Antibodies against nkp46 and constructs thereof for the treatment of cancer and infections

Abstract

The present invention provides monoclonal antibodies that recognize human NKp46 with high affinity and specificity and do not block the binding of NKp46 to its natural ligands. The present invention also provides multispecific antibodies, chimeric antigen receptors (CARs) and their use for the treatment of diseases, in particular malignancies and infections.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy and relates to antibodies and fragments thereof that bind to the human natural killer cell receptor NKp46, polynucleotide sequences encoding these antibodies and fragments, and cells that produce them. The invention also relates to multispecific and chimeric antigen receptor constructs of these antibodies, and the use of said antibodies, their fragments, and constructs in the treatment of diseases, particularly cancer and acute infections. Background Technology

[0002] Cancer immunotherapy is used to generate and enhance anti-tumor immune responses, for example, through the use of antibodies that specifically target antigens on tumor cells, through the use of fusions of antigen-presenting cells with tumor cells, or through the specific activation of anti-tumor NK or T cells. The ability to recruit immune cells that target tumor cells in a patient provides a therapeutic approach against cancer types and metastatic tumors that have been considered incurable until now.

[0003] Natural killer (NK) cells are innate effector lymphocytes capable of killing tumor cells and infected cells without prior stimulation. To date, NK cells have become one of the most critical first-responders in tumor transformation and viral, bacterial, or fungal infections. They are also involved in autoimmune diseases such as type 1 diabetes and rheumatoid arthritis (RA). Due to their large number of germline-encoded activating and inhibitory receptors, NK cells possess the unique ability to recognize many different targets. The balance of signals received by these receptors ultimately determines whether NK cells function to fight a given target cell or remain neutral.

[0004] The three activating receptors found on NK cells—NKp30, NKp44, and NKp46—are collectively referred to as natural cytotoxic receptors (NCRs). These receptors are crucial in NK cell antitumor and antiviral defense. NKp46 has been identified as a key activating receptor because it is expressed almost exclusively by NK cells. Its ligand library ranges from viral ligands such as hemagglutinin (HA) and hemagglutinin-neuraminidase (HN) from influenza virus, Sendai virus, Newcastle disease virus, and poxvirus, to unknown ligands found on bacteria (e.g., Fusobacterium nucleatum), tumors, adipocytes, and human pancreatic β-cells. The identification of these unknown ligands, particularly tumor ligands of NKp46, has been extensively studied for over a decade.

[0005] US Patent Application No. 20180207290 discloses anti-NKp46 antibodies, toxin conjugates, and their therapeutic uses.

[0006] There remains an unmet need for other, more effective, specific, safe, and / or stable agents that can enhance the immune system's cells to attack tumor cells and / or infectious microorganisms, either alone, as part of an immune construct, or in combination with other agents. Summary of the Invention

[0007] In some embodiments, this invention provides antibodies and fragments thereof that recognize the human NKp46 receptor and are suitable for the treatment of cancer and viral diseases. In some embodiments, this invention also provides chimeric antigen receptor (CAR) molecules comprising the binding site of the anti-NKp46 antibodies described herein, and methods for using them in adoptive therapy. In some embodiments, this invention also provides bispecific and trispecific antibodies that are specific to NKp46 and to at least one other receptor or antigen present on tumor cells or infectious microorganisms.

[0008] In advantageous cases, the anti-NKp46 antibodies described herein specifically target the NKp46 juxtamembranous domain (D2 domain, SEQ ID No: 13) and do not block the interaction between NKp46 and its ligands. These antibodies do not internalize or degrade the NKp46 receptor, and are therefore suitable for targeting NK cells in a variety of therapies. These antibodies, their fragments, and constructs are characterized by unique set of CDR sequences, exhibiting high specificity and very high affinity for human NKp46. The antibodies can be used in cancer immunotherapy and also for cancer diagnosis. Constructs containing these unique set of CDR sequences (i.e., the binding sites of these antibodies), such as bispecific and multispecific antibodies, can also be used to treat viral conditions.

[0009] According to one aspect, the present invention provides an antibody or an antibody fragment thereof comprising at least an antigen-binding portion, which specifically binds to a sequence in the D2 domain of human NKp46 without blocking the interaction of NKp46 with its ligand, said antibody having a binding affinity for human NKp46 of at least 5 x 10⁻⁶. -9 The affinity of M. According to some embodiments, the antibody or antibody fragment has at least 10. -10 Affinity of M.

[0010] According to some embodiments, the antibody or antibody fragment comprises a set of 6 CDR sequences, including three CDRs of the heavy chain (HC) variable region of SEQ ID No: 7 and three CDRs of the light chain (LC) variable region containing sequences selected from SEQ ID No: 8 and SEQ ID No: 12, or analogs or derivatives thereof having at least 90% sequence identity with the variable region sequences.

