Novel polypeptides

Abstract

The present invention provides a CD16a binding polypeptide comprising at least one motif binding to CD16a wherein said polypeptide comprises the following structure: [N-terminal moiety]-[helix 1]-[isolated moiety]-[helix 2]-[C-terminal moiety], the CD16a binding motif being moiety [helix 1]-[isolated moiety]-[helix 2]. The invention also provides a pharmaceutical composition comprising the CD16a binding polypeptide, and the use of the CD16a binding polypeptide or pharmaceutical composition as a medicament, in particular for the treatment or prevention of cancer such as 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 a pharmaceutical composition comprising the immune cell engaging polypeptide, and their use in treating and / or preventing cancer. Background Art

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

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

[0004] NK cells are components of the innate immune system, and their functions include cytokine secretion and cell killing by secreting cytolytic granules containing perforin and granzyme. They can produce antibody-dependent cellular toxicity (ADCC) when target cells (such as tumor cells) are bound by IgG antibodies. The combination of Fc antibody region and CD16 receptors (FCγRIII) on NK cells exceeds the inhibitory signal, thereby triggering cytokine secretion and cracking of target cells (Vivier E et al., Functions ofnatural 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 Immunol2016, 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 a form that is mainly expressed on NK cells and found on monocytes. In contrast, CD16b is mainly found on neutrophils, although its expression can also be induced on eosinophils. Despite the high sequence similarity (about 96%) between CD16a and CD16b, CD16a has a much higher binding affinity for IgG. (Roberts JT and Barb AW, A singleamino 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 variation in the CD16a (FcγRIIIa) receptor itself can also alter 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 herein) results in either valine (V) or phenylalanine (F) at position 176, resulting in variable binding phenotypes (F / F: low affinity; V / V or V / F: high affinity, and therefore higher NK cell-mediated ADCC). (Chong KT et al., Distribution of the FcγRIIIa 176F / V polymorphism amongst healthy Chinese, Malaysians and Asian Indians 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 increasing interest in harnessing NK cell responses for cancer immunotherapy. These include the use of checkpoint inhibitor blockers, ex vivo expansion and administration of NK cells, the generation of CAR-NK cells (similar to CAR-T cell therapy), and the use of bivalent or multivalent NK cell conjugates that crosslink 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] Antibody-based NK engagers under development include Affimed’s AFM13 (anti-CD16a / CD30 tetravalent bispecific antibody) and AFM24 (anti-CD16a / EGFR IgG1-scFv fusion antibody). The ability of AFM13 and AFM24 to induce tumor cell killing by macrophages through antibody-dependent cellular phagocytosis (ADCP) was also tested (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 Tetravalent Bispecific Immune Cell Engagers 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 linked to the co-stimulatory molecule IL15.

[0008] Another 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. The properties of the antibody compound are described in Kakiuchi-Kiyota et al. (Leukemia (2022) 36: 1006-1014; https: / / doi.org / 10.1038 / s41375-021-01478-w). Plesner et al. reported the findings of a phase I dose escalation study of RO7297089 in patients with relapsed / refractory multiple myeloma (study registration NCT04434469) (poster abstract 2755, session 653, American Society of Hematology (ASH) meeting, December 12, 2021).

[0009] Despite these advances, antibody-derived NK conjugates still suffer from the inherent difficulties associated with existing antibody cancer therapies, particularly in terms of immunogenicity and tissue penetration. There remains a need for improved cancer immunotherapeutics that can be more effectively delivered to tumors while retaining the target specificity of antibodies and antibody-based drugs and avoiding potential side effects.

[0010] The present invention seeks to address the above needs. 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 comprises the following structure:

[0012] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0013] The CD16a binding motif is part [helix 1]-[detached part]-[helix 2].

[0014] In particular, the present invention provides a CD16a binding polypeptide, wherein:

[0015] 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 ,in

[0016] a) X9 is A, D, F, H, I, K, L, Q, R, T, V or Y;

[0017] X 10 It is Q;

[0018] X 11 Is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V,

[0019] W or Y;

[0020] X 13 is A, Q or V;

[0021] X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y;

[0022] X 17 is Q or R;

[0023] X 18 is A, D, E, F, H, I, K, N, Q, R, S, T, or V;

[0024] X 24 It is H;

[0025] X 25 is A or H;

[0026] X 27 is A, I, K, Q, R, S, T or V;

[0027] X 28 is F or Y;

[0028] X 31 is I or L;

[0029] X 32 is A, E, H, K, L, N, Q or R;

[0030] X 33 is K or S; and

[0031] X 35 is A, H, I, L, M, R or S; or

[0032] b) X9 is V; X 10 It is Q; X 11 It is M; X 13 It is Q; X 14 It is F;X 17 It is R;

[0033] X 18 It is K; X 24 Yes H; X25 Yes H; X 27 Yes S; X 28 It is F;X 31 It's I;

[0034] X 32 It is K; X 33 is S, and X 35 is M, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or

[0035] c) X9 is Q; X 10 It is F;X 11 It is Y; X 13 Yes R; X 14 Yes D; X 17 It is D;

[0036] X 18 Yes L; X 24 Yes E; X 25 Yes D; X 27 It is K; X 28 It is W; X 31 It is Y;

[0037] X 32 It is M; X 33 is S, and X 35 is I, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or

[0038] d) X9 is F; X 10 It is W; X 11 is I; X 13 Yes E; X 14 Yes S; X 17 Yes E; X 18 Yes S; X 24 is I; X 25 It is Y; X 27 It is K; X 28 It is W; X 31 It is K; X 32 It is Y; X 33 is S, and X 35is A, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0039] The present invention also provides a CD16a binding polypeptide, wherein:

[0040] i) Helix 1 contains the sequence X9 X 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 AF X 31 X 32 SL X 35 ,in

[0041] a*)X9 is V; X 10 It is Q; X 11 It is M; X 13 It is Q; X 14 It is F;X 17 Yes R; X 18 It is K; X 24 Yes H; X 25 Yes H; X 27 Yes S; X 28 It is F;X 31 is I; X 32 is K; and X 35 It is M;

[0042] b*)X9 is Q; X 10 It is F;X 11 It is Y; X 13 Yes R; X 14 Yes D; X 17 Yes D; X 18 Yes L; X 24 Yes E; X 25 Yes D; X 27 It is K; X 28 It is W; X 31 It is Y; X 32 is M; and X 35 is I; or

[0043] c*)X9 is F;X 10 It is W; X11 is I; X 13 Yes E; X 14 Yes S; X 17 Yes E; X 18 Yes S; X 24 is I; X 25 It is Y; X 27 It is K; X 28 It is W; X 31 It is K; X 32 is Y; and X 35 It is A;

[0044] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, at least 1 and no more than 3 X n The residue is replaced by an alternative residue and / or at least 1 and no more than 3 residues are not marked with an X n The residues of are replaced by residues;

[0045] The present inventors have surprisingly found that the CD16a binding polypeptides of the present invention based on non-antibody scaffolds are effective in engaging NK cells and triggering ADCC-mediated killing of cancer cells.

[0046] Thus, the present invention provides a novel CD16a binder that shows promise as an anti-cancer immunotherapeutic agent.

[0047] The present invention also provides a CD16a binding polypeptide, which consists of the CD16a binding polypeptide of the present invention; and optionally comprises additional binding moieties (eg, 1, 2, 3 or more additional binding moieties).

[0048] The present invention also provides a CD16a binding oligomer comprising at least two CD16a binding polypeptides of the present invention.

[0049] The invention also provides a CD16a binding polypeptide as defined herein or a CD16a binding oligomer as defined herein, which further comprises additional functional parts (eg at least one, at least two or at least three; eg 1, 2, 3, 4 or 5 additional functional parts).

[0050] Thus, in embodiments wherein the additional functional moiety is an additional binding moiety, the invention also provides a bispecific conjugate or a multispecific conjugate comprising the additional binding moiety.

[0051] The present invention also provides:

[0052] - a nucleic acid molecule encoding a CD16a binding polypeptide or CD16a binding oligomer of the present invention;

[0053] - an expression vector comprising such a nucleic acid molecule;

[0054] - A host cell comprising such a nucleic acid molecule or vector.

[0055] The present invention also provides a method for preparing the CD16a binding polypeptide or CD16a binding oligomer of the present invention.

[0056] 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.

[0057] The present invention also provides a CD16a binding polypeptide, a CD16a binding oligomer, a CD16a binding agent-drug conjugate, a nucleic acid molecule, an expression vector or a pharmaceutical composition of the present invention, which is used in medicine, in particular for treating cancer.

[0058] The present invention also provides 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 drug for treating cancer.

[0059] The present invention also provides a method for treating cancer, wherein the method comprises administering the CD16a binding polypeptide, CD16a binding oligomer, CD16a binding agent-drug conjugate, nucleic acid molecule, expression vector or pharmaceutical composition of the present invention to a patient in need thereof.

[0060] The present invention also provides a kit comprising a CD16a binding polypeptide, a CD16a binding oligomer, a CD16a binding agent-drug conjugate, a nucleic acid molecule, an expression vector or a pharmaceutical composition of the present invention, and optionally, one or more additional therapeutic agents. Such kits are particularly useful for treating and / or preventing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 shows (a) schematic domain construction of binding polypeptides described in the present disclosure; (b) examples of results from phage-ELISA experiments used to screen individual clones present in phage output after selection cycle four. In (b), assay responses to coating antigens (1) HSA, (2) streptavidin, (3) SLAMF7, and (4) hCD16a F158 were measured, and representative results are shown for clones corresponding to CD16a binding polypeptides A10, H09, and A11, respectively.

[0062] Figure 2Shown is SDS-PAGE analysis of expressed and IMAC purified His6-CD16a binding polypeptide-ABDWT constructs corresponding to SEQ ID 78 (lane 1), SEQ ID 76 (lane 2) and SEQ ID 77 (lane 3). Lane M, marker protein with molecular weight expressed in kilodaltons.

[0063] Figure 3 The injection of three purified hCD16a binding variants His6-A10-ABDWT [SEQ ID 76], His6-H09-ABDWT [SEQ ID 78] and His6-A11-ABD at concentrations ranging from 5 nM to 2.56 μM onto the sensor chip flow cell surface with hCD16a F158 or hCD16a V158 immobilized is shown. WT Sensorgrams obtained after [SEQ ID 77].

[0064] Figure 4 Shown are the results from competitive binding studies with hCD16a F158 and hCD16a V158 using the fusion protein His6-A10-ABD WT [SEQ ID 76], His6-H09-ABD WT [SEQ ID 78] and His6-A11-ABD WT Resulting sensorgrams of a pairwise epitope binding assay of [SEQ ID 77].

[0065] Figure 5 shows SDS-PAGE analysis of the various expressed and IMAC purified hCD16a binding constructs. (a) His6-A10-Cys [SEQ ID 79] (lane 1), His6-A11-Cys [SEQ ID 80] (lane 2), A10-A11-His6 [SEQID 83] (lane 3), A11-A10-His6 [SEQ ID 84] (lane 4), His6-A10-A11-Cys [SEQ ID 81] (lane 5), and His6-A11-A10-Cys [SEQ ID 82] (lane 6). The larger molecular weight bands visible in lanes 1, 2, 5, and 6 correspond to the expected presence of dimer species, which are held together by disulfide bonds formed between the C-terminal cysteines present in these constructs. (b) Anti-BCMA-A10-A10-His6 [SEQ ID 86] (lane 1). Lane M, marker protein with molecular weight expressed in kilodaltons.

[0066] Figure 6Shown are sensorgrams obtained after injection of hCD16a F158 or hCD16a V158 onto immobilized hCD16a monomeric binding variants His6-A10-Cys [SEQ ID 79] and His6-A11-Cys [SEQ ID 80] and heterodimeric (bi-para) binding variants His6-A10-A11-Cys [SEQ ID 81] and His6-A11-A10-Cys [SEQ ID 82], respectively.

[0067] Figure 7 Shown are sensorgrams obtained after injection of two purified hCD16a bi-paratopic binding variants A10-A11-His6 [SEQ ID 83] and A11-A10-His6 [SEQ ID 84] at concentrations ranging from 0.5 nM to 32 nM onto the sensor chip flow cell surface containing immobilized hCD16a F158 or hCD16a V158, respectively.

[0068] Figure 8 Shown are sensorgrams obtained from a single cycle kinetic experiment performed with a fusion protein containing the heterodimeric A10-A10CD16a binding polypeptide combination of SEQ ID 86 attached to a 1-E6 BCMA binding polypeptide [SEQ ID 86], injected at five concentrations ranging from 12 nM to 1000 nM onto a sensor chip surface containing immobilized hCD16a F158 or hCD16a V158 protein.

[0069] Fig. 9 SDS-PAGE analysis of the purified potential dual conjugate protein anti-hBCMA-H09-A10-His6 [SEQ ID 85] is shown (lane 1). Lane M, marker protein with molecular weight expressed in kilodaltons.

[0070] Figure 10 shows the experimental setup (a) and resulting sensorgrams (b) obtained after sequential injections of the dual conjugate constructs anti-hBCMA-H09-A10-His6 [SEQ ID 85] (injection I) and hCD16a F158 or V158 (injection III) onto a sensor chip surface containing immobilized hBCMA-rabbit Fc (hBCMA-rFc) fusion protein.

[0071] Fig.11Relative IFNγ secretion in cell culture medium of co-cultures of MM.1S and PBMCs exposed to dual conjugates for 4 hours is shown. The conjugates evaluated were anti-BCMA-A10-His6 [SEQ ID 88], anti-BCMA-H09-A10-His6 [SEQ ID 85], anti-BCMA-A10-A10-His6 [SEQ ID 86], anti-BCMA-A11-A10-His6 [SEQ ID 87] and their corresponding non-BCMA binding counterparts (inactive).

[0072] Fig.12 CD spectra (before and after heating) and thermal melting curves of CD16a binding polypeptides A10, H09 and A11 [SEQ ID 1, 74 and 75], all equipped with a C-terminal His6 tag, are shown.

[0073] Fig.13 Shown are the sensorgrams obtained after injection of 14 alanine substituted variants (in the form of polypeptide-YY-His6, typical concentration 200 nM) on the surface of a sensor chip containing CD16a (F158) protein.

[0074] Fig.14 The CD spectra (before and after heating) of a 14-alanine substituted variant of the CD16a binding polypeptide A10 (polypeptide-YY-His6 form) are shown.

[0075] Fig.15a Thermal melting curves of 14 alanine-substituted variants of the CD16a binding polypeptide A10 (polypeptide-YY-His6 format) are shown.

[0076] Fig.15b The results of the binding experiment of fusion proteins containing the polypeptide having SEQ ID 1 or one of the 13 alanine-substituted variants of the CD16a binding polypeptide A10 variant (polypeptide-YY-His6 format) and CD16a F158 are shown.

[0077] Fig.15c Shows Fig.15b Plot of the respective association rate (M-1s-1) and dissociation rate (s-1) kinetic constants for the compounds analyzed in .

[0078] Fig.16a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0079] Fig.16bEnhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0080] Fig.17a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0081] Fig.17b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0082] Fig.18 Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0083] Fig.19a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0084] Fig.19b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0085] Fig.20a Shown are the presence or absence of BCMA-positive MM.1S cell lines in Jurkat-Lucia TM Responses in the NFAT-CD16 reporter assay. Responses were normalized to the maximal response to elotuzumab.

[0086] Fig.20b Enhancement of NK cell-mediated cell killing of the BCMA-positive MM.1S cell line is shown. Responses were normalized to the maximal response mediated by the belantamab biosimilar.

[0087] Fig.21a Shown are the results of cell killing assays using NK cells and MM.1s cells.

[0088] Figure 21b Shown are the results of an 8-hour flow cytometry-based killing assay using NK cells and MM.1s (E:T 5:1) treated with increasing concentrations of any of the three dual conjugate constructs of the invention.

[0089] Fig. 22 The hBCMA target specificity of the dual conjugate construct with SEQ ID 85 in an in vitro multiple myeloma cell cytotoxicity assay is demonstrated.

[0090] Figure 23 shows sequences and binding data for exemplary compounds of the invention.

[0091] Figure 24 shows the sequences of the binding motifs of the compounds of the invention. DETAILED DESCRIPTION

[0092] The present inventors have found that polypeptides as disclosed herein are effective binding agents for CD16a and effectively engage immune cells. It has been found that polypeptides trigger strong ADCC responses for cancer cells in in vitro models. It is noteworthy that the present inventors have found that such anticancer responses are advantageous in models compared with those obtained using the monoclonal antibody elotuzumab, which is approved for the treatment of multiple myeloma. The present inventors have also found that such anticancer responses are advantageous in models compared with those obtained using the biosimilars of belantamab mafodotin (which is an antibody-drug conjugate for the treatment of multiple myeloma) and those obtained using daratumumab (Daratumumab), a monoclonal antibody approved for the treatment of multiple myeloma.

[0093] In its broadest aspect, the present invention provides a CD16a binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide comprises the following structure:

[0094] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0095] The CD16a binding motif is part [helix 1]-[detached part]-[helix 2].

[0096] The various elements of the polypeptides of the present invention will now be described in more detail:

[0097] Overall structure

[0098] In one embodiment, the polypeptides of the invention may be based on a triple helical scaffold, sometimes referred to as an "affimer". An affibody is a small (about 6.5 kDa) engineered affinity ligand based on a Z domain polypeptide, which is a mutant form of the B domain in the immunoglobulin binding region of Staphylococcal protein A (Nord K et al., Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain, Nature Biotech, 1997: 15: 772, doi: 10.1038 / nbt0897-772). In the full-length affibody, the C-terminal portion includes [second separation portion] - [helix 3] - [C-terminal sequence]. The general structure of the affibody is shown in Figure 1a middle.

[0099] The portions of the molecules of the invention referred to as helix 1 and helix 2 (and when present, helix 3) are generally helical in structure. In some rare embodiments, it may be found that the structures established by sequences with specific residues may not be strictly helical. Such compounds are considered to be within the broadest aspects of the invention. More preferably, the residues in the helix 1 and helix 2 portions do cause those structures to be helical, in the sense of an alpha-helix.

[0100] Sequences of the polypeptides of the present invention

[0101] The sequence of CD16a binding polypeptides as disclosed herein can be expressed according to their constituent amino acids or according to the nucleic acid sequence encoding the polypeptides with those amino acid sequences. In the context of the present disclosure, the term "amino acid" includes any naturally occurring amino acid or non-natural amino acid. The term "non-natural amino acid" as used herein refers to non-proteinogenic (i.e., non-encoded) amino acids, which are found in nature or chemically synthesized (e.g., citrulline, hydroxyproline, β-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid or nitroarginine). It includes α, β, γ and δ amino acids. It includes amino acids of any chiral configuration. In particular, amino acids can be naturally occurring α amino acids. Amino acids can be especially naturally occurring L amino acids. Amino acids can be especially naturally occurring L-α amino acids.

[0102] In a polypeptide chain (e.g., a CD16a binding polypeptide disclosed herein), amino acids are linked by peptide bonds between the carboxyl group of one amino acid and the amine group of the next amino acid in the chain. A single amino acid, once linked in a polypeptide chain, is referred to as a "residue" or "amino acid residue."

[0103] The N-terminus of the amino acid sequences herein is shown on the left, and in the case where the sequence is shown across multiple lines, the N-terminus is at the top left. Unless otherwise indicated, the amino acid residues in the sequence are L-amino acids.

[0104] Amino acid sequences listed in this application are shown using standard letter abbreviations for amino acids.

[0105] Specific sequences given herein are relevant to specific embodiments of the invention.

[0106] The present disclosure also includes derivatives of all sequences described herein (e.g., derivatives of each of the CD16a binding polypeptide and CD16a binding oligomer sequences described herein). Derivatives of sequences described herein are preferably derivatives in which 1 to 5 (e.g., 1, 2, or 3 amino acid residues) can be replaced by alternative residues, such as different naturally occurring amino acids or different non-natural amino acids; or different naturally occurring amino acids or different non-natural amino acids that do not include methionine. Preferably, the non-natural amino acids according to the present invention are amino acids that are isosteric with naturally occurring amino acids (e.g., norleucine).

[0107] For example, in one embodiment, one or more (e.g., each) methionine residues of the sequences described herein may be replaced by different naturally occurring amino acids or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine; and in particular isoleucine and norleucine. For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues or 1 methionine residue may be replaced by different naturally occurring amino acids or non-natural amino acids, such as amino acids selected from isoleucine, leucine, glutamine, and norleucine; and in particular isoleucine and norleucine.

[0108] For example, in which X 11 In embodiments where X is or is methionine, 11 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 35 In embodiments where X is or is methionine, 35 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 11 and X 35 In embodiments where X is or is methionine, 11 and X35 The residue at X can be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine. For example, in which X 32 In embodiments where X is or is methionine, 32 The residue at may be replaced by a different naturally occurring amino acid or a non-natural amino acid, such as an amino acid selected from isoleucine, leucine, glutamine and norleucine; and in particular isoleucine and norleucine.

[0109] Alternatively or additionally, in certain embodiments, the sequences described herein (eg, the CD16a binding polypeptide and CD16a binding oligomer sequences described herein) may contain amino acid substitutions, wherein one or more residues are replaced with a non-natural amino acid.

[0110] For example, in one embodiment, one or more residues of a sequence described herein (e.g., a CD16a binding polypeptide and a CD16a binding oligomer sequence described herein) can be replaced by a non-natural amino acid (e.g., norleucine). For example, 1 to 15 residues can 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), 1 to 3 residues (e.g., 1, 2 or 3) or 1 residue can be replaced by a non-natural amino acid (e.g., norleucine).

[0111] For example, in one embodiment, one or more leucine residues of the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) can be replaced by non-natural amino acids, and preferably norleucine. For example, when present, 1 to 5 leucine residues, 1 to 3 leucine residues (e.g., 1, 2 or 3 leucine residues) or 1 or 2 leucine residues or 1 leucine residue can be replaced by non-natural amino acids (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein), at the positions where leucine residues are listed, the polypeptide has a sequence in which leucine residues are independently replaced by non-natural amino acids, and preferably norleucine.

[0112] Alternatively or additionally, in one embodiment, one or more methionine residues of the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) can be replaced by non-natural amino acids, and preferably norleucine. For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2 or 3 methionine residues) or 1 or 2 methionine residues or 1 methionine residue can be replaced by non-natural amino acids (e.g., norleucine). For example, in certain embodiments, in the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein), at the positions where methionine residues are listed, the polypeptide has a sequence in which methionine residues are independently replaced by non-natural amino acids, and preferably norleucine.

[0113] For example, in which X 11 In embodiments where X is or is methionine, 11 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 35 In embodiments where X is or is methionine, 35 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 11 and X 35 In embodiments where X is or is methionine, 11 and X 35 The residue at position X can be replaced by an unnatural amino acid (e.g., norleucine). 32 In embodiments where X is or is methionine, 32 The residue at may be replaced by an unnatural amino acid (eg, norleucine).

[0114] In certain embodiments, one or more methionine residues of the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may be oxidized, for example, in the form of methionine sulfoxide ("Met(O)"). For example, when present, 1 to 5 methionine residues, 1 to 3 methionine residues (e.g., 1, 2, or 3 methionine residues), or 1 or 2 methionine residues or 1 methionine residue may be oxidized (e.g., may be Met(O)). For example, in which X 11 In embodiments where X is or is methionine, when present, 11 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 35 In embodiments where X is or is methionine, when present, methionine may be oxidized (e.g., Met(O)). 11 and X35 In embodiments where methionine is present, X 11 and X 35 The methionine at the position may be oxidized (e.g., Met(O)). For example, in which X 32 In embodiments where methionine is or may be present, methionine, when present, may be oxidized (e.g., Met(O))

[0115] Alternatively or additionally, in certain embodiments, the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) comprise a peptide purification tag or moiety (e.g., a histidine tag (e.g., a polyhistidine tag optionally comprising 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, e.g., a signal peptide selected from OmpA, DsbA, PhoA, and PelB), a fluorescent tag (e.g., Alexa448), or a tag or moiety that assists conjugation (a cysteine ​​tag (e.g., a single cysteine ​​at the C-terminus or N-terminus)). Such tags and / or moieties may preferably be present at the N-terminus and / or C-terminus of the CD16a binding polypeptides and CD16a binding oligomer sequences described herein.

[0116] Therefore, the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include at least 1 histidine residue (and optionally at least 1 tyrosine residue) and / or at least 1 methionine residue; for example, at least 4, at least 5 or at least 6 histidine residues (and optionally at least 1 tyrosine residue, such as 1, 2 or 3 tyrosine residues) and / or at least 1 methionine residue Additional sequence. In one embodiment, the sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include at least 6 histidine residues and optionally at least 1 tyrosine residue (e.g., 6 histidine residues (e.g., HHHHH) or 6 histidine residues and two tyrosine residues (e.g., YYHHHHHH)) and / or at least 1 methionine residue (e.g., 1 or 2 methionine residues) Additional sequence. Peptide purification tags or moieties (e.g., histidine tags or methionine tags as described above) may preferably be present at the N-terminus and / or C-terminus of the CD16a binding polypeptides and CD16a binding oligomer sequences described herein. For example, additional sequences of at least 6 histidine residues (e.g., 6 histidine residues; or 6 histidine residues and two tyrosine residues) and / or at least 1 methionine residue (e.g., 1 or 2 methionine residues) may be present at the N-terminus and / or C-terminus of the CD16a binding polypeptides and CD16a binding oligomer sequences described herein.

[0117] Sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include additional sequences of at least one cysteine ​​(e.g., one cysteine) at the N-terminus or C-terminus. Sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include fluorophore tags (e.g., 448 Alexa tags) at the N-terminus or C-terminus. Sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include signal peptides at the N-terminus or C-terminus, preferably at the N-terminus, such as selected from OmpA, DsbA, PhoA and PelB. Sequences described herein (e.g., CD16a binding polypeptides and CD16a binding oligomer sequences described herein) may also include additional sequences of at least one glycine (e.g., one glycine) at the N-terminus or C-terminus.

[0118] In certain embodiments, the CD16a binding polypeptide is a polypeptide wherein the CD16a binding efficacy is at least 1%, at least 5%, or preferably at least 10% (more preferably at least 15%, 20%, 25% or 50%) of SEQ ID NO: 1, 74 or 75. When a CD16a binding polypeptide is described herein as having a CD16a binding efficacy of at least X% of a specific peptide (e.g., SEQ ID NO: 1, 74 or 75), it is understood that the IC for a polypeptide binding to a CD16a receptor when measured under the same conditions is 50 The concentration does not exceed the IC of the specific peptide (SEQ ID NO: 1, 74 or 75) for the CD16a receptor. 50 100 / X times the concentration.

[0119] For example, if the binding potency of a CD16a binding polypeptide is at least 5%, 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), that is, the IC of a surrogate polypeptide for binding to a CD16a receptor when measured under the same conditions is 50 The concentration does not exceed the IC of the specific peptide (e.g., SEQ ID NO: 1, 74 or 75) for the CD16a receptor. 50 The concentration is 20 times, and more preferably no more than 10 times, 5 times, 4 times or 2 times.

[0120] In certain embodiments, alternatively or additionally, the CD16a binding polypeptide is a polypeptide that competes with SEQ ID NO: 1, 74 and / or 75.

[0121] The CD16a binding polypeptides of the present invention have binding affinity for the CD16a receptor; and may optionally have binding affinity for the CD16b receptor. For example, in certain embodiments, the CD16a binding polypeptides of the present invention have similar binding affinity for the CD16a receptor and the CD16b receptor. In certain embodiments, the CD16a binding polypeptides of the present invention have a stronger binding affinity for the CD16a receptor than for the CD16b receptor, and in certain embodiments, the CD16a binding polypeptides of the present invention have a stronger binding affinity for the CD16b receptor than for the CD16a receptor.

[0122] The CD16a binding polypeptide of the present invention may have binding affinity for one or two genotype variants FcγRIIIa-176V / F polymorphisms (rs396991) of the CD16a (FcγRIIIa) receptor (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 binding affinity for two genotype variants FcγRIIIa-176V / F polymorphisms (rs396991) of the CD16a (FcγRIIIa) receptor. In certain embodiments, the CD16a binding polypeptide of the present invention has a stronger binding affinity for the valine (V) position 176 phenotype, or has a stronger binding affinity for the phenylalanine (F) position 176 phenotype, or has similar binding to the F and V position 176 phenotypes.

[0123] The present invention provides a CD16a binding polypeptide comprising at least one motif that binds to CD16a, wherein the polypeptide comprises the following structure:

[0124] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0125] The CD16a binding motif is part [helix 1]-[detached part]-[helix 2].

