of the invention in a form and in an amount 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 CD16a-binding polypeptide or oligomer (e.g., in the form of a CD16a-binding agent-drug conjugate) or nucleic acid molecule disclosed herein that is suitable for use according to the invention.

[0343] Cancer The CD16a-binding polypeptide or CD16a-binding oligomer (particularly, CD16a-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 CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate or nucleic acid molecule find use in medicine, for example, in the treatment and / or prevention of cancer in a subject. Non-limiting examples of cancer include cancers such as bladder cancer, breast cancer, colorectal cancer, CNS cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, skin cancer, ovarian pancreas cancer, prostate cancer, or thyroid cancer; and blood cancers such as 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 myeloma (e.g., MGUS, plasmacytoma, smoldering myeloma, multiple myeloma, light chain myeloma, or non-secretory myeloma).

[0344] Preferably, the CD16a-binding polypeptide or CD16a-binding oligomer (especially, the CD16a-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 CD16a-binding polypeptide, oligomer, binding agent-drug conjugate or nucleic acid molecule are found to be useful in the treatment and / or prevention of blood cancers such as 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 myeloma (e.g., MGUS, plasmacytoma, smoldering myeloma, multiple myeloma, light chain myeloma, or non-secretory myeloma). Preferably, the CD16a-binding polypeptide or CD16a-binding oligomer of the present invention, and the pharmaceutical preparation or kit of the present invention containing the CD16a-binding polypeptide or CD16a-binding oligomer are found to be useful 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 CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate or nucleic acid molecule of the present invention, and the pharmaceutical preparation or kit of the present invention containing the CD16a-binding polypeptide, CD16a-binding oligomer, CD16a-binding agent-drug conjugate or nucleic acid molecule are found to be useful in the treatment and / or prevention of multiple myeloma.

[0345] In certain embodiments, the CD16a-binding polypeptide or CD16a-binding oligomer (especially, the CD16a-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 CD16a-binding polypeptide, oligomer, binding agent-drug conjugate or nucleic acid molecule are found to be useful as an anti-cancer immunotherapeutic agent; e.g., for use as an anti-cancer immunotherapeutic agent for the treatment of cancer, and especially blood cancers, such as the blood cancers described herein, and especially multiple myeloma.

[0346] 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 utilized in many different variations which are not specifically illustrated herein.

[0347] In the above description, when an integral invention or element having known, obvious or foreseeable equivalents is recited, such equivalents are incorporated herein as if individually set forth. The true scope of the present invention should be construed to include any such equivalents, and reference should be made to the claims to determine such true scope. It will also be understood by the reader that the whole or features of the present invention described as preferred, advantageous, convenient, etc. are optional and do not limit the scope of the independent claims. Further, it should be understood that such optional integers or features may be possible advantages in some embodiments of the present invention, while being undesirable and thus may not exist in other embodiments.

Examples

[0348] The following examples illustrate the present invention.

[0349] Preparation Example 1: Selection of a binder for hCD16a by phage display using a phage library In this example, human hCD16a 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 hCD16a in monoclonal phage-ELISA (enzyme-linked immunosorbent assay), and ELISA-positive clones were DNA sequenced.

[0350] Materials and methods Affibody library. An M13 phage display library of affibody molecules was prepared based on the phagemid vector pAffi-1 (Gronwall et al. (2007) J. Biotechnol. 128:162-183). This phagemid contains the lac promoter and the OmpA signal peptide and an albumin binding domain (ABD WT) It is designed for phage display of the encoded afibody library members as an in-frame fusion to the amber stop codon and the truncated form (residues 249 - 406) of the M13 phage coat protein 3. A synthetic 121-base-long oligonucleotide (5’-GCGCTTTGGCTTGGGTCATCXXXTAAACTXXXYYYGAAGGCXXXXXXTTGXXXXXXGTTCAGGTTCGGCAGXXXXXXGATCTCXXXXXXCGCXXXXXXXXXTTCTTTGTTGAATTTGTTGT-3’) [SEQ ID NO: 222] encoding amino acids 3 - 41 (reverse complementary strand) of the Z domain (Nilsson et al. (1987) J. Protein Eng. 1:107 - 113), in which a mixture of trinucleotide codon building blocks (XXX = an equimolar mixture of codons for all 20 amino acids except Cys and Pro, YYY = 60% Ile and 10% each of His, Tyr, Lys and Asp) was used during synthesis to randomize the wild-type codons at 14 positions in the domain, was used as a template for PCR amplification using primer forward (5’-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3’) [SEQ ID NO: 223] and reverse (5’-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3’) [SEQ ID NO: 224]. 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 Escherichia coli (E. coli) cells (F’, glnV amber suppressor) (Lucigen, USA) (0.1 cm BioRad cuvette).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, followed by pooling (6 electroporations per pool), incubating at 37 °C for 1 hour with shaking, and then titrating the cell pool by spreading dilution series on Amp (ampicillin) plates. Each was transferred to four 5-liter shake 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 cultures were 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. From the post-electroporation titration and OD600 measurement after overnight culture, the library size (diversity) was approximately 3×10. 10 It was calculated that each 3.5 mL aliquot of cells contained a number of cells corresponding to approximately 10.9-fold the library size. The tubes containing the cells were stored at -80 °C until used for phage stock preparation using M13KO7 helper phage.

[0351] Naive phage library stock production. For the production of the afibody display phage stock, 1.7 mL of the library glycerol stock was aliquoted and inoculated into four Erlenmeyer flasks containing 750 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 OD600 = 1. The cells were infected with 120 μl (multiplicity of infection (MOI) 5) of M13KO7 helper phage (New England Biolabs), gently swirled, incubated at 37 °C for 15 min without shaking, and then at 37 °C for 15 min at 70 rpm. The cells of each culture were centrifuged and resuspended in 750 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 h after inoculation. The cultures were incubated at 37 °C for 16 - 18 h with shaking at 200 rpm. 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 polymerase chain reaction screening was used to analyze the percentage of phage particles carrying the phagemid with the afibody insert.

[0352] Phage display selection of hCD16a binding candidates. Biotinylated hCD16a (biotinylated human hCD16a (F158) corresponding to residues 17 - 208 of Uniprot entry P08637, Avitag, His-tag, Acro Biosystems, catalog number CDA-H82E8) was used at concentrations of 80 nM, 40 nM, 20 nM, and 10 nM, respectively, in two separate selection tracks 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 NaH2PO4·H2O, 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, the phage stock in PBS-T was pre-incubated at room temperature (RT) for 30 min with 0.1% (w / v) BSA and beads under constant end-over-end (eoe) rotation to remove phages carrying binders to SA. The amount of phage stock used was 10 11 colony-forming units (cfu) in cycles 1 and 4, and 10 12It was cfu. In rounds 1-2, the biotinylated target protein was immobilized end-over-end (eoe) for 1 hour at RT on beads of 1 mg or 0.5 mg each. The target-binding beads were incubated end-over-end (eoe) for 30 minutes at RT with 1% (w / v) BSA in PBS-T, then washed with PBS-T and subsequently pre-incubated phage stock was added for selection. In rounds 3-4, the pre-incubated phage stock was added to the biotinylated target protein for selection in solution, and then the phage-antigen complex was captured by incubating end-over-end (eoe) for 30 minutes at RT with SA-beads of 0.5 mg or 0.4 mg each. The selection steps were carried out at RT, and the incubation time for selection was 3 hours (round 1) or 2 hours (rounds 2-4), and then for each subsequent cycle, it was washed with PBS-T end-over-end (eoe) for a total of 5 minutes, 10 minutes, 15 minutes or 20 minutes at RT. 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 one of the selection tracks, the antigen-binding phage was eluted by incubating end-over-end (eoe) for 15 minutes at RT with 0.5 M acetic acid, pH 2.8, and subsequently the eluate was transferred to a new pretreated tube and neutralized with an equal volume of 1 M Tris-HCl, pH 8. For the second selection track, the antigen-binding phage was eluted by incubating end-over-end (eoe) for 30 minutes at RT 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, and then the eluate was transferred to a new pretreated tube.

[0353] Phage stock amplification. 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 OD600 = 0.5 - 0.8, and then infecting them (100 mL after cycles 1 - 2, 50 mL after cycle 3) with phage eluate (total volume after cycle 1, half volume after cycles 2 - 3). The infected cultures were gently swirled and incubated at 37 °C for 25 minutes without shaking, followed by shaking at 70 rpm for 15 minutes at 37 °C. The cultures were 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 and collected in TSB+Y, inoculated into 200 mL of TSB+Y containing 100 μg / mL of Carb, and grown at 37 °C with shaking at 150 rpm until OD600 = 0.5 - 0.8. 30 mL (after cycles 1 - 2) or 20 mL (after cycle 3) were super-infected with M13KO7 helper phage (MOI 10), gently swirled, incubated at 37 °C for 25 minutes without shaking, followed by shaking at 70 rpm for 15 minutes at 37 °C. The cultures were centrifuged and resuspended in 150 mL of TSB+Y containing 100 μg / mL of Carb and 1 mM IPTG. 25 μg / mL of Kan was added 2 hours after inoculation. The cultures were incubated at 37 °C for 16 - 18 hours with shaking at 150 rpm. 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 on Carb plates and the percentage of phage particles carrying phagemids with affibody inserts was analyzed using polymerase chain reaction screening.

