Natural killer cell cement

Abstract

The invention relates to methods and materials for treating cancer. For example, provided herein are cell adhesives capable of binding to natural killer (NK) cells and cancer cells. In some cases, the cell binding agents provided herein may comprise a first antigen binding domain capable of binding to a NK cell group 2 isoform C (NKG2C) polypeptide and a second antigen binding domain capable of binding to a cancer cell surface polypeptide. In some cases, one or more cell binding agents provided herein may be administered to a mammal (e.g., a human) having a cancer (e.g., a leukemia such as acute myelogenous leukemia (AML)) to treat cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. patent application serial number 63 / 428,983, filed on November 30, 2022. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated by reference herein.

[0003] Statement Regarding Federal Funding

[0004] This invention was made with government support under Grants W81XWH-16-1-0380 from the Medical Research and Development Command and CA197292 and CA111412 from the National Institutes of Health. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which has been submitted electronically as an XML file named "09531-0483WO1_SL.xml". This XML file was created on November 22, 2023 and is 68,000 bytes in size. The material in the XML file is incorporated herein by reference in its entirety. Technical Field

[0007] This article relates to methods and materials for treating cancer. For example, provided herein are cell binders that can bind to natural killer (NK) cells and cancer cells. In some cases, provided herein are cell binders that can include a first antigen binding domain that can bind to NK cell group 2 isotype C (NKG2C) polypeptide and a second antigen binding domain that can bind to a cancer cell surface polypeptide. In some cases, one or more cell binders provided herein can be administered to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as acute myeloid leukemia (AML)) to treat cancer. Background Art

[0008] NK cells are innate lymphocytes whose primary function is to detect virus-infected cells and malignant tumors in the body. After recognizing target cells, they become fully activated and trigger a functional response, secreting cytokines and cytolytic molecules, thereby inducing apoptosis in the target cells. However, many tumors maintain relatively normal MHC-I levels, thereby evading NK cell surveillance and clearance. Summary of the Invention

[0009] Provided herein are methods and materials for treating cancer. For example, provided herein are cell engagers that can bind to NK cells and cancer cells. In some cases, the cell engagers provided herein can comprise a first antigen binding domain that can bind to a NKG2C polypeptide and a second antigen binding domain that can bind to a cancer cell surface polypeptide. In some cases, one or more of the cell engagers provided herein can be administered to a mammal (e.g., a human) having a cancer (e.g., a leukemia such as AML) to treat the cancer.

[0010] Also provided herein are cells (e.g., host cells) designed to express one or more cell engagers that can bind to a NKG2C polypeptide and can bind to a cancer cell surface polypeptide, and methods and materials for using such cells to treat a mammal (e.g., a human) having a cancer (e.g., a leukemia such as AML).

[0011] As described herein, one or more cell engagers can be designed to be capable of binding to a NKG2C polypeptide and to a polypeptide (e.g., a CD33 polypeptide) on the surface of a cancer cell. For example, the cell engagers provided herein are capable of binding to a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence of a human NKG2C polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (see, e.g., Example 2), and are capable of binding to a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence of a human CD33 polypeptide as set forth in SEQ ID NO: 3 or SEQ ID NO: 4 (see, e.g., Example 5).

[0012] In some cases, a first antigen binding domain that can bind to a NKG2C polypeptide and a second antigen binding domain that can bind to a cancer cell surface polypeptide can be engineered into a cell engager (such as a bispecific killer engager (e.g., BiKE) and / or a trispecific killer engager) to create a cell engager that is capable of targeting a NKG2C + cell (e.g., a NKG2C + NK cell) and directing the NK cell to a target cell (e.g., a cancer cell) to induce one or more immune responses (e.g., a T cell immune response and / or antibody-dependent cell-mediated cytotoxicity (ADCC)) against the target cell. Notably, when a cell engager provided herein is designed to lack an Fc domain, it can still be referred to as ADCC even in these cases where BiKE and trispecific killer engager-mediated killing is not initiated by an Fc domain.

[0013] As described herein, one or more cell-engaging agents provided herein can be used to treat mammals (e.g., humans) suffering from cancer (e.g., leukemias such as AML). For example, a composition comprising one or more cell-engaging agents described herein can be administered to a mammal (e.g., a human) suffering from cancer (e.g., leukemias such as AML) to reduce the number of cancer cells in the mammal, induce an immune response against cancer cells in the mammal, and / or prolong the survival of the mammal with cancer.

[0014] In general, one aspect described herein describes a cell engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can bind to a NKG2C polypeptide and the second antigen binding domain can bind to a polypeptide expressed on the surface of a cancer cell. The first antigen binding domain comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 5 (or SEQ ID NO: 5 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 6 (or SEQ ID NO: 6 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 7 (or SEQ ID NO: 7 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 8 (or SEQ ID NO: 8 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two or three amino acid additions, deletions or substitutions). The first antigen binding domain comprises (a) a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 19 and (b) a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 20. The first antigen binding domain can be a scFv. The polypeptide expressed on the surface of a cancer cell can be a CD33 polypeptide. The second antigen binding domain comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 24 (or SEQ ID NO: 24 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 28 (or SEQ ID NO: 28 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 29 (or SEQ ID NO: 29 with one, two or three amino acid additions, deletions or substitutions).The second antigen-binding domain comprises (a) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 38 and (b) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 39. The second antigen-binding domain may be an scFv. The cell engaging agent may comprise a linker located between the first antigen-binding domain and the second antigen-binding domain. The linker comprises a linker sequence selected from the group consisting of GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGS (SEQ ID NO: 63), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41), PSGQAGAAASESLFVSNHAY (SEQ ID NO: 64), EASGGPE (SEQ ID NO: 65), EPKSSDKTHTSPPSPEL (SEQ ID NO: 66), RATPSHNSHQVPSAGGPTANSGTSG (SEQ ID NO: 67), and SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 68). The cell engaging agent may comprise an IL-15 polypeptide or a biologically active fragment of an IL-15 polypeptide. The IL-15 polypeptide or biologically active fragment of an IL-15 polypeptide may be positioned between the first antigen-binding domain and the second antigen-binding domain. The IL-15 polypeptide may be separated from the first antigen-binding domain by a first linker, and the IL-15 polypeptide may be separated from the second antigen-binding domain by a second linker. The first linker and the second linker may each comprise GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41).

[0015] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a cell-engaging agent comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain can bind to an NKG2C polypeptide and the second antigen-binding domain can bind to a polypeptide expressed on the surface of a cancer cell. The nucleic acid can be a viral vector. The nucleic acid can be a phagemid.

[0016] In another aspect, described herein are host cells comprising a nucleic acid sequence encoding a cell-engaging agent comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain can bind to an NKG2C polypeptide and the second antigen-binding domain can bind to a polypeptide expressed on the surface of a cancer cell.

[0017] In another aspect, described herein are compositions comprising a cell engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can bind to a NKG2C polypeptide and the second antigen binding domain can bind to a polypeptide expressed on the surface of a cancer cell.

[0018] In another aspect, described herein are methods of treating a mammal having a cancer. The methods can comprise or consist essentially of administering to the mammal having a cancer a cell engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can bind to a NKG2C polypeptide and the second antigen binding domain can bind to a polypeptide expressed on the surface of a cancer cell. The mammal can be a human. The cancer can be a CD33 + cancer. The cancer can be leukemia, lymphoma, myelodysplastic syndrome, or systemic mastocytosis. The number of cancer cells in the mammal can be reduced following the administering step.

[0019] In another aspect, described herein are methods of treating a mammal having a cancer. The methods can comprise or consist essentially of administering to the mammal having a cancer a cell engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can bind to a NKG2C polypeptide and the second antigen binding domain can bind to a polypeptide expressed on the surface of a cancer cell. The mammal can be a human. The cancer can be a CD33 + cancer. The cancer can be leukemia, lymphoma, myelodysplastic syndrome, or systemic mastocytosis. At least a portion of the NK cells can be NKG2C + NK cells. The NKG2C + NK cells can comprise a nucleic acid encoding the NKG2C polypeptide under conditions such that the NKG2C polypeptide is expressed. At least a portion of the NK cells comprise a nucleic acid encoding the DAP12 polypeptide under conditions such that the DAP12 polypeptide is expressed. The number of cancer cells in the mammal can be reduced following the administering steps (a) and (b).

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention relates. Although methods and materials similar or equivalent to those described herein can be used to implement the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. If there is any conflict, this specification (including definitions) shall prevail. In addition, materials, methods and embodiments are only illustrative and are not intended to be limiting.

[0021] The accompanying drawings and the following description further illustrate one or more embodiments of the present invention in detail. Other features, objectives and advantages of the present invention will be apparent from the description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A-1C Adapted NK cells from healthy cytomegalovirus (CMV) seropositive donors with high NKG2C frequencies responded to an anti-NKG2C / IL-15 / anti-CD33 killer conjugate (also referred to herein as NKG2C-KE). + NK cell expression levels <10% or >10% were divided into two groups. Subsequently, these cell populations were incubated with THP1 tumor cell line for 5 hours and stained to detect CD107a degranulation ( Figure 1A ) and IFNγ production ( Figure 1B ). Figure 1C The correlation between NKG2C frequency and degranulation or IFNγ production is shown. The graph represents the correlation between NKG2C frequency and degranulation or IFNγ production using two-way ANOVA. Figure 1A and Figure 1B Analyses were performed as mean ± standard error of the mean (SEM). Linear regression analysis was used Figure 1C The P values ​​are as follows: ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0023] Figures 2A-2G In patients with reactivated CMV, NKG2C-KE demonstrated increased function in peripheral blood mononuclear cells (PBMCs) starting 6 months after transplantation. PBMCs from patients after hematopoietic transplantation were stratified based on whether they were CMV seronegative and had not reactivated CMV, and whether they had reactivated CMV within 100 days of transplantation. The corresponding samples were incubated with THP1 tumor targets in a 5-hour assay and stained for CD107a degranulation ( Figure 2A ) and IFNγ production ( Figure 2B ). The figure shows CD107a degranulation ( Figure 2C ) and IFNγ production (Figure 2D ) and NKG2C expression on NK cells. Representative samples of CMV reactivated and CMV seronegative cells were stained after 7 days of incubation with the indicated treatments ( Figure 2E ). Pooled data on NK cell proliferation in CMV reactivated (n=9) and seronegative (n=4) patients 6 months after transplantation showed the percentage of NKG2C ( Figure 2F ) and NKG2C + :NKG2C - The ratio ( Figure 2G ). The graphs show the differences between the two-way ANOVA and mixed effects analysis ( Figure 2A and Figure 2B ), linear regression( Figure 2C and Figure 2D ) and paired t-test ( Figure 2F and Figure 2G ) The mean ± SEM of the analyzed data. P values ​​are as follows: ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0024] Figures 3A-3E NKG2C-KE controlled tumors as effectively as 161533TriKE, which binds to CD16 on NK cells and CD33 on tumor cells. Figure 3A Schematic diagram of the mouse model. NOD-SCIDγ (NSG) mice were injected with 750,000 HL60-Luc cells, followed three days later by 5 million thawed, expanded NK cells. Mice were injected intraperitoneally (ip) five times weekly for three weeks. Bioluminescence imaging (BLI) was performed on days 6, 13, 20, and 27, and blood was collected on days 14 and 28. Figure 3B ) Thawed expanded NK cells and stained for CD16 and NKG2C. Figure 3C ) Individual bioluminescence images from days 6, 13, 20, 27, and 34, by group. Figure 3D ) Quantification of luminescence measured on days 0, 6, 13, 20, 27 and 34. Figure 3E ) CD16 and NKG2C staining of mouse blood on days 14 and 28. Graphs show mean ± SD using two-way ANOVA. Figure 3C and Figure 3D Repeated measures (RM) analysis was used. P values ​​are as follows: ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0025] Figures 4A-4C : Induced pluripotent stem cell (iPSC)-derived NK (iNK) cells genetically modified to express NKG2C. Figure 4A) iNK cells at end-stage differentiation were stained for CD56 and NKG2C and analyzed by flow cytometry. Figure 4B ) Quantitative analysis of the percentage of NKG2C in 5 batches of end-stage differentiated iNK cells in the indicated cell lines. Figure 4C ) MFI of iNK cells showing NKG2C histograms. Graphs show Figure 4B Mean ± SD using one-way ANOVA and RM analysis. P values are as follows: ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0026] Figures 5A-5E iPSCs transduced with NKG2C and DAP12 and differentiated into NK cells (iNKs) exhibit CD33-specific function against acute myeloid leukemia (AML) targets in the presence of NKG2C-KE. iNKs transduced with no transduction, NKG2C, and NKG2C / DAP12 were incubated with IL-15 or NKG2C-KE for 5 hours and incubated with THP1 Figure 5A and Figure 5B ) or HL60 Figure 5C and Figure 5D ). NK cells were stained for CD 107a degranulation Figure 5A and Figure 5C ) and IFNy production Figure 5B and Figure 5D ) in 3 separate experiments and results are summarized in the graphs. The indicated iNK cell lines were stained with CellTrace and incubated at 37°C for 7 days and proliferation was measured using the indicated treatments Figure 5E ) Graphs show Figures 5A-5D Mean ± SD using two-way ANOVA and RM analysis. P values are as follows: ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0027] Figures 6A-6C Rapid killing kinetics of NKG2C-KE and NKG2C / DAP12 iNKs. Tumor targets stained with CellTrace for tracking were incubated with iNKs and the indicated treatments in an Incucyte live imaging system. Figure 6A ) Representative experimental images of iNKs treated with NKG2C-KE at the indicated times. Figure 6B ) Images were taken every 30 minutes to track the number of cells over time in a representative experiment. Figure 6C) Changes in cell number over time at representative time points from five separate experiments. Graphs show mean ± SD using two-way ANOVA and RM analysis. P values ​​are as follows: ****p < 0.0001, ***p < 0.01, **p < 0.01, *p < 0.05.

