Type I membrane protein heterodimers and methods of use thereof

Abstract

Type I membrane protein heterodimers are provided. Thus, heterodimers are provided that include two polypeptides selected from the group consisting of SIRP alpha, PD1, TIGIT, LILRB2 and SIGLEC10, where each of the two polypeptides can bind to its natural binding partner, and the heterodimer does not include an amino acid sequence of a type II membrane protein that can bind to its natural binding partner. Nucleic acid constructs and systems encoding the heterodimers, host cells expressing the same, and methods of use thereof are also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 63 / 136,687, filed January 13, 2021, and U.S. Patent Application No. 63 / 139,331, filed January 20, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] SEQUENCE LISTING STATEMENT An ASCII file entitled 89962SequenceListing.txt, created on Jan. 13, 2022, containing 303,104 bytes, filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] The present invention, in some embodiments thereof, relates to type I membrane protein heterodimers and methods of use thereof. [Background technology]

[0004] The interaction between cancer and the immune system is complex and multifaceted. Although many cancer patients appear to exhibit anti-tumor immune responses, cancer also exhibits strategies to evade immune detection and destruction. Cancer cells can reduce the expression of tumor antigens on their surface, making them difficult for the immune system to detect, express proteins on their surface that induce the inactivation of immune cells, and / or induce cells in the microenvironment to release substances that suppress the immune response, promoting the growth and survival of tumor cells.

[0005] In recent years, immunotherapies have been developed to enhance immune responses against tumors by stimulating certain components of the immune system or counteracting signals that suppress the immune response produced by cancer cells. Advances in the definition of the mechanisms and molecules that regulate the immune response have provided novel therapeutic targets for treating cancer. Among these targets are co-stimulatory and co-inhibitory molecules that play a central role in controlling T cell immune responses (e.g., CTLA4, PD1), proteins that help control or regulate immune system activity, such as interleukins and interferons, tumor antigens, and components involved in the activity of the innate immune system (e.g., CD47-SIRPα "don't eat-me" signal).

[0006] Additional background art includes: International Publication No. WO 2020 / 146423, International Publication No. WO 201712770, International Publication No. WO 2017152132, International Publication No. WO 2016023001 and International Publication No. WO 2013112986, and U.S. Patent No. 7,569,663 and U.S. Patent No. 8,039,437. Summary of the Invention

[0007] According to an aspect of some embodiments of the present invention there is provided a heterodimer comprising two polypeptides selected from the group consisting of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10, wherein each of the two polypeptides is capable of binding to its natural binding partner, and wherein the heterodimer does not comprise an amino acid sequence of a type II membrane protein capable of binding to its natural binding partner.

[0008] According to some embodiments of the invention, the heterodimer comprises a dimerization moiety attached to two polypeptides.

[0009] According to some embodiments of the invention, the dimerization moiety is an Fc domain of an antibody or fragment thereof.

[0010] According to some embodiments of the invention, the Fc domain is modified to alter its binding to Fc receptors, to reduce its immunostimulatory function, and / or to improve the half-life of the fusion.

[0011] According to some embodiments of the invention, the heterodimer comprises a SIRPα polypeptide and a PD1 polypeptide.

[0012] According to some embodiments of the invention, the heterodimer comprises a SIRPα polypeptide and a LILRB2 polypeptide.

[0013] According to some embodiments of the invention, the heterodimer comprises a SIRPα polypeptide and a SIGLEC10 polypeptide.

[0014] According to some embodiments of the invention, the heterodimer comprises a SIRPα polypeptide and a TIGIT polypeptide.

[0015] According to some embodiments of the present invention, the heterodimer comprises a TIGIT polypeptide and a PD1 polypeptide.

[0016] According to some embodiments of the invention, the heterodimer comprises a TIGIT polypeptide and a LILRB2 polypeptide.

[0017] According to some embodiments of the invention, the heterodimer comprises a TIGIT polypeptide and a SIGLEC10 polypeptide.

[0018] According to some embodiments of the invention, the heterodimer comprises a PD1 polypeptide and a SIGLEC10 polypeptide.

[0019] According to some embodiments of the invention, the heterodimer comprises a LILRB2 polypeptide and a SIGLEC10 polypeptide.

[0020] According to some embodiments of the invention, the heterodimer comprises a PD1 polypeptide and a LILRB2 polypeptide.

[0021] According to some embodiments of the invention, in the heterodimer, each of the polypeptides is a monomer.

[0022] According to some embodiments of the invention, the two polypeptides are comprised in monomers of a heterodimer.

[0023] According to an aspect of some embodiments of the present invention there is provided a composition comprising a heterodimer, wherein the predominant form of the two polypeptides in the composition is the heterodimer.

[0024] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct or system comprising at least one polynucleotide encoding a heterodimer and regulatory elements directing expression of the polynucleotide in a host cell.

[0025] According to an aspect of some embodiments of the present invention there is provided a host cell comprising the heterodimer or the nucleic acid construct or system.

[0026] According to an aspect of some embodiments of the present invention there is provided a method for producing a heterodimer comprising introducing a nucleic acid construct or system into a host cell or culturing the cell.

[0027] According to some embodiments of the invention, the method includes isolating the heterodimer.

[0028] According to an aspect of some embodiments of the present invention, there is provided a method of treating a disease in a subject in need thereof for which heterodimer treatment may be effective, the method comprising administering to the subject a therapeutically effective amount of a heterodimer, composition, nucleic acid construct or system, or cell, thereby treating the disease in the subject.

[0029] According to an aspect of some embodiments of the present invention, there is provided a heterodimer, a composition, a nucleic acid construct or system, or a cell for use in treating a disease in a subject in need thereof that may benefit from heterodimer treatment.

[0030] According to some embodiments of the invention, the disease is one that may benefit from activation of immune cells.

[0031] According to some embodiments of the invention, cells associated with the disease express a natural binding partner.

[0032] According to some embodiments of the invention, the disease is cancer.

[0033] According to some embodiments of the invention, the cancer is selected from the group consisting of lymphoma, leukemia, colon cancer, ovarian cancer, lung cancer, head and neck cancer, and hepatocellular carcinoma.

[0034] According to some embodiments of the invention, the cancer is non-small cell lung cancer (NSCLC) or mesothelioma.

[0035] According to an aspect of some embodiments of the present invention there is provided a method of activating an immune cell comprising activating the immune cell in vitro in the presence of the heterodimer, composition, nucleic acid construct or system, or cell.

[0036] According to some embodiments of the invention, activation is in the presence of cells expressing the natural binding partner.

[0037] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0038] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. In the following, it is emphasized that the details shown, with particular reference to the drawings, are for the purposes of illustration and for the purpose of detailed description of embodiments of the present invention. Similarly, from a reading of the description together with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0039] [Figure 1A] FIG. 1A is a schematic diagram of non-limiting examples of possible arrangements / conformations of a heterodimer. [Figure 1B] FIG. 1B shows a schematic diagram of the composition and configuration of heterodimers contemplated in some embodiments of the present invention. [Diagram 2]FIG. 2A is a schematic diagram of the SIRPα-PD1 heterodimer, referred to herein as "DSP120V1" (SEQ ID NOs: 5 and 7). FIG. 2B-C shows the predicted 3D structure of the SIRPα-PD1 heterodimer DSP120V1 (SEQ ID NOs: 5 and 7). FIG. 2B is a schematic 3D model and FIG. 2C is an all-atom 3D model. SIRPα (in the "knob" chain) is shown in a dark grey ribbon representation (bottom right). PD1 (in the "hole" chain) is shown in a grey ribbon representation (top right). hIgG4 in the "knob" sequence is shown in a white ribbon at the bottom right of the figure. hIgG4 in the "hole" sequence is shown in a grey ribbon at the top right of the figure. "Spacer" / "linker" segments are shown as grey and white ribbons between the structural elements of SIRPα, hIgG4 and PD1. The hinge cysteine ​​residues of the Fc domain of hIgG4, which stabilize the complex, are shown in CPK representation. [Diagram 3] FIG. 3A is a schematic diagram of the SIRPα-LILRB2 heterodimer referred to herein as "DSP216V1" (SEQ ID NOs: 5 and 15). FIG. 3B-C shows the predicted 3D structure of the SIRPα-LILRB2 heterodimer DSP216V1 (SEQ ID NOs: 5 and 15). FIG. 3B is a schematic 3D model and FIG. 3C is an all-atom 3D model. SIRPα (in the "knob" chain) is shown in a dark grey ribbon representation (bottom right). LILRB2 (in the "hole" chain) is shown in a dark grey ribbon representation (top right). hIgG4 in the "knob" sequence is shown in a white ribbon at the bottom right of the figure. hIgG4 in the "hole" sequence is shown in a grey ribbon at the top right of the figure. "Spacer" / "linker" segments are shown as grey and white ribbons between the structural elements of SIRPα, hIgG4 and LILRB2. The hinge cysteine ​​residues of the Fc domain of hIgG4, which stabilize the complex, are shown in CPK representation. [Figure 4]FIG. 4A is a schematic diagram of the TIGIT-SIGLEC10 heterodimer, referred to herein as "DSP404V1" (SEQ ID NOs: 13 and 30). FIG. 4B-C shows the predicted 3D structure of the TIGIT-SIGLEC10 heterodimer DSP404V1 (SEQ ID NOs: 13 and 30). FIG. 4B is a schematic 3D model and FIG. 4C is an all-atom 3D model. TIGIT (in the "knob" chain) is shown in grey surface representation (bottom right). SIGLEC10 (in the "hole" chain) is shown in grey surface representation (top right). hIgG4 in the "knob" sequence is shown in white surface at the bottom right of the figure. hIgG4 in the "hole" sequence is shown in grey surface at the top right of the figure. "Spacer" / "linker" segments are shown as grey and white ribbons between the structural elements of TIGIT, hIgG4 and SIGLEC10. The hinge cysteine ​​residues of the Fc domain of hIgG4, which stabilize the complex, are shown in CPK representation. [Diagram 5] FIG. 5A is a schematic diagram of the TIGIT-PD1 heterodimer, referred to herein as "DSP502V1" (SEQ ID NOs: 13 and 7). FIG. 5B-C shows the predicted 3D structure of the TIGIT-PD1 heterodimer DSP502V1 (SEQ ID NOs: 13 and 7). FIG. 5B is a schematic 3D model and FIG. 5C is an all-atom 3D model. TIGIT (in the "knob" chain) is shown in grey ribbon representation (bottom right). PD1 (in the "hole" chain) is shown in grey ribbon representation (top right). hIgG4 in the "knob" sequence is shown in white ribbon at the bottom right of the figure. hIgG4 in the "hole" sequence is shown in grey ribbon at the top right of the figure. "Spacer" / "linker" segments are shown as grey and white ribbons between the structural elements of TIGIT, hIgG4 and PD1. The hinge cysteine ​​residues of the Fc domain of hIgG4, which stabilize the complex, are shown in CPK representation. [Figure 6-1]Figures 6A-B show SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of several heterodimers produced (see descriptions and sequences in Table 1 below). Figure 6A presents SDS-PAGE images of crude (unpurified) day 5 supernatant samples of Expi293F cells transfected with plasmids encoding the indicated heterodimers, separated under reducing (R) and / or non-reducing (NR) conditions. Control samples are supernatants of day 5 cultures of non-transfected Expi293F cells. Figure 6B presents SDS-PAGE images of samples purified from day 5 supernatants of cells transfected with constructs encoding the indicated heterodimers using the indicated protein A or anion exchange chromatography. [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 7] Figures 7A-C present Western blot analysis of several heterodimers produced (see description and sequences in Table 1 below). Samples shown in the figures are crude (unpurified) day 5 supernatants of Expi293F cells transfected with plasmids encoding the indicated heterodimers. Supernatants were separated on SDS-PAGE under non-reducing (NR) or reducing (R) conditions, followed by immunoblotting with anti-PD1 (Figure 7A), anti-SIRPα (Figure 7B) or anti-LILRB2 (Figure 7C) antibodies. [Figure 8]Figures 8A-B show binding of SIRPα-PD1 heterodimer, herein referred to as "DSP120" (SEQ ID NOs: 1 and 3), to CD47 and PDL1. Supernatants containing the heterodimer or control supernatants (from non-transfected Expi293F cells) were incubated in 96-well plates precoated with CD47 or PDL1. After incubation, detection was performed with anti-PD-1 (for CD47-coated plates) or rabbit anti-human SIRPα antibody (for PDL1-coated plates) followed by incubation with the corresponding HRP-conjugated secondary antibody. Detection was performed using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 620, following standard ELISA protocols, with TMB substrate. Figure 8A shows the concentration-dependent binding of DSP120 to CD47-coated plates, and Figure 8B shows the concentration-dependent binding of DSP120 to PDL1-coated plates. [Figure 9-1] Figures 9A-C show the binding of SIRPα-LILRB2 heterodimers, herein referred to as "DSP216" (SEQ ID NOs: 1 and 11, Figure 9A) and "DSP216V1" (SEQ ID NOs: 5 and 15, Figures 9B-C) to HLA-G. Supernatants containing the heterodimers or control supernatants (from non-transfected Expi293F cells) were incubated in 96-well plates precoated with HLA-G. Binding was detected by incubation with rabbit anti-human SIRPα antibodies followed by incubation with goat anti-rabbit IgG-HRP and TMB substrate according to standard ELISA protocols using a plate reader at 450 nm with a reference wavelength of 620 nm. Figure 9A shows the concentration-dependent binding of DSP216 to plates coated with HLA-G protein. No binding was observed with control supernatants (control). 9B-C show the concentration-dependent binding of crude supernatant containing DSP216V1 (FIG. 9B) or purified DSP216V1 (FIG. 9C) to HLA-G-coated plates. [Figure 9-2] Same as above [Figure 10]Figures 10A-B show the binding of the PD1-TIGIT heterodimer, herein referred to as "DSP502" (SEQ ID NOs: 9 and 3), to its PVR counterpart. Supernatants containing the heterodimer or control supernatants (from non-transfected Expi293F cells) (Figure 10A) or purified protein (Figure 10B) were incubated in 96-well plates precoated with PVR. After incubation, detection was performed with an anti-PD1 antibody followed by incubation with the corresponding HRP-conjugated secondary antibody. Detection was performed with TMB substrate following standard ELISA protocol using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 620. Figures 10A-B show the concentration-dependent binding of DSP502 to PVR-coated plates. [Figure 11-1]Figures 11A-E show the binding of DSP120 and DSP120V1 to cells expressing PDL1 or CD47 as determined by flow cytometry analysis. The indicated MFI values ​​were used to generate binding curve graphs using FlowJo software. Figure 11A is a histogram showing the expression of PDL1 in DLD1-PDL1 overexpressing cell lines. The surface expression level of PDL1 was determined by immunostaining of DLD1 WT and PDL1 overexpressing cell lines (DLD1-PDL1) with a fluorescently labeled anti-PDL1 antibody followed by flow cytometry analysis. Figure 11B is a histogram showing the expression of CD47 receptor in CHO-K1-CD47 HB9 clone cells. The surface expression level of CD47 was determined by immunostaining of CHO-K1 WT and CD47 overexpressing cell lines (clone HB9) with an anti-CD47 antibody followed by flow cytometry analysis. Figure 11C-D shows the binding of DSP120 (Figure 11C) and DSP120V1 (Figure 11D) to DLD1-PDL1 overexpressing cell lines compared to DLD1-WT. The binding of the heterodimer to the cell lines was determined by immunostaining of its SIRPα domain with anti-SIRPα antibody after incubation followed by flow cytometry analysis. Figure 11E shows the binding of DSP120V1 to CHO-K1-CD47 HB9 clone cells. The binding of the heterodimer to hCD47 overexpressing cell lines was determined by immunostaining of its IgG-Fc domain with anti-IgG4 antibody after incubation followed by flow cytometry analysis. CHO-K1 WT cells were used as a negative cell control for the binding assay. [Figure 11-2] Same as above [Figure 11-3] Same as above [Figure 12-1]Figures 12A-F show the binding of DSP216 and DSP216V1 to cells expressing CD47 and HLA-G as determined by flow cytometry analysis. Figures 12A and 12C show the expression of CD47 in HT1080 (Figure 12A) and HT1080-HLA-G (Figure 12C) cell lines. Cell surface expression of CD47 was determined by immunostaining of cell lines with anti-human-CD47 antibody and IgG1 isotype control followed by flow cytometry analysis. Figures 12B and 12D show the expression of HLA-G in HT1080 (Figure 12B) and HT1080-HLA-G (Figure 12D) cell lines. Surface expression levels of HLA-G were determined by immunostaining of cell lines with anti-human-HLA-G antibody and IgG2a isotype control. Figure 12E shows the binding of DSP216 to CD47 expressing cells HT1080. Binding of the heterodimer to the cell line was determined after incubation by immunostaining its LILRB2 domain with LILRB2 antibody followed by flow cytometry analysis. The percentage of cells positive for LILRB2 is shown and used to generate a binding curve graph with GraphPad Prism software. Figure 12F shows the binding of the supernatant containing heterodimer DSP216V1 to HT1080-HLA-G cell line. Binding of the heterodimer to the cell line was determined by immunostaining of the IgG4 backbone with anti-IgG4 antibody followed by flow cytometry analysis. The MFI value is shown and used to generate a binding curve graph with GraphPad Prism software. [Figure 12-2] Same as above [Figure 12-3] Same as above [Figure 13]Figure 13 shows the binding of SIGLEC10-PD1 heterodimer, herein referred to as "DSP402" (SEQ ID NOs: 24 and 3), to DLD1 WT and PDL1 overexpressing cell lines as determined by flow cytometry analysis. After incubating cells with DSP402, the binding of the heterodimer to DLD1 PDL1 overexpressing cell lines was determined by immunostaining of its PD1 domain using an anti-PD1 antibody followed by flow cytometry analysis. DLD-1 WT cells were used as a negative cell control for the binding assay. MFI values ​​are shown and were used to generate binding curve graphs using FlowJo software. [Figure 14-1]Figures 14A-G show the binding of TIGIT-PD1 heterodimer, herein referred to as "DSP502" (SEQ ID NOs: 9 and 3), to cells expressing PVR and PDL1. Figures 14A-C show the expression of PVR in DLD-1 WT (Figure 14A), DLD-1 PDL1 (Figure 14B) and HT1080 (Figure 14C) cell lines. Cell surface expression of PVR was determined by immunostaining of cell lines with APC-labeled anti-PVR antibody followed by flow cytometry analysis. MFI values ​​are shown. Figure 14D shows the expression of PDL1 in HT1080 cells. Surface expression levels of PDL1 were determined by immunostaining of cell lines with APC-labeled anti-PDL1 antibody or isotype control followed by flow cytometry analysis. Figures 14E-F show the binding of DSP502 to DLD1 PDL1 (Figure 14E) or HT1080 (Figure 14F) cells. Binding of the heterodimers to the cell lines was determined by immunostaining of their IgG1-Fc domains with anti-human IgG1 antibodies after incubation, followed by flow cytometry analysis. The specificity of binding to each domain of DSP502 was tested by incubating the cells with blocking antibodies against PVR or PD-L1. Figure 14G shows the specific binding of TIGIT-PD1 heterodimers, herein designated "DSP502V1" (SEQ ID NOs: 13 and 7), "DSP502V2" (SEQ ID NOs: 31 and 7), and "DSP502V3" (SEQ ID NOs: 33 and 7), to PVR, as shown by binding to DLD-1 WT cells expressing PVR and not expressing PD1. Binding of the heterodimers was determined by immunostaining of their IgG4-Fc domains with anti-human IgG4 antibodies, followed by flow cytometry analysis. MFI values ​​are shown and were used to generate binding curve graphs with FlowJo software. [Figure 14-2] Same as above [Figure 14-3] Same as above [Figure 14-4] Same as above [Figure 15]Figure 15 shows SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of several heterodimers produced (see descriptions and sequences in Table 1 below). The figure presents SDS-PAGE images of crude (unpurified) day 5 supernatant samples of Expi293F cells transfected with plasmids encoding the indicated heterodimers, separated under reducing (R) and / or non-reducing (NR) conditions. [Figure 16-1] Figures 16A-F show binding of SIRPα-LILRB2 heterodimers, herein designated "DSP216" (SEQ ID NOs: 1 and 11, Figure 16A), "DSP216V1" (SEQ ID NOs: 5 and 15, Figure 16B), "DSP216V3" (SEQ ID NOs: 138 and 11, Figure 16C), "DSP216V4" (SEQ ID NOs: 140 and 15, Figure 16D), "DSP216V5" (SEQ ID NOs: 142 and 150, Figure 16E) or "DSP216V6" (SEQ ID NOs: 144 and 150, Figure 16F) to cells overexpressing HLA-G (HT1080-HLA-G) compared to HT1080-WT cells. Binding of the heterodimers to cell lines was determined by immunostaining of the IgG backbone with APC-conjugated anti-human IgG1 antibody or the SIRPα domain with anti-SIRPα for DSP216V1 and DSP216V4 after incubation with or without blocking antibodies as indicated, followed by flow cytometry analysis. GMFI values ​​are shown and were used to generate binding curve graphs using GraphPad Prism software. [Figure 16-2] Same as above [Figure 16-3] Same as above [Figure 16-4] Same as above [Figure 17-1]Figures 17A-F show the binding of SIRPα-LILRB2 heterodimers DSP216 (SEQ ID NOs: 1 and 11, Figure 17C), DSP216V3 (SEQ ID NOs: 138 and 11, Figure 17D), DSP216V5 (SEQ ID NOs: 142 and 150, Figure 17E) or DSP216V6 (SEQ ID NOs: 144 and 150, Figure 17F) to JEG-3 cells expressing both CD47 and HLA-G (Figures 17A-B). Binding of the heterodimers to the cell lines was determined by immunostaining of the IgG backbone with anti-human-IgG1 antibody followed by flow cytometry analysis after incubation with or without blocking antibodies as indicated. GMFI values ​​are shown and were used to generate binding curve graphs with GraphPad Prism software. [Figure 17-2] Same as above [Figure 17-3] Same as above [Figure 18-1] Figures 18A-D show the binding of SIRPα-LILRB2 heterodimers DSP216 (SEQ ID NOs: 1 and 11, Figure 18A), DSP216V1 (SEQ ID NOs: 5 and 15, Figure 18B), DSP216V3 (SEQ ID NOs: 138 and 11, Figure 18C) and DSP216V4 (SEQ ID NOs: 140 and 15, Figure 18D) to plate-bound (PB) recombinant human CD47. Supernatants containing heterodimers were incubated in 96-well plates precoated with CD47. Binding was detected by incubation with anti-human IgG1- or IgG4-HRP antibodies and detection with TMB substrate using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 620 nm, following standard ELISA protocols. OD values ​​were used to generate binding curve graphs with GraphPad Prism software. [Figure 18-2]Figures 18E-H show binding of SIRPα-LILRB2 heterodimers DSP216 (SEQ ID NOs: 1 and 11, Figure 18E), DSP216V1 (SEQ ID NOs: 5 and 15, Figure 18F), DSP216V3 (SEQ ID NOs: 138 and 11, Figure 18G) and DSP216V4 (SEQ ID NOs: 140 and 15, Figure 18H) to plate-bound (PB) recombinant human HLA-G. Supernatants containing heterodimers were incubated in 96-well plates precoated with HLA-G. Binding was detected by incubation with anti-human IgG1- or IgG4-HRP antibodies and detection with TMB substrate using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 620 nm, following standard ELISA protocols. OD values ​​were used to generate binding curve graphs with GraphPad Prism software. [Figure 19-1] 19A-I show the binding of TIGIT-PD1 heterodimer DSP502 (SEQ ID NOs: 9 and 3) and the heterodimer referred to herein as "DSP502V4" (SEQ ID NOs: 146 and 148) to cells expressing PDL1 and / or PVR as determined by flow cytometry analysis. Binding curve graphs were generated using FlowJo software using the indicated MFI values. Figure 19A shows the binding of DSP502 to K562PD-L1 cells, Figure 19B shows the binding of DSP502 to K562PD-L1 / PVR cells, Figure 19C shows the binding of DSP502V4 to K562PD-L1 cells, Figure 19D shows the binding of DSP502V4 to K562PD-L1 / PVR cells, Figure 19G shows the binding of DSP502 to K562PVR cells, and Figure 19H shows the binding of DSP502V4 to K562PVR cells. Figures 19E, 19F, and 19I are histograms showing the expression of PDL1 or PVR as indicated in K562PD-L1, K562PVR, and K562PD-L1 / PVR cells. Surface expression levels of PDL1 and PVR were determined by immunostaining of cells with fluorescently labeled anti-PDL1 or anti-PVR antibodies, as indicated, followed by flow cytometric analysis. [Figure 19-2] Same as above [Figure 19-3] Same as above [Figure 20] Figure 20A shows the binding of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3) to SKOV3 cells expressing PVR, as determined by flow cytometry analysis after incubation with or without blocking antibodies as indicated. Binding curve graphs were generated by FlowJo software using the indicated MFI values. Figure 20B shows a histogram showing the expression of PVR in SKOV3 cells. Surface expression levels of PVR were determined by immunostaining of cells with fluorescently labeled anti-PVR antibodies followed by flow cytometry analysis. [Figure 21] Figure 21A shows the binding of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3) to Renca cells expressing mouse PDL1 and PVR as determined by flow cytometry analysis after incubation with or without blocking antibodies as indicated. Binding curve graphs were generated by FlowJo software using the indicated MFI values. Figure 21B shows histograms showing the expression of PDL-1 and PVR in Renca cells. Surface expression levels of PDL1 and PVR were determined by immunostaining of cells with fluorescently labeled anti-PDL1 or anti-PVR antibodies as indicated, followed by flow cytometry analysis. [Figure 22] Figure 22A shows the binding of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3) to mouse cell line AB12 as determined by flow cytometry analysis. Binding curve graphs were generated by FlowJo software using the indicated MFI values. Figure 22B shows histograms showing the surface expression levels of PDL1 and PVR in AB12 cells. Surface expression levels of PDL1 and PVR were determined by immunostaining of cells with fluorescently labeled anti-PDL1 or anti-PVR antibodies as indicated, followed by flow cytometry analysis. [Figure 23]Figure 23A shows the binding of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3) to Jurkat NFAT-CD16 cells as determined by flow cytometry analysis after incubation with or without blocking antibodies as indicated. Binding curve graphs were generated by FlowJo software using the indicated MFI values. Figure 23B shows a histogram showing the expression of CD16 on Jurkat NFAT-CD16 cells. Surface expression levels of CD16 were determined by immunostaining of cells with fluorescently labeled anti-CD16 antibodies followed by flow cytometry analysis as indicated. [Figure 24] 24 shows the luciferase secretion level from Jurkat NFAT-CD16 cells after co-culture with K562-WT cells or PDL1 and PVR overexpressing K562 cells in the presence of various concentrations of DSP502 (SEQ ID NO: 9 and 3). The luciferase secretion level was measured as the luminescence signal generated by the interaction of luciferase with the added substrate (QUANTI-Luc). [Figure 25-1]Figure 25A shows simultaneous binding of TIGIT and PD1 domains of DSP502 (SEQ ID NO: 9 and 3) to their corresponding ligands / receptors. Figure 25A shows binding to plate-bound PDL1 followed by incubation with human CD155 (PVR)-mouse IgG2a Fc. Detection was performed using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 540 nm, following standard ELISA protocol, by adding streptavidin-HRP followed by TMB substrate. Figures 25B-D show doublet formation of NK cells and K562PVR / PD-L1 cells in the presence of various concentrations of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3). Figure 25B is a histogram showing the expression levels of CD16 in NK cells as determined by immunostaining of cells with fluorescently labeled anti-CD16 antibody followed by flow cytometry analysis. Figure 25C shows doublet formation in the presence of DSP502 at the indicated concentrations. Figure 25D shows doublet formation in the presence of DSP502 at the indicated concentrations after incubation with blocking antibody (Fc-blocked, PVR-blocked, or PD-L1-blocked, as indicated). Q1 (top left quarter of each panel) represents K562PVR / PD-L1 CFSE-labeled cells (positive cells on Y-axis), Q3 (bottom right quarter of each panel) represents NK CPD-labeled cells (positive cells on X-axis), and Q2 (top right quarter of each panel) represents NK-K562PVR / PD-L1 cell doublets along with the percentage of doublets. [Figure 25-2] Same as above [Figure 25-3] Same as above [Figure 26] Figure 26 shows the in-vivo antitumor effect of TIGIT-PD1 heterodimer DSP502 (SEQ ID NOs: 9 and 3) in A549-NSCLC xenograft model in humanized NSG mice, as evidenced by reduced tumor volume compared to vehicle control. n=5 in each experimental group. [Figure 27]Figure 27 shows the in-vivo anti-tumor effect of TIGIT-PD1 heterodimer DSP502 (SEQ ID NOs: 9 and 3) in AB12 mesothelioma tumor-bearing mice, as evidenced by increased survival compared to vehicle controls. n=5 for each experimental group. [Figure 28] Figure 28 shows the effect of SIRPα-LILRB2 heterodimer DSP216 (SEQ ID NO: 1 and 11, Figure 16A) on phagocytosis of cancer cells by granulocytes. HT1080 or HT1080-HLA-G cells were labeled and pre-incubated with 0, 1, 2 or 5 μg / mL of DSP216, then co-cultured 1:1 with granulocytes and analyzed by flow cytometry. The percentage of phagocytosis by granulocytes from three donors is shown. [Figure 29] Figure 29A shows the cytotoxic effect of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3). The percentage of dead cells is shown after co-culture of the indicated K562 cells with NK cells at the indicated ratios in the presence of DSP502. The asterisks above the bars represent statistical significance relative to untreated co-cultures. Figure 29B shows the effect of TIGIT-PD1 heterodimer DSP502 (SEQ ID NO: 9 and 3) on the secretion of granzyme B from NK cells after co-culture with the indicated K562 cells. The asterisks above the bars represent statistical significance relative to untreated co-cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present invention, in some embodiments thereof, relates to type I membrane protein heterodimers and methods of use thereof.