[0354] Monoclonal phage supernatant preparation. Following cycle 4, bacterial colonies representing two selection tracks equally and generating PCR products of the correct size were grown individually at 30 °C for 16 - 18 h with shaking at 250 rpm in 500 μl of TSB+Y / Carb in a 96-well deep well plate. 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 with shaking at 250 rpm for 2 h, and then super-infected by the addition of M13K07 helper phage (MOI 7) in 100 μl of TSB+Y / Carb per well. The plate was incubated at 37 °C for 30 min without shaking. Finally, 150 μl of TSB+Y / Carb / IPTG / Kan was added per well. The final concentrations were 100 μg / mL Carb, 1 mM IPTG, and 25 μg / mL Kan. The deep well plate was incubated at 37 °C with shaking at 250 rpm for 16 - 18 h. The next day, the phage supernatant was recovered by centrifugation.

[0355] 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 10 μg / mL biotinylated hCD16a, 20 μg / mL human serum albumin (HSA; Sigma product number SRP6182) (for evaluation of the proper display of an expression cassette containing a tripartite fusion protein comprising an affibody, an affibody albumin-binding domain, and a truncated protein 3), 10 μg / mL SA, or 10 μg / mL of an unrelated control protein (human SLAMF7 / CRACC / CD319, Fc Tag, Acro Biosystems, catalog number SL7-H5256, corresponding to residues 23 - 226 of Uniprot entry Q9NQ25) in 100 mM sodium carbonate buffer, pH 9.6 (1 / 4 of the well volume for each well containing the coating protein) at 4 °C with gentle shaking for 16 - 18 h. The coating solution was removed, and the plates were washed twice with PBS-T and blocked with PBS supplemented with 1% (w / v) BSA at RT for 1 h 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 at RT for 1 h 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 the plates were incubated at RT for 30 min with gentle shaking, then 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 min. Absorbance at 450 nm was measured using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).The candidates considered as ELISA positive clones had high HSA signals and relatively high signals for hCD16a compared to SA and unrelated target controls.

[0356] DNA sequence. After ELISA screening, 26 ELISA positive clones were sent for DNA sequencing by Sanger sequencing (PlateSeq, Eurofins Genomics, Ebersberg, Germany).

[0357] Results To identify polypeptides that bind to hCD16a, 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. After the 4th selection cycle, E. coli cells were infected with phage eluates from the acid and trypsin selection tracks to obtain individual colonies on carbenicillin plates. Forty - eight colonies (clones) randomly picked from each track were subjected to binding analysis using a phage - ELISA experiment using microtiter plates coated with four test antigens (HSA, streptavidin, SLAMF7, and hCD16a F158). Some of the analyzed clones showed strong ELISA signals against HSA and hCD16a and low signals against the streptavidin and SLAMF7 controls used, as shown for representative clones in Figure 1b. When such clones were subjected to DNA sequencing, it was shown that three unique clones were enriched during the selection. The amino acid sequences of these three CD16a - binding polypeptides, A10, A11, and H09, are listed in the Sequence Listing as Table 1 and SEQ ID Nos: 1, 74, and 75 below. Table 1 also lists the sequences of certain other peptides closely based on those also disclosed herein, together with their corresponding SEQ ID references. Throughout this Examples section, an Example compound having the number X is a polypeptide having the amino sequence of the same number, i.e., SEQ ID No: X.

[0358]

Table 2-1

[0359]

Table 2-2

[0360] Biological Example 2: Initial binding study using surface plasmon resonance In this example, the CD16a-binding polypeptides corresponding to SEQ ID NOs: 1, 74, and 75 were subcloned, expressed, and purified as His6-affibody-ABD WT fusion proteins [SEQ ID NOs: 76-78], and their binding to CD16a was first analyzed by surface plasmon resonance.

[0361] Materials and methods Subcloning of CD16a-binding polypeptides DNA fragments encoding three CD16a-binding polypeptides [having SEQ ID NOs: 1, 74, and 75] were amplified from the library vector pAffi-1 using specific primers that introduced overhangs complementary to the ends of the linearized expression vector. Using the In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden), monomeric CD16a-binding polypeptide constructs having an N-terminal His6 tag and a C-terminal ABD WT were cloned. The DNA constructs were sequence-verified using Sanger sequencing (Eurofins Genomics). The amino acid sequences of these three CD16a-binding polypeptides are listed in Table 1 of the Sequence Listing as SEQ ID NOs: 76-78.

[0362] Expression and purification Escherichia coli (E. coli) BL21(DE3) cells were transformed with a plasmid containing the DNA construct 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 at a 1:100 ratio into 200 mL of TSB+Y containing 25 μg / mL of Kan and grown at 37°C with shaking at 150 rpm until OD600 = 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 (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 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 number 89966, Thermo Scientific). The supernatant was incubated with the resin at RT for 30 minutes with rotation. Contaminants were removed by washing three times with wash buffer (7 M guanidinium chloride, 46.6 mM NaH2PO4, 3.4 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), and then the hCD16a-binding polypeptide 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 at RT for 10 minutes with end-over-end rotation. After IMAC purification, the protein buffer was exchanged to PBS using a PD-10 desalting column (catalog number 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA). The purity and concentration were measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein.The low molecular weight marker was from Cytiva (product number 17044601). An hCD16a-binding variant containing an N-terminal His6 and a C-terminal ABDWT was expressed as a soluble gene product in Escherichia coli (E. coli) BL21(DE3).

[0363] N-terminal His6 and C-terminal ABD WT Biosensor analysis of hCD16a-binding variants containing Using a Biacore 8K instrument (Cytiva), the real-time interaction between hCD16a-binding variants (SEQ ID NOs: 76 - 78) and human hCD16a was analyzed. Protein ligands hCD16a F158 (catalog number CDA-H5220, Acro Biosystems), hCD16a V158 (catalog number CD8-H52H4, Acro Biosystems), and human serum albumin (HSA) (Sigma part number SRP6182) as a positive control were diluted in 10 mM NaOAc, pH 4.5 and immobilized on a Series S CM5 sensor chip (Cytiva). The sensor chip consists of a flow channel composed of two flow cells arranged in series. For each protein ligand and the corresponding flow channel, the first flow cell was activated and deactivated and used as a reference surface, and the second flow cell was used to immobilize the protein ligand on the carboxymethylated dextran surface by amine coupling using the manufacturer's instructions. After diluting the hCD16a-binding variant in running buffer PBS-T (composition, pH), the binding analysis was performed at 25 °C and a flow rate of 30 μl / min. After each injection, the flow cell was regenerated by injection of 10 mM HCl. Each hCD16a-binding variant was injected in duplicate onto the hCD16a surface at concentrations in the range of 5 nM to 2.56 μM. The dissociation equilibrium constant (KD value) was calculated using a 1:1 Langmuir binding model in BIA evaluation software (Cytiva).

[0364] Results To examine whether three CD16a-binding polypeptide candidate clones [SEQ ID NOs: 1, 74, and 75] identified by phage-ELISA and DNA sequencing showed target binding when expressed as soluble proteins, they were subcloned and expressed in the cytoplasm of Escherichia coli (E. coli) as His6-CD16a-binding polypeptide-ABD WT fusion proteins [SEQ ID NOs: 76-78] and purified. The ABD WT portion can be used, if desired, for affinity chromatography purification and / or immobilization using human serum albumin as a ligand and / or a small serum albumin-binding protein that can be used for immobilization. Analysis of the immobilized metal ion affinity chromatography (IMAC)-purified fusion proteins by SDS-PAGE using a hexahistidyl (His6) gene fusion partner in the protein showed that all three proteins were of high purity and had approximate molecular weights according to their amino acid sequences (Figure 2). Injection of the three fusion proteins at different concentrations (5 nM to 2.56 μM) onto separate sensor chip surfaces containing immobilized hCD16a F158 and hCD16a V158 allotypes showed that all three fusion proteins showed binding to both the hCD16a F158 and hCD16a V158 allotypes (Figure 3).

[0365] By analyzing the equilibrium response for the interaction, the dissociation equilibrium constant (KD) for the interaction could be determined. The fusion protein containing the A10 CD16a-binding polypeptide [SEQ ID NO: 76] was shown to bind to both the F158 allotype and the V158 allotype with similar affinities, and the KDs were approximately 100 nM and 99 nM, respectively. In contrast, the fusion proteins containing either the H09 or A11 CD16a-binding polypeptide [SEQ ID NO: 77 and 78] both showed binding to the F158 allotype with a higher affinity than the V158 allotype. The dissociation equilibrium constants for the interactions were calculated to be 2.8 μM (A11 / V158), 8.3 μM (A11 / F158), 621 nM (H09 / F158), and 4.9 μM (H09 / V158).