[0028] Figures 7A-7C NKG2C-KE and NKG2C / DAP12iNK can kill primary AML target cells. Primary AML cells from 5 patients were incubated with the indicated iNKs for 5 hours and stained to detect CD107a degranulation ( Figure 7A ) and IFNγ production ( Figure 7B Primary AML cells (n=3) were stained with CellTrace and incubated with iNK cells for 48 hours, and then counted by flow cytometry ( Figure 7C ). Graphs show mean ± SEM using two-way ANOVA and RM analysis in AD. P values ​​are as follows: ****p < 0.0001, ***p < 0.0, **p < 0.01, *p < 0.05.

[0029] Figure 8 NKG2C DAP12 iNKs express more DAP12. At the end of two weeks of expansion, thawed iNKs, untransduced NKG2C, and NKG2C DAP12 were stained for intracellular DAP12.

[0030] Figure 9A –9B. iNK cells transduced with NKG2C and DAP12 do not express an adaptive NK cell phenotype. Figure 9A ) Thawed iNK and adaptive NK cells were stained for intracellular FcεRIγ, EAT2, and PLZF. Figure 9B ) Thawed iNK cells were stained for NKp44, KIR, NKG2D, and NKG2A. The MFI of positive iNK cells is shown below the gate box.

[0031] Figures 10A-10B NKG2C-KE guides NK cells to CD33 + Healthy peripheral blood NK cells are classified as <10% NKG2C + and >10% NKG2C + ( Figure 10A ), and NKG2C DAP12 iNK( Figure 10B ), these NK cells and Raji (CD33 - ) or THP1(CD33 + ) and received corresponding treatment (no drug, rhIL-15, or NKG2C-KE), and were stained for the degranulation marker CD107a.

[0032] Figure 11 Primary AML as well as HL-60 and THP1 cell lines express HLA-E. Cell lines and primary AML samples were thawed and rested overnight prior to HLA-E staining and comparison to control with single fluorescent antibody removed.

[0033] Figure 12 Using NKG2C-KE treatment of humans with cancers including CD33 + Cancer cells (e.g., CD33 + Schematic of an exemplary method of treating a human with a cancer of cancer cells (e.g., CD33

[0034] Figures 13A-13B Schematic of exemplary NKG2C-KE cell engagers designed to bind to (e.g., specifically bind to) NKG2C polypeptides (e.g., human NKG2C polypeptides). Figure 13A NKG2C-KE cell engager designed using an anti-NKG2C scFv linked to an anti-CD33 scFv is shown. Figure 13B NKG2C-KE cell engager is shown, designed using an anti-NKG2C scFv linked to an IL-15 polypeptide, which is linked to an anti-CD33 scFv polypeptide. DETAILED DESCRIPTION

[0035] Provided herein are methods and materials related to treating cancer. For example, provided herein are cell engagers that can bind to NK cells and cancer cells. In some cases, the cell engagers provided herein can comprise a first antigen binding domain that can bind to a NKG2C polypeptide and a second antigen binding domain that can bind to a cancer cell surface polypeptide (e.g., a CD33 polypeptide). In some cases, one or more of the cell engagers provided herein can be administered to a mammal (e.g., a human) with a cancer (e.g., a leukemia such as AML) to treat the cancer.

[0036] As described herein, a cell engager can be designed to comprise at least one antigen binding domain that can bind to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) and at least one other antigen binding domain. The at least one other antigen binding domain can bind to any suitable antigen expressed on the surface of a cancer cell. For example, when a cell engager (such as a BiKE) is designed to engage NKG2C + NK cells and cancer cells, the cell engager can comprise an antigen binding domain that can bind to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) and an antigen binding domain that can bind to a polypeptide (e.g., a CD33 polypeptide) expressed on the surface of a cancer cell.

[0037] In some cases, the cell engaging agents provided herein can comprise a first antigen binding domain that can bind (e.g., specifically binds) to a polypeptide comprising, consisting essentially of, or consisting of amino acids as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (see, e.g., Example 2) and a second antigen binding domain that can bind (e.g., specifically binds) to a polypeptide comprising, consisting essentially of, or consisting of amino acids as set forth in SEQ ID NO: 3 or SEQ ID NO: 4 (see, e.g., Example 5).

[0038] As used herein, the term "cell engager" refers to a polypeptide comprising two or more antigen binding domains (e.g., two, three, or four antigen binding domains) and capable of connecting two cells together. Examples of cell engagers include, but are not limited to, BiTEs, BiKEs, trispecific killer engagers, trispecific NK engagers (TriNKETs), redirected optimized cell killing ( ) binders, diabodies, dual affinity redirecting antibodies (DARTs), and NK cell engagers (NKCEs). Generally speaking, the cell engagers provided herein can be designed to comprise at least one antigen binding domain capable of binding to an NKG2C polypeptide (e.g., a human NKG2C polypeptide) and at least one antigen binding domain capable of binding to a polypeptide (e.g., an antigen) expressed on the surface of a target cell (e.g., a cancer cell). In some cases, the cell engagers described herein can bind to an NKG2C polypeptide through its two or more antigen binding domains. + Cells (e.g., NKG2C + NK cells) is connected to another cell (e.g., a cancer cell). Examples of structures of cell binders provided herein include, but are not limited to, structures as shown in Figure 13. In some cases, the anti-NKG2C scFv shown in Figure 13 can be replaced with different antigen-binding domains that can bind to a polypeptide expressed on the surface of an NK cell. In some cases, the anti-CD33 scFv shown in Figure 13 can be replaced with different antigen-binding domains that can bind to a polypeptide (e.g., an antigen) expressed on the surface of a cell (e.g., a cancer cell).

[0039] The antigen binding domains contained in the cell engaging agents provided herein can comprise the CDRs described herein (e.g., as described in Tables 1 and 2) and can be constructed as human or humanized antigen binding domains. In some cases, the antigen binding domains contained in the cell engaging agents provided herein can comprise the CDRs described herein (e.g., as described in Tables 1 and 2) and can be constructed as scFvs.

[0040] Antigen binding domains capable of binding to NKG2C polypeptides (e.g., human NKG2C polypeptides) can be any appropriate type of antigen binding domain capable of binding to NKG2C polypeptides (e.g., human NKG2C polypeptides). Examples of antigen binding domains capable of binding to polypeptides expressed on the surface of cancer cells that can be used to make the cell engagers (e.g., BiKEs or trispecific killing engagers) provided herein include, but are not limited to, anti-NKG2C scFv, anti-NKG2C single domain antibodies (sdAbs), and HLA-E ectodomain loaded with NKG2C specific peptides.

[0041] In some cases, antigen binding domains capable of binding to NKG2C polypeptides (e.g., human NKG2C polypeptides) can be used to make the cell engagers provided herein, which can comprise a CDR1 of a VH domain, a CDR2 of a VH domain, and a CDR3 of a VH domain, and can also comprise a CDR1 of a VL domain, a CDR2 of a VL domain, and a CDR3 of a VL domain. For example, antigen binding domains capable of binding to NKG2C polypeptides can be used in the cell engagers provided herein, which can comprise the CDR amino acid sequences set forth below:

[0042] Table 1. Exemplary CDR sequences of anti-NKG2C antigen binding domains.

[0043] Sequence SEQ ID NO VH CDR1 GFNIKDT 5 VH CDR2 DPENGY 6 VH CDR3 SRTLFWYFDV 7 VL CDR1 KSSQSVLYSSNQKNYLA 8 VL CDR2 WASTRES 9 VL CDR3 HQYLSSYT 10

[0044] In some cases, an antigen binding domain capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) can be used in the cell engagers provided herein, the antigen binding domain comprising (a) a heavy chain variable domain having a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 5 (or a variant of SEQ ID NO: 5 having one, two, three or four amino acid modifications), a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 6 (or a variant of SEQ ID NO: 6 having one, two, three or four amino acid modifications), and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7 (or a variant of SEQ ID NO: 7 having one, two, three or four amino acid modifications), and / or (b) a light chain variable domain having a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 8 (or a variant of SEQ ID NO: 8 having one, two, three or four amino acid modifications), a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 9 (or a variant of SEQ ID NO: 9 having one, two, three or four amino acid modifications), and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10 (or a variant of SEQ ID NO: 10 having one, two, three or four amino acid modifications), and the variable domain can comprise any appropriate framework region.For example, such antigen binding domains can comprise (a) a heavy chain variable domain comprising a framework region 1 having an amino acid sequence set forth in SEQ ID NO: 11 (or a variant of SEQ ID NO: 11 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 2 having an amino acid sequence set forth in SEQ ID NO: 12 (or a variant of SEQ ID NO: 12 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 3 having an amino acid sequence set forth in SEQ ID NO: 13 (or a variant of SEQ ID NO: 13 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and a framework region 4 having an amino acid sequence set forth in SEQ ID NO: 14 (or a variant of SEQ ID NO: 14 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and / or (b) a light chain variable domain comprising a framework region 1 having an amino acid sequence set forth in SEQ ID NO: 15 (or a variant of SEQ ID NO: 15 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 2 having an amino acid sequence set forth in SEQ ID NO: 16 (or a variant of SEQ ID NO: 16 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 3 having an amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and a framework region 4 having an amino acid sequence set forth in SEQ ID NO: 18 (or a variant of SEQ ID NO: 18 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications).

[0045] In some cases, antigen binding domains that can be used in the cell engagers provided herein that are capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) can comprise a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 20.

[0046] In some cases, antigen binding domains capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 19 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 20. For example, such antigen binding domains can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 19 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 20. In some cases, antigen binding domains capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 19, provided that the heavy chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 5, 6, and 7 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 20, provided that the light chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 8, 9, and 10.

[0047] In some cases, an antigen binding domain capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) can be used in the cell engagers provided herein, which can comprise (a) a heavy chain variable domain having an amino acid sequence as set forth in SEQ ID NO: 19 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) and / or (b) a light chain variable domain having an amino acid sequence as set forth in SEQ ID NO: 20 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, such an antigen binding domain can comprise a heavy chain variable domain having an amino acid sequence as set forth in SEQ ID NO: 19 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 5, 6, and 7, and can comprise a light chain variable domain having an amino acid sequence as set forth in SEQ ID NO: 20 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the light chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 8, 9, and 10.

[0048] In some cases, an antigen binding domain capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 5, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 6, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 7, and / or (b) a light chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 9, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 10.

[0049] When the antigen binding domain is designed as a scFv having a heavy chain variable domain and a light chain variable domain, the two regions can be directly connected or can be connected using any suitable linker sequence. In some cases, a heavy chain variable domain having CDRs of SEQ ID NOs: 5-7 can be directly connected to a light chain variable domain having CDRs of SEQ ID NOs: 8-10. In some cases, a heavy chain variable domain having CDRs of SEQ ID NOs: 5-7 can be connected to a light chain variable domain having CDRs of SEQ ID NOs: 8-10 by a linker sequence. For example, an antigen binding domain targeting a NKG2C polypeptide can comprise a heavy chain variable domain comprising SEQ ID NO: 19, followed by a linker, followed by a light chain variable domain comprising SEQ ID NO: 20. In another example, an antigen binding domain targeting a NKG2C polypeptide can comprise a light chain variable domain comprising SEQ ID NO: 20, followed by a linker, followed by a heavy chain variable domain comprising SEQ ID NO: 19. The linker can be any suitable length. For example, a linker used to connect a heavy chain variable domain and a light chain variable domain can be about 3 to about 100 (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17) amino acid residues in length. Examples of linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain to produce an antigen binding domain include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGS (SEQ ID NO: 63), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41), PSGQAGAAASESLFVSNHAY (SEQ ID NO: 64), EASGGPE (SEQ ID NO: 65), EPKSSDKTHTSPPSPEL (SEQ ID NO: 66), RATPSHNSHQVPSAGGPTANSGTSG (SEQ ID NO: 67), and SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 68).

[0050] The antigen binding domain capable of binding to a polypeptide on the surface of a target cell can bind to any suitable polypeptide on the surface of a cell. In some cases, the target cell can be a cancer cell.

[0051] The antigen binding domain capable of binding to a polypeptide on the surface of a target cell (e.g., a cancer cell) can bind to any suitable polypeptide on the surface of a target cell. In some cases, the polypeptide expressed on the surface of a cancer cell can be a tumor-associated antigen. In some cases, the polypeptide expressed on the surface of a cancer cell can be a tumor-specific antigen. Examples of polypeptides that can be present on the surface of a target cell (e.g., a cancer cell) and can be targeted by the antigen binding domain of a cell engager provided herein include, but are not limited to, a CD33 polypeptide, a B7-H3 polypeptide, a PSMA polypeptide, a TEM-8 polypeptide, a HER2 polypeptide, a mesothelin polypeptide, an EPCAM polypeptide, a CD133 polypeptide, a CSPG4 polypeptide, a CLEC12A polypeptide, a CD19 polypeptide, a CD22 polypeptide, a ROR1 polypeptide, a IGF1R polypeptide, an IRA polypeptide, and a PD-L1 polypeptide. Examples of antigen binding domains capable of binding to a polypeptide expressed on the surface of a cancer cell that can be used to make a cell engager (e.g., a BiKE or a trispecific killing engager) provided herein include, but are not limited to, an anti-CD33 scFv and an anti-CD33 sdAb.

[0052] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) can be used in a cell engager provided herein, which can comprise a CDR1 of a VH domain, a CDR2 of a VH domain, and a CDR3 of a VH domain, and can also comprise a CDR1 of a VL domain, a CDR2 of a VL domain, and a CDR3 of a VL domain. For example, an antigen binding domain capable of binding to a CD33 polypeptide can be used in a cell engager provided herein, which can comprise CDR amino acid sequences as shown in Table 2:

[0053] Table 2. Exemplary CDR sequences of anti-CD33 antigen binding domains.