[0126] Preferably, helix 1 comprises 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 ,in

[0127] a) X9 is A, D, F, H, I, K, L, Q, R, T, V or Y;

[0128] X 10 It is Q;

[0129] X 11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y;

[0130] X 13 is A, Q or V;

[0131] X 14 is A, F, H, I, K, L, N, Q, R, S, T, V, W or Y;

[0132] X 17 is Q or R;

[0133] X 18 is A, D, E, F, H, I, K, N, Q, R, S, T, or V;

[0134] X 24 It is H;

[0135] X 25 is A or H;

[0136] X 27 is A, I, K, Q, R, S, T or V;

[0137] X 28 is F or Y;

[0138] X 31 is I or L;

[0139] X 32 is A, E, H, K, L, N, Q or R;

[0140] X 33 is K or S; and

[0141] X 35 is A, H, I, L, M, R or S; or

[0142] b) X9 is V; X 10 It is Q; X 11 It is M; X 13 It is Q; X 14 It is F;X 17 Yes R; X 18 It is K; X 24 Yes H; X 25 Yes H; X 27 Yes S; X 28 It is F;X 31is I; X 32 It is K; X 33 is S, and X 35 is M, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or

[0143] c) X9 is Q; X 10 It is F;X 11 It is Y; X 13 Yes R; X 14 Yes D; X 17 Yes D; X 18 Yes L; X 24 Yes E; X 25 Yes D; X 27 It is K; X 28 It is W; X 31 It is Y; X 32 It is M; X 33 is S, and X 35 is I, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or

[0144] d) X9 is F; X 10 It is W; X 11 is I; X 13 Yes E; X 14 Yes S; X 17 Yes E; X 18 Yes S; X 24 is I; X 25 It is Y; X 27 It is K; X 28 It is W; X 31 It is K; X 32 It is Y; X 33 is S, and X 35 is A, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0145] In certain embodiments, the CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; X 10 It 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 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 Yes H; X 25 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; and X 35 It is A, H, I, L, M, R or S.

[0146] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, R, T, V or Y; X 10 It is Q; X11 is A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 13 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 Yes 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; and X 35 It is A, H, I, L, M, R or S.

[0147] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, R, T, V or Y; X 10 It 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 A, Q or V; X 14 is F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 17 Yes R; X 18 is A, D, E, F, H, K, N, Q, R, S, T, or V; X 24 Yes H; X 25 Yes H; 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, N, Q or R; X 33 is K or S; and X35 It is A, H, I, L, M, R or S.

[0148] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, R, T, V or Y; X 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 A, Q or V; X 14 is 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 Yes H; X 25 is H or A; 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, L, N, Q, or R; X 33 is K or S; and X 35 It is A, H, I, L, R or S.

[0149] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, R, T, V or Y; X10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 A, Q or V; X 14 is H, I, K, L, N, Q, R, S, T, V, W, or Y; X 17 Yes R; X 18 is A, D, E, F, H, K, N, Q, R, S, T, or V; X 24 Yes H; X 25 Yes 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; and X 35 It is A, H, I, L, R or S.

[0150] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a fast binding or "association" rate to the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, R, T, V or Y; X 10 It is Q; X 11 is A, D, E, F, H, I, K, N, Q, R, S, T, V, W, or Y; X 13 A, Q or V; X 14 is H, I, K, L, N, Q, R, S, V, W, or Y; X 17 Yes R; X 18 is A, D, E, F, H, K, N, Q, R, S, or T; X 24 Yes H; X 25 Yes H; X 27 is A, I, K, Q, R, T or V; X 28 It is F;X 31 Is I or L; X 32is A, E, H, K, N, Q or R; X 33 is K or S; and X 35 It is H, I, L, R or S.

[0151] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a slow dissociation rate or "off" rate for the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is D, F, H, I, K, L, Q, T, V or Y; X 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 A or Q; X 14 is H, I, K, L, Q, R, S, T, V, W, or Y; X 17 Yes R; X 18 is A, D, E, F, H, K, N, Q, R, S, T, or V; X 24 Yes H; X 25 Yes 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; and X 35 It is A, H, I, L or R.

[0152] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a high binding affinity for the CD16a receptor, e.g., a K of 1.0 for the CD16a receptor. D Less than 250 nM. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18and helix 2 contains the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 , where X9 is D, F, H, I, K, L, Q, R, T, V, X 10 It is Q; X 11 is A, D, E, F, H, I, K, N, Q, R, S, T, V, W, or Y; X 13 A or Q; X 14 is H, I, K, L, Q, R, S, V, W, or Y; X 17 Yes R; X 18 is A, F, H, K, N, Q, R, S, or T; X 24 Yes H; X 25 Yes 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; and X 35 It is A, H, I, L, R or S.

[0153] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a particularly fast binding or "association" rate to the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is F, L, Q, T or Y; X 10 It is Q; X 11 is A, F, H, I, L, N, Q, S, or Y; X 13 A or Q; X 14 is I, K, Q, R or V; X 17 Yes R; X 18 is A, E, H, K, Q, R, T, or V; X 24 Yes H; X 25Yes H; X 27 is A, I, K, Q, R or V; X 28 It is F;X 31 is I; X 32 is A, H, K, N, Q or R; X 33 is K or S; and X 35 It is H, I or L.

[0154] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a particularly slow dissociation rate or "off" rate for the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is I, L, Q, T or V; X 10 It is Q; X 11 is A, E, F, H, I, S, V, or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is A, H, K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is I, K, Q, R, T or V; X 28 It is F;X 31 Is I or L; X 32 is K, N or R; X 33 is K or S; and X 35 It is I or L.

[0155] In certain preferred embodiments, the CD16a binding polypeptides of the present invention have a particularly high binding affinity for the CD16a receptor, for example, a KD for the CD16a receptor of less than 100 nM. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18and helix 2 contains the sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 , where X9 is I, L, Q or V; X 10 It is Q; X 11 is A, E, H, I, S, W, or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is A, H, K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is K, Q, R, T or V; X 28 It is F;X 31 is I; X 32 is K, N or R; X 33 is K or S; and X 35 It is I or L.

[0156] In certain preferred embodiments, the CD16a binding polypeptides of the invention have at least two of the following: a fast binding 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, such as a KD of less than 100 nM for the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 , where X9 is I, L, Q or V; X 10 It is Q; X 11 is A, E, H, I, S or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is H, K, Q, R, S or T; X 24Yes H; X 25 Yes H; X 27 is K, Q, R, T or V; X 28 It is F;X 31 is I; X 32 is N or K; X 33 is K or S; and X 35 It is I or L.

[0157] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a particularly slow dissociation rate or "off" rate for the CD16a receptor and a high binding affinity for the CD16a receptor, such as a K of 0.1 for the CD16a receptor. D Less than 100 nM. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 , where X9 is L or V; X 10 It is Q; X 11 is A, I, S or Y; X 13 It is Q; X 14 is K, R or V; X 17 Yes R; X 18 is K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is K, R or V; X 28 It is F;X 31 is I; X 32 is N or K; X 33 is K or S; and X 35 It is I or L.

[0158] In certain preferred embodiments, the CD16a binding polypeptides of the invention bind to CD16a and CD16b receptors. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 and helix 2 contains the sequence X24 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; X 10 It is Q; X 11 is A, D, E, F, H, I, K, N, R, S, V, W, or Y; X 13 It is Q; X 14 is K, L, Q, R, S, V, W or Y; X 17 Yes R; X 18 is A, H, K, N, Q, R, S or T; X 24 Yes H; X 25 Yes 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; and X 35 It is A, I, L or R.

[0159] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a binding preference for the CD16a receptor over the CD16b receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 , where X9 is K, Q or Y; X 10 It is Q; X 11 is I or Q; X 13 It is Q; X 14 W or Y; X 17 Yes R; X 18 is H, K or R; X 24 Yes H; X 25 Yes H; X 27 A, K or T; X 28 It is F;X 31 is I; X32 A, K or Q; X 33 is K or S; and X 35 is I or L;

[0160] In certain preferred embodiments, the CD16a binding polypeptides of the invention have a binding preference for the CD16b receptor over the CD16a receptor. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 X9 is L, V or Y; X 10 It is Q; X 11 is I, N or Q; X 13 It is Q; X 14 is K, R or Q; X 17 Yes R; X 18 is E, A or V; X 24 Yes H; X 25 Yes H; X 27 It is K or Q; X 28 It is F;X 31 is I; X 32 is H, K or Q; X 33 is K or S; and X 35 It is I or L.

[0161] In certain highly preferred embodiments, the CD16a binding polypeptides of the invention are particularly active in functional assays, such as CD16 reporter assays and / or cell killing assays. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 , where X9 is L, Q, T or V; X10 It is Q; X 11 is I, V or Y; X 13 It is Q; X 14 is K, R or Y; X 17 Yes R; X 18 is K, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is I, T or V; X 28 It is F;X 31 Is I or L; X 32 is K or N; X 33 is K or S; and X 35 It is I or L.

[0162] In certain particularly preferred embodiments, the CD16a binding polypeptides of the invention are very particularly active in functional assays, such as CD16 reporter assays and / or cell killing assays. For example, a CD16a binding polypeptide is a polypeptide wherein helix 1 comprises 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 , where X9 is L, T or V; X 10 It is Q; X 11 is I, V or Y; X 13 It is Q; X 14 is K or R; X 17 Yes R; X 18 is R, S or T; X 24 Yes H; X 25 Yes H; X 27 is I or V; X 28 It is F;X 31 Is I or L; X 32 is K or N; X 33 is K; and X 35 It is I or L.

[0163] In certain embodiments of the invention, the CD16a binding polypeptide is at position X 11 or X 35 A polypeptide having no methionine residue at any position of the polypeptide, such as a CD16a binding polypeptide, wherein:

[0164] 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 ,in:

[0165] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y, or an unnatural amino acid (e.g., norleucine); X 13 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 Yes H; X 25 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; and X 35 is A, H, I, L, R, S or an unnatural amino acid (e.g., norleucine); or

[0166] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It 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 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; X24 Yes H; X 25 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; 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

[0167] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 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 Yes H; X 25 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; and X 35 It is A, H, I, L, R or S.

[0168] In another embodiment, at position X 11 or X 35 The CD16a binding polypeptide does not have a methionine residue at the position of the CD16a binding polypeptide, wherein the CD16a binding polypeptide is a polypeptide wherein: Helix 1 comprises 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 ,in

[0169] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, or an unnatural amino acid (e.g., norleucine); X 13 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 Yes H; X 25 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; and X 35 is A, H, I, L, R, S or an unnatural amino acid (e.g., norleucine); or

[0170] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It 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 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 Yes H; X 25 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; 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

[0171] X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 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 Yes H; X 25 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; and X 35 It is A, H, I, L, R or S.

[0172] In another embodiment, at position X 11 or X 35 The CD16a binding polypeptide is a polypeptide having no methionine residue at any position thereof, wherein:

[0173] 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 ,in

[0174] X9 is D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y, or an unnatural amino acid (e.g., norleucine); X 13 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 18is A, D, E, F, H, I, K, N, Q, R, S, T, or V; X 24 Yes 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; and X 35 is A, H, I, L, R, S or an unnatural amino acid (e.g., norleucine); or

[0175] X9 is D, F, H, I, K, L, Q, R, T, V or Y; 10 It 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 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 Yes 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; 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

[0176] X9 is D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 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; X24 Yes 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; and X 35 It is A, H, I, L, R or S.

[0177] In certain embodiments, the CD16a binding polypeptide is a polypeptide wherein the CD16a binding efficacy is at least 1%, at least 5%, or preferably at least 10% (more preferably at least 15%, 20%, 25% or 50%) of SEQ ID NO: 1 (i.e., the binding efficacy of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions). Preferably, the CD16a binding polypeptide is a polypeptide wherein the CD16a binding efficacy is at least 10% of that of SEQ ID NO: 1.

[0178] In certain embodiments, alternatively or additionally, the CD16a binding polypeptide is a polypeptide that competes with SEQ ID NO:1.

[0179] In an advantageous embodiment of the CD16a binding polypeptide of the invention, proline (P) and cysteine ​​(C) are not present in helix 1 or helix 2 of the CD16a binding polypeptide of the invention. Advantageously, glycine (G) is also not present in helix 1 or helix 2 of the CD16a binding polypeptide of the invention.

[0180] In certain embodiments, the CD16a binding polypeptide of the invention is a polypeptide wherein:

[0181] Helix 1 contains the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 , and / or

[0182] Helix 2 contains sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37, wherein X6 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or does not exist (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 does not exist (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 does not exist (e.g., X8 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably E)); X 19 is 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); 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); 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 a naturally occurring amino acid (preferably D, E, N or Q; more preferably D)).

[0183] In certain embodiments, the CD16a binding polypeptide of the invention is a polypeptide wherein:

[0184] Helix 1 contains the sequence X6X7 X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 , and / or helix 2 contains sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 ,

[0185] wherein X6 is D, E, N or Q (preferably N) or absent (e.g., X6 is D, E, N or Q, more preferably N); X7 is H, K or R (preferably K) or absent (e.g., X7 is H, K or R; and more preferably K); X8 is D, E, N or Q (preferably E) or absent (e.g., X8 is D, E, N or Q; and more preferably E);

[0186] X 19 is G, A, V, L or I (preferably L) or absent (eg X 19 is G, A, V, L or I; and more preferably L); X 23 is D, E, N or Q (preferably N) or absent (eg X 23 is D, E, N or Q; and more preferably N); X 36 is D, E, N or Q (more preferably D) or absent (eg X 36 is D, E, N or Q; and more preferably D); X 37 is D, E, N or Q (preferably D) or absent (eg X 37 is D, E, N or Q; and more preferably D).

[0187] In certain embodiments of the invention, such as those described above:

[0188] Helix 1 contains the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 , and / or

[0189] Helix 2 contains sequence X 23 X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 .

[0190] In such embodiments, X6 can be any naturally occurring amino acid or not present; X7 can be any naturally occurring amino acid or not present; X8 can be any naturally occurring amino acid or not present; X 19 Can be any naturally occurring amino acid or absent; X 23 Can be any naturally occurring amino acid or absent; X 36 can be any naturally occurring amino acid or be absent; and X37 It may be any naturally occurring amino group or it may be absent.

[0191] More preferably, X6 can be any naturally occurring amino acid; X7 can be any naturally occurring amino acid; X8 can be any naturally occurring amino acid; X 19 Can be any naturally occurring amino acid; X 23 Can be any naturally occurring amino acid; X 36 can be any naturally occurring amino acid; and X 37 Any naturally occurring amino group may be used.

[0192] In another preferred embodiment, X6 can be D, E, N, Q or absent; X7 can be H, K, R or absent; X8 can be any of D, E, N, Q or absent; X 19 Can be G, A, V, L, I or absent; X 23 Can be G, A, V, L, I or absent; X 36 can be G, A, V, L, I or absent; and X 37 It can be G, A, V, L, I or absent. More preferably, X8 can be E or absent; 19 Can be L or absent; X 23 Can be D or not exist; X 36 can be D or absent; and X 37 Can be D or absent.

[0193] 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 of D, E, N or Q; X 19 Can be G, A, V, L or I; X 23 Can be G, A, V, L or I; X 36 can be G, A, V, L, or I; and X 37 Can be G, A, V, L or I.

[0194] In another preferred embodiment, X6 may be N or absent; X7 may be K or absent; X8 may be E or absent; 19 Can be L or absent; X 23 Can be D or not exist; X 36 can be D or absent; and X 37 Can be D or absent.

[0195] In a particularly preferred embodiment, X6 is N; X7 is K; and X8 is E. In another particularly preferred embodiment, X 36 is D; and X 37It is D.

[0196] In a very particularly preferred embodiment, X6 is N; X7 is K; X8 is E; X 19 Yes L; X 23 Yes D; X 36 is D; and X 37 is D. In such embodiments,

[0197] Helix 1 contains the sequence NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 L, and / or

[0198] Helix 2 contains the sequence DX 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 DD.

[0199] In one embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0200] Helix 1 contains the sequence NKEVQMAQFEIRKL, and helix 2 contains the sequence NHHQSFAFIKSLMDD;

[0201] And optionally wherein at least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5; or for example at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

[0202] In certain embodiments, such CD16a binding polypeptides have 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, the CD16a binding efficacy of such CD16a binding polypeptides is at least 10% of that of SEQ ID NO: 1. In certain embodiments, alternatively or additionally, such CD16a binding polypeptides are polypeptides that compete with SEQ ID NO: 1.

[0203] In one embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0204] Helix 1 contains the sequence NKEQFYARDEIDLL, and helix 2 contains the sequence NEDQKWAFYMSLIDD;

[0205] And optionally wherein at least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5; or for example at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

[0206] In certain embodiments, such CD16a binding polypeptides have 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: 74. Preferably, the CD16a binding efficacy of such CD16a binding polypeptides is at least 10% of that of SEQ ID NO: 74. In certain embodiments, alternatively or additionally, such CD16a binding polypeptides are polypeptides that compete with SEQ ID NO: 74.

[0207] In one embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0208] Helix 1 contains the sequence NKEFWIAESEIESL, and helix 2 contains the sequence NIYQKWAFKYSLADD;

[0209] And optionally wherein at least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5; or for example at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

[0210] In certain embodiments, such CD16a binding polypeptides have 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: 75. Preferably, the CD16a binding efficacy of such CD16a binding polypeptides is at least 10% of that of SEQ ID NO: 75. In certain embodiments, alternatively or additionally, such CD16a binding polypeptides are polypeptides that compete with SEQ ID NO: 75.

[0211] In one embodiment, the CD16a binding motif as part [helix 1]-[isolated part]-[helix 2] is (ie has the sequence):

[0212] X6X7X8X9X 10 X 11 AX 13 X14 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

[0213] in

[0214] X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X 22 is any naturally occurring amino acid, and wherein optionally, X 20 , X 21 or X 22 One or both (eg, optionally 1) of are absent; and the other residues are as defined above.

[0215] More preferably, X6 can be D, E, N, Q or not exist; X7 can be H, K, R or not exist; X8 can be any of D, E, N, Q or not exist; X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 As defined above; X 19 Can be G, A, V, L, I or absent; 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 absent; X 23 Can be G, A, V, L, I or absent; X 36 can be G, A, V, L, I or absent; and X 37 can be G, A, V, L, I or absent; and wherein optionally, X 20 , X 21 , X 22 One or both (eg, optionally 1) of are not present.

[0216] Preferably, X6 can be D, E, N or Q; X7 can be H, K or R; X8 can be any of D, E, N or Q; X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 As defined above; X 19 Can be G, A, V, L or I; X 20 Can be S, T, M, P, F, Y or W (e.g. P or T); X 21 can be D, E, N, or Q; and X 22 Can be G, A, V, L or I; X 23 Can be G, A, V, L or I; X 36 can be G, A, V, L, or I; and X 37 can be G, A, V, L or I; and wherein optionally, X 20 , X 21 , X 22 One or both (eg, optionally 1) of are not present.

[0217] For example, X6 can be D, E, N, or Q; X7 can be H, K, or R; X8 can be any of D, E, N, or Q; X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 As defined above; X 19 Can be G, A, V, L or I; X 20 Can be S, T, M, P, F, Y or W (e.g. P or T); X 21 can be D, E, N, or Q; and X 22 Can be G, A, V, L or I; X 23 Can be G, A, V, L or I; X 36 can be G, A, V, L, or I; and X 37 Can be G, A, V, L or I.

[0218] More preferably, X8 may be E or absent; X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 As defined above; X 19 Can be L or absent; X 23 Can be D or not exist; X 24 , X 25 , X27 , X 28 , X 31 , X 32 , X 33 and X 35 As defined above, and X 36 can be D or absent; and X 37 Can be D or absent.

[0219] In particularly preferred embodiments, X6 is N; X7 is K; X8 is E; X9, X 10 , X 11 , X 13 , X 14 , X 17 and X 18 As defined above; X 19 Yes L; X 20 is S, T, M, P, F, Y, or W (e.g., P or T), X 21 is D, E, N or Q (e.g., N); and X 22 is G, A, V, L or I (e.g. L); X 23 Yes D; X 36 is D; and X 37 is 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 As defined above; X 19 Yes L; X 20 Is P or T; X 21 is N; and X 22 Yes L; X 23 Yes D; X 36 is D; and X 37 It is D.

[0220] For example, the CD16a binding motif is selected from the group consisting of SEQ ID Nos. 150 to 221, as shown in FIG. 24 .

[0221] For example, the CD16a binding motif is selected from the group consisting of:

[0222]

[0223] In the polypeptides of Figure 24 and the above table, the N-terminus of helix 1 may further comprise the sequence X6X7X8; wherein X6, X7 and X8 are as defined above, and are preferably NKE (i.e., X6 is N; X7 is K; X8 is E). In the polypeptides of Figure 24 and the above table, the C-terminus of helix 2 may further comprise the sequence X 36X 37 ; where X 36 and X 37 As defined above, and preferably DD (ie X 36 is D; and X 37 Yes D).

[0224] In the polypeptides of Figure 24 and the above table, optionally, 1 to 5 residues (e.g., 1, 2, 3, 4 or 5), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues. Preferably, in embodiments in which optionally 1 to 5 residues (e.g., 1, 2, 3, 4 or 5, preferably 1, 2 or 3 residues) in the sequence are replaced by alternative residues, the replacement residues are conservative replacements.

[0225] In a preferred embodiment, the sequence is one of SEQ ID 166, 168, 178, 182 or 202 (i.e., as found in one of the exemplary compounds 17, 19, 29, 33 and 53) (and optionally 1 to 5 residues (e.g. 1, 2, 3, 4 or 5), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues, the replacement residues being conservative replacements).

[0226] In another preferred embodiment, the sequence is one of SEQ ID No. 164, 166, 168 or 200 or SEQ ID No. 164, 166 or 168 (i.e., as found in one of the exemplary compounds 15, 17, 19, 51, or as found in one of the exemplary compounds 15, 17, 19) (and optionally, 1 to 5 residues (e.g., 1, 2, 3, 4 or 5), preferably 1, 2 or 3 residues in the sequence are replaced by alternative residues, the replaced residues being conservative replacements).

[0227] In a preferred embodiment, the motif sequence additionally has residues NKE at position X6X7X8 (ie X6 is N; X7 is K; X8 is E). 36 X 37 In addition, there is a residue DD (ie, X 36 is D; and X 37 is D). For example, the motif sequence has NKE at position X6X7X8 and 36 X 37 DD (i.e. X6 is N; X7 is K; X8 is E; X 36 is D; and X 37 is D). Therefore, in a preferred embodiment, the motif sequence may be selected from:

[0228] NKEX9X10 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 ;

[0229] 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

[0230] 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 DD.

[0231] As described above, in embodiments of the present invention, many residues can each be replaced by alternative residues as described herein. For example, in the above embodiments, even when not explicitly mentioned, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5) of the sequence of spiral 1 and / or spiral 2 defined above, and preferably optionally 1 to 3 (e.g., 1, 2 or 3) residues are replaced by alternative residues (e.g., replaced by alternative residues as conservative replacements). In any such alternative polypeptides of the present invention having replacement residues at appropriate positions, binding to the CD16a receptor is maintained. For example, the CD16a binding efficacy is at least 1% of the binding efficacy of the peptide of SEQID NO:1 (i.e., when measured under the same conditions (as described above) , the binding efficacy of the peptide of SEQ ID NO:1 to the CD16a receptor). Alternatively or additionally, for example, in any such alternative polypeptides of the present invention having replacement residues at appropriate positions, the CD16a binding polypeptide is a polypeptide that competes with SEQ ID NO:1.

[0232] In certain preferred embodiments, the binding efficacy is at least 5%, and more preferably at least 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 1 (i.e., the binding efficacy of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions).

[0233] In an advantageous embodiment 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.

[0234] In certain embodiments of the invention, the CD16a binding polypeptide is a polypeptide wherein:

[0235] i) Helix 1 contains 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 ,in

[0236] a*)X9 is V; X 10 It is Q; X 11 It is M; X 13 It is Q; X14 It is F;X 17 Yes R; X 18 It is K; X 24 Yes H; X 25 Yes H; X 27 Yes S; X 28 It is F;X 31 is I; X 32 is K; and X 35 It is M;

[0237] b*)X9 is Q; X 10 It is F;X 11 It is Y; X 13 Yes R; X 14 Yes D; X 17 Yes D; X 18 Yes L; X 24 Yes E; X 25 Yes D; X 27 It is K; X 28 It is W; X 31 It is Y; X 32 is M; and X 35 is I; or

[0238] c*)X9 is F;X 10 It is W; X 11 is I; X 13 Yes E; X 14 Yes S; X 17 Yes E; X 18 Yes S; X 24 is I; X 25 It is Y; X 27 It is K; X 28 It is W; X 31 It is K; X 32 is Y; and X 35 It is A;

[0239] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5; or, for example, at least 1 and no more than 3) of the residues are replaced by alternative residues, and / or are not marked with an X n At least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5; or such as at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0240] In another embodiment, a*)X9 is V; X 10 It is Q; X 11 is I or norleucine; X 13 It is Q; X14 It is F;X 17 Yes R; X 18 It is K; X 24 Yes H; X 25 Yes H; X 27 Yes S; X 28 It is F;X 31 is I; X 32 is K; and X 35 is I or norleucine.

[0241] In one embodiment, helix 1 comprises the sequence KEX9X 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 SLX 35 D, for example, helix 1 contains the sequence NKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 L, and helix 2 contains the sequence NX 24 X 25 QX 27 X 28 AFX 31 X 32 SLX 35 DD.

[0242] As mentioned above, it is not necessary to be exactly the same as the sequence listed in (i) (although in a preferred embodiment of the invention the peptide does have the exact sequence listed). n In certain embodiments, at least one and no more than eight of the residues indicated by the X-mark may be replaced by alternative residues. The replaced residues may be in helix 1 or helix 2, or there may be one or more replaced residues in 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, in the residues indicated by X-mark, n Among the residues represented by the label, there may be 1, 2, 3, 4, 5, 6, 7 or 8 (e.g. 1, 2, 3, 4 or 5, or 1, 2 or 3) replaced residues, such as 1, 2, 3, 4 or 5, such as 1 or 2, or 1. Since there are 15 residues with Xn The peptide with 5 residues replaced has 67% sequence identity to the sequence. For 3 residues replaced, it is 80% sequence identity, and for 1 residue replaced, it is 93% sequence identity. For 2 residues replaced, it is 87% sequence identity, and for 1 residue replaced, it is 93% sequence identity. There are 14 residues with X n In the embodiment marked, the peptide with 5 residues replaced has 64% sequence identity with the sequence. For the replacement of 3 residues, it is 79% sequence identity, and for the replacement of 1 residue, it is 93% sequence identity. For the replacement of 2 residues, it is 86% sequence identity, and for the replacement of 1 residue, it is 93% sequence identity.

[0243] In the fields not marked with an X n Of the residues in the sequence of residues, at least 1 and no more than 5 residues may be replaced by alternative residues. Preferably, at least 1 and no more than 3 residues may be replaced by alternative residues. The replaced residues may be in either helix 1 or helix 2, or there may be 1 or more replaced residues in each of helix 1 and helix 2 (e.g., there may be a replaced residue in one of them and 1 or 2 replaced residues in the other). For example, in the case where X is not used, n Among the residues represented by the label, there may be 1, 2 or 3 substituted residues, in particular 1 or 2, for example 1. Since there are 8 residues without X n The peptide with 3 residues replaced has 63% sequence identity to the sequence labeled, for 2 residues replaced it is 75% sequence identity, and for 1 residue replaced it is 88% sequence identity.

[0244] In one embodiment, the total number of replaced residues in the helix 1 and helix 2 parts is at least 1 and no more than 11 (e.g. no more than 10, such as no more than 9, such as no more than 8, such as no more than 7), such as at least 1 and no more than 6, such as at least 1 and no more than 5, such as at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. In particular there may be 1, 2, 3, 4, 5 or 6 replaced residues in total in those parts.

[0245] For example, represented by X n The residues or not indicated as X n Amino acid residue substitutions of residues of can be conservative substitutions. That is, one residue is replaced by another residue of the same class, for example:

[0246] An aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by another aliphatic residue. A hydroxyl-, sulfur- or selenium-containing residue (serine (S), cysteine ​​(C), selenocysteine ​​(U), threonine (T) or methionine (M)) may be replaced by another hydroxyl-, sulfur- or selenium-containing residue. An aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)) may be replaced by another aromatic residue. A basic residue (histidine (H), lysine (K), arginine (R)) may be replaced by another basic residue. An acidic residue or amide (aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)) may be replaced by another acidic residue or amide.

[0247] Alternatively, denoted as X n The residues or not indicated as X n Amino acid residue substitutions of a residue may be non-conservative substitutions, i.e. a residue is replaced by another residue from a different class, for example an aliphatic residue (glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I)) may be replaced by an aromatic residue (phenylalanine (F), tyrosine (Y) or tryptophan (W)), or for example by an amino acid with opposite characteristics, for example a lysine (K) residue is replaced by an aspartic acid (D) residue.

[0248] In one embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0249] a*) Helix 1 comprises the sequence VQMAQFEIRK (SEQ ID No 230), and Helix 2 comprises the sequence HHQSFAFIKSLM (SEQ ID No 231). For example, Helix 1 comprises the sequence NKEVQMAQFEIRKL (SEQ ID No 232), and Helix 2 comprises the sequence NHHQSFAFIKSLMDD (SEQ ID No 233).