[0041] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0042] Cancer immunotherapy aims to enhance the immune response against tumors by stimulating specific components of the immune system or by counteracting signals produced by cancer cells that suppress the immune response.

[0043] While carrying out certain embodiments of the present invention, the inventors have now generated heterodimers comprising the extracellular portions of two type I membrane proteins selected from the group consisting of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10 (see, e.g., Table 1 below).

[0044] Thus, according to one aspect of the present invention there is provided a heterodimer comprising two polypeptides selected from the group consisting of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10, wherein each of the two polypeptides is capable of binding to its natural binding partner, and wherein the heterodimer does not comprise an amino acid sequence of a type II membrane protein capable of binding to its natural binding partner.

[0045] As used herein, the term "heterodimer" refers to a non-naturally occurring dimeric protein formed by the artificial joining of two different proteins (referred to herein as monomers).

[0046] Methods for determining dimerization, particularly heterodimerization, are well known in the art and include, but are not limited to, NATIVE-PAGE, SEC-HPLC 2D gels, gel filtration, SEC-MALS, analytical ultracentrifugation (AUC) mass spectrometry (MS), and capillary gel electrophoresis (CGE).

[0047] According to certain embodiments, the monomers of the heterodimer are not covalently linked.

[0048] According to other particular embodiments, the monomers of the heterodimer are covalently linked.

[0049] According to other particular embodiments, the monomers of the heterodimer are linked by disulfide bonds.

[0050] According to certain embodiments, the monomers of the heterodimer are linked by multiple disulfide bonds.

[0051] As used herein, the terms "SIRPα polypeptide," "PD1 polypeptide," "TIGIT polypeptide," "LILRB2 polypeptide" and "SIGLEC10 polypeptide" refer to the amino acid sequences of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10 or functional homologs thereof, each of which is capable of binding to at least its natural binding partner, as further described below.

[0052] As used herein, the phrase "functional homolog" refers to fragments, homologs (naturally occurring or synthetically / recombinantly produced) and / or amino acid sequences that contain conservative and non-conservative amino acid substitutions that maintain at least the activity of the full-length protein to bind to its natural binding partner.

[0053] As used herein, the phrase "its natural binding partner" refers to the natural ligand or receptor of the described polypeptide.

[0054] Assays for testing binding are well known in the art and include, but are not limited to, flow cytometry, BiaCore, Biolayer Interferometry Blitz® Assay, HPLC.

[0055] According to certain embodiments, the heterodimer comprises a PD1 polypeptide.

[0056] As used herein, the term "PD1 (also known as programmed death 1, CD279)" refers to a polypeptide encoded by the PDCD1 gene (Gene ID 5133). According to certain embodiments, PD1 is human PD1. According to certain embodiments, PD1 refers to human PD1 as provided in the following GenBank number NP_005009.

[0057] To date, two ligands for PD1 have been identified: PDL1 and PDL2 (also known as B7-DC). According to certain embodiments, the PDL1 protein refers to the human protein as provided under the following GenBank numbers NP_001254635 and NP_054862. According to certain embodiments, the PDL2 protein refers to the human protein as provided under the following GenBank number NP_079515.

[0058] According to a particular embodiment, the PD1 amino acid sequence comprises SEQ ID NO:37.

[0059] According to a particular embodiment, the PD1 amino acid sequence consists of SEQ ID NO:37.

[0060] According to specific embodiments, the PD1 polypeptide binds to PD-L1 with a Kd of 1 nM-100 μM, 10 nM-10 μM, 100 nM-100 μM, or 200 nM-10 μM, as determined by SPR analysis, with each possibility representing a separate embodiment of the present invention.

[0061] According to certain embodiments, the PD1 polypeptide binds to PDL1 with a Kd of about 270 nM as determined by SPR analysis.

[0062] According to certain embodiments, the PD1 polypeptide binds to PDL1 with a Kd of about 8-9 μM as determined by SPR analysis.

[0063] According to certain embodiments, the PD1 polypeptide comprises the extracellular domain of PD1 or a functional homologue (e.g., a fragment) thereof.

[0064] According to certain embodiments, the PD1 polypeptide comprises SEQ ID NO: 41, 42, or 43, or a functional homologue (e.g., a fragment) thereof.

[0065] According to certain embodiments, the PD1 polypeptide comprises SEQ ID NO: 41, 42 or 43.

[0066] According to certain embodiments, the PD1 amino acid sequence consists of SEQ ID NO: 41, 42 or 43.

[0067] The term "PD1 polypeptide" also encompasses functional homologs that exhibit the desired activity (i.e., binding to PD-L1 and / or PD-L2). Such homologs may, for example, resemble polypeptides SEQ ID NO: 37, 41, 42, or 43, or any other PD1 amino acid sequence disclosed herein at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the polypeptide sequence SEQ ID NO: 37, 41, 42, or 43, or any other PD1 amino acid sequence disclosed herein. may be 100% identical or homologous, or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0068] As used herein, "identity" or "sequence identity" refers to global identity, i.e., identity over the entire amino acid or nucleic acid sequence disclosed herein, and not over a portion thereof.

[0069] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW and MUSCLE.

[0070] Homologs can also refer to orthologs, deletion, insertion or substitution variants (including amino acid substitutions), as described further below.

[0071] According to certain embodiments, PD1 polypeptides can include conservative and non-conservative amino acid substitutions, which are known in the art and disclosed, for example, in Maute et al. PNAS, 2015 Nov 24;112(47): E6506-14;Ju Yeon et al. Nature Communications 2016 volume 7, Article number: 13354 (DOI: 10.1038 / ncomms13354);Zack KM et al. Structure. 2015 23(12): 2341-2348 (DOI:10.1016 / j.str.2015.09.010) and US Patent Publication No. 2016 / 0039903, the contents of which are incorporated herein by reference in their entirety.

[0072] According to certain embodiments, the mutations result in an increased affinity of the PD1 polypeptide for PDL1 compared to SEQ ID NO:37.

[0073] According to certain embodiments, the one or more amino acid mutations are located at amino acid residues selected from V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106, and A107, corresponding to the PD1 amino acid sequence set forth in SEQ ID NO:42. According to certain embodiments, the one or more amino acid mutations are located at amino acid residues selected from V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, C68, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106, and A107, corresponding to the PD1 amino acid sequence set forth in SEQ ID NO:42.

[0074] According to certain embodiments, the one or more amino acid changes are, relative to the PD1 amino acid sequence set forth in SEQ ID NO:42, (1) V39H or V39R, (2) L40V or L40I, (3) N41I or N41V, (4) Y43F or Y43H, (5) R44Y or R44L, (6) M45Q, M45E, M45L or M45D, (7) S45Q, S45E, M45L or M45D, or (8) S45Q, S45E, M45L or M45D. 48D, S48L, S48N, S48G or S48V, (8) N49C, N49G, N49Y or N49S, (9) Q50K, Q50E or Q50H, (10) T51V, T51L or T51A, (11) D52F, D52R, D52Y or D52V, (12) K53T or K53L, (13) A56S or A56L, (14) Q63T, Q63I, Q63E, Q63L or Q63P, (15) G65N, G65R, G65I, G65L, G65F or G65V, (16) Q66P, (17) V72I, (18) H82Q, (19) M83L or M83F, (20) R90K, (21) Y96F, (22) L97Y, L97V or L97I, (23) A100I or A100V , (24) S102T or S102A, (25) L103I, L103Y or L103F, (26) A104S, A104H or A104D, (27) P105A, (28) K106G, K106E, K106I, K106V, K106R or K106T, and (29) A107P, A107I or A107V.

[0075] According to certain embodiments, the one or more amino acid changes are, relative to the PD1 amino acid sequence set forth in SEQ ID NO:42, (1) V39H or V39R, (2) L40V or L40I, (3) N41I or N41V, (4) Y43F or Y43H, (5) R44Y or R44L, (6) M45Q, M45E, M45L or M45D, (7) S48 D, S48L, S48N, S48G or S48V, (8) N49C, N49G, N49Y or N49S, (9) Q50K, Q50E or Q50H, (10) T51V, T51L or T51A, (11) D52F, D52R, D52Y or D52V, (12) K53T or K53L, (13) A56S or A56L, (14) Q63T, Q63I , Q63E, Q63L or Q63P, (15) G65N, G65R, G65I, G65L, G65F or G65V, (16) Q66P, (17) C68S(18), V72I, (19) H82Q, (20) M83L or M83F, (21) R90K, (22) Y96F, (23) L97Y, L97V or L97I, (24) A100I or A1 00V, (25) S102T or S102A, (26) L103I, L103Y or L103F, (27) A104S, A104H or A104D, (28) P105A, (29) K106G, K106E, K106I, K106V, K106R or K106T, and (30) A107P, A107I or A107V.

[0076] According to certain embodiments, the amino acid mutation is located at amino acid residue C93 corresponding to the PD1 amino acid sequence set forth in SEQ ID NO: 37 (e.g., corresponding to amino acid residue C68 corresponding to the PD1 amino acid sequence set forth in SEQ ID NO: 42).

[0077] Thus, according to certain embodiments, the PD1 polypeptide comprises SEQ ID NO: 39, or a functional homologue (e.g., a fragment) thereof.

[0078] According to a particular embodiment, the PD1 polypeptide comprises SEQ ID NO:39.

[0079] According to a particular embodiment, the PD1 amino acid sequence consists of SEQ ID NO:39.

[0080] As used herein, the terms "corresponding to the PD1 amino acid sequence set forth in SEQ ID NO:37," "corresponding to SEQ ID NO:37," "corresponding to the PD1 amino acid sequence set forth in SEQ ID NO:42," or "corresponding to SEQ ID NO:42" are intended to include corresponding amino acid residues in the context of any other PD1 amino acid sequence.

[0081] Further description regarding conservative and non-conservative amino acid substitutions is provided above and further below.

[0082] The PD1 polypeptide of some embodiments of the invention has a sequence similar to that of polypeptide SEQ ID NO: 39, 41, 42, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81, which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it, with each possibility representing a separate embodiment of the present invention.

[0083] According to certain embodiments, the PD1 polypeptide does not include any of the amino acid segments P1 to L5 and / or F146 to V150 corresponding to SEQ ID NO:43.

[0084] According to certain embodiments, the PD1 polypeptide does not include any of amino acid residues P1 to L5 and / or F146 to V150 corresponding to SEQ ID NO:43.

[0085] According to specific embodiments, the PD1 polypeptide comprises between 100 and 288 amino acids, between 100 and 200 amino acids, between 120 and 180 amino acids, between 120 and 160 amino acids, between 130 and 170 amino acids, between 130 and 160 amino acids, between 130 and 150 amino acids, between 140 and 160 amino acids, between 145 and 155 amino acids, between 123 and 166 amino acids, between 138 and 145 amino acids, between 123 and 148 amino acids, between 126 and 148 amino acids, between 123 and 140 amino acids, between 126 and 140 amino acids, between 127 and 140 amino acids, or between 130 and 140 amino acids, each possibility representing a separate embodiment of the present invention.

[0086] According to certain embodiments, the PD1 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 42, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81.

[0087] According to certain embodiments, the PD1 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 42, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81.

[0088] According to certain embodiments, the PD1 polypeptide comprises SEQ ID NO: 49, or a functional homologue (e.g., a fragment) thereof.

[0089] According to a particular embodiment, the PD1 polypeptide comprises SEQ ID NO:49.

[0090] According to a particular embodiment, the PD1 polypeptide consists of SEQ ID NO:49.

[0091] According to certain embodiments, a nucleic acid sequence encoding a PD1 polypeptide has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 38, 40, 44, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82, with each possibility representing a separate embodiment of the present invention.

[0092] According to certain embodiments, the nucleic acid sequence encoding the PD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 38, 40, 44, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 and 82.

[0093] According to certain embodiments, the nucleic acid sequence encoding the PD1 polypeptide consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 38, 40, 44, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 and 82.

[0094] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide.

[0095] As used herein, the term "SIRPα (signal regulatory protein alpha, also known as CD172a)" refers to a polypeptide encoded by the SIRPA gene (Gene ID 140885). According to certain embodiments, SIRPα is human SIRPα. According to certain embodiments, SIRPα refers to human SIRPα as provided in the following GenBank numbers NP_001035111, NP_001035112, NP_001317657, or NP_542970.

[0096] According to a particular embodiment, the SIRPα amino acid sequence comprises SEQ ID NO:83.

[0097] According to a particular embodiment, the SIRPα amino acid sequence consists of SEQ ID NO:83.

[0098] A known binding partner of SIRPα is CD47. According to a particular embodiment, the CD47 protein refers to the human protein as provided in the following GenBank numbers NP_001768 or NP_942088.

[0099] According to specific embodiments, the SIRPα polypeptide binds to CD47 with a Kd of 0.1-100 μM, 0.1-10 μM, 1-10 μM, 0.1-5 μM, or 1-2 μM as determined by SPR analysis, each possibility representing a separate embodiment of the present invention.

[0100] According to a particular embodiment, the SIRPα polypeptide comprises the extracellular domain of said SIRPα or a functional homologue (eg, a fragment) thereof.

[0101] According to certain embodiments, the SIRPα polypeptide comprises SEQ ID NO: 85 or a functional homologue (eg, a fragment) thereof.

[0102] According to a particular embodiment, the SIRPα polypeptide comprises SEQ ID NO:85.

[0103] According to a particular embodiment, the SIRPα polypeptide consists of SEQ ID NO:85.

[0104] The term "SIRPα polypeptide" also encompasses functional homologs that exhibit the desired activity (i.e., binding to CD47). Such homologs include, for example, those that resemble at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the polypeptide SEQ ID NO: 83 or 85 or any other SIRPα amino acid sequence disclosed herein. 00% identical or homologous to, or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0105] According to certain embodiments, SIRPα polypeptides can include conservative and non-conservative amino acid substitutions, such as those known in the art and disclosed, for example, in Weiskopf K et al. Science. (2013); 341(6141):88-91, the contents of which are incorporated herein by reference in their entirety.

[0106] According to certain embodiments, the one or more amino acid mutations are located at amino acid residues selected from L4, V6, A21, A27, I31, E47, K53, E54, H56, V63, L66, K68, V92 and F96, corresponding to the SIRPα amino acid sequence set forth in SEQ ID NO: 85.

[0107] According to certain embodiments, the SIRPα polypeptide comprises mutations at amino acid residues selected from the group consisting of L4, A27, E47 and V92, corresponding to the SIRPα amino acid sequence set forth in SEQ ID NO:85.

[0108] According to certain embodiments, the one or more amino acid mutations are selected from the group consisting of L4V or L4I, V6I or V6L, A21V, A27I or A27L, I31F or I31T, E47V or E47L, K53R, E54Q, H56P or H56R, V63I, L66T or L66G, K68R, V92I and F94L or F94V, corresponding to the SIRPα amino acid sequence set forth in SEQ ID NO: 85.

[0109] According to certain embodiments, the SIRPα polypeptide comprises a mutation selected from the group consisting of L4I, A27I, E47V and V92I, corresponding to the SIRPα amino acid sequence set forth in SEQ ID NO:85.

[0110] As used herein, the phrase "corresponding to the SIRPα amino acid sequence set forth in SEQ ID NO: 85" or "corresponding to SEQ ID NO: 85" is intended to include corresponding amino acid residues in the context of any other SIRPα amino acid sequence.

[0111] According to certain embodiments, the SIRPα polypeptide comprises SEQ ID NO: 89 or a functional homologue (eg, a fragment) thereof.

[0112] According to a particular embodiment, the SIRPα polypeptide comprises SEQ ID NO:89.

[0113] According to a particular embodiment, the SIRPα polypeptide consists of SEQ ID NO:89.

[0114] Further description regarding conservative and non-conservative amino acid substitutions is provided above and further below.

[0115] SIRPα polypeptides of some embodiments of the invention are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to polypeptide SEQ ID NO:85, 87, 89, 91 or 93. is homologous to or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it, with each possibility representing a separate embodiment of the present invention.

[0116] According to certain embodiments, the SIRPα polypeptide does not include the amino acid segment K117 to Y343 corresponding to SEQ ID NO:85.

[0117] According to certain embodiments, the SIRPα polypeptide does not include any of amino acid residues K117 to Y343 corresponding to SEQ ID NO:85.

[0118] According to certain embodiments, the SIRPα polypeptide does not include the amino acid segment P118 to Y343 corresponding to SEQ ID NO:85.

[0119] According to certain embodiments, the SIRPα polypeptide does not include any of amino acid residues P118 to Y343 corresponding to SEQ ID NO:85.

[0120] According to certain embodiments, the SIRPα polypeptide comprises between 100 and 504, 100 and 500 amino acids, 150 and 450 amino acids, 200 and 400 amino acids, 250 and 400 amino acids, 300 and 400 amino acids, 320 and 420 amino acids, 340 and 350 amino acids, 300 and 400 amino acids, 340 and 450 amino acids, 100 and 200 amino acids, 100 and 150 amino acids, 100 and 125 amino acids, 100 and 120 amino acids, 100 and 119 amino acids, 105 and 119 amino acids, 110 and 119 amino acids, 115 and 119 amino acids, 105 and 118 amino acids, 110 and 118 amino acids, 115 and 118 amino acids, 105 and 117 amino acids, 110 and 117 amino acids, 115 and 117 amino acids, each possibility representing a separate embodiment of the present invention.

[0121] According to certain embodiments, the SIRPα polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 89, 91 and 93, or a functional homologue (eg, a fragment) thereof.

[0122] According to certain embodiments, the SIRPα polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 89, 91 and 93.

[0123] According to certain embodiments, the SIRPα polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 89, 91 and 93.

[0124] According to certain embodiments, the nucleic acid sequence encoding the SIRPα polypeptide has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 86, 88, 90, 92 or 94, with each possibility representing a separate embodiment of the present invention.

[0125] According to certain embodiments, the nucleic acid sequence encoding the SIRPα polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:86, 88, 90, 92 and 94.

[0126] According to certain embodiments, the nucleic acid sequence encoding the SIRPα polypeptide consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:86, 88, 90, 92 and 94.

[0127] According to a particular embodiment, the heterodimer comprises a TIGIT polypeptide.

[0128] As used herein, the term "TIGIT (T cell immunoreceptor with Ig and ITIM domains)" refers to a polypeptide encoded by the TIGIT gene (Gene ID 201633). According to certain embodiments, TIGIT is human TIGIT. According to certain embodiments, TIGIT refers to human TIGIT as provided in the following GenBank numbers NP_776160 or XP_024309156.

[0129] According to a particular embodiment, the TIGIT amino acid sequence comprises SEQ ID NO:106.

[0130] According to a particular embodiment, the TIGIT amino acid sequence consists of SEQ ID NO:106.

[0131] A known binding partner of TIGIT is CD155 (PVR). According to a particular embodiment, the CD155 protein refers to the human protein as provided in the following GenBank numbers NP_001129240, NP_001129241, NP_001129242, NP_006496.

[0132] According to specific embodiments, the TIGIT polypeptide binds to CD155 with a Kd of 0.01-100 μM, 0.1-100 μM, 0.1-10 μM, or 0.1-5 μM, as determined by SPR analysis, each possibility representing a separate embodiment of the present invention.

[0133] According to certain embodiments, the TIGIT polypeptide comprises the extracellular domain of TIGIT or a functional homologue (eg, a fragment) thereof.

[0134] According to certain embodiments, the TIGIT polypeptide comprises SEQ ID NO: 107, 113, or 115, or a functional homologue (eg, a fragment) thereof.

[0135] According to certain embodiments, the TIGIT polypeptide comprises SEQ ID NO:107, 113 or 115.

[0136] According to certain embodiments, the TIGIT polypeptide consists of SEQ ID NO:107, 113 or 115.

[0137] According to a particular embodiment, the TIGIT polypeptide comprises SEQ ID NO:113.

[0138] According to a particular embodiment, the TIGIT polypeptide consists of SEQ ID NO:113.

[0139] The term "TIGIT polypeptide" also encompasses functional homologs that exhibit a desired activity (i.e., binding to CD155). Such homologs include, for example, those that resemble at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 201%, at least 202%, at least 203%, at least 204%, at least 205%, at least 206%, at least 207%, at least 208%, at least 209%, at least 300%, at least 301%, at least 302%, at least 303%, at least 304%, at least 305%, at least 306%, at least 307%, at least 308%, at least 309%, at least 310%, at least 311%, at least 312%, at least 313%, at least 314%, at least 315%, at least 316%, at least 317%, at least 318%, at least 319%, at least 320%, at least 321%, at least 322%, at least 323%, at least 324%, at least 325%, at least 326%, at least 327%, It may be 9% or 100% identical or homologous, or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0140] According to certain embodiments, the TIGIT polypeptides may contain conservative and non-conservative amino acid substitutions.

[0141] According to a particular embodiment, the one or more amino acid mutations are located at an amino acid residue selected from I42 and C69, corresponding to the TIGIT amino acid sequence set forth in SEQ ID NO:106.

[0142] According to a particular embodiment, the one or more amino acid mutations are selected from the group consisting of I42A and C69S, corresponding to the TIGIT amino acid sequence set forth in SEQ ID NO:106.

[0143] As used herein, the phrase "corresponding to the TIGIT amino acid sequence set forth in SEQ ID NO: 106" or "corresponding to SEQ ID NO: 106" is intended to include corresponding amino acid residues in the context of any other TIGIT amino acid sequence.

[0144] According to certain embodiments, the TIGIT polypeptide comprises SEQ ID NO: 109 or 111, or a functional homologue (eg, a fragment) thereof.

[0145] According to certain embodiments, the TIGIT polypeptide comprises SEQ ID NO:109 or SEQ ID NO:111.

[0146] According to a particular embodiment, the TIGIT polypeptide consists of SEQ ID NO:109 or SEQ ID NO:111.

[0147] Further description regarding conservative and non-conservative amino acid substitutions is provided above and further below.

[0148] According to specific embodiments, the TIGIT polypeptide comprises between 100 and 244 amino acids, between 100 and 200 amino acids, between 100 and 150 amino acids, or between 120 and 140 amino acids, each possibility representing a separate embodiment of the present invention.

[0149] According to certain embodiments, a nucleic acid sequence encoding a TIGIT polypeptide has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 108, 110, 112 or 114.

[0150] According to certain embodiments, the nucleic acid sequence encoding the TIGIT polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 108, 110, 112 and 114.

[0151] According to a particular embodiment, the nucleic acid sequence encoding the TIGIT polypeptide consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 108, 110, 112 and 114.

[0152] According to certain embodiments, the heterodimer comprises a LILRB2 polypeptide.

[0153] As used herein, the term "LILRB2 (leukocyte immunoglobulin-like receptor subfamily B member 2)" refers to a polypeptide encoded by the LILRB2 gene (Gene ID 10288). According to certain embodiments, LILRB2 is human LILRB2. According to certain embodiments, LILRB2 refers to human LILRB2 as provided in the following GenBank numbers NP_001074447, NP_001265332, NP_001265333, NP_001265334, NP_001265335.

[0154] A known binding partner of LILRB2 is major histocompatibility molecules (MHC, e.g., HLA-G). According to certain embodiments, LILRB2 polypeptides bind to MHC (e.g., HLA-G) with a Kd of 0.1 nM to 100 μM, 0.1 nM to 10 μM, 1 nM to 1 μM, 1 to 100 nM, or 1 to 10 nM, as determined by SPR analysis, with each possibility representing a separate embodiment of the present invention.

[0155] According to certain embodiments, the LILRB2 polypeptide comprises the extracellular domain of said LILRB2 or a functional homologue (eg, a fragment) thereof.

[0156] According to certain embodiments, the LILRB2 polypeptide comprises SEQ ID NO: 95 or a functional homologue (eg, a fragment) thereof.

[0157] According to a particular embodiment, the LILRB2 polypeptide comprises SEQ ID NO:95.

[0158] According to a particular embodiment, the LILRB2 polypeptide consists of SEQ ID NO:95.

[0159] The extracellular domain of LILRB2 contains four Ig-like domains, known as D1 to D4.

[0160] Thus, according to a particular embodiment, the amino acid sequence of the LILRB2 polypeptide comprises at least one Ig-like domain.

[0161] According to certain embodiments, the LILRB2 polypeptide comprises at least two Ig-like domains, at least three Ig-like domains or four Ig-like domains.

[0162] According to certain embodiments, the LILRB2 polypeptide comprises domains D1 and D2 of LILRB2, domains D1, D2 and D3 of LILRB2, domains D1, D2 and D4 of LILRB2 or domains D1, D2, D3 and D4 of LILRB2.

[0163] According to certain embodiments, the LILRB2 polypeptide comprises SEQ ID NO: 96 or 98 or a functional homologue (eg, a fragment) thereof.