[0054] Sequence SEQ ID NO VH CDR1 GYTFTDY 24 VH CDR2 YPYNGG 25 VH CDR3 GRPAMDY 26 VL CDR1 RASESVDNYGISFMN 27 VL CDR2 AASNQGS 28 VL CDR3 QQSKEVPWT 29

[0055] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) can be used in the cell engagers provided herein, the antigen binding domain comprising (a) a heavy chain variable domain having a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 24 (or a variant of SEQ ID NO: 24 having one, two, three or four amino acid modifications), a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 25 (or a variant of SEQ ID NO: 25 having one, two, three or four amino acid modifications), and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 26 (or a variant of SEQ ID NO: 26 having one, two, three or four amino acid modifications), and / or (b) a light chain variable domain having a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 27 (or a variant of SEQ ID NO: 27 having one, two, three or four amino acid modifications), a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 28 (or a variant of SEQ ID NO: 28 having one, two, three or four amino acid modifications), and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 29 (or a variant of SEQ ID NO: 29 having one, two, three or four amino acid modifications), and the variable region can comprise any appropriate framework region.For example, such an antigen binding domain can comprise (a) a heavy chain variable domain comprising a framework region 1 having the amino acid sequence of SEQ ID NO: 30 (or a variant of SEQ ID NO: 30 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 2 having the amino acid sequence of SEQ ID NO: 31 (or a variant of SEQ ID NO: 31 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 3 having the amino acid sequence of SEQ ID NO: 32 (or a variant of SEQ ID NO: 32 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and a framework region 4 having the amino acid sequence of SEQ ID NO: 33 (or a variant of SEQ ID NO: 34 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications). NO: 33 (or a variant of SEQ ID NO: 33 with one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and / or (b) a light chain variable domain comprising a framework region 1 having the amino acid sequence as shown in SEQ ID NO: 34 (or a variant of SEQ ID NO: 34 with one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 2 having the amino acid sequence as shown in SEQ ID NO: 35 (or a variant of SEQ ID NO: 35 with one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), a framework region 3 having the amino acid sequence as shown in SEQ ID NO: 36 (or a variant of SEQ ID NO: 36 with one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications), and a light chain variable domain comprising a framework region 1 having the amino acid sequence as shown in SEQ ID NO: 37 (or a variant of SEQ ID NO: 37 with one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications). Framework region 4 of the amino acid sequence shown in (a variant of NO: 37).

[0056] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) useful in the cell engaging agents provided herein can comprise a heavy chain variable domain comprising SEQ ID NO: 38 and a light chain variable domain comprising SEQ ID NO: 39.

[0057] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 38 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 39. For example, such an antigen binding domain can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 38 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 39. In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 38, provided that the heavy chain variable domain comprises an amino acid sequence as set forth in SEQ ID NOs: 24, 25, and 26 and / or (b) a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 39, provided that the light chain variable domain comprises an amino acid sequence as set forth in SEQ ID NOs: 27, 28, and 28.

[0058] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) can be used in the cell engagers provided herein, the antigen binding domain can comprise (a) a heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 38 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) and / or (b) a light chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 39 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, such an antigen binding domain can comprise a heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 38 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 24, 25, and 26, and can comprise a light chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 39 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the light chain variable domain comprises the amino acid sequences as set forth in SEQ ID NOs: 27, 28, and 29.

[0059] In some cases, an antigen binding domain capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide) useful in the cell engagers provided herein can comprise (a) a heavy chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 25, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, and / or (b) a light chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 28, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence as set forth in SEQ ID NO: 29.

[0060] When the antigen binding domain is designed as a scFv having a heavy chain variable domain and a light chain variable domain, the two regions can be directly connected or can be connected using any suitable linker sequence. In some cases, a heavy chain variable domain having CDRs of SEQ ID NOs: 24-26 can be directly connected to a light chain variable domain having CDRs of SEQ ID NOs: 27-29. In some cases, a heavy chain variable domain having CDRs of SEQ ID NOs: 24-26 can be connected to a light chain variable domain having CDRs of SEQ ID NOs: 27-29 by a linker sequence. For example, an antigen binding domain targeting a CD33 polypeptide can comprise a heavy chain variable domain comprising SEQ ID NO: 38, followed by a linker, followed by a light chain variable domain comprising SEQ ID NO: 39. For example, an antigen binding domain targeting a CD33 polypeptide can comprise a light chain variable domain comprising SEQ ID NO: 39, followed by a linker, followed by a heavy chain variable domain comprising SEQ ID NO: 38. The linker can be any suitable length. For example, a linker used to connect a heavy chain variable domain and a light chain variable domain can be about 3 to about 100 (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17) amino acid residues in length. Examples of linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain to produce an antigen binding domain include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGS (SEQ ID NO: 63), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41), PSGQAGAAASESLFVSNHAY (SEQ ID NO: 64), EASGGPE (SEQ ID NO: 65), EPKSSDKTHTSPPSPEL (SEQ ID NO: 66), RATPSHNSHQVPSAGGPTANSGTSG (SEQ ID NO: 67), and SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 68).

[0061] In some cases, the cell-engaging agents provided herein may comprise one or more additional components (e.g., one or more accessory polypeptides). For example, the cell-engaging agents provided herein may comprise a first antigen-binding domain that can bind (e.g., specifically bind) to a polypeptide on the surface of an NK cell (e.g., an NKG2C polypeptide); a second antigen-binding domain that can bind (e.g., specifically bind) to a polypeptide on the surface of a cancer cell (e.g., a CD33 polypeptide); and may comprise one or more accessory polypeptides. Accessory polypeptides that may be included in the cell-engaging agents provided herein include, but are not limited to, polypeptides that can promote NK cell proliferation and / or survival (e.g., an IL-15 polypeptide); signaling polypeptides; and detectable polypeptides.

[0062] In some cases, the cell-engaging agents provided herein can be designed to include an IL-15 polypeptide. The IL-15 polypeptide can be any suitable IL-15 polypeptide. In some cases, the IL-15 polypeptide can be a human IL-15 polypeptide (e.g., a recombinant human IL-15 (rhIL-15) polypeptide). Examples of IL-15 polypeptides that can be included in the cell-engaging agents provided herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO: 40 (see, e.g., Example 8). In some cases, the cell-engaging agents provided herein can be designed to include a functional fragment or variant of the amino acid sequence shown in SEQ ID NO: 40, provided that the variant or fragment retains its essential ability to proliferate, initiate, and survive. For example, the cell-engaging agents provided herein can be designed to include an IL-15 polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO: 40, and having two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.

[0063] In some cases, the cell-engaging agents provided herein can be designed to include a signaling polypeptide. Any suitable signaling polypeptide can be used to design the cell-engaging agents described herein. Signaling polypeptides that can be used to prepare the cell-engaging agents described herein include, but are not limited to, BLK Alb signaling polypeptide, tPA signaling polypeptide, BiP signaling polypeptide, and CD8α signaling polypeptide.

[0064] When a cell engager provided herein comprises one or more additional components (e.g., one or more additional polypeptides), the additional components can be located in any suitable position within the cell engager. In some cases, a cell engager provided herein can have an IL-15 polypeptide located between a first antigen binding domain capable of binding to a NKG2C polypeptide and a second antigen binding domain capable of binding to a polypeptide present on the surface of a cancer cell. For example, a cell engager provided herein can comprise an antigen binding domain capable of binding to a NKG2C polypeptide, followed by an IL-15 polypeptide, followed by an antigen binding domain capable of binding to a polypeptide present on the surface of a cancer cell. For example, a cell engager provided herein can comprise an antigen binding domain capable of binding to a polypeptide present on the surface of a cancer cell, followed by an IL-15 polypeptide, followed by an antigen binding domain capable of binding to a NKG2C polypeptide.

[0065] In some cases, a cell engager provided herein can have a signal polypeptide located N-terminal to a first antigen binding domain capable of binding to a NKG2C polypeptide and a second antigen binding domain capable of binding to a polypeptide present on the surface of a cancer cell (e.g., a CD33 polypeptide). For example, a cell engager provided herein can comprise a signal polypeptide, followed by an antigen binding domain capable of binding to a NKG2C polypeptide, followed by an antigen binding domain capable of binding to a polypeptide present on the surface of a cancer cell (e.g., a CD33 polypeptide).

[0066] When a cell engager provided herein comprises one or more additional components (e.g., one or more additional polypeptides), the one or more additional components can be directly linked or can be linked using any suitable linker sequence. In some cases, an antigen binding domain (e.g., an antigen binding domain capable of binding to a NKG2C polypeptide and / or an antigen binding domain capable of binding to a cancer cell surface polypeptide) can be directly linked to an IL-15 polypeptide. In some cases, an antigen binding domain (e.g., an antigen binding domain capable of binding to a NKG2C polypeptide and / or an antigen binding domain capable of binding to a cancer cell surface polypeptide) can be linked to an IL-15 polypeptide via a linker sequence. For example, a cell engager provided herein can comprise an antigen binding domain capable of binding to a NKG2C polypeptide, followed by a linker, followed by an IL-15 polypeptide, followed by a linker, followed by an antigen binding domain capable of binding to a cancer cell surface polypeptide (e.g., a CD33 polypeptide). A linker can be any suitable length. For example, a linker useful for linking an antigen binding domain (an antigen binding domain capable of binding to a NKG2C polypeptide and / or an antigen binding domain capable of binding to a cancer cell surface polypeptide) and an additional polypeptide (e.g., an IL-15 polypeptide) can be about 3 to about 100 (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17) amino acid residues in length.Examples of linker sequences that can be used to link an IL-15 polypeptide to an antigen binding domain (e.g., an antigen binding domain capable of binding to an NKG2C polypeptide and / or an antigen binding domain capable of binding to a cancer cell surface polypeptide) include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGS (SEQ ID NO: 63), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41), PSGQAGAAASESLFVSNHAY (SEQ ID NO: 64), EASGGPE (SEQ ID NO: 65), EPKSSDKTHTSPPSPEL (SEQ ID NO: 66), RATPSHNSHQVPSAGGPTANSGTSG (SEQ ID NO: 67), and SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 68). NO:68).

[0067] In some cases, the cell-engaging agents provided herein (e.g., BiKE or trispecific killing engagement agents) can be designed to target NKG2C polypeptides and to target polypeptides expressed on the surface of cancer cells (e.g., CD33 polypeptides). For example, the cell-engaging agents provided herein can be designed to comprise an scFv comprising: a heavy chain variable region comprising SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, followed by a linker, followed by another scFv comprising: a heavy chain variable region comprising SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. For example, the cell engaging agents provided herein can be designed to comprise an scFv comprising: a heavy chain variable region comprising SEQ ID NO: 19, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 20, followed by a linker, followed by another scFv comprising: a heavy chain variable region comprising SEQ ID NO: 38, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 39.

[0068] In some cases, the cell engagers (e.g., BiKEs or tri-specific killing engagers) provided herein can be designed to target a NKG2C polypeptide and a polypeptide expressed on the surface of a cancer cell (e.g., a CD33 polypeptide), and further comprise an IL-15 polypeptide. For example, the cell engagers provided herein can be designed to comprise an scFv comprising a heavy chain variable region comprising SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, followed by a linker, followed by an IL-15 polypeptide (e.g., a human IL-15 polypeptide), followed by a linker, followed by another scFv comprising a heavy chain variable region comprising SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. For example, the cell engagers provided herein can be designed to comprise an scFv comprising a heavy chain variable region comprising SEQ ID NO: 19, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 20, followed by a linker, followed by an IL-15 polypeptide (e.g., a human IL-15 polypeptide), followed by a linker, followed by another scFv comprising a heavy chain variable region comprising SEQ ID NO: 38, followed by a linker, followed by a light chain variable region comprising SEQ ID NO: 39.

[0069] As shown herein, the amino acid sequences described herein can include amino acid modifications (e.g., a number of amino acid modifications). Such amino acid modifications can include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof. In some cases, the amino acid modifications can be made to improve binding and / or contact with an antigen, and / or to improve functional activity of the cell engagers provided herein. In some cases, the amino acid substitutions within the sequence identifiers can be conservative amino acid substitutions. For example, a conservative amino acid substitution can be made by replacing one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0070] In some cases, the amino acid substitution within the connecting sequence identifier can be a non-conservative amino acid substitution. A non-conservative amino acid substitution can be made by replacing one amino acid residue with another that has a different side chain. Examples of non-conservative substitutions include, but are not limited to: (a) the substitution of a hydrophilic residue (e.g., serine or threonine) for a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) the substitution of cysteine or proline for any other residue; (c) the substitution of a residue having an acidic side chain (e.g., aspartic acid or glutamic acid) for a residue having a basic side chain (e.g., lysine, arginine, or histidine), and (d) the substitution of a residue having a bulky side chain (e.g., phenylalanine) for a residue having a small side chain (e.g., glycine or other alanine).

[0071] Methods for generating amino acid sequence variants (e.g., amino acid sequences comprising one or more modifications relative to a particular sequence identifier) can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of nucleic acids encoding a cell engager or portion thereof. See, e.g., Zoller, Curr. Opin. Biotechnol. 3:348-354 (1992). Both naturally-occurring and non-naturally-occurring amino acids (e.g., artificially- derived amino acids) can be used to generate the amino acid sequence variants provided herein.

[0072] Example 8 further describes representative cell engagers that are capable of binding to a NKG2C polypeptide (e.g., a human NKG2C polypeptide) and are capable of binding to a CD33 polypeptide (e.g., a human CD33 polypeptide).

[0073] The cell binder provided herein can be produced using any suitable method. For example, the cell binder provided herein can be produced in a recombinant host cell. For example, a nucleic acid encoding the cell binder provided herein can be constructed, introduced into an expression vector, and expressed in a suitable host cell. Example 4, Example 7, and Example 9 are sequence tables of nucleic acid sequences encoding exemplary cell binders described herein. In some cases, the cell binder provided herein can be recombinantly produced in a prokaryotic host (such as Escherichia coli, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei). In some cases, the cell binders provided herein can also be used in eukaryotic hosts such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, lactis or Yarrowia lipolytica), filamentous fungi of the genera Trichoderma (e.g., T. reesei) and Aspergillus (e.g., A. niger and A. oryzae), protozoa (such as Leishmania tarentinois), insect cells, or mammalian cells (e.g., mammalian cell lines such as Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NSO cells, baby hamster kidney (BHK) cells, or the human embryonic kidney cell line HEK293). See, e.g., the reference by Frenzel et al. (Front Immunol., 4:217 (2013)).

