Bispecific CD16A binders

Abstract

The present invention relates to a bispecific antibody construct comprising: (a) a first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6, and (ii) a VH region as set forth in SEQ ID NO: 7; and (b) a second binding domain (B) capable of specifically binding to a second target (B') which is an antigen present on the surface of a target cell, the second target (B') being FOLR1. The present invention also relates to related nucleic acid molecules, vectors, host cells, methods of producing the antibody construct, pharmaceutical compositions, medical uses, and kits.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 369,602, filed July 27, 2022; European Patent Application Nos. EP 22187329.2, filed July 27, 2022, EP 21213774.9, filed December 10, 2021, and EP 21206329.1, filed November 3, 2021, each of which is incorporated by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 5146_002PC01_Seqlisting_ST26.xml; Size: 197,996 bytes; and Creation Date: November 3, 2022) are incorporated by reference herein in their entirety.

[0003] Field of Disclosure The present invention relates to a bispecific antibody construct comprising a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; and a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell, said second target (B') being FOLR1. The present invention also relates to related nucleic acid molecules, vectors, host cells, methods of producing the antibody construct, pharmaceutical compositions, medical uses, and kits. [Background technology]

[0004] background Natural killer cells are cytotoxic, IFN-γ and TNF-α producing innate lymphoid cells that are considered the first line of defense against virus-infected and cancer cells (Cerwenka and Lanier 2001). The cytotoxic potential of NK cells can be exploited in cancer immunotherapy by redirecting NK cytolysis to tumor cells and stimulating the activating receptor CD16A, also known as FcγRIIIA, expressed on the surface of NK cells. NK cells are equipped with multiple activating and inhibitory receptors on their surface that jointly control the activation and induction of effector functions of NK cells. Some of these receptors play a central role for NK cell-mediated recognition, killing, and cytokine secretion of cancer cells. CD16A activation promotes NK cell proliferation and memory-like cytotoxicity against cancer cells (Pahl et al 2018 Cancer Immunol Res; 6(5), 517-27; DOI: 10.1158 / 2326-6066.CIR-17-0550). Upon ligation, CD16A induces a series of potent signals that result in cytokine production and cytotoxic effector activity via antibody-dependent cellular cytotoxicity (ADCC). In this regard, tumor-specific monoclonal antibodies (mABs), such as rituximab, that recognize tumor-selective antigens such as CD20 present on the surface of tumor cells, have been described to induce NK cell-mediated antitumor activity via ADCC (Wang et al., Front. Immunol., 2015, 6:368, doi: 10.3389 / fimmu.2015.00368).

[0005] However, directing NK cells to tumor cell lysis using bispecific or multispecific antibodies is also considered a powerful immunotherapeutic approach, offering the opportunity to increase specificity, efficacy, and utilize novel mechanisms of action. Bispecific antibodies have been developed, consisting of one arm that binds to CD16A and another arm that binds to tumor-associated antigens (e.g., CD19) (Kellner et al 2011 Cancer Lett. 303(2): 128-139). WO 2006 / 125668 and Reusch et al, MABS, 2014, 6:3:728-739 describe antigen-binding proteins, i.e., bispecific tandem diabodies, for binding to CD16A, and their use for natural killer (NK) cell therapy. The cytotoxic activity of NK cells can be enhanced by increasing avidity through multivalent binding to CD16A, for example, using constructs that bind bivalently to CD16A (WO2019 / 198051 Affimed GmbH).

[0006] Activation-induced downregulation / shedding of CD16 on activated NK cells is caused by proteolytic cleavage of its extracellular portion by a disintegrin and metalloproteinase (ADAM17) (Romee at al., Blood, 2013, 121 (18):3599-3608) or membrane type 6 matrix metalloproteinase (MMP25) (Peruzi et al., J. Immunol., 2013, 191:955-957). However, CD16 shedding is not immediately reversed, suggesting that once NK cells are activated and CD16 is downregulated, their ADCC capacity is impaired for several days (Goodier et al., Front. Immunol., 2016, 7:384). Moreover, ADAM17-mediated CD16 shedding has also been described to limit the efficacy of rituximab or trastuzumab antibody therapy with ADCC (Romee at al., Blood, 2013, 121(18):3599-3608). Thus, downregulation of CD16 expression on activated immune effector cells may limit or control their activity and ADCC-mediated cytotoxicity. On the other hand, the use of CD16 inhibitors and NK cells transfected to express a non-cleavable form of CD16 revealed that CD16 shedding upon NK cell activation may be important for NK cell detachment from opsonized target cells, thereby maintaining NK cell survival and increasing continuous target cell binding (Srpan et al., J. Cell. Biol., 2018, 217(9):3267-3283).

[0007] In particular, in solid tumors where the presence of NK cells is limited, CD16 shedding is a major disadvantage to the ability of effector cells to kill multiple tumor cell targets. Overexpression of folate receptor 1 (FOLR1) by a number of solid tumors, including ovarian, breast (especially TNBC), renal, lung, colorectal (CRC), renal (especially ccRCC), pancreatic (especially PDAC), endometrium, and brain, has been described in the art (Hartmann et al., Int J Cancer 2007, 121(5): 938-42; Yang et al., Human Cancer Biology 2007, 13(9): 2557-2567; Scaranti et al., Nat Rev Clin Oncol.2020, 17(6): 349-359). Thus, due to its high expression in solid tumors, FOLR1 may be an attractive therapeutic target for the development of anticancer drugs. A range of FOLR1 targeting approaches, including folate derivatives, folate drug conjugates and small molecules, vaccines, terpolymer-based nanocapsules, T cell therapy, and monoclonal antibodies, have been developed for clinical applications for both imaging and therapeutic purposes (Cheung et al., Oncotarget 2016, 7(32): 52553-52574; Bellotti et al., biomedicines 2021, 9, 1275).

[0008] However, there is still a need in the art for providing a highly efficient therapeutic approach for treating FOLR1-expressing cancer disease, for example, anti-CD16A bispecific antibody constructs for use in immuno-oncology therapy, which induces the activation of immune effector cells by binding to CD16A, thereby enabling high cytokine production and long-lasting target cell killing by activated NK cells.The present invention, as shown herein, addresses this need. Summary of the Invention

[0009] overview The present invention is based at least in part on the surprising finding that a bispecific antibody construct comprising a first binding domain of high affinity anti-CD16A and a second binding domain against an antigen present on the surface of a target cell, the second binding domain being FOLR1, can efficiently activate and redirect immune effector cells for ADCC, thereby avoiding CD16A shedding and immediate inactivation of bound effector cells. Specifically, the inventors have surprisingly observed that the high affinity anti-CD16A binding domain contained in the antibody construct of the present invention strongly stabilizes CD16A expression on NK effector cells after activation, when compared with the low affinity anti-CD16A binding domain, despite the presence of target cells. In this regard, immune effector cells activated by the bispecific antibody construct of the present invention exhibit high cytotoxicity and induce target cell lysis without activation-induced CD16A shedding. This is particularly beneficial for the treatment of solid tumors where NK cells are limited in presence, because CD16 shedding is a major detriment to the ability of effector cells to kill multiple tumor cell targets. As shown in Examples 1, 2, and 12, bispecific antibodies of the invention comprising a specific CD16A binding domain (also referred to herein as CD16a1 anti-CD16A effector domain or CD16a1 domain) exhibit higher affinity for human CD16A when compared to other CD16A binding domains (see Figures 1, 2, and 16A-16C, and Tables 3 and 15). Furthermore, as shown in Example 5, bispecific antibodies of the invention comprising a specific CD16A binding domain exhibit significant CD16 shedding inhibitory effects on stimulated NK cells when compared to the shedding inhibition induced by other CD16A binding domains (see Figures 5A-5C and 6A-6C). Nevertheless, as demonstrated in Example 4, bispecific antibodies of the invention comprising a specific CD16A-binding domain also have EC 50The antibodies exhibit high lytic capacity with CD16A values ​​(see Figures 4 and 17) and low target cell independent activation (see Figures 7 and 18A-18B). In summary, antibody constructs comprising the high affinity anti-CD16A binding domains of the invention surprisingly have high cytotoxic activity, even though these constructs prevent CD16A shedding upon NK cell activation.

[0010] Therefore, the antibody construct of the present invention can be useful for tumor therapy, especially solid tumors, because it can not only activate NK cells through high affinity binding of CD16A receptor, but also achieve long-lasting activation of NK cells without loss of CD16A. Thus, the bispecific antibody construct of the present invention provides high affinity binding of effector NK cells via CD16A and effective killing of target cells by cell-mediated cytotoxicity, thereby enabling sustained effector cell activation. Thus, the antibody construct of the present invention can be useful for efficiently targeting various cancer diseases, especially solid tumors that overexpress FOLR1 on the cell surface, and should be considered superior to antibody constructs that contain low affinity CD16A binding domains (e.g., CD16a2 or CD16a4 anti-CD16A effector domains disclosed herein). Thus, the antibody constructs of the present invention may be useful in treating solid tumors such as ovarian, breast (particularly TNBC), renal, lung, colorectal (CRC), renal (particularly ccRCC), pancreatic (particularly PDAC), endometrial, and brain tumors.

[0011] In particular, the present invention relates to a bispecific antibody construct comprising: (a) a first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6, and (ii) a VH region as set forth in SEQ ID NO: 7 or SEQ ID NO: 94; and (b) a second binding domain (B) capable of specifically binding to a second target (B') which is an antigen present on the surface of a target cell, the second target (B') being FOLR1.

[0012] The present invention also relates to a nucleic acid molecule comprising a sequence encoding an antibody construct of the present invention.

[0013] The present invention also relates to a vector comprising the nucleic acid molecule of the present invention.

[0014] The present invention also relates to a host cell which comprises a nucleic acid molecule of the invention or a vector of the invention.

[0015] The present invention also relates to a method of producing an antibody construct of the present invention, comprising culturing a host cell of the present invention under conditions allowing expression of the antibody construct of the present invention, and optionally recovering the produced antibody construct from the culture.

[0016] The present invention also relates to a pharmaceutical composition comprising an antibody construct of the present invention or an antibody construct produced by a method of the present invention.

[0017] The present invention also relates to an antibody construct of the invention for use in therapy.

[0018] The present invention also relates to a method of treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder, comprising administering to a subject in need thereof an antibody construct of the present invention or an antibody construct produced by the method of the present invention.

[0019] The present invention also relates to a kit comprising an antibody construct of the invention or an antibody construct produced by a method of the invention, a nucleic acid molecule of the invention, a vector of the invention, and / or a host cell of the invention. [Brief description of the drawings]

[0020] [Figure 1] Detection of interaction between CD16A and CD16A-binding domain. Binding of CD123×CD16A ICE to human CD16A158V, CD16A158F, and cynomolgus CD16 was measured by SPR with biotin-captured recombinant CD16A158V, CD16A158F, and cynomolgus CD16 (ligands) and scFv-IgAb_268 (CD16a1×CD123-1), scFv-IgAb_148 (CD16a2×CD123-1), scFv-IgAb_264 (CD16a1×CD123-2) (analytes) using a multivalent multicycle kinetics setup (n=3;2) n=1) at 37° C. Affinity and kinetic parameters were evaluated for interactions with human CD16A and cynomolgus CD16 using a 1:1 binding model. All molecules showed high interactions with human CD16A and cynomolgus CD16, with apparent affinities ranging from KD 0.195 nM to 2.48 nM. [Diagram 2]Binding of CD123xCD16A constructs to cell lines expressing human CD16A. Binding of antibody constructs to huCD16A transfected CHO cells measured by flow cytometry, showing median fluorescence intensity (MFI) of titrated scFv-IgAb_268 (CD123-1xCD16a1, black dots), scFv-IgAb_148 (CD123-1xCD16a2, black triangles) and negative control molecule (scFv-IgAb_139, CD123xRSV, grey dots) relative to total CD16 expression as detected by anti-human CD16 antibody clone 3G8. Experiment no. RHU 066. [Diagram 3] Cell surface retention of anti-CD123 antibodies on NK cells. Enriched primary human NK cells were preloaded with 100 μg / mL of scFv-IgAb_268 (CD123-1×CD16a1), Fc-enhanced anti-CD123 IgG1 (IgAb_338), or scFv-IgAb_148 (CD123-1×CD16a2) on ice, washed, and then incubated at 37° C. for the indicated periods in an excess volume of complete RPMI1640 medium to allow dissociation and prevent reassociation. Residual antibody at each time point was measured by flow cytometry, and the percentage of remaining antibody was analyzed and plotted by nonlinear regression using GraphPad Prism, taking the median fluorescence intensity (MFI) value at time point 0 as 100%. [Figure 4]ADCC against CD123+ EOL-1 cells by anti-CD123 antibodies. Concentration-dependent induction of tumor cell lysis by bispecific antibody constructs scFv-IgAb_268 (CD123-1×CD16a1), scFv-IgAb_267 (CD123-2×CD16a1), scFv-IgAb_265 (CD123-1×CD16a2), and scFv-IgAb_264 (CD123-2×CD16a2) using NK cells as effector cells in a 4-h calcein release cytotoxicity assay. Calcein-labeled EOL-1 target cells were incubated in duplicate with human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the respective antibodies. Target and effector cells without antibody (None) were used as negative controls (Control), and target killing by effectors in the absence of antibody was measured in quadruplicates on each plate. Experiments were performed in biological duplicates, and one representative result figure is shown. All four CD123xCD16A scFv-IgAb constructs induced NK cell-dependent lysis of EOL-1 cells with similar maximal efficacy in the low picomolar range. [Diagram 5] Inhibition of CD16A shedding on activated NK cells. Enriched primary human NK cells were preloaded with 100, 10, or 1 μg / mL of CD123-1×CD16a1 scFv-IgAb_268 (FIG. 5A), CD123-1×CD16a2 scFv-IgAb_148 (FIG. 5B), or Fc-enhanced anti-CD123 IgG1 (IgAb_338) (FIG. 5C) on ice, washed, and then stimulated with PMA / ionomycin (PMA Iono) for 4 hours at 37° C. CD16 expression was measured by flow cytometry and analyzed using FlowJo software. Experiment number: NSC 026. [Figure 6]Inhibition of shedding of CD16A on activated NK cells. Enriched primary human NK cells were preloaded with 100, 10, or 1 μg / mL of CD123-1×CD16a1 scFv-IgAb_268 (FIG. 6A), CD123-1×CD16a2 scFv-IgAb_148 (FIG. 6B), or Fc-enhanced anti-CD123 IgG1 (IgAb_338) (FIG. 6C) on ice, washed, and then stimulated with PMA / ionomycin (PMA Iono) for 4 h at 37°C. Median fluorescence intensity (MFI) values ​​of CD16 expression were measured by flow cytometry and analyzed using FlowJo software. After subtracting the fluorescence intensity values ​​of cells stained with secondary reagents only, MFI values ​​were plotted using GraphPad Prism software. Statistical significance was evaluated using paired Student's t-test. n.s.: p>0.05; *p<0.05. [Figure 7] Target cell-independent activation of NK cells by anti-CD123 antibodies. Enriched primary human NK cells were preloaded with 100, 10, or 1 μg / mL of CD123-1×CD16a1 scFv-IgAb_268 (A), CD123-1×CD16a2 scFv-IgAb_148 (B), or Fc-enhanced anti-CD123 IgG1 (IgAb_338) (C) on ice, washed, and then stimulated with PMA / ionomycin for 4 h at 37°C. CD16 expression was measured by flow cytometry and analyzed using FlowJo software. Experiment number: NSC 026. [Figure 8]Target cell-dependent activation of NK cells by anti-CD123 antibodies. CMFDA-labeled EOL-1 cells were co-cultured with buffy coat-derived allogeneic NK cells (5 × 104 cells) at a 1:1 cell ratio for 24 h in the presence of titrated antibodies (CD123a1 × CD16a1 scFv-IgAb_268, CD123-2 × CD16a1 scFv-IgAb_267, CD123-1 × CD16a2 scFv-IgAb_265, CD123-1 × CD16a2 scFv-IgAb_264) or control molecules (scFv-IgAb_239, scFv-IgAb_238) starting at a concentration of 50 μg / mL followed by six 10-fold serial dilutions. Upregulation of the NK cell activation marker CD137 on NK cells was analyzed by flow cytometry. All four CD123xCD16A scFv-IgAb constructs specifically induced upregulation of the activation marker CD137, with the anti-CD16A CD16a1 domain antibody construct peaking in the percentage of CD137+ NK cells at 0.05 μg / mL, followed by a decrease in the percentage of CD137+ NK cells at higher concentrations. The non-CD123 targeting RSVxCD16A control antibody construct failed to induce NK cell activation in response to EOL-1 cells. [Figure 9]Binding of CD123xCD16A constructs to CD123+ and CD123- tumor cell lines. Binding of four CD123xCD16A antibody constructs, CD123a1xCD16a1 scFv-IgAb_268, CD123-2xCD16a1 scFv-IgAb_267, CD123-1xCD16a2 scFv-IgAb_265, and CD123-1xCD16a2 scFv-IgAb_264, to CD123+ EOL-1, CD123- A-431, and CD123- Karpas-299 cells was analyzed by flow cytometry. All four CD123xCD16A scFv-IgAb constructs showed comparable binding to CD123+ EOL-1 cells (Figure 9A). In contrast, to CD123- A431 cells, scFv-IgAb_268, which contains the CD123-1 and CD16a1 binding domains, showed the lowest potential for non-specific binding (Figure 9B). Overall, across the various antibody construct batches tested, scFv-IgAb_268 showed the least non-specific binding to CD123- A-431 cells, followed by scFv-IgAb_265, followed by scFv-IgAb_267, followed by scFv-IgAb_264. [Figure 10] Structural information and description of preferred bispecific antibody constructs. [Figure 11] Structural information and description of preferred bispecific antibody constructs. [Figure 12]Reduction of CD123+ primary leukemic blasts from peripheral blood and bone marrow of AML patients by anti-CD123 antibodies. Percentage reduction of primary leukemic blasts in PB and BM of AML patients after 24 h of co-culture with buffy coat-derived allogeneic NK cells at a 1:1 effector to target (E:T) cell ratio in the presence of titrated AFM28 (CD123×CD16A scFv-IgAb_268, black squares), Fc-enhanced anti-CD123 IgG talacotuzumab (IgAb_338, grey triangles), negative control molecule (RSV×CD16A scFv-IgAb_239, black circles), or without antibody (black crosses). (FIG. 12A) Representative dose-response data from two AML samples. (FIG. 12B) Data from four AML PB and BM samples (single measurement) at 0.002 μg / mL (10 pM) of antibody constructs. [Figure 13] ADCC against CD123+ BMMC cells from patients diagnosed with AML and HR-MDS by anti-CD123 antibodies. Concentration-dependent induction of tumor cell lysis by the bispecific antibody construct scFv-IgAb_268 (CD123-1×CD16a1) using allogeneic healthy donor NK cells as effector cells in a 24-hour cytotoxicity assay. Bone marrow samples from patients diagnosed with (FIG. 13A) AML or (FIG. 13B) high-risk MDS containing CD123+ target cells were incubated in triplicate with human NK cells as effector cells at an E:T ratio of 1:1 in the presence of serial dilutions of the antibody. Effector target killing in the absence of antibody (0 pM) was measured in triplicate for each sample. Experiments were performed in biological triplicates (AML) and biological duplicates (MDS) and one representative result figure is shown. The scFv-IgAb_268 construct induced NK cell-dependent lysis of CD34+ / CD123+ and CD34neg / CD123+ cells (including leukemic blasts, leukemic stem cells, and BM-MDSCs) in the low picomolar range. The CD34+ / CD123neg hematopoietic stem cell (HSC) compartment remained largely unaffected. [Figure 14]IL-6 release in cynomolgus monkeys at the start of AFM28 (scFv-IgAb_268) infusion. IL-6 release induced by scFv-IgAb_268 in cynomolgus monkeys during repeated weekly iv dosing at three dose levels. Serum collection points are indicated by the time after the start of infusion on each dosing day. [Figure 15] Reduction of CD123+ basophils in peripheral blood of cynomolgus monkeys upon dosing with AFM28 (scFv-IgAb_268). Animals received either vehicle or 4, 20, and 100 mg / kg by 2-hour chair infusion. Blood was collected on two pre-dose occasions, 24 hours after the first dose, pre-dose on days 5, 15, 22, and 29, and on day 43 for recovery animals. Absolute basophil counts (CD3- / CD14- / CD20- / CD159a- / HLA-DR- / FceR1a+) in whole blood were measured by flow cytometry. [Figure 16A] Figures 16A-16C: Binding of FOLR1xCD16A antibody constructs to cell lines expressing human CD16A and cynomolgus CD16. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17] ADCC against FOLR1+ A2780 cells by anti-FOLR1 antibody constructs. [Figure 18] Target cell-independent activation of NK cells by anti-FOLR1 antibodies. Enriched human NK cells were cultured for 24 hours with titrated concentrations of scFv-IgAb_273, scFv-IgAb_274, scFv-IgAb_275, or without antibody (none) as a control. Mean fluorescence intensity (MFI) of CD69 (Figure 18A) and CD137 (Figure 18B) was assessed by flow cytometry and plotted by nonlinear regression using GraphPad Prism. Mean and SD values ​​of three independent experiments are shown. [Figure 19]Binding of FOLR1 x CD16A antibody constructs to cell lines expressing human FOLR1 and cynomolgus monkey FOLR1. Binding of antibody constructs to huFOLR1 (Figure 19A) and cyFOLR1 (Figure 19B) transfected CHO cells measured by flow cytometry showing median fluorescence intensity (MFI) of titrated scFv-IgAb_381 and scFv-IgAb_387. [Figure 20] Structural information of the FOLR1xCD16A bispecific antibody construct. [Figure 21] ScFv-IgAb_387-induced lysis of target cells in the presence of soluble FOLR1. 4-hour calcein release cytotoxicity assay using A2780 and A-549 target cells at a 2.5:1 E:T ratio and enriched human NK cells as effector cells in the presence of scFv-IgAb_387 and 2-, 25-, and 50-fold molar excess of soluble FOLR1 (sFOLR1) or soluble mesothelin (sMSLN). Means and SD of triplicates from one representative experiment are shown. [Figure 22] ADCP induced by scFv-IgAb_387 on FOLR1+ target cells. In vitro differentiated human macrophages were co-cultured with CMFDA-labeled HeLa and HCC-78 target cells at a 5:1 E:T ratio in the presence of serial dilutions of FOLR1 / CD16A scFv-IgAb_387, anti-FOLR1 IgG1 IgAb_355, RSV / CD16A scFv-IgAb_444, and FOLR1 / RSV scFv-IgAb_162, or without antibodies for 4 hours. The percentage of phagocytosed target cells was measured by flow cytometry and plotted. One representative experiment out of three independent experiments is shown. [Diagram 23] Mice were treated with vehicle or scFv-IgAb_387 in either a therapeutic (2101-THE) or prophylactic (2101-PRO) setting. Tumor size was measured twice weekly for 35 days. Mean tumor volume with SEM for each group is shown. [Figure 24]Mice were treated with the indicated treatments and euthanized at the end of the experiment (35 days after tumor cell inoculation). Blood (Figure 24A), spleens (Figure 24B), and tumors (Figures 24C and 24D) were collected for flow cytometry analysis. Absolute numbers (right graph) and percentages (left graph) of the indicated cell populations are shown as mean values ​​with SEM. Each point represents an analyzed tumor. [Diagram 25] MCP-1 and IP-10 induced by scFv-IgAb_387 in 24-h PBMC cultures in the presence and absence of target cells. PBMCs alone (PBMC alone) or 20:1 effector-to-target (E:T) cocultures of PBMCs with FOLR1+ A-549, FOLR1+ OVCAR-3, or FOLR1- Raji as target cells were incubated for 24 h in the presence or absence (none) of increasing concentrations of FOLR1xCD16A scFv-IgAb_387, and MCP-1 and IP-10 in the supernatants were quantified by multiplexing. CD3 / CD28 activator beads were added as a positive control. Means and standard deviations (SD) of three independent experiments are plotted. [Figure 26] Cytokine release induced by FOLR1xCD16A scFv-IgAb in 24-hour cultures of PBMCs in the absence and presence of target cells. PBMCs alone or together with FOLR1+ A-549 cells, FOLR1+ OVCAR-3 cells, or FOLR1- Raji cells at an effector to target (E:T) ratio of 20:1 were cultured in the presence of 100 μg / mL scFv-IgAb_387 for 24 hours. Mean cytokine concentrations in pg / mL from three independent experiments are plotted. [Figure 27A]Figures 27A-27G: Mediated ADCC by scFv-IgAb_387 and control antibodies against various tumor cell lines in a 4-hour calcein release assay. Calcein-labeled target cell lines, including NCI-H2110 (Figure 27A), OVCAR-3 (Figure 27B), IGROV1 (Figure 27C), A2780 (Figure 27D), KARPAS-299 (Figure 27E), MAD-MB-231 (Figure 27F), and SKBR-3 (Figure 27G), were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of FOLR1xCD16A scFv-IgAb_387, anti-FOLR1 IgG1 IgAb_355, RSVxCD16A scFv-IgAb_444, and FOLR1xRSV scFv-IgAb_162 or without antibodies for 4 hours. After incubation, specific target cell lysis was calculated using fluorescent calcein released from lysed target cells. Mean lysis values ​​and SD from duplicates are plotted. For each tumor cell line, results from one representative experiment are shown. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 27D] See legend to Figure 27A. [Figure 27E] See legend to Figure 27A. [Figure 27F] See legend to Figure 27A. [Figure 27G] See legend to Figure 27A. [Figure 28]Binding of scFv-IgAb_387 and control antibody constructs to NK cells in the presence and absence of polyclonal human IgG. NK cells were incubated with increasing concentrations of biotinylated scFv-IgAb_387, biotinylated scFv-IgAb_444 (anti-RSV x CD16A), biotinylated scFv-IgAb_162 (anti-FOLR1 x RSV), or biotinylated anti-FOLR1 IgG1 antibody with wild-type Fc (IgAb_335) and biotinylated 3G8 (mouse anti-human CD16) at 37°C in the presence or absence of 10 mg / mL polyclonal human IgG. Cell surface bound antibodies were detected by streptavidin-FITC followed by flow cytometry analysis. Data shown are from one representative experiment out of four. MFI, median fluorescence intensity. [Figure 29] Binding of scFv-IgAb_387 and control molecules to FOLR1-expressing tumor cells. scFv-IgAb_387 (FOLR1×CD16A) and control antibodies scFv-IgAb_444 (RSV×CD16A), scFv-IgAb_162 (FOLR1×RSV), and IgAb_335 (anti-FOLR1 IgG1) were titrated against IGROV-1 cells expressing high levels of FOLR1 (FIG. 29A) and OVCAR-3 cells expressing intermediate levels of FOLR1 (FIG. 29B) and incubated at 37° C. Anti-FOLR1 mAb (clone LK26) was used as a positive control for all cell lines. Antibody bound to the cell surface was analyzed by flow cytometry and median fluorescence intensity (MFI) was plotted. [Diagram 30] Binding of scFv-IgAb_387 to recombinant human CD16A antigen. One 96-well ELISA plate was coated with (Figure 30A) human CD16A 158V, (Figure 30B) human CD16A 158F, respectively. Antibodies were applied in 3-fold serial dilutions starting from 50 nM. Data shown are one of three (Figure 30A) or four (Figure 30B) replicate experiments. [Diagram 31]Binding of scFv-IgAb_387 to recombinant FOLR1 antigen in ELISA. One 96-well ELISA plate was coated with human FOLR1. Antibodies were applied in 3-fold serial dilutions starting at 5 nM. Data shown is one of three replicate experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition The term "binding domain" in the context of the present invention describes a feature of a domain that can specifically bind to / interact with / recognize a given target epitope or a given target site of a target molecule (antigen), i.e. CD16A present on the surface of immune effector cells and a surface antigen of a target cell, respectively. The structure and / or function of the first binding domain (recognizing CD16A) and also the structure and / or function of the second binding domain (recognizing a target cell surface antigen, in particular FOLR1) are preferably based on the structure and / or function of an antibody, e.g. a full-length or full-length immunoglobulin molecule, and / or derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or a fragment thereof.