[0164] According to certain embodiments, the LILRB2 polypeptide comprises SEQ ID NO: 96 or 98.

[0165] According to a particular embodiment, the LILRB2 polypeptide consists of SEQ ID NO: 96 or 98.

[0166] The term "LILRB2 polypeptide" also encompasses functional homologs that exhibit a desired activity (i.e., binding to MHC, e.g., HLA-G). Such homologs may, for example, resemble the polypeptides SEQ ID NOs: 95, 96, or 98 or any other LILRB2 amino acid sequence disclosed herein at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the LILRB2 amino acid sequence disclosed herein. may be 100% identical or homologous, or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0167] According to certain embodiments, the LILRB2 polypeptide may comprise conservative and non-conservative amino acid substitutions. Further explanation regarding conservative and non-conservative amino acid substitutions is provided further above and below.

[0168] According to specific embodiments, the LILRB2 polypeptide comprises between 100 and 597 amino acids, between 100 and 500 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 300 and 400 amino acids, between 350 and 400 amino acids, or between 150 and 250 amino acids, each possibility representing a separate embodiment of the present invention.

[0169] According to a particular embodiment, the LILRB2 polypeptide comprises SEQ ID NO:96.

[0170] According to a particular embodiment, the LILRB2 polypeptide consists of SEQ ID NO:96.

[0171] According to certain embodiments, a nucleic acid sequence encoding a LILRB2 polypeptide has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 97 or 99.

[0172] According to a particular embodiment, the nucleic acid sequence encoding the LILRB2 polypeptide comprises SEQ ID NO:97.

[0173] According to a particular embodiment, the nucleic acid sequence encoding the LILRB2 polypeptide consists of SEQ ID NO:97.

[0174] According to certain embodiments, the heterodimer comprises a SIGLEC10 polypeptide.

[0175] As used herein, the term "SIGLEC-10 (sialic acid-binding Ig-like lectin 10)" refers to a polypeptide encoded by the SIGLEC10 gene (Gene ID 89790). According to certain embodiments, SIGLEC10 refers to human SIGLEC10 as provided in the following GenBank Nos. NP_001164627, NP_001164628, NP_001164629, NP_001164630, NP_001164632.

[0176] According to a particular embodiment, the SIGLEC10 amino acid sequence comprises SEQ ID NO:100.

[0177] According to a particular embodiment, the SIGLEC amino acid sequence consists of SEQ ID NO:100.

[0178] A known binding partner of SIGLC10 is sialic acid expressed on CD24 and / or CD52. According to particular embodiments, SIGLEC10 polypeptides bind to CD24 or CD52 with a Kd of 1 nM-100 μM, 0.01-100 μM, 0.01-10 μM, 0.1-10 μM, 0.1-5 μM, or 0.1-1 μM, as determined by SPR analysis, with each possibility representing a separate embodiment of the present invention.

[0179] According to certain embodiments, the SIGLEC-10 polypeptide comprises the extracellular domain of SIGLEC10 or a functional homologue (eg, a fragment) thereof.

[0180] According to certain embodiments, the SIGLEC10 polypeptide comprises at least one Ig-like domain.

[0181] According to certain embodiments, the SIGLEC10 polypeptide comprises at least two Ig-like domains.

[0182] According to certain embodiments, the SIGLEC10 polypeptide comprises SEQ ID NO: 105 or a functional homologue (eg, a fragment) thereof.

[0183] According to a particular embodiment, the SIGLEC10 polypeptide comprises SEQ ID NO:105.

[0184] According to a particular embodiment, the SIGLEC10 amino acid sequence consists of SEQ ID NO:105.

[0185] The term "SIGLEC10 polypeptide" also encompasses functional homologs that exhibit the desired activity (i.e., binding to sialic acid expressed on CD24 and / or CD52). Such homologs are, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polypeptide SEQ ID NO: 100 or 105. or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence encoding it (described further below).

[0186] According to certain embodiments, SIGLEC-10 polypeptides may include conservative and non-conservative amino acid substitutions.

[0187] According to a particular embodiment, one mutation is located at amino acid residue C36, which corresponds to the SIGLEC10 amino acid sequence set forth in SEQ ID NO:100.

[0188] According to a particular embodiment, one amino acid mutation is C36S, which corresponds to the SIGLEC10 amino acid sequence set forth in SEQ ID NO:100.

[0189] As used herein, the phrase "corresponding to the SIGLEC10 amino acid sequence set forth in SEQ ID NO: 100" or "corresponding to SEQ ID NO: 100" is intended to include corresponding amino acid residues in the context of any other SIGLEC10 amino acid sequence.

[0190] According to certain embodiments, the SIGLEC10 polypeptide comprises SEQ ID NO: 103 or a functional homologue (eg, a fragment) thereof.

[0191] According to a particular embodiment, the SIGLEC10 polypeptide comprises SEQ ID NO:103.

[0192] According to a particular embodiment, the SIGLEC-10 polypeptide consists of SEQ ID NO:103.

[0193] Further description regarding conservative and non-conservative amino acid substitutions is provided further above and below.

[0194] According to specific embodiments, the SIGLEC10 amino acid sequence comprises between 100 and 639 amino acids, between 100 and 600 amino acids, between 100 and 550 amino acids, between 100 and 300 amino acids, between 100 and 200 amino acids, or between 100 and 150 amino acids, each possibility representing a separate embodiment of the present invention.

[0195] According to certain embodiments, the nucleic acid sequence encoding the SIGLEC10 polypeptide has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 102 or 104.

[0196] According to a particular embodiment, the nucleic acid sequence encoding the SIGLEC10 polypeptide comprises SEQ ID NO:104.

[0197] According to a particular embodiment, the nucleic acid sequence encoding the SIGLEC10 polypeptide consists of SEQ ID NO:104.

[0198] According to particular embodiments, the heterodimer comprises a SIRPα polypeptide and a PD1 polypeptide, a SIRPα polypeptide and a TIGIT polypeptide, a SIRPα polypeptide and a LILRB2 polypeptide, a SIRPα polypeptide and a SIGLEC10 polypeptide, a PD1 polypeptide and a TIGIT polypeptide, a PD1 polypeptide and a LILRB2 polypeptide, a PD1 polypeptide and a SIGLEC10 polypeptide, a TIGIT polypeptide and a LILRB2 polypeptide, a TIGIT polypeptide and a SIGLEC10 polypeptide, or a LILRB2 polypeptide and a SIGLEC10 polypeptide, each possibility representing a separate embodiment of the present invention.

[0199] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide and a PD1 polypeptide.

[0200] According to a particular embodiment, the heterodimer comprises a SIRPα polypeptide set forth in SEQ ID NO:85 and a PD1 polypeptide set forth in SEQ ID NO:49.

[0201] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide and a LILRB2 polypeptide.

[0202] According to a particular embodiment, the heterodimer comprises a SIRPα polypeptide set forth in SEQ ID NO:85 and a LILRB2 polypeptide set forth in SEQ ID NO:96.

[0203] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide set forth in SEQ ID NO:93 and a LILRB2 polypeptide set forth in SEQ ID NO:96.

[0204] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide and a SIGLEC10 polypeptide.

[0205] According to a particular embodiment, the heterodimer comprises a SIRPα polypeptide set forth in SEQ ID NO:85 and a SIGLEC10 polypeptide set forth in SEQ ID NO:103.

[0206] According to a particular embodiment, the heterodimer comprises a SIRPα polypeptide and a TIGIT polypeptide.

[0207] According to a particular embodiment, the heterodimer comprises a SIRPα polypeptide set forth in SEQ ID NO:85 and a TIGIT polypeptide set forth in SEQ ID NO:109.

[0208] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide and a PD1 polypeptide.

[0209] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide set forth in SEQ ID NO: 109, 111 or 113 and a PD1 polypeptide set forth in SEQ ID NO: 49.

[0210] According to a particular embodiment, the heterodimer comprises a TIGIT polypeptide and a LILRB2 polypeptide.

[0211] According to a particular embodiment, the heterodimer comprises a TIGIT polypeptide set forth in SEQ ID NO:109 and a LILRB2 polypeptide set forth in SEQ ID NO:96.

[0212] According to a particular embodiment, the heterodimer comprises a TIGIT polypeptide and a SIGLEC10 polypeptide.

[0213] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide set forth in SEQ ID NO:109 and a SIGLEC10 polypeptide set forth in SEQ ID NO:103.

[0214] According to certain embodiments, the heterodimer comprises a PD1 polypeptide and a SIGLEC10 polypeptide.

[0215] According to certain embodiments, the heterodimer comprises a PD1 polypeptide set forth in SEQ ID NO:49 and a SIGLEC10 polypeptide set forth in SEQ ID NO:103.

[0216] According to certain embodiments, the heterodimer comprises a LILRB2 polypeptide and a SIGLEC10 polypeptide.

[0217] According to certain embodiments, the heterodimer comprises a LILRB2 polypeptide set forth in SEQ ID NO:96 and a SIGLEC10 polypeptide set forth in SEQ ID NO:103.

[0218] According to certain embodiments, the heterodimer comprises a PD1 polypeptide and a LILRB2 polypeptide.

[0219] According to certain embodiments, the heterodimer comprises a PD1 polypeptide set forth in SEQ ID NO:49 and a LILRB2 polypeptide set forth in SEQ ID NO:96.

[0220] According to certain embodiments, the heterodimer does not comprise an amino acid sequence of a type II membrane protein capable of binding to its natural binding partner.

[0221] As used herein, the expression "amino acid sequence of a type II membrane protein" refers to a contiguous amino acid sequence of a type II membrane protein that is capable of binding to at least its natural binding partner. According to certain embodiments, such an amino acid sequence comprises the extracellular domain of a type II membrane protein or a functional fragment thereof.

[0222] As used herein, the phrase "type II membrane protein" refers to a transmembrane protein having a C-terminal extracellular domain.

[0223] Non-limiting examples of such type II membrane proteins include 4-1BBL, FasL, TRAIL, TNF-alpha, TNF-beta, OX40L, CD40L, CD27L, CD30L, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, GITRL, EDAI / 2, lymphotoxin alpha and lymphotoxin beta.

[0224] According to certain embodiments, the heterodimer does not comprise, other than the two polypeptides disclosed herein, an amino acid sequence of a type I membrane protein capable of binding to its natural binding partner.

[0225] As used herein, the expression "amino acid sequence of a type I membrane protein" refers to a contiguous amino acid sequence of a type I membrane protein that is capable of binding to at least a natural binding partner. According to certain embodiments, such an amino acid sequence comprises the extracellular domain of a type I membrane protein or a functional fragment thereof.

[0226] As used herein, the phrase "type I membrane protein" refers to a transmembrane protein having an N-terminal extracellular domain.

[0227] Non-limiting examples of such type I membrane proteins include LAG3, BTN3A1, CD27, CD80, CD86, ENG, NLGN4X, CD84, CD40, IL-8, IL-10, CD164, LY6G6F, CD28, CTLA4, BTLA, LILRB1, TYROBP, ICOS, VEGFA, CSF1, CSF1R, VEGFB, BMP2, BMP3, GDNF, PDGFC, PDGFD, RAET1E, CD155, CD166, MICA, NRG1, HVEM, DR3, TEK, TGFBR (e.g., TGFBR1), LY96, CD96, KIT, CD244, and GFER.

[0228] According to certain embodiments, the heterodimer does not contain any proteinaceous targeting, signaling, immunomodulatory and / or therapeutic moieties other than the two polypeptides disclosed herein and, optionally, a dimerization moiety (e.g., an Fc domain of an antibody or fragment thereof) as further described below.

[0229] Non-limiting examples of such moieties include cytokines, ligands, receptors, immunomodulatory polypeptides and antibody binding domains (eg, ScFv).

[0230] According to certain embodiments, the heterodimer consists of two polypeptides as described herein and, optionally, a dimerization moiety (e.g., an Fc domain of an antibody or fragment thereof) as further described below.

[0231] According to other specific embodiments, the heterodimer is linked to or includes a heterologous therapeutic moiety. The therapeutic moiety can be any molecule, including small molecule compounds and polypeptides.

[0232] Non-limiting examples of therapeutic moieties that may be used with certain embodiments of the present invention include cytotoxic moieties, toxic moieties, cytokine moieties, immunomodulatory moieties, polypeptides, antibodies, drugs, chemicals and / or radioisotopes.

[0233] According to some embodiments of the invention, the therapeutic moiety is attached by translationally fusing a polynucleotide encoding a polypeptide of some embodiments of the invention with a nucleic acid sequence encoding the therapeutic moiety.

[0234] Additionally or alternatively, therapeutic moieties can be chemically conjugated (coupled) to the heterodimers of some embodiments of the present invention using any conjugation method known to one of skill in the art. For example, 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (also called N-succinimidyl 3-(2-pyridyldithio)propionate) ("SDPD") (Sigma, Cat. No. P-3415, see e.g., Cumber et al. 1985, Methods of Enzymology 112: 207-224), glutaraldehyde coupling procedures (see e.g., G.T. Hermanson 1996, "Antibody Modification and Conjugation, in Bioconjugate Techniques, Academic Press, San Diego) or carbodiimide coupling procedures [see e.g., J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985; B. Neises et al. 1978, Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al. 1978, Tetrahedron Lett. 4475; EP Boden et al. 1986, J. Org. Chem. 50:2394 and LJ Mathias 1979, Synthesis 561] can be used to attach the peptide to the drug of interest.

[0235] Therapeutic moieties can be attached to the heterodimers of some embodiments of the invention using any suitable chemical bond, directly or indirectly, for example, by peptide bond (when the functional moiety is a polypeptide) or covalent attachment to an intervening linker element, such as a linker peptide or other chemical moiety (such as an organic polymer), using, for example, standard chemical synthesis techniques widely practiced in the art (see, for example, hypertexttransferprotocol: / / worldwideweb (dot) chemistry (dot) org / portal / Chemistry). Chimeric peptides can be attached by attachment at the carboxy (C) or amino (N) terminus of the peptide, or by attachment to an internal chemical group, such as a linear, branched or cyclic side chain, an internal carbon or nitrogen atom, or the like.

[0236] According to certain embodiments, the heterodimer comprises a detectable tag. Thus, according to certain embodiments, any of the polypeptides contained in the heterodimer may comprise a detectable tag. As used herein, in one embodiment, the term "detectable tag" refers to any moiety that can be detected by a person skilled in the art using techniques known in the art. The detectable tag may be a peptide sequence. Optionally, the detectable tag may be removable by chemicals or enzymatic means such as proteolysis. The detectable tags of some embodiments of the present invention can be used for purification of the polypeptide or heterodimer. For example, the term "detectable tag" includes chitin-binding protein (CBP) tags, maltose-binding protein (MBP) tags, glutathione-S-transferase (GST) tags, poly(His) tags, FLAG tags, epitope tags such as V5 tags, c-myc tags, and HA tags, as well as fluorescent tags such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and cyan fluorescent protein (CFP), as well as derivatives of these tags or any tag known in the art. The term "detectable tag" also includes the term "detectable marker."

[0237] According to certain embodiments, the polypeptide comprises a detectable tag (eg, a poly(His) tag) attached to its N-terminus.

[0238] According to certain embodiments, the polypeptide comprises a detectable tag (eg, a poly(His) tag) attached to its C-terminus.

[0239] According to certain embodiments, the N-terminus of the polypeptide does not comprise a detectable tag (eg, a poly(His) tag).

[0240] According to certain embodiments, the C-terminus of the polypeptide does not comprise a detectable tag (eg, a poly(His) tag).

[0241] According to certain embodiments, the heterodimer comprises a cleavable moiety. Thus, according to certain embodiments, any of the polypeptides comprised in the heterodimer may be fused to a cleavable moiety. Thus, for example, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of some embodiments of the present invention and a fused cleavable moiety. In one embodiment, the polypeptide is designed to be easily isolated by affinity chromatography, for example by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, allowing the peptide to be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site (see, e.g., Booth et al., Immunol. Lett. 19:65-70 (1988) and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)). According to certain embodiments, the heterodimer comprises a dimerization moiety attached to two polypeptides disclosed herein.

[0242] As used herein, the term "dimerization moiety" refers to a moiety that is capable of linking two different monomers to form a heterodimer. Such dimerization moieties are known in the art and include chemical moieties and proteinaceous moieties.

[0243] According to certain embodiments, the dimerization moiety is directly attached to the polypeptide.

[0244] According to certain embodiments, the dimerization moiety is indirectly attached to the polypeptide.

[0245] According to certain embodiments, the dimerization moiety is covalently attached to the polypeptide.

[0246] According to certain embodiments, the dimerization moiety is not covalently attached to the polypeptide.

[0247] According to certain embodiments, the dimerization moiety is heterologous to the polypeptide.

[0248] According to certain embodiments, the dimerization moiety is a composition of at least two different molecules.

[0249] According to certain embodiments, the dimerization moiety is capable of activating an immune response upon binding of the heterodimer to a cell expressing at least one natural binding partner of the two polypeptides and / or a cell expressing a natural binding partner of the two polypeptides.

[0250] As used herein, the term "activate" refers to stimulation of immune cells (e.g., T cells, NK cells, B cells, dendritic cells, macrophages) resulting in cell proliferation, maturation, cytokine production and / or induction of regulatory or effector functions. Methods for assessing immune cell activation or function are well known in the art and include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, expression of activation markers such as CD25, CD69 and CD69 using flow cytometry, and multimer (e.g., tetramer) assays.

[0251] A non-limiting example of such a dimerization moiety that may be used with certain embodiments is the Fc domain of an antibody, as described further below.

[0252] According to certain embodiments, the dimerization moiety is a non-proteinaceous moiety, such as a cross-linker, an organic polymer, a synthetic polymer, a small molecule, and the like.

[0253] Several such non-proteinaceous moieties are known in the art and are commercially available, for example from Santa Cruz, Sigma-Aldrich, Proteochem, etc. According to a particular embodiment, the non-proteinaceous moiety is a heterobifunctional crosslinker. Heterobifunctional crosslinkers have two different reactive ends. Typically, in a first step, a monomer is modified with one reactive group of the heterobifunctional crosslinker and the remaining free reagent is removed. In a second step, the modified monomer is mixed with a second monomer and reacted with the modified group at the other end of the reagent. The most widely used couples proteins via amine and sulfhydryl groups (the most labile amine-reactive NHS esters are coupled first, and the coupling of sulfhydryl groups proceeds after removal of unbound reagent). Sulfhydryl reactive groups are usually maleimide, pyridyl disulfide and alpha-haloacetyl. Other crosslinkers include carbodiimides that link between carboxyl groups (-COOH) and primary amines (-NH2). Another approach is to modify the lysine residues of one monomer to a thiol and the second monomer by the addition of a maleimide group, followed by the formation of a stable thioester bond between the monomers. If one of the monomers naturally has thiols, these groups can be reacted directly with the maleimide attached to the other monomer. There are also heterobifunctional crosslinkers with one photoreactive end, such as bis[2-(4-azidosalicylamido)ethyl)] disulfide (BASED). Photoreactive groups are used when there is no specific group to react with, because they react nonspecifically upon exposure to UV light.Non-limiting examples of such heterobifunctional crosslinkers include, but are not limited to, alkyne-PEG4-maleimide, alkyne-PEG5-N-hydroxysuccinimidyl ester, maleimide-PEG-succinimidyl ester, azido-PEG4-phenyloxadiazole methylsulfone, LC-SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), MPBH (4-(4-N-maleimidophenyl )butyric acid hydrazide hydrogen chloride + 1 / 2 dioxane), PDPH (3-(2-pyridyldithio)propionyl hydrazide), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate), sulfo-KMUS (N-(kappa-maleimido undecanoyloxy)sulfosuccinimide ester), sulfo-SIAB (sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), 3-(maleimido)propionic acid N-Hydroxysuccinimide ester, Methoxycarbonylsulfenyl chloride, Propargyl-PEG-acid, Amino-PEG-t-butyl ester, BocNH-PEG5-acid, BMPH (N-(β-Maleimidopropionic acid) hydrazide, trifluoroacetate), ANB-NOS, BMPS, EMCS, GMBS, LC-SPDP, MBS, SBA, SIA, Sulfo-SIA, SMCC, SMPB, SMPH, SPDP, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SANPAH, Sulfo-SMCC.

[0254] According to other particular embodiments, the dimerization moiety is a proteinaceous moiety.

[0255] According to other particular embodiments, the dimerization moiety is a proteinaceous dimer moiety.

[0256] According to a particular embodiment, the polypeptide is attached to the N-terminus of the dimerizing proteinaceous moiety.

[0257] According to a particular embodiment, the two polypeptides are linked to the N-terminus of the dimerized proteinaceous moiety.

[0258] According to a particular embodiment, the polypeptide is attached to the C-terminus of the dimerizing proteinaceous moiety.

[0259] According to a particular embodiment, the two polypeptides are linked to the C-terminus of the dimerized proteinaceous moiety.

[0260] According to a particular embodiment, one of the two polypeptides is attached to the N-terminus of the dimerized proteinaceous portion and the second of the two polypeptides is attached to the C-terminus of the dimerized proteinaceous portion.

[0261] According to certain embodiments, the dimerization moiety comprises an affinity pair of a polypeptide having two separate affinity moieties for two different affinity complementary tags. Such affinity pairs are known in the art and include, but are not limited to, hemagglutinin (HA), anti-HA, AviTag™, V5, Myc, T7, FLAG, HSV, VSV-G, His, biotin, avidin, streptavidin, resabedin, metal affinity tag, lectin affinity tag. The skilled artisan will know which tag to choose.

[0262] According to a particular embodiment, the dimerization moiety is the Fc domain of any antibody (eg, IgG, IgA, IgD or IgE) or fragment thereof.

[0263] According to particular embodiments, the Fc is IgG, IgA, IgD or IgE.

[0264] According to a particular embodiment, the Fc domain is that of an IgG.

[0265] According to a particular embodiment, the Fc domain is of IgG1 or IgG4.

[0266] According to certain embodiments, the Fc domain is that of human IgG4. A non-limiting example of a human IgG4 Fc domain that can be used with certain embodiments of the present invention is provided in SEQ ID NO: 134.

[0267] According to certain embodiments, the Fc domain is that of human IgG1. Non-limiting examples of human IgG1 Fc domains that can be used with certain embodiments of the present invention are provided in SEQ ID NOs: 137 and 156.

[0268] According to a particular embodiment, the dimerization moiety is an Fc domain monomer.

[0269] According to other particular embodiments, the dimerization moiety is an Fc domain dimer.

[0270] There are several mechanisms that can be used to create heterodimers using the Fc domain of an antibody, including, but not limited to, the knob-into-hole method or the charge pair method (see, e.g., Gunasekaran et al., J. Biol. Chem. (2010) 285(25):19637, incorporated herein by reference in its entirety).

[0271] Thus, according to certain embodiments, the Fc domain may have conservative and non-conservative amino acid substitutions (also referred to herein as mutations).

[0272] When percentages of sequence identity are used with reference to proteins, non-identical residue positions often differ by conservative amino acid substitutions, where an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity), so that the functional properties of the molecule are not altered. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity." Means for making such adjustments are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, a conservative substitution is given a score of 0 to 1, where an identical amino acid has a score of 1 and a non-conservative substitution has a score of 0. Conservative substitution scores are calculated, for example, according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 1992, 89(22): 10915-9].

[0273] Further discussion of conservative and non-conservative amino acid substitutions is provided below.

[0274] Such Fc domain replacements are known in the art.

[0275] A representative example that can be used with certain embodiments of the present invention is the "knob-into-hole" ("KIH") type. Such knob and hole mutations are widely known in the art and are disclosed, for example, in U.S. Pat. No. 8,216,805, Shane Atwell et al. J. Mol. Biol. (1997) 270, 26-35, Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996) and A. Margaret Merchant et.al. Nature Biotechnology (1998) 16 July, the contents of which are incorporated herein by reference. Furthermore, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knob and hole" mutations can be combined with disulfide bonds to prevent heterodimerization.

[0276] Thus, according to a particular embodiment, one of the monomers comprises an Fc domain that includes a knob mutation and the other monomer comprises an Fc domain that includes a hole mutation.

[0277] It is within the skill of one in the art to select a particular immunoglobulin Fc domain from a particular immunoglobulin class and subclass, and to select one Fc variant for knob mutations and the other for hole mutations. Non-limiting examples of substitutions that can be used with certain embodiments include S228P, L235E, T366W, Y349C, T366S, L368A, Y407V and / or E356C [corresponding to human IgG4 as part of a full length antibody, according to EU numbering (Kabat, EA, TT Wu, M. Reid-Miller, HM Perry and KS Gottesman. 1987. Sequences of proteins of Immunological Interest. US. Dept. of Health and Human Services, Bethesda)] or L235A, Y349C, T366W, T354C, D356C, T366S, L368A and / or Y407V [corresponding to human IgG1 as part of a full length antibody, according to EU numbering (Kabat, EA, TT Wu, M. Reid-Miller, HM Perry and KS Gottesman. 1987. Sequences of proteins of Immunological Interest. US. Dept. of Health and Human Services, Bethesda)]. Sequences of proteins of Immunological Interest. US. Dept. of Health and Human Services, Bethesda).

[0278] Non-limiting examples of IgG4 Fc domains containing knob mutations that can be used with certain embodiments of the invention are provided in SEQ ID NOs: 135, 157, 158 and 163.

[0279] Non-limiting examples of IgG4 Fc domains containing hole mutations that can be used with certain embodiments of the present invention are provided in SEQ ID NOs: 136, 159 and 164.

[0280] Non-limiting examples of IgG1 Fc domains containing knob mutations that can be used with certain embodiments of the present invention are provided in SEQ ID NOs: 27, 51, 154 and 160.

[0281] Non-limiting examples of IgG1 Fc domains containing hole mutations that can be used with certain embodiments of the present invention are provided in SEQ ID NOs: 28, 52, 152, 161 and 162.

[0282] According to certain embodiments, the Fc domain has a sequence similar to that of at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or a functional fragment thereof that exhibits a desired activity as disclosed herein. The present invention includes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or homology thereto, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence encoding same.

[0283] According to a particular embodiment, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-136, 27-28 or 51-52.

[0284] According to certain embodiments, the Fc domain is modified to alter its binding to Fc receptors, reduce its immunostimulatory function, and / or improve the half-life of the fusion.

[0285] According to certain embodiments, the Fc domain is modified to reduce its binding to an Fc receptor and / or its immune stimulatory function when the natural binding partner is known to be expressed in healthy cells.

[0286] According to certain embodiments, the SIRPα-LILRB2 heterodimer is modified to reduce its binding to Fc receptors and / or its immunostimulatory function.

[0287] According to other particular embodiments, the Fc domain is not modified to alter its binding to Fc receptors, to reduce its immunostimulatory function, and / or to improve the half-life of the fusion.

[0288] According to certain embodiments, the Fc domain is not modified to alter its binding to an Fc receptor and / or reduce its immunostimulatory function when the natural binding partner is known to be solely expressed or overexpressed in pathological cells (e.g., cancer cells).

[0289] According to certain embodiments, the TIGIT-PD1 heterodimer comprises an Fc domain that has not been modified to alter its binding to Fc receptors and / or reduce its immunostimulatory function.