[0074] In some cases, the cell adhesives provided herein can be substantially pure. As used herein, the term "substantially pure" with respect to a cell adhesive means that the cell adhesive is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acids. Thus, a substantially pure cell adhesive provided herein refers to any cell adhesive that is at least 60% pure. A substantially pure cell adhesive provided herein can have a purity of at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0075] In some cases, provided herein are cell binders that can be fused or coupled (e.g., covalently or non-covalently linked) to another polypeptide or other portion to provide a fusion protein or conjugate. For example, provided herein are cell binders that can be coupled (e.g., covalently or non-covalently linked) to a polymer (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG) and / or polyglutamic acid (PGA) [N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer], hyaluronic acid, fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, drugs, radioisotopes, and / or cytotoxic agents). Any suitable method can be used to couple another polypeptide or other portion (e.g., covalently or non-covalently linked) to a cell binder provided herein. For example, another polypeptide or other portion can be coupled to a binder provided herein using the method described in U.S. Patent No. 8,021,661.

[0076] In some cases, the cell binders provided herein can be modified with a portion to improve their stability and / or retention in the circulation (e.g., blood, serum, or other tissues), for example, by at least 1.5 times, 2 times, 5 times, 10 times, or 50 times. For example, the cell binders provided herein can be attached (e.g., covalently or non-covalently) to a polymer, such as a substantially non-antigenic polymer. Examples of substantially non-antigenic polymers that can be used as described herein include, but are not limited to, polyalkylene oxide and polyethylene oxide. In some cases, the polymer used herein can have any suitable molecular weight. For example, a polymer having an average molecular weight of about 200 daltons to about 35,000 daltons (e.g., about 1,000 daltons to about 15,000 daltons or about 2,000 daltons to about 12,500 daltons) can be used. In some cases, the cell binders provided herein can be attached (e.g., covalently or non-covalently) to a water-soluble polymer. Examples of water-soluble polymers that can be used herein include, but are not limited to, hydrophilic polyethylene polymers, polyvinyl alcohol, polyvinyl pyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene polyols, and copolymers and / or block copolymers thereof, provided that the water solubility of the copolymer or block copolymer is maintained.

[0077] In some cases, the cell-engaging agents provided herein can be linked (e.g., covalently or non-covalently linked) to one or more polyoxyalkylenes (e.g., polyoxyethylene, polyoxypropylene, or a block copolymer of polyoxyethylene and polyoxypropylene), polymethacrylates, carbomers, branched or unbranched polysaccharides, or combinations thereof. For example, the cell-engaging agents provided herein can be covalently linked to polyoxyethylene.

[0078] Also provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) having a nucleic acid sequence encoding at least a portion of a cell bonding agent provided herein. For example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a heavy chain variable domain (such as the heavy chain variable domains shown in Example 3 or Example 5). In some cases, the isolated nucleic acid molecules that can encode the heavy chain variable domains provided herein can be as shown in Example 4 or Example 7. In another example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a light chain variable domain (such as the light chain variable domains shown in Example 3 or Example 5). In some cases, the isolated nucleic acid molecules that can encode the light chain variable domains provided herein can be as shown in Example 4 or Example 7. In some cases, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding (a) heavy chain variable domains and (b) light chain variable domains, wherein the encoding linker polypeptide may be included or not included. The nucleic acid (e.g., isolated nucleic acid molecules) provided herein can be a single-stranded or double-stranded nucleic acid (e.g., DNA, RNA, or DNA / RNA hybrid) of any appropriate type.

[0079] Also provided herein is a nucleic acid construct containing one or more nucleic acids provided herein. In some cases, the nucleic acid construct containing one or more nucleic acids provided herein can be a vector (e.g., a plasmid vector or a viral vector). Examples of plasmid vectors that can be designed to include one or more nucleic acids include, but are not limited to, phagemids, wherein the nucleic acid has a nucleic acid sequence encoding at least a portion of a cell binder provided herein. Examples of viral vectors that can be designed to include one or more nucleic acids include, but are not limited to, retroviral vectors, parvoviral vectors (e.g., adenoviral vectors and adeno-associated virus (AAV) vectors), lentiviral vectors (e.g., herpes simplex virus (HSV) vectors), poxvirus vectors (e.g., vaccinia virus vectors and fowlpox virus vectors), and hybrid or chimeric viral vectors. For example, viral vectors having an adenoviral backbone and lentiviral components (such as those described elsewhere) (Zheng et al., Nat. Biotech., 18(2):176-80 (2000); WO98 / 22143; WO98 / 46778; and WO00 / 17376)) or viral vectors having an adenoviral backbone and AAV components (such as those described elsewhere) (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996))) can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of a cell-engaging agent provided herein.

[0080] In some cases, provided herein is a nucleic acid construct (e.g., a vector, such as a plasmid vector or a viral vector) that can include a nucleic acid sequence encoding a full-length cell binder provided herein. Provided herein is a nucleic acid construct (e.g., a vector, such as a plasmid vector or a viral vector) that can include a nucleic acid sequence encoding a full-length cell binder provided herein. Provided herein is a nucleic acid construct (e.g., a vector, such as a plasmid vector or a viral vector) that can include any suitable promoter and other regulatory sequences (e.g., transcription and translation initiation and termination codons) that are operably connected to a nucleic acid sequence encoding at least a portion of a cell binder provided herein. In some cases, the promoter for driving expression can be a constitutive promoter or a regulatable promoter. Examples of regulatable promoters that can be used as described herein include, but are not limited to, inducible promoters, repressible promoters, and tissue-specific promoters. Examples of viral promoters that can be used as described herein include, but are not limited to, adenovirus promoters, vaccinia virus promoters, CMV promoters (e.g., CMV immediate early promoters), and AAV promoters.

[0081] Any suitable method can be used to prepare the nucleic acid constructs (e.g., vectors, such as plasmid vectors or viral vectors) provided herein, which have at least a portion of the nucleic acid sequence encoding the cell bonding agent provided herein. For example, molecular cloning techniques can be used to prepare the nucleic acid constructs (e.g., vectors, such as plasmid vectors or viral vectors) provided herein, which have at least a portion of the nucleic acid sequence encoding the cell bonding agent provided herein, as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0082] Also provided herein is a host cell comprising a nucleic acid provided herein (e.g., a nucleic acid having a nucleic acid sequence encoding at least a portion of a cell bonding agent provided herein). The host cell that can be designed to comprise one or more nucleic acids provided herein can be a prokaryotic cell or a eukaryotic cell. The example of a prokaryotic cell that can be designed to comprise a nucleic acid provided herein includes, but is not limited to, Escherichia coli (e.g., Tb-1, TG-1, DH5α, XL-BlueMRF (Stratagene), SA2821 or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens or Pseudomonas (e.g., Pseudomonas aeruginosa) cells. The example of a eukaryotic cell that can be designed to comprise a nucleic acid provided herein includes, but is not limited to, insect cells (e.g., Sf9 or Ea4 cells), yeast cells (e.g., Saccharomyces cerevisiae cells) and mammalian cells (e.g., mouse, rat, hamster, monkey or human cells). For example, VERO cell, HeLa cell, 3T3 cell, Chinese hamster ovary (CHO) cell, W138BHK cell, COS-7 cell and MDCK cell all can be designed to comprise nucleic acid provided herein.Can use any suitable method one or more nucleic acid provided herein (for example, carrier, such as plasmid vector or viral vector, it has the nucleic acid sequence of at least part of the cell bonding agent that encodes provided herein) to introduce host cell. For example, calcium chloride-mediated transformation, transduction, conjugation, ternary conjugation, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high-speed bombardment with DNA-coated microparticles, direct microinjection into single cells, electroporation, or a combination thereof can be used to introduce the nucleic acids provided herein into host cells (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Davis et al., Basic Methods in Molecular Biology (1986); and Neumann et al., EMBO J., 1:841 (1982)).

[0083] In some cases, the cell engaging agents provided herein can be produced using a method comprising: (a) introducing a nucleic acid encoding a polypeptide into a host cell; (b) culturing the host cell in a culture medium under conditions sufficient for expression of the polypeptide; (c) harvesting the polypeptide from the cells or culture medium; and (d) purifying the polypeptide (e.g., to a purity of at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99%).

[0084] In some cases, the cell-engaging agents provided herein, the nucleic acids provided herein (e.g., nucleic acids encoding the full-length cell-engaging agents provided herein, the vectors provided herein (e.g., viral vectors designed to express the full-length cell-engaging agents provided herein), and / or the host cells provided herein (e.g., host cells designed to express the full-length cell-engaging agents provided herein) can be formulated into pharmaceutical compositions for administration to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML) to treat the mammal. In some cases, the cell-engaging agents provided herein, the nucleic acids provided herein (e.g., nucleic acids encoding the full-length cell-engaging agents provided herein, the vectors provided herein (e.g., viral vectors designed to express the full-length cell-engaging agents provided herein), and / or the host cells provided herein Cells (e.g., host cells designed to express the full-length cell-engaging agents provided herein) can be formulated into pharmaceutical compositions for administration to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or to increase the survival rate of a mammal suffering from cancer. For example, the cell-engaging agents provided herein that are capable of binding to an NKG2C polypeptide (e.g., a human NKG2C polypeptide) and capable of binding to a polypeptide present on the surface of a cancer cell (e.g., a CD33 polypeptide) can be formulated into pharmaceutical compositions for administration to a mammal (e.g., a human). In some cases, the pharmaceutical compositions provided herein can comprise a pharmaceutically acceptable carrier, such as a buffer, a salt, a surfactant, a sugar, a tonicity modifier, or a combination thereof, as described elsewhere (Gervasi et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131: 8-24 (2018)). Examples of pharmaceutically acceptable carriers that can be used to prepare pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, surfactants (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or sugars (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or combinations thereof. For example, a pharmaceutical composition designed to include a cell binder provided herein (or a nucleic acid, vector, or host cell provided herein) can be formulated to include a buffer (e.g., acetate, citrate, histidine, succinate, phosphate, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar (such as sucrose).Other ingredients that can be included in the pharmaceutical compositions provided herein include, but are not limited to, amino acids (such as glycine or arginine), antioxidants (such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA)), anticancer agents (such as enzalutamide, imanitib, gefitinib, erlotini, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosine, bortezomib, carfilzomib, batimastat, ganetespib, obatoclax, navitoclax, taxol, paclitaxel, or bevacizumab, or combinations thereof.For example, the pharmaceutical compositions provided herein can be formulated to include one or more cell-engaging agents provided herein in combination with one or more checkpoint inhibitors, such as anti-PD-1 antibodies or PD-1 inhibitors (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab,

[0015] These include, but are not limited to, toripalimab, dostarlimab, INCMGA00012, AMP-224, or AMP-514), an anti-PD-L1 antibody or PD-L1 inhibitor (e.g., avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189), and / or an anti-CTLA-4 antibody (e.g., ipilimumab).

[0085] In some cases, when a pharmaceutical composition is formulated to include one or more cell engagers provided herein, any appropriate concentration of the cell engager can be used. For example, a pharmaceutical composition provided herein can be formulated to include about 1 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a cell engager provided herein per milliliter of a liquid. For example, a pharmaceutical composition provided herein can be formulated to include about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a cell engager provided herein per milliliter of a solid or semi-solid. In some cases, when a pharmaceutical composition is formulated to include one or more nucleic acids (e.g., vectors, such as viral vectors) encoding at least a portion of a cell engager provided herein (e.g., a full-length cell engager provided herein), any appropriate concentration of the nucleic acid can be used. For example, a pharmaceutical composition provided herein can be formulated to include about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a nucleic acid provided herein per milliliter of a liquid.For example, the pharmaceutical compositions provided herein can be formulated as a solid or semi-solid comprising about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a nucleic acid provided herein.

[0086] In some cases, the pharmaceutical compositions designed to include cell binders provided herein can be formulated to include reagents that can reduce the aggregation of one or more cell binders after formulation. Examples of such reagents that can be used as described herein include, but are not limited to, methionine, arginine, lysine, aspartic acid, glycine, glutamic acid, and combinations thereof. In some cases, the formulation can include one or more of these amino acids at a concentration of about 0.5 mM to about 145 mM (e.g., about 1 mM to about 145 mM, about 10 mM to about 145 mM, about 100 mM to about 145 mM, about 0.5 mM to about 125 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 75 mM, or about 10 mM to about 100 mM).

[0087] The pharmaceutical compositions provided herein can be in any suitable form. For example, the pharmaceutical compositions provided herein can be designed as liquid, semisolid or solid. In some cases, the pharmaceutical compositions provided herein can be liquid solutions (for example, injectable and / or infusible solutions), dispersions, suspensions, tablets, pills, powders, microemulsions, liposomes or suppositories. In some cases, the pharmaceutical compositions provided herein can be lyophilized. In some cases, the pharmaceutical compositions provided herein (for example, pharmaceutical compositions comprising one or more cell binders provided herein) can be formulated together with a carrier or coating designed to prevent rapid release. For example, the pharmaceutical compositions provided herein can be formulated as controlled release formulations or adjusted release formulations, as described herein (U.S. Patent Application Publication Nos. 2019 / 0241667; 2019 / 0233522; and 2019 / 0233498).

[0088] Also provided herein are methods of administering a composition comprising one or more cell binders as provided herein (or nucleic acids, vectors, or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein) to a mammal (e.g., a human). For example, a composition comprising one or more cell binders as provided herein (or nucleic acids, vectors, and / or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein) can be administered to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia, such as AML) to treat the mammal. In some cases, a composition comprising one or more cell binders as provided herein (or nucleic acids, vectors, and / or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or to increase the survival rate of the cancerous mammal.

[0089] In some cases, a composition comprising one or more cell-engaging agents as provided herein (or a nucleic acid, vector, and / or host cell as provided herein) (e.g., a pharmaceutical composition as provided herein) can be administered to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML) to alleviate or eliminate one or more symptoms of the cancer. Symptoms of cancer (e.g., leukemia such as AML) that can be alleviated using a composition comprising one or more cell-engaging agents described herein include, but are not limited to, fever, bone pain, lethargy and fatigue, shortness of breath, pale skin, frequent infections, easy bruising, abnormal bleeding (e.g., frequent nosebleeds and bleeding gums), and neutropenia.