[0022] The term "specifically binds" as used herein means that the binding domain preferentially binds to or recognizes a target, even when its binding partner is present in a mixture of other molecules or other structures. Binding can be mediated by covalent or non-covalent interactions or a combination of both. In a preferred embodiment, "simultaneous binding to target cells and immune effector cells" includes the physical interaction of the binding domains with their targets on the cells, but also preferably includes the induction of an effect caused by the simultaneous binding to these two cells. Such an effect may be the immune effector function of the immune effector cells, such as a cytotoxic effect.

[0023] The term "antibody construct" refers to a molecule whose structure and / or function is based on that of an antibody, e.g., a full-length or full-length immunoglobulin molecule, and / or derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. Thus, the antibody construct is capable of binding to its specific target or antigen. Furthermore, the binding region of the antibody construct defined in the present invention comprises the minimum structural requirements of an antibody, which allows target binding. For the first binding domain (A), this minimum requirement is defined as the presence of a VL region comprising three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and the presence of a VH region comprising three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). For the second binding domain (B), this minimum requirement can be defined, for example, as the presence of at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs. An alternative approach to define the minimum structural requirements of an antibody is to define the protein domains of the target protein that constitute the antibody epitopes, i.e. epitope regions (epitope clusters), within the structure of the specific target, respectively, or by reference to specific antibodies that compete with the epitopes for the defined antibody. Antibodies on which the constructs defined in the present invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies.

[0024] The first binding domain of the antibody construct defined in the present invention comprises a group of CDRs as described above. These CDRs are contained in the framework of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH). The second binding domain of the antibody construct defined in the present invention may, for example, comprise a group of CDRs as described above. Preferably, these CDRs are contained in the framework of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH); however, they do not have to be contained in both. For example, Fd fragments have two VH regions and often retain some antigen-binding function of the intact antigen-binding region.

[0025] Examples of antibody fragment, antibody variant, or binding domain formats include: (1) a Fab fragment, i.e., a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, i.e., a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge domain; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of one arm of an antibody; (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs); and (7) single chain Fv (scFv), the latter of which is preferred (e.g., derived from an scFv library). Examples for embodiments of antibody constructs according to the invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910, and WO 2015 / 048272.

[0026] The antibody constructs defined in the present invention may comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "rIgG" ("half antibodies"). The antibody constructs defined in the present invention may also comprise modified antibody fragments, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies, which comprise only one variable domain, which may be VHH, VH, or VL, and specifically bind to an antigen or epitope independent of other V regions or V domains.

[0027] As used herein, the term "single chain Fv", "single chain antibody", or "scFv" refers to an antibody fragment of a single polypeptide chain that contains the variable regions from both the heavy and light chains but lacks a constant region. Generally, a single chain antibody further comprises a polypeptide linker between the VH and VL domains that allows it to form the desired structure that allows for antigen binding. A preferred linker for this purpose is a glycine serine linker, which preferably contains about 15 to about 30 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 41), or GGGGS (SEQ ID NO: 46). Such linkers preferably contain 5, 6, 7, 8, 9 and / or 10 repeats of GGS, preferably (GGS)6 (SEQ ID NO: 44) (preferably used for scFvs with the arrangement VH-VL), or preferably (GGS)7 (SEQ ID NO: 45) (preferably used for scFvs with the arrangement VL-VH). Single chain antibodies are discussed in detail by Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods of making single chain antibodies are known and include those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, the single chain antibodies may also be human, and / or humanized, and / or synthetic.The term "bi-scFv" or "ta-scFv" (tandem scFv) as used herein refers to two scFvs fused together. Such bi-scFvs or ta-scFvs may include a linker between the two scFv moieties. Typically, the arrangement of the VH and VL domains in the polypeptide chains within each scFv may be in any order. This means that the "bi-scFv" of a "ta-scFv" may be arranged in the order VH(1)-VL(1)-VH(2)-VL(2), VL(1)-VH(1)-VH(2)-VL(2), VH(1)-VL(1)-VL(2)-VH(2), or VL(1)-VH(1)-VL(2)-VH(2), where (1) and (2) represent the first scFv and the second scFv, respectively.

[0028] The term "double Fab" as used herein refers to two Fab fragments fused together, preferably in a staggered arrangement, where the first chain of the first Fab is fused N-terminally to the first chain of the second Fab, or the second chain of the first Fab is fused N-terminally to the second chain of the second Fab, or both, i.e., the first chain of the first Fab and the second chain of the first Fab are fused to the first and second chains of the second Fab, respectively. A linker may be present between the fused chains of the first and second Fab. The first and second chains of the first and second Fabs may be independently selected from a chain derived from the light chain of the Fab (VL-CL), a chain derived from the heavy chain of the Fab (VH-CH1), as long as each Fab comprises a VH, a VL, a CH1, and a CL. As an illustrative example, a chain derived from the light chain of the first Fab may be fused to a chain derived from the light chain of the second Fab. As another illustrative example, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab. As yet another illustrative example, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the light chain of a second Fab. In some double Fabs, both chains of the two Fabs are fused together. For example, a chain derived from the light chain of a first Fab can be fused to a chain derived from the light chain of a second Fab, and at the same time, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab. Alternatively, a chain derived from the light chain of a first Fab can be fused to a chain derived from the heavy chain of a second Fab, and at the same time, a chain derived from the heavy chain of a first Fab can be fused to a chain derived from the light chain of a second Fab. The fusion of two Fab chains may optionally include a linker. Suitable and preferred linkers include the upper hinge sequence (SEQ ID NO:54) or a glycine serine linker having up to about 20 amino acids, preferably up to 10 amino acids, or most preferably 10 amino acids, such as two repeats of GGGGS (SEQ ID NO:46).The glycine serine linker included in the dual Fab may have one or more repeats, for example 1, 2, 3 or 4 repeats, of GGS, GGGS (SEQ ID NO: 41), or GGGGS (SEQ ID NO: 46).

[0029] As used herein, "diabody" or "Db" refers to an antibody construct comprising two binding domains, which may be constructed using the heavy and light chains disclosed herein and by using the individual CDR regions disclosed herein. Typically, a diabody comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains of the same chain. Preferred linkers for this purpose include glycine-serine linkers having up to about 12 amino acids, preferably up to about 10 amino acids. A preferred glycine-serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 41), or GGGGS (SEQ ID NO: 46). A preferred linker is (GGS)2 (SEQ ID NO: 42). Another preferred linker is (GGS)3 (SEQ ID NO: 43). Thus, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigen-binding sites. A diabody may be formed by two separate polypeptide chains, each comprising a VH and a VL. Alternatively, all four variable domains may be contained in one single polypeptide chain, comprising two VH and two VL domains. In such a case, the diabody may also be referred to as a "single-chain diabody" or "scDb". Typically, an scDb comprises two chains of a non-single-chain diabody, fused together, preferably via a linker. A preferred linker for this purpose is a glycine serine linker, which preferably comprises about 15 to about 30 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 41), or GGGGS (SEQ ID NO: 46). Such linkers preferably contain 5, 6, 7, 8, 9 and / or 10 repeats of GGS, preferably (GGS)6 (SEQ ID NO: 44), or preferably (GGS)7 (SEQ ID NO: 45).In the polypeptide chain, the variable domains of the scDb can be arranged (from N-terminus to C-terminus) in the order VL-VH-VL-VH or VH-VL-VH-VL. Similarly, the spatial arrangement of the four domains in the tertiary / quaternary structure can also be in the order VL-VH-VL-VH or VH-VL-VH-VL. The term diabody does not exclude that further binding domains are fused to the diabody.

[0030] In the present invention, the definition of the term "antibody construct" includes monovalent, bivalent, and multivalent / multivalent constructs, i.e., monovalent, bivalent, trivalent, or even higher valency for the first and second targets to which the first and second binding domains bind, where the antibody construct is necessarily bispecific, i.e., comprises specificity for two different antigens or targets, as described elsewhere herein. The term "valency" means that a determined number of antigen binding domains are present in the antigen-binding protein. Natural IgG has two antigen binding domains and is bivalent. For example, the bispecific antibody construct of the present invention may comprise one, two, or more first antigen binding domains (A) against CD16A, as defined elsewhere herein, and one, two, or more second binding domains (B) against a second target present on the surface of a target cell, preferably a hematological target cell. Furthermore, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain, as well as molecules consisting of multiple polypeptide chains, which chains may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the above-identified antibodies and their variants or derivatives are described, inter alia, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0031] The term "bispecific" as used herein refers to an antibody construct that is "bispecific in nature", i.e., contains specificity for two different antigens or targets, but does not contain further specificity for a third or further antigen or target. Specifically, the bispecific antibody construct of the present invention contains a (first) binding domain that binds to one antigen or target (here, CD16A) and a (second) binding domain that binds to another antigen or target (here, a target cell surface antigen) that is not CD16A. Thus, the antibody construct defined in the present invention contains specificity for two different antigens or targets. For example, the first binding domain preferably binds to an extracellular epitope of one or more NK cell receptors of a species selected from humans, Macaca species, and rodent species, and the second binding domain preferably binds to a cellular epitope of a target cell surface antigen.

[0032] "CD16A" or "CD16a" refers to the activating receptor CD16A, also known as FcγRIIIA, expressed on the cell surface of NK cells. CD16A is an activating receptor that triggers the cytotoxic activity of NK cells. The amino acid sequence of human CD16A is shown in UniProt entry P08637 (version 212 of August 12, 2020) and SEQ ID NO: 50. The affinity of antibodies to CD16A is directly related to their ability to trigger NK cell activation, and thus the higher the affinity to CD16A, the lower the antibody dose required for activation. The antigen-binding site of the antigen-binding protein binds to CD16A, but preferably not to CD16B. For example, an antigen-binding site comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds to CD16A but not to CD16B may be provided by an antigen-binding site that specifically binds to an epitope of CD16A that includes amino acid residues of the C-terminal sequence SFFPPGYQ ​​(positions 201-208 of SEQ ID NO:50) of CD16A and / or residues G147 and / or Y158, which are not present in CD16B.

[0033] "CD16B" refers to the receptor CD16B, also known as FcγRIIIB, which is expressed on neutrophils and eosinophils. This receptor is anchored by glycosylphosphatidylinositol (GPI) and is understood not to induce any type of cytotoxic activity of CD16B-positive immune cells. The amino acid sequence of human CD16B is shown in UniProt entry O75015 (version 212 of August 12, 2020) and SEQ ID NO: 52.

[0034] The term "target cell" describes a cell or cell population that is the target of the mode of action exerted by the antibody construct of the present invention. This cell / cell population includes, for example, pathological cells that are eliminated or inhibited by binding these cells to effector cells via the antibody construct of the present invention. A preferred target cell is a cancer cell.

[0035] The term "CD16A shedding" or "shedding of CD16A" refers to the downregulation / down-regulation / degradation of FcγRIIIA expressed on the cell surface of immune effector cells, such as NK cells, following binding and activation of the immune effector cells by a CD16A binding domain, e.g., an antibody. "CD16A" shedding describes a proteolytic process that is typically mediated by a disintegrin metalloprotease (ADAM17) or membrane type 6 matrix metalloprotease (MMP25) and controls the cell surface density of said surface molecules on immune effector cells. "CD16A shedding" is known as activation-induced downregulation, as described, for example, in Romee at al., Blood, 2013, 121 (18):3599-3608; Peruzi et al., J. Immunol., 2013, 191:955-957; Goodier et al., Front. Immunol., 2016, 7:384; and Srpan et al., J. Cell. Biol., 2018, 217(9):3267-3283, and immune effector cell performance following CD16A shedding may then be impaired for several days.

[0036] The term "target cell surface antigen" refers to an antigenic structure expressed by a cell and present on the cell surface so as to be accessible to the antibody construct described herein. It may be a protein, preferably an extracellular portion of a protein, a peptide presented on the cell surface in association with MHC (including HLA-A2, HLA-A11, HLA-A24, HLA-B44, HLA-C4), or a carbohydrate structure, preferably a carbohydrate structure of a protein such as a glycoprotein. It is preferably a tumor-associated or tumor-restricted antigen. A target cell surface antigen specifically envisaged in the present invention is FOLR1, as defined elsewhere herein. It is envisaged that CD16A is not a target cell surface antigen of the present invention.

[0037] The term "antibody construct" of the present invention is bispecific in nature, i.e. it may not contain additional specificities resulting in an antibody construct such as a trispecific or tetraspecific antibody construct (the latter comprising four or more binding domains or the construct having more than four (e.g. five, six or more) specificities).

[0038] Considering that the antibody constructs defined in the present invention are bispecific, they do not exist in nature and they are significantly different from natural products. Thus, a "bispecific" antibody construct is an artificial hybrid antibody with two different binding sides with different specificities. Bispecific antibody constructs can be produced by various methods including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990).

[0039] The binding and variable domains (VH / VL) of the antibody construct of the present invention may or may not include a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" includes an amino acid sequence by which the amino acid sequence of one (variable and / or binding) domain of the antibody construct as defined herein and the amino acid sequence of another (variable and / or binding) domain of the antibody construct as defined herein are linked to each other. Peptide linkers may also be used to fuse one domain of the antibody construct as defined herein to another domain. In such a case, the peptide linker may also be called a "connector". Such connectors are preferably short linkers, preferably having a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5 nm or less, preferably about 4 nm or less, or even shorter. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers well known to the skilled person. An example of a connector is a glycine-serine linker or a serine linker, preferably comprising about 75 or less amino acids, preferably about 50 or less amino acids. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGGS sequences (SEQ ID NO: 46), such as (GGGGS)2 (SEQ ID NO: 47), (GGGGS)4 (SEQ ID NO: 48), or preferably (GGGGS)6 (SEQ ID NO: 49). Other illustrative examples of linkers are shown in SEQ ID NOs: 42-45. A preferred technical feature of such a peptide linker is that it does not contain any polymerization activity.

[0040] The antibody construct defined in the present invention is preferably "in vitro produced antibody construct". This term refers to the antibody construct according to the above definition, in which all or part of the variable region (e.g., at least one CDR) is produced by a method other than immune cell selection, such as in vitro phage display method, protein chip, or any other method that can test the binding ability of candidate sequence to antigen. Therefore, this term preferably does not include the sequence produced only by using genome rearrangement in animal immune cells. "Recombinant antibody" is an antibody produced by using recombinant DNA technology or genetic engineering.

[0041] The term "monoclonal antibody" (mAb) or monoclonal antibody construct as used herein refers to an antibody obtained from a population of substantially homogenous antibodies. That is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies against different determinants (or epitopes), monoclonal antibodies are highly specific and directed against a single antigenic side or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and therefore are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0042] To prepare monoclonal antibodies, any technique can be used that provides antibodies produced by continuous cell line cultures.For example, the monoclonal antibodies used can be produced by the hybridoma method first described by Koehler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567).Exemplary further techniques for producing human monoclonal antibodies include trioma technology, human B-cell hybridoma technology (Kozbor, Immunology Today 4 (1983), 72), and EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0043] In that case, the hybridomas can be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis to identify one or more hybridomas that produce antibodies that specifically bind to the designated antigen. Any form of the relevant antigen can be used as an immunogen, including recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof. Surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to epitopes of target cell surface antigens (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Another exemplary method of generating monoclonal antibodies includes screening protein expression libraries, such as phage display libraries or ribosome display libraries. Phage display methods are described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991), and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

[0044] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits, or rats). In one embodiment, the non-human animals contain at least a portion of human immunoglobulin genes. For example, it is possible to artificially create mouse strains that are defective in mouse antibody production and have large fragments of the human Ig (immunoglobulin) locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from these genes and with desired specificity can be produced and selected. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0045] Monoclonal antibodies can also be obtained from non-human animals and then modified, e.g., humanized, deimmunized, chimeric, etc., using recombinant DNA techniques known in the art. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with altered effector functions (see, e.g., U.S. Patent No. 5,648,260; Kontermann and Dubel (2010), supra; and Little (2009), supra).

[0046] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for an antigen during an immune response. Repeated exposure to the same antigen induces the host to produce antibodies with increasingly higher affinities. Like the natural prototype, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within the CDRs are introduced using irradiation, chemical mutagens, or error-prone PCR. Additionally, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range.

[0047] A preferred type of amino acid substitution variant of an antibody construct involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sides (e.g., 6-7 sides) are mutated to generate all possible amino acid substitutions on each side. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged inside each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sides for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of antigen-antibody complex to identify the contact points between binding domain and, for example, human target cell surface antigen.Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein.After making such variants, the panel of variants can be subjected to screening as described herein, and the antibody that has superior properties in one or more relevant assays can be selected for further development.

[0048] The monoclonal antibodies and antibody constructs of the present disclosure specifically include "chimeric" antibodies (immunoglobulins) and fragments of such antibodies, so long as they exhibit the desired biological activity, in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). As used herein, chimeric antibodies of interest include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences. Various approaches to making chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. Sci USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0049] Antibodies, antibody constructs, antibody fragments, or antibody variants can also be modified by specific deletion of human T cell epitopes (a method called "deimmunization"), for example by methods disclosed in WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). As described in WO 98 / 52976 and WO 00 / 34317, a computer modeling approach called "peptide threading" can be applied to detect potential T cell epitopes, and further, a database of human MHC class II binding peptides can be searched for motifs present in VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and therefore constitute potential T cell epitopes. Detected potential T cell epitopes can be eliminated by substituting a small number of amino acid residues in the variable domain, or preferably by single amino acid substitutions. Typically, conservative substitutions are made. In many, but not all cases, amino acids that occur frequently at certain positions in human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14: 14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK).These sequences can be used as a source of human sequences, e.g., for the framework regions and CDRs. Consensus human framework regions, e.g., as described in U.S. Patent No. 6,300,064, can also be used.

[0050] "Humanized" antibodies, antibody constructs, variants or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins derived from mostly human sequences, with minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable region (as well as CDRs) are replaced by residues from a hypervariable region (donor antibody) of a non-human (e.g., rodent) species, such as mouse, rat, hamster, or rabbit, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also include residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0051] Humanized antibodies or fragments thereof can be produced by replacing the sequences of Fv variable domains that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for producing humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; and US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences that code for all or part of the immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas that produce antibodies against a given target, as described above, and from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0052] Humanized antibodies may also be produced using transgenic animals such as mice that express human heavy and light chain genes but lack the ability to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace as many CDRs as are necessary for the binding of the humanized antibody to a given antigen.

[0053] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such altered immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and EP 239 400).

[0054] The terms "human antibody", "human antibody construct" and "human binding domain" include antibodies, antibody constructs and binding domains having antibody regions, e.g., variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) supra. A human antibody, antibody construct or binding domain as defined in the present invention may contain, for example, in the CDRs, particularly in CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). A human antibody, antibody construct or binding domain may have at least one, two, three, four, five or more positions substituted with an amino acid residue not encoded by a human germline immunoglobulin sequence. However, the definitions of human antibody, antibody constructs, and binding domains used herein also contemplate "fully human antibodies" that include only non-artificial and / or genetically altered human sequences of antibodies, as may be obtained by using techniques or systems such as Xenomouse. Preferably, a "fully human antibody" does not include amino acid residues that are not encoded by human germline immunoglobulin sequences.

[0055] In some embodiments, the antibody construct as defined herein is an "isolated" or "substantially pure" antibody construct. When used to describe the antibody constructs disclosed herein, "isolated" or "substantially pure" means that the antibody construct has been identified, separated, and / or recovered from components present in its production environment. Preferably, the antibody construct is free or substantially free from all other components from its production environment. Contaminating components present in its production environment, e.g., from transfected recombinant cells, are substances that would typically interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody construct may, for example, comprise at least about 5% or at least about 50% by weight of the total protein in a given sample. It is understood that an isolated protein may comprise 5% to 99.9% by weight of the total protein content, depending on the environment. The polypeptide may be produced at significantly higher concentrations using inducible promoters or high expression promoters to produce at high concentration levels. This definition includes the production of antibody constructs in a wide variety of organisms and / or host cells known in the art. In a preferred embodiment, the antibody construct is purified (1) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. However, usually, isolated antibody constructs are prepared by at least one purification step.

[0056] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may contain proteinaceous and non-proteinaceous parts (e.g., chemical linkers or chemical cross-linking agents, such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) contain two or more amino acids linked together via covalent peptide bonds (resulting in a chain of amino acids).

[0057] The term "polypeptide" or "polypeptide chain" as used herein refers to a group of molecules that usually consists of more than 30 amino acids. The terms "peptide", "polypeptide" and "protein" also refer to naturally modified peptides / polypeptides / proteins that are modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation. As referred to herein, a "peptide", "polypeptide" or "protein" may also be chemically modified, for example pegylated. Such modifications are well known in the art and are described herein below. The above modifications (glycosylation, pegylation, etc.) also apply to the antibody constructs of the present invention.

[0058] Preferably, the binding domain that binds to CD16A and / or the binding domain that binds to target cell surface antigen is a human binding domain.Antibody and antibody constructs that comprise at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have variable and / or constant regions of non-human, e.g., rodents (e.g., mice, rats, hamsters, or rabbits).The presence of such rodent-derived proteins may cause rapid clearance of the antibody or antibody construct, or may result in the patient developing an immune response against the antibody or antibody construct.To avoid the use of rodent-derived antibodies or antibody constructs, human antibodies / antibody constructs or fully human antibodies / antibody constructs can be made by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0059] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline represents a powerful approach for elucidating the functional components of very large or loosely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in the induction and progression of disease.

[0060] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B-cell development. Furthermore, such a strategy may provide an ideal source for the production of fully human monoclonal antibodies (mAbs), a key milestone to realizing the promise of antibody therapy in human diseases. Fully human antibodies or fully human antibody constructs are expected to minimize the immunogenic and allergic responses inherent to mouse or mouse-derivatized mAbs, and thus improve the efficacy and safety of the administered antibodies / antibody constructs. The use of fully human antibodies or fully human antibody constructs may be expected to provide substantial advantages in the treatment of chronic and recurrent human diseases that require repeated compound administration, such as inflammation, autoimmunity, and cancer.

[0061] One approach towards this goal has been to artificially create mouse strains deficient in mouse antibody production, carrying large fragments of the human Ig loci, in the hope that the mice will produce a large repertoire of human antibodies in the absence of mouse antibodies. The large human Ig fragments would maintain the large variable gene diversity and proper regulation of antibody production and expression. By taking advantage of the mechanisms used by mice for antibody diversification and selection and the lack of immune tolerance to human proteins, the recapitulated human antibody repertoires in these mouse strains should yield high affinity antibodies to any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with the desired specificity could be easily produced and selected. This general strategy was demonstrated in conjunction with the creation of the first XenoMouse mouse strains (see Green et al. Nature Genetics 7:13- 21 (1994)). XenoMouse strains were engineered with yeast artificial chromosomes (YACs) containing 245 kb and 190 kb sized germline-configured fragments of the human heavy and kappa light chain loci, respectively, that contain the core sequences of the variable and constant regions. The human Ig-containing YACs proved to be compatible with the mouse system for both antibody rearrangement and expression, and had the ability to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development, produce full human antibodies of the mature human repertoire, and generate antigen-specific human mAbs. These results also suggested that a substantially complete repertoire characteristic of the human humoral response to infection and immunization could be recapitulated by introducing larger portions of the human Ig loci, including a larger number of V genes, additional regulatory elements, and human Ig constant regions. Green et al.'s work has recently been extended to the introduction of more than 80% of the human antibody repertoire by introducing megabase-sized germline-configured YAC fragments of the human heavy and kappa light chain loci, respectively.See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. patent application Ser. No. 08 / 759,620.

[0062] The generation of XenoMouse mice is described in U.S. Patent Application Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, and 08 / 463,191. , 08 / 462,837, 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752, and 08 / 759,620; as well as U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181, and 5,939,598, and as further discussed and described in Japanese Patent Nos. 3068180 B2, 3068506 B2, and 3068507 B2. See also Mendez et al. Nature Genetics 15:146-156(1997) and Green and Jakobovits J. Exp. Med. 188:483-495(1998), EP0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0063] In an alternative approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a γ constant region) form a construct for insertion into an animal. This approach is disclosed in U.S. Patent No. 5,545,807 to Surani et al. and U.S. Patent Nos. 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; and 5,878,220, all of which are incorporated herein by reference. Nos. 5,874,299; and 6,255,458 to Krimpenfort and Berns, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Berns et al., U.S. Pat. Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and U.S. Pat. and U.S. Patent Application Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International. See also EP 0546073 B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884, and U.S. Pat. No. 5,981,175.See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0064] Kirin has also demonstrated the production of human antibodies from mice, where large chromosome fragments or entire chromosomes were introduced by microcell fusion. See European Patent Application Nos. 773288 and 843961. Xenerex Biosciences is developing a technology for potentially producing human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes, such as B cells and / or T cells. The mice are then immunized with an antigen, and the mice can generate an immune response against the antigen. See U.S. Patent Nos. 5,476,996; 5,698,767; and 5,958,765.

[0065] The human anti-mouse antibody (HAMA) response has prompted industry efforts to prepare chimeric or otherwise humanized antibodies. However, it is expected that some human anti-chimeric antibody (HACA) responses will be observed, especially during long-term or multiple use of antibodies. It would therefore be desirable to provide an antibody construct that includes a human binding domain for a target cell surface antigen and a human binding domain for CD16 to eliminate the concerns and / or impact of HAMA or HACA responses.

[0066] The term "epitope" refers to the side on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and thus the term epitope is sometimes referred to herein as an "antigenic structure" or "antigenic determinant." Thus, the binding domain is the "antigen interaction site." It is also understood that the binding / interaction defines the "specific recognition."

[0067] An "epitope" can be formed from both contiguous amino acids or non-contiguous amino acids adjacent to each other due to tertiary folding of a protein. A "linear epitope" is an epitope in which the primary amino acid sequence constitutes the recognized epitope. Typically, a linear epitope contains at least 3 or at least 4, and more usually at least 5 or at least 6 or at least 7, e.g., about 8 to about 10, amino acids in a unique sequence.

[0068] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary sequence of amino acids that constitute the epitope is not the only defining element of the recognized epitope (e.g., the primary sequence of amino acids is not necessarily recognized by the binding domain). Typically, conformational epitopes contain more amino acids than linear epitopes. With respect to the recognition of conformational epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the present invention, the antigenic structure for one of the binding domains is contained within the target cell surface antigen protein). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are in close proximity, thereby allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0069] The interaction of a binding domain with an epitope or an epitope-containing region implies that the binding domain exhibits appreciable affinity for the epitope / epitope-containing region on a particular protein or antigen (here, CD16A and / or a target cell surface antigen, respectively), and typically does not exhibit significant reactivity with proteins or antigens other than CD16a, other antigens on the surface of immune effector cells, and / or target cell surface antigens. "Appreciable affinity" refers to a degree of affinity that is greater than or equal to about 10. -6 M(KD) or stronger. Preferably, the binding is with a binding affinity of about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11~10 -9 M. Whether a binding domain specifically reacts or binds to a target can be readily tested, inter alia, by comparing the reaction of the binding domain with the target protein or antigen to the reaction of the binding domain with other proteins or antigens, such as CD16A and / or target cell surface antigens.