[0366] Biological Example 3: Panning study of hCD16a-binding variants using surface plasmon resonance In this example, SPR experiments were performed to investigate whether three identified anti-CD16 affibodies bind to distinct or shared / overlapping epitopes on hCD16a.

[0367] Materials and methods N-terminal His6 and C-terminal ABD WT Panning of hCD16a-binding variants containing the same. The Biacore 3000 instrument (Cytiva) was used for the competitive binding analysis of hCD16a-binding variants to each other. hCD16a F158 and hCD16a V158 were immobilized on separate flow cell carboxymethylated dextran surfaces on a 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. After diluting the hCD16a-binding variants in running buffer PBS-T, 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.

[0368] Results To examine whether the identified CD16a-binding polypeptides recognize distinct or overlapping / shared epitopes on hCD16a, SPR-based binding experiments were performed using sensor chip surfaces containing immobilized hCD16a F158 and hCD16a V158 proteins, respectively. Here, the hCD16a-binding variant used in the format as His6-A10-ABD WT [SEQ ID NO:76] was injected at 2 μM onto the hCD16a surface, followed by His6-A10-ABD WT [SEQ ID NO:76] at a holding concentration of 2 μM and different double injections of a mixed sample containing either His6-A11-ABD WT [SEQ ID NO:77] or His6-H09-ABD WT [SEQ ID NO:78] at 2 μM. Similar experiments were performed using His6-H09-ABD WT [SEQ ID NO:78] injected at 2 μM in the first pulse, followed by double injections of a mixed sample containing His6-A11-ABD WT [SEQ ID NO:77] also at 2 μM and His6-H09-ABD WT [SEQ ID NO:78] at a holding concentration of 2 μM. As a control, running buffer PBS-T was injected instead of the variant used to hold the surface at 2 μM concentration in the second pulse.

[0369] The results of the epitope-binding assay showed that the initial binding of the fusion protein His6-A10-ABD WT to hCD16a did not block the binding of the subsequently injected fusion proteins His6-H09-ABD WT or His6-A11-ABD WT to hCD16a, indicating non-overlapping epitopes on hCD16a for variant A10 and either of variants H09 and A11 (Figure 4). His6-H09-ABD WT and His6-A11-ABD WTFrom the results obtained in a similar experimental setup using the fusion protein, it was possible to conclude that variants H09 and A11 bind to overlapping epitopes (Figure 4). The results of the mutual epitope binning were independent of the V158 or F158 allotype of hCD16a.

[0370] Biological Example 4: Binding studies of monomeric, heterodimeric, and homodimeric CD16a-binding polypeptide constructs using surface plasmon resonance In this example, SPR experiments were performed to compare the hCD16a-binding properties of a set of monomeric and heterodimeric CD16a-binding polypeptide constructs.

[0371] Materials and methods Subcloning of monomeric, heterodimeric, and homodimeric CD16a-binding polypeptide constructs. DNA fragments encoding CD16a-binding polypeptides were amplified using their respective library pAffi-1 phagemid vectors as templates. For the construction of heterodimeric CD16a-binding polypeptide constructs, codons encoding a flexible (GGGGS)3 CD16a-binding polypeptide linker [SEQ ID NO: 225], as well as specific primers introducing nucleotide overhangs complementary to the cloning termini of a linearized Escherichia coli (E. coli) expression vector, were used. The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to clone monomeric constructs and heterodimeric constructs [SEQ ID NOs: 79 - 82] equipped with both an N-terminal His6 tag and a C-terminal cysteine (Cys), as well as heterodimeric constructs having only a C-terminal His6 tag [SEQ ID NOs: 83 - 84]. In addition, a potential dual-engager construct containing an hBCMA-binding polypeptide (a copy of two tandemly linked A10 CD16a-binding polypeptides) fused to a homodimeric hCD16a-binding arm was cloned using a C-terminal His6 tag [SEQ ID NO: 86]. The DNA constructs were sequence-verified using Sanger sequencing (Eurofins Genomics).

[0372] Expression and Protein Purification of Monomeric, Heterodimeric, and Homodimeric CD16a-Binding Polypeptide Constructs Escherichia coli (E. coli) BL21(DE3) cells were transformed with plasmids containing different DNA constructs 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 OD600 = 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 h with shaking at 150 rpm, and the cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15 mM imidazole, pH 7.4). After cell disruption by sonication, cell debris was removed by centrifugation, and each supernatant was applied to a 3 - 4 mL HisPur packed gravity flow column. Contaminants were removed by washing with 6 column volumes of binding / washing buffer. Subsequently, the hCD16a-binding polypeptide was eluted with elution buffer (PBS containing 300 mM imidazole, pH 7.4). After IMAC purification, the protein buffer was exchanged to PBS (pH 7.4) using a PD-10 desalting column (Cytiva). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen). Purity and concentration were measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein.

[0373] Biosensor Analysis of Monomeric, Heterodimeric, and Homodimeric CD16a-Binding Polypeptide Constructs. Using a Biacore 8K instrument (Cytiva), the generated N-terminal His6 and C-terminal Cys CD16a binding polypeptide constructs [SEQ ID NOs: 79 - 82] were separately immobilized on the second flow cell surface of four flow channels on a Series S CM5 sensor chip (Cytiva) by ligand thiol coupling using the manufacturer's protocol. The first flow cell of each flow channel was activated and deactivated and used as a reference surface. After diluting the analyte with running buffer PBS-T, the binding analysis was performed at 4°C and a flow rate of 30 μl / min. hCD16a F158 and hCD16a V158 proteins were injected at 200 nM onto the CD16a binding polypeptide surface. The reverse setup was used in a second experiment, and hCD16a F158 and hCD16a V158 were separately immobilized on the second flow cell surface of each flow channel on a Series S CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's protocol. The first flow cell of each flow channel was activated and deactivated and used as a reference surface. After diluting the analyte with running buffer PBS-T, the binding analysis was performed at 4°C and a flow rate of 30 μl / min. After each injection, the flow cell was regenerated by injection of 10 mM HCl. The heterodimeric hCD16a binding construct [SEQ ID NOs: 83 - 84] was injected onto the hCD16a surface at concentrations ranging from 0.5 nM to 32 nM. In a third experiment, a Biacore T200 instrument (Cytiva) was used to immobilize hCD16a F158 and hCD16a V158 on the carboxymethylated dextran surface on a CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. A potential dual-engager construct containing an anti-hBCMA polypeptide fused to the homodimeric A10-A10 CD16a binding polypeptide arm [SEQ ID NO: 86] was diluted with running buffer PBS-T. The binding analysis was performed in a single-cycle kinetic setting at 25°C and a flow rate of 30 μl / min at five concentrations ranging from 2.5 to 40 nM. After the last injection, the flow cell was regenerated by injection of 10 mM HCl.

[0374] Results Expression vectors were constructed for a set of monomeric and heterodimeric CD16a-binding polypeptides. Four constructs, His6-A10-Cys, His6-A11-Cys, His6-A10-A11-Cys and His6-A11-A10-Cys [SEQ ID NOs: 79-82], correspond to either monomeric or heterodimeric CD16a-binding polypeptide constructs with both an N-terminal His6 tag and a C-terminal cysteine residue, the latter being used for directed coupling to an SPR sensor chip using thiol chemistry. Two heterodimeric constructs, A10-A11-His6 and A11-A10-His6 [SEQ ID NOs: 83-84], and one dual-engager construct, anti-BCMA-A10-A10-His6 [SEQ ID NO: 86], composed of a BCMA-binding polypeptide fused to the A10-A10 CD16a-binding polypeptide arm, were designed to contain only a C-terminal His6 tag. All seven constructs were expressed intracellularly in Escherichia coli BL21(DE3) cells and the soluble proteins were purified by IMAC. Figure 5 shows the SDS-PAGE analysis of each final protein preparation, demonstrating that these mainly contain bands corresponding to the predicted molecular weights. The larger molecular weight bands seen in lanes 1, 2, 5 and 6 of Figure 5a correspond to the expected presence of dimeric species held together via disulfide bonds formed between the C-terminal cysteines present in these constructs.

[0375] A series of first binding experiments were performed using His6-A10-Cys, His6-A11-Cys, His6-A10-A11-Cys and His6-A11-A10-Cys proteins separately immobilized on the thiol-activated sensor chip surface via their C-terminal cysteine residues. Injection of hCD16a F158 or hCD16a V158 protein at a concentration of 200 nM onto the sensor chip surface containing the monomeric CD16a-binding polypeptide constructs His6-A10-Cys and His6-A11-Cys respectively resulted in a binding response profile similar to that obtained in Example 2 using the reverse setup, and significantly stronger binding was shown by the A10 CD16a-binding polypeptide (Figure 6). Injection of hCD16a F158 or hCD16a V158 protein at a concentration of 200 nM onto the sensor chip surface containing the heterodimeric His6-A10-A11-Cys and His6-A11-A10-Cys proteins resulted in a binding profile characterized by a fairly slow off-rate kinetics for both analytes, indicating that the injected monomeric hCD16a protein bound more tightly here via cooperative binding with contributions from both CD16a-binding polypeptides in the heterodimeric His6-A10-A11-Cys and His6-A11-A10-Cys constructs. Interestingly, the sensorgrams showed that the relative orientation (N-terminus vs C-terminus) A11-A10 was more productive (slower off-rate kinetics) than the A10-A11 orientation using this specific linker length between the segments ((GGGGS)3) [SEQ ID NO: 225]. Considering also the epitope binning results from Example 3, it was shown that the A10 and A11 CD16a-binding polypeptides bind to non-overlapping epitopes on hCD16a, strongly suggesting that both the His6-A10-A11-Cys and His6-A11-A10-Cys proteins are able to bind to hCD16a via a bivalent paratope effect.