[0090] Any suitable cancer can be treated using a composition comprising one or more cell binders as provided herein (or nucleic acids, vectors, or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein). For example, a mammal (e.g., a human) suffering from cancer can be treated by administering a composition comprising one or more cell binders as provided herein (e.g., pharmaceutical compositions). In some cases, the cancer that can be treated as described herein can include one or more solid tumors. In some cases, the cancer that can be treated as described herein can be a blood cancer. Examples of cancers that can be treated as described herein include, but are not limited to, leukemias (e.g., AML), lymphomas, myelodysplastic syndromes (MDS), and systemic mastocytosis. In some cases, a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML) can be administered a composition comprising one or more cell binders as provided herein (e.g., pharmaceutical compositions) to treat the mammal (e.g., to reduce the number of cancer cells in the mammal).

[0091] Any suitable method can be used to administer the compositions (e.g., pharmaceutical compositions) provided herein to mammals (e.g., humans). For example, the compositions (e.g., pharmaceutical compositions comprising one or more cell-engaging agents provided herein) provided herein can be administered intravenously (e.g., by intravenous injection or infusion), subcutaneously (e.g., by subcutaneous injection), intraperitoneally (e.g., by intraperitoneal injection), orally, by inhalation, or intramuscularly (e.g., by intramuscular injection) to mammals (e.g., humans). In some cases, the route and / or mode of administration of the composition (e.g., pharmaceutical composition provided herein) can be adjusted according to the mammal being treated.

[0092] In some cases, an effective amount of a composition comprising a cell-engaging agent as provided herein (or a nucleic acid, vector, or host cell as provided herein) (e.g., a pharmaceutical composition as provided herein) can be an amount that reduces the number of cancer cells in a mammal suffering from cancer (e.g., a leukemia such as AML) without causing significant toxicity to the mammal. In some cases, an effective amount of a composition comprising one or more cell-engaging agents as provided herein (or a nucleic acid, vector, or host cell as provided herein) (e.g., a pharmaceutical composition as provided herein) can be an amount that increases the survival of a mammal suffering from cancer (e.g., a leukemia such as AML) compared to a control mammal suffering from a similar cancer and not treated with the composition. For example, an effective amount of a cell engaging agent provided herein can be from about 0.001 mg / kg to about 100 mg / kg (e.g., from about 0.001 mg / kg to about 90 mg / kg, from about 0.001 mg / kg to about 80 mg / kg, from about 0.001 mg / kg to about 70 mg / kg, from about 0.001 mg / kg to about 60 mg / kg, from about 0.001 mg / kg to about 50 mg / kg, from about 0.001 mg / kg to about 40 mg / kg, from about 0.001 mg / kg to about 30 mg / kg, from about 0.005 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 100 mg / kg, from about 0.05 mg / kg to about 100 mg / kg, from about 0.1 mg / kg to about 100 mg / kg, from about 0.5 mg / kg to about 100 mg / kg, from about 1 mg / kg to about 100 mg / kg, from about 5 mg / kg to about 100 mg / kg, (e.g., about 10 mg / kg to about 30 mg / kg, about 15 mg / kg to about 30 mg / kg, about 20 mg / kg to about 30 mg / kg, about 3 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 25 mg / kg, about 1 mg / kg to about 20 mg / kg, about 2 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 30 mg / kg, about 15 mg / kg to about 30 mg / kg, about 20 mg / kg to about 30 mg / kg, about 3 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 1 mg / kg to about 3 mg / kg). The effective amount can be maintained constant or can be adjusted on a sliding scale or variable dose basis depending on the mammal's response to treatment. Various factors can influence the actual effective amount required for a particular application.For example, when treating a mammal with cancer (e.g., a leukemia such as AML), the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-use with other therapeutic or prophylactic treatments (such as the use of other agents (e.g., checkpoint inhibitors)), and the judgment of the treating physician may require increasing or decreasing the actual effective amount of a composition provided herein (e.g., a pharmaceutical composition containing one or more cell engaging agents provided herein) administered.

[0093] In some cases, the effective frequency of administering a composition comprising one or more cell-engaging agents as provided herein (or a nucleic acid, vector, or host cell as provided herein) (e.g., a pharmaceutical composition as provided herein) can be a frequency that reduces the number of cancer cells in a mammal suffering from cancer (e.g., a leukemia such as AML) without producing significant toxicity to the mammal. In some cases, the effective frequency of administering a composition comprising one or more cell-engaging agents as provided herein (or a nucleic acid, vector, or host cell as provided herein) (e.g., a pharmaceutical composition as provided herein) can be a frequency that increases the survival of a mammal suffering from cancer (e.g., a leukemia such as AML) compared to a control mammal suffering from a similar cancer and not treated with the composition. For example, the effective frequency of administering a pharmaceutical composition as provided herein (e.g., a pharmaceutical composition containing one or more cell-engaging agents as provided herein) can be about twice a day to about once a year (e.g., about twice a day to about once a month, about twice a day to about once a week, about once a day to about once a month, or about once a day to about once a week). In some cases, the frequency of administering a pharmaceutical composition as provided herein (e.g., a pharmaceutical composition containing one or more cell-engaging agents as provided herein) can be once a day. The frequency of administration of the pharmaceutical compositions provided herein (such as, pharmaceutical compositions containing one or more cell binders provided herein) can remain constant or can vary during treatment. Various factors can affect the actual effective frequency required for use in a particular application. For example, the severity of the cancer (e.g., leukemia such as AML), the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-use with other therapeutic or preventive treatments (such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require increasing or decreasing the actual effective frequency of administration of the compositions provided herein (e.g., pharmaceutical compositions containing one or more cell binders provided herein).

[0094] In some cases, the effective duration of administering a composition containing one or more cell binders as provided herein (or nucleic acids, vectors or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein) can be a duration that reduces the number of cancer cells in a mammal without producing significant toxicity to the mammal. In some cases, the effective duration of administering a composition comprising one or more cell binders as provided herein (or nucleic acids, vectors or host cells as provided herein) (e.g., pharmaceutical compositions as provided herein) can be a duration that increases the survival of a mammal suffering from cancer (e.g., leukemia such as AML) compared to a control mammal suffering from a similar cancer and not treated with the composition. For example, the effective duration of administering a pharmaceutical composition as provided herein (e.g., a pharmaceutical composition comprising one or more cell binders as provided herein) can vary from a single administration time point to several weeks to several months (e.g., 4 to 12 weeks). Various factors can affect the actual effective duration for a particular application. For example, the severity of the cancer (e.g., leukemia such as AML), the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-use with other therapeutic or prophylactic treatments (such as the use of other agents (e.g., checkpoint inhibitors)), and the judgment of the treating physician may require increasing or decreasing the actual effective administration duration of a composition provided herein (e.g., a pharmaceutical composition containing one or more cell engaging agents provided herein).

[0095] In some cases, a composition (e.g., a pharmaceutical composition) containing one or more cell-engaging agents as provided herein (or nucleic acids, vectors, and / or host cells as provided herein) can be administered together with a population of NK cells (e.g., in adoptive cell therapy) to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML). Any suitable NK cells can be administered together with one or more cell-engaging agents as provided herein (or nucleic acids, vectors, and / or host cells as provided herein) to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML). In some cases, at least a portion of the NK cells in the NK cell population administered to a mammal can be NKG2C + NK cells (e.g., can be engineered to be NKG2C + NK cells). In some cases, at least a portion of the NK cells in the NK cell population administered to a mammal may be iNK cells. In some cases, at least a portion of the NK cells in the NK cell population administered to a mammal may be engineered to express one or more signaling polypeptides (e.g., DAP12 polypeptide).

[0096] The NK cell population can include any suitable number of NK cells. For example, NK cells (e.g., NKG2C) can be administered to a mammal (e.g., a human) suffering from cancer (e.g., a leukemia such as AML) together with one or more cell-engaging agents as provided herein (or nucleic acids, vectors and / or host cells as provided herein). + An effective amount of NK cells) can be from about 100 million NK cells per kilogram (kg) of body weight of the mammal (cells / kg) to about 900 million NK cells / kg (e.g., from about 100 million to about 800 million NK cells / kg, from about 100 million to about 700 million NK cells / kg, from about 100 million to about 600 million NK cells / kg, from about 100 million to about 500 million NK cells / kg, from about 100 million to about 400 million NK cells / kg, from about 100 million to about 300 million NK cells / kg, from about 100 million to about 200 million NK cells / kg, from about 200 million to about 900 million NK cells / kg, from about 300 million to about 900 million NK cells / kg, from about 400 million to about 900 million NK cells / kg, about 500 million to about 900 million NK cells / kg, about 600 million to about 900 million NK cells / kg, about 700 million to about 900 million NK cells / kg, about 800 million to about 900 million NK cells / kg, about 200 million to about 800 million NK cells / kg, about 300 million to about 700 million NK cells / kg, about 400 million to about 600 million NK cells / kg, about 200 million to about 400 million NK cells / kg, about 300 million to about 500 million NK cells / kg, about 400 million to about 600 million NK cells / kg, about 500 million to about 700 million NK cells / kg or about 600 million to about 800 million NK cells / kg).

[0097] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0098] Example

[0099] Example 1: Anti-NKG2C / IL-15 / anti-CD33 Killer Conjugate Directs Primary and iPSC-Derived NKG2C + NK cells specifically target myeloid leukemia

[0100] This example describes the design and characterization of molecules capable of binding to NKG2C polypeptides. An anti-NKG2C / IL-15 / anti-CD33 killer conjugate (NKG2C-KE) was designed that can direct NKG2C + The cells target CD33 (a tumor-associated antigen expressed by AML cells)-positive cells, thereby eliciting NKG2C + Tumor-targeting responses of NK cells.

[0101] result

[0102] Functional validation of the novel NKG2C-KE

[0103] To determine whether NKG2C engagement could be exploited to specifically activate NK cells, NKG2C-KE was generated using sequences encoding an anti-NKG2C antibody (Cichocki et al., Sci. Transl. Med. 12: eaaz5618 (2020)). The variable heavy and light chain sequences of the anti-NKG2C antibody were cloned into a construct containing sequences encoding an anti-CD33 short chain variable fragment (scFv) component to target AML and a sequence encoding a wild-type IL-15 component to connect the two scFvs. The peripheral blood of healthy CMV seropositive donors was screened for the presence of NKG2C by flow cytometry. + NK cells, then based on NKG2C + The proportion of cells is greater than (NKG2C 高 ) or less than (NKG2C 低 )10% cells were divided into two groups. Peripheral blood mononuclear cells (PBMC) of each donor were isolated and compared with untreated CD33 + AML cell line THP-1, rhIL-15 or NKG2C-KE were co-cultured and functional analysis was performed by flow cytometry for 5 hours. 高 Compared with NKG2C cells co-cultured with NKG2C-KE 高 Cells showed increased degranulation at concentrations of 0.3 nM (18.5% vs. 13.3%, p = 0.05), 3 nM (21.9% vs. 13.9%, p = 0.0001), and 30 nM (22.6% vs. 13.6%, p < 0.0001). Figure 1A ). Compare NKG2C 低 When NKG2C-KE was detected in NKG2C-positive and NKG2C-positive NK cells, NKG2C-KE at concentrations of 3 nM (10.7% vs. 21.9%, p=0.009) and 30 nM (12.2% vs. 22.6%, p=0.02) triggered NKG2C-positive cells. 高 Stronger degranulation response of NK cells ( Figure 1A ). Similar to the degranulation reaction, NKG2C 高 NK cells produced more IFNγ in response to NKG2C-KE than to rhIL-15. Compared with rhIL-15, NKG2C-KE at concentrations of 3 nM (8.1% vs. 16.1%, p=0.0001) and 30 nM (8.0% vs. 15.9%, p=0.0002) increased IFNγ. 低In donors with high NKG2C expression, NKG2C-high NK cells produced more IFNγ after treatment with 3 nM (4.4% vs. 16.1%, p=0.01) and 30 nM (5.5% vs. 15.9%, p=0.04) NKG2C-KE. 低 NK cells, their degranulation and IFNγ production were compared with those of NKG2C cells treated with rhIL-15. 低 There was no statistically significant difference in NK cells ( Figure 1B To evaluate whether NK cell function is associated with NKG2C expression frequency, we performed linear regression analysis. Although there was no correlation between NK cell degranulation and NKG2C expression frequency under rhIL-15 culture conditions, NK cells stimulated with NKG2C-KE showed a significant increase in NKG2C expression frequency at all concentrations (0.3 nM (R 2 =0.7, p=0.0002), 3 nM (R 2 =0.7, p=0.0003) and 30 nM (R 2 =0.6, p=0.0009)) showed a higher frequency of degranulation, and the degranulation frequency was correlated with the NKG2C expression ratio ( Figure 1C When NK cells were stimulated with different concentrations of NKG2C-KE, the frequency of IFNγ production also correlated with NKG2C frequency. Taken together, these data suggest that NKG2C-KE specifically activates NK cells based on NKG2C expression.

[0104] NKG2C-KE activates and expands NKG2C in HCT patients who have experienced CMV reactivation + NK cells

[0105] “Adaptive” NK cells (defined as CD57 + NKG2C + Reconstitution of the NKG2C subset was primarily observed in transplant recipients who were CMV seropositive and underwent CMV reactivation after transplantation. This association suggests that adaptive NKG2C expression is more important than that of typical NK cells that lack NKG2C expression. + NK cells have stronger anti-tumor function. Therefore, we evaluated whether NKG2C-KE would preferentially activate NK cell function in CMV-reactivated transplant recipients (rather than CMV-seronegative patients). PBMCs from patients were collected 6 months after transplantation and co-cultured with THP-1AML cells expressing high levels of CD33 in the presence or absence of NKG2C-KE. Compared with the untreated control group, the degranulation rate of NK cells from CMV-reactivated patients increased by more than 2 times in the presence of NKG2C-KE (16% vs. 48.2%, p < 0.0001) ( Figure 2A), and the production of IFNγ was also significantly increased (4.3% vs. 29.8%, p<0.0001) ( Figure 2B ). However, after transplantation, CMV seronegative and NKG2C + This effect was not observed when NK cells were measured in patients with lower NK cell frequencies ( Figure 2A and 2B ).