[0070] The terms "essentially / substantially does not bind" or "cannot bind" mean that the binding domain of the present invention does not bind to proteins or antigens other than CD16A and / or target cell surface antigens, etc., i.e., when the binding to CD16A and / or target cell surface antigens, etc., is set to 100%, it does not show more than 30% reactivity to proteins or antigens other than CD16A and / or target cell surface antigens, etc., preferably 20% or less, more preferably 10% or less, and particularly preferably 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[0071] Specific binding is believed to be influenced by specific motifs in the amino acid sequences of the binding domain and antigen. Thus, binding is achieved as a result of their primary, secondary, and / or tertiary structures, and as a result of secondary modifications of these structures. The specific interaction of an antigen interaction side with its specific antigen can result in the simple binding of said side to the antigen. Furthermore, the specific interaction of an antigen interaction side with its specific antigen can alternatively or additionally initiate a signal, for example, by inducing a conformational change of the antigen, oligomerization of the antigen, etc.

[0072] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is involved in determining the specificity and binding affinity of an individual antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen-binding side.

[0073] The variability is not evenly distributed throughout the variable domain of an antibody, but is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domains are called "framework" regions (FRMs or FRs), which provide a scaffold in three-dimensional space for the six CDRs to form the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4), most often adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect and in some cases form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding side (Kabat et al., supra).

[0074] The term "CDR" and its plural "CDRs" refer to the complementarity determining regions, three of which make up the binding characteristics of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which make up the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). The CDRs contain most of the residues responsible for the specific interactions of the antibody with the antigen and thus contribute to the functional activity of the antibody molecule: they are the main determinants of antigen specificity.

[0075] The precise boundaries and lengths of CDRs are varied by various classification and numbering systems. Thus, CDRs may be referred to using Kabat, Chothia, contact definitions, or any other boundary definitions, including the numbering systems described herein. Although the boundaries are different, each of these systems has some overlap in what constitutes the so-called "hypervariable regions" in variable sequences. Thus, the definitions of CDRs based on these systems may differ in terms of length and boundary regions with adjacent framework regions. See, for example, Kabat (an approach based on cross-species sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol, 1987, 196: 901-917; and MacCallum et al., J. Mol. Biol, 1996, 262: 732). Yet another standard for characterizing antigen-binding side is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). To the extent that two residue identification techniques define overlapping but not identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

[0076] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation that an antigen-binding (CDR) loop adopts. Comparative structural studies have found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized based on the torsion angle of the polypeptide backbone. Thus, similar loops between antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability found in most of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800). Furthermore, there is a relationship between the adopted loop structure and its surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop and within the conserved framework (i.e., outside the loop), and therefore assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0077] The term "canonical structure" may also include considerations of the linear sequence of an antibody, for example as listed by Kabat (Kabat et al., supra). The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner, and is the preferred scheme applied in the present invention as noted elsewhere herein. Additional structural considerations may also be used to define the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering can be accounted for by the Chothia et al. numbering system and / or can be revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence may be placed into a canonical class that allows, among other things, the identification of an appropriate chassis sequence (e.g., based on a desire to include various canonical structures in a library). The Kabat numbering for the amino acid sequence of an antibody and structural considerations as described by Chothia et al., supra, and their correlation to interpret the canonical appearance of antibody structures are described in the literature. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. A comprehensive reference in immunoinformatics is the IMGT (International ImMunoGenetics Information System) three-dimensional (3D) structure database (Ehrenmann et al., 2010, Nucleic Acids Res., 38, D301-307). IMGT / 3D Structure DB structural data is obtained from the Protein Data Bank (PDB) and annotated based on the IMGT classification concepts using internal tools.Therefore, the IMGT / 3D structure DB provides the closest genes and alleles expressed in the amino acid sequences of the 3D structures by aligning them with the IMGT domain reference directory. In the case of antigen receptors, this directory includes the amino acid sequences of the domains encoded by translation of the constant genes and germline variable and binding genes. The CDR regions of the amino acid sequences of the present inventors are preferably revealed by using the IMGT / 3D structure database.

[0078] The CDR3 of the light chain and especially the CDR3 of the heavy chain may constitute the most important determinant for antigen binding within the variable region of the light and heavy chains. In some antibody constructs, the heavy chain CDR3 is likely to constitute the main contact area between the antigen and the antibody. An in vitro selection scheme that changes only the CDR3 can be used to change the binding properties of the antibody or to determine which residues contribute to antigen binding. Thus, the CDR3 is typically the largest source of molecular diversity within the antibody binding side. For example, H3 can be as short as just 2 amino acid residues or more than 26 amino acids.

[0079] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to VH is usually called CH1. The constant ("C") domain is not directly involved in antigen binding, but exhibits various effector functions such as antibody-dependent cell-mediated cytotoxicity and complement activation. The Fc region of an antibody is contained within the heavy chain constant domain and can interact with Fc receptors located on the cell surface, for example.

[0080] After assembly and somatic mutation, antibody gene sequences show high diversity, and these diversified genes span 10 10It has been estimated that each of the nucleotide sequences encodes a number of different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. These sequences can result from in vivo rearrangement of V, D, and J segments of heavy chains and V and J segments of light chains. Alternatively, these sequences can be generated from cells in which rearrangement occurs, e.g., in response to in vitro stimulation. Alternatively, some or all of these sequences can be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e.g., U.S. Pat. No. 5,565,332. A repertoire can include only one sequence or can include multiple sequences, including sequences in a genetically diverse collection.

[0081] The antibody constructs defined in the present invention may also comprise additional domains, for example, useful for the isolation of the molecule or related to the adapted pharmacokinetic profile of the molecule. The domains useful for the isolation of the antibody constructs may be selected from peptide motifs or secondarily introduced moieties that can be captured in an isolation method, for example, an isolation column. Non-limiting embodiments of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and variants thereof (e.g., StrepII tags), and His tags. All of the antibody constructs disclosed herein that feature identified CDRs may comprise a His tag domain, commonly known as a repeat of consecutive His residues, preferably 5 and more preferably 6 His residues (hexahistidine), in the amino acid sequence of the molecule. The His tag may, for example, be located at the N-terminus or C-terminus of the antibody construct, preferably at the C-terminus. Most preferably, the hexahistidine tag is linked to the C-terminus of the antibody construct according to the invention by a peptide bond. Furthermore, the conjugate system PLGA-PEG-PLGA may be combined with the polyhistidine tag for sustained release applications and improved pharmacokinetic profiles.

[0082] Amino acid sequence modifications of the antibody constructs described herein are also contemplated, as long as the minimal structural constraints of the first binding domain of the antibody construct of the present invention are maintained.For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody construct.Amino acid sequence variants of the antibody constructs are prepared by introducing appropriate nucleotide changes into the antibody construct nucleic acid or by peptide synthesis.Any of the amino acid sequence modifications described below should result in an antibody construct that still retains the desired biological activity (i.e., binding to CD16A and / or target cell surface antigen) of the unmodified parent molecule.

[0083] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, e.g., an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may also be used, if desired. Generally, amino acids can be grouped as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0084] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody construct. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processing of the antibody construct, for example, changing the number or location of glycosylation sites.

[0085] For example, particularly in the second binding domain of the antibody construct, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted, or deleted in each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be inserted, substituted, or deleted in each FR. Preferably, amino acid sequence insertions into the antibody construct include amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertional variants of the antibody construct defined in the present invention include the fusion of an enzyme or a polypeptide to the N- or C-terminus of the antibody construct.

[0086] The most interesting sites for substitution mutagenesis include (but are not limited to) the CDRs of the heavy and / or light chains of the second binding domain, especially the hypervariable regions, but also contemplate changing the FRs of the heavy and / or light chains.These substitutions are preferably conservative substitutions as described herein.Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted in the CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be substituted in the framework region (FR).For example, if the CDR sequence contains 6 amino acids, it is envisaged that 1, 2, or 3 of these amino acids are substituted. Similarly, if a CDR sequence comprises 15 amino acids, it is envisaged that 1, 2, 3, 4, 5 or 6 of these amino acids may be substituted.

[0087] A useful method for identifying specific residues or regions of an antibody construct that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" and is described by Cunningham and Wells in Science, 244: 1081-1085 (1989). Herein, a residue or target residues within the antibody construct are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the epitope.

[0088] The amino acid positions that show functional sensitivity to these substitutions are then more precisely defined by introducing another or other variant at or to the substitution site. Thus, the site or region for introducing the amino acid sequence variant is predetermined, but the nature of the mutation itself does not have to be predetermined. For example, to analyze or optimize the effect of the mutation at a given site, alanine scanning or random mutagenesis may be performed on the target codon or region, and the expressed antibody construct variants are screened for the optimal combination of desired activity. Techniques for making substitution mutations at a predetermined site in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using an assay for antigen binding activity, such as for binding to CD16A and / or target cell surface antigens.

[0089] In general, when amino acids are replaced in one or more or all of the CDRs of the heavy and / or light chain, the resulting "replaced" sequence is preferably at least 60% or at least 65% identical to the "original" CDR sequence, more preferably at least 70% or at least 75%, even more preferably at least 80% or at least 85%, and particularly preferably at least 90% or at least 95% identical. This means that the degree to which the original sequence is identical to the "replaced" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids is preferably at least 80% identical to its replaced amino acid sequence in order for at least one amino acid to be replaced. Thus, the CDRs of an antibody construct may have different degrees of identity to their replaced sequences, for example, CDRL1 may have at least 80% identity and CDRL3 may have at least 90% identity.

[0090] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions) is envisaged, as long as the antibody construct retains the ability to bind, for example, to CD16A via the first binding domain and / or to a target cell surface antigen via the second binding domain, and / or the CDR of the second binding domain has identity to the sequence replaced at that time (at least 60% or at least 65%, more preferably at least 70% or at least 75%, even more preferably at least 80% or at least 85%, and particularly preferably at least 90% or at least 95% identical to the "original" CDR sequence).

[0091] Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. If such substitutions result in altered biological activity, then more extensive changes, referred to as "exemplary substitutions" in Table 1 or further described below for amino acid classes, can be introduced and the products screened for the desired characteristics.

[0092] (Table 1) Amino acid substitutions TIFF2024543828000001.tif107128

[0093] Substantial modification of the biological properties of the antibody constructs of the invention is achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone in the substituted region, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0094] Non-conservative substitutions involve exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody construct may be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).

[0095] For amino acid sequences, sequence identity and / or similarity may be determined by inspection or using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using the default settings. Preferably, the percent identity is calculated by FastDB using the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0096] An example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignments to generate a multiple sequence alignment from a group of related sequences. PILEUP can also plot a tree showing the clustering relationships used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0097] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, taken from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set with the following values: overlap span=1, overlap ratio=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values, determined by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched, however these values ​​can be adjusted to increase sensitivity.

[0098] Another useful algorithm is Gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9; the two-hit method for triggering ungapped extensions imposes a cost of 10+k on the gap length k; Xu is set to 16, and Xg is set to 40 in the database search stage and 67 in the output stage of the algorithm. Gapped alignments are triggered by a score equivalent to approximately 22 bits.

[0099] Generally, the amino acid homology, similarity or identity between each variant CDR or VH / VL sequence is at least 60% with respect to the sequence shown herein, and more typically, the homology or identity is preferably increased to at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and approximately 100%. In a similar manner, the "nucleic acid sequence identity percentage (%)" for the nucleic acid sequence of the binding protein specified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody construct. In one specific method, the BLASTN module of WU-BLAST-2 is used with default parameters, and the overlap span and overlap ratio are set to 1 and 0.125, respectively.

[0100] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequences encoding the individual variant CDRs or VH / VL sequences and the nucleotide sequences set forth herein will be at least 60%, more typically the homology or identity will be at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and preferably close to 100%. Thus, a "variant CDR" or "variant VH / VL region" is one that has a designated homology, similarity or identity to a parent CDR / VH / VL as defined in the present invention and has a biological function, including but not limited to, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0101] In one embodiment, the identity percentage of the antibody construct according to the present invention to human germline is 70% or more or 75% or more, more preferably 80% or more or 85% or more, even more preferably 90% or more, and most preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, or even 96% or more. Identity to human antibody germline gene products is considered an important feature to reduce the risk that the therapeutic protein will cause an immune response to the drug in the patient being treated. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36 (2005) 3-10) demonstrate that reducing the non-human portion of the drug-antibody construct reduces the risk of generating anti-drug antibodies in the patient being treated. A comparison of a comprehensive number of clinically evaluated antibody drugs and their respective immunogenicity data shows that humanization of the V region of an antibody tends to make the protein less immunogenic (5.1% of patients on average) compared to antibodies with unmodified non-human V regions (23.59% of patients on average). Therefore, for V region-based protein therapeutics in the form of antibody constructs, a high degree of identity to human sequences is desirable. For this purpose of measuring germline identity, the V region of the VL can be aligned with the amino acid sequences of human germline V and J segments (vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the percentage of amino acid sequences can be calculated by dividing the identical amino acid residues by the total number of amino acid residues in the VL. The same can be done for the VH segment (vbase.mrc-cpe.cam.ac.uk / ), except that the VH CDR3 may be omitted due to its high diversity and lack of existing human germline VH CDR3s to align with. Recombinant techniques can then be used to increase sequence identity to human antibody germline genes.

[0102] As used herein, the term "CD123" refers to the differentiation cluster 123 protein, also known as the "interleukin-3 receptor", and is an antigenic determinant found on multipotent progenitor cells, basophils, and plasmacytoid dendritic cells (pDCs) and some conventional dendritic cells (cDCs) in peripheral blood mononuclear cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD123 can be found under UniProt / Swiss-Prot accession number P26951, and the nucleotide sequence encoding human CD123 can be found under accession number NM_002183. As used herein, "CD123" includes proteins that contain mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD123. CD123 is a biomarker in hematolymphoid malignancies (El Achi et al., Cancers (Basel)., 2020, 12(11): 3087), and is specifically expressed across acute myeloid leukemia (AML) subtypes, including leukemic stem cells (Seattle Genetics Initiates Phase 1 Trial of SGN-CD123A for Patients with Relapsed or Refractory Acute Myeloid Leukemia Sept 2016).

[0103] As used herein, the term "FOLR1", also known as "folate receptor 1", "folate receptor alpha", "folate binding protein (FBP)" or "ovarian cancer-associated antigen", is a receptor with high affinity for folic acid and some reduced folic acid derivatives, and mediates the delivery of 5-methyltetrahydrofolate to the interior of cells (Henderson, Annu Rev Nutr. 1990; 10:319-35). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human FOLR1 can be found under UniProt / Swiss-Prot accession number P15328, and the nucleotide sequence encoding human FOLR1 can be found under accession number BT007158.1. FOLR1 can be overexpressed by numerous epithelial-derived tumors, including ovarian, breast (especially TNBC), renal, lung, colorectal (CRC), renal (especially ccRCC), pancreatic (especially PDAC), endometrium, and brain (Scaranti et al., Nat Rev Clin Oncol. 2020, 17(6): 349-359). Due to its high expression in some tumors, FOLR1 is an attractive therapeutic target for the development of anticancer drugs to limit toxic side effects to off-target tissues. As used herein, "FOLR1" includes proteins containing mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type FOLR1.

[0104] As used herein with respect to FOLR1, "overexpress" or "overexpression" refers to the increase in the amount of FOLR1 on the surface of a cell in a pathological situation, such as a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder, when compared with the expression of FOLR1 on the surface of a cell of the same tissue in a non-pathological situation.For example, as used herein with respect to FOLR1, "overexpress" or "overexpression" refers to the increase in the amount of FOLR1 on the surface of a neoplastic cell, when compared with the expression of FOLR1 on the surface of a non-neoplastic cell of the same tissue.

[0105] The term "immune effector cell" as used herein may refer to any white blood cell or precursor that is involved in protecting the body against, for example, cancer, diseases caused by infectious agents, foreign substances, or autoimmune reactions. For example, immune effector cells include B lymphocytes (B cells), T lymphocytes (T cells, including CD4+T cells and CD8+T cells), NK cells, NKT cells, monocytes, macrophages, dendritic cells, mast cells, granulocytes, such as neutrophils, basophils, and eosinophils, innate lymphoid cells (ILCs, including ILC-1, ILC-2, and ILC-3), or any combination thereof. Preferably, the term immune effector cell refers to NK cells, ILC-1 cells, NKT cells, macrophages, monocytes, and / or T cells, such as CD8+T cells or γδT cells.

[0106] Natural killer (NK) cells are CD56+CD3- large granular lymphocytes that can kill virus-infected and transformed cells and constitute an essential cell subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells can generate cytotoxicity against tumor cells without the need for prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they avoid potentially lethal complications such as cytokine storm (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target and off-tumor effects.

[0107] Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for approximately 1-3 days and then typically migrate into tissues throughout the body. Monocytes constitute 3-8% of the white blood cells in the blood. Within tissues, monocytes mature into various types of macrophages in various anatomical locations. Monocytes have two main functions in the immune system: (1) recruiting resident macrophages and dendritic cells under normal conditions, and (2) in response to inflammatory signals, monocytes can rapidly migrate (approximately 8-12 hours) to sites of infection in tissues and divide / differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears based on their large bilobed nuclei.

[0108] Macrophages are potent effectors of the innate immune system and can perform at least three distinct antitumor functions: phagocytosis, cell-mediated cytotoxicity, and antigen presentation to orchestrate adaptive immune responses. Whereas T cells require antigen-dependent activation via the T cell receptor or chimeric immune receptors, macrophages can be activated in a variety of ways. Direct macrophage activation is antigen-independent and relies on mechanisms such as pathogen-associated molecular pattern recognition by Toll-like receptors (TLRs). Immune complex-mediated activation is antigen-dependent but requires the presence of antigen-specific antibodies and the absence of inhibitory CD47-SIRPa interactions.

[0109] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), based on the presence of the T cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus gland (although some also mature in the tonsils). There are several subsets of T cells, each with distinct functions.

[0110] T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells divide rapidly and secrete small proteins called cytokines that regulate or assist active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0111] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigens bound to MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, thereby preventing autoimmune diseases.

[0112] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has cleared. Upon re-exposure to the cognate antigen, memory T cells rapidly proliferate into large numbers of effector T cells, thus providing the immune system with a "memory" of past infections. Memory cells can be either CD4+ or CD8+. Typically, memory T cells express the cell surface protein CD45RO.

[0113] Regulatory T cells (Treg cells), previously known as suppressor T cells, are crucial for the maintenance of immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of the immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two main classes of CD4+ Treg cells have been described: natural Treg cells and adaptive Treg cells.

[0114] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) act as a bridge between the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0115] As used herein, the term "half-life prolonging domain" refers to a portion that increases the serum half-life of an antibody construct. The half-life prolonging domain may comprise a portion of an antibody, such as the Fc portion of an immunoglobulin, the hinge domain, the CH2 domain, the CH3 domain, and / or the CH4 domain. Although not optimal, the half-life prolonging domain may also comprise elements that are not contained in an antibody, such as an albumin-binding peptide, an albumin-binding protein, or transferrin, to name just a few. It is preferred that the half-life prolonging domain does not have an immunomodulatory function. When the half-life prolonging domain comprises the hinge domain, the CH2 domain, and / or the CH3 domain, it is preferred that the half-life prolonging domain does not essentially bind to Fc receptors. This can be achieved, for example, by "silencing" the Fcγ receptor binding domain.

[0116] As used herein, "silencing" of an Fc receptor binding domain or an Fcγ receptor binding domain refers to any modification that reduces the binding of the CH2 domain to an Fc receptor, particularly an Fcγ receptor. Such modifications can be made by substitution and / or deletion of one or more amino acids involved in Fc(γ) receptor binding. Such mutations are well known in the art and are described, for example, by Saunders (2019, Front. Immunol. 10:1296). For example, the mutation can be located at any one of positions 233, 234, 235, 236, 237, 239, 263, 265, 267, 273, 297, 329, and 331. Examples of such mutations are: deletion of Glu233→Pro, Glu233, Leu234→Phe, Leu234→Ala, Leu234→Gly, Leu234→Glu, Leu234→Val, deletion of Leu234, Leu235→Glu, Leu235→Ala, Leu235→Arg, Leu235→Phe, deletion of Leu235, deletion of Gly236, Gly237→Ala, Ser239→Lys, Val263→Leu, Asp265→Ala, Ser267→Lys, Val273→Glu, Asn297→Gly, Asn297→Ala, Lys332→Ala, Pro329→Gly, Pro331→Ser, and combinations thereof. Preferably, such modifications include one or both of Leu234→Ala and Leu235→Ala (also known as "LALA" mutations). Preferably, such modifications further include Pro329→Gly mutations, also known as "LALA-PG" mutations (Leu234→Ala, Leu235→Ala, and Pro329→Gly). Preferably, such modifications include one, two, or three of the mutations Leu234→Phe, Leu235→Glu, and Asp265→Ala, more preferably all three of these mutations. The combination of Leu234→Phe, Leu235→Glu, and Asp265→Ala is a preferred modification in the present invention, also known as "FEA" mutations. Preferably, such modifications further include Asn297→Gly.Such preferred modifications include the mutations Leu234→Phe, Leu235→Glu, Asp265→Ala, and Asn297→Gly.

[0117] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of a formulation to the body, isolated tissues, or cells from a patient having a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, for the purpose of curing, healing, mitigating, alleviating, altering, curing, ameliorating, enhancing, or affecting the disease, the symptom of a disease, or the predisposition to a disease.

[0118] The term "amelioration" as used herein refers to any improvement in the disease state of a patient with a tumor or cancer or metastatic cancer, as defined elsewhere herein, by administration of an antibody construct according to the present invention to a subject in need thereof. Such improvement may also be seen as a slowing or halting of progression of the patient's tumor or cancer or metastatic cancer.

[0119] The term "disease" refers to any condition that would benefit from treatment with the antibody constructs or pharmaceutical compositions described herein, including chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disease in question.

[0120] The term "neoplastic disease" or "tumor disorder" refers to a disease characterized by the presence or development of a tumor. A "tumor" is an abnormal proliferation of cells that serves no purpose. Tumors are divided into benign, i.e. non-malignant, and malignant, i.e. cancerous tumors / cancers. Benign tumors grow slowly, have clear borders, and do not invade nearby tissues / spread to other parts of the body, whereas malignant tumors grow rapidly, have irregular borders, and often invade surrounding tissues and spread to other parts of the body, called metastases (Patel, JAMA Oncol, 2020, 6(9):1488).

[0121] "Tumors of the hematopoietic and lymphatic tissues" are tumors that affect the blood, bone marrow, lymphatic fluids, and lymphatic system (Vardiman et al.; Blood, 2009, 114(5): 937-51).

[0122] "Solid tumor" refers to a new growth of tissue, i.e. an abnormal mass of tissue that does not usually contain cysts or liquid areas. They can occur anywhere in the body. Solid tumors can be benign (not cancerous) or malignant (cancerous). A tumor is said to be benign if it does not grow through (invade) the surrounding tissue and does not form secondary tumors (metastasis). On the other hand, malignant solid tumors can destroy the surrounding tissue and spread to other parts of the body. Malignant neoplasms are also known as cancer. It is particularly envisaged that "solid tumor" in the present invention covers malignant solid tumors selected from the group consisting of brain tumor, head and neck cancer, lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, small intestine cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, thyroid cancer, skin cancer, melanoma, and sarcoma, preferably ovarian cancer, breast cancer, kidney cancer, lung cancer, colorectal cancer, and brain tumor. A "neoplasm" is an abnormal growth of tissue, usually, but not always, forming a mass. When it forms a mass, it is also commonly referred to as a "tumor." Neoplasms or tumors can be benign, potentially malignant (precancerous), or malignant. Malignant neoplasms are commonly referred to as cancers. Malignant neoplasms usually invade and destroy surrounding tissues and may form metastases, i.e., spread to other parts, tissues, or organs of the body. Thus, the term "metastatic cancer" encompasses metastases to other tissues or organs other than those of the original tumor. For the purposes of the present invention, they are also encompassed by the terms "tumor" or "cancer."

[0123] "Proliferative disorders" are characterized by excessive proliferation of cells and cell-matrix turnover, as described, for example, in Sporn and Harris, The American Journal of Medicine, 1981, 70(6): 1231-1236.

[0124] A "viral disease" is a disease caused by the invasion of a pathogenic virus, in which infectious viral particles (virions) attach to and enter susceptible cells (Taylor et al., PNAS, 2021, 106(42): 17046-17051). Viruses can have various structural features, including, among others, double-stranded DNA families (such as Adenoviridae, Papillomaviridae, and Polyomaviridae), partially double-stranded DNA viruses (such as Hepadnaviridae), single-stranded DNA viruses (such as Parvoviridae), positive single-stranded RNA families (such as Astroviridae, Caliciviridae, and Picornaviridae), three non-enveloped types, such as virions, partially double-stranded DNA viruses (such as Hepadnaviridae), single-stranded DNA viruses (such as Parvoviridae), and three non-enveloped types, such as Astroviridae, Caliciviridae, and Picornaviridae. These families can include the four enveloped types, such as Coronaviridae, Flaviviridae, Retroviridae, and Togaviridae, negative single-stranded RNA families, such as Arenaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, and Rhabdoviridae, as well as viruses with double-stranded RNA genomes.

[0125] An "immunologic disorder" is a disease or condition caused by a malfunction of the immune system, and includes allergies, asthma, autoimmune diseases, autoinflammatory syndromes, and immune deficiency syndromes.

[0126] The term "subject in need" or a subject "in need of treatment" includes those already with a disorder or disease, as well as those in which a disorder or disease is to be prevented. A subject in need or "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0127] The term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise one or more of the antibody constructs of the present invention, preferably in a therapeutically effective dose. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutical effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, and / or adjuvants. Acceptable components of the composition are preferably non-toxic to the recipient at the dosages and concentrations used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0128] "Pharmaceutically acceptable carrier" refers to any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, such as phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media, and coatings, that are compatible with pharmaceutical administration, particularly parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known conventional methods.

[0129] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially halt the disease and its complications in a patient already suffering from the disease. The amount or dose effective for this use depends on the condition to be treated (indication), the antibody construct delivered, the context and purpose of the treatment, the severity of the disease, previous therapies, the patient's clinical history and response to the therapeutic agent, the route of administration, the size (weight, body surface, or organ size) and / or condition (age and general health) of the patient, and the general state of the patient's own immune system. The appropriate dose can be administered to the patient once or over a series of administrations, and can be adjusted according to the judgment of the attending physician to obtain the optimal therapeutic effect.

[0130] The term "kit" as used herein means two or more components, one of which corresponds to an antibody construct, pharmaceutical composition, vector, or host cell of the present invention, packaged together in a container, receptacle, or other. Thus, a kit can be described as a set of products and / or instruments sufficient to accomplish a particular goal that can be sold as a single unit.

[0131] Detailed Description Innate immune effector cells (e.g., natural killer (NK) cells, macrophages) are activated by a complex mechanism of several different signaling pathways. NK cells and macrophages can be exploited in cancer immunotherapy by redirecting NK cell lysis or macrophage-induced phagocytosis to tumor cells through stimulation of the activating antigen CD16A (FcγRIIIA) expressed on their cell surface. CD16A associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adaptor CD3ζ chain to initiate a signaling cascade that ultimately mediates ADCC and antibody-dependent cellular phagocytosis (ADCP) in NK cells and macrophages, respectively. Signaling through CD16A has been reported to be sufficient to activate the cytotoxic activity of NK cells.

[0132] However, for example in the context of immunosuppressive tumors, stimulation of the microenvironment via CD16A may be suboptimal or insufficient for maximal antitumor activity. Thus, targeting additional surface antigens on NK cells, macrophages, or other immune cell types, such as but not limited to CD8+αβT cells or γδT cells, may improve or maximize antitumor activity.