[0376] Using an inverse assay format, two heterodimer constructs A10-A11-His6 [SEQ ID NO:83] and A11-A10-His6 [SEQ ID NO:84] were separately injected at concentrations in the range of 0.5 nM to 32 nM onto a sensor chip surface containing immobilized hCD16a F158 or hCD16a V158 protein. The results shown in Figure 7 indicate that, also in this assay format, the binding responses obtained for both hCD16a ligands reflect stronger binding (slower off-rate kinetics) than those observed for either the individual A10 or A11 counterparts and monomeric A10 or A11 counterparts analyzed in the same assay format in Example 2 (Figure 3, data for His6-A10-ABDWT and His6-A11-ABDWT). This further shows that the bispecific binding effect to hCD16a can be obtained by combining the A10 and A11 variants into a heterodimer construct. Furthermore, since the H09 CD16a-binding polypeptide was also shown to bind to an epitope that does not overlap with the epitope of the A10 CD16a-binding polypeptide (Example 3), a heterodimer variant based on the combination of the A10 / H09 variants can also be expected from the results of this example to exhibit bispecific binding to hCD16a, which is also demonstrated in Example 5.

[0377] Furthermore, the hCD16a-binding properties of a fusion protein containing a combination of homodimer A10-A10 CD16a-binding polypeptides [SEQ ID NO:86] were also analyzed. Here, single-cycle kinetic analysis was performed, including consecutive injection of the analyte at concentrations in the range of 2.5 to 40 nM onto a sensor chip surface containing immobilized hCD16a F158 or hCD16a V158 protein, respectively. The results show that, compared to a protein containing a single monomeric A10 CD16a-binding polypeptide moiety (Figure 3, His6-A10-ABD WTData for), containing an A10 CD16a-binding polypeptide moiety linked in two tandems, where the protein analyzed here shows a significantly stronger apparent affinity for both hCD16a variants, with apparent dissociation constants calculated under these conditions for hCD16a F158 and hCD16a V158 (e.g., surface density of hCD16a variants on the chip) of 0.4 and 0.3 nM, respectively, demonstrating the effectiveness of the avidity effect from the simultaneous and cooperative binding of a single injected homodimeric analyte protein to two sensor chip-immobilized CD16 protein molecules (Figure 8).

[0378] In summary, this set of hCD16a-binding assays of monomeric, heterodimeric, and homodimeric constructs based on the CD16a-binding polypeptides described herein shows that they can be freely combined in new constructs to yield various novel hCD16a-binding proteins with different binding characteristics, including bivalent paratopes and bivalent bonds. By choosing the CD16a-binding polypeptides and their relative order (from N-terminus to C-terminus), it becomes possible to vary the overall binding properties. In the set of constructs analyzed in this example, a common 15-amino acid linker composed of the GGGSG motif (SEQ ID NO: 145) arranged in three tandems was used to link the homodimeric and heterodimeric combinations. Additional hCD16a-binding constructs based on alternative polypeptides or chemical linkers (including direct linkages; i.e., without a linker), and the incorporation of three or more identical or different CD16a-binding polypeptide moieties, are also possible to fabricate using methods known in the art.

[0379] Biological Example 5: Evaluation of BCMA×hCD16a dual-engager constructs using surface plasmon resonance In this example, an SPR experiment was carried out to analyze the bispecific binding characteristics of a three-component fusion protein containing an hBCMA-binding polypeptide (referred to herein as 1-E6, with the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKLNDAQAPK, having SEQ ID NO: 226) fused to an hCD16a-binding arm consisting of a combination of heterodimer H09-A10 CD16a-binding polypeptides.

[0380] Materials and methods Subcloning of the hBCMA×hCD16a-binding polypeptide bis-engager construct. DNA fragments encoding two of the hBCMA-binding polypeptide and the hCD16a-binding polypeptide [SEQ ID NOs: 1 and 75] were separately amplified from aligned gene fragments (gBlocks gene fragments, Integrated DNA Technologies, Leuven, Belgium) using primers that introduced complementary overhangs at the ends of the linearized expression vector and a flexible (GGGGS)3 linker [SEQ ID NO: 225] between each affinity protein unit. The three gene fragments were assembled using the In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) to construct a three-component BCMA×hCD16a bis-engager with a C-terminal His6 tag [SEQ ID NO: 85]. The DNA construct was sequence-verified using Sanger DNA sequencing (Eurofins Genomics).

[0381] Expression and protein purification of the hBCMA×hCD16a CD16a-binding polypeptide bis-engager construct. Escherichia coli (E. coli) BL21(DE3) cells were transformed with a plasmid containing the 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 OD600 = 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 h with shaking at 150 rpm, and the cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15 mM imidazole, pH 7.4). After cell disruption by sonication, cell debris was removed by centrifugation, and each supernatant was applied to a 3 - 4 mL HisPur packed gravity flow column. Contaminants were removed by washing with 6 column volumes of binding / washing buffer. Subsequently, the hCD16a-binding polypeptide was eluted with elution buffer (PBS containing 300 mM imidazole, pH 7.4). After IMAC purification, the protein buffer was exchanged to PBS using a PD-10 desalting column (Cytiva). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen). The purity and concentration were measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein.

[0382] Evaluation of dual-target binding of the hBCMA×hCD16a CD16a-binding polypeptide dual-engager construct. Using a Biacore T200 instrument (Cytiva), the simultaneous binding of the dual-engager construct to hBCMA and hCD16a was evaluated. Human BCMA-rFc (BCMA protein amino acids 1-54, human, recombinant (ECD, rabbit Fc Tag), catalog number 10620-H15H, Sino Biological, Eschborn, Germany) was immobilized on the carboxymethylated dextran surface on a 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. After diluting the dual-engager construct and hCD16a variant with running buffer PBS-T, the binding analysis was performed at 25 °C and a flow rate of 30 μl / min. After each injection, the flow cell was regenerated by injection of 10 mM HCl.

[0383] Results A BCMA×hCD16a bispecific engager construct containing a BCMA-binding polypeptide genetically fused to an hCD16a-binding arm composed of a heterodimeric H09-A10 affibody combination and a C-terminal His6 tag was constructed and expressed as a soluble gene product in Escherichia coli (E. coli) (DE3). Figure 9 shows an SDS-PAGE analysis of the final protein preparation, which demonstrated that it mainly contained the bispecific engager construct with a band corresponding to the predicted molecular weight. The hBCMA×hCD16a bispecific engager construct was injected at 200 nM onto the surface of a sensor chip containing immobilized hBCMA-rFc protein, followed by successive injections of PBS-T buffer or hCD16a F158 / hCD16a V158 protein at 200 nM as schematically shown in Figure 10a. Since the injected hCD16a variants were expected to bind weakly to the rabbit Fc tag on of the hBCMA-rFc protein, a control successive injection with PBS-T buffer only (without the hBCMA×hCD16a bispecific engager during injection I), followed by injection of the hCD16a F158 or V158 protein at 200 nM, was performed to enable subtraction of any response corresponding to this background binding. Figure 10b shows the resulting sensorgram obtained after subtraction of the signal from the reference flow cell (flow cell 1, activated / inactivated) and the low response signal (<10 RU) obtained from direct hCD16a binding to the rabbit Fc tag of the immobilized hBCMA-rFc ligand. These resulting sensorgrams show that the hBCMA×hCD16a bispecific engager construct can bind simultaneously to both the immobilized hBCMA-rFc ligand and the subsequently injected hCD16a variants. The slow off-rate kinetics observed after injection completion (phase IV in Figure 10b) indicate that, as previously demonstrated for the A10-A11 and A11-A10 combinations (Example 4), the H09-A10 affibody combination is also capable of biparatopic binding to hCD16a.Also, as predicted from the results obtained in Example 2, since it was demonstrated that the H09 affibody variant binds more strongly to the hCD16a F158 allotype, a slower hCD16a dissociation rate (Figure 10b, Phase IV) was observed for the interaction with the hCD16a F158 variant than with the hCD16a V158 variant (Example 2).

[0384] In summary, the results show that here an affibody-based hCD16a binding arm, which is composed of a combination of the heterodimeric H09-A10 affibody, can retain its hCD16a binding ability even when fused to another polypeptide unit that is simultaneously involved in binding to a therapeutically relevant target, as exemplified here by a genetically fused polypeptide that binds to hBCMA, a relevant target in multiple myeloma cancer.