[0106] Since NKG2C is significantly associated with CMV seronegative patients and CMV reactivation patients + NK cell frequency and quality vary, so it is necessary to determine whether responsiveness to NKG2C-KE is associated with NKG2C frequency. 2 =0.6, p<0.0001) and IFNγ production (R 2 =0.9, p<0.0001), further confirming the specificity of NKG2C-KE ( Figure 2C and 2D ). NKG2C-KE was then used to test whether it could induce the selective proliferation of adaptive NK cells using transplanted PBMCs. Compared with rhIL-15, NKG2C-KE was observed to be significantly more effective in patients with CMV reactivation than in those with CMV reactivation. + NK cell-specific proliferation, indicating that NKG2C-KE can target the delivery of IL-15 ( Figure 2E Although rhIL-15 can induce widespread proliferation of all NK cells, IL-15 in the NKG2C-KE environment has a significant effect on NKG2C + When evaluating the proportion of NK cells that proliferated in response to NKG2C-KE, NKG2C + The proportion of proliferating cells in the cells was higher than that in the cells treated with rhIL-15 ( Figure 2F : 65.2% vs. 34.8%, p<0.0001), resulting in a decrease in NKG2C when NKG2C-KE was used. + :NKG2C - Higher ratio ( Figure 2G In samples where NK cells did not contain NKG2C or had a low frequency of NKG2C, rhIL-15 and NKG2C-KE triggered similar proportions of NK cell proliferation.

[0107] NKG2C-KE controls CD33 + AML and leads to NKG2C + Enhanced persistence of adaptive NK cells in vivo

[0108] Based on the display NKG2C +The in vitro data on NK cell and NKG2C-KE specificity were validated in vivo using a myeloid leukemia model in which NOD-SCIDγ (NSG) mice lacking mouse lymphocytes (T, B, and NK cells) were injected with luciferase-labeled CD33 + HL-60 myeloid tumor. Three days after the establishment of AML, animals were treated with expanded PB NK cells from a CMV-positive normal donor, which contained 13.5% NKG2C+ and 91.3% CD16+ NK cells ( Figure 3A and 3B ). NKG2C-KE or CD161533TriKE was administered 5 times a week for 3 weeks, and bioluminescence imaging (BLI) was performed weekly. Blood was collected on days 14 and 28 to assess the persistence and expansion of NK cells. Compared with control animals (mean radiation: 4.8e9+ / -2.1e9), both 161533TriKE or NKG2C-KE combined with NK cells mediated effective tumor control (1.2e8+ / -2.3e8 vs. 1.3e8+ / -1.8e8, p=0.99) ( Figure 3C and 3D ), despite the immunoconjugate's CD16 and NKG2C targets being 7-fold different on expanded PB NK cells ( Figure 3B 28 days after adoptive transfer, NK cells treated with NKG2C-KE showed NKG2C expression compared with 161533TriKE. + The higher persistence of NK cells suggests that adaptive NK cells have preferential persistence / expansion in vivo. The decrease in NKG2C on NK cells in mice treated with NKG2C-KE at day 14 was due to receptor occupancy, as NKG2C was not detectable by flow cytometry in mice treated with active NKG2C-KE due to occupancy ( Figure 3E ).

[0109] Generation and functional characterization of iPSC-derived NK cells transgenically expressing NKG2C

[0110] The frequency of adaptive NK cells expressing NKG2C varies widely in the general population, with a few individuals having frequencies exceeding 10% in their peripheral blood. To expand the application of NKG2C-KE, we developed an ideal off-the-shelf NK cell line using iPSC-derived NK cells and combined it with NKG2C-KE. Two different iNK cell lines were generated using this platform. One iNK cell line was engineered with NKG2C alone, while the other was genetically engineered with both NKG2C and its signaling adaptor molecule DAP12 to enhance NKG2C expression and immune responses. iPSCs were transduced, enriched for NKG2C expression, and stored to produce a renewable starting material. The cells were then differentiated in a staged manner to CD34 + Stage. CD34 + The cells then differentiated along the NK cell lineage. After expansion, the untransduced parental iNK cells expressed low levels of NKG2C. NKG2C-transduced iNK cells showed significantly increased surface NKG2C expression, and expression was further elevated in iNK cells expressing both NKG2C and DAP12 ( Figure 4A This increase in NKG2C-expressing cells was reproducible, with NKG2C and DAP12 iNK cells expressing NKG2C. + The cell population was significantly higher than that of NKG2C cells without transduction and without DAP12 (65.32% vs. 10.86 and 45.52, p>0.01 and 0.001) ( Figure 4B ), and DAP12 also increased ( Figure 8 MFI showed that iNK cells transduced with NKG2C and DAP12 expressed more NKG2C per cell compared to iNK cells transduced with NKG2C alone but not DAP12 ( Figure 4C Although NKG2C with DAP12 did not express intracellular markers of adaptive NK cells (PLZF, EAT2, or FcεRIγ), they did express slightly more of the DAP12-associated receptor NKp44 and KIR at higher densities (Figure 9).

[0111] To test the combined therapeutic potential of NKG2C-KE and NKG2C gene-edited iNK cells, a series of functional assays were performed using two AML cell lines (HL-60 and THP-1) that highly express CD33 as target cells. Untransduced and gene-edited iNK cells were cultured with target cells alone, co-cultured with rhIL-15, or co-cultured with NKG2C-KE, and their degranulation and cytokine production were evaluated ( Figure 5A-D). In transduced iNKs, NKG2C-KE induced significantly higher degranulation against THP-1 targets than rhIL-15 (NKG2C: 16.1% vs. 6.9%, p<0.0001; NKG2C / DAP12: 17.2% vs. 4.6%, p<0.0001), and also significantly increased IFNγ production (NKG2C: 14.4% vs. 6.9%, p=0.003; NKG2C / DAP12: 23.0% vs. 4.9%, p<0.0001) ( Figure 5A and 5B ). and CD33 - Target incubation did not result in increased degranulation or IFNγ production (Figure 10). Functional responses of untransduced cells were not affected by NKG2C-KE, suggesting that low NKG2C frequencies are insufficient to generate enhanced responses. Similar results were observed in HL-60 cells ( Figure 5C and 5D ). Although gene-edited iNK cells (NKG2C+ / -DAP12) showed similar degranulation levels against both tumor cell lines, iNK cells co-transduced with NKG2C and DAP12 produced significantly more IFNγ against THP-1 cells, compared with cells transduced with NKG2C alone, while there was no significant difference against HL-60 cells. Stimulation with rhIL-15 induced strong proliferation signals in both transduced and untransduced iNKs, but NKG2C-KE mediated greater proliferation of iNK cells co-transduced with NKG2C and DAP12 ( Figure 5E The enhanced degranulation, IFNγ production, and proliferation responses suggested that DAP12 plays an important role in NKG2C-mediated iNK cell generation. Therefore, further experiments used iNK cells engineered with both NKG2C and DAP12.

[0112] To directly assess tumor killing, we utilized a dynamic in vitro system that allows continuous quantification of the loss of fluorescently labeled target cells during cell death by live imaging over 24 hours. Untransduced and NKG2C / DAP12-transduced iNK cells were co-cultured with THP-1 target cells in the presence or absence of rhIL-15 or NKG2C-KE and imaged every 30 minutes ( Figure 6A and 6B Untransduced iNK cells exhibited modest natural cytotoxicity against THP-1 cells, whereas the addition of rhIL-15 or NKG2C-KE maintained their toxicity, albeit with statistically significant differences. Differently, the addition of NKG2C-KE to NKG2C / DAP12-transduced iNK cells resulted in increased killing kinetics within the first 18 h post-exposure, with statistically significant increases consistently observed at 6 and 12 h (Figure 6C ).

[0113] NKG2C / DAP12-transduced iNK cells exert cytotoxic effects on primary AML under NKG2C-KE targeting

[0114] The above functional assays tested targeting of AML cell lines. To evaluate the functional capacity of NKG2C / DAP12-transduced iNK cells activated by NKG2C-KE against more physiologically relevant targets, additional functional experiments were performed using primary AML blasts. Five blasts containing 47%-97% CD33 + AML patient samples of blasts were used as targets in flow cytometry-based functional assays, and all samples expressed HLA-E ( Figure 11 ). Regardless of whether rhIL-15 or NKG2C-KE was treated, the degranulation level of the original cells by the non-transduced iNK cells was low ( Figure 7A Similar functional responses were observed with NKG2C / DAP12-transduced iNK cells in the presence or absence of rhIL-15, although rhIL-15 provided a stronger IL-15 signal than NKG2C-KE. However, when used in combination with NKG2C-KE, NKG2C / DAP12-transduced iNK cells enhanced the degranulation response to primary AML blasts by twofold. Although rhIL-15 enhanced IFNγ production by both iNK cell lines, NKG2C / DAP12-transduced cells targeted with NKG2C-KE induced the highest levels of IFNγ ( Figure 7B The killing of primary AML blasts was assessed by measuring the number of viable AML cells after two days of co-culture ( Figure 7C In the absence of rhIL-15 or NKG2C-KE, there was no significant difference in the number of AML cells co-cultured with untransduced and NKG2C / DAP12-transduced iNK cells. rhIL-15 alone induced a robust natural cytotoxic response in both iNK cell lines, resulting in substantial AML killing. However, when NKG2C / DAP12-transduced iNK cells were directed to the CD33 antigen using NKG2C-KE, primary AML targets were almost completely eliminated (rhIL-15, 3298; NKG2C-KE, 486.7; p = 0.02).

[0115] The above results indicate that cell-engaging agents that can simultaneously bind to NKG2C peptide and CD33 peptide can guide NK cells to CD33 + Cancer cells and induce them to produce proteins targeting these CD33 +Cancer Cell Immune Responses As shown herein, such cell-engaging agents can be used to treat mammals (eg, humans) suffering from cancer (eg, leukemias such as AML).

[0116] Materials and methods

[0117] Protein production

[0118] The final construct of NKG2C-KE was spliced ​​into a minicircle plasmid (SBI:MN502A-1) and a CMV promoter was inserted into the multiple cloning site. The NKG2C-KE portion contained a start codon, an output sequence, an anti-NKG2C single-chain antibody (scFv), an 18 amino acid sequence of wild-type IL-15 flanking, an anti-CD33 scFv, and a 10xHis tag. The NKG2C-KE plasmid was transfected into Expi293F (Thermo Fisher, Waltham, Massachusetts: A14527) using ExpiFectimine (Thermo Fisher: A14524). On day 4-5 after transfection, when cell viability dropped below 80%, the supernatant was collected. The supernatant was then incubated with HisPur cobalt-based resin (Thermo Fisher: 89965) for 1 hour. The resin was washed three times, and NKG2C-KE was eluted from the HisPur resin using 250 mM imidazole through the column. The protein was desalted using a PD-10 column (GE Healthcare, Chicago, IL). Protein electrophoresis was performed on a tris-based gel, and purity was assessed using GelCode Blue (Thermo Fisher Scientific; 24592) stain. The preparation method of 161533TriKE is described elsewhere (Hermanson et al., Stem Cells, 34:93–101 (2016)). Briefly, the plasmid was transformed into Escherichia coli strain BL21 (DE3) (EMD), and the precipitate was harvested by centrifugation after 2 hours. The bacteria were then resuspended and the inclusion bodies were harvested, followed by washing to remove endotoxins. The protein was then refolded and purified using FPLC ion exchange chromatography.

[0119] Healthy donor and patient samples

[0120] Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using a density gradient of Ficoll-Paque (GE Healthcare). PBMCs were either cryopreserved in liquid nitrogen or used fresh. AML blasts were obtained from a single sample obtained from a patient with de novo (chemotherapy-naive) AML with a normal karyotype and cryopreserved; the frequency of AML blasts in this sample ranged from 45% to 93%. All cells were cultured in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum and penicillin / streptomycin at 37°C and 5% CO2. PBMC and AML samples were thawed and allowed to rest overnight before use.

[0121] Expanding NK cells

[0122] Human peripheral blood mononuclear cells (PBMCs) obtained from the above protocol were enriched with NK cells (Stemcell). The cells were then cultured for 14 days in RPMI supplemented with irradiated K562 cells expressing 4-1BBL and membrane-bound IL-21 in 10% heat-inactivated FBS, penicillin-streptomycin, and 50 IU / mL IL-2. The medium was changed every 2-3 days. Feeder cells were added on days 0 and 7.

[0123] Generation of NKG2C from iPSCs + NK cells

[0124] Culture of human iPSCs and their differentiation into iCD34 + For the differentiation of iNK cells, see Cichocki et al. (Sci. Transl. Med., 12: eaaz5618 (2020)). At the beginning of iNK cell differentiation culture, iCD34 + Cells were seeded onto stromal cells and cultured in B0 medium supplemented with cytokines that support NK cell differentiation from hematopoietic progenitor cells. Following directed iNK cell differentiation, iNK cells were harvested and co-cultured with modified K562 cells in B0-supplemented medium for expansion. K562 cells were propagated in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Hyclone).

[0125] Cell lines, antibodies, and reagents

[0126] THP-1 and HL-60 cells were cultured in RPMI-1640 (Gibco) with 10% heat-inactivated fetal bovine serum and penicillin / streptomycin supplement. THP-1 and HL-60 cells were cultured to a density between 0.2-2 million cells / mL. Cell lines were purchased from ATCC. Fluorochrome conjugated antibodies were purchased from BioLegend (San Diego, CA): anti-CD56 (clone HCD56), anti-IFNy (clone XMG1.2), anti-CD45 (clone HI30), anti-CD34 (clone 561), anti-NKp44 (clone P44-8), anti-KIR (clone HP-MA4, DX27 and DX9), anti-NKG2D (clone 1D11), anti-HLA-E (clone 3D12); BD Biosciences (San Jose, CA): anti-CD3 (clone UCHT1); Thermo Fisher (Waltham, MA): Live / Dead Aqua (Cat# L34966), CellTrace Violet (C34557), Live / Dead NearIR (L34976), CellTrace FarRed (C34564); R&D Systems (Minneapolis, MN): anti-NKG2C (clone 134591), anti-PLZF (clone 6318100), anti-DAP12 (clone 406288); Millipore (Burlington, MA): anti-FcsRIy (polyclonal); Proteintech (Rosemont, IL): anti-EAT2 (SH2D1B) (polyclonal); Beckman Coulter (Indianapolis, IN): anti-NKG2A (clone z199); Sartorius (France): Caspase 3 / 7 Apoptosis Detection Reagent FITC (Sartorius 4440).