[0133] However, even though it is known that activation-induced downregulation / shedding of CD16, especially CD16A, on activated NK cells impairs their activity, thereby reducing NK cell responses at individual cell-cell contacts, CD16 shedding has recently been described as beneficial for NK cell detachment from opsonized target cells, which may preserve NK cell survival and reduce activation-induced death (Srpan et al., J. Cell. Biol., 2018, 217(9):3267-3283). Contrary to this teaching, the present invention aims to provide an antibody construct capable of activating immune effector cells, such as NK cells, via binding to CD16A present on the surface of said effector cells, without the risk of activation-induced downregulation / shedding of CD16A. This can be achieved by the specific high affinity anti-CD16A binding domain (herein referred to as CD16a1 anti-CD16A effector domain or CD16a1 domain) contained by the antibody construct of the present invention. This may be achieved by a) inhibiting shedding below a threshold that provides a compromise to inhibit shedding sufficiently to result in increased NK cell activation while avoiding impairment of NK cell activity, and / or b) avoiding apoptotic death of NK cells due to excessive inhibition of CD16A shedding. Thus, the antibody constructs of the present invention can specifically activate immune effector cells for ADCC-induced phagocytosis against target cell antigens, specifically FOLR1, thereby resulting in efficient lysis of said target cells without loss of activity and efficiency due to activation-induced CD16A degradation.

[0134] Thus, the present invention envisages an antibody construct comprising a specific first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell, and a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell, said second target (B') being FOLR1, thereby reducing the rate of activation-induced CD16A shedding on the surface of the effector cell.

[0135] The inventors of the present application believe that the specific CD16A binding domain contained by the antibody construct of the present invention is particularly beneficial when compared to known low affinity CD16A binding domains (such as the CD16a2 or CD16a4 effector domains, also referred to as CD16a2 or CD16a4 domains, as described herein). This is because the CD16A binding domain of the present invention provides high affinity binding to CD16A present on the surface of immune effector cells (see Figures 1, 2, and 16A-16C, and Tables 3 and 15), but does not result in stronger induction of CD16A loss in the presence of target cells (such as CD123 positive (+) cells). As demonstrated in Figures 5A-5C and 6A-6C of the present application, the CD16a1 binding domain results in stabilization of CD16A receptor levels at various antibody concentrations, regardless of the presence of target cells. Whereas the presence of circulating target cells (such as CD123+ cells in peripheral blood) resulted in rapid activation of NK cells and loss of CD16A from the cell surface upon infusion of bispecific CD123×CD16A targeting antibodies comprising low affinity anti-CD16A binding domain variants (such as CD16a2), the bispecific CD123×CD16A antibody constructs of the invention with high affinity anti-CD16A binding domains resulted in stabilization of CD16A receptor levels regardless of the presence of (CD123+) target cells.

[0136] Furthermore, the antibody constructs of the present invention comprising the CD16a1 anti-CD16A effector domain described herein exhibit substantially longer retention on NK cells when compared to the CD16a2 anti-CD16A effector domain described herein (see FIG. 3 and Table 5). Furthermore, the antibody constructs of the present invention comprising the CD16a1 or CD16a3 anti-CD16A effector domain described herein induce NK cell-dependent lysis on target cells with similar maximum potency when compared to other antibody constructs comprising low affinity anti-CD16A binding domains (see FIG. 4 and 17). In addition, the inventors of the present application observed that the antibody constructs of the present invention comprising the CD16a1 or CD16a3 anti-CD16A effector domain described herein exhibit the lowest non-specific activity, i.e., upregulation of the activation marker CD137 on NK cells in the absence of target cells (see FIG. 7 and 18A-18B). Furthermore, the antibody constructs of the present invention comprising the CD16a1 anti-CD16A effector domain described herein induce specific CD137 upregulation on NK cells in response to target cells (Figure 8), thereby exhibiting the lowest potential for non-specific binding (Figures 9A-9B).

[0137] However, the prior art has recently reported a clear advantage of CD16 shedding on activated NK cells, which may maintain NK cell survival (Srpan et al., J. Cell. Biol., 2018, 217(9):3267-3283). Contrary to this, the inventors of the present application found that the anti-CD16A binding domain contained by the bispecific antibody construct of the present invention did not result in activation-induced death of CD16A+ immune effector cells, despite a low degree of CD16A shedding. Instead, CD16A+ NK cells activated by the CD123×CD16A bispecific antibody construct containing the specific anti-CD16A binding domain of the present invention stably expressed CD16A, but did not show activation-induced death. Instead, CD16A+ NK cells activated by the bispecific antibody construct of the present invention were available for effective target cell killing.

[0138] Thus, the antibody construct according to the present invention, comprising the high affinity anti-CD16A binding domain described herein, shows some advantages when compared with the anti-CD16A binding domain that shows lower affinity to CD16A.Nevertheless, the use of high affinity anti-CD16A binding domain (including the CD16a1 or CD16a3 binding domain described herein) to stabilize CD16A must be considered counterintuitive and not obvious to those skilled in the art, because high affinity binding typically leads to greater NK cell activation and loss of CD16A.Therefore, typically, one would try to reduce the efficacy to prevent CD16A loss by using a lower affinity domain, in an attempt to exploit the selectivity of ADCC toward cells that express higher levels of FOLR1 and away from cells that have lower levels of expression. Contrary to this expectation, lower affinity anti-CD16A binding domain variants (e.g. CD16a2 as described herein) did not stabilize CD16A when compared to the anti-CD16A binding domain CD16a1, resulting in increased CD16 shedding. However, this is expected to have a detrimental effect on the therapeutic activity of the low affinity bispecific antibody constructs, since CD16A is rapidly shed on circulating NK cells before these cells can reach their intended tumor target. This is relevant in disease contexts, since we want to avoid immediate inactivation of NK cells by CD16 shedding, especially when treating solid tumors characterized by a limited presence of NK cells with bispecific FOLR1 x CD16A antibodies.

[0139] Thus, the antibody construct of the present invention provides a novel approach that allows tumor cell targeting and sustained NK cell survival in NK cell-mediated ADCC without inducing CD16A loss. In summary, the present invention is based at least in part on the surprising finding that a bispecific antibody construct comprising a first binding domain of high affinity anti-CD16A and a second binding domain against an antigen present on the surface of a target cell, specifically FOLR1, can efficiently kill the target cell, thereby avoiding CD16A shedding and immediate inactivation of bound NK cells. Thus, the antibody construct of the present invention can be useful for tumor therapy, particularly for solid tumors overexpressing FOLR1, since it can not only activate NK cells through high affinity binding of CD16A receptor, but also achieve long-lasting activation of NK cells without CD16A degradation.

[0140] The antibody construct of the present invention is characterized in that it induces low or no CD16A shedding on the surface of immune effector cells, preferably NK cells, to which the antibody binds in the presence of target cells. Peripheral blood derived NK cells have approximately 10 CD16A shedding on their surface. 6It is understood that CD16A receptors are present on the buffy coat of the NK cell line (Peipp et al., Oncotarget, 2015, vol 6, no 31: 32075-32088). Without wishing to be bound by theory, the inventors of the present application believe that up to about 50% CD16 shedding appears to be significant for sustaining effector cell activity, particularly NK cell activity, after binding to the anti-CD16A binding domain. The extent of CD16A shedding can be measured by flow cytometry, essentially as described in Example 5. Such an assay is preferably performed as follows: PBMCs are isolated from the buffy coat by density gradient centrifugation. The buffy coat sample is diluted with 2-3 volumes of PBS, layered on a cushion of Lymphoprep, and centrifuged at 800×g for 25 minutes at room temperature without brake. The PBMCs located at the interface are harvested and washed three times with PBS, then cultured overnight in complete RPMI1640 medium without stimulation. For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells and the Big Easy EasySep™ Magnet according to the manufacturer's instructions. NK cells were then cultured in pre-chilled complete RPMI 1640 medium at 10–15 × 10 6The cells are suspended in a volume of 1 mL at a density of 100, 10, and 1 μg / mL. Antibody constructs are added at concentrations of 100, 10, and 1 μg / mL and incubated on ice for 45 minutes. The cells are then washed with complete RPMI1640 medium and transferred to a 96-well round-bottom plate. The NK cells are incubated with or without 50 ng / mL phorbol-12-myristate-13-acetate (PMA) and 0.5 μM ionomycin at 37° C. for 4 hours. After stimulation, the cells are washed with FACS buffer (PBS containing 2% heat-inactivated FCS and 0.1% sodium azide). To detect CD16 levels, the cells are re-stained with 100 μg / mL of the respective anti-CD16A antibody, followed by incubation with 15 μg / mL of FITC-conjugated goat anti-mouse IgG and stained with fixable viability stain eFluor™ 780 to exclude dead cells. After the final washing step, the cells are resuspended in 0.2 mL of FACS buffer, the fluorescence of the cells is measured using a flow cytometer, and the median fluorescence intensity of the cell samples is calculated. After subtracting the fluorescence intensity values ​​of cells stained with the secondary reagent only, the MFI values ​​are plotted using GraphPad Prism software. Figures are generated using FlowJo software.

[0141] In some embodiments, a "low degree of CD16A shedding" on the surface of immune effector cells, preferably NK cells, means that the degree of CD16A shedding using a test molecule, such as a bispecific antibody construct comprising an anti-CD16A binding domain of the present invention, does not exceed about 50%. The degree of CD16A shedding on effector cells caused by an antibody construct of the present invention, preferably determined at a concentration of 100 μg / mL, is preferably about 45% or less, more preferably about 40% or less, more preferably about 35% or less, more preferably about 30% or less, more preferably about 25% or less, more preferably about 20% or less, more preferably about 18% or less, more preferably about 16% or less, more preferably about 14% or less, more preferably about 12% or less, more preferably about 11% or less, more preferably about 10% or less. In some even more preferred embodiments, the degree of CD16A shedding using the antibody constructs of the present invention, preferably determined at a concentration of 100 μg / mL, is even lower, for example, preferably about 9% or less, more preferably about 8% or less, more preferably about 7% or less, more preferably about 6% or less, more preferably about 5% or less, more preferably about 4% or less, more preferably about 3% or less, more preferably about 2% or less, or more preferably about 1% or less, or most preferably, is undetectable in an assay essentially as described herein, preferably as defined above.

[0142] In some embodiments, the antibody constructs of the present invention induce a lower degree of CD16A shedding, preferably determined at a test antibody and control antibody concentration of 100 μg / mL, compared to control antibody constructs such as scFv-IgAb_148 (SEQ ID NOs: 92-93), scFv-IgAb_264 (SEQ ID NOs: 82-83), and scFv-IgAb_265 (SEQ ID NOs: 84-85), which contain low affinity anti-CD16A binding domains.

[0143] In some embodiments, the antibody constructs of the present invention induce a lower degree of CD16A shedding, preferably determined at a test antibody and control antibody concentration of 100 μg / mL, compared to control antibody constructs such as scFv-IgAb_381 (SEQ ID NOs: 140-141), scFv-IgAb_273 (SEQ ID NOs: 134-135), and scFv-IgAb_274 (SEQ ID NOs: 136-137), which comprise low affinity anti-CD16A binding domains.

[0144] The antibody construct of the present invention may further be characterized in that it induces a low degree of apoptotic death of immune effector cells, preferably NK cells, or does not induce any apoptotic death of said effector cells, when bound to said immune effector cells, preferably NK cells, in the presence of target cells. Thus, the antibody construct of the present invention is characterized in that it avoids induction of apoptosis of immune effector cells, preferably NK cells, due to excessive inhibition of CD16A shedding.

[0145] In some embodiments, "low degree of apoptotic death" means that the degree of immune effector cell apoptosis using a test molecule, such as a bispecific antibody construct comprising an anti-CD16A binding domain of the present invention, does not exceed about 50%. The degree of immune effector cell apoptosis caused by the antibody construct of the present invention, preferably determined at a concentration of 100 μg / mL, is preferably about 45% or less, more preferably about 40% or less, more preferably about 35% or less, more preferably about 30% or less, more preferably about 25% or less, more preferably about 20% or less, more preferably about 18% or less, more preferably about 16% or less, more preferably about 14% or less, more preferably about 12% or less, more preferably about 11% or less, more preferably about 10% or less. In some even more preferred embodiments, the extent of immune effector cell apoptosis of the antibodies of the invention, preferably determined at a concentration of 100 μg / mL, is even lower, e.g., preferably about 9% or less, more preferably about 8% or less, more preferably about 7% or less, more preferably about 6% or less, more preferably about 5% or less, more preferably about 4% or less, more preferably about 3% or less, more preferably about 2% or less, or more preferably about 1% or less, or most preferably undetectable.

[0146] In some embodiments, the antibody constructs of the present invention induce a lower degree of immune effector cell apoptosis as compared to control antibody constructs such as scFv-IgAb_148 (SEQ ID NOs: 92-93), scFv-IgAb_264 (SEQ ID NOs: 82-83), and scFv-IgAb_265 (SEQ ID NOs: 84-85), which contain low affinity anti-CD16A binding domains, preferably determined at a test antibody and control antibody concentration of 100 μg / mL.

[0147] In some embodiments, the antibody constructs of the present invention induce a lower degree of immune effector cell apoptosis, preferably determined at a test antibody and control antibody concentration of 100 μg / mL, as compared to control antibody constructs such as scFv-IgAb_381 (SEQ ID NOs: 140-141), scFv-IgAb_273 (SEQ ID NOs: 134-135), and scFv-IgAb_274 (SEQ ID NOs: 136-137), which contain low affinity anti-CD16A binding domains.

[0148] As indicated herein above, the present invention relates to a bispecific antibody construct comprising: (a) a first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6, and (ii) a VH region as set forth in SEQ ID NO: 7 or SEQ ID NO: 94; and (b) a second binding domain (B) capable of specifically binding to a second target (B') which is an antigen present on the surface of a target cell, the second target (B') being FOLR1.

[0149] The first binding domain (A) can specifically bind to CD16A, and preferably includes the ability to distinguish between CD16A and CD16B. In other words, the first binding domain (A) preferably binds to CD16A with higher affinity than CD16B, which may be at least about 10 times higher, at least about 100 times higher, or at least about 1000 times higher. More preferably, the first binding domain does not essentially bind to CD16B. Therefore, it is understood that the first binding domain is preferably not a non-silenced CH2 domain, i.e., a CH2 domain that can bind to both CD16A and CD16B.

[0150] Thus, the first binding domain preferably binds to an epitope of CD16A, comprising amino acid residues of the C-terminal sequence SFFPPGYQ ​​(positions 201-209 of SEQ ID NO: 50) of CD16A, which are not present in CD16B, and / or residues G147 and / or Y158. It is preferred in the present invention that the first binding domain that binds to CD16A present on the surface of an effector cell binds to an epitope on CD16A that is closer to the membrane than the physiological Fcγ receptor binding domain of CD16A. A binding domain that specifically binds to an epitope comprising Y158 is preferred, since this epitope is closer to the cell membrane and thus further contributes to reducing the possibility of simultaneous binding to a second immune effector cell.

[0151] In some preferred embodiments, the first binding domain (A) comprises a pair of VH and VL chains having sequences set forth in a pair of sequences selected from the group consisting of SEQ ID NOs: 7 and 8, and SEQ ID NOs: 94 and 95.

[0152] In some preferred embodiments, the first binding domain (A) comprises a VL region depicted in SEQ ID NO:8 or SEQ ID NO:95 and a VH region depicted in SEQ ID NO:7 or SEQ ID NO:94.

[0153] The first binding domain (A) is preferably derived from an antibody. The first binding domain (A) preferably comprises the VH and VL domains of an antibody. Preferred structures of the first binding domain (A) include a pair of VL and VH that may be comprised in an Fv, scFv, Fab, or a diabody (Db), scDb, or bi-Fab. Preferably, the first binding domain (A) is an scFv. Equally preferred, the first binding domain (A) is an Fv. Equally preferred, the first binding domain (A) is an Fab. Equally preferred, the first binding domain (A) is a Db. Equally preferred, the first binding domain (A) is an scDb. Equally preferred, the first binding domain (A) is a bi-Fab. Most preferably, the first binding domain (A) is an scFv.

[0154] In some preferred embodiments, the first binding domain (A) of the antibody construct of the present invention is an scFv having the amino acid sequence shown in SEQ ID NO: 9. In some preferred embodiments, the first binding domain (A) of the antibody construct of the present invention is an scDb having the amino acid sequence shown in SEQ ID NO: 10.

[0155] In some preferred embodiments, the first binding domain (A) of the antibody construct of the present invention is an scFv having the amino acid sequence shown in SEQ ID NO: 96. In some preferred embodiments, the first binding domain (A) of the antibody construct of the present invention is an scDb having the amino acid sequence shown in SEQ ID NO: 97.

[0156] A control antibody construct comprising a low affinity anti-CD16A binding domain as described herein may comprise a first binding domain (A) capable of specifically binding to CD16A present on the surface of immune effector cells, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as set forth in SEQ ID NO: 11, CDR-L2 as set forth in SEQ ID NO: 12, and CDR-L3 as set forth in SEQ ID NO: 13, and (ii) a VH region as set forth in SEQ ID NO: 17 or 104; and (b) a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell. The first binding domain (A) of the control antibody construct may comprise a pair of VH and VL chains having sequences as set forth in a pair of sequences selected from the group consisting of SEQ ID NO: 17 or 104 and 18 or 105. The control antibody construct may have a first binding domain (A) of the scFv depicted in SEQ ID NO: 19 or 106. The control antibody construct may also have a first binding domain (A) of the scDb depicted in SEQ ID NO: 20 or 107.

[0157] In the present invention, the second binding domain (B) of the antibody construct of the present invention, which is specific for a second target (B'), which is an antigen present on the surface of a target cell, specifically a tumor-associated antigen, is specific for FOLR1, i.e., FOLR1 is the target (B').

[0158] The cell surface antigen FOLR1 present on the surface of target cells is associated with certain disease entities, which are described elsewhere herein.Specifically, FOLR1 is a cell surface antigen characteristic of solid tumors, such as ovarian cancer (e.g., high-grade serous ovarian cancer or epithelial ovarian cancer), breast cancer (particularly TNBC), renal cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) or mesothelioma), colorectal cancer (CRC), renal cancer (particularly ccRCC), pancreatic cancer (particularly PDAC), endometrial cancer (e.g., non-malignant endometrial cancer) and brain tumors.

[0159] Antibodies against FOLR1 are well known in the art and are described, for example, in US9522196B2 and WO2014104270. Specific examples of anti-human FOLR1 antibodies are farletuzumab (INN number 9067, the sequence is also disclosed under drugbank.ca / drugs / DB05595 / polypeptide_sequences.fasta and the IMTG database (imtg.org, INN number 9067)) and mirvetuximab (INN number 10187, the sequence is disclosed in the IMTG database (imtg.org, INN number 10187)).

[0160] In the present invention, the second binding domain (B) specific for FOLR1 preferably comprises a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 114 or 124, CDR-H2 as shown in SEQ ID NO: 115 or 125, CDR-H3 as shown in SEQ ID NO: 116 or 126, CDR-L1 as shown in SEQ ID NO: 117 or 127, CDR-L2 as shown in SEQ ID NO: 118 or 128, and CDR-L3 as shown in SEQ ID NO: 119 or 129.

[0161] Thus, it is particularly envisaged that the second binding domain (B) specific for FOLR1 comprises a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 114, CDR-H2 as shown in SEQ ID NO: 115, CDR-H3 as shown in SEQ ID NO: 116, CDR-L1 as shown in SEQ ID NO: 117, CDR-L2 as shown in SEQ ID NO: 118 and CDR-L3 as shown in SEQ ID NO: 119.

[0162] It is also specifically envisaged that the second binding domain (B) specific for FOLR1 comprises a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 124, CDR-H2 as shown in SEQ ID NO: 125, CDR-H3 as shown in SEQ ID NO: 126, CDR-L1 as shown in SEQ ID NO: 127, CDR-L2 as shown in SEQ ID NO: 128, and CDR-L3 as shown in SEQ ID NO: 129.

[0163] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises one of the following: A pair of VH and VL chains having sequences as set forth in a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 120 and 121, respectively, or a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 130 and 131, respectively. Includes.

[0164] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises a pair of VH and VL chains having sequences set forth in the pair of sequences SEQ ID NOs: 120 and 121 or SEQ ID NOs: 130 and 131.

[0165] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises a pair of VH and VL chains having sequences set forth in a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 120 and 121, respectively.

[0166] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises a pair of VH and VL chains having the sequences shown in SEQ ID NOs: 120 and 121.

[0167] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises a pair of VH and VL chains having sequences set forth in a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 130 and 131, respectively.

[0168] In some preferred embodiments, the second binding domain (B) specific for FOLR1 comprises a pair of VH and VL chains having the sequences shown in SEQ ID NOs: 130 and 131.

[0169] In some preferred embodiments, the second binding domain (B) specific for FOLR1 is an scFv having an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 122 or 132, respectively. In some preferred embodiments, the second binding domain (B) specific for FOLR1 is an scFv having an amino acid sequence shown in SEQ ID NO: 122 or 132. In some preferred embodiments, the second binding domain (B) specific for FOLR1 is an scFv having an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 123 or 133, respectively. In some preferred embodiments, the second binding domain (B) specific for FOLR1 is a scDb having the amino acid sequence shown in SEQ ID NO: 123 or 133.

[0170] The second binding domain (B) specific for CD123 disclosed herein preferably comprises a VH domain comprising the following three heavy chain CDRs and a VH domain comprising the following three light chain CDRs: CDR-H1 as depicted in SEQ ID NO: 21, CDR-H2 as depicted in SEQ ID NO: 22, CDR-H3 as depicted in SEQ ID NO: 23, CDR-L1 as depicted in SEQ ID NO: 24, CDR-L2 as depicted in SEQ ID NO: 25, CDR-L3 as depicted in SEQ ID NO: 26.

[0171] The second binding domain (B) specific for CD123 may comprise a pair of VH and VL chains having the sequences shown in the pair of sequences SEQ ID NOs: 27 and 28.

[0172] The second binding domain (B) specific for CD123 may be an scFv having the amino acid sequence shown in SEQ ID NO: 29. The second binding domain (B) specific for CD123 may be an scDb having the amino acid sequence shown in SEQ ID NO: 30.

[0173] The second binding domain (B) of the antibody construct specific for CD123 may comprise a VH domain comprising the three heavy chain CDRs and the three light chain CDRs: CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 as shown in SEQ ID NO: 35, CDR-L3 as shown in SEQ ID NO: 36.

[0174] The second binding domain (B) specific for CD123 may comprise a pair of VH and VL chains having the sequences shown in the pair of sequences SEQ ID NOs: 37 and 38.

[0175] The second binding domain (B) specific for CD123 may be an scFv having the amino acid sequence shown in SEQ ID NO: 39. The second binding domain (B) specific for CD123 may be an scDb having the amino acid sequence shown in SEQ ID NO: 40.

[0176] The second binding domain (B) is also preferably derived from an antibody. The second binding domain (B) preferably comprises the VH and VL domains of an antibody. Preferred structures of the second binding domain (B) include a pair of VL and VH that may be comprised in an Fv, scFv, Fab, or a diabody (Db), scDb, or double Fab. Preferably, the second binding domain (B) is an scFv. Equally preferred, the second binding domain (B) is an Fv. Equally preferred, the second binding domain (B) is a Fab. Equally preferred, the second binding domain (B) is a Db. Equally preferred, the second binding domain (B) is an scDb. Equally preferred, the second binding domain (B) is a double Fab. Most preferably, the second binding domain (B) is an scFv.

[0177] In the present invention, it is specifically envisaged that the antibody construct binds simultaneously to a target cell and an immune effector cell.

[0178] The antibody construct of the present invention may comprise a third domain (C), which comprises a half-life prolonging domain as described herein. The half-life prolonging domain may comprise a CH2 domain, the Fcγ receptor binding domain of the CH2 domain being silenced. The half-life prolonging domain may comprise two such CH2 domains. Whenever the half-life prolonging domain comprises a CH2 domain, the Fcγ receptor binding domain of the CH2 domain is silenced. The half-life prolonging domain may comprise one CH3 domain. The half-life prolonging domain may comprise two CH3 domains. The half-life prolonging domain may comprise one hinge domain. The half-life prolonging domain may comprise two hinge domains. The half-life prolonging domain may comprise one CH2 domain and one CH3 domain. In such a case, the CH2 domain and the CH3 domain are preferably fused to each other, preferably in the order (amino to carboxyl) of CH2 domain-CH3 domain. Non-limiting examples of such fusions are shown in SEQ ID NOs: 66-81. The half-life prolonging domain may comprise one hinge domain and one CH2 domain. In such cases, the hinge domain and the CH2 domain are preferably fused to each other, preferably in the order (amino to carboxyl) as hinge domain-CH2 domain. The half-life prolonging domain may comprise one hinge domain, one CH2 domain, and one CH3 domain. In such cases, the hinge domain, the CH2 domain, and the CH3 domain are preferably fused to each other, preferably in the order (amino to carboxyl) as hinge domain-CH2 domain-CH3 domain. The half-life prolonging domain may comprise two hinge domain-CH2 domain elements, two CH2 domain-CH3 domain elements, or two hinge domain-CH2 domain-CH3 domain elements. In such cases, the two fusions may be located on two different polypeptide chains. Alternatively, the fusions may be located on the same polypeptide chain.An illustrative example when two hinge domain-CH2 domain-CH3 domain elements are arranged on the same polypeptide chain is the "single chain Fc" or "scFc" format. In this case, both hinge-CH2-CH3 subunits are fused together via a linker that allows assembly of the Fc domain. A preferred linker for this purpose is a glycine serine linker, which preferably contains about 20 to about 40 amino acids. A preferred glycine serine linker may have one or more repeats of GGS, GGGS (SEQ ID NO: 41), or GGGGS (SEQ ID NO: 46). Such a linker preferably contains 4 to 8 repeats of GGGGS (e.g., 4, 5, 6, 7, or 8 repeats). Such a linker is preferably (GGGGS)6 (SEQ ID NO 49). Further scFc constant domains are known in the art and are described, inter alia, in WO 2017 / 134140.

[0179] In general, the antibody construct of the present invention can be monovalent, bivalent, trivalent, or even higher valency for any one of the first target (A') and the second target (B'). Thus, the antibody construct of the present disclosure may comprise one, two, three, or even more of any one of the first binding domain (A) and the second binding domain. For the antibody construct of the present invention, it is preferred that the antibody construct is at least monovalent for the first target (A') and at least monovalent for the second target (B'). For the antibody construct of the present invention, it is also preferred that the antibody construct is at least monovalent for the first target (A') and bivalent for the second target (B'). For the antibody construct of the present invention, it is further preferred that the antibody construct is at least bivalent for the first target (A') and at least bivalent for the second target (B'). It is also preferred for the antibody construct of the present invention that the antibody construct is at least bivalent for the first target (A') and at least trivalent for the second target (B'). It is also preferred for the antibody construct of the present invention that the antibody construct is at least bivalent for the first target (A') and at least monovalent for the second target (B'). Most preferably, the antibody construct of the present invention is bivalent for the first target (A') and bivalent for the second target (B').

[0180] Therefore, for the antibody construct of the present invention, it is preferred that the antibody construct comprises at least one first binding domain (A) and at least one second binding domain (B). For the antibody construct of the present invention, it is further preferred that the antibody construct comprises at least one first binding domain (A) and at least two second binding domains (B). For the antibody construct of the present invention, it is further preferred that the antibody construct comprises at least two first binding domains (A) and at least two second binding domains (B). For the antibody construct of the present invention, it is further preferred that the antibody construct comprises at least two first binding domains (A) and at least three second binding domains (B). For the antibody construct of the present invention, it is further preferred that the antibody construct comprises at least two first binding domains (A) and at least one second binding domain (B). Most preferably, the antibody construct of the present invention comprises two first binding domains (A) and two second binding domains (B).