[0385] Biological Example 6: Anti-BCMA engagers induce immune cell activation in the presence of tumor cells In a co-culture of human PBMC and the BCMA-positive multiple myeloma cell line MM.1S, experiments were conducted to evaluate the tendency of different anti-BCMA engagers to induce an IFNγ response. PBMC (4W-270, Lonza) thawed and rested overnight were co-cultured with MM.1S cells (CRL-2974, ATCC) in a V-bottom 96-well plate (249935, Thermo Scientific) at an effector-to-target ratio of 25:1. A total of 4×10 in 200 μl / well 5Cells were treated with 200 nM of an anti-BCMA engager or 200 nM of elotuzumab (300 mg of Empliciti, Bristol-Myers Squibb) for 4 hours at 37°C in a humidified 5% CO2 incubator. Supernatants were harvested and subsequently centrifuged at 600 g for 5 minutes. IFN-γ levels were determined by enzyme-linked immunosorbent assay (ELISA) using the Human IFN-γ ELISA MAX™ Deluxe Set (430104, BioLegend). Data were processed using Excel and plotted using GraphPad Prism 9.

[0386] An anti-BCMA engager construct containing a BCMA-binding polypeptide (referred to herein as 1-E6) genetically fused to an hCD16a-binding arm composed of any of monomer A10 (SEQ ID NO: 88), homodimer A10 (SEQ ID NO: 86), heterodimer H09-A10 (SEQ ID NO: 85), or heterodimer A11-A10 (SEQ ID NO: 87) affibodies was evaluated. Corresponding constructs carrying a null non-BCMA-binding polypeptide with high sequence identity to the BCMA-binding polypeptide were also evaluated. The SLAMF7 monoclonal antibody elotuzumab was used as a positive control, and single cultures of PBMC and MM.1S were used as negative controls. All dual engagers containing the BCMA-binding polypeptide induced an IFNγ response in co-cultures of PBMC and MM.1S cells, which was greater for all engagers than the response of the positive control elotuzumab (Figure 11). Dual engager constructs lacking BCMA binding showed no or limited IFNγ response in co-cultures of PBMC and MM.1S cells. These experiments demonstrate BCMA-dependent activation of PBMC against BCMA-positive MM.1S myeloma cell lines induced by the anti-BCMA engagers described herein.

[0387] Biological Example 7: Evaluation of the secondary structure content and thermal denaturation profile of CD16a-binding polypeptides using circular dichroism In this example, three CD16a-binding polypeptides A10, H09, and A11 [SEQ ID NOs: 1, 74, and 75] were subcloned, expressed, and purified as polypeptide-His6 fusion proteins, and their secondary structure contents and their thermal denaturation profiles were evaluated by circular dichroism (CD) spectroscopy.

[0388] Materials and Methods Cloning of CD16a-Binding Polypeptides DNA fragments encoding three CD16a-binding polypeptides [SEQ ID NOs: 1, 74, and 75] were amplified from their respective pAffi-1 library vectors using specific primers designed to introduce complementary overhangs at the ends of the linearized expression vectors. Monomeric polypeptide constructs with a C-terminal His6 tag were cloned using the In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden). The DNA constructs were sequence-verified using Sanger sequencing (Eurofins Genomics).

[0389] Expression and Purification Escherichia coli (E. coli) BL21(DE3) cells were transformed with the plasmid containing the 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 OD 600It was grown at 37°C with shaking at 150 rpm from 0.6 to 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 (7 M guanidinium chloride, 47 mM NaH2PO4, 2.65 mM NaH2PO4, 10 mM Tris-HCl, 100 mM NaCl, pH 8). 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 at RT for 30 minutes with rotation with the resin. Contaminants were removed by washing three times with wash buffer (7 M guanidinium chloride, 46.6 mM Na2HPO4, 3.4 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), and then the CD16a-binding polypeptide variant 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 incubation at RT for 10 minutes with 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 the purity and concentration of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA). The purity and concentration were measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein.

[0390] CD analysis The secondary structure content of three CD16a-binding polypeptides was determined by recording CD spectra in the range of 195 - 260 nm at 20 °C using a Chirascan CD Spectrometer (Applied Photophysics, Leatherhead, United Kingdom), and their thermal denaturation profiles were determined by recording CD ellipticity at 221 nm during heating from 20 °C to 92 °C.

[0391] Results The CD spectra obtained showed that all of the polypeptides analyzed had a high α-helix secondary structure content at 20 °C. The thermal denaturation profiles (Figure 12) showed melting temperatures (Tm) of 49 °C for clone A10-His6, 48 °C for clone H09-His6, and 50 °C for clone A11-His6, determined by variable temperature measurements. Furthermore, the CD profiles of the heated (92 °C) and then cooled (20 °C) samples showed almost complete overlap. Collectively, these results support that the three CD16a-binding polypeptides generated mainly contain an α-helix secondary structure and can refold after thermal denaturation.

[0392] Biological Example 8: Alanine Scan of CD16a-Binding Clone A10 In this example, an alanine scan of the polypeptide variant A10 [SEQ ID NO: 1] was performed to examine the relative importance of amino acid occupancy at positions randomized during library construction with respect to the binding interaction between A10 and CD16a.

[0393] Materials and Methods Site-Directed Mutagenesis of CD16a-Binding Clone A10. The DNA fragment encoding the CD16a-binding polypeptide clone A10 [SEQ ID NO:1] was subjected to site-directed mutagenesis by using an expression plasmid containing the A10-His6 construct as a template in individual mutagenic PCR reactions, where each of the original codons at 14 variable positions was replaced with an alanine codon using primers designed for each position. The resulting 14 alanine-substituted variants were sequence-verified using Sanger sequencing (Eurofins Genomics).

[0394] Expression and purification Escherichia coli (E. coli) BL21(DE3) cells were transformed with plasmids encoding 14 alanine variants each with a C-terminal His6 tag as well as the wild-type A10, 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 from 0.6 to 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 while shaking at 150 rpm, and the cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15 mM imidazole, pH 7.4). After cell disruption by sonication, cell debris was removed by centrifugation, and each 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 at RT for 30 minutes while rotating gently with the resin. Contaminants were removed by washing three times with the washing buffer, and subsequently, the A10 variant was eluted with elution buffer (PBS containing 300 mM imidazole, pH 7.4) by incubating at RT for 10 minutes while rotating gently. After IMAC purification, buffer exchange was performed to PBS using a PD - 10 desalting column (catalog number 17085101, Cytiva, Uppsala, Sweden). SDS - PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4 - 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA). The purity and concentration were measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence of the purified protein.

[0395] Biosensor analysis of alanine variant The interaction between CD16a and the His6-tagged wild-type A10 polypeptide and 14 alanine variants was analyzed using a Biacore T200 instrument (Cytiva). The protein ligand hCD16a F158 (catalog number CDA-H5220, Acro Biosystems) was immobilized (1430 RU) onto a Series S CM5 sensor chip (Cytiva) using the manufacturer's instructions. One flow cell was activated and deactivated and used as a reference cell. After diluting the analyte in running buffer PBS-T, 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. The wild-type A10-His6 construct and the 14 alanine variants were injected onto the CD16a (F158) surface at a common concentration of 200 nM.

[0396] Results: When 14 alanine substitution variants (polypeptide-YY-His6 format) were separately injected at a common concentration of 200 nM onto the surface of a sensor chip containing CD16a(F158) protein, a series of sensorgrams were obtained from which the importance of the original amino acids in the anti-CD16a A10 polypeptide for binding to CD16a could be estimated. The results are shown in Figure 13. Substitution of alanine at positions 9, 11, 13, 14, 18, 27, 32 [SEQ ID NOs: 2, 3, 4, 5, 6, 8 and 9] did not significantly affect binding as the resulting sensorgrams were similar to those obtained for the original A10 polypeptide with respect to response level and curve morphology. In contrast, alanine substitutions at positions 10, 17, 24, 28 and 31 resulted in variants showing significantly lower binding responses. Some variants, H25A and M35A [SEQ ID NOs: 7 and 10], were moderately affected by the alanine substitution. Analysis by circular dichroism spectroscopy showed that all variants had a profile characteristic of proteins with a high content of α-helix having distinct minima at 221 and 205 nm (see data in Figure 14). Thermal melting experiments showed that all except one variant (I31A) exhibited a maintained thermal melting point of approximately 50 °C (see data in Figure 15). Taken together, this indicated that the lower binding responses seen for some variants were not associated with a corresponding loss of overall structure, but rather that important residues for interaction with CD16a had been addressed.

[0397] Preparation Example 9: Second-generation library construction, selection and phage ELISA In this example, second-generation libraries of the CD16a-binding polypeptide A10 were constructed to various extents based on the re-randomization of certain variable positions. The libraries were used for the identification of second-generation variants of the CD16a-binding A10 polypeptide, while retaining the ability to bind CD16a at different strengths. The libraries were methionine-deficient. Individual unique clones obtained after four rounds of phage display selection cycles and subsequent DNA sequencing were assayed for binding to CD16a in monoclonal phage-ELISA.