[0290] According to certain embodiments, the Fc domain is modified to reduce or prevent binding to Fc receptors (e.g., Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII) in vivo. Such modifications have been described, for example, by Clark et al., who designed and described a series of mutant IgG1, IgG2 and IgG4 Fc domains and their Fc, gamma, R binding properties (Armour et al., 1999; Armour et al., 2002, the contents of which are incorporated herein by reference in their entireties). Additional or alternative modifications in the Fc of human IgG1 that reduce its binding to Fc receptors have been described by CHAPPEL et al. (Proc. Natl. Acad. Sci (1991) 88: 9036-9040, the contents of which are incorporated herein by reference in their entirety), who identified amino acids L234 and L235 [according to EU numbering corresponding to full-length antibodies (Kabat et al.)] as essential for Fc receptor binding. An additional substitution of P329 to G further weakens binding, and this LALA-PG substitution combination has been described, for example, by Schlothauer, T., et al. (2016) Protein Eng. Des. Sel. 29, 457-466 and WO 2012 / 130831, the contents of which are incorporated herein by reference in their entirety. Additional or alternative modifications in the Fc of human IgG4 that prevent Fab arm exchange and reduce its binding to Fc receptors have been described by John-Paul Silva et al. (THE JOURNAL OF BIOLOGICAL CHEMISTRY (2015), 290: 9, 5462-5469, the contents of which are incorporated herein by reference in their entirety) and Newman et al. (Clinical Immunology (2001) 98:2, the contents of which are incorporated herein by reference in their entirety), who identified S228P and L235E [according to EU numbering corresponding to full-length antibodies (Kabat et al.)], respectively.

[0291] According to certain embodiments, the Fc domain is modified to maximize FcγRIIIa binding. Such modifications are described, for example, by Shields RL J Biol Chem. (2001) 276:6591; Smith P, Proc Natl Acad Sci USA. (2012) 109:6181; Stavenhagen et al., Cancer Res (2007) 67:8882; Lazar et al., Proc Natl Acad Sci UCA (2006) 103:4005; Richards et al., 2008 Cancer Ther 7:2517 and Mimoto et al., (2013) MAbs 5:229, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of such modifications that can be used with certain embodiments include substitutions at one or more amino residues selected from S298, E333 and K334 (e.g., S298A, E333A, K334A), G236A, S239A, A330L and I332E, F243L, R292P, Y300L, V305I and P396L, S239D, I332E and A330L, 236A, S239D and I332E (according to EU numbering corresponding to a full length antibody (Kabat et al.)) and asymmetric substitutions (L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the opposite heavy chain).

[0292] According to certain embodiments, the Fc domain is modified to provide altered effector function (e.g., reduced complement binding) and / or reduced or abolished complement dependent cytotoxicity. Such modifications are described, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260, U.S. Pat. No. 6,194,551, WO 99 / 51642, Wines et al., 2000, Idusogie et al. (2000) J. Immunol. 164:4178; Tao et al. (1993) J. Exp. Med. 178:661, and Canfield & Morrison (1991) J. Exp. Med. 173: 1483, the contents of which are incorporated herein by reference in their entireties. Non-limiting examples of such modifications that can be used with certain embodiments include substitutions of one or more amino acids at positions (according to EU numbering (Kabat et al.) corresponding to a full-length antibody) selected from: 234, 235, 236, 237, 297, 318, 320 and 322; 329, 331 and 322; L234 and / or L235 (e.g., L234A and / or L235A); D270, K322, P329 and P331 (e.g., D270A, K322A, P329A and P331A).

[0293] According to certain embodiments, the Fc domain is modified to result in an improvement in the half-life of the fusion protein. Such modifications are described, for example, in US Pat. Nos. 5,869,046 and 6,121,022, the contents of which are incorporated herein by reference in their entirety. For example, the substitution of one or more amino acids at positions (according to EU numbering corresponding to full-length antibodies (Kabat et al.)) selected from 252 (e.g., to introduce Thr), 254 (e.g., to introduce Ser) and 256 (e.g., to introduce Phe). Another modification to improve half-life can be by modifying the CH1 or CL region to introduce a salvage receptor motif, such as that found in the two loops of the CH2 domain of the Fc region of IgG.

[0294] Maximizing FcRn binding and extending half-life is also described, for example, in Stapleton NM, Nat Commun. (2011) 2:599, Shields RL. J Biol Chem. (2001) 276:6591, Dall'acqua WF J Immunol. (2002) 169:5171, Zalevsky J, Nat Biotechnol. (2010) 28:157, Ghetie V,.Nat. Biotechnol. (1997) 15:637, and Monnet C, MAbs. (2014) 6:422, the contents of which are incorporated by reference in their entireties. Non-limiting examples of such modifications that can be used with certain embodiments include substitution of one or more amino acid residues selected from Arg435His; Asn434Ala; Met252Tyr, Ser254Thr and Thr256Glu; Met428Leu and Asn434Ser; Thr252Leu, Thr253Ser and Thr254Phe; Glu294delta, Thr307Pro and Asn434Tyr; Thr256Asn, Ala378Val, Ser383Asn and Asn434Tyr (according to EU numbering (Kabat et al.) corresponding to a full-length antibody).

[0295] According to certain embodiments, the dimerization moiety comprises a leucine zipper or a helix-loop-helix.

[0296] According to certain embodiments, each of the moieties included in the heterodimer may include a linker separating the moieties, for example, between the polypeptide (e.g., SIRPα, PD1, TIGIT, LILRB2, SIGLEC10) and the dimerization moiety.

[0297] According to other particular embodiments, the heterodimer does not include a linker between the polypeptide (e.g., SIRPα, PD1, TIGIT, LILRB2, SIGLEC10) and the dimerization moiety.

[0298] Any linker known in the art may be used with certain embodiments of the present invention.

[0299] According to certain embodiments, the linker may be derived from a naturally occurring multidomain protein or may be an empirical linker, for example, as described in Chichili et al., (2013), Protein Sci. 22(2): 153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker may be designed using linker design databases and computer programs, such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are incorporated herein by reference.

[0300] According to certain embodiments, the linker is a synthetic linker, such as PEG.

[0301] According to certain embodiments, the linker may be functional. For example, but not limited to, the linker may function to result in improved folding and / or stability, improved expression, improved pharmacokinetics, and / or improved biological activity of the heterodimer. In another example, the linker may function to target the heterodimer to a particular cell type or location.

[0302] According to certain embodiments, the linker is a polypeptide.

[0303] Non-limiting examples of polypeptide linkers include the sequences LE, GGGGS (SEQ ID NO: 124), (GGGGS) n(n = 1 to 4) (SEQ ID NO: 123), GGGGSGGGG (SEQ ID NO: 122), (GGGGS) x2 (SEQ ID NO: 125), (GGGGS) x2 + GGGG (SEQ ID NO: 121), (GGGGS) x3 (SEQ ID NO: 117), (GGGGS) x4 (SEQ ID NO: 118), (Gly) 8 (SEQ ID NO: 119), (Gly) 6 (SEQ ID NO: 120), (EAAAK) n (n = 1 to 3) (SEQ ID NO: 126), A (EAAAK) n A (n = 2 to 5) (SEQ ID NO: 127), AEAAAKEAAAKA (SEQ ID NO: 128), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 129), PAPAP (SEQ ID NO: 130), K ESGSVSS EQ LAQ FRS LD (SEQ ID NO: 131), EGKSSGSGSESKST (SEQ ID NO: 132), GSAGSAAGSGEF (SEQ ID NO: 133), and (XP) n (X designates any amino acid, e.g., Ala, Lys, or Glu).

[0304] According to certain embodiments, the linker comprises SEQ ID NO: 117 (for example and without limitation, as a linker between the LILRB2 polypeptide and the Fc domain).

[0305] According to certain embodiments, the linker comprises SEQ ID NO: 125 (for example and without limitation, as a linker between a SIRPα, PD1, TIGIT or SIGLEC10 polypeptide and an Fc domain).

[0306] According to a particular embodiment, the linker is 1 to 6 amino acids in length.

[0307] According to certain embodiments, the linker is substantially composed of glycine and / or serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or 100% glycine and serine).

[0308] According to certain embodiments, the linker is a single amino acid linker.

[0309] In some embodiments of the invention, one amino acid is glycine.

[0310] According to certain embodiments, the linker is not an Fc domain or hinge region of an antibody or fragment thereof.

[0311] The heterodimers disclosed herein comprise two polypeptides selected from the group consisting of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10. Non-limiting examples of possible configurations of such heterodimers are shown diagrammatically in FIG. 1A.

[0312] According to certain embodiments, the heterodimer configuration is selected from those shown in panels 1-6 of FIG. 1A, each possibility representing a separate embodiment of the present invention.

[0313] According to certain embodiments, the two polypeptides are comprised in monomers of a heterodimer. Non-limiting examples of such heterodimer configurations that may be used with certain embodiments of the present invention are shown in panels 1-2 of Figure 1A.

[0314] Thus, according to a particular embodiment, one of the monomers of the heterodimer is a fusion polypeptide comprising two polypeptides.

[0315] According to a particular embodiment, one of the monomers of the heterodimer is a fusion polypeptide comprising two polypeptides linked by a proteinaceous dimerization moiety (eg, the Fc domain of an antibody or fragment thereof).

[0316] As used herein, the term "fusion polypeptide" refers to an amino acid sequence having two or more moieties that are not naturally found together in a single amino acid sequence.

[0317] According to certain embodiments, one of the monomers of the heterodimer is a fusion polypeptide comprising two polypeptides linked by an Fc domain of an antibody or fragment thereof comprising a knob mutation, and the other monomer comprises an Fc domain of an antibody or fragment thereof comprising a hole mutation.

[0318] According to certain embodiments, one of the monomers of the heterodimer is a fusion polypeptide comprising two polypeptides linked by an Fc domain of an antibody or fragment thereof comprising a hole mutation, and the other monomer comprises an Fc domain of an antibody or fragment thereof comprising a knob mutation.

[0319] According to other particular embodiments, each of the two polypeptides is a monomer in the heterodimer. Non-limiting examples of such heterodimer configurations that may be used with certain embodiments of the present invention are shown in panels 3-6 of Figure 1A. According to particular embodiments, the heterodimer configuration is as shown in panel 5 of Figure 1A.

[0320] According to certain embodiments, the heterodimer comprises a first monomer comprising one of the two polypeptides linked (e.g., as a translational fusion) to a proteinaceous dimerization moiety (e.g., the Fc domain of an antibody or fragment thereof) and a second monomer comprising the second of the two polypeptides linked (e.g., as a translational fusion) to a proteinaceous dimerization moiety (e.g., the Fc domain of an antibody or fragment thereof).

[0321] According to certain embodiments, the heterodimer comprises a first monomer comprising one of the two polypeptides linked (e.g., as a translational fusion) to the Fc domain of an antibody or fragment thereof that comprises a knob mutation, and a second monomer comprising the second of the two polypeptides linked (e.g., as a translational fusion) to the Fc domain of an antibody or fragment thereof that comprises a hole mutation.

[0322] According to certain embodiments, the composition and configuration of the heterodimer is selected from the heterodimers depicted generally in FIG. 1B, each possibility representing a separate embodiment of the present invention.

[0323] Non-limiting examples of heterodimers that can be used with certain embodiments of the present invention are shown in Table 1 below.

[0324] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1 or 5. The present invention also includes monomers comprising a PD1 polypeptide having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:3 or 7.

[0325] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:1 and a monomer comprising SEQ ID NO:3.

[0326] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:1 and a monomer set forth in SEQ ID NO:3.

[0327] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:5 and a monomer comprising SEQ ID NO:7.

[0328] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:5 and a monomer set forth in SEQ ID NO:7.

[0329] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9 or 13. and monomers comprising a LILRB2 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 11 or 15.

[0330] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:9 and a monomer comprising SEQ ID NO:11.

[0331] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:9 and a monomer set forth in SEQ ID NO:11.

[0332] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:13 and a monomer comprising SEQ ID NO:15.

[0333] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:13 and a monomer set forth in SEQ ID NO:15.

[0334] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1, 5, 17, 138, 140, 142 or 144. and monomers comprising an LLIRB2 polypeptide having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 11, 15, 19 or 150.

[0335] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1, 5, or 17. and monomers comprising an LLIRB2 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 11, 15 or 19.

[0336] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:1 and a monomer comprising SEQ ID NO:11.

[0337] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:1 and a monomer set forth in SEQ ID NO:11.

[0338] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:5 and a monomer comprising SEQ ID NO:15.

[0339] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:5 and a monomer set forth in SEQ ID NO:15.

[0340] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:17 and a monomer comprising SEQ ID NO:19.

[0341] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:17 and a monomer set forth in SEQ ID NO:19.

[0342] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:138 and a monomer comprising SEQ ID NO:11.

[0343] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:138 and a monomer set forth in SEQ ID NO:11.

[0344] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:140 and a monomer comprising SEQ ID NO:15.

[0345] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:140 and a monomer set forth in SEQ ID NO:15.

[0346] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:142 and a monomer comprising SEQ ID NO:150.

[0347] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:142 and a monomer set forth in SEQ ID NO:150.

[0348] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:144 and a monomer comprising SEQ ID NO:150.

[0349] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:144 and a monomer set forth in SEQ ID NO:150.

[0350] According to certain embodiments, the heterodimer comprises a LILRB2 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 21 or 22. The present invention also includes monomers comprising a peptide and monomers comprising a PD1 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:3 or 7.

[0351] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:21 and a monomer comprising SEQ ID NO:3.

[0352] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:21 and a monomer set forth in SEQ ID NO:3.

[0353] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:22 and a monomer comprising SEQ ID NO:7.

[0354] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:22 and a monomer set forth in SEQ ID NO:7.

[0355] According to certain embodiments, the heterodimer comprises a SIGLEC10 polynucleotide having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 24 or 25. These include monomers comprising peptides and monomers comprising PD1 polypeptides comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:3 or 7.

[0356] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:24 and a monomer comprising SEQ ID NO:3.

[0357] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:24 and a monomer set forth in SEQ ID NO:3.

[0358] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:25 and a monomer comprising SEQ ID NO:7.

[0359] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:25 and a monomer set forth in SEQ ID NO:7.

[0360] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1 or 5. and monomers comprising a SIGLEC10 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:29 or 30.

[0361] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:1 and a monomer comprising SEQ ID NO:29.

[0362] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:1 and a monomer set forth in SEQ ID NO:29.

[0363] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:5 and a monomer comprising SEQ ID NO:30.

[0364] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:5 and a monomer set forth in SEQ ID NO:30.

[0365] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9 or 13. and monomers comprising a SIGLEC10 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:29 or 30.

[0366] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:9 and a monomer comprising SEQ ID NO:29.

[0367] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:9 and a monomer set forth in SEQ ID NO:29.

[0368] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:13 and a monomer comprising SEQ ID NO:30.

[0369] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:13 and a monomer set forth in SEQ ID NO:30.

[0370] According to certain embodiments, the heterodimer comprises a SIGLEC10 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:24 or 25. The present invention also includes monomers comprising a LILRB2 polypeptide having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 11 or 15.

[0371] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:24 and a monomer comprising SEQ ID NO:11.

[0372] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:24 and a monomer set forth in SEQ ID NO:11.

[0373] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:25 and a monomer comprising SEQ ID NO:15.

[0374] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:25 and a monomer set forth in SEQ ID NO:15.

[0375] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 9, 13, 31, 33 or 146. and monomers comprising a PD1 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:3, 7 or 148.

[0376] According to certain embodiments, the heterodimer comprises a TIGIT polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 9, 13, 31 or 33. and monomers comprising a PD1 polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:3 or 7.

[0377] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:9 and a monomer comprising SEQ ID NO:3.

[0378] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:9 and a monomer set forth in SEQ ID NO:3.

[0379] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:13 and a monomer comprising SEQ ID NO:7.

[0380] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:13 and a monomer set forth in SEQ ID NO:7.

[0381] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:31 and a monomer comprising SEQ ID NO:7.

[0382] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:31 and a monomer set forth in SEQ ID NO:7.

[0383] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:33 and a monomer comprising SEQ ID NO:7.

[0384] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:33 and a monomer set forth in SEQ ID NO:7.

[0385] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:146 and a monomer comprising SEQ ID NO:148.

[0386] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:146 and a monomer set forth in SEQ ID NO:148.

[0387] According to certain embodiments, the heterodimer comprises a SIRPα polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1 or 5. and monomers comprising a TIGIT polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 35 or 36.

[0388] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:1 and a monomer comprising SEQ ID NO:35.

[0389] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:1 and a monomer set forth in SEQ ID NO:35.

[0390] According to a particular embodiment, the heterodimer comprises a monomer comprising SEQ ID NO:5 and a monomer comprising SEQ ID NO:36.

[0391] According to a particular embodiment, the heterodimer comprises a monomer set forth in SEQ ID NO:5 and a monomer set forth in SEQ ID NO:36.

[0392] According to certain embodiments, the heterodimers disclosed herein are soluble (ie, not immobilized on a synthetic or naturally occurring surface).

[0393] According to certain embodiments, the heterodimers disclosed herein are immobilized on a synthetic or naturally occurring surface.

[0394] According to additional or alternative aspects of the present invention, there is provided a composition comprising a heterodimer disclosed herein, wherein the heterodimer is the predominant form of the two polypeptides in said composition.

[0395] Methods for determining dimerization, and in particular heterodimerization, are well known in the art and are further described above and below.

[0396] According to certain embodiments, the predominant form accounts for at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%, with each possibility representing a separate embodiment of the present invention.

[0397] According to certain embodiments, the manufacturing yield, stability, activity, selectivity and / or safety of a heterodimer described herein or a composition comprising same is greater than the manufacturing yield, stability, activity, selectivity and / or safety of a composition comprising a homodimer comprising the same two polypeptides, the homodimer being the predominant form of the two polypeptides in the composition, and the isolated monomer comprises the same two polypeptides and / or each of the two polypeptides as a single agent.

[0398] According to certain embodiments, the production yield, stability, activity, selectivity and / or safety of the heterodimers described herein, or compositions comprising same, is greater than the production yield, stability, activity, selectivity and / or safety of antibodies, e.g., bispecific antibodies, that target the natural binding partners of the two polypeptides described herein.

[0399] According to certain embodiments, increased selectivity and / or safety may be manifested by selective activity only when the heterodimer binds to the natural binding partners of both polypeptides (e.g., cells expressing the natural binding partners of both polypeptides of the heterodimer compared to cells expressing only one of the natural binding partners). In certain embodiments, where the dimerization moiety is an Fc domain, selectivity and / or safety may be manifested by binding and / or activation of Fc receptors only when the heterodimer binds to the natural binding partners of both polypeptides.

[0400] According to certain embodiments, the term "higher" refers to a statistically significant increase.

[0401] According to certain embodiments, the term "higher" refers to an increase of at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, or at least 5-fold.

[0402] According to certain embodiments, the heterodimers described herein or compositions comprising same have improved combined activity compared to each of the two polypeptides as single agents. As used herein, the phrase "improved combined activity" refers to at least additive, but preferably synergistically improved activity.

[0403] According to certain embodiments, the amount of aggregates of the heterodimers described herein or compositions comprising same is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% less than the amount of aggregates of a composition comprising a homodimer comprising the same two polypeptides, wherein the homodimer is the predominant form of the two polypeptides in the composition, and the isolated monomer comprises the same two polypeptides and / or each of the two polypeptides as a single agent.

[0404] Because the heterodimers of some embodiments of the present invention comprise two polypeptides selected from SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10, the heterodimers may be used in methods of activating immune cells in-vitro, ex-vivo and / or in-vivo.

[0405] Thus, according to one aspect of the present invention there is provided a method of activating an immune cell comprising activating the immune cell in vitro in the presence of the heterodimer, a composition comprising same, a nucleic acid construct or system encoding same, or a host cell comprising same.

[0406] According to certain embodiments, the immune cells comprise peripheral blood mononuclear cells (PBMCs).

[0407] As used herein, the term "peripheral blood mononuclear cells (PBMCs)" refers to mononuclear blood cells, and includes lymphocytes, monocytes and dendritic cells (DCs).

[0408] According to certain embodiments, the PBMCs are selected from the group consisting of dendritic cells (DCs), T cells, B cells, NK cells and NKT cells.

[0409] According to certain embodiments, the PBMCs comprise T cells, B cells, NK cells and NKT cells.

[0410] Methods for obtaining PBMCs are well known in the art, such as drawing whole blood from a subject and collecting it in a container containing an anticoagulant (e.g., heparin or citrate) and apheresis. Then, according to certain embodiments, at least one type of PBMCs is purified from the peripheral blood. There are several methods and reagents known to those skilled in the art for purifying PBMCs from whole blood, such as leukapheresis, sedimentation, density gradient centrifugation (e.g., ficoll), centrifugal elutriation, fractionation, such as chemical lysis of red blood cells (e.g., by ACK), selection of specific cell types using cell surface markers (e.g., using FACS sorters or magnetic cell separation technology available from Invitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, Miltenyi Biotec, etc.) and methods for removing specific cell types based on eradication (e.g., killing) using specific antibodies, and affinity-based purification by negative selection (e.g., using magnetic cell separation technology, FACS sorters and / or capture ELISA labeling). Such methods are described, for example, in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes 1 to 4, (DN Weir, editor) and FLOW CYTOMETRY AND CELL SORTING (A. Radbruch, editor, Springer Verlag, 2000).

[0411] According to certain embodiments, the immune cells comprise tumor infiltrating lymphocytes.

[0412] As used herein, the term "tumor infiltrating lymphocytes (TILs)" refers to mononuclear leukocytes that have migrated away from the bloodstream and into a tumor.

[0413] According to certain embodiments, the TILs are selected from the group consisting of T cells, B cells, NK cells and monocytes.

[0414] Methods for obtaining TILs are well known in the art, such as obtaining a tumor sample from a subject by biopsy or autopsy and preparing a single cell suspension thereof. The single cell suspension can be obtained in any suitable manner, such as mechanical (e.g., disaggregation of tumors using a GentleMACS™ Dissociator (Miltenyi Biotec, Auburn, Calif.)) or enzymatic (e.g., collagenase or DNase). At least one type of TIL can then be purified from this cell suspension. To purify the desired type of TILs, there are several methods and reagents known to those skilled in the art, such as selection of specific cell types using cell surface markers (e.g., using a FACS sorter or magnetic cell separation technology available from Invitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, Miltenyi Biotec, etc.) and removal of specific cell types based on eradication (e.g., killing) using specific antibodies, and affinity-based purification by negative selection (e.g., using magnetic cell separation technology, FACS sorter, and / or capture ELISA labeling). Such methods are described, for example, in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes 1 to 4, (DN Weir, editor) and FLOW CYTOMETRY AND CELL SORTING (A. Radbruch, editor, Springer Verlag, 2000).

[0415] According to certain embodiments, the immune cells include phagocytes.

[0416] As used herein, the term "phagocyte" refers to a cell capable of performing phagocytosis, and includes both professional and non-professional phagocytes. Methods for analyzing phagocytosis are well known in the art and include, for example, killing assays, flow cytometry and / or microscopic evaluation (live cell imaging, fluorescence microscopy, confocal microscopy, electron microscopy). According to certain embodiments, the phagocyte is selected from the group consisting of monocytes, dendritic cells (DCs) and granulocytes.

[0417] According to certain embodiments, the phagocytes include granulocytes.

[0418] According to certain embodiments, the phagocytes include monocytes.

[0419] According to certain embodiments, the immune cells comprise monocytes.

[0420] According to certain embodiments, the term "monocytes" refers to both circulating monocytes and macrophages (also known as mononuclear phagocytes) present in tissues.

[0421] According to certain embodiments, the monocytes comprise macrophages, whose cell surface phenotype typically comprises CD14, CD40, CD11b, CD64, F4 / 80 (mouse) / EMR1 (human), lysozyme M, MAC-1 / MAC-3 and CD68.

[0422] According to certain embodiments, the monocytes comprise circulating monocytes. Typically, the cell surface phenotype of circulating monocytes comprises CD14 and CD16 (e.g., CD14++CD16-, CD14+CD16++, CD14++CD16+).

[0423] According to a particular embodiment, the immune cells comprise DCs.

[0424] As used herein, the term "dendritic cell (DC)" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. DCs are a class of professional antigen-presenting cells with a high capacity to sensitize HLA-restricted T cells. DCs include, for example, plasmacytoid dendritic cells, myeloid dendritic cells (including immature and mature dendritic cells), Langerhans cells, interdigitating cells, and follicular dendritic cells. Dendritic cells can be recognized by function or phenotype, particularly cell surface phenotype. These cells are characterized by a distinctive morphology with veil-like projections on the cell surface, intermediate to high levels of surface HLA class II expression, and the ability to present antigen to T cells, particularly naive T cells (see Steinman R, et al., Ann. Rev. Immunol. 1991; 9:271-196). Typically, the cell surface phenotype of DC includes CD1a+, CD4+, CD86+ or HLA-DR. The term DC includes both immature and mature DC.

[0425] According to certain embodiments, the immune cells include granulocytes.

[0426] As used herein, the term "granulocyte" refers to a polymorphonuclear leukocyte, characterized by the presence of granules within the cytoplasm.

[0427] According to certain embodiments, the granulocytes include neutrophils.

[0428] According to certain embodiments, the granulocytes comprise mast cells.

[0429] According to certain embodiments, the immune cells comprise T cells.

[0430] As used herein, the term "T cell" refers to a CD3+, T cell receptor (TCR)+ differentiated lymphocyte with either a CD4+ or CD8+ phenotype. T cells can be either effector T cells or regulatory T cells.

[0431] As used herein, the term "effector T cell" refers to a T cell that activates or directs other immune cells, e.g., by producing cytokines, or a T cell that has cytotoxic activity, e.g., CD4+, Th1 / Th2, CD8+ cytotoxic T lymphocytes.

[0432] As used herein, the term "regulatory T cells" or "Treg" refers to T cells that negatively regulate the activation of other T cells, including effector T cells, and cells of the innate immune system. Treg cells are characterized by sustained suppression of effector T cell responses. According to certain embodiments, Treg are CD4+CD25+Foxp3+ T cells.

[0433] According to a particular embodiment, the T cells are CD4+ T cells.

[0434] According to other particular embodiments, the T cells are CD8+ T cells.

[0435] According to certain embodiments, the T cells are memory T cells. Non-limiting examples of memory T cells include effector memory CD4+ T cells with a phenotype of CD3+ / CD4+ / CD45RA- / CCR7-, central memory CD4+ T cells with a phenotype of CD3+ / CD4+ / CD45RA- / CCR7+, effector memory CD8+ T cells with a phenotype of CD3+ / CD8+CD45RA- / CCR7-, and central memory CD8+ T cells with a phenotype of CD3+ / CD8+CD45RA- / CCR7+.

[0436] According to certain embodiments, the T cells include engineered T cells transduced with a nucleic acid sequence encoding an expression product of interest.

[0437] According to particular embodiments, the expression product of interest is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0438] As used herein, the phrases "transduced with a nucleic acid sequence encoding a TCR" or "transducing a nucleic acid sequence encoding a TCR" refer to cloning the variable α- and β-chains from a T cell with specificity for a desired antigen presented in the context of MHC. Methods for transducing TCRs are known in the art and are disclosed, for example, in Nicholson et al. Adv Hematol. 2012; 2012:404081, Wang and Riviere Cancer Gene Ther. 2015 Mar;22 (2):85-94) and Lamers et al, Cancer Gene Therapy (2002) 9, 613-623.