[0181] Disclosed herein are antibody constructs comprising one first binding domain (A) that specifically binds to CD16A and one second binding domain (B) that specifically binds to CD123. These antibody constructs may comprise one first binding domain (A) that specifically binds to CD16A and two second binding domains (B) that specifically bind to CD123. These antibody constructs may also comprise two first binding domains (A) that specifically bind to CD16A and one second binding domain (B) that specifically binds to CD123. These antibody constructs may also comprise two first binding domains (A) that specifically bind to CD16A and two second binding domains (B) that specifically bind to CD123.

[0182] It is particularly preferred for the antibody construct of the present invention that the antibody construct comprises one first binding domain (A) that specifically binds to CD16A and one second binding domain (B) that specifically binds to FOLR1. It is also preferred for the antibody construct of the present invention that the antibody construct comprises one first binding domain (A) that specifically binds to CD16A and two second binding domains (B) that specifically bind to FOLR1. It is also preferred for the antibody construct of the present invention that the antibody construct comprises two first binding domains (A) that specifically bind to CD16A and one second binding domain (B) that specifically binds to FOLR1. It is also preferred for the antibody construct of the present invention that the antibody construct comprises two first binding domains (A) that specifically bind to CD16A and two second binding domains (B) that specifically bind to FOLR1.

[0183] In a preferred embodiment, the first binding domain (A) is fused to the C-terminus of the Fc region. Such a fusion format is exemplarily shown in FIG. 10. The first binding domain (A) may be fused to the constant domain of the antibody via a linker. Such a linker is preferably a short linker, preferably having a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5 nm or less, preferably about 4 nm or less, or even shorter. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers well known to those skilled in the art. An example of a suitable linker is a glycine-serine linker or a serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) GGGGS sequences (SEQ ID NO: 46), such as (GGGGS)2 (SEQ ID NO: 47), (GGGGS)4 (SEQ ID NO: 48), or preferably (GGGGS)6 (SEQ ID NO: 49). Other illustrative examples for linkers are shown in SEQ ID NOs: 42-45. The first binding domain (A) is preferably an scFv fragment, which is preferably fused to the C-terminus of the Fc domain via the VL domain of the scFv. Thus, the configuration of the polypeptide chains (from N to C) is preferably ...-CH2-CH3-VL-VH, optionally with a linker between the Fc and the scFv. The second binding domain can be located at any suitable position of the antibody construct. If the antibody construct comprises an Fc region, the second binding domain (B) can be located at the N-terminus of the Fc region, either directly or via at least a portion of a hinge domain. Other linkers disclosed herein can also be used to link the third binding domain to the Fc domain. However, a hinge domain is preferred for this purpose.The second binding domain (B) can be of any suitable structure as disclosed herein, although an scFv structure is preferred.

[0184] The antibody construct of the present invention is preferably in a format essentially as shown in FIG. 10 and also referred to herein as "scFv-IgAb". Such an antibody construct comprises an immunoglobulin with one scFv fragment fused to the C-terminus of each of two heavy chains, optionally via a linker, preferably a connector as disclosed herein. The scFv forms the first binding domain (A). Two second binding domains (B) are formed by the binding sites of the immunoglobulin. The scFv-IgAb format may comprise four polypeptide chains: two light chains of the configuration VL(B)-CL, and two heavy chains each fused to an scFv of the configuration VH(B)-CH1-hinge-CH2-CH3-VL(A)-VH(A) (or less preferably VH(B)-CH1-hinge-CH2-CH3-VH(A)-VL(A)). The letters in brackets represent the first binding domain (A) and the second binding domain (B), respectively. For example, VL(A) represents the VL domain of the first binding domain (A) and VH(B) represents the VH domain of the second binding domain (B). Illustrative examples of such antibody constructs are shown in SEQ ID NOs: 86-87 and 88-98.

[0185] In another preferred embodiment, two first binding domains (A) are fused to the two C-terminus of the Fc region, and the two first binding domains (A) are preferably fused together in the form of a diabody or single-chain diabody, preferably via the VL domain of the first binding domain (A). Such a fusion format is exemplarily shown in FIG. 11. The first binding domain (A) may be fused to the constant domain of the antibody via a linker. Such a linker is preferably a short linker, preferably having a length of about 10 nm or less, preferably about 9 nm or less, preferably about 8 nm or less, preferably about 7 nm or less, preferably about 6 nm or less, preferably about 5 nm or less, preferably about 4 nm or less, or preferably even shorter. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers well known to those skilled in the art. An example of a suitable linker is a glycine-serine linker or a serine linker, preferably comprising about 75 amino acids or less, preferably about 50 amino acids or less. In an illustrative example, a suitable linker comprises one or more GGGGS sequences (SEQ ID NO: 46), such as (GGGGS)2 (SEQ ID NO: 47), (GGGGS)4 (SEQ ID NO: 48), or preferably (GGGGS)6 (SEQ ID NO: 49). Other illustrative examples of linkers are shown in SEQ ID NOs: 42-45. The first binding domain (A) is preferably an scDb fragment, preferably fused to the two C-terminus of the Fc domain via the VL domain of the scDb. Thus, the configuration of the polypeptide chain (from N to C) is preferably ...-CH2-CH3-VL-VH-VL-VH, optionally with a linker between the Fc and the scDb. The second binding domain can be located at any suitable position of the antibody construct. If the antibody construct comprises an Fc region, the second binding domain (B) can be located at the N-terminus of the Fc region, either directly or linked via at least a portion of the hinge domain.Other linkers disclosed herein can also be used to link the third binding domain to the Fc domain. However, a hinge domain is preferred for this purpose. The second binding domain (B) can be of any suitable structure disclosed herein, but a Fab structure is preferred.

[0186] The antibody construct of the present invention is also preferably in the format essentially as shown in Figure 11 and also called "scDb-IgAb". Such an antibody construct comprises an immunoglobulin with one scDb fragment fused to the C-terminus of each of the two heavy chains, optionally via a linker, preferably a connector as disclosed herein. The scFv forms the first binding domain (A). The two second binding domains (B) are formed by the binding sites of the immunoglobulin. The scDb-IgAb format may comprise four polypeptide chains, two light chains of the arrangement VL(B)-CL, and two heavy chains each fused to an scDb of the arrangement VH(B)-CH1-hinge-CH2-CH3-VL(A)-VH(A)-VL(A)-VH(A) (or less preferred VH(B)-CH1-hinge-CH2-CH3-VH(A)-VL(A)-VH(A)-VL(H)). The arrangement VH(B)-hinge-CH2-CH3-VL(A)-VH(A)-VL(A)-VH(A) (or less preferred VH(B)-hinge-CH2-CH3-VH(A)-VL(A)-VH(A)-VL(H)) is also envisaged. The letters in brackets represent the first binding domain (A) and the second binding domain (B), respectively. For example, VL(A) represents the VL domain of the first binding domain (A) and VH(B) represents the VH domain of the second binding domain (B).

[0187] In general, the hinge domain comprised in the antibody construct of the present disclosure may comprise a full-length hinge domain, for example the hinge domain shown in SEQ ID NO: 53. The hinge domain may also comprise a shortened and / or modified hinge domain. The shortened hinge domain may comprise the upper hinge domain, for example the hinge domain shown in SEQ ID NO: 54, or the middle hinge domain, for example the hinge domain shown in SEQ ID NO: 55, but not the entire hinge domain, the latter being preferred. The hinge domain preferred in the present invention exhibits a modulated flexibility compared to antibody constructs with wild-type hinge domains, as described in Dall'Acqua et al (J Immunol. 2006 Jul 15;177(2):1129-38) or WO 2009 / 006520. Furthermore, the preferred hinge domain is characterized in that it consists of less than 25 amino acid residues. More preferably, the length of the hinge is between 10 and 20 amino acid residues. The hinge domain included in the antibody construct of the present disclosure may also comprise an IgG2 subtype hinge sequence. TIFF2024543828000002.tif4128, IgG3 subtype hinge sequence TIFF2024543828000003.tif12128, and / or IgG4 subtype hinge sequence TIFF2024543828000004.tif4128. Further hinge domains that can be used in the present invention are known to those skilled in the art and are described, for example, in WO 2017 / 134140.

[0188] The antibody construct of the present invention preferably comprises: (a) a first binding domain (A) capable of specifically binding to a first target (A') present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, and a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94, the first binding domain (A) being an scFv; (b) a second binding domain (B') capable of specifically binding to a second target (B') present on the surface of a target cell, the first binding domain (A) comprising CDR-H1 as shown in SEQ ID NO: 114, CDR-H2 as shown in SEQ ID NO: 115, CDR-H3 as shown in SEQ ID NO: 116, CDR-L1 as shown in SEQ ID NO: 117, CDR-H4 as shown in SEQ ID NO: 118, CDR-H5 as shown in SEQ ID NO: 119, CDR-L6 as shown in SEQ ID NO: 200, CDR-L7 as shown in SEQ ID NO: 210, CDR-L8 as shown in SEQ ID NO: 211, CDR-L9 as shown in SEQ ID NO: 212, CDR-L1 as shown in SEQ ID NO: 213, CDR-L1 as shown in SEQ ID NO: 214, CDR-L2 as shown in SEQ ID NO: 215, CDR-H3 as shown in SEQ ID NO: 216, CDR-L1 as shown in SEQ ID NO: 217, CDR-L1 as shown in SEQ ID NO: 218, CDR-L2 as shown in SEQ ID NO: 219, CDR-L3 as shown in SEQ ID NO: 220, C and (c) a third binding domain, preferably comprising two hinge domain-CH2 domain-CH3 domain elements, as depicted in SEQ ID NOs: 53 and 67; wherein the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain.

[0189] Alternatively, the antibody construct of the present invention preferably comprises: (a) a first binding domain (A) capable of specifically binding to a first target (A') present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region comprising CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, and a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94, the first binding domain (A) being an scFv; (b) a second binding domain (B') capable of specifically binding to a second target (B') present on the surface of a target cell, the first binding domain (A) comprising CDR-H1 as shown in SEQ ID NO: 124, CDR-H2 as shown in SEQ ID NO: 125, CDR-H3 as shown in SEQ ID NO: 126, CDR-L1 as shown in SEQ ID NO: 127, CDR-L2 as shown in SEQ ID NO: 128, CDR-L3 as shown in SEQ ID NO: 129, CDR-L4 as shown in SEQ ID NO: 200, CDR-L5 as shown in SEQ ID NO: 201, CDR-L6 as shown in SEQ ID NO: 210, CDR-L7 as shown in SEQ ID NO: 211, CDR-L8 as shown in SEQ ID NO: 212, CDR-L9 as shown in SEQ ID NO: 213, CDR-L1 as shown in SEQ ID NO: 214, CDR-L1 as shown in SEQ ID NO: 215, CDR-H3 as shown in SEQ ID NO: 216, CDR-L1 as shown in SEQ ID NO: 217, CDR-L1 as shown in SEQ ID NO: 218, CDR-L2 as shown in SEQ ID NO: 219 and (c) a third binding domain, preferably comprising two hinge domain-CH2 domain-CH3 domain elements as depicted in SEQ ID NOs: 53 and 67; wherein the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain.

[0190] In some embodiments, the antibody construct of the present invention preferably comprises: (a) a first binding domain (A) capable of specifically binding to a first target (A') present on the surface of an immune effector cell, the first binding domain (A) comprising (i) a VL region as depicted in SEQ ID NO:8 or SEQ ID NO:95 and a VH region as depicted in SEQ ID NO:7 or SEQ ID NO:94, the first binding domain (A) being an scFv; (b) a second binding domain capable of specifically binding to a second target (B') present on the surface of a target cell, the second binding domain comprising a VL region as depicted in SEQ ID NO:121 and a VH region as depicted in SEQ ID NO:120, the second binding domain being a Fab; and (c) preferably a second binding domain as depicted in SEQ ID NO: 53 and 67, and a third binding domain comprising two hinge domain-CH2 domain-CH3 domain elements; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain. Alternatively, the second binding domain of the antibody construct, which can specifically bind to a second target (B') which is FOLR1 present on the surface of a target cell, comprises a VL region as shown in SEQ ID NO: 131 and a VH region as shown in SEQ ID NO: 130, and the second binding domain is a Fab.

[0191] The antibody construct of the present invention is preferably an antibody construct selected from the group consisting of SEQ ID NOs: 142-143 and 138-139, i.e. an antibody construct having the amino acid sequence of SEQ ID NOs: 142-143 or SEQ ID NOs: 138-139, with SEQ ID NOs: 142-143 being preferred in the present invention. In this regard, it is envisaged that the antibody comprises two of the listed heavy and light chains so as to form an IgAb.

[0192] The antibody construct of the present invention is preferably a variant of an antibody construct selected from the group consisting of SEQ ID NOs: 142-143 and 138-139, having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% sequence identity to any one of these aforementioned antibody constructs, provided that the CDR-L1-L3 sequence and the VH region of the first binding domain and the CDR sequence of the second binding domain contained in these antibody constructs are not modified.

[0193] The present invention also relates to nucleic acid molecules (DNA and RNA) that comprise nucleotide sequences encoding the antibody constructs disclosed herein. The present disclosure also encompasses vectors that comprise the nucleic acid molecules of the present invention. The present invention also encompasses host cells that comprise said nucleic acid molecules or said vectors. Because the degeneracy of the genetic code allows some codes to be replaced by other codons that designate the same amino acid, the present disclosure is not limited to a particular nucleic acid molecule that encodes the antibody constructs described herein, but encompasses any nucleic acid molecule that comprises a nucleotide sequence that encodes a functional polypeptide. In this regard, the present disclosure also relates to nucleotide sequences that encode the antibody constructs of the present disclosure.

[0194] The nucleic acid molecules disclosed in the present application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of the nucleic acid molecule.

[0195] A nucleic acid molecule, such as DNA, is said to be "capable of expressing a nucleic acid molecule" or "allowing for expression of a nucleotide sequence" if it contains sequence elements that contain information for transcriptional and / or translational regulation and such sequences are "operably linked" to a nucleotide sequence that encodes a polypeptide. An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are joined in a manner that allows gene expression. The exact nature of the regulatory regions necessary for gene expression can vary from species to species, but generally these regions include promoters, which in prokaryotes include both the promoter itself, i.e., the DNA elements that direct transcription initiation and the DNA elements that, when transcribed into RNA, signal translation initiation. Usually, such promoter regions include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno elements in prokaryotes, or the TATA box, CAAT sequence, and 5' capping element in eukaryotes. These regions may also contain enhancer or repressor elements, as well as translated signal or leader sequences for targeting the native polypeptide to a particular compartment of the host cell.

[0196] In addition, the 3' non-coding sequences may also contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences are not fully functional in a particular host cell, the termination sequences may be replaced with signals functional in that cell.

[0197] Therefore, the nucleic acid molecule of the present disclosure can include regulatory sequences such as promoter sequences.In some embodiments, the nucleic acid molecule of the present disclosure includes promoter sequences and transcription termination sequences.The example of the promoter that is useful for expression in eukaryotic cells is SV40 promoter or CMV promoter.

[0198] The nucleic acid molecules of the disclosure can also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0199] Such cloning vehicles can contain, apart from the aforementioned regulatory sequences and nucleic acid sequences encoding the antibody constructs described herein, replication and control sequences derived from a species compatible with the host cell used for expression, as well as a selection marker which confers a selectable phenotype on transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available.

[0200] The present invention also relates to a method for producing the antibody construct of the present disclosure, which is produced starting from a nucleic acid encoding the antibody construct or any subunit thereof. This method can be carried out in vivo, for example, the polypeptide can be produced in a bacterial or eukaryotic host organism and then isolated from this host organism or its culture. It is also possible to produce the antibody construct of the present disclosure in vitro, for example, using an in vitro translation system.

[0201] When producing antibody constructs in vivo, the nucleic acid encoding such polypeptides is introduced into suitable bacterial or eukaryotic host organisms using recombinant DNA technology.For this purpose, host cells can be transformed with cloning vectors that contain the nucleic acid molecules encoding the antibody constructs described herein using established standard methods.The host cells can then be cultured under conditions that allow the expression of heterologous DNA and thus the synthesis of the corresponding polypeptide or antibody construct.Subsequently, the polypeptide or antibody construct is recovered from either the cells or the culture medium.

[0202] Suitable host cells can be eukaryotic, for example, an immortalized mammalian cell line (eg, HeLa cells or CHO cells) or primary mammalian cells.

[0203] The antibody constructs of the present disclosure described herein do not necessarily have to be made or produced solely by genetic engineering. Rather, such polypeptides can also be obtained by chemical synthesis, such as Merrifield solid-phase polypeptide synthesis, or by in vitro transcription and translation. Methods for solid-phase and / or solution-phase synthesis of proteins are well known in the art (see, for example, Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

[0204] The antibody constructs of the present disclosure may be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0205] The present invention also provides a composition, preferably a pharmaceutical composition, comprising the antibody construct of the present invention.

[0206] Certain embodiments provide pharmaceutical compositions comprising an antibody construct as defined in the present invention and one or more other excipients, such as those exemplarily described in this section and elsewhere herein.In this regard, excipients can be used in the present invention for a variety of purposes, such as for adjusting the physical, chemical, or biological properties of the formulation, for example adjusting the viscosity, and / or for the process of one aspect of the present invention to enhance the efficacy and / or stabilize such formulation, as well as for the process of combating degradation and damage due to pressures that occur during manufacture, transportation, storage, preparation before use, administration, and thereafter.

[0207] In certain embodiments, pharmaceutical compositions may include formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulation materials include, but are not limited to, the following: Charged amino acids, preferably lysine, lysine acetate, arginine, glutamic acid, and / or histidine, including amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine Antimicrobial agents, such as antibacterial and antifungal agents ·Antioxidants such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite; Buffers, buffer systems, and buffering agents used to maintain compositions at physiological pH or slightly lower pH; examples of buffering agents are borates, bicarbonates Tris-HCI, citrate, phosphate, or other organic acids, succinate, phosphate, and histidine; for example, Tris buffer at a pH of about 7.0 to 8.5; · Non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; Aqueous carriers, including water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media; · Biodegradable polymers such as polyester; · Bulking agents such as mannitol or glycine; · Chelating agents such as ethylenediaminetetraacetic acid (EDTA); ·Tonicity and absorption retarding agents; Complexing agents such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin Fillers; Monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrins); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol; · a (low molecular weight) protein, polypeptide or proteinaceous carrier, preferably of human origin, such as human or bovine serum albumin, gelatin or immunoglobulins; Colouring and flavouring agents; Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; · Diluents; ·emulsifier; · Hydrophilic polymers such as polyvinylpyrrolidone; · A salt-forming counterion, such as sodium; · preservatives such as antimicrobials, antioxidants, chelating agents, and inert gases; examples are benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide; · Metal complexes such as Zn-protein complexes; · Solvents and co-solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol, or xylitol stachyose, mannose, sorbose, xylose, ribose, myo-inositose, galactose, lactitol, ribitol, myo-inositol, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol, and polyhydric sugar alcohols; ·Suspending agents; Surfactants or wetting agents, e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be surfactants, preferably with a molecular weight greater than 1.2 KD, and / or polyethers, preferably with a molecular weight greater than 3 KD; non-limiting examples of preferred surfactants are Tween 20, Tween 40, Tween 60, Tween 80, and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000, and PEG 5000; ·Stability enhancers such as sucrose or sorbitol; · an alkali metal halide, preferably sodium chloride or potassium chloride, an osmolality increasing agent such as mannitol sorbitol; Parenteral delivery vehicles, including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils; Intravenous delivery vehicles, including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0208] It will be apparent to one of skill in the art that various components of a pharmaceutical composition (e.g., those listed above) may have different effects, e.g., amino acids may act as buffers, stabilizers, and / or antioxidants; mannitol may act as a bulking agent and / or osmolality increasing agent; sodium chloride may act as a delivery vehicle and / or osmolality increasing agent, etc.

[0209] It is envisaged that the compositions of the present invention may contain, in addition to the polypeptides of the present invention as defined herein, further biologically active substances, depending on the intended use of the composition.

[0210] In certain embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended administration route, delivery mode, and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra.For example, suitable vehicle or carrier can be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other substances that are common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin is yet another exemplary vehicle.

[0211] Additional pharmaceutical compositions, including formulations that contain the antibody constructs of the present invention in sustained or controlled delivery / release formulations, will be clear to those skilled in the art.Technologies for formulating various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.See, for example, International Patent Application No. PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.Slow-release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Publication No. EP 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyrate (European Patent Publication No. EP 133,988). The sustained release composition can also include liposomes, which can be prepared by any of several methods known in the art.See, for example, Eppstein et al., 1985, Proc.Natl.Acad.Sci.USA 82:3688-3692; European Patent Application Publication No. EP 036,676; European Patent Application Publication No. EP 088,046 and European Patent Application Publication No. EP 143,949.

[0212] The antibody constructs may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0213] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations.Sterilization can be achieved by filtration through a sterile filtration membrane.If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution.Compositions for parenteral administration can be stored in lyophilized form or in solution.Parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0214] In one embodiment of the pharmaceutical composition according to one aspect of the present invention, the composition is administered to the patient intravenously.

[0215] Methods and protocols for intravenous (iv) administration of the pharmaceutical compositions described herein are well known in the art.

[0216] The antibody construct of the present invention and / or the pharmaceutical composition of the present invention are preferably used in the prevention, treatment, or amelioration of a disease selected from a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder. In the present invention, the disease is preferably characterized by FOLR1 overexpression. Preferably, the neoplastic disease is a malignant disease, preferably a cancer.

[0217] The neoplastic disease that the present invention deals with is solid tumor.It is particularly assumed that said solid tumor is selected from the group consisting of ovarian cancer (e.g. high-grade serous ovarian cancer or epithelial ovarian cancer), breast cancer (particularly TNBC), renal cancer, lung cancer (e.g. non-small cell lung cancer (NSCLC) or mesothelioma), colorectal cancer (CRC), renal cancer (particularly ccRCC), pancreatic cancer (particularly PDAC), endometrial cancer (e.g. non-malignant endometrial cancer) and brain tumor.

[0218] In a preferred embodiment, the neoplastic disease is a metastatic tumor.

[0219] The present invention also provides a method for treating or ameliorating a disease, comprising administering to a subject in need thereof an antibody construct according to the present invention.

[0220] In one embodiment of the method for treating or ameliorating a disease, the subject suffers from a proliferative disease, a neoplastic disease, an infectious disease, such as a viral disease, or an immunological disorder.Preferably, the neoplastic disease is a malignant disease, preferably a cancer, as defined elsewhere herein.

[0221] The antibody constructs of the present invention are generally designed with a range of bioavailability and durability, for a particular route and method of administration, for a particular dosage and frequency of administration, for a particular treatment of a particular disease, among others. The materials of the composition are preferably formulated at concentrations that are acceptable to the site of administration.

[0222] Thus, formulations and compositions may be designed in accordance with the present invention for delivery by any suitable route of administration, including, but not limited to, the following: ·Topical routes (dermal, inhalation, nasal, ocular, auricle / ear, vaginal, mucous membranes, etc.); Enteral routes (oral, gastrointestinal, sublingual, sublabial, buccal, rectal, etc.); and Parenteral routes (intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, subarachnoid, subcutaneous, intraperitoneal, extra-amniotic, intra-articular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intracavity, etc.).

[0223] The pharmaceutical compositions and antibody constructs of the present invention are particularly useful for parenteral administration, e.g., subcutaneous or intravenous delivery, e.g., by injection, such as a bolus injection, or by infusion, such as continuous infusion. The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163. As described elsewhere herein, pharmaceutical compositions according to the present invention are preferably administered intravenously.

[0224] In particular, the present invention provides for uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e. continuous administration, can be achieved by a miniature pump system worn by the patient to meter the influx of therapeutic agent into the patient's body. The pharmaceutical composition comprising the antibody construct of the present invention can be administered by using said pump system. Such pump systems are generally known in the art and generally rely on periodic replacement of a cartridge containing the therapeutic agent to be infused. When replacing the cartridge in such a pump system, a temporary interruption of the otherwise uninterrupted flow of therapeutic agent into the patient's body may result. In such a case, the administration step before cartridge replacement and the administration step after cartridge replacement are still considered within the meaning of the pharmaceutical means and methods of the present invention, and together constitute one "uninterrupted administration" of such therapeutic agent.

[0225] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration. The lyophilized material can be reconstituted, for example, in bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was before lyophilization.

[0226] The composition of the present invention can be administered to the subject in appropriate dosage.Dosage regimen is determined by attending physician and clinical factors.As is well known in the medical field, the therapeutically effective dosage for any one patient depends on many factors, including the size, body surface area, age, the specific compound administered, sex, administration time and route, general health, and other drugs administered at the same time.

[0227] The therapeutically effective amount or dosage of the antibody construct of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of impairment or functional impairment due to disease suffering. For treating neoplastic diseases, the therapeutically effective amount of the antibody construct of the present invention preferably inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of the compound to inhibit tumor growth can be evaluated in animal models predictive of efficacy in human tumors.

[0228] The present invention also relates to a kit comprising the antibody construct of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, or the host cell of the present invention. The kit may comprise one or more receptacles (vials, ampoules, containers, syringes, bottles, bags, etc.) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) that contain the antibody construct of the present invention or pharmaceutical composition in a dosage suitable for administration. The kit may additionally contain instructions for use (e.g., in the form of a leaflet or instruction manual), a means for administering the antibody construct of the present invention, e.g., a syringe, pump, injector, etc., a means for reconstituting the antibody construct of the present invention, and / or a means for diluting the antibody construct of the present invention. The present invention also provides kits for single-dose administration units. The kit of the present invention may also contain a first receptacle that contains the dried / lyophilized antibody construct, and a second receptacle that contains an aqueous formulation. In a particular embodiment of the present invention, a kit is provided that contains single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes). Kits of the present invention typically include a container which contains an antibody construct of the invention, a nucleic acid molecule of the invention, a vector of the invention, or a host cell of the invention, and optionally may include one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0229] In some aspects, the invention can be characterized by the following:

[0230] Item 1. Below: (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell, (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94; a first binding domain (A) comprising: (b) a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell, wherein the second target (B') is FOLR1; 2. A bispecific antibody construct comprising:

[0231] Item 2. The antibody construct of item 1, wherein the first binding domain (A) comprises a VL region as depicted in SEQ ID NO: 8 or SEQ ID NO: 95 and a VH region as depicted in SEQ ID NO: 7 or SEQ ID NO: 94.

[0232] Item 3. The antibody construct of item 1 or 2, wherein the first binding domain (A) is a variable domain (Fv), a single chain Fv (scFv), a Fab, a single chain diabody (scDb), a diabody (Db), or a bi-Fab, preferably an scFv.

[0233] Item 4. The antibody construct of any one of items 1 to 3, wherein the second binding domain (B) comprises an antibody VH domain and a VL domain.

[0234] Item 5. The antibody construct of any one of items 1 to 4, wherein the second binding domain (B) is a variable domain (Fv), a single chain Fv (scFv), a Fab, a single chain diabody (scDb), a diabody (Db), or a bi-Fab, preferably a bi-Fab.

[0235] Item 6. The antibody construct of any one of items 1 to 5, which simultaneously binds to a target cell and an immune effector cell.

[0236] Item 7. The first binding domain is An epitope on CD16A that is C-terminal to the physiological Fcγ receptor binding domain Binds to The epitope preferably comprises Y158 of SEQ ID NO: 50; The antibody construct of any one of items 1 to 6.

[0237] Item 8. The antibody construct of any one of items 1 to 7, further comprising a third domain (C) comprising a half-life extending domain.

[0238] Item 9. The antibody construct of any one of items 1 to 8, wherein the half-life extending domain comprises a CH2 domain and the Fcγ receptor binding domain is silenced.

[0239] Item 10. The antibody construct of any one of items 1 to 8, wherein the half-life extending domain comprises a CH3 domain.

[0240] Item 11. The antibody construct of any one of items 1 to 10, comprising at least one hinge domain and a CH3 domain fused to a CH2 domain in the following order from amino to carboxyl: hinge domain-CH2 domain-CH3 domain.