[0398] Materials and Methods Second-generation library of CD16a-binding polypeptide A10 An M13 phage display second-generation selection library was prepared based on the A10 polypeptide. The phagemid vector used was pAffi-1 (Gronwall et al. (2007) J. Biotechnol. 128:162-183). The pAffi-1 phagemid containing the lac promoter and the OmpA signal peptide was designed for the phage display of the encoded polypeptide library members 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).

[0399] Two synthetic oligonucleotides based on the A10 sequence were used for gene assembly overlap extension PCR (OE-PCR) and subsequent amplification by PCR: A10-fwd, 65 bp in length, encoding positions 3 to 23 and positions 19 to 41 (reverse complementary strand) corresponding to the Z domain numbering (Nilsson et al. (1987) Protein Eng. 1: 107-113) (5’-AC AAC AAA TTC AAC AAA GAA Z01 Z02 Z03 GCG Z01 Z01 GAG ATC Z04 Z01 CTG CCG AAC CTG AAC[SEQ ID NO: 227]-’3’), and A10-rev, 69 bp in length (5’-ACT CTG GCT CGG ATC ATC Z03 CAG Z05 Z01 Z06 GAA TGC Z07 Z01 CTG Z08 Z09 GTT CAG GTT CGG CAG[SEQ ID NO: 228]-3’), in which 15 original A10 codons were re-randomized based on the data obtained in the alanine scan experiment (Example 8) during synthesis using a mixture of trinucleotide codon building blocks (amino acid codons for Met, Cys, Pro and Gly were excluded); Z01 = 10% Ala and 90% equally distributed among the remaining 15 allowed amino acids, Z02 = 10% Gln and 90% equally distributed among the remaining 15 allowed amino acids, Z03 = 15% Ile and 85% equally distributed among the remaining 15 allowed amino acids, Z04 = 90% Arg and 10% equally distributed among the remaining 15 allowed amino acids, Z05 = 80% Ser and 20% Lys, Z06 = 90% Ile and 10% equally distributed among the remaining 15 allowed amino acids, Z07 = 90% Phe and 10% equally distributed among the remaining 15 allowed amino acids, Z08 = 65% His and 35% equally distributed among the remaining 15 allowed amino acids, Z09 = 90% His and 10% equally distributed among the remaining 15 allowed amino acids). Following OE-PCR, primer forward: (5’-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3’)[SEQ ID NO: 223] And reverse: (5’-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3’)[SEQ ID NO: 224] was used for PCR amplification.

[0400] Approximately 5.5 μg of double-digested and purified (Qiagen, Germany) PCR product of the assembled gene with XhoI and NheI was ligated to the pAffi-1 phagemid vector that had been 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 22 portions, and used to electroporate 25 μl of electrocompetent ER2738 E. coli cells (F’, glnV amber suppressor) (Lucigen, USA) (0.1 cm BioRad cuvette). 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, followed by pooling (11 electroporations per pool), incubation at 37 °C for 1 hour with shaking, after which the pool of cells was titrated by spreading dilution series on ampicillin plates, and each was transferred to two 5-liter shaking 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% (w / v) glucose and 100 μg / mL ampicillin. The overnight culture of the cells was pelleted by centrifugation, resuspended in 25 mL of cold 40% glycerol to a final volume of approximately 37 ml, and subsequently distributed into 20 tubes of approximately 1 ml of cell-glycerol solution per tube and 5 tubes of approximately 3 ml of cell-glycerol solution per tube. Titration after electroporation and OD after overnight culture 600From the measurement, it was calculated that the library size (diversity) was approximately 5×10^6, and each 1 mL aliquot of cells contained a cell number corresponding to approximately 14,000× the library size. A portion of the cells was used directly for phage stock preparation using M13KO7 helper phage, and the remaining tubes containing cells were stored at -80 °C.

[0401] Phage stock preparation. For the production of the affibody display phage stock, 1.14 mL of the library glycerol stock was aliquoted and inoculated into two baffled E-flasks each containing 750 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 = 1.30 mL. The cells per culture flask were infected with M13KO7 helper phage (New England Biolabs) at a multiplicity of infection (MOI) of 5, gently swirled, incubated at 37 °C for 15 minutes without shaking, and subsequently incubated at 37 °C at 70 rpm for 15 minutes. The cells of each culture were centrifuged and resuspended in two baffled E-flasks each containing 500 mL of TSB+Y, 100 μg / mL Carb, and 1 mM 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 with shaking at 90 rpm. The phage library stock was recovered by precipitation twice with 20% (w / v) PEG6000 / 2.5 M NaCl. The titer of the stock was measured by spot titration, and the percentage of clones carrying phagemids with gene inserts of the correct size was analyzed using polymerase chain reaction screening.

[0402] Selection from the secondary library of the CD16a-binding clone A10. Biotinylated hCD16a (biotinylated human hCD16a (F158), Avitag, His tag, Acro Biosystems, catalog number CDA-H82E8, corresponding to residues 17 - 208 of Uniprot entry P08637) was used, with 100 nM in 4 tracks in cycle 1, 50 nM in 3 tracks in cycle 2 (pooling 2 tracks from cycle 1 together), and 50 nM in 5 tracks in cycles 3 and 4 (splitting 2 tracks from cycle 2 into 2), for 4 cycles of panning. Further, in cycle 3, a molar excess of 25 μM of non-biotinylated target [CD16a F158 Y140H]-[Z963]-[AVI-tag]-[His6]: (GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFHIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGSGVDNKFNKETQEASWEIFTLPNLNGRQVAAFISSLLDDPSQSANLLAEAKKLNDAQAPKGGGSGGLNDIFEAQKIEWHEHHHHHH)[SEQ ID NO: 229] was used in the competition. Streptavidin (SA)-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 NaH2PO4·H2O, pH 7.4). To avoid enrichment 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, the phage stock in PBS-T was pre-incubated at room temperature (RT) for 30 minutes with 0.1% (w / v) BSA and beads under constant end-over-end (eoe) rotation to remove phages carrying binders to SA. The amount of phage stock used was 10 10 colony forming units (cfu) in cycle 1, 10 11 cfu in cycles 2 and 3, and 10 11~12 cfu in cycle 4.

[0403] For tracks involving solid-phase selection, biotinylated target protein was immobilized end-over-end (eoe) at RT for 1 h on 1 mg of beads in cycles 1 and 4, or on 0.5 mg of beads in cycles 2 and 3. Target-containing beads were incubated with 1% (w / v) BSA in PBS-T at RT for 30 min, then washed with PBS-T and pre-incubated phage stock was added, and selection was performed by eoe for 3 h in cycles 1 and 4, 2 h in cycle 2, and 30 min at RT in cycle 3. For tracks involving liquid-phase selection, pre-incubated phage stock was added to biotinylated target protein for selection in solution, and then the phage-antigen complex was captured by incubating with SA-beads at RT for 30 min by eoe. Cycle 3 included competition with non-biotinylated target (off, further incubated for 1 h or ON (18 h) competition with non-biotinylated target (off-rate selection). SA-bead-captured phage-antigen complexes were washed with PBS-T by eoe for a total of 5 min at RT (cycles 1 and 4), 10 min or 30 min (cycle 2), or 20 min (cycle 3). 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. After incubation with 0.5 M acetic acid, pH 2.8 at RT for 15 min by eoe, the eluate was transferred to a new pretreated tube and neutralized with an equal volume of 1 M Tris-HCl, pH 8, or by supplementing with 1 mM CaCl2 before transferring the eluate to a new pretreated tube, and the antigen-binding phage was eluted by incubating with 0.25 mg / mL trypsin (Gibco Life Technologies) in TBS-T (TRIS-buffered saline, 0.1% (v / v) Tween-20) at RT for 30 min by eoe.

[0404] Following cycle 1, a new phage stock for selection round 2 was prepared at OD 600It was generated by growing E. coli XL-1 Blue cells (Agilent) in TSB+Y containing 10 μg / mL Tet at 37 °C while shaking at 150 rpm from 0.5 to 0.8. Infect 50 mL of bacteria with the total phage eluate volume, gently swirl, incubate without shaking at 37 °C for 25 minutes, and then shake at 70 rpm at 37 °C for 15 minutes. Then, centrifuge the culture and resuspend it in TSB+Y, and plate it on a blood agar plate (40 g / l blood agar) containing 100 μg / mL Carb and 1% (w / v) glucose, and incubate at 37 °C for 16 - 18 hours. Collect in TSB+Y by scraping bacterial colonies, inoculate 200 mL of TSB+Y containing 100 μg / mL of Carb, and OD 600 Grow at 37 °C while shaking at 150 rpm until OD = 0.5. Superinfect 30 mL with M13KO7 helper phage (MOI 5), gently swirl, incubate without shaking at 37 °C for 25 minutes, and then shake at 70 rpm at 37 °C for 15 minutes. Centrifuge the culture and resuspend it in 150 mL of TSB+Y containing 100 μg / mL of Carb and 1 mM IPTG. Add 25 μg / mL of Kan 2 hours after inoculation. Incubate the culture at 37 °C while shaking at 150 rpm for 16 - 18 hours. The phage 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 polymerase chain reaction screening was used to analyze the percentage of phage particles carrying phagemids with affibody inserts.