[0250] In such embodiments, optionally, at least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5; or for example, at least 1 and no more than 3 (e.g., 1, 2, or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution);

[0251] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0252] b*) Helix 1 comprises the sequence QFYARDEIDL (SEQ ID No 234), and Helix 2 comprises the sequence EDQKWAFYMSLI (SEQ ID No 235). For example, Helix 1 comprises the sequence NKEQFYARDEIDLL (SEQ ID No 236), and Helix 2 comprises the sequence NEDQKWAFYMSLIDD (SEQ ID No 237).

[0253] In such embodiments, optionally, at least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5; or for example, at least 1 and no more than 3 (e.g., 1, 2, or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution);

[0254] In another embodiment, the CD16a binding polypeptide is a polypeptide wherein:

[0255] c*) Helix 1 comprises the sequence FWIAESEIES (SEQ ID No 238), and Helix 2 comprises the sequence IYQKWAFKYSLA (SEQ ID No 239). For example, Helix 1 comprises the sequence NKEFWIAESEIESL (SEQ ID No 240), and Helix 2 comprises the sequence NIYQKWAFKYSLADD (SEQ ID No 241).

[0256] In such embodiments, optionally, at least 1 and no more than 5 (e.g., 1, 2, 3, 4, or 5; or for example, at least 1 and no more than 3 (e.g., 1, 2, or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution);

[0257] As noted above, a number of residues may each be substituted with alternative residues.

[0258] The CD16a binding polypeptide of the present invention has an overall structure of [N-terminal portion]-[helix 1]-[separating portion]-[helix 2]-[C-terminal portion].

[0259] The separating portion can be a sequence of 1 to 5 (e.g., 1, 2, 3, 4, or 5) naturally occurring amino acids. Preferably, the separating portion is a sequence of 2 to 5 (e.g., 2, 3, 4, or 5) naturally occurring amino acids. For example, the separating portion is a sequence of 3 to 5 (e.g., 3, 4, or 5) naturally occurring amino acids. Preferably, the separating portion is a sequence of 3 amino acids.

[0260] In certain embodiments, the separation portion has the sequence X20 X 21 X 22 , where X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X 22 is any naturally occurring amino acid; and wherein optionally, X 20 , X 21 or X 22 One or both of the two do not exist. For example, X 20 , X 21 or X 22 None of them does not exist, X 20 , X 21 or X 22 One of the two does not exist, or X 20 , X 21 or X 22 More preferably, X 20 , X 21 or X 22 None of them does not exist, or X 20 , X 21 or X 22 Most preferably, X 20 , X 21 or X 22 None of them does not exist, that is, the separated part has the sequence X 20 X 21 X 22 , where X 20 is any naturally occurring amino acid, X 21 is any naturally occurring amino acid; and X 22 Any naturally occurring amino acid.

[0261] 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 optionally, X 20 , X 21 or X 22 One or both (for example, one) of does not exist.

[0262] 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 X22 is L. For example, the isolated portion has the sequence PNL or TNL.

[0263] The N-terminal portion may be absent or may be a sequence of 1 to 15 naturally occurring amino acids. For example, the N-terminal portion may be absent or may be a sequence of 1 to 10, 1 to 8, 1 to 6, or 1 to 5 naturally occurring amino acids (e.g., 1, 2, 3, 4, or 5 naturally occurring amino acids).

[0264] In certain embodiments, the N-terminal portion has the sequence X1X2X3X4X5, wherein 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.

[0265] Preferably, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is G, A, V, L, I or absent, X2 is D, E, N, Q or absent, X3 is D, E, N, Q or absent, X4 is H, K, R or absent, and X5 is F, Y, W or absent. More preferably, X1 is V, G or absent (e.g., V or absent); X2 is D or absent; X3 is N or absent; X4 is K or absent; and X5 is F or absent.

[0266] In certain embodiments, preferably, none of X1, X2, X3, X4 and X5 is absent; X1 is absent and the other residues are not absent; X1 and X2 are absent and the other residues are not absent; X1, X2 and X3 are absent and the other residues are not absent; X1, X2, X3 and X4 are absent and the other residues are not absent; or X1, X2, X3, X4 and X5 are all absent.

[0267] In certain particularly preferred embodiments, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent. For example, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, that is, the N-terminal portion has the sequence VDNKF, GDNKF or does not exist; and more preferably has the sequence VDNKF or does not exist.

[0268] In a preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, ie the N-terminal portion has the sequence VDNKF or GDNKF; and more preferably the N-terminal portion has the sequence VDNKF.

[0269] In a preferred embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent, ie, the N-terminal portion is absent.

[0270] The C-terminal portion may not exist or may be a sequence of 1 to 50 naturally occurring amino acids. For example, the C-terminal portion may not exist 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 not exist or may be a sequence of 10 to 35, 10 to 30, 15 to 25, 18 to 22, 1 to 21 naturally occurring amino acids (e.g., 18, 19, 20, 21 or 22 naturally occurring amino acids). Preferably, the C-terminal portion has a sequence such that it enhances target binding through the CD16a binding motifs of helix 1 and helix 2.

[0271] In certain embodiments, the C-terminal portion is absent or has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is a sequence of 1 to 14 naturally occurring amino acids, X 39 is any naturally occurring amino acid, X 56is any naturally occurring amino acid or is absent, X 57 is any naturally occurring amino acid or is absent, and X 58 is any naturally occurring amino acid or is absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example by alternative residues that are conservative substitutions.

[0272] 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 38 is any naturally occurring amino acid (ie any single (one) naturally occurring amino acid residue).

[0273] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQ X 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W, X 56 G, A, V, L, I or not present, X 57 is P or not present, and X 58 is H, K, R or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. In another preferred embodiment, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 G, A, V, L, I or not present, X 57 is P or not present, and X 58 is H, K, R or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example as conservative substitutions.

[0274] In a particularly preferred embodiment, the C-terminal portion has X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 It is P, X 39 It is S, X 56 A or not, X 57 is P or not present, and X 58 is K or absent, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues as conservative substitutions. In another particularly preferred embodiment, the C-terminal portion has PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 A or not, X 57 is P or not present, and X 58 is K or absent, and optionally wherein 1 to 5 (eg 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0275] For example, in certain embodiments, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is P, and X 39 is S; and:

[0276] X 56 It's A, X 57 is P, and X 58 is K; or X 56 A, or X 57 is P, and X 58 does not exist; or X 56 It's A, X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. Alternatively, for example, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 It's A, X 57 is P, and X 58 is K; or X 56 A, or X 57 is P, and X 58 does not exist; or X 56 It's A, X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0277] In a preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 It is P, X 39 is S; and X 56 It's A, X 57 is P, and X 58 is K. In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions. Alternatively, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 It's A, X 57 is P, and X 58is K. In such embodiments, optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions.

[0278] In an alternative preferred embodiment, the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 is P, and X 39 is S; and:

[0279] X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, such as alternative residues that are conservative substitutions. Alternatively, for example, the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0280] In one embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F, or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent); and the C-terminal portion has the sequence X38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W, X 56 G, A, V, L, I or not present, X 57 is P, and X 58 is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions; and more preferably X 38 Yes P, X 39 It is S, X 56 It's A, X 57 is P, and X 58 is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, eg, as conservative substitutions.

[0281] In another embodiment, the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is F; (e.g., X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F), or X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent); and the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 , where X 56 G, A, V, L, I or not present, X 57 is P, and X 58is H, K or R, and optionally wherein 1 to 5 (e.g. 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions; and more preferably X 56 It's A, X 57 is P, and X 58 is K, and optionally, 1 to 5 (eg, 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are substituted by alternative residues, eg, as conservative substitutions.

[0282] In a very preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; and X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K).

[0283] In an alternative highly preferred embodiment, X1 is absent, X2 is absent, X3 is absent, X4 is absent, and X5 is absent; and X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K).

[0284] 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 , where X 38 Yes P, X 39 is S, T, M, P, F, Y or W (preferably S); and X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 Does not exist; X56 is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 More preferably, X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); or X 56 Does not exist, X 57 does not exist, and X 58 In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ are replaced by alternative residues, for example, by alternative residues that are conservative substitutions.

[0285] 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 , where X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 Does not exist; X 56 is G, A, V, L or I (preferably A), X 57 does not exist, and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 More preferably, X 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K); or X 56 Does not exist, X 57 does not exist, and X 58In such embodiments, optionally, 1 to 5 (e.g., 1, 2, 3, 4 or 5; preferably 1, 2 or 3) residues in the sequence PSQSANLLAEAKKLNDAQ are replaced by alternative residues, for example, as conservative substitutions.

[0286] In a very preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; and X 56 Does not exist, X 57 does not exist, and X 58 Does not exist.

[0287] In an alternative highly preferred embodiment, X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; 56 is G, A, V, L or I (preferably A), X 57 is P, and X 58 is H, K or R (preferably K).

[0288] In certain embodiments, (i) the separation portion has a sequence X 20 X 21 X 22 ; and / or the N-terminal portion has the sequence X1X2X3X4X5; and / or the C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 ;

[0289] Wherein, in the separation part, X 20 Is P or T; X 21 Yes N; X 22 It is L;

[0290] wherein in the N-terminal portion, 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;

[0291] and wherein in the C-terminal portion, X 56 A or not; X 57 Is P or does not exist; X 58 It is K or not present.

[0292] Alternatively, (ii) the separating part, the N-terminal part and the C-terminal part are as defined in (i), wherein optionally

[0293] (a) within each portion, 1, 2 or 3 residues are replaced by alternative residues; or

[0294] (b) within those portions, at least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5) residues are collectively replaced by the alternative residues.

[0295] In such embodiments, preferably, the separating portion has the sequence PNL or TNL.Alternatively or additionally, in such embodiments, the N-terminal portion has the sequence VDNKF.

[0296] In one embodiment, the N-terminal portion may comprise the sequence X1X2X3X4X5, wherein:

[0297] X1 is V or does not exist; X2 is D or does not exist; X3 is N or does not exist; X4 is K or does not exist; X5 is F or does not exist.

[0298] For example, the N-terminal portion may comprise the sequence VDNKF.

[0299] The isolated portion may contain the sequence X 20 X 21 X 22 ,in:

[0300] X 20 Is P or T; X 21 Yes N; X 22 It's L.

[0301] For example, the isolated portion may comprise the sequence PNL.

[0302] The C-terminal portion may comprise the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 ,in:

[0303] X 56 A or not; X 57 Is P or does not exist; X 58 It is K or not present.

[0304] For example, the C-terminal portion may comprise the sequence PSQSANLLAEAKKLNDAQAPK or PSQSANLLAEAKKLNDAQ. The C-terminal portion may have the structure [second separation portion]-[helix 3]-[C-terminal sequence]. For example:

[0305] - the second separated part comprises the sequence PS;

[0306] - Helix 3 contains the sequence QSANLLAEAKKLNDAQ;

[0307] - and the C-terminal sequence contains the sequence APK or is absent.

[0308] In such embodiments, in those five parts (N-terminal part, separation part, second separation part, helix 3 and C-terminal sequence), at least 1 and no more than 8 (e.g., 5) residues can be replaced together by alternative residues. For example, the number of replaced residues is at least 1 and no more than 7, at least 1 and no more than 6, at least 1 and no more than 5, at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. In particular, there can be 1, 2, 3, 4 or 5 replacement residues in total in those parts.

[0309] Thus, in such embodiments, in the CD16a binding polypeptide of the invention (i.e., in the overall structure [N-terminal portion]-[helix 1]-[separation portion]-[helix 2]-[C-terminal portion]), at least 1 and no more than 11 residues can be replaced by alternative residues. For example, the number of replaced residues is at least 1 and no more than 10, such as at least 1 and no more than 9, such as at least 1 and no more than 8, such as at least 1 and no more than 7, such as at least 1 and no more than 6. In particular, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 replacement residues in total in the overall structure [N-terminal portion]-[helix 1]-[separation portion]-[helix 2]-[C-terminal portion]. In an advantageous embodiment of the CD16a binding polypeptide of the invention, wherein the C-terminal portion comprises a helical or substantially helical region (e.g., helix 3 just described above; e.g., sequence QSANLLAEAKKLNDAQ), proline (P) and cysteine ​​(C) are not present in the helical or substantially helical region. In another embodiment, additionally, glycine (G) is also not present in the helical or substantially helical region (e.g., helix 3 just described above; e.g., sequence QSANLLAEAKKLNDAQ).

[0310] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.1 to 75, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.1 to 75. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.1 to 75.

[0311] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.1 to 73, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.1 to 73. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.1 to 73.

[0312] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.11 to 73, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.11 to 73. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.11 to 73.

[0313] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.1 to 67, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.11 to 67. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.1 to 67.

[0314] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO. 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, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO. 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 polypeptide has a sequence selected from the group consisting of SEQ ID NO. 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.

[0315] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.34, 45 and 51, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.34, 45 and 51. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.34, 45 and 51.

[0316] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO. 26, 35 and 47, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO. 26, 35 and 47. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO. 26, 35 and 47.

[0317] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.12, 17, 19, 29, 33, 49, 51 and 53, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.12, 17, 19, 29, 33, 49, 51 and 53. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.12, 17, 19, 29, 33, 49, 51 and 53.

[0318] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.17, 19, 29, 33 and 53, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.17, 19, 29, 33 and 53. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.17, 19, 29, 33 and 53.

[0319] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.15, 17, 19 and 51, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.15, 17, 19 and 51. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.15, 17, 19 and 51.

[0320] In one aspect of the invention, the CD16a binding polypeptide comprises a sequence selected from SEQ ID NO.15, 17 and 19, as 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, and preferably by residues that are conservatively replaced. In one embodiment, the CD16a polypeptide comprises a sequence selected from SEQ ID NO.15, 17 and 19. In one embodiment, the CD16a polypeptide has a sequence selected from the group consisting of SEQ ID NO.15, 17 and 19.

[0321] In one aspect of the invention, the CD16a binding polypeptide comprises the sequence:

[0322] a*)VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK[SEQ IDNO:1].

[0323] In another aspect of the invention, the CD16a binding polypeptide comprises the sequence:

[0324] b*)VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPK[SEQ IDNO:2].

[0325] In another aspect of the invention, the CD16a binding polypeptide comprises the sequence:

[0326] c*)VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK[SEQ IDNO:3].

[0327] As described above, many residues in the polypeptide of the present invention can be replaced by alternative residues. For example, in the polypeptide listed in a*), b*) or c*), at least 1 and no more than 5 residues can be replaced by alternative residues. For example, the number of replaced residues is at least 1 and no more than 4, such as at least 1 and no more than 3, such as at least 1 and no more than 2. In particular, there can be 1, 2, 3, 4 or 5 replacement residues in total in those parts. Since there are 58 residues in the polypeptide of this aspect of the present invention, the peptide in which 5 residues are replaced has 91% sequence identity (91.4%) with the sequence. For the replacement of 4 residues, it is 93% sequence identity (93.1%), for the replacement of 3 residues, it is 95% sequence identity (94.8%), for the replacement of 2 residues, it is 97% sequence identity (96.6%), and for the replacement of 1 residue, it is 98% sequence identity (98.3%).

[0328] As mentioned above, in one embodiment, a*) Helix 1 comprises the sequence VQMAQFEIRK, and Helix 2 comprises the sequence HHQSFAFIKSLM. In that embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptides of the invention having replacement residues in appropriate positions, binding to the CD16a receptor is maintained. For example, when measured under the same conditions, the CD16a binding efficacy is at least 1% of the binding efficacy of the peptide of SEQ ID NO: 1 to the CD16a receptor.

[0329] At 1% binding efficacy, it is understood that the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is 50 The concentration of the peptide of SEQ ID NO: 1 does not exceed the IC of the CD16a receptor 50 100 times the concentration.

[0330] More preferably, the binding efficacy is at least 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 1 to the CD16a receptor when measured under the same conditions. That is, the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is at least 5%, 10%, 20%, 25% or 50%. 50 The concentration of the peptide of SEQ ID NO: 1 does not exceed the IC of the CD16a receptor 50 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0331] As also mentioned above, in one embodiment, b*) Helix 1 comprises the sequence QFYARDEIDL and Helix 2 comprises the sequence EDQKWAFYMSLI. In that embodiment, as in other embodiments, a number of residues may each be substituted with alternative residues as described herein. In any such alternative polypeptide of the invention with replacement residues in appropriate positions, binding to the CD16a receptor is maintained. For example, the CD16a binding efficacy is at least 1% of the binding efficacy of the peptide of SEQ ID NO: 74 to the CD16a receptor when measured under the same conditions.

[0332] At 1% binding efficacy, it is understood that the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is 50 The concentration of the peptide of SEQ ID NO: 74 does not exceed the IC of the CD16a receptor 50 100 times the concentration.

[0333] More preferably, the binding efficacy is at least 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 74 to the CD16a receptor when measured under the same conditions. That is, the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is at least 5%, 10%, 20%, 25% or 50%. 50 The concentration of the peptide of SEQ ID NO: 74 does not exceed the IC of the CD16a receptor 50 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0334] As also mentioned above, in one embodiment, c*) Helix 1 comprises the sequence FWIAESEIES, and Helix 2 comprises the sequence IYQKWAFKYSLA. In that embodiment, as in other embodiments, a number of residues can each be substituted with alternative residues as described herein. In any such alternative polypeptides of the invention having replacement residues in appropriate positions, binding to the CD16a receptor is maintained. For example, when measured under the same conditions, the CD16a binding efficacy is at least 1% of the binding efficacy of the peptide of SEQ ID NO: 75 to the CD16a receptor.

[0335] At 1% binding efficacy, it is understood that the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is 50 The concentration of the peptide of SEQ ID NO: 75 does not exceed the IC of the CD16a receptor 50 100 times the concentration.

[0336] More preferably, the binding efficacy is at least 5%, 10%, 20%, 25% or 50% of the binding efficacy of the peptide of SEQ ID NO: 75 to the CD16a receptor when measured under the same conditions. That is, the IC of the surrogate polypeptide for binding to the CD16a receptor when measured under the same conditions is at least 5%, 10%, 20%, 25% or 50%. 50 The concentration of the peptide of SEQ ID NO: 75 does not exceed the IC of the CD16a receptor 50 20 times, 10 times, 5 times, 4 times or 2 times the concentration.

[0337] The present invention further provides a CD16a binding polypeptide, wherein the CD16a binding polypeptide consists of a motif that binds to CD16a, wherein the polypeptide consists of the following structure:

[0338] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0339] The CD16a binding motif is the portion [helix 1]-[separating portion]-[helix 2] (and optionally, the peptide further comprises one or more additional functional portions as described below (e.g. 1, 2, 3, 4, 5, 6 or more); e.g. one, two or three additional functional portions).

[0340] The present invention also provides a CD16a binding polypeptide of the present invention, wherein the CD16a binding polypeptide consists of a CD16a binding polypeptide (and optionally further comprises one or more additional functional parts as described below (e.g. 1, 2, 3, 4, 5, 6 or more); e.g. one, two or three additional functional parts).

[0341] In a very preferred embodiment, a CD16a binding polypeptide (e.g., a CD16a binding polypeptide of the invention described above or below) consists of a motif that binds to CD16a, wherein the polypeptide consists of the following structure:

[0342] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0343] The CD16a binding motif is the portion [helix 1]-[separating portion]-[helix 2] (and optionally further comprises one or more additional functional portions (e.g. 1, 2, 3, 4, 5, 6 or more); e.g. one, two or three additional functional portions) as described below.

[0344] In a very preferred embodiment, the CD16a binding polypeptide consists of a CD16a binding polypeptide of the invention (and optionally further comprises one or more additional functional parts as described below (e.g. 1, 2, 3, 4, 5, 6 or more); e.g. one, two or three additional functional parts).

[0345] For the avoidance of doubt, in embodiments wherein the CD16a binding polypeptide consists of a CD16a binding motif and / or a CD16a binding polypeptide of the invention, the CD16a binding polypeptide is not linked to another CD16a binding polypeptide, i.e. the CD16a binding polypeptide is not part of a CD16a binding oligomer of the invention.

[0346] In another very preferred embodiment, the CD16a binding polypeptide (e.g., the CD16a binding polypeptide of the invention described above or below) consists of a motif that binds to CD16a, wherein the polypeptide consists of the following structure:

[0347] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0348] The CD16a binding motif is the portion [helix 1]-[separating portion]-[helix 2], and wherein the polypeptide further comprises one or more additional functional portions as described below (e.g. 1, 2, 3, 4, 5, 6 or more; for example one, two or three additional functional portions). In another very preferred embodiment, the CD16a binding consists of a CD16a binding polypeptide of the invention and further comprises one or more additional functional portions as described below (e.g. 1, 2, 3, 4, 5, 6 or more); for example 1, 2 or 3 additional functional portions (preferably 1 or 2 additional functional portions).

[0349] Multimeric CD16a binding peptides: CD16a binding oligomers

[0350] The present invention also provides CD16a binding oligomers comprising at least two (i.e., two or more, such as 2, 3, 4, 5, 6 or more; preferably 2, 3 or 4) 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 5 or at least 6 CD16a binding polypeptides of the present invention, such as 3, 4, 5 or 6 or more CD16a binding polypeptides of the present invention.

[0351] The CD16a binding polypeptides of the CD16a binding oligomers of the present invention may optionally be connected via one or more linkers. The CD16a binding oligomers of the present invention are multimeric binding agents because they have multiple CD16a binding motifs, i.e., they have a CD16a binding motif ([helix 1]-[separated portion]-[helix 2]) in each CD16a binding polypeptide contained therein.

[0352] This aspect of the invention may also be defined as such that the CD16a binding polypeptide constitutes two or more CD16a binding moieties or CD16a binding peptides, such as two, three, four, five, six or more CD16a binding moieties or CD16a binding peptides, such as two, three or four CD16a binding moieties, optionally connected by one or more linkers. This definition is used in the numbered embodiments of the invention to refer to the CD16a binding oligomer aspect of the invention.

[0353] The CD16a binding oligomer of the present invention comprises at least a first CD16a binding polypeptide as a CD16a binding polypeptide of the present invention and a second CD16a binding polypeptide as a CD16a binding polypeptide of the present invention. The first CD16a binding polypeptide and the second CD16a binding polypeptide may have the same sequence. Alternatively, the first CD16a binding polypeptide and the second CD16a binding polypeptide may have different sequences. The CD16a binding oligomer of the present invention may optionally further comprise a third CD16a binding polypeptide as a CD16a binding polypeptide of the present invention. The third CD16a binding polypeptide may have a sequence identical to the first CD16a binding polypeptide sequence and / or the second CD16a binding polypeptide sequence. Alternatively, the third CD16a binding polypeptide may have a sequence different from the first CD16a binding polypeptide and the second CD16a binding polypeptide. The CD16a binding oligomer of the present invention may optionally further comprise a fourth CD16a binding polypeptide as a CD16a binding polypeptide of the present invention. The fourth CD16a binding polypeptide may have a sequence identical to the first CD16a binding polypeptide sequence and / or the second CD16a binding polypeptide sequence and / or the third CD16a binding polypeptide sequence. Alternatively, the fourth CD16a binding polypeptide may have a sequence different from the first CD16a binding polypeptide, the second CD16a binding polypeptide and the third CD16a binding polypeptide.

[0354] 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 the following: SEQ ID NO.150-221) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions); and a second CD16a binding polypeptide comprising a second binding motif selected from the following: SEQ ID NO.150-221) (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions). The first CD16a binding motif and the second CD16a binding motif may have the same sequence or different sequences.

[0355] In a preferred embodiment, the CD16a binding oligomer of the present invention comprises: a first CD16a binding polypeptide selected from the following sequences: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions); and a second CD16a binding polypeptide having a sequence selected from the following sequences: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions). The first CD16a binding polypeptide and the second CD16a binding polypeptide may have the same sequence or different sequences.

[0356] In another embodiment, the CD16a binding oligomer of the present invention comprises: a first CD16a binding polypeptide selected from the following sequences: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues that are conservatively replaced); a second CD16a binding polypeptide having a sequence selected from the following sequences: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues that are conservatively replaced); and a third CD16a binding polypeptide having a sequence selected from the following sequences: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues that are conservatively replaced). The first CD16a binding polypeptide, the second CD16a binding polypeptide and the third CD16a binding polypeptide may have the same sequence, or each may have a different sequence, or two sequences may be the same and one may be different. Optionally, there may be a fourth CD16a binding polypeptide having a sequence selected from the following: SEQ ID NO.1-75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservatively replaced); it may be the same as any one of the first sequence, the second sequence or the third sequence, or may be different.

[0357] In a preferred embodiment, the CD16a binding oligomer of the present invention comprises: a first CD16a binding polypeptide selected from the following sequences: SEQ ID NO.1, 74 or 75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions); and a second CD16a binding polypeptide having a sequence selected from the following sequences: SEQ ID NO.1, 74 or 75 (and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by residues, and preferably replaced by residues as conservative substitutions). The first CD16a binding polypeptide and the second CD16a binding polypeptide may have the same sequence or different sequences (e.g., the first CD16a binding polypeptide may have / have SEQ ID NO.1; and the second CD16a binding polypeptide may have SEQ ID NO.1; or the first CD16a binding polypeptide may have SEQ ID NO.74; and the second CD16a binding polypeptide may have SEQ ID NO.1; the first CD16a binding polypeptide may have SEQ ID NO.75; and the second CD16a binding polypeptide may have SEQ ID NO.1).

[0358] The CD16a binding polypeptides in the CD16a binding oligomers of the present invention can be separated by a linker. For example, each CD16a binding polypeptide in the CD16a binding oligomers of the present invention can be separated by a linker. Preferably, the linker is a linker as defined herein, such as a flexible amino acid linker, a rigid amino acid linker, or a cleavable amino acid linker or a non-amino acid linker. In the case where the CD16a binding oligomers of the present invention contain more than one linker, the linkers can be the same, or can be different. Preferably, the linker used in the CD16a binding oligomer of the present invention comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0359] In a preferred embodiment, the linker used in the CD16a binding oligomer of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSG GGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0360] In a preferred embodiment, the linker used in 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 used in 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.

[0361] Alternatively, the CD16a binding polypeptides in the CD16a binding oligomers of the invention may not be separated by a linker (ie, they may be directly attached to each other).

[0362] In a preferred embodiment, the CD16a binding oligomer does not comprise a linker.

[0363] In one embodiment, the CD16a binding oligomer of the present invention comprises at least two (eg, two) CD16a binding polypeptides, and the CD16a binding oligomer comprises the following structure:

[0364] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0365] wherein each N-terminal portion in the oligomer can have the same sequence or a different sequence; each C-terminal portion in the oligomer can have the same sequence or a different sequence; each separation portion in the oligomer can have the same sequence or a different sequence; each helix 1 portion in the oligomer can have the same sequence or a different sequence; and each helix 2 portion in the oligomer can have the same sequence or a different sequence.

[0366] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS GGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GSGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0367] Alternatively, the linker may be absent, ie the CD16a binding oligomer comprises the following structure:

[0368] [N-terminal part]-[helix 1]-[detached part]-[helix 2]-[C-terminal part]-[N-terminal part]-[helix 1]-[detached part]-[helix 2]-[C-terminal part].

[0369] In one embodiment, the CD16a binding oligomer of the present invention comprises at least 3 (eg, 3) CD16a binding polypeptides, and the CD16a binding oligomer comprises the following structure:

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

[0371] and wherein each linker portion in the oligomer can have the same sequence or have a different sequence; each N-terminal portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each C-terminal portion can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each separation portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; each helix 1 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; and each helix 2 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence.

[0372] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S, and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. Furthermore, for example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS, GGGSGGGGSGGGGSGGGGS, GGSGG, GGGSGGGGSGG, GGS GGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG or SGGGGSGGGGSGGGGSGGGG.

[0373] Alternatively, one or both linkers may be absent, i.e. the CD16a binding oligomer comprises the following structure:

[0374] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part];

[0375] [N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]-[linker]-[N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[N-terminal portion]-[helix 1]-[isolated portion]-[helix 2]-[C-terminal portion]; or

[0376] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[helix 1]-[isolated part]-[helix 2]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part].

[0377] In one embodiment, the CD16a binding oligomer comprises the sequence VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO.242], and wherein optionally 1, 2 or 3 residues in the sequence are replaced by alternative residues, and preferably by residues that are conservative substitutions.

[0378] In another embodiment, the CD16a binding oligomer comprises the sequence VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO.243], and wherein optionally, 1, 2 or 3 residues in the sequence are replaced by alternative residues, and preferably by residues that are conservative substitutions.

[0379] In another embodiment, the CD16a binding oligomer comprises the sequence VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO.244], and wherein optionally, 1, 2 or 3 residues in the sequence are replaced by alternative residues, and preferably by residues that are conservative substitutions.

[0380] In another embodiment, the CD16a binding oligomer of the invention may comprise two 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 described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, each of the two CD16-a binding polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1].

[0381] In an alternative example, a CD16a binding oligomer of the invention may comprise two 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 of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, one of the two CD16a binding polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 2], and

[0382] Another may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1].

[0383] In an alternative example, a CD16a binding oligomer of the 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 described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, one of the two polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1], and

[0384] Another may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 75].