[0241] Item 12. The antibody construct of any one of items 1 to 11, comprising at least two hinge domain-CH2 domain-CH3 domain elements.

[0242] Item 13. The antibody construct of any one of items 1 to 12, wherein the first binding domain (A) is fused to the C-terminus of the CH3 domain and the second binding domain (B) is fused to the N-terminus of the hinge region.

[0243] Item 14. The antibody construct of any one of items 1 to 13, which is monovalent with respect to the first binding domain (A) and monovalent with respect to the second binding domain (B).

[0244] Item 15. The antibody construct of any one of items 1 to 14, which is bivalent with respect to the first binding domain (A) and bivalent with respect to the second binding domain (B).

[0245] Item 16. (a) A first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1, an antigen present on the surface of a target cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 117, a CDR-L2 as set forth in SEQ ID NO: 118, and a CDR-L3 as set forth in SEQ ID NO: 119; (ii) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 114, CDR-H2 as set forth in SEQ ID NO: 115, and CDR-H3 as set forth in SEQ ID NO: 116; wherein the second binding domain is a Fab; and (c) a third binding domain, Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Includes two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of any one of items 1 to 13 or 15.

[0246] Item 17. (a) A first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1, an antigen present on the surface of a target cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 127, a CDR-L2 as set forth in SEQ ID NO: 128, and a CDR-L3 as set forth in SEQ ID NO: 129; (ii) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 124, CDR-H2 as set forth in SEQ ID NO: 125, and CDR-H3 as set forth in SEQ ID NO: 126; wherein the second binding domain is a Fab; and (c) a third binding domain, Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Includes two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of any one of items 1 to 13 or 15.

[0247] Item 18. (a) A first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, (i) a VL region as depicted in SEQ ID NO: 8 or SEQ ID NO: 95; (ii) a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1 present on the surface of a target cell; (i) a VL region as depicted in SEQ ID NO: 121; (ii) the VH region shown in SEQ ID NO: 120; wherein the second binding domain is a Fab; and (c) a third binding domain, Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Includes two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of any one of items 1 to 13, 15, or 16.

[0248] Item 19. (a) A first binding domain (A) capable of specifically binding to a first target (A') which is CD16A present on the surface of an immune effector cell, (i) a VL region as depicted in SEQ ID NO: 8 or SEQ ID NO: 95; (ii) a VH region as shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1 present on the surface of a target cell; (i) a VL region as depicted in SEQ ID NO: 131; (ii) the VH region shown in SEQ ID NO: 130; wherein the second binding domain is a Fab; and (c) a third binding domain, Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Includes two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of any one of items 1 to 13, 15, or 17.

[0249] Item 20A. The second binding domain (B) specific for FOLR1 comprises: A pair of VH and VL chains having sequences as set forth in a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 120 and 121, respectively, or a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 130 and 131, respectively. 18. The antibody construct of any one of items 1 to 17, comprising:

[0250] Item 20B. The antibody construct of any one of items 1 to 13, 15, 16, or 18, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 142-143 and 138-139, with SEQ ID NOs: 142-143 being preferred.

[0251] Item 21. The antibody construct of any one of items 1 to 20B, which induces less CD16A shedding compared to a control construct having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-135, 136-137, and 140-142.

[0252] Item 22. A nucleic acid molecule comprising a sequence encoding the antibody construct of any one of items 1 to 21.

[0253] Item 23. A vector comprising the nucleic acid molecule of Item 22.

[0254] Item 24. A host cell comprising the nucleic acid molecule of Item 22 or the vector of Item 23.

[0255] Item 25. A method for producing an antibody construct according to any one of items 1 to 21, comprising culturing a host cell according to item 24 under conditions allowing expression of the antibody construct according to any one of items 1 to 21, and recovering the produced antibody construct from the culture.

[0256] Item 26. A pharmaceutical composition comprising the antibody construct of any one of items 1 to 21 or the antibody construct produced by the method of item 25.

[0257] Item 27. The antibody construct of any one of items 1 to 21 for use in therapy.

[0258] Item 28. The antibody construct of any one of items 1 to 21 or the antibody construct produced by the method of item 25 for use in the prevention, treatment, or amelioration of a disease selected from a proliferative disease, a tumor disease, a viral disease, or an immunological disorder.

[0259] Item 29. The antibody construct for use according to item 28, wherein the neoplastic disease is a solid tumor, preferably a malignant solid tumor.

[0260] Item 30. The antibody construct for use in accordance with item 29, wherein the solid tumor is selected from the group consisting of ovarian cancer, breast cancer (particularly TNBC), renal cancer, lung cancer, colorectal cancer (CRC), renal cancer (particularly ccRCC), pancreatic cancer (particularly PDAC), endometrial cancer, and brain cancer.

[0261] Item 31. A method for treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder, comprising administering to a subject in need thereof an antibody construct according to any one of items 1 to 21 or an antibody construct produced by the method of item 25.

[0262] Item 32. A kit comprising the antibody construct of any one of items 1 to 21 or the antibody construct produced by the method of item 25, the nucleic acid of item 22, the vector of item 23, and / or the host cell of item 24.

[0263] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context specifically dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or used in place of the methods described herein.

[0264] Unless otherwise specified, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0265] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term".

[0266] As used herein, the term "about" or "approximately" means within 10% (plus (+) or minus (-)), preferably within 5%, more preferably within 2%, and even more preferably within 1% of a given value or range. However, the term also includes the actual number, for example, about 20 includes 20.

[0267] The terms "less than" or "greater than" are inclusive of the actual number. For example, less than 20 means "less than" or "equal to." Similarly, "greater than" or "greater than" means "greater than" or "equal to," or "greater than" or "equal to," respectively.

[0268] Throughout this specification and the claims which follow, unless the context dictates otherwise, the word "comprise," and variations such as "comprises" and "comprising," are understood to mean the inclusion of a recited integer or step, or group of integers or steps, but not the exclusion of any other integers and steps or groups of integers and steps. As used herein, the term "comprising" can be replaced by the terms "containing" or "including," or, as sometimes used herein, the term "having."

[0269] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0270] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. For example, disclosure of the term "comprising" includes disclosure of the term "consisting essentially of" as well as disclosure of the term "consisting of."

[0271] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0272] All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, and the like, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, the present specification shall take precedence over any such material.

[0273] A further understanding of the present invention and its advantages will be gained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0274] Example 1: Detection of interaction between CD16A and CD16A-binding domain method Human CD16A 158V , CD16A 158FMonovalent interaction kinetics of CD123×CD16A ICE to cynomolgus CD16 and CD123×CD16A ICE to cynomolgus CD16 were analyzed at 37°C using a Biacore T200 instrument (GE Healthcare) equipped with a research-grade sensor chip CAP (Biotin CAPture Kit, GE Healthcare) pre-equilibrated in HBS-P+ running buffer. For multivalent interaction analysis, biotinylated mFc.silencing / Avi-tagged antigen was captured (FC2, FC4) to a density of 120-200 RU, after which CD123×CD16A ICE (concentrations: 0-60 nM) was injected at a flow rate of 40 μL / min for 240 s and the complex was allowed to dissociate at the same flow rate for 300 s.

[0275] After each cycle, the chip surface was regenerated with 6 M guanidine-HCl, 0.25 M NaOH and reloaded with biotin capture reagent. Interaction kinetics were determined by fitting data from multi-cycle kinetic experiments to a simple 1:1 interaction model using the local data analysis options (Rmax and RI) available within the Biacore T200 evaluation software (v3.1). Reference measurements were performed on flow cells without ligand capture (Fc2-Fc1, Fc4-Fc3).

[0276] result Human CD16A 158V , CD16A 158F Binding of CD123×CD16A ICE to cynomolgus monkey CD16 was measured using biotin-captured recombinant CD16A. 158V , CD16A 158F , and cynomolgus CD16 (ligand), as well as scFv-IgAb_268 (CD16a1 × CD123-1), scFv-IgAb_148 (CD16a2 × CD123-1), and scFv-IgAb_264 (CD16a1 × CD123-2) (analytes) in a multivalent multicycle kinetic setting at 37°C (n = 3; 2)n=1) were measured by SPR. Affinity and kinetic parameters were evaluated for interactions with human CD16A and cynomolgus CD16 using a 1:1 binding model. All molecules showed high interactions with human CD16A and cynomolgus CD16, with apparent affinities of K D The range was 0.195 nM to 2.48 nM (Figure 1).

[0277] Example 2: Binding of CD123xCD16A constructs to cell lines expressing human CD16A method

[0278] (Table 2) Antibody constructs TIFF2024543828000005.tif49128

[0279] Flp-In CHO host cell culture Flp-In CHO cells (Life Technologies, R75807), a derivative of CHO-K1 Chinese hamster ovary cells, were adapted to growth in suspension in HyClone CDM4CHO medium (Cytiva, catalog SH30557.02) supplemented with L-glutamine (Invitrogen, catalog 25030-024), HT supplement (Thermo Fisher Scientific, catalog 41065012), penicillin / streptomycin (Invitrogen, catalog 1540-122), and 100 μg / mL Zeocin (Thermo Fisher Scientific, catalog R250-01). Single-cell derived clonal lines were obtained by limiting dilution cloning in a medium mixture of standard culture medium and Ham's F-12 (Thermo Fisher Scientific, Cat. 11500586) supplemented with InstiGRO CHO supplement (Solentim, Cat. RS-1105), expanded, and cryopreserved in medium containing 10% DMSO (Sigma, Cat. D2650). Cultures were routinely subcultured after 2 or 3 days, diluted into fresh medium to 3E+5 viable cells / mL for the subsequent 2-day passages, or 2E+5 viable cells / mL for the 3-day passages, and grown in shake flasks or tubes at 37°C, 5% CO2, and 120–200 rpm depending on vessel type.

[0280] Generation of stably transfected antigen-expressing cells (cAg) Suspension-adapted Flp-In CHO host cells were subcultured in standard medium without Zeocin one day before transfection. Recombinant CHO cells were generated by transfection of 2E+6 cells in 2 mL of CHO-S-SFMII medium (Thermo Fisher Scientific, catalog 12052-114) with an expression plasmid encoding a recombinant cell anchor antigen sequence (cAg) in a modified version of the pcDNA5 / FRT vector mediating puromycin or hygromycin resistance and an expression plasmid encoding Flp recombinase (pOG44, Thermo Fisher, V600520) using a total of 2.5 μg of DNA at a DNA:PEI ratio of 1:2.5 (μg / μg) and Transporter 5 transfection reagent. DNA and transfection reagent were mixed in 100 μL of 0.9% NaCl solution (Sigma, catalog S8776) and incubated for 20 min before addition to the cells. As a negative control (mock), cells were transfected with a control plasmid that does not mediate resistance. After 4 hours, the transfected cells were diluted in 8 mL of a 1:1 medium mixture of standard culture medium and Ham's F-12. Selection of stably transfected cells was initiated the next day by the addition of 3.2 μg / mL puromycin dihydrochloride (Thermo Fisher Scientific, Cat. A1113803) as the selection antibiotic and increased to 6.3 μg / mL on the second day, or 500 μg / mL hygromycin B (Thermo Fisher Scientific, Cat. 10687010). Viable cell density was measured twice a week, and cells were centrifuged and resuspended in fresh selection medium containing the selection antibiotic at a maximum density of 2-4E+5 viable cells / mL. The concentration of puromycin dihydrochloride was increased to 7.0 μg / mL on the 10th day after transfection. Stably transfected cell pools regained proliferation and viability after approximately 2-3 weeks, were expanded in standard culture medium, and were cryopreserved in freezing medium containing 7.5% DMSO.For analysis of antigen expression, cultures were expanded in shake flasks or tubes, subcultured after 2 or 3 days, and then diluted into fresh medium to 6E+5 viable cells / mL for 2-day passages or 3E+5 viable cells / mL for 3-day passages, and grown at 37°C, 5% CO2, and 120-200 rpm depending on vessel type.

[0281] Flow cytometry analysis To analyze the binding of the different antibody constructs to human CD16A (cAg_34)-transfected CHO cells relative to CD16 expression by flow cytometry, 1–2 × 10 5 Cells were resuspended in 100 μL of FACS buffer (PBS (Invitrogen, Cat: 14190-169) containing 2% heat-inactivated FCS (Invitrogen, Cat: 10270-106) and 0.1% sodium azide (Roth, Karlsruhe, Germany, Cat: A1430.0100)) in a round-bottom 96-well microtiter plate. After washing in FACS buffer, cells were incubated for 30 min on ice in the dark in 50 μL of FACS buffer containing either no antibody or titrated antibody starting at a concentration of 100 μg / mL followed by ten 5-fold serial dilutions. After two washes, cells were incubated with APC-conjugated goat anti-human IgG (H+L)-APC (Dianova, Cat: 109-136-088) for 30 min on ice in the dark. As a control, cells were incubated with anti-human CD16-BV421 (clone 3G8, Biolegend, catalog 302038) alone. After washing, binding was measured by flow cytometry and mean fluorescence intensity (MFI) of cell samples was calculated and corrected for background staining with control cells stained with secondary antibody only.

[0282] statistical analysis The equilibrium dissociation constant (K D), mean and standard deviation (SD) were calculated by plotting MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism for Windows (v9; GraphPad Software; La Jolla California USA).

[0283] result The apparent avidity of scFv-IgAb_268 (CD123×CD16A) and scFv-IgAb_148 (CD123×CD16A) for human (hu)CD16A was measured. CHO cells expressing recombinant huCD16A (cAg_34) were incubated with increasing concentrations of scFv-IgAb_268, scFv-IgAb_148 and binding was assessed by flow cytometry compared to a control molecule (scFv-IgAb_139). CD16 expression on huCD16A CHO cells was confirmed using the anti-human CD16A antibody clone 3G8 (Figure 2). The antibody construct scFv-IgAb_268 had an average K D 16.3 nM, demonstrating higher dose-dependent binding to huCD16A compared to scFv-IgAb_148, which yielded an average K D (Figure 2, Table 3). No binding was detected by a negative control molecule (CD123xRSV, scFv-IgAb_139), which contains the same antibody scaffold and CD123 targeting domain as scFv-IgAb_268, but contains an irrelevant anti-RSV domain instead of CD16A. Thus, these results confirm the higher binding specificity of scFv-IgAb_268, which contains the CD16a1 anti-CD16 effector domain, to human CD16A, compared to scFv-IgAb_148, which contains the CD16a2 anti-CD16 effector domain.

[0284] Table 3. Mean apparent avidity (KD) of scFv-IgAb_268, scFv-IgAb_148, and control antibodies for human CD16A expressed on CHO cells. Binding of antibody constructs to huCD16A transfected CHO cells measured by flow cytometry for titrated scFv-IgAb_268 (CD123×CD16A), scFv-IgAb_148 (CD123×CD16A), and negative control molecules (scFv-IgAb_139, CD123×RSV). The equilibrium dissociation constants (KD) of antibody binding were calculated by plotting the MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism. SD, standard deviation; na, not applicable. TIFF2024543828000006.tif80141

[0285] Example 3: Evaluation of cell surface retention of anti-CD123 antibodies on NK cells method

[0286] (Table 4) Antibody constructs TIFF2024543828000007.tif47135

[0287] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0288] Flow cytometric detection of cell surface retention on NK cells NK cells were cultured in pre-chilled complete RPMI 1640 medium at 10–15 × 10 6The NK cells were suspended in a volume of 1 mL at a density of 100 μg / mL cells / mL. Antibody constructs were added to a concentration of 100 μg / mL and incubated on ice for 45 minutes. 10 mL of complete RPMI 1640 medium was then added, the cell suspension was divided into two equal volumes, washed twice with complete RPMI 1640 medium, and each NK cell suspension was resuspended in 10 mL of complete RPMI 1640 medium. For each dissociation time, an aliquot of 1 mL of NK cell suspension was then transferred into a single tube containing 9 mL of pre-warmed complete RPMI 1640 medium. The diluted NK cell suspension was then placed in a 37°C water bath for the respective duration to dissociate bound antibodies and placed on ice to stop dissociation. The "0 min" sample was transferred directly onto ice. Cell aliquots were washed once with FACS buffer (PBS (Invitrogen, Cat: 14190-169) containing 2% heat-inactivated FCS (Invitrogen, Cat: 10270-106) and 0.1% sodium azide (Roth, Karlsruhe, Germany, Cat: A1430.0100)) and transferred to 96-well round-bottom plates for detection of cell surface-retained antibodies by flow cytometry. The cell surface bound scFv-IgAb_268, scFv-IgAb_148, and IgAb_338 were detected by staining with 10 μg / mL of anti-anti-CD123 mAb (clone 8-1-1), followed by incubation with 15 μg / mL of FITC goat anti-mouse IgG (Dianova, catalog 115-095-062), and staining with the fixable viability stain eFluor™ 780 (Fisher Scientific, catalog: 65-0865-14) to exclude dead cells. After a final washing step, the cells were resuspended in 0.2 mL of FACS buffer, the fluorescence of the cells was measured using a flow cytometer, and the median fluorescence intensity of the cell samples was calculated. After subtracting the fluorescence intensity values ​​of cells stained with only the secondary and / or tertiary reagents, the MFI value at time point 0 was taken as 100%, and the percentage of remaining antibody was analyzed by nonlinear regression using GraphPad Prism for Windows (v9; GraphPad Software; La Jolla California USA).

[0289] result To assess the retention of constructs on the surface of NK cells, primary human NK cells were preloaded with anti-CD123 antibody constructs containing various effector domains against CD16A. The Fc-enhanced anti-CD123 IgG1 antibody (IgAb_338) dissociated very rapidly from NK cells, reaching a lower plateau after the first 5-10 min. CD123xCD16A scFv-IgAb_148, containing the CD16a2 effector domain, showed lower dissociation, reaching a plateau of 20% remaining antibody after 48 h (Figure 3). In contrast to scFv-IgAb_148, which contains Fc-enhanced IgG1 and a CD16a2 anti-CD16A domain, AFM28 (CD123xCD16A scFv-IgAb_268), which contains a CD16a1 anti-CD16A effector domain, showed substantially longer retention (approximately 60%) on NK cells after dissociation for 24 and 48 hours at 37°C (Figure 3, Table 5).

[0290] Table 5. Percentage of remaining antibodies on NK cells after 24 hours [%] Enriched primary human NK cells were preloaded with 100 μg / mL of CD123 / CD16A scFv-IgAb_268, Fc-enhanced anti-CD123 IgG1 (IgAb_338), or CD123 / CD16A scFv-IgAb_148 on ice, washed, and then incubated at 37° C. for the indicated periods in an excess volume of complete RPMI1640 medium to allow dissociation and prevent reassociation. Residual antibodies after 24 h were measured by flow cytometry, the median fluorescence intensity (MFI) value at time point 0 was taken as 100%, and the percentage of remaining antibodies was analyzed using GraphPad Prism. SD, standard deviation. TIFF2024543828000008.tif71141

[0291] Example 4: ADCC against CD123+ EOL-1 cells by anti-CD123 antibodies method

[0292] (Table 6) Antibody constructs TIFF2024543828000009.tif69135

[0293] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0294] Culture of EOL-1 tumor cell line The EOL-1 cell line was cultured in complete RPMI medium (RPMI 1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, 100 μg / mL streptomycin sulfate) in a humidified atmosphere at 37 °C and 5% CO2 under standard conditions recommended by the supplier (DSMZ, catalog: ACC-386).

[0295] Calcein release cytotoxicity assay Antibody-mediated target cell lysis by NK cells in vitro was assessed by quantifying the release of calcein from calcein-labeled target cells into the cell culture supernatant. For this, target cells were labeled with 10 μM calcein AM for 30 min at 37° C. in RPMI 1640 medium without FCS. After gentle washing, calcein-labeled cells were diluted in complete RPMI medium at 1 × 10 5 The cells were then resuspended at a density of 1 × 10 4 Target cells were seeded into individual wells of round-bottom 96-well microtiter plates and mixed with enriched human NK cells at an effector to target cell (E:T) ratio of 5:1 unless otherwise stated. Cultures of NK cells with target cells were performed in duplicate without antibody addition or in the presence of titrated antibodies starting at a concentration of 25 μg / mL followed by 10 two-fold serial dilutions. After centrifugation at 200×g for 2 min, the microtiter plates were incubated for 4 h at 37° C. in a humidified atmosphere containing 5% CO2. Spontaneous calcein release, maximum release, and killing of targets by effectors in the absence of antibody were measured in quadruplicate on each plate. Spontaneous release was measured by incubation of target cells in the absence of effectors and antibodies. Maximum release was achieved by adding Triton X-100 to a final concentration of 1% in the absence of effector cells and antibodies. Following incubation, 100 μL of cell-free cell culture supernatant was collected from each well after centrifugation at 500×g for 5 min and transferred to a black flat-bottom 96-well microplate. The fluorescence counts of released calcein were measured at 520 nm using a multimode plate reader. Specific cell lysis was calculated according to the following formula: [fluorescence(sample)−fluorescence(spontaneous)] / [fluorescence(maximum)−fluorescence(spontaneous)]×100%, where "fluorescence(spontaneous)" and "fluorescence(maximum)" are defined as the fluorescence in the absence of effector cells and antibodies, and the fluorescence induced by the addition of Triton X-100, respectively.

[0296] result All four CD123xCD16A scFv-IgAb constructs induced NK cell-dependent lysis against EOL-1 cells with similar maximal efficacy in the low picomolar range (Fig. 4).

[0297] Example 5: Evaluation of inhibition of CD16A shedding on activated NK cells in the presence of AFM28 method

[0298] (Table 7) Antibody constructs TIFF2024543828000010.tif43130

[0299] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0300] Flow cytometric detection of CD16A expression on NK cells NK cells were cultured in pre-chilled complete RPMI 1640 medium at 10–15 × 10 6 The NK cells were suspended in a volume of 1 mL at a density of 100, 10, and 1 μg / mL. Antibody constructs were added at concentrations of 100, 10, and 1 μg / mL and incubated on ice for 45 minutes. The cells were then washed with complete RPMI 1640 medium and transferred to a 96-well round-bottom plate. The NK cells were incubated with or without 50 ng / mL PMA and 0.5 μM ionomycin for 4 hours at 37° C. After stimulation, the cells were washed with FACS buffer (PBS (Invitrogen, Catalog: 14190-169) containing 2% heat-inactivated FCS (Invitrogen, Catalog: 10270-106) and 0.1% sodium azide (Roth, Karlsruhe, Germany, Catalog: A1430.0100)). To detect CD16 levels, cells were re-stained with 100 μg / mL scFv-IgAb_268, scFv-IgAb_148, or IgAb_338, followed by incubation with 15 μg / mL FITC-conjugated goat anti-mouse IgG (Dianova, Cat. 115-095-062) and staining with fixable viability stain eFluor™ 780 (Fisher Scientific, Cat.: 65-0865-14) to exclude dead cells. After a final washing step, cells were resuspended in 0.2 mL FACS buffer, the fluorescence of cells was measured using a flow cytometer, and the median fluorescence intensity of the cell samples was calculated. After subtracting the fluorescence intensity values ​​of cells stained with the secondary reagent only, the MFI values ​​were plotted using GraphPad Prism software (v8.0 / 9.06.0 / 7.0; GraphPad Software; La Jolla California USA). Figures were generated using FlowJo software (v10.6 / 10.8, FlowJo Software, BD Ashland USA).

[0301] statistical analysis Quantitative variables were compared using the paired Student's t-test. Statistical significance was assessed with GraphPad Prism software (v9.0). A p-value <0.05 was considered significant.

[0302] result Primary human NK cells were preloaded with anti-CD123 constructs containing various effector domains against CD16A and stimulated with PMA / ionomycin. CD16 expression levels were assessed by flow cytometry. As explained, NK cells stimulated with PMA / ionomycin show no expression of CD16 compared to non-stimulated cells (Figures 5A-5C, 6A-6C). This phenomenon was described in the literature as shedding of CD16 in response to NK cell stimulation (Romee R. et al. 2013). NK cells incubated with various concentrations of Fc-enhanced anti-CD123 IgG1 antibody (IgAb_338) followed by PMA / ionomycin stimulation showed a similar effect (Figures 5C, 6C). Interestingly, high concentrations of CD123 / CD16A scFv-IgAb_268 (100 μg / mL), which contains the CD16a1 anti-CD16A effector domain, showed significantly higher levels of CD16 expression after stimulation compared to unstimulated NK cells (Figure 5A, Figure 6A). Furthermore, we could observe concentration-dependent shedding inhibition by scFv-IgAb_268, and to a lesser extent by scFv-IgAb_148 (CD123 / CD16A), which contains the CD16a2 anti-CD16A effector domain (Figure 5A-5B, Figure 6A-5B). However, the CD16 shedding inhibitory effect on stimulated NK cells was more strongly induced by scFv-IgAb_268 compared to the shedding inhibition induced by scFv-IgAb_148.

[0303] Example 7: Target cell-independent activation of NK cells by anti-CD123 antibodies method

[0304] (Table 8) Antibody constructs TIFF2024543828000011.tif47128

[0305] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0306] Culture and flow cytometric analysis Buffy coat-derived NK cells (5 × 10 4 (1000 cells) were cultured overnight in complete RPMI medium in 96-well microtiter plates with or without titrated antibodies starting at a concentration of 40 μg / mL followed by five 10-fold serial dilutions. Upregulation of the NK cell activation marker CD137 on CD56+ CD45+ CD3- CD19- NK cells was then assessed by flow cytometry after extracellular staining with fluorescently conjugated mouse anti-human antibodies diluted in 50 μL of FACS buffer. The percentage of CD137 positive NK cells is shown.

[0307] result Of the four anti-CD123 antibodies, the CD123xCD16A scFv-IgAb construct comprising the anti-CD16A CD16a1 domain showed the lowest activity in upregulating the activation marker CD137 on NK cells in the absence of CD123+ target cells. At the highest tested concentration of 40 μg / mL, scFv-IgAb_268 appeared to have the lowest nonspecific activity in activating NK cells, followed by scFv-IgAb_267, scFv-IgAb_265, and scFv-IgAb_264 (Figure 7).

[0308] Example 7: Target cell-dependent activation of NK cells by anti-CD123 antibodies method

[0309] (Table 9) Antibody constructs TIFF2024543828000012.tif67135

[0310] Cultivation of tumor cell lines The EOL-1 cell line was cultured in complete RPMI medium (RPMI 1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, 100 μg / mL streptomycin sulfate) in a humidified atmosphere at 37 °C and 5% CO2 under standard conditions recommended by the supplier (DSMZ, catalog: ACC-386).

[0311] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0312] Co-culture and flow cytometry analysis CMFDA-labeled EOL-1 cells (5 × 10 4 buffy coat-derived allogeneic NK cells (5 × 10 cells) were cultured in complete RPMI medium in 96-well microtiter plates in the presence of titrated antibodies or control molecules starting at a concentration of 50 μg / mL followed by six 10-fold serial dilutions. 4 (cells) at a 1:1 cell ratio for 24 h. Afterwards, upregulation of the NK cell activation marker CD137 on CD56+ CD45+ CD3- CD19- NK cells was assessed by flow cytometry after extracellular staining with fluorescently conjugated mouse anti-human antibodies diluted in 50 μL of FACS buffer. The percentage of CD137 positive NK cells is shown.

[0313] result All four CD123xCD16A scFv-IgAb constructs specifically induced upregulation of the activation marker CD137 on NK cells in response to CD123+ EOL-1 cells (Figure 8). Notably, the antibody constructs comprising the anti-CD16A CD16a1 domain peaked in the percentage of CD137+ NK cells at 0.05 μg / mL, followed by a decrease in the percentage of CD137+ NK cells at higher concentrations. In contrast, the antibody constructs comprising the anti-CD16A CD16a2 domain led to a continuous increase in the percentage of CD137+ NK cells up to the highest tested concentration of 50 μg / mL. A non-CD123 targeting RSVxCD16A control antibody construct in which CD123 was replaced by a non-binding RSV domain was unable to induce NK cell activation in response to EOL-1 cells.