[0405] In the next cycles 2 - 3, in TSB+Y containing 10 μg / mL Tet and 1% (w / v) D-glucose, at 37 °C while shaking at 150 rpm and OD 600= 0.5 - 0.8. A new phage stock was generated by growing E. coli XL-1 Blue cells until a volume of 30 mL was reached. The grown E. coli XL-1 Blue bacteria were infected with half the phage eluate volume and incubated for 30 minutes at 37°C with shaking at 150 rpm. An equal volume of TSB+Y containing 10 μg / mL Tet and 100 μg / mL Carb was added, and then the bacteria were incubated for 1.5 hours at 37°C with shaking at 150 rpm for infection. Subsequently, they were super-infected with M13KO7 helper phage (MOI 76 - 382) and further incubated for 1.5 hours at 37°C with shaking at 150 rpm for phenotype determination. The culture was centrifuged and resuspended in 100 mL of TSB+Y containing 100 μg / mL Carb, 25 μg / mL Kan, and 0.5 mM IPTG. The culture was incubated at 30°C for 16 - 18 hours with shaking at 150 rpm. The phage 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 polymerase chain reaction screening was used to analyze the percentage of phage particles carrying phagemids with affibody inserts.

[0406] DNA sequence. Following phage display selection, 25 and 129 individually grown colonies after infecting E. coli XL-1 Blue with phage eluate after cycles 3 and 4 respectively were sent for sequencing by Sanger sequencing (PlateSeq, Eurofins Genomics, Ebersberg, Germany). 67 unique and new sequences were identified.

[0407] Monoclonal phage-ELISA screening. Preparation of monoclonal phage supernatants and monoclonal phage-ELISA screening were performed as described in Example 1 using 67 unique and novel sequences identified by Sanger sequencing. In this example, MaxiSorp ELISA plates (Clear Flat-Bottom Immuno Nonsterile 384-Well Plates, catalog number 464718, Thermo Fisher Scientific) were coated at 4 °C for 16 - 18 h with gentle shaking with the following antigens: 30 μl of 4 μg / mL biotinylated hCD16a in 100 mM sodium carbonate buffer, pH 9.6 (1 / 4 of the wells containing each coating protein), 20 μg / mL human serum albumin (HSA; Sigma product number SRP6182) (for evaluation of the proper display of an expression cassette containing a three-component fusion protein including an affibody, an affibody albumin-binding domain, and a truncated protein 3), 10 μg / mL SA or 10 μg / mL BSA. 53 binding candidates were identified as ELISA positive.

[0408] Results The positions (Q10, R17, H24, F28, and I31) in the A10 peptide identified as important for CD16a binding were conservatively randomized. For these positions, the trinucleotide codon mixes used for the synthesis of mutagenic oligonucleotides provided a 90% likelihood for the reinsertion of the amino acids found in the A10 peptide and were biased to leave a 10% likelihood for the insertion of a different amino acid (any of the 20 natural amino acids minus the A10 amino acid or methionine, cysteine, proline, or glycine). For the six positions (9, 13, 14, 18, 27, and 32) in the A10 peptide where the original amino acids were not found to be important for CD16a binding, the trinucleotide codon mixes used for the synthesis of mutagenic oligonucleotides provided only a 10% likelihood for the reinsertion of the amino acids found in the A10 peptide and were biased to leave a 90% likelihood for the insertion of a different amino acid (any of the 20 natural amino acids minus the A10 amino acid or methionine, cysteine, proline, or glycine). For position 25 occupied by histidine in the A10 peptide, the trinucleotide codon mix used for the synthesis of mutagenic oligonucleotides provided a 65% likelihood for the reinsertion of histidine and was biased to leave a 35% likelihood for the insertion of a different amino acid (any of the 20 natural amino acids minus histidine, methionine, cysteine, proline, or glycine). For position 33 occupied by serine in the A10 peptide, the trinucleotide codon mix used for the synthesis of mutagenic oligonucleotides provided an 80% likelihood for the reinsertion of serine and was biased to leave a 20% likelihood for the insertion of lysine.For positions 11 and 35 (both occupied by oxidation-prone methionines in the A10 peptide), the trinucleotide codon mixes used for the synthesis of mutagenic oligonucleotides were biased to provide a 15% probability for the insertion of isoleucine and an 85% probability for the insertion of a different amino acid (any of the 20 natural amino acids excluding methionine, cysteine, proline, or glycine).

[0409] Therefore, selection for CD16a-binding polypeptides from this library has the potential to yield novel A10-related CD16a-binding polypeptides with different affinities (the A10 shown * ). It also has the potential to yield CD16a-binding polypeptides that do not contain methionine.

[0410] Selection against the CD16a (F158) target protein was performed by phage display as described above (Materials and Methods). Analysis of the selection outputs from different selection tracks by monoclonal phage-ELISA and DNA sequencing identified 53 unique ELISA-positive clones. Their sequences are shown in the table of Figure 23 as SEQ ID NOs: 11 to 63. Three additional clones of interest (SEQ ID NOs: 64 to 66) were also identified.

[0411] Biological Example 10A: Expression, protein purification, and SPR analysis of the CD16a-binding polypeptides identified in Example 9 Materials and Methods Subcloning of the hBCMA×hCD16a-binding polypeptide dual-engager construct. The 53 CD16a-binding polypeptides [SEQ ID NOs: 11 to 63] identified in Example 9 (A10 *) And a DNA fragment encoding the hBCMA-binding polypeptide designated 1-E6 herein was amplified using their respective library pAffi-1 phagemid vectors as templates and primers that introduced an overhang complementary to the ends of the linearized expression vector and a linker encoding a flexible (GGGGS)3 between each polypeptide unit. The gene fragment was assembled into 53 different BCMA×hCD16a bispecific engager constructs with a C-terminal His6 tag as anti-BCMA-A10 * -His6. Here, A10 * corresponds to the CD16a-binding polypeptides identified in Example 9 [SEQ ID NOs: 11-63]. The DNA constructs were sequence-verified using Sanger DNA sequencing (Eurofins Genomics).

[0412] Expression and protein purification of the hBCMA×hCD16a-binding polypeptide bispecific engager constructs. E. coli BL21(DE3) cells were transformed with a plasmid containing 53 DNA constructs and cultured at 37 °C for 16 - 18 hours with shaking at 150 rpm in 500 μl of TSB+Y containing 25 μg / mL of Kan. Also, the cells were transformed with a DNA construct encoding the BCMA×hCD16a dual engager construct anti-BCMA-A10-His6 [SEQ ID NO:88] described in Example 5 above. The culture was inoculated 1:100 into 2.5 mL of TSB+Y containing 25 μg / mL of Kan and grown at 37 °C with shaking at 150 rpm until OD600 = 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 (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 hours with shaking at 150 rpm, the cells were centrifuged, and the denatured protein from the supernatant of the cell lysate supplemented with approximately 15 mM imidazole was added to a tube containing HisPur Cobalt IMAC Resin (Catalog No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT for 30 minutes with rotation. Contaminants were removed by washing three times with wash buffer (PBS containing 15 mM imidazole, pH 7.4), and subsequently, the BCMA×hCD16a dual engager was eluted with elution buffer (PBS containing 400 mM imidazole, pH 7.64). The concentration was measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence. SDS-PAGE analysis was performed on a subset of the purified protein to confirm the purity and concentration (Mini-PROTEAN TGX, Tris / Glycine / SDS, Bio-Rad).

[0413] Evaluation of CD16a binding of the hBCMA×hCD16a binding polypeptide dual engager construct. The interaction between the hCD16a-binding variant and human hCD16a was analyzed in real time using a Biacore 8K instrument (Cytiva). The protein ligands hCD16a F158 (catalog number CDA-H5220, Acro Biosystems) and hCD16a V158 (catalog number CD8-H52H4, Acro Biosystems) were diluted in 10 mM NaOAc, pH 4.5 and immobilized on separate sensor chip surfaces of a Series S CM5 sensor chip (Cytiva). The sensor chip consists of eight flow channels (two of which are used here), each composed of two flow cells arranged in series. For eac...

Claims

1. A CD16a-binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide has the following structure: It includes [N-terminal portion] - [Helix 1] - [Separation portion] - [Helix 2] - [C-terminal portion], A CD16a-binding polypeptide in which the CD16a-binding motif is a [helix 1]-[separation portion]-[helix 2] portion.