[0385] For example, such a CD16a 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 described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, each of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0386] In an alternative example, a CD16a binding oligomer of the 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 of which has a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, two of the three polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 74], and

[0387] The other one may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1].

[0388] In an alternative example, a CD16a binding oligomer of the 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 two of which have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, one of the three polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 74], and

[0389] The other two may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1].

[0390] In an alternative example, a CD16a binding oligomer of the 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 two of which have a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, one of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1], and

[0391] The other two may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 75].

[0392] In an alternative example, a CD16a binding oligomer of the 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 of which has a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, two of the three polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1], and

[0393] The other one may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 75].

[0394] For example, such a CD16a binding oligomer of the 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 described elsewhere, such sequences may have a plurality of residues substituted with alternative residues. For example, each of the four polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1].

[0395] In an alternative example, a CD16a binding oligomer of the 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 two of which have a sequence in which helix 1 comprises the sequence VQMAQFEIRK and helix 2 comprises the sequence HHQSFAFIKSLM. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, two of the four polypeptides may have the sequence: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 74], and

[0396] The other two may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 1].

[0397] In an alternative example, a CD16a binding oligomer of the 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 of which have a sequence in which helix 1 comprises the sequence FWIAESEIES and helix 2 comprises the sequence IYQKWAFKYSLA. As described elsewhere, such sequences may have multiple residues substituted with alternative residues. For example, two of the four polypeptides may have the sequence: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLN DAQAPK [SEQ ID NO: 1], and

[0398] The other two may have the sequence: VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO: 75].

[0399] In the above-mentioned 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 by 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 linkers may be amino acid sequences, and they may have the same amino acid sequence or different amino acid sequences.

[0400] Connectors

[0401] When present, the linker connects two or more functional parts of the polypeptide of the present invention (further defined below). For example, a linker can connect two CD16a binding polypeptides of the present invention together (e.g., in a CD16a binding oligomer of the present invention), or a linker can connect a CD16a binding polypeptide of the present invention and an additional functional part of the present invention together; in embodiments where there is more than one additional functional part, a linker can also connect an additional functional part of the present invention and an additional functional part of the present invention together. Those skilled in the art know that different types of linkers with different properties are known in the art, and suitable linkers can be selected according to the desired properties. Types of linkers include, for example, flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers; non-amino acid linkers can also be used. For example, a linker can be selected to increase stability or improve folding, to increase expression, to improve biological activity, to be able to target or to change pharmacokinetics. Non-amino acid linkers can be referred to as synthetic linkers.

[0402] For the avoidance of doubt, in certain embodiments, the linker may not connect two CD16a binding polypeptides of the invention together (e.g., in a CD16a binding oligomer of the invention), and / or the linker may not connect a CD16a binding polypeptide of the invention and an additional functional moiety of the invention together; and / or in embodiments where there is more than one additional functional moiety, the linker may not connect an additional functional moiety of the invention and an additional functional moiety of the invention together. For example, in certain embodiments, two CD16a binding polypeptides of the invention (e.g., in a CD16a binding oligomer of the invention) may be directly connected; and / or a CD16a binding polypeptide or oligomer of the invention may be directly connected to an additional functional moiety of the invention; and / or an additional functional moiety of the invention may be directly connected to an additional functional moiety of the invention.

[0403] In one embodiment, the CD16a binding polypeptide according to any aspect disclosed herein or the CD16a binding oligomer according to any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from a flexible amino acid linker, a rigid amino acid linker, and a cleavable amino acid linker. In one embodiment, the linker is between two or more CD16a binding polypeptides, such as in the CD16a binding oligomers of the present invention, or between CD16a binding polypeptides or CD16a binding oligomer portions, such as immune signaling molecules or additional binding moieties (e.g., as described in further detail below).

[0404] In one embodiment, the CD16a binding oligomer according to any aspect disclosed herein comprises at least one linker, such as at least one linker selected from a flexible amino acid linker, a rigid amino acid linker and a cleavable amino acid linker. The linker is between two or more CD16a binding polypeptides. Additional linkers between CD16a binding polypeptides or CD16a binding oligomers and additional functional parts (e.g., immune signaling molecules or additional binding moieties (e.g., as described in further detail below)) may also be present in the CD16a binding oligomer according to any aspect disclosed herein. Additional linkers between additional functional parts (e.g., immune signaling molecules or additional binding moieties (e.g., as described in further detail below)) and additional functional parts (e.g., immune signaling molecules or additional binding moieties (e.g., as described in further detail below)) may also be present in the CD16a binding oligomer according to any aspect disclosed herein.

[0405] In embodiments and aspects of the invention comprising more than one linker, each linker can be the same, or can be different, or some linkers can be the same and some can be different (e.g., in embodiments having 3 or more linkers (e.g., 3, 4, 5, 6, 7, 8 or more linkers)).

[0406] When the domain of connection needs some distance and conformational freedom, flexible linkers can be used, and can be advantageous in some embodiments of the present invention.Such linkers are usually composed of small non-polar (e.g., G) or polar (e.g., S or T) amino acids.Some flexible linkers are mainly composed of segments of G and S residues, such as (GGGGS)p or (GGGSG)p.Other examples include (GGSGG)p, (GSGGG)p or (SGGGG)p.Regulating copy number "p" allows optimizing linkers to achieve appropriate separation between functional parts, or to maintain necessary part interactions.In a preferred embodiment, linkers are (GGGSG)p, such as (GGGSG)1, (GGGSG)2 or (GGGSG)3, such as (GGGSG)3.In one embodiment, linkers are (GGGGS)p, such as (GGGGS)1, (GGGGS)2 or (GGGGS)3, such as (GGGGS)3. In one embodiment, the linker is (GGSGG)p, such as (GGSGG)1, (GGSGG)2, or (GGSGG)3, such as (GGSGG)3. In one embodiment, the linker is (GSGGG)p, such as (GSGGG)1, (GSGGG)2, or (GSGGG)3, such as (GSGGG)3. In one embodiment, the linker is (SGGGG)p, such as (SGGGG)1, (SGGGG)2, or (SGGGG)3, such as (SGGGG)3. In another embodiment, the linker is G.

[0407] In one embodiment, the linker of the invention comprises or has 1 to 50 (e.g., 1 to 40, 1 to 30 or 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T. In one embodiment, the linker of the invention comprises or has 1 to 50 (e.g., 1 to 25, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; or 1 to 20, such as 1, 5, 10, 15, or 20; or 1 to 15, such as 1, 5, 10, or 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G and S. For example, the linker of the invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, or GGGSGGGGSGGGGSGGGGSG.

[0408] Furthermore, for example, the linker of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG. For example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGG GGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0409] In a preferred embodiment, the linker of the present invention is G or comprises or has the sequence GGGSG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. 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.

[0410] In addition to G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues (such as T and A) to maintain flexibility and containing polar amino acid residues to improve solubility. In general, it is known in the art that linker sequence and length can affect the characteristics of the connected parts, so the skilled person will be able to select a suitable linker for use in binding polypeptides as described herein.

[0411] Other types of linkers (such as rigid and / or cleavable linkers) can also be used to connect domains in multi-domain constructs to improve or control their biological activity. Such linkers are known in the art (Chen X et al., Fusion protein linkers: Property, design and functionality, Adv Drug Deliv Rev 2013: 65: 10: 1357, doi: 10.1016 / j.addr.2012.09.039 ).

[0412] Alternatively, different binding moieties of binding polypeptides as described herein (e.g., two or more CD16a binding polypeptides or CD16a binding polypeptides and additional functional moieties, such as immune signaling molecules or additional binding moieties) can be covalently linked by chemical linkers. Such chemical linkers can be produced, for example, by maleimide or "click" chemistry. Those skilled in the art will know other linkers suitable for binding polypeptides as described herein.

[0413] With regard to the description of the binding polypeptides comprising the CD16a binding polypeptides according to the present disclosure above, it should be noted that the designation of the first part, the second part and the further part is for reasons of clarity, so as to distinguish the CD16a binding polypeptides according to the present invention on the one hand, and to distinguish the binding parts that exhibit other functions on the other hand. These nomenclatures are not intended to refer to the actual order of the different regions of the binding polypeptides. Similarly, the naming of the first part and the second part (or monomeric unit) is for reasons of clarity to distinguish between the units. Thus, for example, the first part (or monomeric unit) may appear at the N-terminus, the middle or the C-terminus of the binding polypeptide without restriction.

[0414] The CD16a binding polypeptides in the CD16a binding oligomers of the present invention can be separated by a linker. For example, each CD16a binding polypeptide in the CD16a binding oligomers of the present invention can be separated by a linker. Preferably, the linker is a linker as defined herein. In the case where the CD16a binding oligomers of the present invention contain more than one linker, the linkers can be the same, or can be different.

[0415] Preferably, the linker used in the CD16a binding oligomer of the present invention comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0416] In a preferred embodiment, the linker used for the CD16a binding oligomer of the present invention is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker used 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.

[0417] In embodiments of the present invention comprising additional functional moieties, the CD16a binding polypeptide of the present invention and the additional functional moieties in the CD16a binding polypeptide or CD16a binding oligomer can be separated by a joint. For example, each additional functional moiety in the CD16a binding polypeptide or CD16a binding oligomer of the present invention and the CD16a binding polypeptide can be separated by a joint. Preferably, the joint is a joint as defined herein. In embodiments of the present invention comprising two or more additional functional moieties, the additional functional moieties can be separated by a joint. Preferably, the joint is a joint as defined herein.

[0418] In the case where a CD16a binding polypeptide or CD16a binding oligomer of the invention comprises more than one linker (i.e., where there are at least two CD16a binding polypeptides and at least one additional functional moiety; or where there are at least one CD16a binding polypeptide and at least two additional functional moieties), the linkers may be the same, or may be different. Preferably, the linker for a CD16a binding polypeptide or CD16a binding oligomer comprising an additional functional moiety comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0419] In a preferred embodiment, the linker for CD16a binding polypeptide or CD16a binding oligomer comprising an additional functional part is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG or GGGSGGGGSGGGGSGGGGSG. In another embodiment, the linker for the 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.

[0420] Additional features section

[0421] The CD16a binding polypeptide disclosed herein may be attached to one or more additional functional moieties, for example, via a linker as described above. The CD16a binding oligomer as disclosed herein may be attached to one or more additional functional moieties, for example, via a linker as 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 as described herein.

[0422] For the avoidance of doubt, a CD16a binding polypeptide or CD16a binding oligomer of the invention may be directly attached to one or more additional functional moieties (ie not via a linker).

[0423] As used herein, "functional portion" refers to a component or "part" that possesses a specific desired biological activity.

[0424] The one or more additional components (ie one or more additional functional parts) may for example be a signalling molecule.Examples of suitable signalling molecules include immune signalling molecules, such as cytokines, for example IL-15 and derivatives thereof.

[0425] The one or more additional components (i.e., one or more additional functional parts) can be one or more additional binding parts, such as one or more binding partners that recognize cell surface proteins or antigens, such as immune cell surface proteins or cell surface tumor antigens (also known as cancer cell surface antigens). Cell surface tumor antigens can be, for example, tumor-associated antigens or tumor-specific antigens.

[0426] Examples of additional components (i.e., additional functional parts) include additional binding parts that are engagement partners that recognize the following: B cell maturation antigen (BCMA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), disintegrin and metalloproteinase 17 (ADAM17), programmed death ligand 1 (PD-L1), SLAM family member 7 (SLAMF7), epithelial cell adhesion molecule (EPCAM), epidermal growth factor receptor (EGFR / ErbB-1), epidermal growth factor receptor variant 3 (EGFRvIII), erb-b2 tyrosine kinase 2 (ERBB2 / HER2 / CD340), prostate specific membrane antigen (PSMA), Claudin8.2 (CLDN18.2), delta-like protein 3 (DLL3), 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 NKG2D. Examples of additional components (i.e., additional functional parts) include additional binding parts, which are binding partners that recognize cell surface tumor antigens or cancer cell surface targets selected from the group consisting of: BCMA, ADAM17, SLAMF7, PD-L1, EPCAM, EGFR / ErbB-1, EGFRvIII, ERBB2 / HER2 / CD340, PSMA, CLDN18.2, DLL3, MUC16, MUC17, MUC1, TPBG / 5T4 / WAIF1, and B7-H4 / VTCN1 / B7x / B7S1. Examples of additional components (i.e., additional functional parts) include additional binding parts, which are binding partners that recognize immune cell surface proteins or immune cell surface targets 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 (e.g., BCMA, CD20, CD19, CD22, or CD30) expressed in hematological malignancies. (For the avoidance of doubt, examples of additional binding moieties (e.g., binding partners that recognize cell surface proteins or antigens) are non-limiting and are described in detail herein by way of example.) The additional binding moieties mentioned herein are not CD16a binding polypeptides of the present invention.

[0427] In one embodiment, the CD16a binding polypeptide or CD16a binding oligomer of the present invention is optionally attached to one or more additional functional parts (e.g., one or more additional binding parts and / or signaling molecules) via a linker as described above. For example, at least one CD16a binding polypeptide or CD16a binding oligomer is attached to one, two, three, four or more additional functional parts (e.g., one, two, three, four or more additional binding parts 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 parts (e.g., one or two additional binding parts or signaling molecules). In certain embodiments, at least one CD16a binding polypeptide or CD16a binding oligomer is attached to an additional functional part (e.g., an additional binding part or signaling molecule).

[0428] In certain preferred embodiments, the additional functional part is a signal transduction molecule.Signal transduction molecules (e.g., immune signal transduction molecules, such as cytokines, such as IL-15 or derivatives thereof) can be optionally attached to the N-terminal or C-terminal of a CD16a binding polypeptide or a CD16a binding oligomer by a joint as described above. Alternatively, one or more signal transduction molecules can be attached between two CD16a binding polypeptides in a CD16a binding oligomer, optionally separated by one or more connecting sequences as described above. Preferably, a signal transduction molecule can be optionally attached to the N-terminal or C-terminal of a CD16a binding polypeptide or a CD16a binding oligomer by a joint as described above. Preferably, a signal transduction molecule can be optionally attached to the C-terminal of a CD16a binding polypeptide or a CD16a binding oligomer by a joint as described above.

[0429] In certain preferred embodiments, the additional functional part is an additional binding part. For example, the additional functional part is an additional binding part that recognizes a binding partner of 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 portion is an additional binding portion 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.

[0430] In certain preferred embodiments, the additional binding moiety is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In certain preferred embodiments, the additional binding moiety is specific for an immune cell surface target (e.g., a NK cell target, such as NKp46, NKG2D, PD-1, etc.).

[0431] Additional binding moieties (e.g., binding partners that recognize cell surface tumor antigens BCMA) may be optionally attached to the N-terminus or C-terminus of a CD16a binding polypeptide or CD16a binding oligomer via a linker as 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 connecting sequences as described above. Preferably, additional binding moieties (e.g., binding partners that recognize cell surface tumor antigens BCMA) may be optionally attached to the N-terminus or C-terminus of a CD16a binding polypeptide or CD16a binding oligomer via a linker as described above. More preferably, additional binding moieties (e.g., binding partners that recognize cell surface tumor antigens BCMA) may be optionally attached to the N-terminus of a CD16a binding polypeptide or CD16a binding oligomer via a linker as described above.

[0432] The inventors have surprisingly found that a "dual binder" polypeptide comprising a CD16a binding polypeptide or CD16a binding oligomer as disclosed herein can retain its CD16a binding ability when fused to an additional binding moiety targeting the myeloma antigen BCMA. A "dual binder" polypeptide comprising a CD16a binding polypeptide as disclosed herein fused to a BCMA binding moiety is also surprisingly able to activate NK cells in the presence of tumor cells expressing BCMA.

[0433] In a preferred embodiment, the CD16a binding polypeptide or CD16a binding oligomer of the present invention further comprises one or more additional functional parts, such as at least one, at least two or at least three or at least four additional functional parts. For example, in a specific embodiment, the CD16a binding polypeptide or CD16a binding oligomer of the present invention further comprises 1, 2, 3, 4 or 5 additional functional parts. 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 parts, most preferably two.

[0434] In embodiments in which the CD16a binding polypeptide or CD16a binding oligomer comprises at least two, at least three or at least four additional functional moieties (e.g., 2, 3, 4 or 5 additional functional moieties), each additional functional moiety may be the same, or may be different, or some additional functional moieties may be the same while some additional functional moieties are different (e.g., in embodiments having at least 3 or at least 4 additional functional moieties (e.g., 3, 4, 5, 6 or more additional functional moieties)).

[0435] 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., 2, 3, 4 or 5 additional functional moieties), each additional functional moiety may have the same function, or may have a different function, or some additional functional moieties may have the same function and some may have different functions (e.g., in embodiments having at least 3 or at least 4 additional functional moieties (e.g., 3, 4, 5, 6 or more additional functional moieties)).

[0436] 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., 2, 3, 4 or 5 additional functional moieties), the first additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target); and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof. For example, in a preferred embodiment, the first additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof. In particular, the first additional functional moiety may comprise an additional binding moiety specific for BCMA; and the second additional functional moiety may comprise a cytokine, such as IL-15 or a derivative thereof. Alternatively, for example, in a preferred embodiment, the first additional functional portion may include an additional binding portion specific for an immune cell target (e.g., an NK cell target), and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof. In particular, the first additional functional portion may include an additional binding portion specific for an NK cell target; and the second additional functional portion may include a cytokine, such as IL-15 or a derivative thereof.

[0437] In a very preferred embodiment, the first additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target); and the second additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target). For example, the first additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the second additional functional moiety may comprise an additional binding moiety specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In particular, the first additional functional moiety may comprise an additional binding moiety specific for BCMA; and the second additional functional moiety may comprise an additional binding moiety specific for BCMA.

[0438] Alternatively, for example, the first additional functional moiety may include an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the second additional functional moiety may include an additional binding moiety that is 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 that is specific for BCMA; and the second additional functional moiety may include an additional binding moiety that is specific for an NK cell target.

[0439] In another embodiment, the first additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; and the second additional functional moiety may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

[0440] When present (e.g., in embodiments in which the CD16a binding polypeptide or CD16a binding oligomer comprises at least three or at least four additional functional moieties (e.g., 3, 4, or 5)), the third additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target), or an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof. Preferably, when present, the third additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target; and in particular an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA).

[0441] When present (e.g., in embodiments in which the CD16a binding polypeptide or CD16a binding oligomer comprises at least four additional functional moieties (e.g., 4 or 5)), the fourth additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target), or comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof. Preferably, when present, the fourth additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding moiety specific for an immune cell target, such as an NK cell target; and in particular, an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA).

[0442] When present (e.g., in embodiments in which the CD16a binding polypeptide or CD16a binding oligomer comprises at least five additional functional moieties (e.g., 5 or 6)), the fifth additional functional moiety may comprise an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, e.g., a myeloma cell surface antigen, e.g., BCMA, or an additional binding moiety specific for an immune cell target, e.g., an NK cell target), or comprises an immune signaling molecule, e.g., a cytokine, e.g., IL-15, or a derivative thereof.

[0443] In a preferred embodiment, the CD16a binding polypeptide or CD16a binding oligomer comprises at least three (e.g., 3, 4 or 5) additional functional parts, the first additional functional part may comprise an additional binding part (e.g., an additional binding part that is specific to a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding part that is specific to an immune cell target, such as an NK cell target); and the second additional functional part may comprise an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; and the third additional functional part may comprise an additional binding part (e.g., an additional binding part that is specific to a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding part that is specific to an immune cell target, such as an NK cell target).

[0444] In one embodiment, the first additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the second additional functional part may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA) or an immune cell target (e.g., an NK cell target). In particular, the first additional functional part may include an additional binding portion that is specific for BCMA; and the second additional functional part may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In another embodiment, the first additional functional part may include an additional binding portion that is specific for BCMA; and the second additional functional part may include a cytokine, such as IL-15 or a derivative thereof; and the third additional functional part may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target).

[0445] For example, the first additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the second additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA); and the third additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA) or an immune cell target (e.g., an NK cell target). In particular, the first additional functional part may include an additional binding portion that is specific for BCMA; and the second additional functional part may include an additional binding portion that is specific for BCMA; and the third additional functional part may include an additional binding portion that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). In another embodiment, the first additional functional part may include an additional binding portion that is specific for BCMA; and the second additional functional part may include an additional binding portion that is specific for BCMA; and the third additional functional part may include an additional binding portion that is specific for an immune cell target (e.g., an NK cell target).

[0446] In one embodiment, the CD16a binding polypeptide or CD16a binding oligomer comprising an additional binding portion of the invention has an additional binding portion 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.

[0447] Preferably, the linker for a CD16a binding polypeptide or CD16a binding oligomer comprising a binding functional portion comprises or has 1 to 50 (e.g., 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S).

[0448] In a preferred embodiment, the linker for a 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 GGGSGGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS GS, GGGGSGGGSGGGGSGGGGS, GGSGG, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GGSGGGGSGGG, GGGGGSGGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGSGGGGSGGGGSGGGG.

[0449] In a preferred embodiment, the linker used in 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 used in 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.

[0450] Alternatively, in one embodiment, the CD16a binding polypeptide or CD16a binding oligomer comprising an additional binding moiety of the invention has the additional binding moiety 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).

[0451] In a preferred embodiment, the CD16a binding polypeptide or CD16a binding oligomer comprising the additional binding moiety of the invention does not comprise a linker. In a preferred embodiment, the CD16a binding polypeptide or CD16a binding oligomer is directly attached to the additional binding moiety.

[0452] In certain embodiments, the CD16a binding polypeptide of the invention comprising an additional functional moiety comprises the following structure:

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

[0454] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part];

[0455] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0456] [additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

[0457] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part];

[0458] Wherein, when there is more than one additional functional part, each additional function may be the same, or may be different.

[0459] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15 or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS S, GGSGG, GGSGGGSGGGSGG, GGSGGGSGGGGGSGGGGSGGGGSGG, GSGGG, GGSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGGSGGGG, SGGGGSGGGGSGGGG, or SGGGGGSGGGGSGGGGSGGGG.

[0460] Alternatively, the linker may be absent, ie, the CD16a binding polypeptide or CD16a binding oligomer comprises the following structure:

[0461] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[additional functional part];

[0462] [Additional functional part]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

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

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

[0465] [Additional functional part]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Additional functional part].

[0466] In such embodiments, more preferably, the CD16a binding polypeptide comprises the following structure:

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

[0468] [additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

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

[0470] (or contain the following structure

[0471] [Additional functional part]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0472] [Additional functional part]-[Additional functional part]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]; or

[0473] [Additional functional part]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Additional functional part]).

[0474] In such embodiments, preferably, each additional functional moiety is an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). Most preferably, each additional functional moiety is an additional binding moiety that is specific for BCMA.

[0475] Even more preferably, the CD16a binding polypeptide comprises the following structure:

[0476] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]; or

[0477] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0478] In such embodiments, preferably, each additional functional moiety is an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). Most preferably, each additional functional moiety is an additional binding moiety that is specific for BCMA.

[0479] In a very particularly preferred embodiment of the present invention, the CD16a binding polypeptide comprises the following structure:

[0480] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0481] In such embodiments, preferably, each additional functional moiety is an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). Most preferably. Each additional functional moiety is an additional binding moiety that is specific for BCMA. Optionally, in such embodiments, the linker may not be present.

[0482] In another very particularly preferred embodiment of the present invention, the CD16a binding polypeptide consists of the following structure:

[0483] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0484] In such embodiments, preferably, each additional functional moiety is an additional binding moiety that is specific for a cancer cell surface target (e.g., a myeloma cell surface antigen, such as BCMA). Most preferably. Each additional functional moiety is an additional binding moiety that is specific for BCMA. Optionally, in such embodiments, the linker may not be present.

[0485] In certain embodiments, the CD16a binding oligomer of the invention comprising an additional functional moiety comprises the following structure:

[0486] [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[additional functional part];

[0487] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0488] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part];

[0489] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0490] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0491] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part];

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

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

[0494] [N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separation part]-[helix 2]-[C-terminal part];

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

[0496] In such embodiments, the linker preferably comprises or has 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) naturally occurring amino acids; and preferably 1 to 20 (e.g., 1, 5, 10, 15, or 20; more preferably 1 to 15) naturally occurring amino acids, such as a sequence selected from the group consisting of G, S, and T (preferably G and S). For example, the linker is G or comprises or has the sequence GGGSG, GGGGS, GGSGG, GSGGG, and / or SGGGG. Furthermore, for example, the linker is G or contains or has the sequence GGGSG, GGGGSGGGGSG, GGGGSGGGSGGGGSG, GGGGSGGGGSGGGGSGGGGSG, GGGGS, GGGSGGGGS, GGGGSGGGGSGGGGS, GGGSGGGGSGGGGSGGGGS, GGSGG, GGGSGGGGSGG, GGS GGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGGGGSGG, GSGGG, GSGGGGSGGG, GGSGGGSGGGGSGGG, GSGGGGSGGGGSGGGGSGGG, SGGGG, SGGGGSGGGG, SGGGGSGGGGSGGGG or SGGGGSGGGGSGGGGSGGGG.

[0497] In such embodiments, one or more linkers may be absent.

[0498] In such embodiments, more preferably, the CD16a binding oligomer comprises the following structure:

[0499] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

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

[0501] [additional functional moiety]-[linker]-[N-terminal moiety]-[helix 1]-[separating moiety]-[helix 2]-[C-terminal moiety]-[linker]-[helix 1]-[separating moiety]-[helix 2]-[C-terminal moiety]-[linker]-[additional functional moiety]; or

[0502] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0503] Even more preferably, the CD16a binding oligomer comprises the following structure:

[0504] [additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

[0505] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0506] In certain embodiments, the CD16a binding oligomer of the invention comprising an additional functional moiety comprises at least three CD16a binding polypeptides and comprises the following structure:

[0507] [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part];

[0508] [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Isolation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Isolation part]-[Helix 2]-[C-terminal part];

[0509] [N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part];

[0510] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Separation part]-[Helix

[0511] 2]-[C-terminal portion]-[linker]-[N-terminal portion]-[helix 1]-[separating portion]-[helix 2]-[C-terminal portion]; wherein each linker portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each N-terminal portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each C-terminal portion can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each separating portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; each helix 1 portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence; and each helix 2 portion in the oligomer can have the same sequence, have different sequences, or two can have the same sequence and one can have a different sequence.

[0512] In such embodiments, the linker is preferably a linker as defined above. In such embodiments, one or more linkers may be absent.

[0513] In such embodiments, more preferably, the CD16a binding oligomer comprises the following structure:

[0514] [additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 2]-[separating part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or

[0515] [Additional functional part]-[Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 2]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part].

[0516] In the above embodiments defining the structure of a CD16a binding polypeptide or CD16a binding oligomer comprising an additional functional moiety, each additional functional moiety may be any one of those described herein. For example, each additional functional moiety may be independently selected from

[0517] Additional binding moieties (e.g., additional binding moieties specific for cancer cell surface targets, e.g., myeloma cell surface antigens such as BCMA, or immune cell targets, e.g., NK cell targets); and

[0518] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0519] For example, each additional functional moiety can be independently selected from

[0520] Additional binding moieties that are specific for cancer cell surface targets (e.g., myeloma cell surface antigens such as BCMA);

[0521] Additional binding moieties specific for immune cell targets (e.g., NK cell targets); and

[0522] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0523] For example, each additional functional moiety can be independently selected from

[0524] an additional binding moiety specific for BCMA;

[0525] Additional binding moieties specific for NK cell targets; and

[0526] Immune signaling molecules, such as cytokines, such as IL-15 or derivatives thereof.

[0527] For example, each additional functional moiety can be independently selected from

[0528] an additional binding moiety specific for BCMA; and

[0529] Additional binding moieties specific for NK cell targets; and

[0530] Cytokines, such as IL-15 or its derivatives.

[0531] For example, each additional functional moiety can be independently selected from

[0532] an additional binding moiety specific for BCMA; and

[0533] Additional binding moieties specific for NK cell targets; and

[0534] IL-15 or its derivatives.

[0535] In a preferred embodiment, each additional functional moiety can be independently selected from

[0536] an additional binding moiety specific for BCMA; and

[0537] Additional binding moieties specific for NK cell targets; and

[0538] Cytokines, such as IL-15 or its derivatives.

[0539] In a preferred embodiment, each additional functional moiety can be independently selected from

[0540] an additional binding moiety specific for BCMA; and

[0541] Additional binding moieties with specificity for NK cell targets.

[0542] In a preferred embodiment, each additional functional moiety is an additional binding moiety specific for BCMA.

[0543] In the above embodiments defining the structure of a CD16a binding polypeptide or CD16a binding oligomer comprising an additional functional moiety, each additional functional moiety can be an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA). For example, each additional functional moiety can be an additional binding moiety for BCMA.

[0544] In a preferred embodiment, the additional binding moiety for BCMA is an hBCMA binding polypeptide comprising at least one hBCMA binding motif, wherein the polypeptide comprises the following structure:

[0545] [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]

[0546] The hBCMA binding motif is part [helix 1]-[isolated part]-[helix 2].