[0439] As used herein, the phrase "transduced with a nucleic acid sequence encoding a CAR" or "transducing a nucleic acid sequence encoding a CAR" refers to the cloning of a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen recognition portion and a T cell activation portion. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains an antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to a T cell signaling domain or a T cell activation domain. Methods for transducing CARs are known in the art and have been disclosed, for example, in Davila et al. Oncoimmunology. 2012 Dec 1;1 (9):1577-1583, Wang and Riviere Cancer Gene Ther. 2015 Mar;22 (2):85-94), Maus et al. Blood. 2014 Apr 24;123 (17):2625-35, Porter DL The New England journal of medicine. 2011, 365 (8):725-733, Jackson HJ, Nat Rev Clin Oncol. 2016;13 (6):370-383, and Globerson-Levin et al. Mol Ther. 2014;22 (5):1029-1038.

[0440] According to certain embodiments, the immune cells comprise B cells.

[0441] As used herein, the term "B cell" refers to a lymphocyte with a B cell receptor (BCR)+, CD19+ and / or B220+ phenotype. B cells are characterized by their ability to bind to a specific antigen and elicit a humoral response.

[0442] According to certain embodiments, the immune cells comprise NK cells.

[0443] As used herein, the term "NK cells" refers to differentiated lymphocytes with a CD16+CD56+ and / or CD57+TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that fail to express "self" MHC / HLA antigens by activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0444] According to certain embodiments, the immune cells comprise NKT cells.

[0445] As used herein, the term "NKT cells" refers to a specialized T cell population that expresses the semi-invariant αβT cell receptor, but also expresses various molecular markers typically associated with NK cells (e.g., NK1.1). NKT cells include NK1.1+ and NK1.1- as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR of NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC-I-like molecule CD1d. NKT cells can have protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance.

[0446] According to certain embodiments, the immune cells are obtained from a healthy subject.

[0447] According to certain embodiments, the immune cells are obtained from a subject suffering from a disease (eg, cancer).

[0448] According to a particular embodiment, the activation is in the presence of a cell expressing at least one natural binding partner of the two polypeptides or at least one exogenous binding partner of the two polypeptides.

[0449] According to a particular embodiment, the activation is in the presence of a natural binding partner of the two polypeptides or a cell expressing an exogenous binding partner of the two polypeptides.

[0450] According to certain embodiments, the exogenous binding partner is soluble.

[0451] According to other particular embodiments, the exogenous binding partner is immobilized on a solid support.

[0452] According to certain embodiments, the cells expressing the binding partner include pathological (diseased) cells, such as cancer cells.

[0453] According to certain embodiments, activation is performed in the presence of a stimulatory agent capable of delivering at least a primary activation signal (e.g., ligation of a major histocompatibility complex (MHC) / peptide complex with a T cell receptor (TCR) on an antigen presenting cell (APC)) that leads to cell proliferation, maturation, cytokine production, phagocytosis and / or induction of immune cell regulatory or effector functions. According to certain embodiments, the stimulatory agent is also capable of delivering a secondary co-stimulatory signal.

[0454] The method for determining the amount of stimulating agent and the ratio between stimulating agent and immune cells is well within the capabilities of a person skilled in the art and will not be specified herein.

[0455] Stimulatory agents can activate immune cells in an antigen-dependent or antigen-independent (ie, polyclonal) manner.

[0456] According to certain embodiments, the stimulating agent comprises an antigen-non-specific stimulating factor.

[0457] Non-specific stimulatory factors are known to those skilled in the art. Thus, when the immune cells include T cells, non-limiting examples of antigen non-specific stimulatory factors can be agents capable of binding to T cell surface structures and inducing polyclonal stimulation of T cells, such as, but not limited to, anti-CD3 antibodies in combination with costimulatory proteins, such as anti-CD28 antibodies. Other non-limiting examples include anti-CD2, anti-CD137, anti-CD134, Notch ligands (e.g., Delta-like 1 / 4), Jagged 1 / 2, alone or in various combinations with anti-CD3. Other agents capable of inducing polyclonal stimulation of T cells include, but are not limited to, mitogens, PHA, PMA-ionomycin, CEB, and CytoStim (Miltenyi Biotech). According to certain embodiments, the antigen non-specific stimulatory factors include anti-CD3 antibodies and anti-CD28 antibodies. According to certain embodiments, the T cell stimulator comprises anti-CD3 and anti-CD28 coated beads, such as CD3CD28 MACSiBeads available from Miltenyi Biotec.

[0458] According to certain embodiments, the stimulating agent comprises an antigen-specific stimulating factor.

[0459] Non-limiting examples of antigen-specific T cell stimulators include antigen-loaded antigen-presenting cells (APCs, e.g., dendritic cells) and peptide-loaded recombinant MHC. Thus, for example, the T cell stimulator can be a dendritic cell preloaded with a desired antigen (e.g., a tumor antigen) or transfected with mRNA encoding the desired antigen.

[0460] According to a particular embodiment, the antigen is a cancer antigen.

[0461] As used herein, the term "cancer antigen" refers to an antigen that is overexpressed or exclusively expressed by cancerous cells compared to non-cancerous cells. Cancer antigens can be known cancer antigens or new specific antigens (i.e., neo-antigens) that arise in cancer cells.

[0462] Non-limiting examples of known cancer antigens include MAGE-AI, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE-AIO, MAGE-All, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAG E-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl / CT7, MAGE-C2, NY-ES0-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and XAGE, melanocyte differentiation antigen, p53, ras, CEA, MUCI, PMSA, PSA, tyrosinase, Melan A, MART-I, gplOO, gp75, alpha actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn 2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-All, hsp70-2, KIAA0205, Mart2, Mum-2 and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17 and TRP2-Int2, (MART-I ), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, plSOerbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29 / BCAA), CA195, CA242, CA-50, CAM43, CD68 / KP1, C0-029, FGF-5, 0250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 170K, NYCO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, tyrosinase-related protein, TRP-1 or TRP-2.

[0463] Other tumor antigens that can be expressed are known in the art (see, for example, WO 00 / 20581, Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge). The sequences of these tumor antigens are readily available from public databases, but can also be found in WO 1992 / 020356 A1, WO 1994 / 005304 A1, WO 1994 / 023031 A1, WO 1995 / 020974 A1, WO 1995 / 023874 A1 and WO 1996 / 026214 A1.

[0464] Alternatively or additionally, tumor antigens can be identified using cancer cells obtained from the subject, for example by biopsy.

[0465] Thus, according to certain embodiments, the stimulatory agent comprises cancer cells.

[0466] According to certain embodiments, the activation is carried out in the presence of an anti-cancer drug.

[0467] According to certain embodiments, the immune cells are purified after activation.

[0468] Thus, the present invention also contemplates isolated immune cells obtainable by the methods of the present invention.

[0469] According to certain embodiments, the immune cells used and / or obtained in the present invention may be freshly isolated, stored (e.g., cryopreserved) (i.e., frozen) at any stage, e.g., at liquid nitrogen temperatures, for extended periods (e.g., months, years) for future use, and cell lines.

[0470] Methods of cryopreservation are widely known to those of skill in the art and are disclosed, for example, in WO 2007 / 054160 and WO 2001 / 039594 and U.S. Patent Application Publication No. 2012 / 0149108.

[0471] According to certain embodiments, the cells obtained according to the invention may be stored in a cell bank, or repository, or storage facility.

[0472] As a result, the present teachings further suggest, but are not limited to, uses and methods of the isolated immune cells of the present invention as a source of adoptive immune cell therapy for diseases in which activation of immune cells can be effective (e.g., hyperproliferative diseases), as well as diseases associated with immunosuppression and infectious diseases.

[0473] Thus, according to certain embodiments, the methods of the invention comprise adoptively transferring immune cells after activation to a subject in need thereof.

[0474] According to a particular embodiment, immune cells obtainable by the method of the invention are provided for use in adoptive cell therapy.

[0475] Cells used in certain embodiments of the invention may be autologous or non-autologous, syngeneic or non-syngeneic, and allogeneic or xenogeneic to the subject, with each possibility representing a separate embodiment of the present invention.

[0476] The present teachings also contemplate the use of the compositions of the invention (e.g., the heterodimers, compositions comprising same, nucleic acid constructs or systems encoding same, or host cells expressing same) in methods of treating diseases in which heterodimer treatment may be effective.

[0477] Thus, according to one aspect of the present invention, there is provided a method for treating a disease in which treatment with a heterodimer can be effective, comprising administering to a subject in need thereof a heterodimer, a composition comprising the same, a nucleic acid construct or system encoding the same, or a host cell comprising the same, thereby treating the disease in the subject.

[0478] According to further or alternative aspects of the present invention, there is provided a heterodimer, a composition comprising same, a nucleic acid construct or system encoding same, or a host cell comprising same, for use in treating a disease in which heterodimer treatment may be effective.

[0479] The term "treating" or "treatment" refers to inhibiting, preventing or arresting the onset of a condition (disease, disorder or medical condition) and / or causing a reduction, amelioration or relief of a condition or a symptom of a condition. Those skilled in the art will understand that a variety of methodologies and assays can be used to assess the onset of a condition, and similarly a variety of methodologies and assays can be used to assess a reduction, amelioration or relief of a condition.

[0480] As used herein, the term "subject" includes mammals of any age and any sex, such as humans. According to certain embodiments, the term "subject" refers to a subject suffering from a condition (disease, disorder, or medical condition). According to certain embodiments, the term includes individuals at risk of developing the condition.

[0481] According to certain embodiments, cells associated with the disease (eg, cancer cells) express at least one natural binding partner of the two polypeptides.

[0482] According to certain embodiments, cells associated with the disease (eg, cancer cells) express natural binding partners of the two polypeptides.

[0483] According to certain embodiments, the disease may benefit from activation of immune cells.

[0484] As used herein, the phrase "disease that may benefit from immune cell activation" refers to a disease in which the activity of a subject's immune response may be sufficient to at least ameliorate or delay the onset of symptoms of the disease, but in which the activity of the subject's immune response to do so is, for some reason, less than optimal.

[0485] Non-limiting examples of diseases in which immune cell activation may be effective include hyperproliferative diseases, diseases associated with immunosuppression, immunosuppression caused by drugs (e.g., mTOR inhibitors, calcineurin inhibitors, steroids), and infectious diseases.

[0486] According to certain embodiments, the disease comprises a hyperproliferative disease.

[0487] According to certain embodiments, the hyperproliferative disease comprises sclerosis, fibrosis, idiopathic pulmonary fibrosis, psoriasis, systemic sclerosis / scleroderma, primary biliary cholangitis, primary sclerosing cholangitis, hepatic fibrosis, prevention of radiation-induced pulmonary fibrosis, myelofibrosis, or retroperitoneal fibrosis.

[0488] According to other particular embodiments, the hyperproliferative disease comprises cancer.

[0489] As used herein, the term cancer includes both malignant and pre-malignant cancers.

[0490] With respect to pre-malignant or benign forms of cancer, the compositions and methods can optionally be applied to halt the progression of pre-malignant cancer to a malignant form.

[0491] The cancer that may be treated by the methods of some embodiments of the present invention may be any solid or non-solid cancer and / or metastatic cancer.

[0492] According to certain embodiments, the cancer comprises a malignant cancer.

[0493] Cancers that can be treated by the methods of some embodiments of the present invention are any solid or non-solid cancer and / or metastatic cancer. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer) (gastric cancer or stomach cancer), and pulmonary cancer. cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), high-grade lymphoblastic NHL, high-grade small non-resectable These include: infiltrated cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia), T-cell lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), hairy cell leukemia, chronic myeloblastic leukemia (CML) and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation, edema (e.g. associated with brain tumors) and Meigs' syndrome associated with phacomatosis.

[0494] According to certain embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Cancerous conditions suitable for treatment according to the present invention include metastatic cancer.

[0495] According to certain embodiments, the cancer comprises a pre-malignant cancer.

[0496] Premalignant cancers (or precancers) are well characterized and known in the art (see, e.g., Berman JJ. and Henson DE., 2003. Classifying the precancers: a metadata approach. BMC Med Inform Decis Mak. 3:8). Classes of premalignant cancers suitable for treatment by the methods of the present invention include acquired small or microscopic premalignant cancers, acquired large lesions with nuclear atypia, precursor lesions occurring with hereditary hyperplastic syndromes that progress to cancer, and acquired diffuse hyperplasia and diffuse dysplasia. Examples of small or microscopic premalignant cancers include HGSIL (high-grade squamous intraepithelial lesions of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, aberrant crypt foci (of the colon), and PIN (prostatic intraepithelial neoplasia). Examples of acquired large lesions with nuclear atypia include tubular adenoma, AILD (angioimmunoblastic lymphadenopathy with dysproteinemia), atypical meningioma, gastric polyp, large plaque-type psoriasis, myelodysplasia, papillary transitional intraepithelial carcinoma, refractory anemia with excess blast cells, and Schneider's papilloma. Examples of precursor lesions that occur with inherited hyperplastic syndromes that progress to cancer include atypical mole syndrome, C cell adenomatosis, and MEA. Examples of acquired diffuse hyperplasia and diffuse dysplasia include AIDS, atypical lymphoproliferation, Paget's disease of bone, post-transplant lymphoproliferative disease, and ulcerative colitis.

[0497] According to certain embodiments, the cancer is acute myeloid leukemia, anal cancer, basal cell carcinoma, B-cell non-Hodgkin's lymphoma, bile duct cancer, bladder cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fallopian tube cancer, follicular lymphoma, gastric cancer, esophagogastric (GE) junction carcinoma, germ cell tumor, germinoma (seminomatosis), germ cell tumor, glioblastoma multiforme (GBM), gliosarcoma, head and neck cancer, hepatocellular carcinoma. , Hodgkin's lymphoma, hypopharyngeal cancer, laryngeal cancer, leiomyosarcoma, mantle cell lymphoma, melanoma, Merkel cell carcinoma, multiple melanoma, neuroendocrine tumors, non-Hodgkin's lymphoma, non-small cell lung cancer, oral (mouth) cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, peripheral nerve sheath tumor (neurofibrosarcoma), peripheral T-cell lymphoma (PTCL), peritoneal cancer, prostate cancer, renal cell carcinoma, salivary gland cancer, skin cancer, small cell lung cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic carcinoma, thyroid cancer, ureteral cancer, urethral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0498] According to certain embodiments, the cancer is acute myeloid leukemia, bladder cancer, breast cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, diffuse large B-cell lymphoma, ovarian epithelial cancer, epithelial tumors, fallopian tube cancer, follicular lymphoma, glioblastoma multiforme, hepatocellular carcinoma, head and neck cancer, leukemia, lymphoma, mantle cell lymphoma, melanoma, mesothelioma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cell carcinoma.

[0499] According to certain embodiments, the cancer is selected from the group consisting of lymphoma, leukemia and carcinoma (eg, colon cancer, ovarian cancer, lung cancer, head and neck cancer, hepatocellular carcinoma).

[0500] According to certain embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0501] According to certain embodiments, the cancer is mesothelioma (e.g., malignant pleural mesothelioma (MPM)).

[0502] According to certain embodiments, the leukemia is selected from the group consisting of acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonic cell leukemia, eosinophilic leukemia, gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, The present invention is directed to a method for treating or preventing a myeloma, comprising administering to a patient a therapeutically effective amount of ...

[0503] According to certain embodiments, the leukemia is promyelocytic leukemia, acute myeloid leukemia, or chronic myeloid leukemia.

[0504] According to certain embodiments, the cancer is lymphoma.

[0505] According to certain embodiments, the lymphoma is a B-cell lymphoma, a T-cell lymphoma, a Hodgkin's lymphoma or a non-Hodgkin's lymphoma.

[0506] According to certain embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of aggressive NHL, transformed NHL, indolent NHL, relapsed NHL, refractory NHL, low-grade non-Hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, B cell lymphoma, T cell lymphoma, mantle cell lymphoma, Burkitt lymphoma, NK cell lymphoma, diffuse large B cell lymphoma, acute lymphoblastic lymphoma, and cutaneous T cell carcinomas including mycosis fungoides / Sezry syndrome.

[0507] According to a particular embodiment, the cancer is multiple myeloma.

[0508] According to at least some embodiments, the multiple myeloma is selected from the group consisting of multiple myeloma cancers that produce kappa and / or lambda light chains, aggressive multiple myeloma including primary plasma cell leukemia (PCL), benign plasma cell disorders such as MGUS (monoclonal gammopathy) that can progress to multiple myeloma, Waldenstrom's macroglobulinemia (WM, also known as lymphoplasmacytic lymphoma), smoldering multiple myeloma (SMM), indolent multiple myeloma, pre-malignant forms of multiple myeloma that can also progress to multiple myeloma, and primary amyloidosis.

[0509] According to certain embodiments, the cancer is defined by the presence of tumors that have tumor infiltrating lymphocytes (TILs) in the tumor microenvironment and / or tumors with relatively high expression of natural binding partners in the tumor microenvironment.

[0510] According to certain embodiments, the disease comprises a disease associated with immunosuppression or immunosuppression caused by a drug (e.g., mTOR inhibitors, calcineurin inhibitors, steroids).

[0511] According to certain embodiments, the disease comprises that caused by HIV, measles, influenza, LCCM, RSV, human rhinovirus, EBV, CMV or parvovirus.

[0512] According to certain embodiments, the disease comprises an infectious disease.

[0513] As used herein, the term "infectious disease" or "infectious disease" refers to a disease caused by a pathogen. Particular examples of pathogens include viral pathogens, bacterial pathogens, such as intracellular mycobacterial pathogens (e.g., Mycobacterium tuberculosis), intracellular bacterial pathogens (e.g., Listeria monocytogenes), or intracellular protozoan pathogens (e.g., Leishmania and Trypanosoma).

[0514] Particular types of viral pathogens causing infectious diseases treatable according to the teachings of the present invention include, but are not limited to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, and filoviruses.

[0515] Specific examples of viral infections that may be treated in accordance with the teachings of the present invention include, but are not limited to, human immunodeficiency virus (HIV)-induced acquired immune deficiency syndrome (AIDS), influenza, rhinovirus infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B or C virus infection, measles, papillomavirus infection / warts, cytomegalovirus (CMV) infection, herpes simplex virus infection, yellow fever, Ebola virus infection, rabies, and the like.

[0516] According to certain embodiments, the compositions disclosed herein (e.g., the heterodimer, a composition comprising same, a nucleic acid construct or system encoding same, and / or a host cell expressing same) can be administered to a subject in combination with other established or experimental therapeutic regimens for treating a disease, including, but not limited to, analgesics, chemotherapeutic agents, radiotherapeutic agents, cytotoxic therapy (conditioning), hormonal therapy, antibodies, and other therapeutic regimens known in the art (e.g., surgery).

[0517] According to certain embodiments, the therapeutic agent administered in combination with the compositions of some embodiments of the present invention comprises an antibody.

[0518] According to certain embodiments, the compositions disclosed herein (e.g., the heterodimer, compositions comprising same, nucleic acid constructs encoding same and / or host cells expressing same) can be administered to a subject in combination with adoptive cell transfer, such as, but not limited to, transplantation of bone marrow cells, hematopoietic stem cells, PBMCs, umbilical cord blood stem cells and / or induced pluripotent stem cells.

[0519] According to certain embodiments, the therapeutic agent administered in combination with the compositions of some embodiments comprises an anti-cancer agent.

[0520] According to certain embodiments, therapeutic agents administered in combination with the compositions of some embodiments include anti-infective agents (eg, antibiotics and antivirals).

[0521] According to certain embodiments, therapeutic agents administered in combination with the compositions of some embodiments include immunosuppressants (eg, GCSF and other bone marrow stimulating factors, steroids).

[0522] According to certain embodiments, the combination therapy has an additive effect.

[0523] According to certain embodiments, the combination therapy has a synergistic effect.

[0524] According to another aspect of the present invention, there is provided an article of manufacture comprising packaging material for packaging a therapeutic agent for treating a disease, and the heterodimer, a composition comprising same, a nucleic acid construct or system encoding same, or a host cell comprising same.

[0525] In certain embodiments, the article is identified for the treatment of a disease that may benefit from treatment with the heterodimer, for example a disease that may benefit from activation of immune cells.

[0526] According to certain embodiments, the therapeutic agent for treating a disease and the heterodimer, a composition comprising same, a nucleic acid construct or system encoding same, or a host cell expressing same are contained in separate containers.

[0527] In certain embodiments, the therapeutic agent for treating the disease and the heterodimer, a composition comprising it, a nucleic acid construct or system encoding it, or a host cell expressing it are contained as a combination preparation.

[0528] As used herein, the terms "amino acid sequence", "protein", "peptide", "polypeptide" and "proteinaceous moiety" are used interchangeably herein and include native peptides (either degradation products, artificially synthesized peptides or recombinant peptides) and peptidomimetics (generally artificially synthesized peptides) as well as peptoids and semipeptoids, which are peptide analogs that may have modifications that render the peptide more stable in the body or more permeable into cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details on this are provided below in this specification.

[0529] The peptide bond (-CO-NH-) in a peptide can be, for example, an N-methylated bond (-N(CH3)-CO-), an ester bond (-C(R)HCOOC(R)-N-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), an α-aza bond (-NH-N(R)-CO-) (wherein R is any alkyl, e.g., methyl), an amine bond (-CH2-NH-), a sulfide bond (- The aryl group may be substituted by an aryl group such as aryl, aryl, or aryl groups, such as aryl, aryl, or aryl groups. The aryl group may be substituted by an aryl group such as aryl, aryl, or aryl groups.

[0530] These modifications can occur at any bond along the peptide chain and can even occur simultaneously at several (2-3) bonds.

[0531] The natural aromatic amino acids Trp, Tyr and Phe can be replaced with synthetic non-natural acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylamine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.

[0532] Peptides in some embodiments of the invention may also contain one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0533] The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids, amino acids that are frequently post-translationally modified in vivo, such as hydroxyproline, phosphoserine, and phosphothreonine, and other uncommon amino acids, such as, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Additionally, the term "amino acid" includes both D- and L-amino acids.

[0534] Tables 2 and 3 below list natural amino acids (Table 2) and unconventional or modified amino acids (eg, synthetic, Table 3) that can be used in some embodiments of the invention.

[0535] [Table 1]

[0536] [Table 2-1]

[0537] [Table 2-2]

[0538] [Table 2-3]

[0539] [Table 2-4]

[0540] The polypeptides of some embodiments of the present invention are preferably used in a linear form, although it will be understood that cyclic forms of the peptides may be used where cyclization does not significantly interfere with the characteristics of the peptide.

[0541] Because the heterodimers of the present invention are preferably used in therapies that require the heterodimer to be in a soluble form, the polypeptides in some embodiments of the present invention contain one or more unnatural or natural polar amino acids, including, but not limited to, serine and threonine, whose hydroxyl-containing side chains can increase the solubility of the peptide.

[0542] Amino acids in the polypeptides of the present invention may be substituted in a conservative or non-conservative manner.

[0543] The term "conservative substitution" as used herein refers to the replacement of an amino acid present in a native sequence in a peptide with a natural or non-natural amino acid or peptidomimetic having similar steric properties. If the side chain of the natural amino acid to be replaced is polar or hydrophobic, the conservative substitution should be with a natural amino acid, non-natural amino acid or peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the substituted amino acid).

[0544] Conservative substitutions with natural amino acids can be easily determined since natural amino acids are generally classified according to their properties and, according to the present invention, the substitution of a charged amino acid with a sterically similar uncharged amino acid is considered to be a conservative substitution.

[0545] It is also possible to use amino acid analogs (synthetic amino acids) well known in the art to make conservative substitutions with non-natural amino acids. Peptide mimetics of natural amino acids are well described in the literature known to those skilled in the art.

[0546] When making a conservative substitution, the substituting amino acid should have the same or a similar substituent in the side chain as the original amino acid.

[0547] Conservative substitution tables providing functionally similar amino acids are well known in the art. Guidance regarding which amino acid changes are likely to be phenotypically silent can also be found in Bowie et al., 1990, Science 247: 1306 1310. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. Exemplary conservative substitutions include, but are not limited to, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids can be substituted based on properties associated with their side chains. For example, substitutions of amino acids with polar side chains, such as serine (S) and threonine (T), amino acids based on the charge of the side chain, such as arginine (R) and histidine (H), and amino acids with hydrophobic side chains, such as valine (V) and leucine (L) are possible. As indicated, typically the changes are minor in nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.

[0548] The phrase "non-conservative substitution" as used herein refers to the replacement of an amino acid present in the parent sequence with another natural or non-natural amino acid having different electrochemical and / or steric properties. Thus, the side chain of the substituting amino acid may have a functional group that is significantly larger (or smaller) than the side chain of the natural amino acid being replaced and / or has significantly different electronic properties compared to the amino acid being replaced. Examples of this type of non-conservative substitution include the replacement of alanine with phenylalanine or cyclohexylmethylglycine, glycine with isoleucine, or aspartic acid with -NH-CH[(-CH2)5-COOH]-CO-. These non-conservative substitutions that are within the scope of the present invention are those that still constitute a peptide with antibacterial properties.

[0549] The N- and C-termini of the peptides of the invention may be protected by functional groups. Suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate the transport of the compound attached thereto into cells, for example by reducing the hydrophilicity of the compound and increasing the lipophilicity of the compound.

[0550] In certain embodiments, one or more amino acids may be modified by the addition of a functional group, e.g., conceptually "chemically modified". For example, the side chain amino acid residues present in the native sequence may be optionally modified, but alternatively, as described below, other parts of the protein may be optionally modified in addition to or instead of the side chain amino acid residues. If a chemical synthesis process is followed, the modification may optionally be performed during the synthesis of the molecule, e.g., by adding a chemically modified amino acid. However, chemical modification of amino acids already present in the molecule ("in situ" modification) is also possible. Modifications to peptides or proteins may be introduced, for example, by gene synthesis, site-directed (e.g., PCR-based) mutagenesis by exonuclease deletion or random mutagenesis (e.g., EMS), chemical modification, or fusion of polynucleotide sequences encoding heterologous domains or binding proteins.

[0551] As used herein, the term "chemical modification" refers to a peptide in which at least one of its amino acid residues has been modified, either by natural processes such as processing or other post-translational modifications, or by chemical modification techniques known in the art. Non-limiting representative modifications include carboxymethylation, acetylation, acylation, phosphorylation, glycosylation, amidation, ADP-ribosylation, fatty acid acylation, farnesyl group, isofarnesyl group, carbohydrate group, fatty acid group, addition of linker for binding, functionalization, GPI anchor formation, covalent attachment of lipid or lipid derivative, methylation, myristylation, PEGylation, prenylation, phosphorylation, ubiquitination or any similar process as well as known protecting / blocking groups. Optionally, an ether bond may be used to link the hydroxyl of serine or threonine to the hydroxyl of a sugar. Amide bonds can optionally be used to link the carboxyl group of glutamate or aspartate to the amino group of a sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds can optionally be formed between amino acids and carbohydrates. For example, fatty acid acyl derivatives can optionally be made by acylation of free amino groups (e.g., lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).

[0552] According to certain embodiments, the modifications include the addition of cycloalkane moieties to the peptides, as described in International Application No. 2006 / 050262, which is incorporated by reference as if fully set forth herein. These moieties are designed for use with biomolecules and can optionally be used to confer various properties to the proteins.