2. Helix 1 includes array X ９ X １０ X １１ AX １３ X １４ EIX １７ X １８ and Helix 2 includes array X ２４ X ２５ QX ２７ X ２８ AFX ３１ X ３２ X ３３ LX ３５ [SEQ ID NO: 120], wherein a) X ９ However, it is A, D, F, H, I, K, L, Q, R, T, V, or Y. X １０ However, Q is, X １１ However, it is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y, X １３ However, it is A, Q, or V. X １４ However, it is A, F, H, I, K, L, N, Q, R, S, T, V, W, or Y, X １７ However, it is either Q or R, X １８ However, it is A, D, E, F, H, I, K, N, Q, R, S, T or V, X ２４ However, it is H, X ２５ However, it is either A or H, X ２７ However, it is A, I, K, Q, R, S, T, or V, X ２８ However, it is either F or Y. X ３１ However, it is either I or L. X ３２ However, it is A, E, H, K, L, N, Q, or R. X ３３ However, it is either K or S. X ３５ However, it is A, H, I, L, M, R or S or b) X ９ However, V is X １０ However, Q is X １１ However, M is X １３ However, Q is X １４ However, F is X １７ However, R is X １８ However, K is X ２４ However, H is X ２５ However, H is X ２７ However, S is X ２８ However, F is X ３１ However, I is X ３２ However, K is X ３３ However, S is X ３５ However, M is, and optionally, within helix 1 and helix 2, X ｎ At least one and five or fewer residues are replaced by substitute residues, and / or X ｎ At least one and up to five of the unlabeled residues are replaced by substitute residues, or c) X ９ However, Q is X １０ However, F is X １１ However, Y is X １３ However, R is X １４ However, D is X １７ However, D is X １８ However, L is X ２４ However, E is X ２５ However, D is X ２７ However, K is X ２８ However, W is X ３１ However, Y is X ３２ However, M is X ３３ However, S is X ３５ However, I is, and optionally, within helix 1 and helix 2, X ｎ At least one and five or fewer residues are replaced by substitute residues, and / or X ｎ At least one and five or fewer residues that are not labeled as are replaced by alternative residues, or d) X ９ However, F is X １０ However, W is X １１ However, I is X １３ However, E is X １４ However, S is X １７ However, E is X １８ However, S is X ２４ However, I is X ２５ However, Y is X ２７ However, K is X ２８ However, W is X ３１ However, K is X ３２ However, Y is X ３３ However, S is X ３５ However, A is, and optionally, within helix 1 and helix 2, X ｎ At least one and up to five residues are replaced by substitute residues, and / or X ｎ The CD16a-binding polypeptide according to claim 1, wherein at least one and five or fewer unlabeled residues are replaced by substitute residues.

3. helix 1 includes the said array X ９ X １０ X １１ AX １３ X １４ EIX １７ X １８ includes [SEQ ID NO: 127], and helix 2 includes the said array X ２４ X ２５ QX ２７ X ２８ AFX ３１ X ３２ X ３３ LX ３５ includes [SEQ ID NO: 120], wherein X ９ is A, D, F, H, I, K, L, Q, R, T, V or Y, and X １０ is Q, and X １１ is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y, and X １３ is A, Q or V, and X １４ is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y, and X １７ is Q or R, and X １８ is A, D, E, F, H, I, K, N, Q, R, S, T or V, and X ２４ is H, and X ２５ is A or H, and X ２７ is A, I, K, Q, R, S, T or V, and X ２８ is F or Y, and X ３１ is I or L, and X ３２ is A, E, H, K, L, N, Q or R, and X ３３ is K or S, and X ３５ is A, H, I, L, M, R or S, the CD16a-binding polypeptide according to claim 2.

4. X ９ However, it is K, Q, or Y, and X １０ However, Q is X １１ However, it is either I or Q, and X １３ However, Q is X １４ However, it is either W or Y, and X １７ However, R is X １８ However, it is H, K, or R, and X ２４ However, H is X ２５ However, H is X ２７ However, it is A, K, or T, and X ２８ However, F is X ３１ However, I is X ３２ However, it is A, K, or Q, and X ３３ However, it is either K or S, and X ３５ The CD16a-conjugated polypeptide according to claim 3, wherein the polyparticle is I or L.

5. The CD16a-binding polypeptide according to claim 1, wherein the CD16a-binding efficacy is at least 10% of that of SEQ ID NO: 1, and / or the CD16a-binding polypeptide competes with SEQ ID NO:

1.

6. The CD16a-binding polypeptide according to claim 1, wherein the separated portion is a sequence of 1 to 5 naturally occurring amino acids.

7. The aforementioned N-terminal portion It does not exist, or The CD16a-binding polypeptide according to claim 1, wherein is a sequence of 1 to 15 naturally occurring amino acids.

8. The C-terminal portion is It does not exist, or The CD16a-binding polypeptide according to claim 1, wherein the sequence of amino acids is 1 to 50 naturally occurring amino acids.

9. The separated portion is arranged in the array X ２０ X ２１ X ２２ Having and / or The N-terminal portion is the sequence X １ X ２ X ３ X ４ X ５ Having and / or The C-terminal portion is the sequence PSQSANLLAEAKKLNDAQX ５６ X ５７ X ５８ [Sequence ID 137] In the separation section, X ２０ However, P or T, X ２１ However, N is X ２２ However, it is L, In the aforementioned N-terminal portion, X １ However, it is either V or G, or does not exist, X ２ However, it is either D or does not exist, X ３ However, if N is true, or if X does not exist, 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 CD16a-conjugated polypeptide according to claim 1, wherein K is present or absent.

10. The CD16a-conjugated polypeptide according to claim 1, wherein the separated portion has the sequence PNL or TNL, and / or the N-terminal portion has the sequence VGNKF [SEQ ID NO: 138].

11. The sequence of the CD16a-bound polypeptide is, VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence ID 1], VDNKFNKEQQIAQYEIRKLPNLNHHQTFAFIKSLLLDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 51], VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 74], or A CD16a-conjugated polypeptide according to claim 10, selected from VDNKFNKEFWIIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLLAEAKKLNDAQAPK [SEQ ID NO: 75].

12. A CD16a-conjugated oligomer comprising at least two CD16a-conjugated polypeptides according to claim 1.

13. The CD16a-conjugated oligomer according to claim 12, wherein each of the CD16a-conjugated polypeptides is separated by a linker.

14. VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK[Sequence ID 242], VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGGGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK [Sequence ID 243], or, A CD16a-conjugated oligomer according to claim 12, comprising a sequence selected from VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLLAEAKKLNDAQAPKGGGGGSGGGGGGGGGGVGVGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLLAEAKKLNDAQAPK [SEQ ID NO: 244].

15. The CD16a-conjugated polypeptide according to claim 1, further comprising additional functional components.

16. The CD16a-conjugated oligomer according to claim 12, further comprising an additional functional part.

17. The CD16a-binding polypeptide according to claim 15 or the CD16a-binding oligomer according to claim 16, wherein the additional functional portion comprises an immune signaling molecule.

18. The CD16a-conjugated polypeptide according to claim 15 or the CD16a-conjugated oligomer according to claim 16, wherein the additional functional portion includes an additional binding portion.

19. The CD16a-binding polypeptide or CD16a-binding oligomer according to claim 18, wherein the additional binding portion is specific to a cancer cell surface target.

20. The CD16a-binding polypeptide or CD16a-binding oligomer according to claim 19, wherein the additional binding portion is specific to myeloma cell surface antigen, for example, BCMA.

21. The CD16a-conjugated polypeptide or CD16a-conjugated oligomer according to claim 15, wherein the CD16a-conjugated polypeptide or CD16a-conjugated oligomer comprises at least two additional functional parts, each of which may be the same or different.

22. The CD16a-conjugated polypeptide or CD16a-conjugated oligomer according to claim 15, wherein the additional functional portion is separated from the CD16a-conjugated polypeptide or CD16a-conjugated oligomer by a linker.

23. A nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or encoding a CD16a-binding oligomer according to any one of claims 12 to 14 or 16.

24. An expression vector comprising a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16.

25. A host cell comprising an expression vector containing a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, or a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16.

26. A method for producing a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, comprising: maintaining a host cell containing an expression vector comprising a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, or a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, under conditions optimal for nucleic acid expression; and isolating the CD16a-binding polypeptide or CD16a-binding oligomer.

27. A CD16a-binding agent-drug conjugate comprising a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, and an additional therapeutic agent.

28. The CD16a conjugate-drug conjugate according to claim 27, wherein the additional therapeutic agent is a cytotoxic drug.

29. The CD16a-binding polypeptide is linked to the additional therapeutic agent via a linker, as described in Claim 27.

30. A pharmaceutical composition comprising an expression vector comprising a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a nucleic acid molecule encoding a CD16a-binding oligomer according to any one of claims 12 to 14 or 16, or a nucleic acid molecule encoding a CD16a-binding polypeptide according to any one of claims 1 to 11 or 15, or a nucleic acid molecule encoding a CD16a-binding oligomer according to any one of claims 12 to 14 or 16.

31. A pharmaceutical composition according to claim 30 for use in the treatment of cancer.

32. The pharmaceutical composition according to claim 31, wherein the cancer is multiple myeloma.

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