[0547] For example, it is an hBCMA-binding polypeptide as defined in a PCT application filed on May 31, 2023 in the name of Oncopeptides Innovation 1AB claiming priority to GB application number 2208027.9 and GB application number 2214718.5. The contents of this PCT application are incorporated herein by reference.

[0548] In particular, an hBCMA-binding polypeptide is a polypeptide wherein:

[0549] i) Helix 1 contains the sequence X9X10 X 11 ADX 14 EIX 17 X 18 , and helix 2 contains the sequence FX 25 QKWAFX 31 RX 33 LX 35 , where, independently of each other,

[0550] a) X9 and X 10 is any naturally occurring amino acid; X 11 is E, F, H, Q, T or Y; X 14 is any naturally occurring amino acid; X 17 is A, E, Q, S, T, or V; X 18 is any naturally occurring amino acid; X 25 is F or Y; X 31 is I, M or V; X 33 is K or S; X 35 is I, L, M or V;

[0551] or ii) helix 1 and helix 2 are as defined in i), wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

[0552] For example, an hBCMA binding polypeptide is a polypeptide having an hBCMA binding potency of at least 1% of the following sequence:

[0553] VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLA

[0554] E AKKLNDAQAPK [SEQ ID 226].

[0555] In a very preferred embodiment, the hBCMA binding polypeptide is the polypeptide of SEQ ID 226.

[0556] CD16a binder-drug conjugates

[0557] Alternatively or additionally, a CD16a binding polypeptide or CD16a binding oligomer as disclosed herein can be attached to a therapeutic agent to form a CD16a binding agent-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 one or more linkers as described above. Non-limiting examples of such therapeutic agents include cytotoxic drugs, such as mitomycin C, desmethyltopotecan, SN-38, MMAE, MMAF, doxorubicin, pyrrolobenzodiazepines, Amanitin, maytansinoid (eg maytansinoid DM1 or maytansinoid DM4) or duostatin (eg dolastatin 5.2).

[0558] For the avoidance of doubt, during the production of a CD16a binder-drug conjugate according to the invention, one or more therapeutic agents (e.g., MMAF) must be modified by reaction between a functional group at the point of attachment (e.g., -NH, -OH, or -SH (e.g., in Cys)) and any attachment group or linker used. Thus, one skilled in the art will appreciate that the therapeutic agent in a CD16a binder-drug conjugate (e.g., a CD16a binder-MMAF conjugate) comprises such modifications.

[0559] Peptide production

[0560] The polypeptides of the present invention can be produced using methods known in the art. For example, they can be prepared by chemical synthesis or by recombinant protein production technology in, for example, bacteria, yeast, insects, fungi, plants or mammalian cells.

[0561] The polypeptide of the present invention can also be fused with different molecules with therapeutic potential, such as immunoglobulins with therapeutic potential, through the above-mentioned recombinant or chemical synthesis techniques.

[0562] Modulation of peptide properties

[0563] The pharmacokinetic properties of the polypeptides of the present invention can be adjusted by methods known in the art. For example, they can be linked to a portion that prolongs the plasma half-life, such as a polyethylene glycol polymer, an unstructured polypeptide (such as XTEN or PAS), or an FcRn binding ligand, such as the Fc domain of a serum albumin or an immunoglobulin.

[0564] preparation

[0565] The polypeptide according to the present invention (eg, a CD16a binding polypeptide or CD16a binding oligomer, or a CD16a binding agent-drug conjugate of the present invention) may be present in a formulation, and in particular a pharmaceutical formulation.

[0566] In certain embodiments, the present invention provides nucleic acid molecules encoding CD16a binding polypeptides or CD16a binding oligomers of the present invention. Nucleic acid molecules encoding CD16a binding polypeptides or CD16a binding oligomers of the present invention can be used as medicines, for example, can be used to treat cancer. Nucleic acid molecules can be, for example, DNA or RNA molecules, such as mRNA molecules. Nucleic acid molecules according to the present invention can be present in preparations, and in particular in pharmaceutical preparations. Therefore, the present invention also provides preparations of nucleic acid molecules (e.g., DNA or RNA, and in particular mRNA molecules) encoding CD16a binding polypeptides or CD16a binding oligomers of the present invention, and in particular pharmaceutical preparations.

[0567] Pharmaceutical formulations include, for example, those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary) or intraperitoneal administration, although the most appropriate route may depend, for example, on the condition and disorder of the subject being treated.

[0568] 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, e.g., an mRNA molecule of the invention) according to the invention is administered as a pharmaceutical formulation suitable for oral or parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion) and intramedullary) administration.

[0569] Pharmaceutical formulations suitable for oral administration may be in the form of discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of active ingredient; powders or granules; solutions or suspensions in aqueous liquids or non-aqueous liquids; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. The peptides of the present invention may also be in the form of boluses, electuaries or pastes. Various pharmaceutically acceptable carriers and their formulations are described in, for example, EW Martin's Remington's Pharmaceutical Sciences standard formulations. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supplement 42: 2S, 1988.

[0570] Preparations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may include antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Preferably, the preparation may be present in a unit dose or separate dose container, such as a sealed ampoule and vial. The preparation can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as saline, a physiologically acceptable solution, or water for injection, immediately before use.

[0571] Ready-to-use injection and infusion solutions and suspensions can be prepared from sterile powders, granules or other dry compositions. Exemplary compositions for parenteral administration include injectable solutions or suspensions that may contain, for example, suitable nontoxic, parenterally acceptable diluents or solvents (such as mannitol, 1,3-butylene glycol, water, Ringer's solution, isotonic sodium chloride solution); or other suitable dispersants or wetting agents and suspending agents (including synthetic monoglycerides or diglycerides) and fatty acids (including oleic acid or Cremaphor).

[0572] Dosage regimen

[0573] The CD16a binding polypeptides or CD16a binding oligomers of the present invention (particularly CD16a binding agent-drug conjugates), and pharmaceutical preparations comprising such polypeptides, oligomers or binding agent-drug conjugates or nucleic acid molecules of the present invention (e.g., DNA or RNA molecules of the present invention, such as mRNA molecules of the present invention), and pharmaceutical preparations comprising those nucleic acid molecules, can be used to treat and / or prevent cancer, such as multiple myeloma.

[0574] The amount of CD16a binding polypeptide or CD16a binding oligomer (particularly CD16a binding agent-drug conjugate) or nucleic acid molecule required to achieve a therapeutic effect will vary with the specific route of administration and the characteristics of the subject being treated, such as species, age, weight, sex, medical condition, specific disease and its severity, and other relevant medical and physical factors. An ordinary technician can easily determine and administer the effective amount of CD16a binding polypeptide, CD16a oligomer, CD16a binding agent-drug conjugate and / or compositions comprising them, or nucleic acid molecules and / or compositions comprising them required for the treatment and / or prevention of cancer.

[0575] Depending on the subject to be treated and the severity of the cancer, the CD16a binding polypeptides, CD16a binding oligomers (particularly CD16a binding agent-drug conjugates) or nucleic acid molecules of the invention or pharmaceutical formulations thereof can be administered, for example, daily, weekly, every two weeks, every three weeks or every four weeks or even as a high single dose.

[0576] The CD16a binding polypeptides, CD16a binding oligomers (particularly CD16a binding agents-drug conjugates) or nucleic acid molecules of the present invention or their pharmaceutical preparations can be administered, for example, as parenteral or oral doses. Parenteral administration includes intravenous (into a vein, such as a central or peripheral vein, bolus or infusion), intraarterial (into an artery, such as a central or peripheral artery), intraosseous infusion (into the bone marrow), intramuscular (into the muscle), intradermal (into the dermis) and subcutaneous (under the skin) administration. In a preferred embodiment, the dose of the present invention is administered intravenously or intraarterially, and more preferably by intravenous infusion (e.g., central vein infusion or peripheral vein infusion). In another preferred embodiment, the dose of the present invention is administered by subcutaneous injection. Therefore, pharmaceutical preparations particularly useful for the present invention are those suitable for intravenous administration, more particularly intravenous infusion or subcutaneous administration.

[0577] The CD16a binding polypeptide, CD16a binding oligomer (particularly CD16a binding agent-drug conjugate) or nucleic acid molecule and its pharmaceutical preparation of the present invention can be administered as part of a treatment cycle. In a treatment cycle, the polypeptide can be administered on the first day of the cycle, wherein the cycle lasts for X days, and the CD16a binding polypeptide or CD16a binding oligomer of the present invention is no longer administered on the next X-1 day. X can be, for example, 1 day to 42 days, such as 2 days to 14 days. Alternatively, the CD16a binding polypeptide or CD16a binding oligomer can be administered in divided doses, such as on the first and second days of the cycle.

[0578] Depending on the type, category or stage of the cancer to be treated, the cycle can be repeated once or several times. For example, the cycle can be repeated 1 to 100 times, such as 2 to 50 times, such as 8 to 40 times, such as 8 or 16 times. For example, the CD16a binding polypeptide or CD16a binding oligomer or nucleic acid molecule of the present invention can be administered 8 times of repeated 7-day cycles, followed by 16 times of repeated 14-day cycles, optionally followed by further repeated 28-day cycles. Ordinary skilled physicians or clinicians can easily determine the number of cycles of CD16a binding polypeptides (e.g., CD16a binding agents-drug conjugates) required to prevent, combat or prevent cancer progression.

[0579] Combination therapy

[0580] Although the CD16a binding polypeptides or CD16a binding oligomers (particularly CD16a binding agent-drug conjugates) or nucleic acid molecules disclosed herein can be used as the sole active ingredient in the present invention, they can also be used in combination with one or more additional therapeutic agents, and the use of such combinations provides an embodiment of the present invention. Such additional therapeutic agents may be agents or other pharmaceutically active materials suitable for treating and / or preventing cancer. Such agents are known in the art.

[0581] Non-limiting examples of additional therapeutic agents for use in the present invention may include proteasome inhibitors (PIs) (e.g., carfilzomib, bortezomib, or ixazomib), immunomodulatory agents (IMiDs) (e.g., lenalidomide, thalidomide, or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, bendamustine, or melflufen )), 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 a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflurane), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

[0582] One or more additional therapeutic agents can be used simultaneously, sequentially or separately with the administration of a dose of a CD16a binding polypeptide, CD16a binding oligomer, CD16a binding agent-drug conjugate or nucleic acid molecule of the invention. The individual components of such a combination can be administered separately at different times during the course of treatment, or simultaneously in separate or single combination forms.

[0583] In one embodiment of the invention, one or more additional therapeutic agents are selected from PI, IMiD and steroids. For example, one or more additional therapeutic agents are PI (e.g., bortezomib or carfilzomib), IMiD (e.g., lenalidomide, thalidomide and pomalidomide) and steroids (e.g., prednisone, prednisolone and dexamethasone). Preferably, one or more therapeutic agents are bortezomib, thalidomide and dexamethasone.

[0584] In one embodiment of the invention, one or more additional therapeutic agents are selected from PIs, alkylating agents, and steroids. For example, one or more additional therapeutic agents are PIs (e.g., bortezomib or carfilzomib), alkylating agents (e.g., cyclophosphamide, melphalan, melflufen, or bendamustine), and steroids (e.g., prednisone, prednisolone, and dexamethasone). Preferably, one or more therapeutic agents are bortezomib, melphalan, melflufen, and prednisone.

[0585] In one embodiment of the invention, one or more additional therapeutic agents are selected from PIs and steroids. For example, 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.

[0586] In one embodiment of the invention, one or more additional therapeutic agents are selected from IMiDs and steroids. For example, one or more therapeutic agents are IMiDs (e.g., lenalidomide, thalidomide, and pomalidomide) and steroids (e.g., prednisone, prednisolone, and dexamethasone). Preferably, IMiDs are lenalidomide, and steroids are dexamethasone.

[0587] In another embodiment of the invention, the one or more therapeutic agents may be selected from NK cell-based or T cell-based therapies.

[0588] Alternatively, the CD16a binding polypeptide or CD16a binding oligomer (particularly CD16a binding agent-drug conjugate) or nucleic acid molecule according to the present invention can be combined with a therapeutic method, such as a stem cell transplantation procedure, for example, an autologous stem cell transplantation or an allogeneic stem cell transplantation. Therefore, in one embodiment of the present invention, the CD16a binding polypeptide disclosed herein can be combined with an autologous stem cell transplantation procedure. In another embodiment of the present invention, the CD16a binding polypeptide disclosed herein can be combined with an allogeneic stem cell transplantation procedure.

[0589] Simultaneous, sequential or separate administration of one or more additional therapeutic agents or therapeutic procedures with the CD16a binding polypeptides, CD16a binding oligomers, CD16a binding agent-drug conjugates or nucleic acid molecules of the invention further enhances their effectiveness in treating and / or preventing cancer.

[0590] Reagent test kit

[0591] The present invention provides a kit comprising a CD16a binding polypeptide, a CD16a binding oligomer (particularly a CD16a binding agent-drug conjugate) or a nucleic acid molecule as disclosed herein, and one or more additional therapeutic agents useful for treating and / or preventing cancer.

[0592] Non-limiting examples of additional therapeutic agents for use in the present invention may include proteasome inhibitors (PIs) (e.g., carfilzomib, bortezomib, or ixazomib), immunomodulators (IMiDs) (e.g., lenalidomide, thalidomide, or pomalidomide), alkylating agents (e.g., cyclophosphamide, melphalan, melflufenamic acid, 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. The one or more additional therapeutic agents may, for example, be selected from a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflurane), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

[0593] In one embodiment of the present invention, the kit of the present invention can be used for treating and / or preventing cancer.

[0594] For the avoidance of doubt, the CD16a binding polypeptides, CD16a binding oligomers (particularly CD16a binding agent-drug conjugates) or nucleic acid molecules disclosed herein are present in a form and amount suitable for use according to the present invention in a kit according to the present invention. Suitable pharmaceutical formulations are described herein. The skilled person can easily determine the amount of CD16a binding polypeptides or oligomers (e.g., in the form of CD16a binding agent-drug conjugates) or nucleic acid molecules disclosed herein suitable for use according to the present invention.

[0595] cancer

[0596] The CD16a binding polypeptides, CD16a binding oligomers (particularly CD16a binding agent-drug conjugates) or nucleic acid molecules of the present invention, and the pharmaceutical preparations or kits of the present invention comprising the CD16a binding polypeptides, CD16a binding oligomers, CD16a binding agent-drug conjugates or nucleic acid molecules can be used in medicine, for example, for treating and / or preventing cancer in a subject. Non-limiting examples of cancer include solid cancers such as bladder cancer, breast cancer, colorectal cancer, CNS cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, or thyroid cancer; and blood cancers such as leukemias (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia), lymphomas (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, small lymphocytic lymphoma and other high-grade B-cell lymphomas), or plasmacytomas and myelomas (e.g., MGUS, plasmacytoma, latent myeloma, multiple myeloma, light chain myeloma, or non-secretory myeloma).

[0597] Preferably, the CD16a binding polypeptide or CD16a binding oligomer of the present invention (particularly a CD16a binding agent-drug conjugate) or the nucleic acid molecule of the present invention and the pharmaceutical preparation or kit of the present invention comprising the CD16a binding polypeptide, oligomer, binding agent-drug conjugate or nucleic acid molecule can be used to treat and / or prevent blood cancers, such as leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, small lymphocytic lymphoma and other high-grade B-cell lymphomas) or plasmacytoma and myeloma (e.g., MGUS, plasmacytoma, oligomyeloma, 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 comprising the CD16a binding polypeptide or CD16a binding oligomer can be used to treat and / or prevent 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 comprising the CD16a binding polypeptide, CD16a binding oligomer, CD16a binding agent-drug conjugate or nucleic acid molecule can be used to treat and / or prevent multiple myeloma.

[0598] In certain embodiments, the CD16a binding polypeptides or CD16a binding oligomers (particularly CD16a binding agent-drug conjugates) of the present invention or the nucleic acid molecules of the present invention, and the pharmaceutical preparations or kits of the present invention comprising the CD16a binding polypeptides, oligomers, binding agent-drug conjugates or nucleic acid molecules can be used as anti-cancer immunotherapeutics; for example, anti-cancer immunotherapeutics for treating cancer, and particularly blood cancers, such as the blood cancers described herein, and particularly multiple myeloma.

[0599] While the present invention has been described and illustrated with reference to specific embodiments, it will be appreciated by those skilled in the art that the present invention is susceptible to many different variations not specifically described herein.

[0600] In the foregoing description, when reference is made to integers or elements with known, obvious or foreseeable equivalents, such equivalents are incorporated herein as if set forth separately. Reference should be made to the claims to determine the true scope of the invention, which should be interpreted as encompassing any such equivalents. The reader will also understand that integers or features of the invention described as preferred, advantageous, convenient, etc. are optional and do not limit the scope of the independent claims. In addition, it should be understood that although such optional integers or features may have benefits in certain embodiments of the invention, in other embodiments, the optional integers or features may be undesirable and therefore may not exist.

[0601] Example

[0602] The following examples illustrate the invention.

[0603] Preparation Example 1: Selection of binders to hCD16a using a phage library by phage display

[0604] In this example, human hCD16a was used as a target in phage display selection using a phage-based affinity library. Individual clones obtained after four selection cycles were assayed for binding to hCD16a in a monoclonal phage-ELISA (enzyme-linked immunosorbent assay), and DNA sequencing was performed on ELISA-positive clones.

[0605] Materials and methods

[0606] Affibody Library .

[0607] Based on the phagemid vector pAffi-1 ( et al. (2007) J. Biotechnol. 128: 162-183) prepared an M13 phage display library of affibody molecules. This phagemid containing the lac promoter and OmpA signal peptide was designed for phage display of the encoded affibody library members as albumin binding domains (ABD WT ), an amber stop codon and an in-frame fusion of a truncated form of the M13 phage coat protein 3 (residues 249-406). A synthetic 121 base oligonucleotide (5'-GCGCTTTGGCTTGGGTCATCXXXTAAACTXXXYYYGAAGGCXXXXXXTTG) encoding amino acids 3-41 of the Z domain (reverse complement) during synthesis

[0608] XXXXXXGTTCAGGTTCGGCAGXXXXXXGATCTCXXXXXXCGCXXXXXXXXXTTCTTTGTTGAATTTGTTGT-3') [SEQ ID 222] (Nilsson et al. (1987) Protein Eng. 1: 107-113) (wherein a mixture of trinucleotide codons were used to build the blocks during synthesis (XXX = an equal mixture of codons for all 20 amino acids except Cys and Pro, YYY = 60% Ile and 10% each for His, Tyr, Lys and Asp) and the wild-type codons at 14 positions in the domain were randomized) was used as a template for PCR amplification using forward primer (5'-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3') [SEQ ID 223] and reverse primer (5'-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3') [SEQ ID 224]. Approximately 6.4 μg of XhoI and NheI double cut and gel purified (Qiagen, Germany) PCR product was ligated with 35 μg of XhoI and NheI double cut and gel purified pAffi-1 phagemid vector using T4 DNA ligase. The resulting ligation mixture was desalted using column purification (Qiagen, Germany), divided into 24 portions and used for electroporation (0.1 cm BioRad cuvette) of 25 μl electrocompetent ER2738 E. coli cells (F', glnV amber suppressor) (Lucigen, USA). 970 μl recovery medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) was added to the electroporated cells, which were then combined (six electroporations per pool) and incubated at 37°C for 1 hour under shaking, after which the cell pool was titrated by spreading a dilution series on an Amp (ampicillin) plate and transferred to four 5-liter shake flasks, each containing one liter of tryptic soy broth + yeast extract medium (30 g / l tryptic soy broth, 5 g / l yeast extract; TSB+Y) supplemented with 2% glucose and 100 μg / ml Amp. Overnight cell cultures were pelleted by centrifugation and resuspended in 50 ml of cold 40% glycerol, then distributed to 29 tubes, approximately 3.5 ml of cells / glycerol solution per tube. Based on the titration after electroporation and OD600 measurement after overnight culture, the library size (diversity) was calculated to be approximately 3 × 10 10, and each 3.5 ml cell aliquot contains a cell number corresponding to approximately 10,9x library size. The tubes containing cells were stored at -80 °C until used for phage stock preparation using M13KO7 helper phage.

[0609] Production of primary phage library stock .

[0610] To produce phage stocks displaying Affimers, 1.7 ml of the library glycerol stock was distributed and inoculated into four E flasks containing 750 ml TSB+Y, 1% (w / v) glucose, 10 μg / ml Tet (tetracycline) and 100 μg / ml Carb (carbenicillin) and grown at 37°C with shaking at 150 rpm until the culture reached OD600=1. Cells were infected with 120 μl (multiplicity of infection (MOI) of 5) of M13KO7 helper phage (New England Biolabs), gently swirled and incubated at 37°C without shaking for 15 minutes, followed by incubation at 37°C at 70 rpm for 15 minutes. Cells from each culture were centrifuged and resuspended in 750 ml TSB+Y, 100 μg / ml Carb and 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). 25 μg / ml Kan (kanamycin) was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 200 rpm for 16 to 18 hours. The phage library stock was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titer of the stock was measured by spot titration, and the percentage of phage particles carrying phagemids with affibody inserts was analyzed using polymerase chain reaction-screening.

[0611] Phage display selection of hCD16a binding candidates .

[0612] Four cycles of panning were performed in two separate selection tracks using biotinylated hCD16a (biotinylated human hCD16a (F158), Avitag, His tag, Acro Biosystems, catalog number CDA-H82E8, corresponding to residues 17-208 of Uniprot entry P08637) at concentrations of 80 nM, 40 nM, 20 nM, and 10 nM, respectively. 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 nonspecific binders, all tubes used for biopanning were pretreated with 1% (w / v) bovine serum albumin (BSA) in PBS-T (PBS supplemented with 0.05% (v / v) Tween-20, pH 7.4). In addition, phage stocks in PBS-T were preincubated with 0.1% (w / v) BSA and beads for 30 min at room temperature (RT) under constant end-over-end (eoe) rotation to remove phage carrying binders to SA. The amount of phage stock used was 10 in cycles 1 and 4. 11 colony forming units (cfu), and 10 in cycles 2-3 12cfu. In rounds 1-2, the biotinylated target protein was fixed on 1 mg or 0.5 mg beads at RT and end-to-end (eoe) for 1 hour. The target-bound beads were incubated with PBS-T containing 1% (w / v) BSA at RT and eoe for 30 minutes, then washed with PBS-T, and then pre-incubated phage stock solution was added for selection. In rounds 3-4, the pre-incubated phage stock solution was added to the biotinylated target protein for selection in solution, and then the phage antigen complex was captured by incubating with 0.5 mg or 0.4 mg SA beads at RT and eoe for 30 minutes. The selection step was performed at RT, and the incubation time for selection was 3 hours (round 1) or 2 hours (rounds 2-4), followed by washing with PBS-T eoe for a total of 5 minutes, 10 minutes, 15 minutes or 20 minutes at RT for each subsequent cycle. The final wash volume was transferred to a new 1% (w / v) BSA pretreated tube to remove sticky binders attached to the tube wall. For one of the selection tracks, antigen-bound phages were eluted by incubation with 0.5 M acetic acid (pH 2.8) at eoe, RT for 15 minutes, followed by transfer of the eluate to a new pretreated tube and neutralization with an equal volume of 1 M Tris-HCl (pH 8). For the second selection track, antigen-bound phages were eluted by incubation with 0.25 mg / ml trypsin (Gibco Life Technologies) in TBS-T (TRIS buffered saline, 0.1% (v / v) Tween-20) supplemented with 1 mM CaCl2 for 30 minutes at eoe, RT, and then the eluate was transferred to a new pretreated tube.

[0613] Phage stock amplification .

[0614] New phage stocks (total volume after cycle 1, half volume after cycle 2-3) were generated by growing E. coli XL-1 Blue cells (Agilent) in TSB + Y with 10 μg / ml Tet at 150 rpm at 37°C until OD600 = 0.5-0.8, and then infecting them with phage eluate (100 ml after cycle 1-2, 50 ml after cycle 3). The infected culture was gently vortexed, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was then centrifuged and resuspended in TSB + Y, and then plated on blood agar plates (40 g / l blood agar) with 100 μg / ml Carb and 1% (w / v) glucose and incubated at 37°C for 16 to 18 hours. Bacterial colonies were collected by scraping into TSB+Y and inoculated into 200 ml TSB+Y containing 100 μg / ml 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 superinfected with M13KO7 helper phage (MOI of 10), swirled gently, incubated at 37°C without shaking for 25 minutes, and then at 37°C with shaking at 70 rpm for 15 minutes. The culture was centrifuged and resuspended in 150 ml TSB+Y containing 100 μg / ml and 1 mM IPTG. 25 μg / ml Kan was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 150 rpm for 16 to 18 hours. The phage library stock was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titers of the stocks were measured by spot titration on Carb plates and the percentage of phage particles carrying phagemids with the affibody insert was analyzed using polymerase chain reaction screening.

[0615] Monoclonal phage supernatant preparation .

[0616] After cycle 4, the PCR products of the correct size and the bacterial colonies representing the two selection tracks were grown in 500 μl TSB+Y / Carb in a 96-well deep-well plate at 30°C with 250 rpm shaking for 16 to 18 hours. 30 μl of overnight culture was inoculated into 720 μl TSB+Y / Carb in a new 96-well deep-well plate and incubated at 37°C, 250 rpm for 2 hours, and then super-infected by adding M13K07 helper phage (MOI 7) in 100 ul TSB+Y / Carb per well. The plate was incubated at 37°C without shaking for 30 minutes. Finally, 150 μl TSB+Y / Carb / IPTG / Kan was added to each well. The final concentration was 100 μg / ml Carb, 1 mM IPTG and 25 μg / ml Kan. The deep-well plate was incubated at 37°C, 250 rpm for 16 to 18 hours. The next day, phage supernatants were collected by centrifugation.

[0617] Monoclonal phage-ELISA screening of candidates .

[0618] MaxiSorp ELISA plates (clear flat bottom immunonon-sterile 384-well plates, catalog number 464718, ThermoFisher Scientific) were coated with 30 μl of 10 μg / ml biotinylated hCD16a, 20 μg / ml human serum albumin (HSA; Sigma catalog number SRP6182) (for assessing the correct display of the expression cassette containing the three-part fusion protein including the affibody, the affibody albumin binding domain and the truncated protein 3), 10 μg / ml SA or 10 μg / ml irrelevant 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 / well (1 / 4 of the well with each coating protein) at 4°C with slow shaking for 16 to 18 hours. The coating solution was removed and the plate was washed twice with PBS-T, and blocked with PBS supplemented with 1% (w / v) BSA for 1 hour at RT under slow shaking. The blocking solution was discarded and the wells were incubated with 10 μl phage supernatant diluted with 20 μl PBS-T 1:3 at RT under slow shaking for 1 hour. The supernatant was removed and the plate was washed three times with PBS-T. After adding 30 μl PBS-T containing 1:5000 α-M13-HRP (Sigma-Aldrich, Stockholm, Sweden) and incubating for 30 minutes at RT under slow shaking, the plate was washed twice with PBS-T and once with PBS. 30 μl TMB substrate (TMB substrate kit, Thermo Fisher Scientific) was added to each well. The reaction was terminated by adding 30 μl 2M H2SO4 after 15 to 25 minutes. The absorbance at 450 nm was measured using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).Candidates considered as ELISA-positive clones had a high HSA signal and a relatively high signal for hCD16a compared to SA and irrelevant target controls.

[0619] DNA Sequencing. After ELISA screening, 26 ELISA-positive clones were sent for DNA sequencing by Sanger sequencing (PlateSeq, Eurofins Genomics, Ebersberg, Germany).

[0620] result

[0621] To identify polypeptides that bind to hCD16a, an affinity phage library was constructed and used as input for the first cycle of a four-cycle phage display selection campaign as described in Materials and Methods (above). After the fourth cycle of selection, phage eluates from the acid and trypsin selection tracks were used to infect E. coli cells to obtain single colonies on carbenicillin plates. Using a phage-ELISA experiment, forty-eight randomly selected colonies (clones) from each track were subjected to binding analysis using microtiter plates coated with four test antigens (HSA, streptavidin, SLAMF7, and hCD16a F158). Several of the clones analyzed showed strong ELISA signals for HSA and hCD16a, as well as low signals for the streptavidin and SLAMF7 controls used, such as Figure 1b . Such clones were subjected to DNA sequencing, which showed 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 as SEQ ID 1, 74 and 75 in Table 1 and the sequence table below. Table 1 also lists the sequences of certain other peptides, which are closely based on the sequences disclosed herein with the corresponding SEQ ID references. Throughout this example section, the exemplary compounds numbered X are polypeptides having the same numbered amino acid sequence (i.e., SEQ ID X).

[0622] Table 1

[0623]

[0624]

[0625] Table 1 shows the sequences of certain peptides of the present disclosure and their assigned SEQ ID NOs.