[0553] In addition, any part of the peptide may be optionally modified. For example, PEGylation of glycosylated portions of the protein may be optionally performed, as described in International Application No. 2006 / 050247, which is incorporated by reference as if fully set forth herein. One or more polyethylene glycol (PEG) groups may be optionally added to O-linked and / or N-linked glycosylation. The PEG groups may be optionally branched or linear. Optionally, any type of water-soluble polymer may be attached to the glycosylation site of the protein via a glycosyl linker.

[0554] A "PEGylated protein" refers to a biologically active protein or fragment thereof having a polyethylene glycol (PEG) moiety covalently attached to an amino acid residue of the protein.

[0555] "Polyethylene glycol" or "PEG" refers to a polyalkylene glycol compound or derivative thereof, with or without derivatization with a coupling agent or coupling or activation moiety (e.g., with a thiol, triflate, tresylate, aziridine, oxirane, or preferably with a maleimide moiety). Compounds such as maleimide monomethoxy PEG are exemplary or activated PEG compounds of the invention. Other polyalkylene glycol compounds may be used in the invention, such as polypropylene glycol. Other suitable polyalkylene glycol compounds include, but are not limited to, the following types of charged or neutral polymers: dextran, colominic acid or other carbohydrate-based polymers, polymers of amino acids, and biotin derivatives.

[0556] According to certain embodiments, the peptides may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, "altered" refers to the deletion of one or more carbohydrate moieties and / or the addition of at least one glycosylation site to the original protein.

[0557] Glycosylation of proteins is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used).

[0558] Addition of glycosylation sites to a peptide is conveniently accomplished by altering the amino acid sequence of the peptide to contain one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by the addition of, or substitution by, one or more serine or threonine residues in the original peptide sequence (for O-linked glycosylation sites). The amino acid sequence of the peptide can also be altered by introducing changes at the DNA level.

[0559] Another means of increasing the number of carbohydrate moieties on a peptide is the chemical or enzymatic coupling of glycosides to amino acid residues of the peptide. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston, CRC Crit. Rev. Biochem., 22: 259-306 (1981).

[0560] Removal of any carbohydrate moieties present in a peptide can be accomplished chemically, enzymatically, or by introducing changes at the DNA level. Chemical deglycosylation requires exposure of the protein to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence unchanged.

[0561] Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem. Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins can be achieved by using a variety of endo- and exoglycosidases, as described in Thotakura et al., Meth. Enzymol., 138: 350 (1987).

[0562] The polypeptides of some embodiments of the present invention and heterodimers comprising same may be synthesized and purified by any technique known to those skilled in the art of peptide synthesis, including but not limited to solid phase techniques and recombinant techniques.

[0563] According to certain embodiments, the preparation of the polypeptides and / or heterodimers comprises solid phase peptide synthesis.

[0564] For solid phase peptide synthesis, an overview of many techniques can be found in JM Stewart and JD Young, Solid Phase Peptide Synthesis, WH Freeman Co. (San Francisco), 1963, and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For conventional solution synthesis, see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

[0565] In general, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to a growing peptide chain. Usually, the amino or carboxyl group of the first amino acid is protected with a suitable protecting group. The protected or derivatized amino acid is then added to the next amino acid in the sequence that has a suitably protected complementary (amino or carboxyl) group, either attached to an inert solid support or used in solution under conditions suitable for the formation of an amide bond. The protecting group is then removed from this newly added amino acid residue and the next (suitably protected) amino acid is added, and so on. Once all of the desired amino acids have been coupled in the correct order, the remaining protecting groups (and solid support) are removed sequentially or simultaneously to obtain the final peptide compound. Simple modifications of this general method are also possible, for example by coupling a protected tripeptide with a properly protected dipeptide (under conditions that do not racemize the chiral center) so that after deprotection a pentapeptide, etc. are formed, to add multiple amino acids to the growing chain at once. Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505.

[0566] Large scale peptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.

[0567] According to a particular embodiment, the polypeptide or a heterodimer comprising same is synthesized using an in vitro expression system.

[0568] Thus, any of the polypeptides described herein can be encoded from polynucleotides that can be used per se or can be used in the recombinant production of the polypeptides disclosed herein.

[0569] A "recombinant" polypeptide refers to a polypeptide produced by recombinant DNA techniques, ie, produced from cells transformed with an exogenous DNA construct encoding the desired polypeptide.

[0570] Thus, according to another aspect of the present invention there is provided a nucleic acid construct or system comprising at least one polynucleotide encoding the heterodimer and regulatory elements directing expression of the polynucleotide in a host cell.

[0571] Non-limiting examples of polynucleotide sequences that may be used with certain embodiments of the present invention are described above and in Table 1 below.

[0572] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0573] According to certain embodiments, any polynucleotide and nucleic acid sequence disclosed herein may have conservative nucleic acid substitutions. A conservatively modified polynucleotide refers to a nucleic acid that encodes the same or substantially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to a substantially identical or related (e.g., naturally contiguous) sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, this codon can be modified to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. All polynucleotide and nucleic acid sequences herein that encode a polypeptide also refer to silent variations of that nucleic acid. It will be recognized by those skilled in the art that, in certain contexts, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to result in a functionally identical molecule. Accordingly, silent variations of a nucleic acid which encodes a polypeptide are implicit in the described sequence of the expression product.

[0574] To express an exogenous polypeptide in a mammalian cell, a polynucleotide sequence encoding the polypeptide is preferably ligated into a nucleic acid construct suitable for expression in a mammalian cell, such a nucleic acid construct comprising a promoter sequence that directs transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0575] According to certain embodiments, the regulator is a heterologous regulator.

[0576] The nucleic acid constructs (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. By way of example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof.

[0577] The nucleic acid constructs of some embodiments of the invention typically include a signal sequence for secretion of the peptide from a host cell that the construct is contained in. Preferably, the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variant of some embodiments of the invention.

[0578] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and the upstream promoter element. The TATA box, located 25 to 30 base pairs upstream from the transcription initiation site, is thought to be involved in the initiation of RNA synthesis by RNA polymerase. The other upstream promoter element determines the frequency of transcription initiation.

[0579] Promoters used in the nucleic acid constructs of some embodiments of the present invention are preferably those that are active in the particular cell population transformed. Examples of cell type-specific and / or tissue-specific promoters include promoters such as albumin, which is liver-specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275], in particular promoters of T cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulin promoters [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters, such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreatic-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters, such as the whey promoter (U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166).

[0580] Enhancer elements can increase transcription from linked homologous or heterologous promoters up to 1,000-fold. Enhancers are active whether downstream or upstream from the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the invention include those derived from polyoma virus, human or mouse cytomegalovirus (CMV), and long terminal repeat sequences from various retroviruses, such as murine leukemia virus, mouse or Rous sarcoma virus, and HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1983, incorporated herein by reference.

[0581] In constructing an expression vector, a promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting, although, as is known in the art, some variation in this distance can be tolerated without loss of promoter function.

[0582] To increase the efficiency of mRNA translation, polyadenylation sequences can also be added to the expression vector. Accurate and efficient polyadenylation requires two characteristic sequence elements: a GU-rich or U-rich sequence located downstream of the polyadenylation site and a highly conserved six-nucleotide AAUAAA sequence located 11-30 nucleotides upstream. Termination and polyadenylation signals suitable for some embodiments of the invention include those derived from SV40.

[0583] In addition to the elements already described, the expression vectors of some embodiments of the present invention may contain other specialized elements, typically intended to increase the expression level of the cloned nucleic acid or to facilitate the identification of cells carrying recombinant DNA. For example, some animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying the replicons of such viruses are replicated episomally as long as the appropriate factors are provided by genes on the plasmid or on the genome of the host cell.

[0584] A vector may or may not contain a eukaryotic replicon. If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using a suitable selectable marker. If a vector does not contain a eukaryotic replicon, episomal amplification is not possible. Instead, recombinant DNA is integrated into the genome of engineered cells, and the promoter expresses the desired nucleic acid.

[0585] The expression vectors of some embodiments of the invention can further comprise additional polynucleotide sequences, such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide, for example, allowing translation of several proteins from a single mRNA.

[0586] Thus, according to certain embodiments, both monomers contained in the heterodimer are expressed from one construct.

[0587] According to other particular embodiments, each of the monomers comprised in the heterodimer is expressed from a different construct.

[0588] It will be understood that the individual elements contained in an expression vector may be arranged in a variety of configurations. For example, enhancer elements, promoters, etc., and even polynucleotide sequences encoding monomers of a heterodimer may be arranged in a "head to tail" configuration, may exist as reverse complements or complementary strands in antiparallel strands. Although such configurational variations are more likely to occur with non-coding elements of an expression vector, alternative configurations of coding sequences in an expression vector are also contemplated.

[0589] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech and derivatives thereof.

[0590] Expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses can also be used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters reported to be effective for expression in eukaryotic cells.

[0591] As mentioned above, viruses are highly specialized infectious agents that have often evolved to evade host defense mechanisms. Typically, viruses infect and spread to specific cell types. The targeting specificity of viral vectors introduces recombinant genes into infected cells by using natural specificity to specifically target a given cell type. Thus, the type of vector used by some embodiments of the present invention depends on the cell type to be transformed. The ability to select a suitable vector depending on the cell type to be transformed is well within the capabilities of those skilled in the art, and therefore no overview of selection considerations is provided herein. For example, bone marrow cells can be targeted using human T-cell leukemia virus type I (HTLV-I), and kidney cells can be targeted using a heterologous promoter present in the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51-60).

[0592] Recombinant viral vectors offer advantages such as horizontal infection and targeting specificity, making them useful for in vivo expression of monomers and heterodimers. Horizontal infection, for example, is inherent in the life cycle of retroviruses, and is a process in which a single infected cell buds and generates many progeny virions that infect neighboring cells. This results in rapid infection of a wide area, most of which were not initially infected by the original viral particle. This is in contrast to vertical infection, where infectious agents are only transmitted through daughter progeny. It is also possible to generate viral vectors that cannot be transmitted horizontally. This feature can be useful when the desired goal is to introduce a particular gene into only a limited number of target cells.

[0593] A variety of methods can be used to introduce the expression vectors of some embodiments of the present invention into cells. Such methods are reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see US Pat. Nos. 5,464,764 and 5,487,992 regarding positive-negative selection methods.

[0594] Introduction of nucleic acids by viral infection has several advantages over other methods such as lipofection and electroporation because higher transfection efficiencies can be obtained due to the infectious nature of viruses.

[0595] Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs such as adenovirus, lentivirus, herpes simplex virus type I or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated gene transfer are, for example, DOTMA, DOPE and DC-Chol [Tonkinson et al., Cancer Investigation, 14 (1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenovirus, AAV, lentivirus or retrovirus. Viral constructs, such as retroviral constructs, contain at least one transcription promoter / enhancer or locus determining element or other elements that control gene expression by other means such as alternative messenger splicing, nuclear RNA transport or post-translational modification. Such vector constructs also contain packaging signals, long terminal repeats (LTRs) or portions thereof, and plus and minus primer binding sites appropriate for the virus used, unless already present in the viral construct. In addition, such constructs typically include a signal sequence for secretion of the peptide from the host cell in which the construct resides. Preferably, the signal sequence for this purpose is a mammalian signal sequence or a signal sequence of the polypeptide variant of some embodiments of the present invention. Optionally, the construct may include a signal that provides polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof. Other non-viral vectors may be used, such as cationic lipids, polylysine, and dendrimers.

[0596] As already mentioned, in addition to containing the elements necessary for the transcription and translation of the inserted coding sequence, the expression constructs of some embodiments of the invention can also contain sequences engineered to enhance the stability, production, purification, yield or toxicity of the expressed monomer or heterodimer. For example, one can engineer the expression of a fusion protein or a cleavable fusion protein comprising the monomer or heterodimer of some embodiments of the invention and a heterologous protein. Such a fusion protein can be designed so that the fusion protein can be easily isolated by affinity chromatography, for example by immobilization on a column specific for the heterologous protein. If a cleavage site is engineered between the monomer or heterodimer of some embodiments of the invention and the heterologous protein, the monomer or heterodimer can be released from the chromatography column by treatment with an appropriate enzyme or agent that destroys the cleavage site (see, e.g., Booth et al. (1988) Immunol. Lett. 19:65-70 and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859).

[0597] The present invention also contemplates cells containing the constructs described herein.

[0598] Thus, according to one aspect of the present invention there is provided a host cell comprising the heterodimer or a nucleic acid construct or system encoding the heterodimer.

[0599] As previously described herein, various prokaryotic or eukaryotic cells can be used as host expression systems for expressing the heterodimers of some embodiments of the present invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequence, yeast transformed with recombinant yeast expression vectors containing the coding sequence, and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors such as Ti plasmids, containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of some embodiments of the present invention.

[0600] Examples of bacterial constructs include the pET series of E. coli expression vectors (Studier et al. (1990) Methods in Enzymol. 185:60-89).

[0601] Examples of eukaryotic cells that can be used in conjunction with the teachings of the present invention include, but are not limited to, mammalian cells, fungal cells, yeast cells, insect cells, algae cells, or plant cells.

[0602] In yeast, several vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Patent Application No. 5,932,447. Alternatively, vectors that promote integration of foreign DNA sequences into the yeast chromosome can be used.

[0603] When using plant expression vectors, the expression of the coding sequence can be driven by several promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514] or the coat protein promoter of TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B [Gurley et al. (1986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to those of skill in the art, see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0604] Other expression systems, such as insect and mammalian host cell systems, which are well known in the art, may also be used in accordance with some embodiments of the present invention.

[0605] According to certain embodiments, the cell is a mammalian cell.

[0606] According to certain embodiments, the cell is a human cell.

[0607] According to certain embodiments, the cell is a cell line.

[0608] According to another particular embodiment, the cells are primary cells.

[0609] The cells may be derived from any suitable tissue, including, but not limited to, blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testes, ovaries, hair, skin, bone, breast, uterus, bladder, spinal cord, or various types of body fluids. The cells may be derived from any developmental stage, including embryonic, fetal and adult, and from any developmental origin, i.e., ectodermal, mesodermal and endodermal origin.

[0610] Non-limiting examples of mammalian cells include SV40 transformed monkey kidney CV1 (COS, e.g., COS-7, ATCC CRL 1651), human embryonic kidney (HEK293 cells or HEK293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol., 36:59 1977), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HeLa, ATCC CCL 2), NIH3T3, Jurkat, canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 1982), MRC 5 cells, FS4 cells, and human hepatoma lines (Hep G2), PER.C6, K562, and Chinese hamster ovary cells (CHO).

[0611] According to some embodiments of the invention, the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK293 cells, PER.C6 cells, HT1080 cells, NS0 cells, Sp2 / 0 cells, BHK cells, Namalwa cells, COS cells, HeLa cells and Vero cells.

[0612] According to some embodiments of the invention, the host cell comprises a Chinese Hamster Ovary (CHO) cell, a PER.C6 cell or a 293 (eg, Expi293F) cell.

[0613] According to another aspect of the present invention there is provided a method for producing a heterodimer comprising introducing a nucleic acid construct or system as described herein into a host cell, or culturing a cell expressing a nucleic acid construct or system as described herein.

[0614] According to a particular embodiment, production involves incubation at 32-37° C. and 5-10% CO2 for 5-13 days.

[0615] Non-limiting examples of manufacturing conditions that can be used with certain embodiments of the present invention are disclosed in the Examples section below.

[0616] Thus, for example, an expression vector encoding the heterodimer is introduced into mammalian cells such as Expi293F, ExpiCHO cells, CHO-K1 or CHO-DG44. The transduced cells are then cultured in cell-specific medium at 32-37° C. and 5-10% CO2 according to the manufacturer's instructions for Expi293F, ExpiCHO, CHO-K1 or CHO-DG44 cells (Thermo), and after at least 5 days of culture, the protein is collected from the supernatant and purified.

[0617] According to certain embodiments, the cultivation is carried out in batch, split-batch, fed-batch or perfusion mode.

[0618] According to certain embodiments, the cultivation is carried out under fed-batch conditions.

[0619] According to a particular embodiment, the culturing is carried out at 36.5°C.

[0620] According to a particular embodiment, the culture is carried out at 36.5° C., with a temperature shift to 32° C. This temperature shift can be carried out in order to slow down the metabolism of the cells before reaching a steady state.

[0621] According to certain embodiments, the method comprises adding a dimerization moiety to the expressed polypeptide.

[0622] According to certain embodiments, the method comprises isolating the heterodimer.

[0623] According to certain embodiments, the recombinant heterodimer is harvested after culturing for an appropriate time. According to certain embodiments, the recombinant heterodimer is harvested by collecting the entire culture medium containing the heterodimer, and does not necessarily imply a further separation or purification step. According to certain embodiments, the heterodimer of some embodiments of the present invention can be purified using various standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, mixed mode chromatography, metal affinity chromatography, lectin affinity chromatography, chromatofocusing, and differential solubilization.

[0624] According to certain embodiments, following production and purification, the therapeutic efficacy of the heterodimers can be detected either in vivo or in vitro by methods known in the art and can include, for example, cell viability, survival of transgenic mice, and expression of activation markers.

[0625] The compositions of some embodiments of the present invention (e.g., the heterodimers, compositions comprising same, nucleic acid constructs or systems and / or cells encoding same) can be administered to an organism by themselves or as a pharmaceutical composition in which the composition is mixed with a suitable carrier or excipient.

[0626] Thus, in some embodiments, the present invention features a pharmaceutical composition comprising a therapeutically effective amount of the compositions disclosed herein.

[0627] As used herein, the term "active ingredient" refers to a composition that is responsible for a biological effect (e.g., a heterodimer described herein, a composition, a nucleic acid construct or a system and / or a cell comprising same).

[0628] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0629] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the compound being administered. These terms include adjuvants.

[0630] As used herein, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The active compound may include one or more pharmaceutically acceptable salts depending on the route of administration. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0631] Pharmaceutical compositions according to at least some embodiments of the present invention may include pharma- ceutically acceptable antioxidants. Examples of pharma-ceutically acceptable antioxidants include (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium disulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Pharmaceutical compositions according to at least some embodiments of the present invention may include additives such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)) and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).

[0632] Examples of suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions according to at least some embodiments of the present invention include water, various buffer components (e.g., Tris-HCl, acetate, phosphate), buffered saline solutions of various pH and ionic strengths, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

[0633] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0634] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by both sterilization procedures (see above) and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, can bring about prolonged absorption of the injectable pharmaceutical form.

[0635] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharma-ceutical active substances is known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical composition according to at least some embodiments of the present invention is contemplated.Supplementary active compounds can also be incorporated into the composition.

[0636] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include an isotonic agent, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, for example, monostearate salts and gelatin in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of active compound in an appropriate solvent, with one or a combination of ingredients as enumerated above, as needed, followed by sterile microfiltration. Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other necessary components listed above.For the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization).By these methods, the powder of the active ingredient and any additional desired ingredient can be obtained from the solution of the active ingredient and any additional desired ingredient that has been previously sterilized and filtered.

[0637] Sterile injection solution can be prepared by incorporating the required amount of active compound into a suitable solvent, with one or a combination of the above-listed components as necessary, followed by sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization).By these methods, the powder is obtained from the solution of active ingredient and any additional desired ingredient that has been previously sterilized and filtered.

[0638] The amount of active ingredient that can be combined with carrier materials to create a single dosage form varies depending on the subject being treated and the specific mode of administration.The amount of active ingredient that can be combined with carrier materials to create a single dosage form is generally the amount of the composition that produces a therapeutic effect.This amount generally ranges from about 0.01 percent to about 99 percent of active ingredient, preferably about 0.1 percent to about 70 percent of active ingredient combined with a pharma-ceutically acceptable carrier, most preferably about 1 percent to about 30 percent, out of 100 percent.

[0639] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, it may be a single bolus administration, or several divided doses may be administered over time, or the dose may be relatively reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous for parenteral compositions to be formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unit dosages for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to provide the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage form according to at least some embodiments of the present invention are determined and directly influenced by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the constraints inherent in the technical field of compounding such active compounds to treat individual susceptibility.

[0640] Techniques for formulation and administration of drugs may be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0641] Pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0642] The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for the therapeutic agents according to at least some embodiments of the present invention include intravascular (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal and sublingual), intravesical, intravitreal, intraperitoneal, intravaginal, brain (e.g., intraventricular, intracerebral and convection enhanced diffusion), CNS delivery (e.g., intrathecal, perispinal and intraspinal) or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous (IV) and intradermal), transdermal (either passively or using iontophoresis or electroporation), transmucosal (e.g., sublingual, nasal, vaginal, rectal or sublingual) or by implant, or other parenteral routes of administration, for example by injection or infusion, or other delivery routes and / or forms of administration known in the art. The term "parenteral administration" as used herein refers to a mode of administration other than enteral administration and topical administration, usually by injection. It can be formulated in a dosage form suitable for each administration route, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and substernal injection and infusion or the use of biodegradable inserts. In certain embodiments, the protein, therapeutic agent or pharmaceutical composition according to at least some embodiments of the present invention can be administered intraperitoneally or intravenously.

[0643] According to certain embodiments, the compositions disclosed herein are administered by parenteral injection as an aqueous solution. The formulation may be in the form of a suspension or emulsion. In general, pharmaceutical compositions for parenteral injection are provided that contain an effective amount of the compositions disclosed herein, and optionally contain pharma- ceutical acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more of additives such as diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., water soluble antioxidants such as ascorbic acid, sodium disulfite, cysteine ​​hydrochloride, sodium hydrogen sulfate, sodium disulfite, sodium sulfite, oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid), and preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are ethanol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate.The formulation can be freeze-dried (lyophilized) or vacuum-dried and redissolved / resuspended immediately before use.The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, incorporating a sterilizing agent into the composition, irradiating the composition, or heating the composition.

[0644] Various compositions (e.g., polypeptides) disclosed herein can be applied locally, which is not effective for most peptide formulations, but may be effective when applied to the mucous membranes of the lungs, nasal passages, oral cavity (sublingual, buccal), vagina, or rectum.

[0645] The compositions of the present invention, when delivered as either aerosols or spray-dried particles with an aerodynamic diameter of less than about 5 microns, can be delivered to the lungs during inhalation and cross the pulmonary epithelial layer to enter the bloodstream. A variety of mechanical devices designed to deliver therapeutic formulations to the lungs can be used. These mechanical devices include, but are not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are well known to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, MO), the Acorn II nebulizer (Marquest Medical Products, Englewood, CO), the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, NC), and the Spinhaler powder inhaler (Fisons Corp, Bedford, MA). Nektar, Alkermes, and Mannkind have inhalable insulin powder preparations approved or are in clinical trials where this technology can be applied to the formulations described herein.

[0646] Formulations for administration to mucosa are typically spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any pharmaceutical company. Oral formulations can be in the form of chewing gum, gel pieces, tablets or lozenges.

[0647] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal formulations require the inclusion of a penetration enhancer. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an effective amount of the active ingredient to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in conjunction with the particular composition used, the age, sex, weight, condition, overall health and past medical history of the patient being treated, and similar factors well known in the medical field.

[0648] According to certain embodiments, the compositions disclosed herein are administered to a subject in a therapeutically effective amount.As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or otherwise produce a desired pharmacological and / or physiological effect.The determination of a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0649] For any formulation used in the method of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell cultures, or in experimental animals by standard pharmaceutical procedures. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition (see, for example, Fingl et al. (1975) in "The Pharmacological Basis of Therapeutics," Ch. 1 p.1).

[0650] Dosage and intervals can be adjusted individually to provide a level of active ingredient sufficient to induce or suppress a biological effect (minimal effective concentration, MEC). The MEC varies with each formulation but can be estimated from in vitro data. Dosages required to achieve the MEC vary with individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0651] Depending on the severity and responsiveness of the condition to be treated, dosage may be of single or multiple administrations over the course of treatment lasting from several days to several weeks, or until a cure is achieved or the disease state is eliminated.

[0652] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0653] In certain embodiments, the composition (e.g., the heterodimer, a composition comprising it, a nucleic acid construct or system, or a cell) is administered locally, for example, by direct injection into the site to be treated. Typically, injection increases the local concentration of the composition to a higher concentration than can be achieved by systemic administration. Combining the heterodimer composition with the aforementioned matrix can help to increase the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide out of the site to be treated.

[0654] The pharmaceutical composition of the present invention can be administered using medical devices known in the art.For example, in optional embodiments, the pharmaceutical composition of at least some embodiments of the present invention can be administered using needleless hypodermic injection devices, such as the devices disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implants and modules useful in the present invention include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate, U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin, U.S. Patent No. 4,447,233, which discloses an infusion pump for delivering drugs at a precise infusion rate, U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for sustained drug delivery, U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment, and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0655] Active compound can be prepared with carrier that prevents the rapid release of compound, for example, sustained release formulation, including implant, transdermal patch and microencapsulated delivery system.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid.Many methods for preparing such formulations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0656] Sustained release polymeric devices can be manufactured for extended systemic release after implantation of the polymeric device (rods, cylinders, films, disks) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres with the peptide dispersed in a solid polymer matrix or microcapsules with the peptide dispersed or suspended within the core (which core can be substantially liquid or solid) with a core made of a different material than the polymer shell. Unless expressly defined herein, microparticles, microspheres and microcapsules are used interchangeably. Alternatively, the polymer can be cast as thin slabs or films ranging from a few nanometers to 4 centimeters, can be a powder made by milling or other standard techniques, or can be a gel such as a hydrogel.

[0657] Either non-biodegradable or biodegradable matrices can be used to deliver the active ingredients disclosed herein, but biodegradable matrices are preferred. They can be natural or synthetic polymers, but synthetic polymers are preferred because they have better degradation and release profile characteristics. The polymer is selected based on the period of time over which release is desired. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight), and may be optionally crosslinked with multivalent ions or polymers.

[0658] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, such as those described in, for example, Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl Polymer ScL, 35:755-774 (1988).

[0659] These devices may be formulated for local release to treat the implanted or injected area, typically delivering a dosage significantly lower than that for whole body treatment, or may be formulated for systemic delivery. These devices may be implanted or injected subcutaneously, intramuscularly, intrafatty, or may be swallowed.

[0660] In certain embodiments, to ensure that therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), these therapeutic compounds can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that selectively transport to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., Low et al., U.S. Pat. No. 5,416,016), mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134), p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090). See also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0661] The composition of some embodiments of the present invention can be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, contain metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may contain a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals. This notice reflects that the form of the composition or its administration to humans or animals has been approved by that agency. Such notice may, for example, be on a prescription drug label approved by the U.S. Food and Drug Administration or an approved product insert. A composition comprising the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indication as detailed above.

[0662] As used herein, the term "about" refers to ±10%.

[0663] The terms "include," "including," "containing," "including," "having" and conjugations thereof mean "including but not limited to."

[0664] The term "consisting of" means "including and limited to."

[0665] The term "consisting essentially of" indicates that a composition, method, or structure may include additional ingredients, steps, and / or moieties, provided that the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0666] As used herein, the singular forms "a," "an," and "the" include the plural references unless the context clearly indicates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds, including mixtures thereof.

[0667] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and is not an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to specifically disclose all of the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 is intended to specifically disclose not only the subranges 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., but also the individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0668] Whenever a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "range between" a first indicated number and a second indicated number and "range from" a first indicated number to a second indicated number are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractional and integer numbers therebetween.