[0127] NK cell functional assays

[0128] Effector cells were co-incubated with target cells at a 2:1 effector:target (E:T) ratio, with or without the addition of NKG2C-KE. Anti-CD107a antibody was added at the start of the co-culture. After a one-hour incubation, GolgiStop and GolgiPlug were added to each well and incubated for an additional 4 hours. After a 5-hour incubation, cells were first stained with Live / Dead Aqua to assess viability, followed by surface staining for CD3 and CD56. Cells were then fixed with 2% paraformaldehyde in PBS for 20 minutes, permeabilized with 0.1% Triton X for 5 minutes, and finally intracellularly stained for IFNγ. Samples were analyzed using an LSRII flow cytometer (BD) and FlowJo software (BD, Ashland, OR). For the proliferation assay, cells were pre-stained with CellTrace Violet and then incubated with NKG2C-KE or recombinant human IL-15 (R&D Systems, Minneapolis, MN) at 37°C, 5% CO2 for 7 days. After 7 days, cells were washed and stained with Live / Dead NearIR, followed by surface staining for CD3, CD56, and NKG2C. THP-1 cells for real-time imaging were stained with CellTrace Far Red before inoculation. Caspase 3 / 7 apoptosis detection reagent was added to each well. iNK cells were added to the wells at an E:T ratio of 5:1. The culture plates were placed in an IncuCyte S3 (Satorus Inc., France) for 24 hours. Images were collected every 30 minutes. Graphs are plotted based on the number of surviving THP-1 cells (normalized to the no effector cell control group and the initial inoculation number). All conditions were repeated three times. Primary AML cells were pre-stained with CellTrace Violet and co-cultured with iNK cells and NKG2C-KE for 2 days. Subsequently, they were stained with antibodies against CD45 and CD34 and analyzed by flow cytometry. + 、CD45 中等表达 、CD34 + The remaining AML cells were counted.

[0129] In vivo studies in mice

[0130] The HL-60-Luc mouse model is described elsewhere (Dezell et al., Biol. Blood Marrow Transplant., 18:536–545 (2012); Miller et al., Blood, 83:2594–2601 (1994); Hermanson et al., Stem Cells, 34:93–101 (2016)). Briefly, female NOD-SCID-γ (NSG) mice were injected intravenously with 750,000 HL60-luc cells. Three days later, the mice were imaged with bioluminescence (BLI) and divided into groups based on tumor burden. Five million expanded NK cells were then injected intravenously, and drug treatment was initiated five times a week for three weeks. Treatment was administered by intraperitoneal injection. BLI imaging was performed on days 6, 13, 27, and 35, and facial vein blood samples were collected on days 14 and 28. After erythrocyte lysis, cells were stained with antibodies including hCD45, mCD45, CD3, CD56, NKG2C, and CD16.

[0131] Statistics

[0132] GraphPad Prism (GraphPad Prism Software, Inc., La Jolla, CA) was used to plot graphs, with error bars representing mean ± SEM or ± SD (where appropriate) and associated curves representing 95% CI. GraphPad was also used to calculate linear regression t-tests, one-way ANOVA, and two-way ANOVA with or without repeated measures (RM) analysis where appropriate, and to determine statistical significance, with the following criteria: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The specific statistical analysis results used for each graph are indicated in the figure legends.

[0133] Example 2: Exemplary NKG2C polypeptides

[0134] This example provides the amino acid sequence of the human NKG2C polypeptide (SEQ ID NO: 1). The underlined and bold amino acid sequences of the human NKG2C polypeptide represent the NKG2C extracellular domain (SEQ ID NO: 2).

[0135]

[0136] Example 3: Exemplary scFvs capable of binding to NKG2C polypeptides

[0137] This example provides the amino acid sequences of the heavy chain variable region and the light chain variable region of exemplary scFv. The respective CDRs, framework sequences and constant regions are also provided and described.

[0138] Anti-NKG2C VH (CDRs underlined):

[0139] EVQLQQSGAELVKPGASVTLSCTASGFNIKDTYMHWVQQRPEQGLEWI GRI DPENGY TKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYHCAR SR TLFWYFDV WGAGTTVTVSS (SEQ ID NO: 19)

[0140] Framework Region 1 of the Heavy Chain Variable Domain:

[0141] EVQLQQSGAELVKPGASVTLSCTAS (SEQ ID NO: 11)

[0142] CDR1 of the Heavy Chain Variable Domain:

[0143] GFNIKDT (SEQ ID NO: 5)

[0144] Framework Region 2 of the Heavy Chain Variable Domain:

[0145] YMHWVQQRPEQGLEWIGRI (SEQ ID NO: 12)

[0146] CDR2 of the Heavy Chain Variable Domain:

[0147] DPENGY (SEQ ID NO: 6)

[0148] Framework Region 3 of the Heavy Chain Variable Domain:

[0149] TKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYHCAR (SEQ ID NO: 13)

[0150] CDR3 of the Heavy Chain Variable Domain:

[0151] SRTLFWYFDV (SEQ ID NO: 7)

[0152] Framework Region 4 of the Heavy Chain Variable Domain:

[0153] WGAGTTVTVSS (SEQ ID NO: 14)

[0154] Anti-NKG2C VL (CDRs underlined):

[0155] NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLA WYQQKPGQ SPKLLIY WASTRES GVPDRFTGSGSGTDFTLTITNIQAEDLAVYYC HQYLSSY T FGGGTKLEIKRA (SEQ ID NO: 20)

[0156] Framework Region 1 of the light chain variable domain:

[0157] NIMMTQSPSSLAVSAGEKVTMSC (SEQ ID NO: 15)

[0158] CDR1 of the light chain variable domain:

[0159] KSSQSVLYSSNQKNYLA (SEQ ID NO: 8)

[0160] Framework Region 2 of the light chain variable domain:

[0161] WYQQKPGQSPKLLIY (SEQ ID NO: 16)

[0162] CDR2 of the light chain variable domain:

[0163] WASTRES (SEQ ID NO: 9)

[0164] Framework Region 3 of the light chain variable domain:

[0165] GVPDRFTGSGSGTDFTLTITNIQAEDLAVYYC (SEQ ID NO: 17)

[0166] CDR3 of the light chain variable domain:

[0167] HQYLSSYT (SEQ ID NO: 10)

[0168] Framework Region 4 of the light chain variable domain:

[0169] FGGGTKLEIKRA (SEQ ID NO: 18)

[0170] Example 4: Nucleic acids encoding exemplary NKG2C scFv

[0171] The nucleic acid sequence of the exemplary NKG2C scFv shown in Example 3.

[0172] Nucleic acid encoding SEQ ID NO: 19 (NKG2C scFv heavy chain):

[0173] GAGGTACAGCTGCAGCAGTCTGGAGCCGAGCTGGTCAAACCCGGCGCTTCTGTCACTCTGAGCTGCACCGCATCTGGGTTCAACATTAAGGACACATACATGCACTGGGTTCAGCAGCGCCCAGAGCAGGGACTGGAATGGATTGGCAGAATCGACCCCGAAAACGGATACACGAAGTATGACCCCAATTTCCAGGGAAAGGCAACCATCACGGCTGATACTTCCTCAAACACCGCATATTTGCAACTGTCATCACTGACCAGTGAGGATACTGCTGTATATCACTGTGCTCGCAGCCGTACCCTGTTCTGGTATTTCGACGTCTGGGGTGCCGGCACAACTGTTACTGTTAGTTCG (SEQ ID NO:42)

[0174] Nucleic acid encoding SEQ ID NO: 20 (NKG2C scFv light chain):

[0175] AACATAATGATGACCCAGTCGCCTTCTTCCCTAGCCGTGAGCGCAGGCGAAAAGGTCACAATGAGTTGCAAATCAAGCCAGTCCGTCCTGTACTCGAGCAACCAGAAAAATTACCTCGCATGGTATCAACAGAAGCCTGGACAATCACCTAAGCTGCTGATATATTGGGCTTCCACACGCGAGTCTGGTGTTCCCGACCGATTTACTGGGTCCGGGAGCGGCACAGACTTTACCTTGACAATCACCAATATCCAGGCCGAGGACCTAGCTGTATATTATTGTCACCAGTATCTTTCTAGTTATACTTTCGGCGGAGGGACTAAACTCGAGATTAAGAGAGCC (SEQ ID NO:43)

[0176] Example 5: Exemplary CD33 Polypeptides

[0177] This example provides an amino acid sequence of a human CD33 polypeptide (SEQ ID NO: 3). The underlined and bolded amino acid sequence of the human CD33 polypeptide represents the extracellular domain of the CD33 polypeptide (SEQ ID NO: 4).

[0178]

[0179]

[0180] GAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSK

[0181] LHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0182] Example 6: Exemplary scFv capable of binding CD33 polypeptides

[0183] This example provides the amino acid sequences of the heavy chain variable region and the light chain variable region of an exemplary scFv. The respective CDRs, framework sequences, and constant regions are also provided and described.

[0184] Anti-CD33 VH (CDRs are underlined):

[0185] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFTDY NMHWVRQAPGQGLE WIGYI YPYNGG TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCAR GRPAMDY WGQGTLVTVSS (SEQ ID NO: 38)

[0186] Framework region 1 of the heavy chain variable domain:

[0187] QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 30)

[0188] CDR1 of the heavy chain variable domain:

[0189] GYTFTDY (SEQ ID NO: 24)

[0190] Framework region 2 of the heavy chain variable domain:

[0191] NMHWVRQAPGQGLEWIGYI (SEQ ID NO: 31)

[0192] CDR2 of the heavy chain variable domain:

[0193] YPYNGG (SEQ ID NO: 25)

[0194] Framework region 3 of the heavy chain variable domain:

[0195] TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCAR(SEQ ID NO:32)

[0196] CDR3 of the heavy chain variable domain:

[0197] GRPAMDY (SEQ ID NO: 26)

[0198] Framework region 4 of the heavy chain variable domain:

[0199] WGQGTLVTVSS (SEQ ID NO: 33)

[0200] Anti-CD33 VL (CDRs are underlined):

[0201] DIQMTQSPSSLSASVGDRVTITC RASESVDNYGISFMN WFQQKPGKAPK LLIY AASNQGS GVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSKEVPWT F GQGTKVEIK (SEQ ID NO: 39)

[0202] Framework region 1 of the light chain variable domain:

[0203] DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:34)

[0204] CDR1 of the light chain variable domain:

[0205] RASESVDNYGISFMN (SEQ ID NO: 27)

[0206] Framework region 2 of the light chain variable domain:

[0207] WFQQKPGKAPKLLIY (SEQ ID NO: 35)

[0208] CDR2 of the light chain variable domain:

[0209] AASNQGS (SEQ ID NO: 28)

[0210] Framework region 3 of the light chain variable domain:

[0211] GVPSRFSGSGSGTDFTLTISSLQPDDFATYYC(SEQ ID NO:36)

[0212] CDR3 of the light chain variable domain:

[0213] QQSKEVPWT (SEQ ID NO: 29)

[0214] Framework region 4 of the light chain variable domain:

[0215] FGQGTKVEIK (SEQ ID NO: 37)

[0216] Example 7: Nucleic Acids Encoding Exemplary CD33 scFvs

[0217] This example provides the nucleic acid sequence of the exemplary CD33 scFv shown in Example 6.

[0218] Nucleic acid encoding SEQ ID NO: 38 (CD33 scFv heavy chain):

[0219] CAGGTGCAACTGGTTCAATCTGGCGCCGAGGTAAAAAAACCAGGCTCATCTGTAAAAGTGAGCTGTAAGGCTTCCGGATACACTTTCACTGACTACAACATGCATTGGGTTAGGCAAGCACCCGGACAAGGACTCGAGTGGATCGGGTACATATACCCTTACAACGGGGGAACAGG GTACAATCAGAAGTTCAAAAGTAAGGCTACTATTACAGCCGATGAGAGCACTAACACCGCCTACATGGAGCTTAGCAGTCTGAGATCTGAAGATACCGCCGTGTACTATTGCGCACGGGGCAGACCCGCCATGGATTACTGGGGCCAGGGCACCCTGGTTACGGTGTCTAGT(SEQ ID NO:44)

[0220] Nucleic acid encoding SEQ ID NO: 39 (CD33 scFv light chain):

[0221] GACATTCAGATGACTCAGTCCCCCAGCTCACTGTCTGCTTCCGTGGGCGACCGCGTGACTATTACGTGTCGCGCCTCTGAATCAGTGGACAATTATGGCATATCCTTCATGAACTGGTTCCAGCAGAAACCAGGCAAGGCTCCCAAGCTGCTTATATACGCGGCGTCTAATCAAGGCAGTGGTGTGCCTTCCCGATTCAGTGGTTCAGGGAGTGGGACTGATTTCACTCTGACAATTTCAAGCCTCCAGCCAGATGATTTCGCTACATACTACTGTCAACAGTCTAAGGAAGTGCCATGGACATTCGGGCAGGGTACCAAGGTGGAGATCAAG (SEQ ID NO: 45)

[0222] Example 8: Exemplary Cell Engagers

[0223] This example provides amino acid sequences of anti-NKG2C cell engagers that also bind to CD33 polypeptides and comprise IL-15 polypeptides. Various components (e.g., domains and linkers) of the cell engagers are also provided and described.