[0626] Biological Example 2 :

[0627] Initial binding studies using surface plasmon resonance

[0628] In this example, the CD16a binding polypeptides corresponding to SEQ ID 1, 74 and 75 were subcloned, expressed and purified as His6-Affibody-ABD WT The fusion proteins [SEQ ID NO. 76-78] were initially analyzed for binding to CD16a by surface plasmon resonance.

[0629] Materials and methods

[0630] Subcloning of CD16a binding peptides

[0631] DNA fragments encoding three CD16a binding polypeptides [having SEQ ID 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. The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to clone the DNA fragments with an N-terminal His6 tag and a C-terminal ABD WT The monomeric CD16a binding polypeptide constructs were constructed. 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 as SEQ ID 76-78 in the sequence table.

[0632] Expression and purification

[0633] Escherichia coli BL21 (DE3) cells were transformed with plasmids containing DNA constructs and cultured with 150 rpm shaking at 37 ° C for 16 to 18 hours in 10 ml TSB + Y containing 25 μg / ml Kan. The culture was inoculated with 1: 100 in 200 ml TSB + Y containing 25 μg / ml Kan and grown with 150 rpm shaking at 37 ° C until OD600 = 0.6-1. IPTG was added to a final concentration of 1 mM to induce protein expression. The culture was cultured with 150 rpm shaking at 25 ° C for 16 to 18 hours and harvested by centrifugation. The cell pellet was resuspended in a denaturing lysis buffer (7M guanidine hydrochloride, 47mM Na2HPO4, 2.65mMNaH2PO4, 10mM TRIS-HCl, 100mM NaCl, pH 8). After incubation at 37 ℃ with 150rpm shaking for 2 hours, the cells were centrifuged and the denatured proteins from the supernatant of the cell lysate were added to the tube containing 3ml HisPur Cobalt IMAC resin (Cat. No. 89966, Thermo Scientific). The supernatant was incubated with the resin for 30 minutes at RT and rotated end to end. The pollutants were removed by washing three times with washing buffer (7M guanidinium chloride salt, 46.6mM NaH2PO4, 3.4mM NaH2PO4, 300mM NaCl, 10mM imidazoles, pH 8), and then by incubating 10 minutes at RT under end to end rotation with elution buffer (6M urea, 50mM NaH2PO4, 100mM NaCl, 30mM glacial acetic acid, 70mM sodium acetate, pH 5) eluting the hCD16a binding polypeptide. After IMAC purification, the protein buffer was replaced with PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA), which was measured by the absorbance at 280 nm using an extinction coefficient calculated from the amino acid sequence. Low molecular weight markers were from Cytiva (Product No. 17044601). hCD16a binding variants with N-terminal His6 and C-terminal ABDWT were expressed as soluble gene products in E. coli BL21 (DE3).

[0634] With N-terminal His6 and C-terminal ABD WT Biosensor analysis of hCD16a binding variants

[0635] Biacore 8K instrument (Cytiva) is used to analyze the real-time interaction of hCD16a binding variants (SEQ ID NO 76-78) and human hCD16a. Protein ligand hCD16a F158 (Cat. No. CDA-H5220, Acro Biosystems), hCD16a V158 (Cat. No. CD8-H52H4, Acro Biosystems) and human serum albumin (HSA) (Sigma article number SRP6182) as positive control are diluted in 10mM NaOAc (pH 4.5) and are fixed on Series S CM5 sensor chip (Cytiva). The sensor chip is composed of flow channel, and flow channel is composed of two flow cells arranged in series. For every protein ligand and corresponding flow channel, the first flow cell is activated and deactivated to be used as a reference surface, and the second flow cell is used to fix the protein ligand to the carboxymethylated dextran surface by amine coupling using the manufacturer's instructions. The hCD16a binding variants were diluted in running buffer PBS-T (composition, pH) and then subjected to binding analysis at 25°C and a flow rate of 30 μl / min. After each injection, the flow cell was regenerated by injecting 10 mM HCl. Each hCD16a binding variant was injected in duplicate onto the hCD16a surface at a concentration of 5 nM to 2.56 μM. The dissociation equilibrium constant (KD value) was calculated using a 1:1 Langmuir binding model in BIAevaluation software (Cytiva).

[0636] result

[0637] To investigate whether the three CD16a binding polypeptide candidate clones [SEQ ID 1, 74 and 75] identified by phage-ELISA and DNA sequencing showed target binding when expressed as soluble proteins, these clones were subcloned, cytoplasmically expressed in E. coli and purified as His6-CD16a binding polypeptide-ABD WT Fusion protein [SEQ ID 76-78]. ABD WT The fusion proteins purified by immobilized metal ion affinity chromatography (IMAC) using the six histidyl (His6) gene fusion partners in the protein were analyzed by SDS-PAGE, showing that all three proteins were highly pure and had an approximate molecular weight consistent with their amino acid sequences ( Figure 2Injection of different concentrations (5 nM to 2.56 μM) of the three fusion proteins on separate sensor chip surfaces containing immobilized hCD16a F158 and hCD16a V158 allotypes, respectively, showed that all three fusion proteins showed binding to both hCD16a F158 and hCD16a V158 allotypes ( Figure 3 ).

[0638] Analysis of the equilibrium response of the interaction allows determination of the dissociation equilibrium constant (KD) of the interaction. Fusion proteins containing the A10 CD16a binding polypeptide [SEQ ID NO.76] were shown to bind to both F158 and V158 allotypes with similar affinity, with KD' of approximately 100 nM and 99 nM, respectively. In contrast, fusion proteins containing either the H09 or A11 CD16a binding polypeptides [SEQ ID 77 and 78] were shown to bind to the F158 allotype with a higher affinity than the V158 allotype. The dissociation equilibrium constants of the interaction were calculated to be 2.8 μM (A11 / V158), 8.3 μM (A11 / F158), 621 nM (H09 / F158), and 4.9 μM (H09 / V158).

[0639] Biological Example 3 :

[0640] Binning studies of hCD16a binding variants using surface plasmon resonance

[0641] In this example, SPR experiments were performed to investigate whether the three identified anti-CD16 affibodies bind to different or common / overlapping epitopes on hCD16a.

[0642] Materials and methods

[0643] With N-terminal His6 and C-terminal ABD WT Binning of hCD16a binding variants .

[0644] Biacore 3000 instrument (Cytiva) is used for competitive binding analysis of hCD16a binding variants with each other. Using the manufacturer's instructions, hCD16a F158 and hCD16a V158 are fixed to the individual flow cell carboxymethylated dextran surface on the CM5 sensor chip (Cytiva) by amine coupling. A flow cell is activated and deactivated to be used as a reference pool. The hCD16a binding variant is diluted in running buffer PBS-T, and then the binding analysis is performed at a flow rate of 25°C and 30 μl / min. After each injection series, the flow cell is regenerated by injecting 10mM HCl.

[0645] result

[0646] To investigate whether the identified CD16a binding polypeptides recognize distinct or overlapping / shared epitopes on hCD16a, SPR-based binning experiments were performed using sensor chip surfaces containing immobilized hCD16a F158 and hCD16a V158 proteins, respectively. WT [SEQ ID 76]) was injected at 2 μM onto the hCD16a surface, followed by a double injection of His6-A10-ABD containing 2 μM concentration. WT [SEQID 76] and His6-A11-ABD WT [SEQ ID 77] or His6-H09-ABD WT [SEQ ID 78] (both at 2 μM). His6-H09-ABD injected at 2 μM in the first pulse WT [SEQ ID 78] was subjected to a similar experiment, followed by a double injection of His6-H09-ABD containing a retention concentration of 2 μM WT [SEQ ID 78] and His6-A11-ABD WT [SEQ.ID 77] (also at 2 μM) was mixed sample. As a control, running buffer PBS-T was injected instead of the variant used to retain the surface at 2 μM concentration in the second pulse.

[0647] The results of epitope binning assay showed that the fusion protein His6-A10-ABD WT The first binding to hCD16a does not block the subsequently injected fusion protein His6-H09-ABD containing H09 and A11 CD16a binding peptides, respectively WT or His6-A11-ABD WT Binding to hCD16a indicates that variant A10 and either of variants H09 and A11 have non-overlapping epitopes on hCD16a ( Figure 4 ). According to the use of His6-H09-ABD WT and His6-A11-ABD WT The results obtained in a similar experimental setting for fusion proteins allow us to infer that variants H09 and A11 bind to overlapping epitopes ( Figure 4 ). The reciprocal epitope binning results were independent of the V158 or F158 allotype of hCD16a.

[0648] Biological Example 4 :

[0649] Binding of monomeric, heterodimeric and homodimeric CD16a binding polypeptide constructs using surface plasmon resonance Joint research

[0650] In this example, SPR experiments were performed to compare the hCD16a binding properties of a panel of monomeric and heterodimeric CD16a binding polypeptide constructs.

[0651] Materials and methods

[0652] 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. To construct heterodimeric CD16a binding polypeptide constructs, specific primers that introduced codons encoding a flexible (GGGGS)3CD16a inter-binding polypeptide linker [SEQ ID 225] and nucleotide overhangs complementary to the cloning ends of the linearized E. coli expression vector were used. The In-Fusion HD cloning kit (Takara Bio, Gothenburg, Sweden) was used to clone monomeric and heterodimeric constructs equipped with both an N-terminal His6 tag and a C-terminal cysteine ​​(Cys) [SEQ ID 79-82] and heterodimeric constructs with only a C-terminal His6 tag [SEQ ID 83-84]. In addition, a potential double conjugate construct containing an hBCMA binding polypeptide fused to a homodimeric hCD16a binding arm (two tandemly linked copies of an A10 CD16a binding polypeptide) was cloned with a C-terminal His6 tag [SEQ ID 86]. DNA constructs were sequence verified using Sanger sequencing (Eurofins Genomics).

[0653] Expression and protein purification of monomeric, heterodimeric and homodimeric CD16a binding polypeptide constructs

[0654] E. coli BL21 (DE3) cells were transformed with plasmids containing different DNA constructs and cultured with 150 rpm shaking at 37 ° C for 16 to 18 hours in 10 ml TSB + Y containing 25 μg / ml Kan. The culture was inoculated with 1: 100 in 200 ml TSB + Y containing 25 μg / ml Kan and grown with 150 rpm shaking at 37 ° C until OD600 = 0.6-1. IPTG was added to a final concentration of 1 mM to induce protein expression. The culture was cultured with 150 rpm shaking at 25 ° C for 16 to 18 hours and cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15 mM imidazole, pH 7.4). After the cells were broken by ultrasound, cell debris was removed by centrifugation, and each supernatant was applied to a gravity flow column filled with 3 ml-4 ml HisPur. Pollutants were removed by washing with six times the column volume of binding / washing buffer. The hCD16a binding polypeptide was then eluted with elution buffer (PBS with 300 mM imidazole, pH 7.4). After IMAC purification, the protein buffer was replaced with 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% to 12%, Bis Tris, Invitrogen), which was measured by the absorbance at 280 nm using an extinction coefficient calculated according to the amino acid sequence.

[0655] Biosensor analysis of monomeric, heterodimeric, and homodimeric CD16a binding peptide constructs .

[0656] Using a Biacore 8K instrument (Cytiva), the second flow cell surface of each of the four flow channels on a Series S CM5 sensor chip (Cytiva) was immobilized with the generated N-terminal His6 and C-terminal Cys CD16a binding polypeptide constructs [SEQ ID 79-82] by ligand thiol coupling using the manufacturer's protocol. The first flow cell of each flow channel was activated and deactivated to serve as a reference surface. The analytes were diluted in running buffer PBS-T and then the binding analysis was performed at 4°C and a flow rate of 30μl / min. hCD16a F158 and hCD16a V158 proteins were injected onto the CD16a binding polypeptide surface at 200nM. A reverse setup was used in a second experiment, in which the second flow cell surface of each flow channel on a Series S CM5 sensor chip (Cytiva) was immobilized with hCD16a F158 and hCD16a V158 by amine coupling using the manufacturer's protocol. The first flow cell of each flow channel was activated and deactivated to serve as a reference surface. The analytes were diluted in running buffer PBS-T and then subjected to binding analysis 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 constructs [SEQ ID 83-84] were injected onto the hCD16a surface at concentrations ranging from 0.5 nM to 32 nM. In a third experiment, hCD16aF158 and hCD16a V158 were immobilized on the surface of carboxymethylated dextran on a CM5 sensor chip (Cytiva) by amine coupling using a Biacore T200 instrument (Cytiva) using the manufacturer's instructions. Potential dual conjugate constructs containing anti-hBCMA polypeptides fused to homodimeric A10-A10 CD16a binding polypeptide arms [SEQ ID 86] were diluted in running buffer PBS-T. Binding analysis was performed in a single cycle kinetics setup at 25°C and a flow rate of 30 μl / min at five concentrations ranging from 2.5 nM to 40 nM. After the last injection, the flow cell was regenerated by injection of 10 mM HCl.

[0657] result

[0658] A set of expression vectors for monomeric and heterodimeric CD16a binding polypeptides were constructed. Four constructs (His6-A10-Cys, His6-A11-Cys, His6-A10-A11-Cys, and His6-A11-A10-Cys [SEQ ID 79-82]) correspond to monomeric or heterodimeric CD16a binding polypeptide constructs equipped with both an N-terminal His6 tag and a C-terminal cysteine ​​residue, the latter for direct coupling to an SPR sensor chip using thiol chemistry. Two heterodimeric constructs (A10-A11-His6 and A11-A10-His6 [SEQ ID 83-84]) and one dual conjugate construct consisting of a BCMA binding polypeptide fused to a homodimeric A10-A10 CD16a binding polypeptide arm, anti-BCMA-A10-A10-His6 [SEQ ID 86], were designed to contain only a C-terminal His6 tag. All seven constructs were expressed intracellularly in E. coli BL21 (DE3) cells and soluble proteins were purified by IMAC. Figure 5 shows SDS-PAGE analysis of each final protein preparation, which confirms that these contain primarily bands corresponding to the expected molecular weight. Figure 5a The larger molecular weight bands visible in lanes 1, 2, 5, and 6 in FIG correspond to the expected dimeric species held together by disulfide bonds formed between the C-terminal cysteines present in these constructs.

[0659] A first series of binding experiments were performed using His6-A10-Cys, His6-A11-Cys, His6-A10-A11-Cys and His6-A11-A10-Cys proteins, which were immobilized to the thiol-activated sensor chip surface via their C-terminal cysteine ​​residues, respectively. Injection of 200 nM concentrations of hCD16aF158 or hCD16a V158 proteins onto the sensor chip surface containing the monomeric CD16a binding polypeptide constructs His6-A10-Cys and His6-A11-Cys, respectively, produced binding response curves similar to those obtained using the reverse setup in Example 2, with the A10 CD16a binding polypeptide showing significantly stronger binding ( Figure 6). Injection of 200 nM concentration of hCD16a F158 or hCD16aV158 protein on the sensor chip surface containing heterodimeric His6-A10-A11-Cys and His6-A11-A10-Cys proteins resulted in binding curves characterized by significantly slower off-rate kinetics for both analytes, indicating that the injected monomeric hCD16a protein binds more tightly here by synergistic binding with contributions from both CD16a binding polypeptides in the heterodimeric His6-A10-A11-Cys and His6-A11-A10-Cys constructs. Interestingly, the sensorgram indicates that the relative orientation of A11-A10 (N-terminus to C-terminus) is more productive (slower off-rate kinetics) than the A10-A11 orientation using this particular linker length between the moieties ((GGGGS)3) [SEQ ID 225]. When the epitope binning results from Example 3 are also considered, showing that the A10 and A11 CD16a binding polypeptides bind to non-overlapping epitopes on hCD16a, the results strongly suggest that both His6-A10-A11-Cys and His6-A11-A10-Cys proteins are able to bind to hCD16a via a bi-paratopic effect.

[0660] Using the reverse assay format, two heterodimeric constructs A10-A11-His6 [SEQ ID 83] and A11-A10-His6 [SEQ ID 84] were injected at concentrations ranging from 0.5 nM to 32 nM, respectively, onto the sensor chip surface containing immobilized hCD16a F158 or hCD16aV158 protein. Figure 7 The results presented in also show that in this assay format, the binding responses obtained for both hCD16a ligands reflect stronger binding (slower off-rate kinetics) than observed for either of the individual and monomeric A10 or A11 counterparts analyzed in the same assay format in Example 2 ( Figure 3 , His6-A10-ABDWT and His6-A11-ABDWT). This further indicates that by combining the A10 and A11 variants into a heterodimeric construct, a dual epitope binding effect on hCD16a can be obtained. In addition, since the H09 CD16a binding polypeptide also shows binding to an epitope that does not overlap with the A10 CD16a binding polypeptide (Example 3), based on the results of this example, heterodimeric variants based on the combination of A10 / H09 variants are also expected to show dual paratope binding to hCD16a, which is also confirmed in Example 5.

[0661] In addition, the hCD16a binding properties of fusion proteins containing the homodimeric A10-A10 CD16a binding polypeptide combination [SEQ ID 86] were also analyzed. Here, a single cycle kinetic analysis was performed, involving continuous injection of analyte in a concentration range of 2.5 nM-40 nM on the sensor chip surface containing immobilized hCD16a F158 or hCD16a V158 protein, respectively. The results showed that compared with proteins containing a single monomeric A10 CD16a binding polypeptide portion ( Figure 3 , His6-A10-ABD WT ), the proteins containing two tandemly linked A10 CD16a binding polypeptide moieties analyzed here all showed significantly stronger apparent affinities for the hCD16a variants, with the calculated apparent dissociation constants for hCD16a F158 and hCD16a V158 under these conditions (e.g., surface density of hCD16a variants on the chip) being 0.4 nM and 0.3 nM, respectively, indicating that the avidity effect resulting from the simultaneous and cooperative binding of a single injection of homodimeric analyte protein to two sensor chip-immobilized CD16 protein molecules is effective ( Figure 8 ).

[0662] In summary, hCD16a binding analysis based on this set of monomeric, heterodimeric and homodimeric constructs of CD16a binding polypeptides described herein shows that these constructs can be freely combined in new constructs to produce a variety of novel hCD16a binding proteins with different binding characteristics (including bi-parametric and bivalent binding). The selection of CD16a binding polypeptides and their relative order (N-terminal to C-terminal) enables the overall binding properties to be varied. In the construct group analyzed in this example, a common 15 amino acid linker consisting of three tandemly arranged GGGSG motifs (SEQ ID 145) was used to connect homodimers and heterodimer combinations. Using methods known in the art, it is also possible to generate additional hCD16a binding constructs based on alternative polypeptides or chemical linkers (including direct connection; i.e., no linker) and including two or more identical or different CD16a binding polypeptide portions

[0663] Biological Example 5 :

[0664] Evaluation of BCMA×hCD16a dual conjugate constructs using surface plasmon resonance

[0665] In this example, SPR experiments were performed to analyze the bispecific binding characteristics of a tripartite fusion protein containing a hBCMA binding polypeptide (referred to herein as 1-E6, having the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAK KL NDAQAPK, SEQ ID 226) fused to an hCD16a binding arm consisting of a heterodimeric H09-A10 CD16a binding polypeptide combination.

[0666] Materials and methods

[0667] Subcloning of hBCMA×hCD16a binding polypeptide dual conjugate constructs .

[0668] DNA fragments encoding both hBCMA binding polypeptide and hCD16a binding polypeptide [SEQ ID 1 and 75] were amplified separately from ordered gene fragments (gBlocks Gene Fragments, Integrated DNA Technologies, Leuven, Belgium) using primers that introduced overhangs complementary to the ends of the linearized expression vector and a flexible (GGGGS)3 linker [SEQ ID 225] between each affimer unit. The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to assemble the three gene fragments to construct a three-part BCMA×hCD16a double junction with a C-terminal His6 tag [SEQ.ID 85]. The DNA constructs were sequence verified using Sanger DNA sequencing (Eurofins Genomics).

[0669] Expression and protein purification of hBCMA×hCD16a CD16a binding polypeptide dual conjugate constructs .

[0670] Escherichia coli BL21 (DE3) cells were transformed with plasmids containing DNA constructs and cultured with shaking at 150 rpm for 16 to 18 hours at 37°C in 10 ml TSB + Y containing 25 μg / ml Kan. The culture was inoculated with 1: 100 in 200 ml TSB + Y containing 25 μg / ml Kan and grown with shaking at 150 rpm at 37°C until OD600 = 0.6-1. IPTG was added to a final concentration of 1 mM to induce protein expression. The culture was cultured with shaking at 150 rpm at 25°C for 16 to 18 hours and cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15 mM imidazole, pH 7.4). After the cells were broken by ultrasound, cell debris was removed by centrifugation, and each supernatant was applied to a gravity flow column filled with 3 ml-4 ml HisPur. Contaminants were removed by washing with 6 column volumes of binding / washing buffer. The hCD16a binding polypeptide was then eluted with elution buffer (PBS with 300 mM imidazole, pH 7.4). After IMAC purification, the protein buffer was replaced with 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% to 12%, Bis Tris, Invitrogen), which was measured by the absorbance at 280 nm using an extinction coefficient calculated according to the amino acid sequence.

[0671] Evaluation of dual target binding of hBCMA x hCD16a CD16a binding polypeptide dual binder constructs. The BiacoreT200 instrument (Cytiva) was used to evaluate the simultaneous binding of the dual conjugate construct to hBCMA and hCD16a. Human BCMA-rFc (BCMA protein aa 1-54, human, recombinant (ECD, rabbit Fc tag), catalog number 10620-H15H, Sino Biological, Eschborn, Germany) was fixed to the carboxymethylated dextran surface on the CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. A flow cell was activated and deactivated to serve as a reference pool. The dual conjugate construct and hCD16a variant were diluted in running buffer PBS-T and then subjected to binding analysis at a flow rate of 25°C and 30 μl / min. After each injection, the flow cell was regenerated by injecting 10mM HCl.

[0672] result

[0673] A BCMA×hCD16a dual conjugate construct containing a BCMA-binding polypeptide genetically fused to an hCD16a binding arm consisting of a heterodimeric H09-A10 affibody combination and a C-terminal His6 tag was constructed and expressed as a soluble gene product in E. coli (DE3). Fig. 9 SDS-PAGE analysis of the final protein preparation is shown, which confirms that it contains mainly dual conjugate constructs with bands corresponding to the expected molecular weight. The hBCMA×hCD16a dual conjugate construct was injected at 200 nM onto the sensor chip surface containing the immobilized hBCMA-rFc protein and then Fig.10a PBS-T buffer or hCD16a F158 / hCD16a V158 protein were injected consecutively at 200 nM as schematically shown in Figure 1. Since the injected hCD16a variants were expected to bind weakly to the rabbit Fc tag on the hBCMA-rFc protein, a control series of injections with PBS-T buffer alone (no hBCMA×hCD16a dual conjugate during injection I) was performed, followed by injections of 200 nM of hCD16a F158 or V158 protein to allow subtraction of any response corresponding to this background binding. Fig.10b The resulting sensorgrams obtained after subtracting the signal from the reference flow cell (flow cell 1, activated / deactivated) and the low response signal (<10 RU) obtained from direct hCD16a binding to the rabbit Fc tag of the immobilized hBCMA-rFc ligand are shown. These resulting sensorgrams show that the hBCMA×hCD16a dual binder construct is able to bind simultaneously to the immobilized hBCMA-rFc ligand and the subsequently injected hCD16a variant. The results of the slow dissociation rate kinetics observed after the injection was completed ( Fig.10b IV phase in ) indicates that the H09-A10 affibody combination is also able to bind bi-paratopically to hCD16a, as previously demonstrated for the A10-A11 and A11-A10 combinations (Example 4). In addition, as expected from the results obtained in Example 2, a slower hCD16a dissociation rate was observed for the interaction with the hCD16a F158 variant than for the interaction with the hCD16a V158 variant ( Fig.10b , Phase IV), since the H09 affibody variant has been shown to bind more strongly to the hCD16a F158 allotype (Example 2).

[0674] In summary, the results show that the affimer-based hCD16a binding arm (here composed of the heterodimeric H09-A10 affimer combination) can also retain its hCD16a binding ability when fused to another polypeptide unit that is simultaneously involved in binding to a therapeutically relevant target, here exemplified by a gene fusion polypeptide that binds to hBCMA, a relevant target in multiple myeloma cancer.

[0675] Biological Example 6 :

[0676] Anti-BCMA conjugates induce immune cell activation in the presence of tumor cells .

[0677] Experiments were performed to evaluate the propensity of different anti-BCMA conjugates to elicit an IFNγ response in co-cultures of human PBMCs and the BCMA-positive multiple myeloma cell line MM.1S. Thawed and overnight rested PBMCs (4W-270, Lonza) were co-cultured with MM.1S cells (CRL-2974, ATCC) in V-shaped 96-well plates (249935, Thermo Scientific) at an effector to target ratio of 25:1. A total of 4×10 5 Cells were treated with 200 nM anti-BCMA conjugate or 200 nM elotuzumab (300 mg Empliciti, Bristol-Myers Squibb) in a humidified 5% CO2 incubator at 37°C for 4 hours. The supernatant was harvested and then centrifuged at 600 g for 5 minutes. Human IFN-γ ELISA MAX TM IFN-γ levels were measured by enzyme-linked immunosorbent assay (ELISA) using the Bio-Rad Biosystems Deluxe Set (430104, BioLegend). Data were processed using Excel and graphed using GraphPad Prism 9.

[0678] Anti-BCMA conjugate constructs containing a BCMA binding polypeptide (referred to herein as 1-E6) genetically fused to an hCD16a binding arm consisting of monomeric A10 (Seq ID 88), homodimeric A10 (Seq ID 86), heterodimeric H09-A10 (Seq ID 85), or heterodimeric A11-A10 (Seq ID 87) affibodies were evaluated. Corresponding constructs containing an inactive 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 a single culture of PBMCs and MM.1S was used as a negative control. Dual conjugates containing the BCMA binding polypeptide all elicited an IFNγ response in a co-culture of PBMCs and MM.1S cells that was greater than the response of the positive control elotuzumab for all conjugates ( Fig.11 ) Dual conjugate constructs lacking BCMA binding showed no or limited IFNγ responses in co-cultures of PBMC and MM.1S cells. This experiment demonstrates BCMA-dependent activation of PBMCs against BCMA-positive MM.1S myeloma cell lines induced by the anti-BCMA conjugates described herein.

[0679] Biological Example 7 :

[0680] Evaluation of secondary structure content and thermal denaturation curves of CD16a binding peptides using circular dichroism

[0681] In this example, three CD16a binding polypeptides A10, H09 and A11 [SEQ ID 1, 74 and 75] were subcloned, expressed and purified as polypeptide-His6 fusion proteins to evaluate the secondary structure content and their thermal denaturation curves by circular dichroism (CD) spectroscopy.

[0682] Materials and methods

[0683] Cloning of CD16a binding peptides

[0684] DNA fragments encoding three CD16a binding polypeptides [SEQ ID 1, 74 and 75] were amplified from their respective pAffi-1 library vectors using specific primers designed to introduce overhangs complementary to the ends of the linearized expression vector. The In-Fusion HD cloning kit (Takara Bio, Gothenburg, Sweden) was used to clone monomeric polypeptide constructs with a C-terminal His6 tag. Sequence verification of the DNA constructs was performed using Sanger sequencing (Eurofins Genomics).

[0685] Expression and purification

[0686] E. coli BL21 (DE3) cells were transformed with the plasmid containing the DNA construct and grown in 10 ml TSB + Y containing 25 μg / ml Kan at 37°C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated 1:100 into 200 ml TSB + Y containing 25 μg / ml Kan and grown at 37°C with shaking at 150 rpm until OD 600=0.6-1. Add IPTG to a final concentration of 1mM to induce protein expression. The culture was shaken at 150rpm at 25°C for 16 to 18 hours and the cells were harvested by centrifugation. The cell pellet was resuspended in denaturing lysis buffer (7M guanidine hydrochloride, 47mM NaH2PO4, 2.65mMNaH2PO4, 10mM TRIS-HCl, 100mM NaCl, pH 8). After incubation at 37°C with 150rpm shaking for 2 hours, the cells were centrifuged and the denatured protein from the supernatant of the cell lysate was added to a tube containing 3ml HisPur Cobalt IMAC resin (Catalog No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT under eoe rotation for 30 minutes. Contaminants were removed by washing three times with wash buffer (7M guanidinium chloride, 46.6mM NaHPO4, 3.4mM NaHPO4, 300mMNaCl, 10mM imidazole, pH 8), and then eluted with elution buffer (6M urea, 50mM NaHPO4, 100mM NaCl, 30mM glacial acetic acid, 70mM sodium acetate, pH 5) by incubating at RT for 10 minutes under eoe rotation. After IMAC purification, the protein buffer was exchanged for PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA) as measured by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence.

[0687] CD analysis

[0688] A Chirascan CD spectrometer (Applied Photophysics, Leatherhead, United Kingdom) was used to determine the secondary structure content of the three CD16a binding polypeptides by recording CD spectra in the range of 195 nm-260 nm at 20°C, and their thermal denaturation curves by recording CD ellipticity at 221 nm during heating from 20°C to 92°C.