[0669] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine or those that can be readily developed by practitioners from known manners, means, techniques, and procedures. When referring to a particular sequence listing, it should be understood that such reference also encompasses sequences that substantially correspond to complementary sequences containing minor sequence variations, e.g., due to sequencing errors, cloning errors, or other modifications resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0670] It is recognized that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for clarity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as preferred in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be regarded as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0671] Various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0672] The following examples provide experimental evidence for various embodiments and aspects of the present invention as recited above and claimed below.

[0673] The nomenclature used herein and the laboratory procedures used in the present invention generally include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), methods described in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057, “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994), “Culture of Animal Cells - A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition, “Current Protocols in Immunology” Volumes I-III Coligan JE, ed.(1994), Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Available immunoassays have been widely described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, and in the "Oligonucleotide Immunoassays" section of the US Pat. Synthesis” Gait, MJ, ed. (1984), “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985), “Transcription and Translation” Hames, BD, and Higgins SJ, Eds. (1984), “Animal Cell Culture” Freshney, RI, ed. (1986), “Immobilized Cells and Enzymes” IRL Press, (1986), “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). All of the above references are fully incorporated herein by reference. Other general references are provided throughout the specification. The procedures described therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0674] Materials and Methods Reagents - ExcelBand™ 3-color broad range protein marker or ExcelBand™ 3-color extra range protein marker (SMOBIO, Cat#PM2600 or PM2800, respectively), Sample buffer (GenScript, Cat#M00676), Polyacrylamide gel 8% or 4-20% (GenScript, Cat#M00662 or M00656, respectively), ECL Plus Western Blotting substrate (Pierce, Cat#32132), TMB-ELISA substrate solution (Sigma, Cat#T0440), TMB stop solution (Southern Biotech, Cat#0412-01), Streptavidin-HRP (Pierce, Cat#TS21126). FuGENE® HD Transfection Reagent (Promega, Cat#TM328). Vybrant DiD cell labeling solution (Thermo Fisher, Cat# V22887), Lymphoprep™ density gradient medium (StemCells Technologies, Cat#07801).

[0675] Antibodies - LEAFTM purified mouse anti-human PD-L1 (CD274) B7H1 clone 29E.2A3, BioLegend, Cat#329711, anti-human PD1 (GenScript, Cat#A01829-40), APC-labeled anti-PD1 (Biolegend, Cat#329908), APC mouse IgG2b, κIC (Biolegend, Cat#400322), biotinylated rabbit anti-human SIRPα (LsBio, Cat#LS-C370337), rabbit anti-human SIRPα antibody (anti-drug antibody DSP107) batch #104, anti-human LILRB2 (R&D systems, Cat#MAB2078), APC anti-human CD155 antibody (Biolegend, Cat#337618), APC mouse IgG1 k (Biolegend, Cat#400120), APC anti-human CD47 antibody (Biolegend, Cat#323124), APC anti-human CD274 (Biolegend, Cat#329708), APC anti-human CD172a / b SIRPα clone SE5A5 antibody, mouse IgG1, κ (Biolegend, Cat#323809), PE mouse anti-human IgG1-Fc (SouthernBiotech, Cat#9054-09), PE mouse anti-human IgG4 (SouthernBiotech, Cat#9190-09), AF647 anti-human IgG4-Fc (SouthernBiotech, Cat#9200-31), APC anti-human CD85d (ILT4) antibody clone 41D1 (Biolegend, Cat#338708), goat anti-rabbit IgG(H+L)-HRP conjugate (R&D systems, Cat#170-6515), goat anti-mouse IgG HRP conjugate (Bio-Rad, Cat#170-6516), APC anti-human HLA-G (from US Patent Application Publication No. 2020 / 0102390A1), APC human IgG4 (Biolegend, Cat#403706), CD47 blocking antibody (from WO 2011 / 143624A2), HLA-G blocking antibody (from US Patent Application Publication No. 2020 / 0102390A1), mouse anti-human IgG1 HRP conjugate (SouthernSouthern Biotech, Cat#9054-05), Mouse anti-human IgG4 HRP conjugate (Southern Biotech, Cat#9200-05), PE mouse anti-human IgG1 FC (Southern Biotech, Cat#9054-09), PE mouse anti-human IgG4-Fc (Southern Biotech, Cat#9190-09), anti-human PVR (NOVUS, Cat#NB6001241), APC mouse anti-human IgG1 hinge (Southern Biotech, Cat#9054-09), APC anti-human TIGIT, mIgG2a clone A15153G (Biolegend, Cat#372706), APCmIgG2a MOPC-173 (Biolegend, Cat#400220), PE anti-human PD-L1, 29E2A3, mIgG2b (Biolegend, Cat#329706), APC anti-human CD16, mIgG1 clone ICRF44 (Biolegend, Cat#301310).

[0676] Recombinant proteins - human recombinant HLA-G protein (His tag), (Abcam, Cat# ab225660), human PDL-1 Fc tag (Acro, Cat# PD1-H5258), human PDL-1-Fc (GenScript, U3420DK140-1), human CD47 protein HIS tag (Acro, Cat# CD7-H5227), human CD155 (PVR) protein Fc tag (Acro, Cat# CD5-H5251 and CD5-H5254).

[0677] Cell lines - Expi293F (manufactured by Gibco, Cat#A14257), DLD1-WT cell line (ATCC, CCL-221), DLD1-PDL1 cell line (Hendriks et al 2016), HT1080 WT (ATCC, CCL-121), CHO-K1, CHO-K1-CD47, CHO-K1-CD47 (Cell Bank Australia, CBA-0146), HT1080-HLA-G (cells generated by viral infection with an HLA-G expression plasmid), JEG-3 cells (ATCC, HTB-36), K562 (ATCC, CCL-243), K562PVR, K562PD-L1, K562PVR / PD-L1 (generated by transfection of K562 cells with PDL1 and / or PVR expression plasmids and selection of stable expressing cells), AB12 (ECACC, 10092306), Renca (ATCC, CRL-2947), Jurkat NFAT-CD16 (Promega, jktl-nfat-cd16), SK-OV-3 (ATCC, HTB-77).

[0678] Media and tissue culture reagents - Expi269 medium (Gibco, Cat#A-14351-01), RPMI 1640 (Biological Industries, Cat#01-100-1A), FCS (Gibco, Cat#12657-029), BSA, Sigma, A7030, EDTA, Sigma, E7889, Cell dissociation buffer, Gibco, 13151-014, EMEM (Biological Industries, Cat#01-040-1A), DMEM (Biological Industries, Cat#01-055-1A), TrypLE Express (Gibco, Cat# 12604-13), Glutamax (Gibco, Cat# 35050-038), penicillin-streptomycin (Gibco, Cat# 151140-122), sodium pyruvate (Biological Industries, Cat# 03-042-1B), IMDM (Biological Industries, Cat# 01-058-1A).

[0679] Equipment – ​​FACS device, Stratadigm Cytometry S1000EXI, ELISA reader, Thermoscience Multiskan FC, ELISA software, SkanIt software 4.1 for microplate reader RE, ver.4.1.0.43.

[0680] Structural analysis of heterodimer-protein - Based on the X-ray structure of the homologue, homology modelling was carried out for each part. For PD1: PDB IDs: 3RRQ, 5GGR, 5GGS, 5JXE and 4ZQK were used as templates. For hIgG4: PDB IDs: 4C54, 4C55, 5W5M and 5W5N were used as templates. For SIRPα: PDB IDs: 2UV3, 2WNG, 4CMM, 6BIT, 2JJS and 2JJT were used as templates. For LILRB2: PDB IDs: 2GW5, 4LLA, 2DYP and 6BCP were used as templates. For SIGLEC10: PDB IDs: 2N7A and 2N7B of SIGLEC8 homologue were used as templates. For TIGIT: PDB IDs: 3QOH, 3RQ3, 3UCR, 3UDW and 5V52 were used as templates.

[0681] The linker segments were modeled primarily using loop modeling in CHARMM to avoid structural violations and allow plausible estimation of possible "spacer" lengths.

[0682] Heterodimer Production - For comparative functional analysis and production evaluation, several heterodimers (referred to herein as "DSPs") were designed and / or produced using the "knobs" into "holes" method (e.g., as described in U.S. Pat. No. 8,216,805). See Table 1 below. Production was performed in Expi293F cells transfected with pcDNA3.4 expression vectors cloned with coding sequences for the desired Fc fusion proteins (see Table 1 below).

[0683] The sequences were cloned into the vector using restriction enzymes such as EcoRI and HindIII or XbaI and EcoRV, with the addition of an artificial signal peptide (MESPAQLLFLLLLWLPDGVHA, SEQ ID NO: 116) above the Kozak sequence and stop codon. Proteins were collected from cell culture supernatants and optionally purified by one-step purification using Protein A (PA) Poros MabCapture A resin or anion exchange High Trap Q FF resin.

[0684] [Table 3-1]

[0685] [Table 3-2]

[0686] SDS-PAGE analysis - 35 μl of cell culture supernatant or 3 μg of purified protein from each heterodimer sample was mixed with loading buffer with or without β-mercaptoethanol (reducing and non-reducing conditions, respectively), heated at 95°C for 5 min, and separated by 8% or 4-20% gradient polyacrylamide gel electrophoresis SDS-PAGE. Protein migration on the gel was visualized by e-Stain apparatus (GenScript) according to the manufacturer's instructions.

[0687] Western blot analysis - Samples containing the prepared heterodimers (50-500ng / lane) were treated under reducing or non-reducing conditions (in loading buffer with or without β-mercaptoethanol, respectively), heated to 95°C for 5 min, and separated by 8% or 4-20% gradient SDS-PAGE. Proteins were then transferred onto a PVDF membrane and incubated with primary antibodies for 1 h or overnight, followed by incubation with HRP-conjugated secondary antibodies for 1 h. Signals were detected after ECL development.

[0688] Binding of heterodimers to their corresponding ligands / receptors by sandwich ELISA - Flat-bottom 96-well plates were precoated with one arm receptor / ligand of the heterodimer to be analyzed by overnight incubation at 4°C with recombinant counterpart proteins such as CD47, PDL1, HLA-G or PVR proteins, followed by blocking and washing. Serially diluted cell culture supernatants or purified protein samples containing the prepared heterodimers were added to the corresponding precoated wells. Following an additional washing step, biotinylated unlabeled or HRP-labeled antibodies against the second arm or IgG backbone of the heterodimer were added and allowed to bind to the captured proteins. Then, HRP-conjugated secondary antibodies or streptavidin-HRP were added if the detected antibody was not HRP-labeled. Detection was performed using a plate reader (Multiscan FC from Thermo Scientific) at 450 nm with a reference wavelength of 620 nm, following standard ELISA protocols, with TMB substrate. OD values ​​were used to generate binding curve graphs with GraphPad Prism software.

[0689] Binding of heterodimers to their counterparts-ligands / receptors expressed on the cell surface - Cells expressing one of the counterparts of the analyzed heterodimers were incubated with serial dilutions of the produced heterodimers for 30 min at 4°C, then immunostained with fluorescently labeled antibodies specific for the second component of the heterodimer (i.e., not binding to the counterpart expressed by the cells) or specific for the IgG backbone and analyzed by flow cytometry. Optionally, if the cells express both counterparts, the cells were pre-incubated with a blocking antibody against one of them before incubation with the heterodimer. OD values ​​were used to generate binding curve graphs with GraphPad Prism software.

[0690] Effect of heterodimers containing SIRPα and LILRB2 domains on macrophages and PMN cells - To test the phagocytosis of cancer cells by granulocytes, HT1080 expressing human CD47 or HT1080-HLA-G cells expressing human CD47 and human HLA-G were labeled with DiD and preincubated with 0, 1, 2 or 5 μg / mL DSP216 at room temperature for 15 min. Then, the cells were co-cultured with granulocytes at an effector-to-target (E:T) ratio of 1:1 overnight at 37°C and analyzed by flow cytometer. The MFI values ​​were used to generate binding curve graphs by GraphPad Prism software.

[0691] Cytotoxicity assay - Target cell killing by NK cells (effector cells) was tested in a co-culture assay. After overnight incubation, the percentage of killed target cells was analyzed by flow cytometry analysis (FACS). Pre-labeled target cells [K562WT, K562 overexpressing PVR (herein K562PVR) cells, K562 overexpressing PDL1 (herein K562PDL1) cells or K562 overexpressing PVR / PDL1 (herein K562PVR / PDL1) cells] were placed in 96-well plates and incubated with primary NK cells at various effector-target (E:T) ratios in the presence of different concentrations of TIGIT-PD1 heterodimer. After ON incubation at 37°C, cells were analyzed by flow cytometry. K562WT target cells were used as a reference for inhibition of killing effect in the presence of PVR / PD-L1 ligand expressed on target cells. The reference for TIGIT-PD1 heterodimer treatment was untreated target cells.

[0692] Granzyme B secretion - Granzyme B is a serine protease most commonly found in the granules of NK cells and cytotoxic T cells. Granzyme B is secreted by these cells along with the pore-forming protein perforin and mediates apoptosis of target cells. Prelabeled target cells (K562WT, K562PVR, K562PDL1 and K562PVR / PDL1 overexpressing cells) were placed in 96-well plates and incubated with primary NK cells at various effector-target (E:T) ratios in the presence of different concentrations of TIGIT-PD1 heterodimer. After ON incubation at 37°C, supernatants were collected and analyzed for granzyme B levels using ELISA.

[0693] FcγRIIIa binding using luciferase activity assay with reporter gene system - Activation of FcγRIIIa by IgG1-Fc was detected using a reporter system (Promega) in Jurkat NFAT CD16 overexpressing cells. In this system, binding to FcγRIIIa induces a cascade of signaling events including an increase in intracellular calcium levels and activation of calcineurin, a calcium-sensitive phosphatase that rapidly dephosphorylates NFAT protein. Dephosphorylated NFAT translocates into the nucleus and induces the expression and secretion of luciferase. The level of secreted luciferase was measured as a luminescent signal generated by the interaction of luciferase with an added substrate (QUANTI-Luc). The binding of the IgG1-Fc arm of the TIGIT-PD1 heterodimer to FcγRIIIa (CD16) on Jurkat CD16-overexpressing cells was tested in a co-culture assay with K562PVR / PDL-1 cells (target cells) and Jurkat NFAT-CD16-overexpressing cells (effector cells). For this purpose, K562PVR / PD-L1 were cultured in 96-well plates and incubated in the presence of different concentrations of TIGIT-PD1 heterodimer. After 1 h of incubation at 37°C, Jurkat NFAT CD16-overexpressing cells were added to the target cells at an E:T ratio of 2:1. After 6 h of incubation at 37°C, luciferase activity was analyzed by luminometer. K562WT target cells were used as a reference for luciferase activity in the presence of PVR / PD-L1 ligand expressed on the target cells. The control for TIGIT-PD1 heterodimer treatment was untreated target cells.

[0694] Simultaneous binding of TIGIT-PD1 heterodimers to their counterparts as determined by ELISA - Flat-bottom 96-well plates were precoated with hrPDL1-Fc protein, followed by blocking and washing. Serially diluted purified TIGIT-PD1 heterodimer samples were added to the corresponding precoated wells. Following incubation and additional washing steps, the detection protein - human CD155 (PVR) conjugate to mouse IgG2a Fc was added and allowed to bind to the captured proteins. Following additional washing, peroxidase-conjugated AffiniPure goat anti-mouse IgG was added and after incubation and washing, detection was performed using TMB substrate following standard ELISA protocol using a plate reader (Thermo Scientific Multiscan FC) at 450 nm with a reference wavelength of 620 nm.

[0695] Simultaneous binding of TIGIT-PD1 heterodimer to NK cells and tumor cells - Simultaneous binding of TIGIT and PD1 to NK and tumor cells was tested in a co-culture cell system of primary NK cells and K562PVR / PD-L1 cells in the presence of TIGIT-PD1 heterodimer. Simultaneous binding of the two moieties to different cell types resulted in the formation of doublets, which were detected by flow cytometry as double positive stained cell populations. To allow detection of doublet cell formation, co-cultures of primary NK cells pre-labeled with CPD dye were mixed with K562PVR / PD-L1 cells pre-labeled with CFSE dye in a 2:1 ratio. After 2 hours of incubation at 4°C, doublet cell formation was tested in the presence of different concentrations of TIGIT-PD1 heterodimer. In addition, the specificity of doublet cell formation was tested after 1 hour of incubation with blocking antibodies: Fc blockers, PVR antibodies or PD-L1 antibodies before adding the heterodimer.

[0696] In vivo syngeneic model: Effect of TIGIT-PD1 heterodimer in AB12 mouse mesothelioma cancer model - 7-week-old female BALB / c mice were inoculated intraperitoneally (ip) with AB12 mouse malignant pleural mesothelioma (MPM) cells and then treated with TIGIT-PD1 heterodimer or vehicle control. The effect of the heterodimer was assessed by its effect on mouse survival.

[0697] Treatment Protocol: 10 on day 0 of the study 5 AB12 cells were inoculated ip.

[0698] The study was divided into two groups: Group 1 - Following inoculation, mice were treated ip with 200 μl vehicle / mouse starting on day 6. Vehicle was administered every 3 days. Group 2 - Following inoculation, mice were treated ip with 150 μg of TIGIT-PD1 heterodimer in a volume of 200 μl starting on day 6. A total of four doses were administered every 3 days.

[0699] Animals were assessed daily for morbidity and mortality.

[0700] In vivo NSG model: Effect of TIGIT-PD1 heterodimer in A549 human lung adenocarcinoma model - 11-13 week old NSG mice were subcutaneously (sc) inoculated with human lung adenocarcinoma cells and then treated with TIGIT-PD1 heterodimer or vehicle control. The effect of the heterodimer was evaluated by assessing tumor volume and calculating tumor growth inhibition (TGI).

[0701] Treatment Protocol: 5.0 × 10 on day 0 of the study 6 A549 cells were inoculated sc. On day 9, mice were irradiated and injected with human PBMCs (5 × 10 6 ) intravenously, followed by administration of human PBMCs (5 × 10 6 ) was administered as a second injection.

[0702] The study was divided into two groups: Starting from groups 1-9, mice were injected ip with 200 μl / mouse of PBS every other day (EOD). Mice were treated ip with 150 μg TIGIT-PD1 heterodimer in a volume of 200 μl / mouse EOD starting from groups 2-9. A total of four doses were administered.

[0703] Tumor volumes were measured at EOD during the follow-up period from the day of the first treatment (day 9). All animals were sacrificed on day 18.

[0704] Example 1 Selection, design, production and characterization of heterodimers containing two Fc fusion proteins Several heterodimers were designed (see Table 1 above) comprising two proteins selected from SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10, where each of the proteins is fused to the Fc domain of IgG1 or IgG4 using the "knobs" into "holes" method (e.g., as described in U.S. Pat. No. 8,216,805).

[0705] The structural analysis of the heterodimer protein was carried out to optimize the following parameters: Folding: Proper folding to enable binding to the target, minimizing potential disulfide scrambling. Integrity: no exposed proteolytic sites; High expression in mammalian expression systems, and Low immunogenicity.

[0706] Figures 2A-5C present schematics of the heterodimers referred to herein as "DSP120V1", "DSP216V1", "DSP404V1" and "DSP502V1" (see descriptions and sequences in Table 1 above) and 3D models generated for the identified domains and segments. This analysis predicted possible binding to ligands and no interference between the different domains.

[0707] Several heterodimers were subsequently produced and analyzed. As shown in Figures 6A-B and 15, the majority of the protein in the predicted heterodimeric molecular weight form was observed under non-reducing conditions, and expression of the two subunits was confirmed under reducing conditions. Only low levels of homodimers (dimers containing either two "knob" fragments or two "hole" fragments) were detected by SDS-PAGE.

[0708] Furthermore, the heterodimers produced contain all of the domains designed; for example, DSP120V1 contains both the PD1 and SIRPα domains (Figures 7A-B and 8A-B), DSP216 and DSP216V1 contain both the LILRB2 and SIRPα domains (Figures 7C and 9A-C), DSP402 contains the PD1 domain (Figure 7A), and DSP502 contains both the PD1 and TIGIT domains (Figures 7A and 10A-B).

[0709] In a next step, the produced heterodimers are further analyzed according to their composition according to Examples 2-8 below.

[0710] Example 2 Heterodimers bind to their ligand / receptor counterparts Binding analysis of SIRPα-PD1 heterodimer to cell surface expressed CD47 and PDL1 Binding of the PD1 domain of the SIRPα-PD1 heterodimer, referred to herein as "DSP120" and "DSP120V1," to human PDL1 expressed on cells was determined using the DLD1-PDL1 cell line (FIG. 11A), which overexpresses PDL1, with anti-SIRPα as the detection antibody. DLD1-WT cells, which express low levels of endogenous PDL1 (FIG. 11A), served as a control. As shown in FIG. 11C-D, DSP120 and DSP120V1 bound to DLD1 PDL1-overexpressing cells in a dose-dependent manner.

[0711] The binding of the SIRPα domain of the SIRPα-PD1 heterodimer DSP120 to human CD47 expressed on cells was determined using a CHO-K1 cell line overexpressing human CD47 (Figure 11B) with a PE-conjugated anti-IgG4 antibody as the detection antibody. CHO-K1 cells served as a control since they do not express human CD47 (Figure 11B). As shown in Figure 11E, DSP120 bound to CHO-K1 CD47-overexpressing cells in a dose-dependent manner.

[0712] Binding analysis of SIRPα-PD1 heterodimer to plate-bound CD47 or PDL1 Binding of the PD1 and SIRPα domains of the SIRPα-PD1 heterodimer DSP120 to plate-bound PDL1 or CD47 was determined after incubation with anti-human PD-1 (for CD47-coated plates) or anti-human SIRPα antibody (for PDL1-coated plates) followed by incubation with the corresponding HRP-conjugated secondary antibody. Detection was performed using TMB substrate. Figure 8A shows the concentration-dependent binding of DSP120 to CD47-coated plates, and Figure 8B shows the concentration-dependent binding of DSP120 to PDL1-coated plates.

[0713] Binding analysis of SIRPα-LILRB2 heterodimer to cell surface expressed CD47 and HLA-G Binding of the SIRPα domain of the SIRPα-LILRB2 heterodimer, referred to herein as "DSP216," to human CD47 expressed on cells was determined using the HT1080 cell line, which expresses human CD47 (FIG. 12A) and does not express HLA-G (FIG. 12B), and APC-conjugated anti-LILRB2 as the detection antibody. As shown in FIG. 12E, DSP216 bound to CD47-expressing cells in a dose-dependent manner.

[0714] Binding of the SIRPα and LILRB2 domains of the SIRPα-LILRB2 heterodimer, referred to herein as "DSP216V1," to HLA-G and / or human CD47 expressed on cells was determined using the HLA-G-overexpressing HT1080 cell line (FIG. 12D) and an anti-human IgG4 antibody. As shown in FIG. 12F, DSP216V1 bound to HLA-G and human CD47-expressing cells in a dose-dependent manner.

[0715] Since HT1080-HLA-G expresses both HLA-G and CD47, the specific binding to human CD47 and HLA-G was further tested using blocking antibodies against each of the ligands. As shown in Figure 16A, the specific dose-dependent binding of DSP216 to HLA-G expressed on HT1080-HLA-G cell line was confirmed using anti-human HLA-G blocking antibody (APC-conjugated anti-human IgG1 antibody was used as the detection antibody). Similarly, the specific dose-dependent binding of DSP216V1 to human CD47 on HT1080 cell line and human CD47 and human HLA-G expressed on HT1080-HLA-G cell line was confirmed using anti-human CD47 and anti-human HLA-G blocking antibodies (Figure 16B, APC-conjugated anti-SIRPα antibody was used as the detection antibody).

[0716] Similarly, specific dose-dependent binding of the LILRB2 domain to human HLA-G and specific dose-dependent binding of the SIRPα domain to CD47 of other SIRPα-LILRB2 heterodimers, referred to herein as "DSP216V3," "DSP216V4," "DSP216V5," and "DSP216V6," was determined using HLA-G-overexpressing HT1080-HLA-G or JEG3 cell lines (both lines express both human CD47 and human HLA-G) and anti-human HLA-G or anti-human CD47 blocking antibodies (Figures 16C-17F, APC-bound anti-human IgG1 or APC-bound anti-SIRPα antibodies were used as detection antibodies).

[0717] Binding analysis of SIRPα-LILRB2 heterodimers to plate-bound human CD47 or human HLA-G Binding of the SIRPα and LILRB2 domains of SIRPα-LILRB2 heterodimers DSP216, DSP216V1, DSP216V3 and DSP216V4 to plate-bound CD47 or HLA-G was determined after incubation with anti-human SIRPα antibody (for HLA-G coated plates) followed by incubation with the corresponding HRP-conjugated secondary antibody or with anti-human IgG1-HRP or anti-human IgG4-HRP. Detection was performed with TMB substrate. As shown in Figures 9A-C and 18A-H, all tested heterodimers bound both plate-bound CD47 and plate-bound HLA-G in a concentration-dependent manner.

[0718] Binding analysis of SIGLEC-10-PD1 heterodimer to cell surface expressed CD24 and PDL1 The binding of the PD1 domain of the SIGLEC-10-PD1 heterodimer, referred to herein as "DSP402", to human PDL1 was determined using DLD1-PDL1 cell lines overexpressing PDL1 and APC-conjugated anti-PD1 as a detection antibody (Figure 11A). DLD1-WT cells expressing low levels of endogenous PDL1 served as a control (Figure 11A). As shown in Figure 13, DSP402 bound to DLD1 PDL1-overexpressing cells in a dose-dependent manner.

[0719] The binding of SIGLEC-10 domains to their receptors is tested using CD24 expressing cells.

[0720] Binding analysis of TIGIT-PD1 heterodimer to cell surface expressed PVR, PDL1, and FcγRIIIa (CD16) The binding of the PD1 domain of the TIGIT-PD1 heterodimer, referred to herein as "DSP502", to human PDL1 was determined using DLD1-PDL1 cell line overexpressing PDL1 (Figure 11A) and HT1080 cell line endogenously expressing PD-L1 (Figure 14D). The binding of the TIGIT domain of DSP502 to human PVR was determined using DLD1-PDL1 and HT1080 cell lines, both of which express high levels of endogenous PVR (14B and 14C). A PE-conjugated anti-human IgG1 antibody was used as the detection antibody in both assays.

[0721] As shown in Figure 14E-F, DSP502 bound to PDL1-expressing cells in a dose-dependent manner, and the binding was blocked by an anti-human PD-L1 blocker antibody.

[0722] Figures 14E-F also show dose-dependent binding of the TIGIT domain of DSP502 to PVR-expressing cells, as demonstrated by blocking binding of DSP502 to these cells with an anti-human PVR blocking antibody.

[0723] Figure 14G shows the dose-dependent binding of the TIGIT domains of other TIGIT-PD1 heterodimers (herein referred to as "DSP502V1," "DSP502V2," and "DSP502V3") to DLD-1 WT cells expressing PVR and not PD1 (Figures 14A and 11A). A PE-conjugated anti-human IgG4 antibody was used as the detection antibody. The binding patterns of these three proteins are similar.

[0724] In addition, dose-dependent binding of the PD1 and TIGIT domains of DSP502 and another TIGIT-PD1 heterodimer (containing the LALA mutation on the Fc-IgG1 domain), referred to herein as "DSP502V4," to human PDL1 and human PVR was determined using K562 PD-L1 cell lines overexpressing PD-L1, K562 cell lines overexpressing PVR, and K562 PVR / PD-L1 cell lines overexpressing both PVR and PD-L1, respectively (Figures 19A-I).