[0314] Example 8: Binding of CD123xCD16A constructs to CD123+ and CD123- tumor cell lines method

[0315] (Table 10) Antibody constructs TIFF2024543828000013.tif47128

[0316] Cultivation of tumor cell lines The EOL-1 (DSMZ, catalog: ACC-386) and Karpas-299 (DSMZ, catalog: ACC-31) cell lines were cultured in complete RPMI medium (RPMI 1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, 100 U / mL penicillin G sodium, 100 μg / mL streptomycin sulfate) in a humidified atmosphere at 37 °C and 5% CO2 under standard conditions recommended by the supplier. Adherent A-431 cells were detached by accutase treatment and maintained in complete DMEM medium (Dulbecco's modified Eagle's medium with 10% hi FCS, 2 mM L-glutamine, 100 U / mL penicillin G sodium, 100 μg / mL streptomycin sulfate) in a humidified atmosphere at 37 °C and 5% CO2 under standard conditions recommended by the supplier (DSMZ, catalog: ACC-91).

[0317] Flow cytometry analysis For analysis of binding of the CD123xCD16A antibody construct to CD123+ EOL-1 cells, CD123- A-431 cells, and CD123- Karpas-299 cells by flow cytometry, 1 × 10 5 Cells were resuspended in 100 μL of FACS buffer in a round-bottom 96-well microtiter plate. After washing in FACS buffer, cells were incubated for 45 min on ice in the dark in 100 μL of FACS buffer with either no antibody or titrated antibody starting at a concentration of 100 μg / mL followed by eight 10-fold serial dilutions. After two washes, cells were incubated with APC-conjugated goat anti-human IgG(H+L)-APC (1 / 200 dilution) for 30 min on ice in the dark. After washing, binding was measured by flow cytometry and mean fluorescence intensity (MFI) of cell samples was calculated and corrected for background staining using control cells stained with secondary antibody only.

[0318] result All four CD123xCD16A scFv-IgAb constructs showed comparable binding to CD123+ EOL-1 cells (Figure 9A). In contrast, to CD123- A431 cells, scFv-IgAb_268, composed of the CD123-1 domain and the CD16a1 binding domain, showed the lowest potential for non-specific binding. Overall, across the various antibody construct batches tested, scFv-IgAb_268 showed the least non-specific binding to CD123- A-431 cells, followed by scFv-IgAb_265, followed by scFv-IgAb_267, followed by scFv-IgAb_264 (Figure 9B).

[0319] Example 9: NK cell-dependent depletion of primary leukemic blasts mediated by anti-CD123 antibodies method

[0320] (Table 11) Antibody constructs TIFF2024543828000014.tif48145

[0321] Table 12. Primary samples from AML patients TIFF2024543828000015.tif75168

[0322] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) of healthy donors by density gradient centrifugation using SepMate-50 tubes. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 775×g for 20 min at ambient temperature with brake. PBMCs located at the interface were harvested and washed three times with PBS. PBMCs were maintained in RPMI 1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (referred to as complete RPMI medium, all components from Invitrogen) at 37°C and 5% CO2 in a humidified atmosphere until use.

[0323] For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions. NK cells were resuspended in complete RPMI medium and used immediately.

[0324] Thawing of primary AML patient material Cryopreserved peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from AML patients were obtained from commercial biobanks (Cureline, USA; Tissue Solutions, UK) and thawed according to the manufacturer's instructions, briefly outlined as follows: Cureline: Cells in cryovials were thawed for 1-2 min at 37°C, then quickly transferred to pre-warmed (37°C) complete RPMI medium, washed, and immediately subjected to functional assays. Tissue Solutions: Cells in cryovials were thawed for 1-2 min at 37°C, then quickly transferred to chilled (4°C) complete RPMI medium, washed, and immediately subjected to functional assays. The number of viable cells was determined by trypan blue exclusion.

[0325] Calcein release cytotoxicity assay PB (PBMC) or BM (BMMC) from AML patients containing 49-86% leukemic blasts (0.5 × 10 5 ) in complete RPMI medium in 96-well microtiter plates in the presence of titrated AFM28 (CD123xCD16A scFv-IgAb_268), control molecules, or in the absence of antibody constructs, with buffy coat-derived allogeneic NK cells (0.5x10 5AML samples were co-cultured with 1:1 cells (1:1 cells) for 24 hours. To support survival of leukemic blasts of patient-derived AML samples, the co-cultures were supplemented with 20ng / mL GM-CSF (PeproTech, Cat:300-03). In one experiment with AML 1 sample, allogeneic NK cells were fluorescently labeled with CMFDA prior to the assay to guide the discrimination between tumor cells and NK cells. The cell suspension was then subjected to extracellular staining of NK cell surface markers and markers to aid in the determination of AML blasts within PBMCs and BMMCs from AML patients, followed by Annexin V staining to distinguish viable tumor cells from pre-apoptotic (Annexin V+dead cell marker-) and dead (Annexin V+dead cell marker+) cells. The percentage of NK cell-dependent AFM28-mediated tumor cell reduction was assessed by flow cytometry and compared to tumor cell reduction by NK cells in the absence of AFM28.

[0326] Flow cytometry analysis Extracellular staining of NK cell and tumor cell surface markers was performed in round-bottom 96-well microtiter plates with the indicated fluorescently labeled antibodies diluted in 50 μL of FACS buffer for 30 min on ice in the dark. Cells were then washed once and twice in FACS buffer, followed by measurement on a CytoFlex S flow cytometer (Beckman Coulter) and analysis with CytExpert software (v2.4, Beckman Coulter). Leukemic blasts in AML PB and BM samples were identified using a combination of markers CD45 (Biolegend, Cat: 304048), CD33 (Biolegend, Cat: 366612), and CD34 (Biolegend, Cat: 343534), as shown in the table of primary AML samples above. To describe the percentage of leukemic blasts positive for CD123 (BD ​​Bioscience, Cat: 563599), the cutoff for CD123 negativity was determined based on the percentage of lymphocyte subpopulations (CD45 high CD33- CD34- SSC lowCD3+ (Biolegend, Cat: 300448) CD19+ (Biolegend, Cat: 302242) cells).

[0327] result CD123 is overexpressed in many hematological malignancies and has been identified as one of the characteristic markers overexpressed on the surface of primary leukemic blasts and leukemic stem cells in AML patients, whereas in healthy tissues, CD123 expression is rather restricted to hematopoietic cell types, e.g., basophils (Testa, 2019, Cancers, ncbi.nlm.nih.gov / pmc / articles / PMC6769702 / ). Here, we investigated whether AFM28 (CD123×CD16A scFv-IgAb_268) could induce NK cell-mediated reduction of matched peripheral blood and bone marrow primary leukemic blasts from AML patients.

[0328] Buffy coat-derived NK cells were incubated with PBMCs or BMMCs from allogeneic AML patients in the presence of titrated increasing concentrations of AFM28 and control molecules in a 24-h flow cytometry-based tumor cell reduction assay. After co-culture with allogeneic NK cells, the reduction of primary leukemic blasts was significantly and dose-dependently increased in the presence of AFM28. Notably, the Fc-enhanced anti-CD123 IgG, talacuzumab (IgAb_338), showed a lower level of potency than AFM28 and required higher concentrations to reach comparable antitumor activity against primary leukemic blasts (Figures 12A-12B). In conclusion, AFM28 can induce NK cell cytotoxic responses against CD123-positive primary leukemic blasts from peripheral blood and bone marrow of AML patients.

[0329] Example 10: CD34 + / CD123 - Compartment-sparing CD123 from bone marrow samples + Depletion of primary AML blasts and MDS cells by scFv-IgAb_268 Co-culture and flow cytometry analysis Freshly thawed bone marrow cells (5 × 10 4 buffy coat-derived allogeneic NK cells (5 × 10 cells) were cultured in complete RPMI medium in 96-well microtiter plates in the presence of titrated antibodies starting at a concentration of 1000 pM followed by five dilutions (500 pM, 100 pM, 50 pM, 10 pM, 5 pM). 4 (cells) at a 1:1 cell ratio for 24 hours in triplicate. + CD123 from mononuclear cell fraction + Target cell depletion was assessed by flow cytometry after extracellular staining with fluorescently conjugated mouse anti-human antibodies diluted in 50 μL of FACS buffer. Absolute cell numbers (viable, gated on CD45) are shown in the figures.

[0330] result The CD123×CD16A scFv-IgAb_268 construct is + Specifically, it induced a decrease in BMMCs (Figures 13A-13B). Of note, in the combined samples, normal hematopoietic stem cells (CD34 + / CD123 neg No lysis of CD123neg bystander cells, including HSCs, was observed. NK cells alone did not induce lysis of bone marrow cells in the absence of antibody.

[0331] The high specificity and affinity of binding to effector cells via CD16A and to target cells via CD123 allows the binding of CD16A to tumor cells and potentially target positive immunosuppressive cells (e.g., CD34 neg / CD33 + / CD123 + This promotes efficient depletion of BM-MDSCs, resulting in the depletion of other bystander cells and normal hematopoietic stem cells (CD34 + / CD123 neg Limit non-specific lysis of HSCs (Figures 13A-13B).

[0332] Example 11: Preclinical toxicity models in cynomolgus monkeys suggest that scFv-IgAb_268 is well tolerated and pharmacologically active, as demonstrated by reduction in peripheral blood basophils method Ten naive cynomolgus monkeys of Mauritian origin were dosed weekly by 2-h infusion for 4 weeks with a 2-week recovery period (q7d x 28d). Animals were assigned to 4 groups summarized in Table 13.

[0333] (Table 13) TIFF2024543828000016.tif44163

[0334] Toxicity assessment was based on clinical observations, body weight, temperature, clinical pathology and anatomic pathology. Additional endpoints included determination of serum cytokine levels of IL-2, IL-6, IL-8, TNF-α, GM-CSF and INF-γ, as well as flow cytometric assessment of lymphocyte subsets (CD45, CD3, CD4, CD8, CD20, CD16, CD159a). In addition, quantification of basophils and plasmacytoid dendritic cells (pDC) in peripheral blood was incorporated as a pharmacodynamic endpoint (Busfield et al., 2014). Blood was collected for toxicokinetic evaluation of scFv-IgAb_268 and anti-drug antibodies were measured using an electrochemiluminescence immunoassay based on the MSD® platform. A complete necropsy was performed on all animals, organ weights were measured, followed by gross and microscopic examination of all tissues.

[0335] result In this intravenous repeated dose-ranging study, scFv-IgAb_268 did not induce systemic or local toxicity. All animals were clinically well, and no effects on body weight, temperature, or clinical pathology were observed up to the highest tested dose level of 100 mg / kg.

[0336] A notable finding was a transient, non-dose-dependent increase in IL-6 levels 2-4 hours after the start of the infusion. IL-6 levels returned to normal after 24 hours (Figure 14). scFv-IgAb_268 had no effect on IL-2, IL-8, IFN-γ, GM-CSF, and TNF-α levels at any dose. Furthermore, at 100 mg / kg, scFv-IgAb_268 caused a transient reduction in absolute NK cell counts (CD3-CD20-CD159+ positive) after the first dose, as well as a reduction in neutrophil counts on days 22 and 29.

[0337] Four of eight scFv-IgAb_268 treated animals showed marginal or marked splenic enlargement. The test article at 20 or 100 mg / kg induced an increase in hematopoietic cellularity (mild or marked) in sternal and femoral bone marrow and an increase in extramedullary hematopoiesis in the spleen in two female animals.

[0338] Absolute basophil count and pDC count (CD123 + A decrease in IgAb_268 (Figure 15) was observed in peripheral blood at all dose levels 24 hours after the first dose, demonstrating the expected pharmacodynamic effect of scFv-IgAb_268.

[0339] All animals treated with scFv-IgAb_268 were challenged systemically. TK parameters were measured after the first dose and serum t 1 / 2 ranged from 27 to 78 hours. Half-lives are likely underestimated because the β-elimination phase was not fully reached before the end of the dosing interval. Dose-proportional PK was observed that was greater than expected for an IgG-like molecule, as determined by area under the curve (AUC). Five of eight treated animals tested positive for ADA, with 4 / 5 showing response to exposure.

[0340] Example 12: Binding of FOLR1xCD16A constructs to cell lines expressing human CD16A and cynomolgus CD16 method

[0341] (Table 14) Antibody constructs TIFF2024543828000017.tif43158

[0342] Flp-In CHO host cell culture Flp-In CHO cells (Life Technologies, R75807), a derivative of CHO-K1 Chinese hamster ovary cells, were adapted to growth in suspension in HyClone CDM4CHO medium (Cytiva, catalog SH30557.02) supplemented with L-glutamine (Invitrogen, catalog 25030-024), HT supplement (Thermo Fisher Scientific, catalog 41065012), penicillin / streptomycin (Invitrogen, catalog 1540-122), and 100 μg / mL Zeocin (Thermo Fisher Scientific, catalog R250-01). Single-cell derived clonal lines were obtained by limiting dilution cloning in a medium mixture of standard culture medium and Ham's F-12 (Thermo Fisher Scientific, Cat. 11500586) supplemented with InstiGRO CHO supplement (Solentim, Cat. RS-1105), expanded, and cryopreserved in medium containing 10% DMSO (Sigma, Cat. D2650). Cultures were routinely subcultured after 2 or 3 days, diluted into fresh medium to 3E+5 viable cells / mL for the subsequent 2-day passages, or 2E+5 viable cells / mL for the 3-day passages, and grown in shake flasks or tubes at 37°C, 5% CO2, and 120–200 rpm depending on vessel type.

[0343] Generation of stably transfected antigen-expressing cells (cAg) Suspension-adapted Flp-In CHO host cells were subcultured in standard medium without Zeocin one day before transfection. Recombinant CHO cells were generated by transfection of 2E+6 cells in 2 mL of CHO-S-SFMII medium (Thermo Fisher Scientific, catalog 12052-114) with an expression plasmid encoding a recombinant cell anchor antigen sequence (cAg) in a modified version of the pcDNA5 / FRT vector mediating puromycin or hygromycin resistance and an expression plasmid encoding Flp recombinase (pOG44, Thermo Fisher, V600520) using a total of 2.5 μg of DNA at a DNA:PEI ratio of 1:2.5 (μg / μg) and Transporter 5 transfection reagent. DNA and transfection reagent were mixed in 100 μL of 0.9% NaCl solution (Sigma, catalog S8776) and incubated for 20 min before addition to the cells. As a negative control (mock), cells were transfected with a control plasmid that does not mediate resistance. After 4 hours, the transfected cells were diluted in 8 mL of a 1:1 medium mixture of standard culture medium and Ham's F-12. Selection of stably transfected cells was initiated the next day by the addition of 3.2 μg / mL puromycin dihydrochloride (Thermo Fisher Scientific, Cat. A1113803) as the selection antibiotic and increased to 6.3 μg / mL on the second day, or 500 μg / mL hygromycin B (Thermo Fisher Scientific, Cat. 10687010). Viable cell density was measured twice a week, and cells were centrifuged and resuspended in fresh selection medium containing the selection antibiotic at a maximum density of 2-4E+5 viable cells / mL. The concentration of puromycin dihydrochloride was increased to 7.0 μg / mL on the 10th day after transfection. Stably transfected cell pools regained proliferation and viability after approximately 2-3 weeks, were expanded in standard culture medium, and were cryopreserved in freezing medium containing 7.5% DMSO.For analysis of antigen expression, cultures were expanded in shake flasks or tubes, subcultured after 2 or 3 days, and then diluted into fresh medium to 6E+5 viable cells / mL for 2-day passages or 3E+5 viable cells / mL for 3-day passages, and grown at 37°C, 5% CO2, and 120-200 rpm depending on vessel type.

[0344] Flow cytometry analysis To analyze the binding of the various antibody constructs to CHO cells transfected with human CD16A(158F) (cAg_34), human CD16A(158V) (cAg_35), and cynomolgus monkey CD16 (cAg_36) relative to CD16 expression by flow cytometry, 1–5 × 10 5 Cells were resuspended in 100 μL of FACS buffer (PBS (BioWest, Catalog: L0615-500) containing 2% heat-inactivated FCS (Invitrogen, Catalog: 10500-064) and 0.1% sodium azide (Sigma, Catalog: S8032_100G)) in a round-bottom 96-well microtiter plate. After washing in FACS buffer, cells were incubated for 40-50 min on ice in the dark in 50 μL of FACS buffer containing no antibody or titrated antibody starting at a concentration of 1000 nM followed by eleven 3-fold serial dilutions. After two washes, cells were incubated with FITC-conjugated goat anti-human IgG (H+L) (Jackson Immunologies, Catalog: 109-096-088) for 40-50 min on ice in the dark. As a control, cells were incubated with anti-human CD16-FITC (clone 3G8, Biolegend, catalog 302006) alone. After washing, binding was measured by flow cytometry and mean fluorescence intensity (MFI) of cell samples was calculated and corrected for background staining with control cells stained with secondary antibody only.

[0345] statistical analysis The equilibrium dissociation constant (K D), mean and standard deviation (SD) were calculated by plotting MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism for Windows (v9; GraphPad Software; La Jolla California USA).

[0346] result The apparent avidity of scFv-IgAb_381 and scFv-IgAb_387 for human (hu)CD16A transfected CHO cells (both 158F and 158V allotypes) and cynomolgus monkey (cy)CD16 transfected CHO cells was measured. CHO cells expressing recombinant huCD16A(158F)(cAg_34), huCD16A(158V)(cAg_35), and cyCD16(cAg_36) were incubated with increasing concentrations of scFv-IgAb_381 and scFv-IgAb_387, and binding was assessed by flow cytometry relative to a control molecule (scFv-IgAb_162). The expression of human CD16A and cynomolgus CD16 on transfected CHO cells was confirmed using anti-human CD16A-FITC antibody clone 302006 (Biolegend) (Figures 16A, 16B, and 16C). The antibody construct scFv-IgAb_387 showed higher concentration-dependent binding to huCD16A(158F), huCD16A(158V), and cyCD16 than scFv-IgAb_381 (Figures 16A, 16B, 16C, and Table 15). No binding was detected by a negative control molecule (FOLR1 x RSV, scFv-IgAb_162), which contains the same antibody scaffold and FOLR1 targeting domain as scFv-IgAb_381 and scFv-IgAb_387, but contains an irrelevant anti-RSV domain instead of CD16A. Thus, these results confirm the higher binding specificity of scFv-IgAb_387, which contains the CD16a3 anti-CD16A effector domain, to human CD16A and cynomolgus CD16 compared to scFv-IgAb_381, which contains the CD16a4 anti-CD16A effector domain.

[0347] Table 15. Mean apparent avidity (K) of scFv-IgAb_381, scFv-IgAb_387, and control antibodies for human CD16A (both 158F and 158V allotypes) and cynomolgus monkey CD16 expressed on CHO cells. D ) Binding of antibody constructs to huCD16A (158F and 158V) and cyCD16 transfected CHO cells was measured by flow cytometry. The equilibrium dissociation constant (KD) of antibody binding was calculated by plotting the MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism. SD, standard deviation; na, not applicable. TIFF2024543828000018.tif61156

[0348] Example 13: ADCC against FOLR1+ A2780 cells by anti-FOLR1 antibodies method

[0349] (Table 16) Antibody constructs TIFF2024543828000019.tif56145

[0350] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0351] Cultivation of A2780 tumor cell line The A2780 cell line was cultured in complete RPMI medium (RPMI 1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, 100 U / mL sodium penicillin G, 100 μg / mL streptomycin sulfate) in a humidified atmosphere at 37 °C and 5% CO2 under standard conditions recommended by the supplier.

[0352] Calcein release cytotoxicity assay Antibody-mediated target cell lysis by NK cells in vitro was assessed by quantifying the release of calcein from calcein-labeled target cells into cell culture supernatants. For this, target cells were labeled with 10 μM calcein AM in RPMI 1640 medium without FCS for 30 min at 37 °C. After gentle washing, calcein-labeled cells were diluted in complete RPMI medium at 1 × 10 5 The cells were then resuspended at a density of 1 × 10 4 Target cells were seeded into individual wells of round-bottom 96-well microtiter plates and mixed with enriched human NK cells at an effector to target cell (E:T) ratio of 1.25:1 unless otherwise stated. Cultures of NK cells with target cells were performed in duplicate without the addition of antibody or in the presence of increasing concentrations of antibody. After centrifugation at 200×g for 2 min, the microtiter plates were incubated for 4 h at 37° C. in a humidified atmosphere containing 5% CO2. Spontaneous calcein release, maximum release, and killing of targets by effectors in the absence of antibody were measured in quadruplicate on each plate. Spontaneous release was measured by incubation of target cells in the absence of effectors and in the absence of antibodies. Maximum release was achieved by adding Triton X-100 to a final concentration of 1% in the absence of effector cells and in the absence of antibodies. Following incubation, 100 μL of cell-free cell culture supernatant was collected from each well after centrifugation at 500×g for 5 min and transferred to a black flat-bottom 96-well microtiter plate. Fluorescence counts of released calcein were measured at 520 nm using a multimode plate reader. Specific cell lysis was calculated according to the following formula: [fluorescence(sample)−fluorescence(spontaneous)] / [fluorescence(maximum)−fluorescence(spontaneous)]×100%, where "fluorescence(spontaneous)" and "fluorescence(maximum)" are defined as the fluorescence in the absence of effector cells and antibodies, and the fluorescence induced by the addition of Triton X-100, respectively.

[0353] result All three FOLR1xCD16A scFv-IgAb constructs induced NK cell-dependent lysis against A2780 cells with similar maximal efficacy (Figure 17).

[0354] Example 14: Target cell-independent activation of NK cells by anti-FOLR1 antibodies method

[0355] (Table 17) Antibody constructs TIFF2024543828000020.tif38153

[0356] Isolation of PBMCs from buffy coat and enrichment of human NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS and then cultured overnight without stimulation in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 IU / mL sodium penicillin G and 100 μg / mL streptomycin sulfate (all components from Invitrogen)). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0357] Culture and flow cytometric analysis Buffy coat-derived NK cells (5 × 104 ) were cultured for 24 hours in the presence or absence of titrated antibodies starting at a concentration of 660 nM followed by seven 10-fold serial dilutions in complete RPMI 1640 medium in 96-well round-bottom microtiter plates. Then, upregulation of NK cell activation markers CD137 and CD69 was confirmed by staining with anti-CD16-FITC (Biolegend, Cat: 302006), anti-CD69 PE (Biolegend, Cat: 310906), anti-CD45 PerCP-Cy5.5 (Biolegend, Cat: 304028), anti-CD56 PE-Cy7 (Biolegend, Cat: 318318), anti-CD137 APC (Biolegend, Cat: 309810), and viability dye (Thermo NK cells were assessed by extracellular staining with CD4+ (Fisher, Cat: 65-0865-18) followed by flow cytometry analysis. NK cells were gated as live, CD56+, and CD45+. Mean fluorescence intensity (MFI) of CD137 and CD69 on NK cells is shown. MFI values ​​were analyzed by nonlinear regression using GraphPad Prism for Windows (v9; GraphPad Software; La Jolla California USA).

[0358] result Among the three anti-FOLR1 antibodies, the FOLR1 / CD16A scFv-IgAb construct comprising the anti-CD16A CD16a3 domain inhibited FOLR1 +In the absence of target cells, scFv-IgAb_275 showed the lowest tendency for upregulation of activation markers CD69 and CD137 on NK cells. At the highest tested concentration of 660 nM, scFv-IgAb_275 appeared to induce the lowest target-independent NK cell activation, followed by scFv-IgAb_274 and scFv-IgAb_273 (Figures 18A-18B).

[0359] Example 15: Binding of FOLR1xCD16A constructs to cell lines expressing human FOLR1 and cynomolgus monkey FOLR1 method

[0360] (Table 18) Antibody constructs TIFF2024543828000021.tif34158

[0361] Flp-In CHO host cell culture Flp-In CHO cells (Life Technologies, R75807), a derivative of CHO-K1 Chinese hamster ovary cells, were adapted to growth in suspension in HyClone CDM4CHO medium (Cytiva, catalog SH30557.02) supplemented with L-glutamine (Invitrogen, catalog 25030-024), HT supplement (Thermo Fisher Scientific, catalog 41065012), penicillin / streptomycin (Invitrogen, catalog 1540-122), and 100 μg / mL Zeocin (Thermo Fisher Scientific, catalog R250-01). Single-cell derived clonal lines were obtained by limiting dilution cloning in a medium mixture of standard culture medium and Ham's F-12 (Thermo Fisher Scientific, Cat. 11500586) supplemented with InstiGRO CHO supplement (Solentim, Cat. RS-1105), expanded, and cryopreserved in medium containing 10% DMSO (Sigma, Cat. D2650). Cultures were routinely subcultured after 2 or 3 days, diluted into fresh medium to 3E+5 viable cells / mL for the subsequent 2-day passages, or 2E+5 viable cells / mL for the 3-day passages, and grown in shake flasks or tubes at 37°C, 5% CO2, and 120–200 rpm depending on vessel type.

[0362] Generation of stably transfected antigen-expressing cells (cAg) Suspension-adapted Flp-In CHO host cells were subcultured in standard medium without Zeocin one day before transfection. Recombinant CHO cells were generated by transfection of 2E+6 cells in 2 mL of CHO-S-SFMII medium (Thermo Fisher Scientific, catalog 12052-114) with an expression plasmid encoding a recombinant cell anchor antigen sequence (cAg) in a modified version of the pcDNA5 / FRT vector mediating puromycin or hygromycin resistance and an expression plasmid encoding Flp recombinase (pOG44, Thermo Fisher, V600520) using a total of 2.5 μg of DNA at a DNA:PEI ratio of 1:2.5 (μg / μg) and Transporter 5 transfection reagent. DNA and transfection reagent were mixed in 100 μL of 0.9% NaCl solution (Sigma, catalog S8776) and incubated for 20 min before addition to the cells. As a negative control (mock), cells were transfected with a control plasmid that does not mediate resistance. After 4 hours, the transfected cells were diluted in 8 mL of a 1:1 medium mixture of standard culture medium and Ham's F-12. Selection of stably transfected cells was initiated the next day by the addition of 3.2 μg / mL puromycin dihydrochloride (Thermo Fisher Scientific, Cat. A1113803) as the selection antibiotic and increased to 6.3 μg / mL on the second day, or 500 μg / mL hygromycin B (Thermo Fisher Scientific, Cat. 10687010). Viable cell density was measured twice a week, and cells were centrifuged and resuspended in fresh selection medium containing the selection antibiotic at a maximum density of 2-4E+5 viable cells / mL. The concentration of puromycin dihydrochloride was increased to 7.0 μg / mL on the 10th day after transfection. Stably transfected cell pools regained proliferation and viability after approximately 2-3 weeks, were expanded in standard culture medium, and were cryopreserved in freezing medium containing 7.5% DMSO.For analysis of antigen expression, cultures were expanded in shake flasks or tubes, subcultured after 2 or 3 days, and then diluted into fresh medium to 6E+5 viable cells / mL for 2-day passages or 3E+5 viable cells / mL for 3-day passages, and grown at 37°C, 5% CO2, and 120-200 rpm depending on vessel type.