[0689] result

[0690] The obtained CD spectra showed that the analyzed peptides all had high α-helical secondary structure content at 20°C. Fig.12) showed that the melting temperature (Tm) of clone A10-His6 was 49°C, the melting temperature (Tm) of clone H09-His6 was 48°C, and the melting temperature (Tm) of clone A11-His6 was 50°C, determined by variable temperature measurement. In addition, the CD curves of the heated (92°C) and subsequently cooled (20°C) samples showed almost perfect overlap. In summary, the results support that the three CD16a binding polypeptides generated mainly contain α-helical secondary structures and are able to refold after thermal denaturation.

[0691] Biological Example 8 :

[0692] Alanine scanning of CD16a binding clone A10

[0693] In this example, alanine scanning of polypeptide variant A10 [SEQ ID 1] was performed to investigate the relative importance of the amino acid occupancy at the positions randomized during library construction for the binding interaction of A10 with CD16a.

[0694] Materials and methods

[0695] Site-directed mutagenesis of CD16a-binding clone A10 .

[0696] The DNA fragment encoding the CD16a binding polypeptide clone A10 [SEQ ID 1] was subjected to site-directed mutagenesis in a single mutagenesis PCR reaction using an expression plasmid containing the A10-His6 construct as a template, wherein each original codon in the 14 variable positions was substituted with an alanine codon using primers designed for each position. The resulting 14 alanine substitution variants were sequence verified using Sanger sequencing (Eurofins Genomics).

[0697] Expression and purification

[0698] E. coli BL21 (DE3) cells were transformed with plasmids encoding 14 alanine variants and wild-type A10, each equipped with a C-terminal His6 tag, and cultured in 10 ml TSB + Y containing 25 μg / ml Kan at 37 ° C with shaking at 150 rpm for 16 to 18 hours. The culture was inoculated 1:100 in 200 ml TSB + Y containing 25 μg / ml Kan and grown at 37 ° C with shaking at 150 rpm until OD 600=0.6-1. Add IPTG to a final concentration of 1mM to induce protein expression. The culture was shaken at 150rpm for 16 to 18 hours at 25°C and the cells were harvested by centrifugation. The cell pellet was resuspended in binding / washing buffer (PBS containing 15mM imidazole, pH 7.4). After ultrasonic cell disruption, cell debris was removed by centrifugation, and each supernatant of the cell lysate was added to a tube containing 3ml HisPur Cobalt IMAC resin (Catalog No. 89966, Thermo Scientific). The supernatant was incubated with the resin at RT under eoe rotation for 30 minutes. Contaminants were removed by washing three times with washing buffer, and the A10 variant was eluted with elution buffer (PBS containing 300mM imidazole, pH 7.4) for 10 minutes under eoe rotation at RT. After IMAC purification, the polypeptide buffer was exchanged into PBS using a PD-10 desalting column (Cat. No. 17085101, Cytiva, Uppsala, Sweden). SDS-PAGE analysis was performed to confirm the purity and concentration of the purified protein (NuPAGE, 4% to 12%, Bis Tris, Invitrogen, Waltham, Massachusetts, USA), which was measured by the absorbance at 280 nm using an extinction coefficient calculated from the amino acid sequence.

[0699] Biosensor analysis of alanine variants

[0700] Biacore T200 instrument (Cytiva) is used to analyze the interaction between wild-type A10 polypeptide and 14 alanine variants of CD16a and His6 tags. Protein ligand hCD16a F158 (catalog number CDA-H5220, Acro Biosystems) is fixed (1430RU) on S series CM5 sensor chip (Cytiva) by amine coupling using the manufacturer's instructions. A flow cell is activated and deactivated to be used as a reference cell. The analyte is diluted in running buffer PBS-T and then subjected to binding analysis at a flow rate of 25°C and 30 μl / min. After each injection series, the flow cell is regenerated by injecting 10mM HCl. Wild-type A10-His6 construct and 14 alanine variants are injected onto the CD16a (F158) surface at a common concentration of 200nM.

[0701] result :

[0702] A series of sensorgrams were generated by injecting 14 alanine-substituted variants (peptide-YY-His6 format, common concentration 200 nM) on the surface of a sensor chip containing CD16a (F158) protein, from which the importance of the original amino acid in the anti-CD16a A10 peptide for binding to CD16a can be inferred. The sensorgrams are shown in Fig.13 Substitution of alanine at positions 9, 11, 13, 14, 18, 27 and 32 [SEQ ID 2, 3, 4, 5, 6, 8 and 9] did not significantly affect binding, as the resulting sensorgrams were similar to those obtained from the original A10 polypeptide in terms of response level and curve form. In contrast, alanine substitutions at positions 10, 17, 24, 28 and 31 resulted in variants that showed a significantly lower binding response. Some variants, H25A and M35A [SEQ ID 7 and 10], were moderately affected by alanine substitutions. Analysis by circular dichroism spectrometry showed that all variants had curves characteristic of proteins with high alpha helical content, with distinct minima at 221 nm and 205 nm (see Fig.14 ). Thermal melting experiments showed that all variants except one (I31A) showed a retained thermal melting point of about 50°C (see data in FIG15 ). Overall, this indicates that the lower binding response observed for some variants is not associated with a corresponding loss of overall structure, but rather has proposed key residues for interaction with CD16a.

[0703] Preparation Example 9 :

[0704] Second-generation library construction, selection and phage ELISA

[0705] In this example, a second generation library of CD16a binding polypeptide A10 is constructed based on re-randomization and different degrees of certain variable positions. The library is used to identify second generation variants of CD16a binding A10 polypeptide, but retains the ability to bind CD16a with different strengths. The library does not contain methionine. Single and unique clones obtained after four phage display selection cycles and subsequent DNA sequencing are determined in monoclonal phage-ELISA for binding to CD16a.

[0706] Materials and methods

[0707] The second generation library of CD16a binding peptide A10

[0708] The M13 phage display second generation selection library was prepared based on the A10 polypeptide. The phagemid vector used was pAffi-1 ( et al., (2007) J. Biotechnol. 128:162-183). The pAffi-1 phagemid containing the lac promoter and OmpA signal peptide was designed for phage display of the encoded polypeptide library members as in-frame fusions with the albumin binding domain (ABD), an amber stop codon and a truncated form of the M13 phage coat protein 3 (residues 249-406).

[0709] Two synthetic oligonucleotides based on the A10 sequence were used for gene assembly overlap extension PCR (OE-PCR) and subsequent PCR amplification: A10-fwd (5′-AC AAC AAA TTC AAC AAA GAA Z01 Z02 Z03 GCG Z01Z01 GAG ATC Z04 Z01 CTG CCG AAC CTG AAC [SEQ ID 227]-3′) with a length of 65 bp and A10-rev (5′-ACT CTG GCT CGG ATC ATC Z03 CAG Z05 Z01 Z06 GAA TGC Z07 Z01 CTG Z08Z09GTT CAG GTT CGG CAG [SEQ ID 228]-3′) with a length of 69 bp, encoding amino acid positions 3–23 and 19–41 (reverse complementary strand) corresponding to the Z domain numbering (Nilsson et al. (1987) Protein Eng. 1:107-113), wherein based on the data obtained in the alanine scanning experiment (Example 8), during the synthesis using a mixture of trinucleotide codon building blocks, 15 original A10 codons were re-randomized (amino acid codons excluding Met, Cys, Pro and Gly; Z01 = 10% Ala and 90% evenly distributed among the remaining 15 allowed amino acids, Z02 = 10% Gln and 90% evenly distributed among the remaining 15 allowed amino acids, Z03 = 15% Ile and 85% evenly distributed among the remaining 15 allowed amino acids, Z04 = 90% Arg and 10% evenly distributed among the remaining 15 allowed amino acids, Z05 = 80% Ser and 20% Lys, Z06 = 90% Ile and 10% evenly distributed among the remaining 15 allowed amino acids, Z07 = 90% Phe and 10% evenly distributed among the remaining 15 allowed amino acids, Z08 = 65% His and 35% evenly distributed among the remaining 15 allowed amino acids, Z09 = 90% His and 10% evenly distributed among the remaining 15 allowed amino acids). After OE-PCR, forward primer: (5'-GATGAAGCCCTCGAGGTAGACAACAAATTCAACAAAGAA-3') [SEQ ID 223]

[0710] Reverse primer: (5'-TTAGCTTCTGCTAGCAAGTTAGCGCTTTGGCTTGGGTCATC-3') [SEQ ID 224] was used for PCR amplification.

[0711] Approximately 5.5 μg of XhoI and NheI double-cut and gel-purified (Qiagen, Germany) PCR product of the assembled gene was ligated with 30 μg of XhoI and NheI double-cut and gel-purified pAffi-1 phagemid vector using T4 DNA ligase. The resulting ligation mixture was desalted using column purification (Qiagen, Germany), divided into 22 portions and used for electroporation (0.1 cm BioRad cuvette) 25 μl electrocompetent ER2738 Escherichia coli (E. coli) cells (F', glnV amber repressor) (Lucigen, USA). 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, which were then pooled (seven electroporations per pool) and incubated at 37°C with shaking for 1 hour, after which the cell pools were titered by plating a dilution series on ampicillin plates and transferred to two 5-liter shake flasks, each containing 500 ml of tryptic soy broth + yeast extract medium (30 g / l tryptic soy broth, 5 g / l yeast extract; TSB+Y) supplemented with 1.5% (w / v) glucose and 100 μg / ml ampicillin. Overnight cell cultures were pelleted by centrifugation and resuspended in 25 ml of cold 40% glycerol to a final volume of about 37 ml, then distributed into 20 tubes of about 1 ml of cell-glycerol solution per tube and 5 tubes of about 3 ml of cell-glycerol solution per tube. 600 The library size (diversity) was calculated to be about 5×10 6 , and each 1 ml cell aliquot contained a cell number corresponding to approximately 14000× the library size. A portion of the cells was used directly for phage stock preparation using M13KO7 helper phage, while the remaining tubes with cells were stored at -80°C.

[0712] Preparation of phage stock solution .

[0713] To generate phage stocks displaying affimers, 1.14 ml of the library glycerol stock was dispensed and inoculated into two baffled E flasks, each containing 750 ml TSB+Y, 1% (w / v) glucose, 10 μg / ml Tet (tetracycline) and 100 μg / ml Carb (carbenicillin) and grown at 37°C with shaking at 150 rpm until the culture reached an OD of 600 =1. 30 ml cells / flask were infected with M13KO7 helper phage (New England Biolabs) at an infection efficiency (MOI) of 5, gently swirled and incubated at 37°C without shaking for 15 minutes, then incubated at 37°C at 70 rpm for 15 minutes. Cells from each culture were centrifuged and resuspended in two baffled E flasks containing 500 ml TSB+Y, 100 μg / ml Carb and 1 mM IPTG (isopropyl β-D-1-thiogalactoside). 25 μg / ml Kan (kanamycin) was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 90 rpm for 16 to 18 hours. The phage library stock solution was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titers of the stocks were measured by spot titration, and polymerase chain reaction screening was used to analyze the percentage of clones carrying phagemids with gene inserts of the correct size.

[0714] Selection from the secondary library of CD16a binding clone A10 .

[0715] Four cycles of panning were performed using biotinylated hCD16a (biotinylated human hCD16a (F158), Avitag, His tag, AcroBiosystems, catalog number CDA-H82E8, corresponding to residues 17-208 of Uniprot entry P08637) at 100 nM in four tracks of cycle 1, 50 nM in three tracks of cycle 2 (the two tracks from cycle 1 were combined together), and 50 nM in five tracks of cycles 3 and 4 (the two tracks from cycle 2 were divided into two). In addition, in cycle 3, a molar excess of 25 μM non-biotinylated target [CD16aF158 Y140H]-[Z963]-[Avitag]-[His6]:

[0716] (GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFH IPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGSGVDNKFNKETQEASWEIFTLPNLNGRQVAAFISSLLDDPSQSANLLAEAKKLNDAQAPKGGGSGGLNDIFEAQKIEWHEHHHHHH)[SEQ IDNO.229] for competition. The paramagnetic beads (Dynabeads M-280 streptavidin, catalog number 11205D, Invitrogen, Waltham, Massachusetts, USA) coated with SA (streptavidin) were washed twice with PBS (150 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4·H2O, pH 7.4). To avoid the enrichment of nonspecific binders, all tubes used for bio-panning were pretreated with 1% (w / v) bovine serum albumin (BSA) in PBS-T (PBS supplemented with 0.05% (v / v) Tween-20, pH 7.4). In addition, the phage stock solution in PBS-T was pre-incubated with 0.1% (w / v) BSA and beads at room temperature (RT) under constant end-over-end (eoe) rotation for 30 minutes to remove phage carrying binders to SA. The amount of phage stock used in cycle 1 was 10 10 Colony forming units (cfu), 10 in cycles 2 and 3 11 cfu, and in cycle 4 it is 10 11-12 cfu, it depends on the trajectory.

[0717] For tracks using solid phase selection, biotinylated target proteins were immobilized on 1 mg beads in cycles 1 and 4 and on 0.5 mg beads in cycles 2 and 3 for 1 hour at RT and end-on-end (eoe). The beads containing the target were incubated with PBS-T containing 1% (w / v) BSA at RT and eoe for 30 minutes, then washed with PBS-T, and then pre-incubated phage stocks were added and selected at RT and eoe for 3 hours in cycles 1 and 4, 2 hours in cycle 2 and 3 for 30 minutes. For tracks using liquid phase selection, pre-incubated phage stocks were added to the biotinylated target proteins for selection in solution, followed by capture of phage-antigen complexes by incubation with SA beads at RT and eoe for 30 minutes. Cycle 3 included competition with non-biotinylated target (off-rate selection), which was added after the above incubation and incubated for another 1 hour or association (ON) (18 hours). The phage antigen complex captured by the SA beads was washed with PBS-Te for a total of 5 minutes (cycles 1 and 4), 10 minutes or 30 minutes (cycle 2) or 20 minutes (cycle 3) at RT. The final wash volume was transferred to a new 1% (w / v) BSA pretreated tube to remove sticky binders attached to the tube wall. Antigen-bound phages were eluted by incubating with 0.5M acetic acid (pH 2.8) eoe for 15 minutes at RT, then transferring the eluate to a new pretreated tube and neutralizing with an equal volume of 1M Tris-HCl (pH 8) or by incubating with 0.25mg / ml trypsin (Gibco LifeTechnologies) at RT in TBS-T (TRIS buffered saline, 0.1% (v / v) Tween-20) supplemented with 1mM CaCl2 eoe for 30 minutes, then transferring the eluate to a new pretreated tube.

[0718] After cycle 1, E. coli XL-1 Blue cells (Agilent) were grown in TSB+Y with 10 μg / ml Tet at 37°C with shaking at 150 rpm until OD 600=0.5-0.8 to generate a new phage stock for the second round of selection. 50 ml of bacteria were infected with the total phage eluate volume, vortexed gently, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was then centrifuged and resuspended in TSB+Y, then plated on blood agar plates (40 g / l blood agar) with 100 μg / ml Carb and 1% (w / v) glucose and incubated at 37°C for 16 to 18 hours. Bacterial colonies were collected into TSB+Y by scraping and inoculated into 200 ml TSB+Y containing 100 μg / ml Carb and grown at 37°C with shaking at 150 rpm until OD 600 =0.5. 30 ml was superinfected with M13KO7 helper phage (MOI 5), swirled gently, incubated at 37°C without shaking for 25 minutes, and then shaken at 70 rpm for 15 minutes at 37°C. The culture was centrifuged and resuspended in 150 ml TSB+Y containing 100 μg / ml and 1 mM IPTG. 25 μg / ml Kan was added 2 hours after inoculation. The culture was incubated at 37°C with shaking at 150 rpm for 16 to 18 hours. The phage stock solution was harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titer of the stock solution was measured by spot titration, and the percentage of phage particles carrying phagemids with affinity body inserts was analyzed using polymerase chain reaction screening.

[0719] For subsequent cycles 2-3, E. coli XL-1 Blue cells were grown in TSB+Y with 10 μg / ml Tet and 1% (w / v) D-glucose at 37°C with shaking at 150 rpm until OD 600=0.5-0.8 to produce new phage stock solution.The Escherichia coli XL-1Blue bacteria of 30ml growth are infected by half phage eluate volume and incubated at 37 ℃ with 150rpm shaking for 30 minutes.Add isopykalemic TSB+Y with 10 μ g / ml Tet and 100 μ g / ml Carb, then by incubating at 37 ℃ with 150rpm shaking for 1.5 hours, then superinfect with M13KO7 helper phage (MOI 76-382) and incubate extra 1.5 hours at 37 ℃ with 150rpm shaking, bacteria are infected phenotype analysis.Culture is centrifuged and resuspended in 100ml TSB+Y with 100 μ g / ml Carb, 25 μ g / ml Kan and 0.5mM IPTG.Culture is incubated at 30 ℃ with 150rpm shaking for 16 hours to 18 hours. Phage stocks were harvested by precipitation twice with 20% (w / v) PEG6000 / 2.5M NaCl. The titers of the stocks were measured by spot titration and the percentage of phage particles carrying phagemids with affibody inserts was analyzed using polymerase chain reaction screening.

[0720] DNA sequencing. After phage display selection, 25 and 129 individually grown colonies were sent for DNA sequencing by Sanger sequencing (PlateSeq, Eurofins Genomics, Ebersberg, Germany) after infection of E. coli XL-1 Blue with phage eluates after cycle 3 and cycle 4, respectively. 67 unique and novel sequences were identified.

[0721] Monoclonal phage-ELISA screening. Using 67 unique and novel sequences identified by Sanger sequencing, monoclonal phage supernatant preparation and monoclonal phage-ELISA screening were performed as described in Example 1. In this example, MaxiSorp ELISA plates (clear flat-bottom immunonon-sterile 384-well plates, catalog number 464718, Thermo Fisher Scientific) were coated with the following antigens: 30 μl of 4 μg / ml biotinylated hCD16a, 20 μg / ml human serum albumin (HSA; Sigma product number SRP6182) (for evaluating the correct display of the expression cassette containing the three-part fusion protein including the affibody, the affibody albumin binding domain and the truncated protein 3), 10 μg / ml SA or 10 μg / ml BSA in 100 mM sodium carbonate buffer pH 9.6 / well (1 / 4 of the well with each coating protein) at 4°C with slow shaking for 16 to 18 hours. 53 binding candidates were identified as ELISA positive.

[0722] result

[0723] The positions identified as important for CD16a binding in the A10 peptide were conservatively randomized (Q10, R17, H24, F28, and I31). For these positions, the trinucleotide codon mixture used to synthesize the mutagenic oligonucleotides was biased to provide a 90% possibility of reinsertion of the amino acid found in the A10 peptide, leaving a 10% possibility of 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 six positions (9, 13, 14, 18, 27, and 32) where the original amino acid in the A10 peptide was found to be not critical for CD16a binding, the trinucleotide codon mixture used to synthesize the mutagenic oligonucleotides was biased to provide only a 10% possibility of reinsertion of the amino acid found in the A10 peptide, leaving a 90% possibility of 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 mixture used to synthesize the mutagenic oligonucleotide was biased to provide a 65% probability of reinsertion of histidine, leaving a 35% probability of 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 mixture used to synthesize the mutagenic oligonucleotide was biased to provide an 80% probability of reinsertion of serine, leaving a 20% probability of insertion of lysine. For positions 11 and 35, both occupied by methionine, which is easily oxidized, in the A10 peptide, the trinucleotide codon mixture used to synthesize the mutagenic oligonucleotide was biased to provide a 15% probability of insertion of isoleucine, and an 85% probability of insertion of a different amino acid (any of the 20 natural amino acids, minus methionine, cysteine, proline, or glycine).

[0724] Therefore, selecting CD16a binding polypeptides from this library will have the potential to generate new A10-related CD16a binding polypeptides (denoted as A10*) of different affinities. It also has the potential to generate CD16a binding polypeptides that do not contain methionine.

[0725] The selection of CD16a (F158) target protein was carried out by phage display as described above (materials and methods). The analysis of the selection output from different selection tracks by monoclonal phage-ELISA and DNA sequencing resulted in the identification of 53 unique and ELISA-positive clones. Their sequences are shown as SEQ ID 11-63 in the table of Figure 23. Three other interested clones (SEQ ID 64-66) were also identified.

[0726] Biological Example 10A :

[0727] Expression, protein purification and SPR analysis of the CD16a binding polypeptide identified in Example 9

[0728] Materials and methods

[0729] Subcloning of hBCMA x hCD16a binding polypeptide dual conjugate constructs.

[0730] DNA fragments encoding the 53 CD16a binding polypeptides [SEQ ID 11-63] (A10*) identified in Example 9 and the hBCMA binding polypeptides referred to herein as 1-E6 were amplified using their respective library pAffi-1 phagemid vectors as templates, using primers that introduced overhangs complementary to the ends of the linearized expression vector and a flexible (GGGGS)3 encoding linker between each polypeptide unit. The In-Fusion HD Cloning Kit (Takara Bio, Gothenburg, Sweden) was used to assemble the gene fragments into 53 different BCMA×hCD16a dual conjugate constructs with a C-terminal His6 tag as anti-BCMA-A10*-His6, where A10* corresponds to the CD16a binding polypeptides [SEQ ID 11-63] identified in Example 9. The DNA constructs were sequence verified using Sanger DNA sequencing (Eurofins Genomics).

[0731] Expression and protein purification of hBCMA×hCD16a binding polypeptide dual conjugate constructs .

[0732] E. coli BL21 (DE3) cells were transformed with plasmids containing 53 DNA constructs and cultured in 500 μl TSB + Y containing 25 μg / ml Kan at 37 ° C with shaking at 150 rpm for 16 hours to 18 hours. Cells were also transformed with the DNA construct encoding the BCMA × hCD16a dual conjugate construct anti-BCMA-A10-His6 [SEQ ID 88] described in Example 5 above. The culture was inoculated 1: 100 in 2.5 ml TSB + Y containing 25 μg / ml 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 cultured at 25 ° C with shaking at 150 rpm for 16 hours to 18 hours and the cells were harvested by centrifugation. The cell pellet is resuspended in denaturing lysis buffer (7M guanidine hydrochloride, 47mM Na2HPO4, 2.65mM NaH2PO4, 10mM TRIS-HCl, 100mM NaCl, pH 8). After incubation at 37°C with 150rpm shaking for 2 hours, the cells are centrifuged and the denatured protein of the supernatant from the cell lysate supplemented with about 15mM imidazole is added to a tube containing HisPur Cobalt IMAC resin (Cat. No. 89966, ThermoScientific). The supernatant is incubated with the resin at RT for 30 minutes and rotated end to end. Contaminants are removed by washing three times with a wash buffer (PBS with 15mM imidazole, pH 7.4), and the BCMA × hCD16a double conjugate is subsequently eluted with an elution buffer (PBS with 400mM imidazole, pH 7.64). The concentration is measured by the absorbance at 280nm using the extinction coefficient calculated according to the amino acid sequence. A subset of the purified protein was subjected to SDS-PAGE analysis to confirm purity and concentration (Mini-PROTEAN TGX, Tris / Glycine / SDS, Bio-Rad).

[0733] Evaluation of CD16a binding of hBCMA×hCD16a binding polypeptide dual conjugate constructs .

[0734] The interaction of hCD16a binding variants with human hCD16a was analyzed in real time using Biacore 8K instrument (Cytiva). Protein ligands hCD16a F158 (Catalog No. CDA-H5220, Acro Biosystems) and hCD16a V158 (Catalog No. CD8-H52H4, Acro Biosystems) were diluted in 10mM NaOAc (pH 4.5) and fixed on the surface of a separate sensor chip of S series CM5 sensor chip (Cytiva). The sensor chip consists of eight flow channels (two are used here), and each flow channel consists of two flow cells arranged in series. For each protein ligand and corresponding flow channel, the first flow cell is activated and deactivated to be used as a reference surface, and the second flow cell is used to fix the protein ligand to the carboxymethylated dextran surface by amine coupling using the manufacturer's instructions. BCMA × hCD16a dual conjugate His6 fusion protein is diluted in running buffer PBS-T.

[0735] result

[0736] The genes encoding 53 polypeptides identified in Example 9 were re-cloned for soluble expression as 1-E6-(GGGSG)3-A10*-YY-His6 fusion proteins. The polypeptide designated 1-E6 herein is an hBCMA binding polypeptide having the sequence VDNKFNKENQFADEEIAALPNLNFYQKWAFIRKLMDDPSQSANLLAEAKKL NDAQAPK (SEQ ID 226). A10* is used here to represent a single CD16a binding polypeptide identified in Example 9. The A10* sequence is the sequence shown in SEQ ID 11 to 63 in the table of Figure 23.

[0737] Expression was performed at a 2.5 ml culture scale and polypeptide purification was performed by IMAC. A Biacore 8K instrument was used to study the binding of different 1-E6-(GGGSG)3-A10*-YY-His6 polypeptides. In the analysis, polypeptides hav...

Claims

1. A CD16a binding polypeptide, comprising at least one motif that binds to CD16a, wherein the polypeptide comprises the following structure: [N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part] The CD16a binding motif is part [helix 1]-[detached part]-[helix 2].

2. The CD16a binding polypeptide according to claim 1, wherein: 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 [SEQ ID NO 120], wherein b) X9 is A, D, F, H, I, K, L, Q, R, T, V or Y; X 10 It 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 It 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; And X 35 is A, H, I, L, M, R or S; or b) X9 is V; X 10 It is Q; X 11 It is M; X 13 It is Q; X 14 It is F;X 17 It is R; X 18 It is K; X 24 Yes H; X 25 Yes H; X 27 Yes S; X 28 It is F;X 31 is I; X 32 It is K; X 33 is S, and X 35 is M, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or c) X9 is Q; X 10 It is F;X 11 It is Y; X 13 Yes R; X 14 Yes D; X 17 It is D; X 18 Yes L; X 24 Yes E; X 25 Yes D; X 27 It is K; X 28 It is W; X 31 It is Y; X 32 It is M; X 33 is S, and X 35 is I, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g. at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues; or d) X9 is F; X 10 It is W; X 11 is I; X 13 Yes E; X 14 Yes S; X 17 It is E; X 18 Yes S; X 24 is I; X 25 It is Y; X 27 It is K; X 28 It is W; X 31 It is K; X 32 It is Y; X 33 is S, and X 35 is A, and optionally wherein within helix 1 and helix 2, X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues are replaced by alternative residues and / or are not marked with an X n At least 1 and no more than 5 (e.g., at least 1 and no more than 3) of the residues in the protein are replaced by alternative residues.

3. The CD16a binding polypeptide according to claim 1 or claim 2, wherein: Helix 1 contains the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 [SEQ ID NO 127], and helix 2 comprises sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 [SEQ ID NO 120], 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, M, N, Q, R, S, T, V, W, or Y; X 13 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 It is A, D, E, F, H, I, K, N, Q, R, S, T, or V; X 24 Yes H; X 25 A or H; X 27 It 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; and X 35 It is A, H, I, L, M, R or S.

4. A CD16a binding polypeptide according to any preceding claim, wherein: Helix 1 contains the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 [SEQ ID NO 127], and helix 2 comprises sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 [SEQ ID NO 120], 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 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 Yes 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; and X 35 It is A, H, I, L, M, R or S.

5. A CD16a binding polypeptide according to any preceding claim, wherein: Helix 1 contains the sequence X9X 10 X 11 AX 13 X 14 EIX 17 X 18 [SEQ ID NO 127], and helix 2 comprises sequence X 24 X 25 QX 27 X 28 AFX 31 X 32 X 33 LX 35 [SEQ ID NO 120], 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, 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, 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 Yes H; X 25 is H or A; 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, L, N, Q, or R; X 33 is K or S; and X 35 It is A, H, I, L, R or S.

6. The CD16a binding polypeptide according to claim 5, 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 Yes H; X 25 It is H; X 27 is A, I, K, Q, R, T or V; X 28 It 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; and X 35 It is H, I, L, R or S.

7. The CD16a binding polypeptide according to claim 5, wherein: X9 is D, F, H, I, K, L, Q, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, or Y; X 13 A or Q; X 14 is H, I, K, L, Q, R, S, T, V, W, or Y; X 17 Yes R; X 18 is A, D, E, F, H, K, N, Q, R, S, T, or V; X 24 Yes H; X 25 Yes 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; and X 35 It is A, H, I, L or R.

8. The CD16a binding polypeptide according to claim 5, wherein: X9 is D, F, H, I, K, L, Q, R, T, V or Y; 10 It is Q; X 11 is A, D, E, F, H, I, K, N, Q, R, S, T, V, W, or Y; X 13 A or Q; X 14 is H, I, K, L, Q, R, S, V, W, or Y; X 17 Yes R; X 18 is A, F, H, K, N, Q, R, S, or T; X 24 Yes H; X 25 Yes 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; and X 35 It is A, H, I, L, R or S.

9. The CD16a binding polypeptide according to claim 5, wherein: X9 is F, L, Q, T or Y; X 10 It is Q; X 11 is A, F, H, I, L, N, Q, S, or Y; X 13 A or Q; X 14 is I, K, Q, R or V; X 17 Yes R; X 18 is A, E, H, K, Q, R, T, or V; X 24 Yes H; X 25 Yes H; X 27 is A, I, K, Q, R or V; X 28 It is F;X 31 is I; X 32 is A, H, K, N, Q or R; X 33 is K or S; and X 35 It is H, I or L.