[0725] PE-conjugated anti-human IgG1 antibody or APC-conjugated anti-TIGIT and anti-human IgG1 antibodies were used as detection antibodies in the binding assays, as indicated in the figures.

[0726] We also observed dose-dependent binding of the TIGIT domain of DSP502 to the SKOV3 cell line, which expresses high levels of endogenous PVR (FIGS. 20A-B, PE-conjugated anti-human IgG1 antibody was used as the detection antibody).

[0727] We also observed dose-dependent binding of the PD1 and TIGIT domains of DSP502 to mouse PDL1 and mouse PVR expressed on cells using the AB12 cell line, which endogenously expresses PVR (Figure 22A-B, PE-conjugated anti-human IgG1 antibody was used as the detection antibody) or the Renca cell line, which endogenously expresses both PD-L1 and PVR (Figure 21A-B, PE-conjugated anti-human IgG1 antibody was used as the detection antibody). Specific binding of DSP502 to its ligands (PDL1 and PVR), respectively, expressed on Renca cells was demonstrated using anti-mPDL1 and anti-mPVR blocking antibodies.

[0728] Furthermore, binding of the Fc domain of DSP502 to the human Fc receptor CD16 was demonstrated using a Jurkat NFAT cell line overexpressing CD16 (Figure 23B). A PE-conjugated anti-human IgG1 antibody was used as the detection antibody. As shown in Figure 23A, DSP502 bound to the cells in a dose-dependent manner. The specificity of binding to CD16 was demonstrated using an Fc blocking agent that completely blocked the binding of the heterodimer to cells.

[0729] To further test the effect of heterodimer binding to Fc receptors, a co-culture assay of K562PVR / PDL-1 cells (target cells) and Jurkat NFAT cells overexpressing CD16 (effector cells) was followed by analysis of luciferase signal after 6 hours of incubation. Jurkat-NFAT-CD16 cells stably express the Lucia luciferase reporter gene under the control of the ISG54 promoter fused to the NFAT element. FcγRIIIa binding was measured as a bioluminescent signal generated by luciferase upon addition of detection reagent. As shown in Figure 24, co-culturing the cells in the presence of DSP502 increased the secretion of luciferase. Importantly, no secretion of luciferase was detected when Jurkat NFAT-CD16 cells were incubated as a mono-culture assay with DSP502 (i.e., in the absence of K562PVR / PDL-1 cells). Thus, binding of DSP502 to FcγRIIIa expressed on Jurkat cells is not sufficient to initiate signaling unless anchored to PVR and / or PDL1.

[0730] Binding analysis of TIGIT-PD1 heterodimers to plate-bound PVR Binding of the TIGIT domain of the TIGIT-PD1 heterodimer DSP502 to plate-bound PVR was determined after incubation with an anti-human PD1 antibody, followed by incubation with the corresponding HRP-conjugated secondary antibody. Detection was performed with TMB substrate. As shown in Figure 10A-B, DSP502 bound to plate-bound PVR in a concentration-dependent manner.

[0731] Example 3A Heterodimers bind simultaneously to their ligand / receptor counterparts as determined by ELISA The binding of both sides of the heterodimer to their counterparts, i.e. PD1 to PDL1, LILRB2 to HLA-G, SIRPα to CD47, TIGIT to PVR and SIGLEC-10 to CD24, is tested by a sandwich ELISA-based assay, which is also used to compare the functional properties of different variants of the heterodimeric protein.

[0732] Flat-bottom 96-well plates are precoated with the receptor / ligand of one arm by incubating with recombinant counterpart proteins such as CD47 protein, PDL1 protein, HLA-G protein, PVR or CD24, followed by blocking and washing. Serially diluted cell culture supernatants or purified protein samples containing the produced heterodimers are added to the corresponding precoated wells. Following an additional washing step, a biotinylated or unlabeled soluble receptor / ligand for the second arm of the heterodimer is added and bound to the captured protein. Then, streptavidin-HRP or HRP-conjugated antibody for the second receptor / ligand is added and detection is performed with TMB substrate according to standard ELISA protocols using a plate reader (Multiscan FC from Thermo Scientific) at 450 nm with a reference wavelength of 620 nm.

[0733] Alternatively, plates are coated with a mixture of equimolar amounts of the two proteins and binding is detected using an IgG-specific antibody.

[0734] As shown in Figure 25A, the TIGIG-PD1 heterodimer DSP502 bound simultaneously to both PVR and PDL1.

[0735] Example 3B Heterodimers simultaneously bind their ligand / receptor counterparts as determined by flow cytometry The simultaneous binding of TIGIT-PD1 heterodimer DSP502 to all its counterparts (i.e., for example, IgG1-Fc binding to FcγRIIIa, TIGIT binding to PVR, and PD1 binding to PDL1) was examined by flow cytometry using primary NK cells expressing FcγRIIIa (Figure 25B) and K562PVR / PD-L1 cells expressing PVR and PDL1 (Figure 19F). Specifically, NK cells pre-labeled with CPD dye were co-cultured with K562PVR / PD-L1 cells pre-labeled with CFSE dye at a ratio of 2:1 in the presence of different concentrations of DSP502, respectively. The formation of doublets was analyzed using flow cytometry and appeared as double staining events. As shown in Figure 25C, DSP502 mediated doublet formation, indicating that DSP binds to both cell types simultaneously. Furthermore, these doublet formation-mediated activities by DSP502 were blocked by specific blocking antibodies against PVR, PD-L1 or FcγRIII (Figure 25D), indicating that optimal conditions for doublet formation involve three receptors.

[0736] Example 4 Effect of heterodimers on blocking ligand-receptor binding The heterodimers are designed to block the interaction of the endogenous ligand / receptor expressed on the target cell with the native receptor / ligand.

[0737] Thus, for example, the PD1 portion of the relevant heterodimer is designed to block the interaction between endogenous PD1 expressed on T cells and PDL1 expressed on tumor cells. To this end, the effectiveness of the manufactured heterodimer as a blocker of this interaction is evaluated. Plates are coated with recombinant human PDL1. The plates are then washed and incubated for 1 hour with different concentrations of the manufactured PD1-containing heterodimer (see, for example, Table 1 above) or a positive control anti-PD1 blocker antibody. Biotinylated PD1 is added, followed by an additional incubation, and then the plates are washed and blotted with streptavidin-HRP and TMB substrate according to standard ELISA protocols. The plates are analyzed using a plate reader (Multiscan FC from Thermo Scientific) at 450 nm with a reference wavelength of 620 nm.

[0738] Similarly, the blocking activity of related heterodimers will be studied to assess their effectiveness for blocking PVR-TIGIT, SIGLEC10-CD24, LILRB2-HLA-G and CD47-SIRPα binding.

[0739] Example 5 In vivo antitumor effect of the heterodimer Experimental Design: To test the efficacy of the produced heterodimers in treating cancer, three different in-vivo mouse models are used. 1. NSG mice inoculated with human stem cells, or human PBMCs, or fixed human PBMCs and human tumor cells expressing the heterodimer target. 2. Nude-SCID mice inoculated with human tumor cells. 3. Syngeneic mouse tumor models using surrogate mouse proteins of the heterodimers tested.

[0740] In all models, mice are inoculated with tumor cells intravenously (IV), intraperitoneally (IP), subcutaneously (SC) or orthotopically. 3), mice were treated IV, IP, SC or orthotopically with the prepared heterodimers at different doses and in different regimens.

[0741] Mice were followed for weight and clinical signs. Tumors were measured with calipers several times a week and tumor volume was calculated according to the formula: V = length x width. 2 The tumor growth was calculated based on the mean tumor size and tumor survival rate (TFS) of 1000 mg / kg / day. Mice were weighed daily. Tumor growth and survival were monitored throughout the experiment.

[0742] Immune cell infiltration and subtyping within the tumors was examined by tumor resection or lymph node sampling, digestion and staining with specific antibodies and immunophenotyping using flow cytometry analysis. Additionally or alternatively, tumors were excised, embedded in paraffin, sectioned and immunohistologically stained with specific antibodies to determine immune cell infiltration or tumor necrosis grade.

[0743] At the time of sacrifice, mouse organs were harvested and embedded in paraffin blocks for H&E and IHC staining.

[0744] Blood samples were taken from mice at different time points following a general procedure for the following studies: PK analysis, measurement of cytokines in plasma, FACS profiling of circulating blood cell subpopulations, hematology tests, serum chemistry tests, anti-drug antibody (ADA) analysis and neutralizing antibody analysis (Nab).

[0745] result: Antitumor effect of TIGIT-PD1 heterodimer in NSG mice bearing human NSCLC tumors The antitumor in vivo efficacy of the TIGIT-PD1 heterodimer DSP502 was evaluated in 11-13 week-old male NSG mice inoculated with A549 cells, a human lung adenocarcinoma cell line. Tumor volumes in mice treated with DSP502 showed little increase over 18 days after tumor inoculation, whereas tumor volumes in control mice increased to 400 mm 3 The tumors reached a volume of 100 μg / mL, indicating that treatment with DSP502 resulted in significant inhibition of tumor growth (FIG. 26).

[0746] Antitumor effect of TIGIT-PD1 heterodimer in mice bearing murine mesothelioma tumors The antitumor in-vivo effect of TIGIT-PD1 heterodimer DSP502 was evaluated in 7-week-old female BALB / c mice inoculated with AB12 cells, a mouse malignant pleural mesothelioma (MPM) cell line. Treatment with DSP502 significantly extended the survival of mice compared to control mice (Figure 27). For example, 87.5% of control-treated mice died by day 33 after tumor inoculation, whereas 62% of DSP502-treated mice survived more than 80 days after tumor inoculation.

[0747] Example 6 Effect of the LILRB2 arm of related heterodimers on M-CSF-dependent macrophage maturation The LILRB2 arm of the heterodimer is designed to compete for or block the interaction of endogenous LILRB2 expressed on APCs such as macrophages and dendritic cells with HLA-G expressed on tumor or immune cells, thereby blocking the induced immunosuppressive signal. M1-like macrophages have been reported to exhibit antitumor activity, whereas M2 macrophages have been reported to promote tumor progression. Blockade of LILRB2 with antagonistic antibodies during M-CSF-dependent macrophage maturation was shown to result in a rounder, more tightly bound M1-like (antitumor) phenotype with lower expression of CD14 and CD163. After stimulation of the generated macrophages with LPS, enhanced secretion of the pro-inflammatory cytokine TNFα and reduced secretion of the anti-inflammatory IL-10 were detected.

[0748] To this end, the effect of the manufactured heterodimers containing LILRB2 on M-CSF-dependent macrophage maturation is assessed by flow cytometry-based detection of CD14 and CD163 and measurement of TNFα and IL-10 release after stimulation of LPS-pretreated macrophages.

[0749] Example 7 Effects of heterodimers containing SIRPα or LILRB2 domains on macrophages and polymorphonuclear cells The SIRPα portion of the heterodimer of some embodiments is designed to block the "don't-eat-me" signal induced by CD47-expressing tumor cells towards endogenous SIRPα expressed on APCs such as macrophages and granulocytes by competing for and blocking the interaction of endogenous SIRPα with CD47 on the tumor cells. This blocking of the "don't-eat-me" signal induces phagocytosis of tumor cells.

[0750] The LILRB2 portion of the heterodimer of some embodiments is designed to block immunosuppressive signals induced by HLA-G expressed on tumor or immune cells toward endogenous LILRB2 expressed on APCs such as macrophages and DCs, in part by competing for and blocking the interaction of HLA-G with endogenous LILRB2 on tumor and immune cells. This blockade of the "don't-eat-me signal" of HLA-G induces phagocytosis of tumor cells and prevents the transmission of inhibitory HLA-G-LILRB2 signals between immune cells, resulting in enhanced phagocytosis.

[0751] The effect of the manufactured SIRPα-containing heterodimers on the phagocytosis of tumor cells by human macrophages or polymorphonuclear cells (PMN) and the effect of LILRB2-containing heterodimers on the phagocytosis of tumor cells by human macrophages or DCs will be assessed using flow cytometry-based assays or fluorescence microscopy.

[0752] To this end, the effect of the SIRPα-LILRB2 heterodimer DSP216 on the phagocytosis of tumor cells by human PMNs (granulocytes) was evaluated using a flow cytometry-based assay. Granulocytes from three different donors were incubated with 1, 2 or 5 μg / mL of DSP216 and then co-cultured with the tumor cell line HT1080 expressing human CD47 or HT1080-HLA-G expressing both human CD47 and human HLA-G at a 1:1 E:T ratio (Figures 12A and 12D). As shown in Figure 28, DSP216 treatment increased the phagocytosis percentage of HT1080 cells and the phagocytosis percentage of HT1080-HLA-G to a greater extent.

[0753] Example 8 Cytotoxic activity of NK cells by heterodimers containing the TIGIT domain Natural killer (NK) cells induce direct cytotoxicity or secretion of cytokines / chemokines without recognizing specific antigens as B cells and T cells do. NK cytotoxicity plays an important role in immune responses against infected cells, malignant tumors, and stressed cells, and is involved in the pathological processes of various diseases.

[0754] To determine the effect of the produced heterodimers on NK activation, a number of assays known in the art were used, including but not limited to the following:

[0755] · Cytotoxicity assay: Killing of target cells by NK cells (effector cells) in a co-culture assay. The percentage of killing is analyzed by flow cytometric analysis (FACS). Pre-labeled target cells (e.g. K562PVR / PD-L1 cells or K562WT cells) are placed in 96-well plates and incubated at 37°C with pre-labeled primary NK cells at different effector-target (E:T) ratios. Optionally, NK cells are cultured with 1000 U / mL IL-2 for 48 hours prior to the assay. Cells are harvested after 4, 12 and / or 24 hours and assayed by flow cytometry. The number of target cells recovered from cultures without NK cells is used as a reference.

[0756] Cytotoxicity assay: killing of target cells by NK cells (effector cells) in a co-culture assay. The percentage of killing is determined on an Incucyte instrument using labeled target cells and a caspase-sensitive fluorescent substrate.

[0757] Secretion of inflammatory cytokines: Primary NK cells are stimulated with different ratios of different target cells for 24 hours. Levels of interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in cell-free culture supernatants are determined by ELISA or Cytometric Bead Array (CBA).

[0758] Granzyme B secretion: Primary NK cells are stimulated with different ratios of different target cells for 12 hours. Granzyme B levels in cell-free culture supernatants are determined by ELISA.

[0759] result: Killing of K562WT target cells by NK cells was higher compared to killing of K562 cells expressing PVR and / or PD-L1 (Figure 29A). Addition of TIGIT-PD1 heterodimer DSP502 to co-cultures containing K562WT cells as target cells did not increase NK cytotoxicity (data not shown). However, addition of DSP502 to co-cultures containing K562 cells expressing PVR and / or PD-L1 as target cells significantly increased NK cytotoxicity compared to untreated cells at all E:T ratios tested (Figure 29A). The most significant cytotoxic effect was observed in K562 cells expressing both PVR and PD-L1 treated with DSP502 heterodimer.

[0760] Consistent with the cytotoxicity assay described above, granzyme B secretion from NK cells co-cultured with K562WT as target cells was higher than that from K562 cells expressing PVR and / or PD-L1 as target cells (Figure 29B). Addition of DSP502 to co-cultures containing K562 cells expressing PVR and / or PD-L1 as target cells significantly increased granzyme B secretion compared to untreated cells at all E:T ratios tested (Figure 29B). As with the cytotoxicity assay, the most significant increase in granzyme B secretion was observed in K562 cells expressing both PVR and PD-L1.

[0761] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0762] It is the intention of the applicants that all publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, any citation or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, any priority documents of this application are incorporated herein by reference in their entirety. [Sequence List Free Text]

[0763] SEQ ID NO: 1: SIRP Fc (IgG1 knob) 585AA 1st monomer of DSP120, DSP216, DSP403, DSP503 SEQ ID NO:2: Nucleic acid #1 SEQ ID NO:3: PD1 Fc (IgG1 whole) 382AA 2nd monomer of DSP120, DSP220, DSP402, DSP502 SEQ ID NO:4: Nucleic acid #3 SEQ ID NO: 5: SIRP alpha (IgG4 knob) 582AA 1st monomer of DSP120V1, DSP216V1, DSP403V1, DSP503V1 SEQ ID NO:6: Nucleic acid #5 SEQ ID NO: 7: PD1 (IgG4 Hole) 379AA 2nd monomer of DSP120V1, DSP402V1, DSP502V2, DSP502V3 SEQ ID NO: 8: Nucleic acid #7 SEQ ID NO: 9: TIGIT Fc (IgG1 knob) 354AA 1st monomer of DSP205, DSP404, DSP502 SEQ ID NO: 10: Nucleic acid #9 SEQ ID NO: 11: LILRB Fc (IgG1 whole) 645AA 2nd monomer of DSP205, DSP216, DSP412 SEQ ID NO: 12: Nucleic acid #11 SEQ ID NO: 13: TIGIT Fc (IgG4 knob) 351AA 1st monomer of DSP205V1 SEQ ID NO: 14: Nucleic acid #13 SEQ ID NO: 15: LILRB Fc (IgG4 Hole) 642AA 2nd monomer of DSP205V1, DSP216V1, DSP412V1 SEQ ID NO: 16: Nucleic acid #15 SEQ ID NO: 17: SIRP alpha Fc (IgG1 knob as IgG4) 585AA 1st monomer of DSP216V2 SEQ ID NO: 18: Nucleic acid #17 SEQ ID NO: 19: LILRB2 FC (IgG1 hole as IgG4) 645AA 2nd monomer of DSP216V2 SEQ ID NO: 20: Nucleic acid #19 SEQ ID NO: 21: LILRB2 Fc (IgG1 knob) 645AA 1st monomer of DSP220 SEQ ID NO: 22: LILRB2 Fc IgG4 knob 642AA 1st monomer of DSP220V1 SEQ ID NO: 23: Nucleic acid #22 SEQ ID NO: 24: SIGLEC10 Fc (IgG1 knob) 375AA DSP402, 1st monomer of DSP412 SEQ ID NO: 25: SIGLEC10 Fc(IgG4) 367AA DSP402V1, 1st monomer of DSP412V1 SEQ ID NO: 26: Nucleic acid #25 SEQ ID NO: 27: hIgG1 Fc linker (only knob V1 in DSP216V2) 232aa SEQ ID NO: 28: hIgG1 Fc linker (only knob V1 in DSP216V2) 232aa SEQ ID NO: 29: SIGLEC10 Fc (IgG1 whole) 475AA DSP404, 1st monomer of DSP403 SEQ ID NO: 30: SIGLEC10 Fc (IgG4 whole) DSP404V1, second monomer of DSP403V1 SEQ ID NO: 31: TIGIT No I42D Fc (IgG4 knob) 351AA 1st monomer of DSP502V2 SEQ ID NO: 32: Nucleic acid #31 SEQ ID NO: 33: TIGIT WT Fc (IgG4 knob) 355AA 1st monomer of DSP502V3 SEQ ID NO: 34: Nucleic acid #33 SEQ ID NO: 35: TIGIT Fc (IgG1 whole) 354AA 2nd monomer of DSP503 SEQ ID NO: 36: TIGIT Fc (IgG4 Hole) 351AA Second monomer of DSP503V1 SEQ ID NO: 37: Amino acid sequence of full-length PD1 SEQ ID NO: 38: Nucleic acid sequence of full length PD1 SEQ ID NO: 39: PD1 ECD full length with CYS93>Ser substitution SEQ ID NO: 40: Nucleic acid sequence encoding SEQ ID NO: 39 SEQ ID NO: 41: PD1 ECM known fragment SEQ ID NO: 42: PD1 ECM known fragment SEQ ID NO: 43: 150aa native PD1 domain SEQ ID NO: 44: nucleic acid of SEQ ID NO: 43 SEQ ID NO: 45: 150aa native PD1 domain with CYS93>Ser substitution SEQ ID NO: 46: nucleic acid of SEQ ID NO: 45 SEQ ID NO: 47: 140aa -5-5 of PD1 domain SEQ ID NO: 48: nucleic acid of SEQ ID NO: 47 SEQ ID NO: 49: 140aa -5-5 of PD1 domain with CYS93>Ser substitution SEQ ID NO: 50: nucleic acid of SEQ ID NO: 49 SEQ ID NO: 51: hIgG1 Fc linker (knob) 232aa SEQ ID NO: 52: hIgG1 Fc linker (whole) 232aa SEQ ID NO: 53: 128aa PD1 segment SEQ ID NO:54: Nucleic acid #53 SEQ ID NO: 55: 135aa PD1 segment SEQ ID NO: 56: Nucleic acid #55 SEQ ID NO: 57: PD1 segment of 133aa SEQ ID NO: 58: Nucleic acid #57 SEQ ID NO: 59: PD1 -10-5 (with CYS93>Ser substitution) SEQ ID NO: 60: Nucleic acid #59 SEQ ID NO: 61: PD1 -10-12 (with CYS93>Ser substitution) SEQ ID NO: 62: Nucleic acid #61 SEQ ID NO: 63: PD1 -5-12 (novel, derived from V20) (with CYS93>Ser substitution) SEQ ID NO: 64: Nucleic acid #63 SEQ ID NO: 65: PD 1-11-12 SEQ ID NO: 66: Nucleic acid #65 SEQ ID NO: 67: PD1 -11-12 (novel, derived from V18) (with CYS93>Ser substitution) SEQ ID NO: 68: Nucleic acid #67 SEQ ID NO: 69: PD1 -11-5 SEQ ID NO: 70: Nucleic acid of #69 SEQ ID NO: 71: PD1 -11-5 (novel, derived from V19) (with CYS93>Ser substitution) SEQ ID NO: 72: Nucleic acid of #71 SEQ ID NO: 73: PD1 -5-7 SEQ ID NO: 74: Nucleic acid #73 SEQ ID NO: 75: PD1 -5-7 (novel, derived from V21) (with CYS93>Ser substitution) SEQ ID NO: 76: Nucleic acid of #75 SEQ ID NO: 77: PD1 -5-9 derived from (without CYS93>Ser substitution) SEQ ID NO: 78: Nucleic acid #77 SEQ ID NO: 79: PD1 -0-5 derived from (without CYS93>Ser substitution) SEQ ID NO: 80: Nucleic acid #79 SEQ ID NO: 81: PD1 -0-7 143aa from (without CYS93>Ser substitution) SEQ ID NO: 82: Nucleic acid of #81 SEQ ID NO: 83: Amino acid sequence of full length SIRP alpha SEQ ID NO: 84: Nucleic acid #83 SEQ ID NO: 85: Native SIRPa domain (343AA) SEQ ID NO: 86: Nucleic acid #85 SEQ ID NO: 87: 116aa SIRPa segment SEQ ID NO: 88: Nucleic acid #87 SEQ ID NO: 89: 343 amino acid sequence of SIRPa with 4 point mutations SEQ ID NO: 90: Nucleic acid of #89 SEQ ID NO: 91: 116 amino acid sequence of SIRPa with 4 point mutations SEQ ID NO: 92: Nucleic acid #91 SEQ ID NO: 93: aa SIRPa segment - SEQ ID NO: 87 (117aa SIRPa segment) with K added to the C-terminus SEQ ID NO: 94: Nucleic acid #93 SEQ ID NO: 95: LILRB2 full length ECD amino acid sequence SEQ ID NO: 96: LILRB2 D1-4, AA22-419 (Q8N423-1 UniParc) amino acid sequence SEQ ID NO: 97: LILRB2 D1-4, AA22-419 (Q8N423-1 UniParc) nucleic acid sequence SEQ ID NO: 98: LILRB2 D1-2, AA22-219 (Q8N423-1 UniParc) amino acid sequence SEQ ID NO: 99: LILRB2 D1-2, AA22-219 (Q8N423-1 UniParc) nucleic acid sequence SEQ ID NO: 100: Siglec 10 full length (Q96LC7-1 Uniprot) amino acid sequence SEQ ID NO: 101: Siglec 10, AA18-145 mutation-free (Q96LC7-1 Uniprot) amino acid sequence SEQ ID NO: 102: Siglec 10, AA18-145 mutation free (Q96LC7-1 Uniprot) nucleic acid sequence SEQ ID NO: 103: Siglec 10, AA18-145 and C36S (Q96LC7-1 Uniprot) amino acid sequence SEQ ID NO: 104: Siglec 10, AA18-145 and C36S (Q96LC7-1 Uniprot) nucleic acid sequence SEQ ID NO: 105: Siglec 10 full length ECD, AA17-550 (Q96LC7-1 Uniprot) amino acid sequence SEQ ID NO: 106: TIGIT full length (Q495A1 Uniprot ID) amino acid sequence SEQ ID NO: 107:...

Claims

1. 1. A heterodimer comprising two polypeptides selected from the group consisting of SIRPα, PD1, TIGIT, LILRB2 and SIGLEC10, wherein each of the two polypeptides is capable of binding to its natural binding partner, and wherein the heterodimer does not contain an amino acid sequence of a type II membrane protein capable of binding to its natural binding partner.

2. The heterodimer of claim 1 , comprising a dimerization moiety attached to the two polypeptides.

3. The heterodimer of claim 2 , wherein the dimerization moiety is an Fc domain of an antibody or fragment thereof.

4. The heterodimer of claim 3, wherein the Fc domain is modified to alter its binding to an Fc receptor, to reduce its immune stimulatory function, and / or to improve the half-life of the fusion.

5. The heterodimer of any one of claims 1 to 3, comprising the SIRPα polypeptide and the LILRB2 polypeptide.

6. The heterodimer according to any one of claims 1 to 3, comprising the TIGIT polypeptide and the PD1 polypeptide.

7. The heterodimer according to any one of claims 1 to 6, wherein in the heterodimer, each of the polypeptides is a monomer.

8. The heterodimer according to any one of claims 1 to 6, wherein the two types of polypeptides are contained in a monomer of the heterodimer.

9. A composition comprising a heterodimer according to any one of claims 1 to 8, wherein the predominant form of the two polypeptides in the composition is the heterodimer.

10. A nucleic acid construct or system comprising at least one polynucleotide encoding a heterodimer described in any one of claims 1 to 8 and a control factor directing expression of the polynucleotide in a host cell.

11. A host cell comprising a heterodimer described in any one of claims 1 to 8 or a nucleic acid construct or system described in claim 10.

12. A method for producing a heterodimer, comprising introducing a nucleic acid construct or system described in claim 10 into a host cell or culturing a cell described in claim 11.

13. The method of claim 12, comprising isolating the heterodimer.

14. The heterodimer according to any one of claims 1 to 8, for use in treating a disease in a subject in need thereof, in which treatment with the heterodimer can be effective.

15. The heterodimer for use according to claim 14, wherein the disease is one for which activation of immune cells can be effective.

16. 16. The heterodimer for use according to claim 14 or 15, wherein cells associated with the disease express the natural binding partner.

17. The heterodimer for use according to any one of claims 14 to 16, wherein the disease is cancer.

18. A method of activating an immune cell, comprising activating the immune cell in vitro in the presence of a heterodimer according to any one of claims 1 to 8, a composition according to claim 9, a nucleic acid construct or system according to claim 10, or a cell according to claim 11.

19. 20. The method of claim 18, wherein the activation is in the presence of a cell expressing the natural binding partner.

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