[0363] Flow cytometry analysis To analyze the binding of various antibody constructs to CHO cells transfected with human FOLR1 (cAg_125) and cynomolgus monkey FOLR1 (cAg_126), 1–5 × 10 5 Cells were resuspended in 100 μL of FACS buffer (PBS (BioWest, Catalog: L0615-500) containing 2% heat-inactivated FCS (Invitrogen, Catalog: 10500-064) and 0.1% sodium azide (Sigma, Catalog: S8032_100G)) in a round-bottom 96-well microtiter plate. After washing in FACS buffer, cells were incubated for 40-50 min on ice in the dark in 50 μL of FACS buffer containing no antibody or titrated antibody starting at a concentration of 1000 nM followed by eleven 3-fold serial dilutions. After two washes, cells were incubated with FITC-conjugated goat anti-human IgG (H+L) (Jackson Immunologies, Catalog: 109-096-088) for 40-50 min on ice in the dark. After washing, binding was measured by flow cytometry and the mean fluorescence intensity (MFI) of cell samples was calculated and corrected for background staining using control cells stained with secondary antibody only.

[0364] statistical analysis The equilibrium dissociation constant (K D), mean and standard deviation (SD) were calculated by plotting MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism for Windows (v9; GraphPad Software; La Jolla California USA).

[0365] result The apparent avidity of scFv-IgAb_381 and scFv-IgAb_387 for human (hu)FOLR1 transfected CHO cells and cynomolgus monkey (cy)FOLR1 transfected CHO cells was measured. CHO cells expressing recombinant huFOLR1 (cAg_125) and cyFOLR1 (cAg_126) were incubated with increasing concentrations of scFv-IgAb_381 and scFv-IgAb_387, and binding was evaluated by flow cytometry. Both scFv-IgAb_381 and scFv-IgAb_387 antibody constructs showed similar concentration-dependent binding to huFOLR1 and cyFOLR1, which may be attributed to the same anti-FOLR1 binding domain present in both antibody constructs (Figures 19A, 19B, and Table 19).

[0366] Table 19. Mean apparent avidity (K) of scFv-IgAb_381 and scFv-IgAb_387 for human FOLR1 and cynomolgus monkey FOLR1 expressed on CHO cells. D ) Binding of antibody constructs to huFOLR1-transfected and cyFOLR1-transfected CHO cells was measured by flow cytometry. The equilibrium dissociation constant (KD) of antibody binding was calculated by plotting the MFI values ​​and fitting a nonlinear regression model of one-site binding to the hyperbolic dose-response curve using GraphPad Prism. SD, standard deviation; na, not applicable. TIFF2024543828000022.tif42145

[0367] Example 16: FOLR1 x CD16A innate immune cell engager selectively targets FOLR1-expressing tumor cells to safely harness potent anti-cancer responses background Innate cell engager (ICE®) molecules are designed that bivalently bind to CD16A+ natural killer (NK) cells and macrophages, as well as tumor cell surface antigens, and induce potent tumor-directed cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). scFv-IgAb_387 (Figure 20) is a tetravalent bispecific ICE® that crosslinks folate receptor alpha (FOLR1) on tumor cells with innate immune cells to induce potent and selective targeted tumor cell killing. Herein, we describe the structure, mechanism of action, and preliminary safety data of scFv-IgAb_387.

[0368] (Table 20) Antibody constructs TIFF2024543828000023.tif29145

[0369] method Binding of scFv-IgAb_387 to CD16A and human FOLR1 was assessed by ELISA. Binding of scFv-IgAb_387 to NK cells was assessed by flow cytometry in the presence of physiological levels of IgG (10 mg / mL). ADCC was assessed using a 4-hour calcein release assay with purified NK cells from healthy donors against a panel of tumor cell lines with various expression levels of FOLR1. ADCP was assessed using a flow cytometry-based method with macrophages derived from healthy donor monocytes and target cell lines with various levels of FORL1 expression. Quantification of cytokines secreted by healthy donor PBMC cultures in the presence of scFv-IgAb_387 and target cells was assessed after 24 hours of incubation using multiplex cytokine quantification.

[0370] result scFv-IgAb_387 binds to CD16A (both 158V and 158F variants) with an apparent avidity of 0.1 nM and to hFOLR1 with an apparent avidity of 0.05 nM. Physiological levels of competing IgG do not alter binding efficacy. scFv-IgAb_387 induces potent and selective ADCC even against cells with low FOLR1. scFv-IgAb_387 is also shown to induce efficient ADCP in vitro. Furthermore, scFv-IgAb_387 is shown to be more efficient and potent in both ADCC and ADCP assays than farletuzumab, an Fc-competent monoclonal antibody targeting FOLR1 that shares the same VH / VL sequence as scFv-IgAb_387. Co-cultures of PBMCs, tumor cells, and scFv-IgAb_387 demonstrated concentration-dependent release of pro-inflammatory cytokines (IFNγ, IP-10, TNFα) and minimal off-target cytokine release.

[0371] conclusion We demonstrate that scFv-IgAb_387 selectively and potently kills tumor cells with a range of expression levels of FOLR1 by two complementary mechanisms: ADCC and ADCP. The high avidity for CD16A confers increased potency and efficacy compared to Fc-competent FOLR1 targeting antibodies with the same VH / VL sequence. Because scFv-IgAb_387 selectively binds to CD16A outside the IgG binding epitope, physiological levels of IgG do not compete for binding. We show that scFv-IgAb_387 induces moderate concentration-dependent proinflammatory cytokine release in a target-restricted manner, confirming the potent yet safe in vitro profile of scFv-IgAb_387.

[0372] Example 17: Evaluation of ADCC induced by FOLR1×CD16A scFv-IgAb in the presence of soluble FOLR1 To evaluate the effect of soluble FOLR1 (sFOLR1) on FOLR1×CD16A scFv-IgAb_387-induced lysis of target cells by NK cells, 4-hour calcein release cytotoxicity assays were performed against A2780 and A-549 target cells with enriched primary human NK cells as effector cells at an E:T ratio of 2.5:1 and a fixed, non-saturating concentration of scFv-IgAb_387 in the presence of 2-, 25-, and 50-fold molar excesses of sFOLR1 and soluble mesothelin (sMSLN) as a negative control.

[0373] method Isolation of PBMCs from buffy coat and enrichment of NK cells PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (Invitrogen, Cat: 14190-169), layered on a cushion of Lymphoprep (Stem Cell Technologies, Cat: 07861) and centrifuged at 800 × g for 25 min at room temperature without brake. PBMCs located at the interface were harvested, washed three times with PBS, and then cultured overnight without stimulation in RPMI1640 medium (Invitrogen, Catalog: 21875-091) supplemented with 10% heat-inactivated (hi) FCS (Invitrogen, Catalog: 10270-106), 2 mM L-glutamine (Invitrogen, Catalog: 25030-024), and 100 U / mL penicillin G sodium / 100 μg / mL streptomycin sulfate (Invitrogen, Catalog: 15140-122) (referred to herein as complete RPMI1640 medium). For enrichment of NK cells, PBMCs were harvested from overnight cultures and used for one round of negative selection using the EasySep™ Human NK Cell Enrichment Kit for Immunomagnetic Isolation of Intact Human NK Cells (Stem Cell Technologies, Catalog: 17055) and the Big Easy EasySep™ Magnet (Stem Cell Technologies, Catalog: 18001) according to the manufacturer's instructions.

[0374] Cultivation of tumor cell lines A-549 cells (CLS, Cat: 30014) were cultured in advanced DMEM / F12 (Invitrogen, Cat: 12634010) supplemented with 5% heat-inactivated (hi) FCS (Invitrogen, Cat: 10270-106), 2 mM L-glutamine (Invitrogen, Cat: 25030-024), and 100 U / mL penicillin G sodium / 100 μg / mL streptomycin sulfate (Invitrogen, Cat: 15140-122). The ovarian cancer cell line A2780 was purchased from Sigma (Cat: 93112519-1VL) and cultured in complete RPMI1640 medium. All cell lines were cultured under standard conditions at 37 °C and 5% CO2 in a humidified atmosphere.

[0375] 4-hour calcein release cytotoxicity assay For calcein release cytotoxicity assay, target cells were labeled with 10 μM of the fluorescent dye calcein AM (Invitrogen, Cat: C3100MP) for 30 min in RPMI 1640 medium without FCS and then incubated at 1 × 10 4 Aliquots of calcein-labeled target cells were seeded into individual wells of a round-bottom 96-well microtiter plate with freshly isolated and enriched primary human NK cells at an effector:target (E:T) ratio of 2.5:1. 10 pM scFv-IgAb_387 was added to A2780 target cells in the assay, and 50 pM scFv-IgAb_387 was added to A-549 target cells in the assay in the presence of 2-fold, 25-fold, or 50-fold molar excess of soluble FOLR1 (R&D Systems, Catalog: 5646-FR-050), soluble mesothelin (R&D Systems, Catalog: 3265-MS-050), or without soluble antigen, in triplicate. scFv-IgAb_387 was added to the wells after soluble antigen.

[0376] Spontaneous calcein release, maximum release, and killing of targets by effectors in the absence of antibody were measured in quadruplicates on each plate. Spontaneous release was measured by incubation of target cells in the absence of effector cells and in the absence of antibody. Maximum release was achieved by adding Triton X-100 (Roth, Cat: 3051.2) to a final concentration of 1% in the absence of effector cells and in the absence of antibody. After centrifugation at 200×g for 2 min, the microtiter plates were incubated at 37° C. in a humidified atmosphere containing 5% CO2 for 4 h. Following incubation, 100 μL of cell-free cell culture supernatant was taken from each well after centrifugation at 500×g for 5 min and transferred to a black flat-bottom 96-well microplate. Fluorescence counts of released calcein were measured at 520 nm using a multimode plate reader (EnSight, Perkin Elmer). Specific cell lysis was calculated according to the following formula: [fluorescence (sample)-fluorescence (spontaneous)] / [fluorescence (maximum)-fluorescence (spontaneous)]×100%, where "fluorescence (spontaneous)" and "fluorescence (maximum)" are defined as the fluorescence in the absence of effector cells and antibodies, and the fluorescence induced by the addition of Triton X-100, respectively. The mean and standard deviation (SD) of specific target cell lysis (%) were plotted using GraphPad Prism (v9; GraphPad Software, La Jolla California USA).

[0377] result FOLR1×CD16A scFv-IgAb_387 induced lysis of A2780 and A-549 target cells by NK cells at an E:T ratio of 2.5:1 in a 4-hour calcein release cytotoxicity assay, which clearly exceeded the lysis induced by NK cells in the absence of antibody. Addition of 2-, 25-, or 50-fold molar excess of soluble FOLR1 compared to the scFv-IgAb_387 concentration did not reduce antibody-mediated cytotoxicity. As expected, soluble mesothelin used as a negative control also did not alter the efficacy of scFv-IgAb_387-mediated target cell lysis (FIG. 21).

[0378] Example 18: Evaluation of ADCP induced by FOLR1×CD16A scFv-IgAb To evaluate the potency and efficacy of FOLR1 x CD16A scFv-IgAb_387 to induce antibody-dependent cellular phagocytosis (ADCP), macrophages were differentiated from enriched primary human monocytes and used as effector cells in a 4-hour flow-based ADCP assay with CMFDA-labeled HeLa, HCC-78, and DK-MG target cells in the presence of serial dilutions of scFv-IgAb_387, anti-FOLR1 IgG1 IgAb_355 (SEQ ID NO: 158+159), RSV / CD16A scFv-IgAb_444 (SEQ ID NO: 156+157), and FOLR1 / RSV scFv-IgAb_162.

[0379] method Isolation of PBMCs from buffy coat and enrichment of monocytes PBMCs were isolated from buffy coats (Transfusion department, University Hospital Pilsen, Czech Republic) by density gradient centrifugation. Buffy coat samples were diluted with 2-3 volumes of PBS (BioWest, Cat: L0615-500), layered on a cushion of Lymphoprep (Scintila, Cat: 07811) and centrifuged at 800×g for 25 min at room temperature without brake. PBMCs located at the interface were harvested and washed three times with PBS. PBMCs were then used for enrichment of monocytes by positive selection using the EasySep™ Human CD14 Positive Selection Kit II (Stem Cell Technologies, Cat: 17858) and Big Easy EasySep™ Magnet (Stem Cell Technologies, Cat: 18001) according to the manufacturer's instructions.

[0380] Differentiation of macrophages from monocytes The enriched monocytes were cultured in RPMI1640 medium (Life Technologies, Catalog: 21875-034) supplemented with 10% heat-inactivated (hi) FCS (Invitrogen, Catalog: 10500-064), 2 mM L-glutamine (Invitrogen, Catalog: 25030-024), and 100 U / mL penicillin G sodium and 100 μg / mL streptomycin sulfate (BioWest, Catalog: L0022-100) in the presence of 50 ng / mL M-CSF (ThermoFisher, Catalog: PHC9501) for 5 days at 37° C. and 5% CO2 in a humidified atmosphere (referred to herein as complete RPMI1640 medium). After medium change, the cells were cultured for an additional 2 days in complete RPMI1640 medium supplemented with M-CSF.

[0381] Cultivation of tumor cell lines HeLa cells were cultured in DMEM medium (Fisher Scientific, Catalog: 41965062) supplemented with 10% hi FCS, 2 mM L-glutamine, and 100 U / mL penicillin G sodium / 100 μg / mL streptomycin sulfate. HCC-78 (DSMZ, Catalog: ACC 563) and DK-MG (DSMZ, Catalog: ACC 277) cell lines were cultured in RPMI1640 medium supplemented with 10% hi FCS, 2 mM L-glutamine, and 100 U / mL penicillin G sodium / 100 μg / mL streptomycin sulfate. All cell lines were cultured under standard conditions at 37°C and 5% CO2 in a humidified atmosphere.

[0382] 4-h ADCP assay For ADCP assay, 5 x 10 4 Aliquots of in vitro differentiated macrophages were seeded into individual wells of a 96-well UpCell microplate (ThermoFisher, Cat: 174897) in 100 μL / well of complete RPMI1640 medium and cultured overnight at 37° C. and 5% CO2 in a humidified atmosphere.

[0383] On the same day, target cells were labeled with 0.5 μM CMFDA (Invitrogen, catalog: C7025) in RPMI1640 medium without FCS for 30 min at 37° C., washed once with RPMI1640 medium without FCS, then resuspended in complete RPMI1640 medium and cultured overnight at 37° C. and 5% CO2 in a humidified atmosphere.

[0384] On the day of the ADCP assay, CMFDA-labeled target cells were harvested, counted, and then plated at 1 × 10 per well. 4Aliquots of target cells were seeded on top of precultured adherent macrophages to yield an E:T ratio of 5:1. The indicated antibodies were added to the cocultures in ten serial 1:10 dilutions starting at 500 nM. As controls, macrophages alone, target cells alone, and macrophages with target cells were seeded in the absence of antibody. After centrifugation at 300×g for 1 min at room temperature, the plates were incubated at 37° C. and 5% CO2 in a humidified atmosphere for 4 h.

[0385] After incubation, cultures were washed twice with chilled PBS (Invitrogen, Cat: 392-0434) containing 2% hi FCS (Invitrogen, Cat: 10500-064) and 0.1% sodium azide (Sigma, Cat: S8032) (herein referred to as FACS buffer) and then stained for 30 min in the dark with AlexaFluor700-conjugated mAb anti-CD11b (Biolegend, Cat: 101222) and eFluor780 viability dye (ThermoFisher, Cat: 65-0865-14) in FACS buffer supplemented with 1 mg / mL polyclonal human IgG (Cutaquig, Octapharma, Cat: K939D8143). After an additional washing step, cells were resuspended in FACS buffer and viable CMFDA in a defined sample volume was isolated. + Events and CD11b + The number of events was quantified using a flow cytometer (MACSQuant X, Miltenyi Biotec). The percentage of phagocytosis was calculated using the following formula: (viable CMFDA + / CD11b + Number of target cells) / (viable CMFDA + number of target cells) × 100%.

[0386] Plot the phagocytosis values ​​(%) and, if applicable, the in vitro potency (EC 50 ) and efficacy (E top) was determined by fitting a nonlinear regression model to a sigmoidal dose-response curve (variable slope) using GraphPad Prism (v9; GraphPad Software, La Jolla California USA).

[0387] result Results from an independent ADCP assay demonstrated efficient phagocytosis of HeLa cells expressing high levels of FOLR1 (mean SABC: 354,206) and HCC-78 target cells expressing intermediate levels of FOLR1 (mean SABC: 96,969) induced by FOLR1 / CD16A scFv-IgAb_387 in the presence of human macrophages (Figure 22). Anti-FOLR1 IgG1 IgAb_355 induced phagocytosis with lower efficacy (Table 21), with FOLR1 still phagocytosed in the presence of scFv-IgAb_387. low It did not induce measurable phagocytosis of DK-MG target cells. Two control antibody constructs, RSV / CD16A scFv-IgAb_444 and FOLR1 / RSV scFv-IgAb_162, in which either the anti-FOLR1 Fv domain or the anti-CD16A Fv domain was replaced by the anti-RSV Fv domain, did not induce ADCP under the same experimental conditions, demonstrating the specificity of scFv-IgAb_387-mediated ADCP and the requirement of both anti-FOLR1 Fab and anti-CD16A scFv for efficient and potent ADCP.

[0388] Table 21. Efficacy (% phagocytosis) and potency (EC 50 ) average TIFF2024543828000024.tif144142

[0389] Example 19: scFv-IgAb_387 exhibits anti-tumor efficacy in vivo in a xenograft model of ovarian cancer method cell culture Tumor IGROV-1 cell lines were maintained in vitro as monolayer cultures in appropriate culture medium supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5% CO2. Tumor cells were routinely subcultured by trypsin-EDTA treatment 2-3 times a week depending on growth rate. Cells in exponential growth phase were harvested and counted for inoculation. Cells were tested free of pathogens and mycoplasma.

[0390] mouse CB-17 SCID-Fcgr4tm2(FCGR3A) / Bcgen mice were used in this study. Briefly, in B-hCD16A mice (CB-17 SCID), the control region and exons 1-5 of the mouse Fcgr4 gene, which encodes the full-length protein CD16, were replaced by human FCGR3A exons 1-4 and the control region.

[0391] Tumor inoculation IGROV-1 cells were harvested in exponential growth phase, centrifuged at 335×g in a refrigerated centrifuge, and the medium was aspirated. The cell pellet was resuspended in 10 volumes of serum-free medium, filtered through a 70 μm nylon mesh cell strainer, and counted using a NucleoCounter® NC-200™. The cell suspension was centrifuged again as above, resuspended in serum-free medium to obtain twice the final concentration, and then diluted 1:1 with Matrigel. Thus, each 100 μL delivered the number of cells required per inoculation. Each mouse was inoculated subcutaneously in the lower right flank (near the dorsal thigh region). Mice were lightly anesthetized during the implantation procedure.

[0392] group The study groups and the number of animals per group are shown in Table 22.

[0393] In the prevention group, treatment was initiated one day after inoculation (day 1), with the day of inoculation designated as day 0. In the treatment group, treatment was initiated when tumor volumes (TV) reached approximately 50 mm 3Based on TV, mice were grouped into respective groups using the Matched Distribution randomization method in Studylog software (Studylog Systems, San Francisco, USA).

[0394] (Table 22) Various groups are shown along with the number of mice per group (N), dose, route of administration (ROA), frequency, and dosing schedule. IV, intravenous; BIW, twice a week. TIFF2024543828000025.tif25160Dosing volume is 5mL / kg for IV. 1 For BIW, the second dose can be either day 3 or 4 after the first. The actual dosing schedule can vary depending on the treatment initiation date. 2 Treatment in the Tx cohort included patients with a TV of approximately 50 mm 3 That is, approximately 10±1 days after inoculation.

[0395] Flow cytometry analysis Cells were resuspended in FcR blocking solution, stained with FACS antibodies, resuspended in live / dead stain, fixed with fixative solution, and analyzed by flow cytometer. Raw data was analyzed using FlowJo software. Cells were gated on live and single cells (FSC-A vs. FSC-H) using live-dead dyes, followed by gating on lineage markers. Median fluorescence intensity (MFI) and percentage of cells positive for specific markers were exported and plotted using GraphPad prism software. Fluorescence minus one (FMO) controls were used as references to set gates.

[0396] statistical analysis All data were analyzed using GraphPad software Prism 9, and P<0.05 was considered statistically significant.

[0397] result Tumor growth analysis Mice treated with scFv-IgAb_387 showed reduced tumor growth compared to vehicle-treated mice in both prophylactic and therapeutic settings. After 35 days of tumor growth, the mean tumor volume in the vehicle-treated group was 366 mm 3 whereas the therapeutically and prophylactically treated groups had 157 mm 3 and 133mm 3 had significantly smaller neoplastic lesions (Figure 23).

[0398] Flow cytometry analysis Mice were sacrificed at the end of the experiment, 35 days after tumor inoculation. Blood, spleens, and tumors were harvested and analyzed by flow cytometry. Signs of peripheral pharmacodynamic activity of scFv-IgAb_387 were observed in blood and CD16 in treated mice. + The number of cells increased, but the frequency did not increase (Figure 24A). A similar phenomenon was observed in the spleen as well. Specifically, both therapeutic and prophylactic scFv-IgAb_387 treatment increased the number of CD16 + The absolute number and frequency of CD16 cells was significantly increased in the tumors (Fig. 24B). + A red...

Claims

1. below: (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region shown in SEQ ID NO: 7 or SEQ ID NO: 94; a first binding domain (A) comprising: (b) a second binding domain (B) capable of specifically binding to a second target (B'), which is an antigen present on the surface of a target cell, wherein the second target (B') is FOLR1; 1. A bispecific antibody construct comprising:

2. The antibody construct of claim 1, wherein the first binding domain (A) comprises a VL region as shown in SEQ ID NO: 8 or SEQ ID NO: 95 and a VH region as shown in SEQ ID NO: 7 or SEQ ID NO:

94.

3. 2. The antibody construct of claim 1, wherein the first binding domain (A) is a variable domain (Fv), a single-chain Fv (scFv), a Fab, a single-chain diabody (scDb), a diabody (Db), or a double Fab, preferably an scFv.

4. The antibody construct of claim 1, wherein the second binding domain (B) comprises the VH and VL domains of an antibody.

5. 2. The antibody construct of claim 1, wherein the second binding domain (B) is a variable domain (Fv), a single-chain Fv (scFv), a Fab, a single-chain diabody (scDb), a diabody (Db), or a double Fab, preferably a double Fab.

6. The antibody construct of claim 1, which simultaneously binds to a target cell and an immune effector cell.

7. the first binding domain comprises An epitope on CD16A C-terminal to the physiological Fcγ receptor binding domain binds to The epitope preferably comprises Y158 of SEQ ID NO: 50; The antibody construct of claim 1.

8. 2. The antibody construct of claim 1, further comprising a third domain (C) comprising a half-life extending domain.

9. The antibody construct of claim 1, wherein the half-life extending domain comprises a CH2 domain and the Fcγ receptor binding domain is silenced.

10. 2. The antibody construct of claim 1, wherein the half-life extending domain comprises a CH3 domain.

11. 2. The antibody construct of claim 1, comprising at least one hinge domain and a CH3 domain fused to a CH2 domain in the order of hinge domain-CH2 domain-CH3 domain from amino to carboxyl.

12. The antibody construct of claim 1, comprising at least two hinge domain-CH2 domain-CH3 domain elements.

13. 2. The antibody construct of claim 1, wherein the first binding domain (A) is fused to the C-terminus of the CH3 domain and the second binding domain (B) is fused to the N-terminus of the hinge region.

14. 2. The antibody construct of claim 1, which is monovalent with respect to the first binding domain (A) and monovalent with respect to the second binding domain (B).

15. 2. The antibody construct of claim 1, which is bivalent with respect to the first binding domain (A) and bivalent with respect to the second binding domain (B).

16. (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1, an antigen present on the surface of a target cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 117, a CDR-L2 as set forth in SEQ ID NO: 118, and a CDR-L3 as set forth in SEQ ID NO: 119; (ii) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 114, CDR-H2 as set forth in SEQ ID NO: 115, and CDR-H3 as set forth in SEQ ID NO: 116; wherein the second binding domain is a Fab; and (c) a third domain: Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Contains two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of claim 8.

17. (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5, and a CDR-L3 as set forth in SEQ ID NO: 6; (ii) a VH region shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1, an antigen present on the surface of a target cell; (i) a VL region comprising a CDR-L1 as set forth in SEQ ID NO: 127, a CDR-L2 as set forth in SEQ ID NO: 128, and a CDR-L3 as set forth in SEQ ID NO: 129; (ii) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 124, CDR-H2 as set forth in SEQ ID NO: 125, and CDR-H3 as set forth in SEQ ID NO: 126; wherein the second binding domain is a Fab; and (c) a third domain: Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Contains two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of claim 8.

18. (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; (i) a VL region as set forth in SEQ ID NO: 8 or SEQ ID NO: 95; (ii) a VH region shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1 present on the surface of a target cell; (i) a VL region as set forth in SEQ ID NO: 121; (ii) the VH region shown in SEQ ID NO: 120; wherein the second binding domain is a Fab; and (c) a third domain: Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Contains two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of claim 8.

19. (a) a first binding domain (A) capable of specifically binding to a first target (A'), which is CD16A present on the surface of an immune effector cell; (i) a VL region as set forth in SEQ ID NO: 8 or SEQ ID NO: 95; (ii) a VH region shown in SEQ ID NO: 7 or SEQ ID NO: 94; wherein the first binding domain is an scFv; (b) a second binding domain capable of specifically binding to a second target (B'), which is FOLR1 present on the surface of a target cell; (i) a VL region as set forth in SEQ ID NO: 131; (ii) the VH region shown in SEQ ID NO: 130; wherein the second binding domain is a Fab; and (c) a third domain: Preferably, the hinge domain-CH2 domain-CH3 domain elements shown in SEQ ID NOs: 53 and 67. Contains two; the first binding domain (A) is fused to the C-terminus of the CH3 domain of the third domain, and the second binding domain (B) is fused to the N-terminus of the hinge region of the third domain; The antibody construct of claim 8.

20. A second binding domain (B) specific for FOLR1, comprising: a pair of VH and VL chains having sequences set forth in a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 120 and 121, respectively, or a pair of sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 130 and 131, respectively; 2. The antibody construct of claim 1, comprising:

21. 2. The antibody construct of claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 142-143 and 138-139, with SEQ ID NOs: 142-143 being preferred.

22. 2. The antibody construct of claim 1, which induces less CD16A shedding compared to a control construct having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-135, 136-137, and 140-142.

23. A nucleic acid molecule comprising a sequence encoding the antibody construct of any one of claims 1 to 22.

24. 24. A vector comprising the nucleic acid molecule of claim 23.

25. 24. A host cell comprising the nucleic acid molecule of claim 23, or a vector comprising said nucleic acid molecule.

26. 26. A method for producing an antibody construct according to any one of claims 1 to 22, comprising culturing a host cell according to claim 25 under conditions allowing expression of the antibody construct according to any one of claims 1 to 22, and recovering the produced antibody construct from the culture.

27. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 22.

28. 28. The pharmaceutical composition of claim 27 for use in therapy.

29. 28. The pharmaceutical composition of claim 27 for use in the prevention, treatment, or amelioration of a disease selected from a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder.

30. 30. The pharmaceutical composition of claim 29, wherein the neoplastic disease is a solid tumor, preferably a malignant solid tumor.

31. 31. The pharmaceutical composition of claim 30, wherein the solid tumor is selected from the group consisting of ovarian cancer (optionally high-grade serous ovarian cancer or epithelial ovarian cancer), breast cancer (optionally TNBC), renal cancer, lung cancer (optionally non-small cell lung cancer (NSCLC) or mesothelioma), colorectal cancer (optionally CRC), renal cancer (optionally ccRCC), pancreatic cancer (optionally PDAC), endometrial cancer (optionally non-malignant endometrial cancer), and brain tumor.

32. 23. Use of an antibody construct according to any one of claims 1 to 22 in the manufacture of a medicament for treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immunological disorder.

33. A kit comprising an antibody construct according to any one of claims 1 to 22, a nucleic acid molecule according to claim 23, a vector according to claim 24, and / or a host cell comprising said nucleic acid molecule or said vector.

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