[0224] EVQLQQSGAELVKPGASVTLSCTASGFNIKDTYMHWVQQRPEQGLEWIGRIDPENGYTKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYHCARSRTLFWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSNIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTITNIQAEDLAVYYCHQYLSSYTFGGGTKLEIKRAGSTSGSGKPGSGEGSTKGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKVDEHHHHHHHHHH

[0225] (SEQ ID NO:46)

[0226] BLK Alb信号多肽:

[0227] KWVTFISLLFLFSSAYS(SEQ ID NO:47)

[0228] 抗-NKG2C VH(CDR以下划线标示):

[0229] EVQLQQSGAELVKPGASVTLSCTASGFNIKDTYMHWVQQRPEQGLEWI GRI DPENGY TKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYHCAR SR TLFWYFDVWGAGTTVTVSS (SEQ ID NO: 19)

[0230] Connector:

[0231] GGGGSGGGGSGGGGS (SEQ ID NO: 21)

[0232] NKG2C VL (CDRs are underlined):

[0233] NIMMTQSPSSLAVSAGEKVTMSC KSSQSVLYSSNQKNYLA WYQQK

[0234] GQSPKLLIY WASTRES GVPDRFTGSGSGTDFTLTITNIQAEDLAVYYC HQYLSSYT FGGGTKLEIKRA (SEQ ID NO: 20)

[0235] Whitlow connector:

[0236] GSTSGSGKPGSGEGSTKG(SEQ ID NO:41)

[0237] IL-15 peptide:

[0238] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:40)

[0239] Anti-CD33 VH (CDRs are underlined):

[0240] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFTDY NMHWVRQAPGQGLEWIGYI YPYNGG TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCAR GRPAMDY WGQGTLVTVSS (SEQ ID NO: 38)

[0241] Connector:

[0242] GGGGSGGGGSGGGGS (SEQ ID NO: 21)

[0243] Anti-CD33 VL (CDRs are underlined):

[0244] DIQMTQSPSSLSASVGDRVTITC RASESVDNYGISFMN WFQQKPGKAPKLLIY AASNQGS GVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSKEVPWT DPENGY SR TLFWYFDV KSSQSVLYSSNQKNYLA WASTRES HQYLSSYT GYTFTDY YPYNGG GRPAMDY RASESVDNYGISFMN AASNQGS QQSKEVPWT FGQGTKVEIK (SEQ ID NO: 39)

[0245] Spacer:

[0246] VDE

[0247] 10x His peptide tag:

[0248] HHHHHHHHHH (SEQ ID NO: 48)

[0249] Example 9: Nucleic Acids Encoding Exemplary Anti-NKG2C Cell Engaging Agents

[0250] This example provides the nucleic acid sequences of the exemplary NKG2C cell engagers presented in Example 5. The nucleic acid sequences of the individual components (eg, domains and linkers) of the cell engagers are provided and described.

[0251]

[0252] GCCGCCACC

[0253] Start codon:

[0254] ATG

[0255] Nucleic acid sequence encoding BLK Alb signal polypeptide:

[0256] AAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCC(SEQ ID NO:50)

[0257] Nucleic acid sequence encoding anti-NKG2C VH:

[0258] GAGGTACAGCTGCAGCAGTCTGGAGCCGAGCTGGTCAAACCCGGCGCTTCTGTCACTCTGAGCTGCACCGCATCTGGGTTCAACATTAAGGACACATACATGCACTGGGTTCAGCAGCGCCCAGAGCAGGGACTGGAATGGATTGGCAGAATCGACCCCGAAAACGGATACACGAAGTAT GACCCCAATTTCCAGGGAAAGGCAACCATCACGGCTGATACTTCCTCAAACACCGCATATTTGCAACTGTCATCACTGACCAGTGAGGATACTGCTGTATATCACTGTGCTCGCAGCCGTACCCTGTTCTGGTATTTCGACGTCTGGGGTGCCGGCACAACTGTTACTGTTAGTTCG(SEQ ID NO:51)

[0259] Nucleic acid sequence encoding the linker:

[0260] GGCGGTGGCGGCTCTGGTGGTGGCGGTAGTGGCGGAGGTGGTAGC (SEQ ID NO:52)

[0261] Nucleic acid sequence encoding anti-NKG2C VL:

[0262] AACATAATGATGACCCAGTCGCCTTCTTCCCTAGCCGTGAGCGCAGGCGAAAAGGTCACAATGAGTTGCAAATCAAGCCAGTCCGTCCTGTACTCGAGCAACCAGAAAAATTACCTCGCATGGTATCAACAGAAGCCTGGACAATCACCTAAGCTGCTGATATATTGGGCTTCCACACGCGAGTCTGGTGTTCCCGACCGATTTACTGGGTCCGGGAGCGGCACAGACTTTACCTTGACAATCACCAATATCCAGGCCGAGGACCTAGCTGTATATTATTGTCACCAGTATCTTTCTAGTTATACTTTCGGCGGAGGGACTAAACTCGAGATTAAGAGAGCC(SEQID NO:53)

[0263] 编码瘭疽接头的核酸序列:

[0264] GGCAGTACCAGCGGGTCAGGGAAACCTGGCAGTGGGGAAGGTTCCA CAAAAGGT(SEQ ID NO:54)

[0265] 编码IL-15多肽的核酸序列:

[0266] AACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT(SEQ ID NO:55)

[0267] 编码抗-CD33 VH的核酸序列:

[0268] CAGGTGCAACTGGTTCAATCTGGCGCCGAGGTAAAAAAACCAGGCTCATCTGTAAAAGTGAGCTGTAAGGCTTCCGGATACACTTTCACTGACTACAACATGCATTGGGTTAGGCAAGCACCCGGACAAGGACTCGAGTGGATCGGGTACATATACCCTTACAACGGGGGAACAGGGTACAATCAGAAGTTCAAAAGTAAGGCTACTATTACAGCCGATGAGAGCACTAACACCGCCTACATGGAGCTTAGCAGTCTGAGATCTGAAGATACCGCCGTGTACTATTGCGCACGGGGCAGACCCGCCATGGATTACTGGGGCCAGGGCACCCTGGTTACGGTGTCTAGT(SEQ ID NO:56)

[0269] 编码接头的核酸序列:

[0270] GGCGGAGGTGGGAGCGGTGGGGGGGGTAGCGGGGGAGGCGGCTCT(SEQ ID NO:57)

[0271] 编码抗-CD33 VL的核酸序列:

[0272] GACATTCAGATGACTCAGTCCCCCAGCTCACTGTCTGCTTCCGTGGGCGACCGCGTGACTATTACGTGTCGCGCCTCTGAATCAGTGGACAATTATGGCATATCCTTCATGAACTGGTTCCAGCAGAAACCAGGCAAGGCTCCCAAGCTGCTTATATACGCGGCGTCTAATCAAGGCAGTGGTGTGCCTTCCCGATTCAGTGGTTCAGGGAGTGGGACTGATTTCACTCTGACAATTTCAAGCCTCCAGCCAGATGATTTCGCTACATACTACTGTCAACAGTCTAAGGAAGTGCCATGGACATTCGGGCAGGGTACCAAGGTGGAGATCAAG(SEQ ID NO:58)编码间隔物的核酸序列:

[0273] GTCGACGAG

[0274] 编码10x His多肽标签的核酸序列:

[0275] CATCATCATCATCACCACCACCACCACCAC(SEQ ID NO:59)

[0276] Stop codon:

[0277] TGA

[0278] Example 10: Nucleic Acids Encoding Exemplary NKG2C Polypeptides

[0279]

[0280] Example 11: Exemplary DAP12 sequences

[0281] Exemplary DAP12 polypeptide sequences

[0282] GGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0283] (SEQ ID NO: 61)

[0284] Nucleic acids encoding exemplary DAP12 polypeptide sequences

[0285] CCACGCGTCCGCGCTGCGCCACATCCCACCGGCCCTTACACTGTGGTGTCCAGCAGCATCCGGCTTCATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAATGAGCCCGAATCATGACAGTCAGCAACATGATACCTGGATCCAGCCATTCCTGAAGCCCACCCTGCACCTCATTCCAACTCCTACCGCGATACAGACCCACAGAGTGCCATCCCTGAGAGACCAGACCGCTCCCCAATACTCTCCTAAAATAAACATGAAGCACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 62)

[0286] Example 12: Treating cancer

[0287] One or more cell-engaging agents capable of binding to (a) an NKG2C polypeptide and (b) a CD33 polypeptide are administered to a patient diagnosed with leukemia (e.g., AML). The one or more cell-engaging agents capable of binding to the NKG2C polypeptide are administered intravenously. After administration of the one or more cell-engaging agents capable of binding to the NKG2C polypeptide, the number of cancer cells in the patient is reduced. After administration of the one or more cell-engaging agents capable of binding to the NKG2C polypeptide, the size of one or more tumors in the patient is reduced.

[0288] Example 13: Treatment of Cancer

[0289] One or more cell-engaging agents capable of binding to (a) NKG2C polypeptide and (b) CD33 polypeptide and NKG2C + A population of NK cells is administered (e.g., as adoptive cell therapy) to a patient diagnosed with leukemia (e.g., AML). (1) one or more cell-engaging agents capable of binding to (a) an NKG2C polypeptide and (b) a CD33 polypeptide and (2) an NKG2C polypeptide. + The NK cell populations are administered intravenously (e.g., as a single injection). Following administration of one or more cell-engaging agents that bind to NKG2C polypeptides and CD33 polypeptides, the number of cancer cells in the patient is reduced. Following administration of one or more cell-engaging agents that bind to NKG2C polypeptides and CD33 polypeptides, the size of one or more tumors in the patient is reduced.

[0290] Other Implementations

[0291] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the claims.

Claims

1. A cell-engaging agent comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds to an NKG2C polypeptide, and wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a cancer cell.

2. The cell engaging agent of claim 1 , wherein the first antigen-binding domain comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 5 (or SEQ ID NO: 5 having one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 6 (or SEQ ID NO: 6 having one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 7 (or SEQ ID NO: 7 having one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 (or SEQ ID NO: 8 having one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 9 (or SEQ ID NO: 9 having one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 10 (or SEQ ID NO: 10 having one, two or three amino acid additions, deletions or substitutions).

3. The cell engaging agent of any one of claims 1-2, wherein the first antigen binding domain comprises (a) a heavy chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 19, and (b) a light chain variable domain comprising the amino acid sequence as shown in SEQ ID NO:

20.

4. The cell engaging agent of any one of claims 1-3, wherein the first antigen binding domain comprises a scFv. The cell-engaging agent according to any one of claims 1 to 4, wherein the polypeptide expressed on the surface of the cancer cells is CD33 polypeptide.

6. The cell engaging agent of any one of claims 1 to 5, wherein the second antigen-binding domain comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 24 (or SEQ ID NO: 24 having one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 25 (or SEQ ID NO: 25 having one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 26 (or SEQ ID NO: 26 having one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 27 (or SEQ ID NO: 27 having one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 28 (or SEQ ID NO: 28 having one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 29 (or SEQ ID NO: 29 having one, two or three amino acid additions, deletions or substitutions).

7. The cell engaging agent of any one of claims 1 to 6, wherein the second antigen binding domain comprises (a) a heavy chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 38, and (b) a light chain variable domain comprising the amino acid sequence as shown in SEQ ID NO:

39.

8. The cell engaging agent of any one of claims 1-7, wherein the second antigen binding domain comprises a scFv.

9. The cell engaging agent of any one of claims 1 to 8, wherein the cell engaging agent comprises a linker located between the first antigen binding domain and the second antigen binding domain.

10. The cell engager of claim 9, wherein the linker comprises a linker sequence selected from the group consisting of GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGS (SEQ ID NO: 63), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41), PSGQAGAAASESLFVSNHAY (SEQ ID NO: 64), EASGGPE (SEQ ID NO: 65), EPKSSDKTHTSPPSPEL (SEQ ID NO: 66), RATPSHNSHQVPSAGGPTANSGTSG (SEQ ID NO: 67), and SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 68).

11. The cell engaging agent of any one of claims 1 to 10, wherein the cell engaging agent further comprises an IL-15 polypeptide or a biologically active fragment of the IL-15 polypeptide.

12. The cell engaging agent of claim 11, wherein the IL-15 polypeptide or the biologically active fragment of the IL-15 polypeptide is located between the first antigen binding domain and the second antigen binding domain.

13. The cell engager of claim 12, wherein the IL-15 polypeptide is separated from the first antigen binding domain by a first linker, and wherein the IL-15 polypeptide is separated from the second antigen binding domain by a second linker. The cell engager of claim 13 , wherein the first linker and the second linker each comprise GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41).

15. A nucleic acid construct comprising a nucleic acid sequence encoding the cell engaging agent according to any one of claims 1 to 14.

16. The nucleic acid construct of claim 15, wherein the nucleic acid is a viral vector.

17. The nucleic acid construct of claim 15, wherein the nucleic acid is a phagemid.

18. A host cell comprising the nucleic acid of any one of claims 15-17.

19. A composition comprising the cell engaging agent of any one of claims 1-14.

20. A method of treating a mammal suffering from cancer, wherein the method comprises administering to the mammal the cell engaging agent of any one of claims 1-14 or the composition of claim 19.

21. The method of claim 20, wherein the mammal is a human.

22. The method of any one of claims 20-21, wherein the cancer is CD33 + cancer.

23. The method of any one of claims 20-22, wherein the cancer is selected from the group consisting of leukemia, lymphoma, myelodysplastic syndrome, and systemic mastocytosis.

24. The method of any one of claims 20-23, wherein the number of cancer cells in the mammal is reduced after the administering step.

25. A method of treating a mammal suffering from cancer, wherein the method comprises: (a) administering to the mammal a cell-engaging agent according to any one of claims 1 to 14 or a composition according to claim 19, and (b) administering a population of natural killer (NK) cells to the mammal.

26. The method of claim 25, wherein the mammal is a human.

27. The method of any one of claims 25-26, wherein the cancer is CD33 + cancer.

28. The method of any one of claims 25-27, wherein the cancer is selected from the group consisting of leukemia, lymphoma, myelodysplastic syndrome, and systemic mastocytosis.

29. The method according to any one of claims 25-28, wherein at least a portion of the NK cells are NKG2C + NK cells.

30. The method of claim 29, wherein the NKG2C + The NK cell comprises a nucleic acid encoding the NKG2C polypeptide under conditions that express the NKG2C polypeptide.

31. The method of any one of claims 25-30, wherein at least a portion of the NK cells comprises a nucleic acid encoding a DAP12 polypeptide under conditions that express the DAP12 polypeptide.

32. The method of any one of claims 25-31, wherein the number of cancer cells in the mammal is reduced following the administering of steps (a) and (b).

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