10. The CD16a binding polypeptide according to claim 5, wherein: X9 is I, L, Q, T, or V; X 10 It is Q; X 11 is A, E, F, H, I, S, V, or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is A, H, K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is I, K, Q, R, T or V; X 28 It is F;X 31 Is I or L; X 32 is K, N or R; X 33 is K or S; and X 35 It is I or L.

11. The CD16a binding polypeptide according to claim 5, wherein: X9 is I, L, Q or V; X 10 It is Q; X 11 is A, E, H, I, S, W or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is A, H, K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is K, Q, R, T or V; X 28 It is F;X 31 is I; X 32 is K, N or R; X 33 is K or S; and X 35 is I or L; For example, where: X9 is I, L, Q or V; X 10 It is Q; X 11 is A, E, H, I, S or Y; X 13 It is Q; X 14 is K, L, R, V, W or Y; X 17 Yes R; X 18 is H, K, Q, R, S or T; X 24 Yes H; X 25 Yes H; X 27 is K, Q, R, T or V; X 28 It is F;X 31 is I; X 32 is N or K; X 33 is K or S; and X 35 It is I or L.

12. The CD16a binding polypeptide according to claim 5, wherein: X9 is L or V; X 10 It is Q; X 11 is A, I, S or Y; X 13 It is Q; X 14 is K, R or V; X 17 Yes R; X 18 is K, Q, R, S or T; X 24 Yes H; X 25 It is H; X 27 is K, R or V; X 28 It is F;X 31 is I; X 32 is N or K; X 33 is K or S; And X 35 It is I or L.

13. The CD16a binding polypeptide according to claim 5, wherein: X9 is D, H, I, K, L, Q, T or V; X 10 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 Yes H; X 25 Yes 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; and X 35 is A, I, L or R; or X9 is K, Q or Y; X 10 It is Q; X 11 is I or Q; X 13 It is Q; X 14 W or Y; X 17 Yes R; X 18 is H, K or R; X 24 Yes H; X 25 Yes H; X 27 A, K or T; X 28 It is F;X 31 is I; X 32 A, K or Q; X 33 is K or S; and X 35 is I or L; or X9 is L, V or Y; X 10 It is Q; X 11 is I, N or Q; X 13 It is Q; X 14 is K, R or Q; X 17 Yes R; X 18 is E, A or V; X 24 Yes H; X 25 Yes H; X 27 It is K or Q; X 28 It is F;X 31 is I; X 32 is H, K or Q; X 33 is K or S; and X 35 It is I or L.

14. The CD16a binding polypeptide according to claim 5, wherein: X9 is L, Q, T or V; X 10 It is Q; X 11 is I, V or Y; X 13 It is Q; X 14 is K, R or Y; X 17 Yes R; X 18 is K, R, S or T; X 24 Yes H; X 25 It is H; X 27 is I, T or V; X 28 It is F;X 31 Is I or L; X 32 is K or N; X 33 is K or S; and X 35 is I or L; For example, where X9 is L, T or V; 10 It is Q; X 11 is I, V or Y; X 13 It is Q; X 14 is K or R; X 17 Yes R; X 18 is R, S or T; X 24 Yes H; X 25 It is H; X 27 is I or V; X 28 It is F;X 31 Is I or L; X 32 is K or N; X 33 is K; and X 35 It is I or L.

15. The CD16a binding polypeptide according to claims 2 to 14, wherein the CD16a binding efficacy is at least 10% of SEQ ID NO: 1; and / or wherein the CD16a binding polypeptide competes with SEQ ID NO:

1.

16. The CD16a binding polypeptide according to claims 2 to 14, wherein: Helix 1 contains the sequence X6X7X8X9X 10 X 11 AX 13 X 14 EIX 17 X 18 X 19 , and / or helix 2 contains sequence X 23 X 24 X 25 QX27X 28 AFX 31 X 32 X 33 LX 35 X 36 X 37 [SEQ ID NO 128], wherein X6 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or absent; X7 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or absent; X8 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably E) or absent; X 19 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably L) or is absent; X 23 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or is absent; X 36 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or is absent; and X 37 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or is absent; and preferably wherein X6 is N; X7 is K; and X8 is E; and / or wherein X 36 is D; and X 37 It is D; For example, where X6 is N; X7 is K; X8 is E; X 19 Yes L; X 23 Yes N; X 36 is D; and X 37 It is D.

17. The CD16a binding polypeptide according to any one of claims 2 to 4, wherein: Helix 1 contains the sequence NKEVQMAQFEIRKL [SEQ ID NO 129], and Helix 2 contains the sequence NHHQSFAFIKSLMDD [SEQ ID NO 130]; and optionally wherein, At least 1 and no more than 5 (e.g., 1, 2, 3, 4 or 5) (e.g., at least 1 and no more than 3 (e.g., 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

18. The CD16a binding polypeptide of claim 17, wherein the CD16a binding efficacy is at least 10% of SEQ ID NO: 1; and / or wherein the CD16a binding polypeptide competes with SEQ ID NO:

1.

19. The CD16a binding polypeptide according to claim 2, wherein: Helix 1 contains the sequence NKEQFYARDEIDLL [SEQ ID NO 131], and Helix 2 contains the sequence NEDQKWAFYMSLIDD [SEQ ID NO 132]; and optionally wherein, At least 1 and no more than 5 (e.g., 1, 2, 3, 4 or 5) (e.g., at least 1 and no more than 3 (e.g., 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

20. The CD16a binding polypeptide of claim 19, wherein the CD16a binding efficacy is at least 10% of SEQ ID NO:74; and / or wherein the CD16a binding polypeptide competes with SEQ ID NO:

74.

21. The CD16a binding polypeptide according to claim 2, wherein: Helix 1 contains the sequence NKEFWIAESEIESL [SEQ ID NO 133], and Helix 2 contains the sequence NIYQKWAFKYSLADD [SEQ ID NO 134]; and optionally wherein, At least 1 and no more than 5 (e.g., 1, 2, 3, 4 or 5) (e.g., at least 1 and no more than 3 (e.g., 1, 2 or 3)) residues in the sequence of helix 1 and / or helix 2 are replaced by an alternative residue (e.g., by an alternative residue that is a conservative substitution).

22. The CD16a binding polypeptide of claim 21, wherein the CD16a binding efficacy is at least 10% of SEQ ID NO:75; and / or wherein the CD16a binding polypeptide competes with SEQ ID NO:

75.

23. A CD16a binding polypeptide according to any preceding claim, wherein the isolated portion is a sequence of 1 to 5 (preferably 2 to 5, such as 2, 3, 4 or 5; or such as 3 to 5, such as 3, 4 or 5, and more preferably 3) naturally occurring amino acids.

24. A CD16a binding polypeptide according to any preceding claim, wherein the isolated portion has the sequence X 20 X 21 X 22 ,in X 20 is any naturally occurring amino acid (preferably S, T, M, P, F, Y or W; more preferably P or T); X 21 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N); X 22 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably L); and wherein optionally X 20 , X 21 , X 22 One or both (e.g., optionally 1) of are absent; and preferably wherein X 20 Is P or T; X 21 is N; and X 22 is L (eg, the isolated portion has the sequence PNL or TNL).

25. A CD16a binding polypeptide according to any preceding claim, wherein the N-terminal portion is absent or It is a sequence of 1 to 15 (preferably 1 to 10 or 1 to 8, more preferably 1 to 5, such as 1, 2, 3, 4 or 5) naturally occurring amino acids.

26. A CD16a binding polypeptide according to any preceding claim, wherein the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is any naturally occurring amino acid (preferably G, A, V, L, or I; more preferably V or G; and most preferably V) or absent; X2 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably D) or absent; X3 is any naturally occurring amino acid (preferably D, E, N or Q; more preferably N) or absent; X4 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or is absent; and X5 is any naturally occurring amino acid (preferably F, Y or W; more preferably F) or absent; And preferably wherein X1 is V, G or absent (preferably V or absent), X2 is D or absent, X3 is N or absent, X4 is K or absent, and X5 is F or absent.

27. The CD16a binding polypeptide according to any preceding claim, wherein the N-terminal portion is absent, or the N-terminal portion has the sequence X1X2X3X4X5, wherein X1 is V or G (preferably V), X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is D, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is N, X4 is K, and X5 is F; or X1 is absent, X2 is absent, X3 is absent, X4 is K, and X5 is F; or X1 does not exist, X2 does not exist, X3 does not exist, X4 does not exist, and X5 is F.

28. A CD16a binding polypeptide according to any preceding claim, wherein the C-terminal portion is absent or is a sequence of 1 to 50 (eg, 1 to 35, 1 to 30, 15 to 25, or 18 to 22) naturally occurring amino acids.

29. The CD16a binding polypeptide of claim 28, wherein the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 [SEQ ID NO 135], wherein X 38 is a sequence of 1 to 14 (preferably 1 to 9 or 1 to 7, more preferably 1 to 4, such as 1, 2, 3 or 4) naturally occurring amino acids (and preferably X 38 is any naturally occurring amino acid (most preferably X 38 is P)); X 39 is any naturally occurring amino acid (preferably S, T, M, P, F, Y or W; more preferably S); X 56 is any naturally occurring amino acid (preferably G, A, V, L or I; more preferably A) or is absent; X 57 is any naturally occurring amino acid (preferably P) or is absent; and X 58 is any naturally occurring amino acid (preferably H, K or R; more preferably K) or is absent; (For example, X 38 It is P; X 39 Yes S; X 56 A or not; X 57 Is P or does not exist; X 58 is K or does not exist) and optionally wherein, At least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5) (preferably at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence QSANLLAEAKKLNDAQ [SEQ ID NO 136] are replaced by alternative residues; And preferably optionally wherein at least 1 and no more than 5 (e.g. 1, 2, 3, 4 or 5) (preferably at least 1 and no more than 3 (e.g. 1, 2 or 3)) residues in the sequence QSANLLAEAKKLNDAQ [SEQ ID NO 136] are replaced by alternative residues as conservative substitutions.

30. The CD16a binding polypeptide of claim 29, wherein the C-terminal portion has the sequence X 38 X 39 QSANLLAEAKKLNDAQX 56 X 57 X 58 [SEQ ID NO 135], wherein X 56 It's A, X 57 is P, and X 58 It is K; X 56 A; or X 57 is P, and X 58 does not exist; X 56 It's A, X 57 does not exist; and X 58 does not exist; or X 56 Does not exist, X 57 does not exist, and X 58 does not exist; and optionally wherein, At least one and no more than three (e.g., one, two or three) residues in the sequence QSANLLAEAKKLNDAQ [SEQ ID NO 136] are replaced by alternative residues; and preferably optionally wherein at least 1 and no more than 3 (e.g. 1, 2 or 3) residues in the sequence QSANLLAEAKKLNDAQ [SEQ ID NO 136] are replaced by alternative residues as conservative substitutions; and Preferably, X 38 is P, and X 39 It’s S.

31. A CD16a binding polypeptide according to any preceding claim, wherein: (i) the isolated portion has a sequence X 20 X 21 X 22 ; and / or The N-terminal portion has the sequence X1X2X3X4X5; and / or The C-terminal portion has the sequence PSQSANLLAEAKKLNDAQX 56 X 57 X 58 [SEQ ID NO 137]; Wherein, in the separation part, X 20 Is P or T; X 21 Yes N; X 22 It is L; wherein in the N-terminal portion, 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; and wherein in the C-terminal portion, X 56 A or not; X 57 Is P or does not exist; X 58 It is K or not present; or (ii) the separation portion, the N-terminal portion and the C-terminal portion are as defined in (i), wherein optionally (a) within each portion, 1, 2 or 3 residues are replaced by alternative residues; or (b) within those portions, at least 1 and no more than 10 (eg no more than 5, such as 1, 2, 3, 4 or 5) residues are collectively replaced by the alternative residues.

32. A CD16a binding polypeptide according to any preceding claim, wherein the isolated portion has the sequence PNL or TNL; and / or wherein the N-terminal portion has the sequence VDNKF [SEQ ID NO 138].

33. A CD16a binding polypeptide according to any preceding claim, consisting of a CD16a binding polypeptide as defined in any preceding claim; and optionally comprising an additional binding moiety.

34. A CD16a binding polypeptide according to any preceding claim, wherein the CD16a binding motif sequence is selected from SEQ ID NOs. 150 to 221; Preferably selected from SEQ ID NO. 166, 168, 178, 182 or 202; or Preferably selected from SEQ ID NO. 164, 166, 168 or 200 (eg 164, 166 or 168); And wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably by residues that are conservative substitutions.

35. The CD16a binding polypeptide according to claim 1 or 2, comprising a sequence selected from SEQ ID NO. 1-75, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions; or A CD16a binding polypeptide according to any one of claims 1 to 3, comprising a sequence selected from SEQ ID NO. 1-73, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions; or A CD16a binding polypeptide according to any one of claims 1 to 4, comprising a sequence selected from SEQ ID NO. 1 or 11 to 67, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions; or The CD16a binding polypeptide according to any one of claims 1 to 5, comprising a sequence selected from SEQ ID NO. 11 to 67, and wherein optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues, and preferably optionally, 1 to 5 (preferably 1, 2 or 3) residues in the sequence are replaced by alternative residues as conservative substitutions.

36. The CD16a binding polypeptide according to claims 1 to 4, wherein the sequence of the CD16a binding polypeptide is selected from: VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEA KKLNDAQAPK[SEQ ID NO:1], or VDNKFNKEQQIAQYEIRKLPNLNHHQTFAFIKSLLDDPSQSANLLAEA KKLNDAQAPK [SEQ ID NO: 51].

37. The CD16a binding polypeptide of claim 1 or claim 2, wherein the sequence of the CD16a binding polypeptide is selected from: VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAE AKKLNDAQAPK [SEQ ID NO: 74]; and VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPK[SEQ ID NO:75].

38. The CD16a binding polypeptide of claim 1 or claim 2, wherein the sequence of the CD16a binding polypeptide is selected from SEQ ID NO. 12, 17, 19, 29, 33, 49, 51 and 53; for example, SEQ ID NO. 17, 19, 29, 33 and 53; or wherein the sequence of the CD16a binding polypeptide is selected from SEQ ID NO. 19, 33, 17, 29, 53, 16, 25, 15, 51, 36, 49, 55, 43, 24, 56, 12, 28, 21, 59, 52, 32, 18, 27, 35 or 11; or 34, 45 or 51; or 26, 35 or 47.

39. The CD16a binding polypeptide of claim 1 or claim 2, wherein the sequence of the CD16a binding polypeptide is selected from SEQ ID NO. 15, 17, 19 and 51; for example 15, 19 or 17.

40. The CD16a binding polypeptide according to any preceding claim, wherein the CD16a binding polypeptide does not comprise methionine.

41. A CD16a binding polypeptide having a sequence according to any preceding claim, wherein At the positions where methionine residues are listed, the polypeptide has a sequence in which the methionine residues are independently substituted with a different naturally occurring amino acid or a non-natural amino acid (e.g., a different naturally occurring amino acid or norleucine); and preferably, each methionine residue is independently substituted with an amino acid selected from isoleucine (I), leucine (L), glutamine (Q) and norleucine; and more preferably, each methionine residue is independently substituted with norleucine or isoleucine.

42. A CD16a binding polypeptide according to any preceding claim, wherein one or more residues (e.g., 1 to 5 residues, such as 1, 2, 3, 4 or 5) of the CD16a binding polypeptide are substituted with an unnatural amino acid, such as norleucine; and / or wherein one or more methionine residues when present (e.g., 1 or 2 methionine residues when present) are substituted with an unnatural amino acid, such as norleucine; and / or wherein one or more leucine residues when present (e.g., 1 to 5 leucine residues when present) are substituted with norleucine; and / or wherein one or more methionine residues when present (eg 1 or 2 methionine residues when present) are oxidized (eg are Met(O)).

43. A CD16a binding oligomer comprising at least two CD16a binding polypeptides as defined in any one of claims 1 to 42, such as 2, 3, 4 or 5 CD16a binding polypeptides as defined in any one of claims 1 to 42.

44. The CD16a binding oligomer according to claim 43, comprising at least two CD16a binding polypeptides, wherein the first CD16a binding polypeptide is as defined in any one of claims 2 to 42, and the second CD16a binding polypeptide is as defined in any one of claims 2 to 42.

45. The CD16a binding oligomer of claim 44, comprising at least two CD16a binding polypeptides, wherein the first CD16a binding polypeptide and the second CD16a binding polypeptide have the same sequence.

46. ​​The CD16a binding oligomer of claim 44, comprising at least two CD16a binding polypeptides, wherein the first CD16a binding polypeptide and the second CD16a binding polypeptide have different sequences.

47. The CD16a binding oligomer of claim 43, comprising at least two CD16a binding polypeptides, wherein: The first CD16a binding polypeptide comprises a first CD16a binding motif selected from SEQ ID No. 150-221 or comprises a sequence selected from SEQ ID NO. 1-75; And the second CD16a binding polypeptide comprises a second CD16a binding motif selected from SEQ ID NO.150-221 or comprises a sequence selected from SEQ ID NO.1-75.

48. The CD16a binding oligomer of claim 47, comprising at least two CD16a binding polypeptides, wherein the first CD16a binding polypeptide and the second CD16a binding polypeptide have the same sequence, or the selected first CD16a binding motif and the second CD16a binding motif have the same sequence.

49. The CD16a binding oligomer according to claim 47, comprising at least two CD16a binding polypeptides, wherein the selected first binding motif and the second binding motif or the first CD16a binding polypeptide and the second CD16a binding polypeptide have different sequences.

50. The CD16a binding oligomer according to any one of claims 43 to 49, wherein the CD16a binding polypeptides are each separated by a linker.

51. The CD16a binding oligomer according to claim 50, wherein the linker is a sequence of 1 to 50 (e.g., 1 to 25) naturally occurring amino acids; preferably a sequence of 1 to 25 (e.g., 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S).

52. The CD16a binding oligomer of claim 50 or 51, wherein the linker is G or comprises the sequence GGGSG [SEQ ID NO 139], GGGGS [SEQ ID NO 140], GGSGG [SEQ ID NO 141], GSGGG [SEQ ID NO 142] and / or SGGGG [SEQ ID NO 143]; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG [SEQ ID NO 144], GGGSGGGGSGGGGSG [SEQ ID NO 145], GGGSGGGGSGGGGSGGGGSG [SEQ ID NO 146], GGSGG, GGSGGGGSGG [SEQ ID NO 147], GGSGGGGSGGGGSGG [SEQ ID NO 148] or GGSGGGGSGGGGSGGGGSGG [SEQ ID NO 149]. 149].

53. The CD16a binding oligomer according to any one of claims 50 to 52, wherein the CD16a binding oligomer comprises at least 2 (e.g., 2) CD16a binding polypeptides, and the CD16a binding oligomer comprises the following structure: [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]; wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (e.g. 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S), for example, wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG or GGSGGGGSGGGGSGGGGSGG; wherein each N-terminal portion in the oligomer can have the same sequence or have a different sequence; Each C-terminal portion in the oligomer can have the same sequence or a different sequence; Each separate portion of the oligomer can have the same sequence or a different sequence; Each helix 1 portion in the oligomer can have the same sequence or a different sequence; and Each helix 2 portion in the oligomer can have the same sequence or a different sequence.

54. The CD16a binding oligomer according to any one of claims 50 to 53, wherein the CD16a binding oligomer comprises at least 3 (e.g., 3) CD16a binding polypeptides, and the CD16a binding oligomer comprises the following structure: [N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]-[linker]-[N-terminal part]-[helix 1]-[isolated part]-[helix 2]-[C-terminal part]; wherein each linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (e.g. 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S), for example, wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG or GGSGGGGSGGGGSGGGGSGG; and wherein each linker moiety in the oligomer can have the same sequence or have a different sequence; Each N-terminal portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each C-terminal portion can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each separate portion of the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence; Each helix 1 portion in the oligomer can have the same sequence, can have a different sequence, or two can have the same sequence and one can have a different sequence; and Each helix 2 portion in the oligomer can have the same sequence, have a different sequence, or two can have the same sequence and one can have a different sequence.

55. The CD16a binding oligomer according to claim 43, 44 or 53, comprising the sequence VDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK [SEQ ID NO 242].

56. The CD16a binding oligomer according to claim 43, 44 or 53, comprising the sequence VDNKFNKEQFYARDEIDLLPNLNEDQKWAFYMSLIDDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO. 243].

57. The CD16a binding oligomer according to claim 43, 44 or 53, wherein the CD16a binding oligomer comprises the sequence VDNKFNKEFWIAESEIESLPNLNIYQKWAFKYSLADDPSQSANLLAEAKKLNDAQAPKGGGSGGGGSGGGGSGVDNKFNKEVQMAQFEIRKLPNLNHHQSFAFIKSLMDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO. 244].

58. The CD16a binding polypeptide according to any one of claims 2 to 42, or the CD16a binding oligomer according to any one of claims 43 to 57, further comprising additional functional parts (e.g., at least one, at least two or at least three; e.g., 1, 2, 3, 4 or 5 additional functional parts).

59. The CD16a binding polypeptide or CD16a binding oligomer according to claim 58, wherein the additional functional part comprises an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof.

60. The CD16a binding polypeptide or CD16a binding oligomer of claim 58, wherein the additional functional moiety comprises an additional binding moiety.

61. The CD16a binding polypeptide or CD16a binding oligomer of claim 60, wherein the additional binding moiety is specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or is specific for a NK cell target.

62. The CD16a binding polypeptide or CD16a binding oligomer according to claim 58, wherein the CD16a binding polypeptide or CD16a binding oligomer comprises at least two (e.g., 2, 3, 4 or 5) additional functional parts, wherein each additional functional part can be the same or can be different.

63. The CD16a binding polypeptide or CD16a binding oligomer according to claim 58, wherein the CD16a binding polypeptide or CD16a binding oligomer comprises at least two (eg, 2, 3, 4 or 5) additional functional moieties, wherein the first additional functional moiety comprises an additional binding moiety (e.g., an additional binding moiety specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or for a NK cell target); and wherein the second additional functional moiety comprises an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; or wherein the first additional functional portion comprises an additional binding portion (e.g., an additional binding portion that is specific to a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding portion that is specific to a NK cell target); and wherein the second additional functional portion comprises an additional binding portion (e.g., an additional binding portion that is specific to a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or an additional binding portion that is specific to a NK cell target).

64. The CD16a binding polypeptide or CD16a binding oligomer according to claim 63, which comprises additional functional parts, wherein the CD16a binding polypeptide or CD16a binding oligomer comprises at least 3 (e.g., 3, 4 or 5) additional functional parts, wherein the third additional functional part comprises an additional binding part (e.g., an additional binding part that is specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or is specific for a NK cell target), or comprises an immune signaling molecule, such as a cytokine, such as IL-15 or a derivative thereof; and preferably wherein the third additional functional part comprises an additional binding part (e.g., an additional binding part that is specific for a cancer cell surface target, such as a myeloma cell surface antigen, such as BCMA, or is specific for a NK cell target).

65. The CD16a binding polypeptide or CD16a binding oligomer according to any one of claims 58 to 64, wherein the additional functional moiety is separated from the CD16a binding polypeptide or CD16a binding oligomer by a linker, For example, wherein the linker is a sequence of 1 to 50 naturally occurring amino acids, preferably 1 to 25 (eg 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S).

66. A CD16a binding polypeptide or CD16a binding oligomer according to claim 65, wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG or GGSGGGGSGGGGSGGGGSGG.

67. The CD16a binding polypeptide or CD16a binding oligomer according to claim 65 or 66, comprising an additional functional part, wherein the CD16a binding polypeptide comprises the following structure: [N-terminal part] - [helix 1] - [isolation part] - [helix 2] - [C-terminal part] - [linker] - [additional functional part]; or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]; or [N-terminal part]-[helix 1]-[isolation part]-[helix 2]-[C-terminal part]-[linker]-[additional functional part]-[additional functional part]; [additional functional part]-[additional functional part]-[linker]-[N-terminal part]-[helix 1]-[separating part]-[helix 2]-[C-terminal part]; or [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]-[Additional functional part]; (and preferably [Additional functional part]-[Linker]-[N-terminal part]-[Helix 1]-[Separation part]-[Helix 2]-[C-terminal part]; [Additional functional part] - [Additional functional part] - [Linker] - [N-terminal part] - [Helix 1] - [Separation part] - [Helix 2] - [C-terminal part];) wherein each linker is a sequence of 1 to 50 (e.g. 1 to 25) naturally occurring amino acids, preferably 1 to 25 (e.g. 1 to 20) naturally occurring amino acids selected from the group consisting of G, S and T (preferably G and S) (e.g., wherein the linker is G or comprises the sequence GGGSG, GGGGS, GGSGG, GSGGG and / or SGGGG; for example, wherein the linker is G or comprises or has the sequence GGGSG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGSGG, GGSGGGGSGGGGSGG, GGSGG, GGSGGGGSGGGGSGG or GGSGGGGSGGGGSGGGGSGG). And among them, When there is more than one additional functional part, each additional functional part can be the same or different.

68. A nucleic acid molecule encoding a CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67; or encoding a CD16a binding oligomer according to any one of claims 43 to 67.

69. An expression vector comprising the nucleic acid molecule according to claim 68.

70. A host cell comprising the nucleic acid molecule according to claim 68 or the expression vector according to claim 69.

71. A method for preparing a CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67 or a CD16a binding oligomer according to any one of claims 43 to 67, the method comprising maintaining the host cell according to claim 70 under optimal conditions for expressing nucleic acid, and isolating the CD16a binding polypeptide.

72. A CD16a binding agent-drug conjugate comprising a CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67 or a CD16a binding oligomer according to any one of claims 43 to 67, and an additional therapeutic agent.

73. The CD16a binding agent-drug conjugate of claim 72, wherein the additional therapeutic agent is a cytotoxic drug, such as MMAF, MMAE, doxorubicin, pyrrolobenzoic acid, nitrogen Amanitine, maytansinoids, dolastatin, mitomycin C, desmethyltopotecan, or SN-38.

74. The CD16a binding agent-drug conjugate of claim 72 or claim 73, wherein the hBCMA binding polypeptide is linked to the additional therapeutic agent via a linker.

75. A pharmaceutical composition comprising a CD16a binding polypeptide as defined in any one of claims 1 to 42 or 58 to 67, a CD16a binding oligomer according to any one of claims 43 to 67, a nucleic acid molecule according to claim 68 or an expression vector according to claim 67.

76. The CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, the CD16a binding oligomer according to any one of claims 43 to 67, The nucleic acid molecule according to claim 68, the expression vector according to claim 69, the CD16a binding agent-drug conjugate according to any one of claims 72 to 74, and / or The pharmaceutical composition according to claim 75, for use in medicine.

77. The CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, the CD16a binding oligomer according to any one of claims 43 to 67, The nucleic acid molecule according to claim 68, the expression vector according to claim 69, the CD16a binding agent-drug conjugate according to any one of claims 72 to 74, and / or The pharmaceutical composition according to claim 75, for use in treating cancer.

78. The CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, the CD16a binding oligomer according to any one of claims 43 to 67, The nucleic acid molecule according to claim 68, the expression vector according to claim 69, the CD16a binding agent-drug conjugate according to any one of claims 72 to 74, and / or The pharmaceutical composition according to claim 75, for use according to claim 77, wherein the cancer is multiple myeloma.

79. The CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, the CD16a binding oligomer according to any one of claims 43 to 67, The nucleic acid molecule according to claim 68, the expression vector according to claim 69, the CD16a binding agent-drug conjugate according to any one of claims 72 to 74, and / or Use of the pharmaceutical composition according to claim 75 in the manufacture of a medicament for treating cancer.

80. A method of treating cancer, the method comprising administering to a patient in need thereof a CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, a CD16a binding oligomer according to any one of claims 43 to 67, The nucleic acid molecule according to claim 68, the expression vector according to claim 69, the CD16a binding agent-drug conjugate according to any one of claims 72 to 74 and / or the pharmaceutical composition according to claim 75.

81. A kit comprising a CD16a binding polypeptide according to any one of claims 1 to 42 or 58 to 67, a CD16a binding oligomer according to any one of claims 43 to 67, a CD16a-binding agent-drug conjugate according to any one of claims 72 to 74, or The pharmaceutical composition of claim 75, and optionally one or more additional therapeutic agents.

82. The kit of claim 81, wherein the one or more additional therapeutic agents are selected from a proteasome inhibitor (e.g., carfilzomib or bortezomib), an immunomodulator (e.g., lenalidomide or thalidomide), an alkylating agent (e.g., melphalan or melflurane), a steroid (e.g., dexamethasone or prednisone), an anti-CD38 agent (e.g., daratumumab), an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor), and an ADAM17 inhibitor.

83. A kit according to claim 81 or claim 82 for use in treating cancer, such as multiple myeloma.

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