[0011] Several methods for determining the CDR sequence of a given antibody molecule are known in the art, but there is no standard, definitive method. Determining the CDR sequence from the variable regions of the antibody heavy and light chains can be performed using any method known in the art, including but not limited to methods known as KABAT, Chothia, and IMGT. A selected set of CDRs may include sequences identified by more than one method; for example, some CDR sequences may be determined using KABAT, and others using IMGT. According to some embodiments, the CDR sequence of the mAb variable region is determined using the IMGT method.

[0012] According to some embodiments, the antibody or fragment comprises the CDR sequence of a monoclonal antibody named clone K3, namely, the three CDR sequences contained in the heavy chain variable region set forth in SEQ ID No: 7 and the three CDR sequences contained in the light chain variable region set forth in SEQ ID No: 12, or comprises the CDR sequence of a monoclonal antibody named clone P4, namely, the three CDR sequences contained in the heavy chain variable region set forth in SEQ ID No: 7 and the three CDR sequences contained in the light chain variable region set forth in SEQ ID No: 8.

[0013] According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR1 containing the sequence EYSMH (SEQ ID No: 1). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR2 containing the sequence GISPNSGGTSYNQKFKG (SEQ ID No: 2). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR3 containing the sequence RDFHSSFDY (SEQ ID No: 3).

[0014] According to certain embodiments, the antibody or antibody fragment comprises: (i) HC CDR1 comprising the sequence EYSMH (SEQ ID No: 1); (ii) HC CDR2 comprising the sequence GISPNSGGTSYNQKFKG (SEQ ID No: 2); and (iii) HC CDR3 comprising the sequence RDFHSSFDY (SEQ ID No: 3).

[0015] According to some embodiments, the antibody or antibody fragment comprises a light chain CDR1 containing the sequence RASQSISDYLH (SEQ ID No: 4). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR2 containing the sequence YASQSIS (SEQ ID No: 5). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR3 containing the sequence QNGHSFPLT (SEQ ID No: 6).

[0016] According to certain embodiments, the antibody or antibody fragment comprises: (i) LC CDR1 comprising the sequence RASQSISDYLH (SEQ ID No: 4); (ii) LC CDR2 comprising the sequence YASQSIS (SEQ ID No: 5); and (iii) HC CDR3 comprising the sequence QNGHSFPLT (SEQ ID No: 6).

[0017] According to certain specific embodiments, the antibody or fragment comprises: a heavy chain CDR1 sequence comprising the sequence EYSMH (SEQ ID No: 1), a heavy chain CDR2 comprising the sequence GISPNSGGTSYNQKFKG (SEQ ID No: 2), a heavy chain CDR3 comprising the sequence RDFHSSFDY (SEQ ID No: 3), a light chain CDR1 comprising the sequence RASQSISDYLH (SEQ ID No: 4), a light chain CDR2 comprising the sequence YASQSIS (SEQ ID No: 5), and a light chain CDR3 comprising the sequence QNGHSFPLT (SEQ ID No: 6), or analogues thereof comprising no more than 5% amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence.

[0018] According to certain specific embodiments, the antibody or fragment comprises a set of 6 CDR sequences, which consist of the following sequences:

[0019] i. Heavy chain CDR1 having the sequence described in SEQ ID No: 1;

[0020] ii. Heavy chain CDR2 having the sequence described in SEQ ID No: 2;

[0021] iii. A heavy chain CDR3 having the sequence described in SEQ ID No: 3;

[0022] iv. A light chain CDR1 having the sequence described in SEQ ID No: 4;

[0023] v. A light chain CDR2 having the sequence set forth in SEQ ID No: 5; and

[0024] vi. A light chain CDR3 having the sequence set forth in SEQ ID No: 6.

[0025] According to some embodiments, the antibody or a fragment thereof comprises the heavy chain variable region set forth in SEQ ID No: 7, or an analogue or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

[0026] According to some embodiments, the antibody or a fragment thereof comprises the light chain variable region set forth in SEQ ID No: 8 or an analogue thereof having at least 90% sequence identity with the sequence of the light chain variable region.

[0027] According to some embodiments, the antibody or a fragment thereof comprises the light chain variable region set forth in SEQ ID No: 12 or an analogue thereof having at least 90% sequence identity with the sequence of the light chain variable region.

[0028] According to a particular embodiment, the antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID No: 7 and a light chain variable region having the sequence set forth in SEQ ID No: 8, or an analogue thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

[0029] According to a particular embodiment, the antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID No: 7 and a light chain variable region having the sequence set forth in SEQ ID No: 12, or an analogue thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

[0030] According to a particular embodiment, the antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID No: 7 and two distinct light chain variable regions having the sequences set forth in SEQ ID No: 8 and SEQ ID No: 12, or analogues thereof having at least 90% sequence identity with the light chain and / or heavy chain sequences.

[0031] According to some implementations, the antibody is an isolated monoclonal antibody.

[0032] According to some embodiments, the antibody or fragment thereof is at least 5x10 -9 M has an affinity for human NKp46. According to other embodiments, the antibody or antibody fragment is at least 10... -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 10 - 12 M, 10 -13 M or even higher affinity binds to human NKp46. According to some embodiments, the antibody or antibody fragment is in the form of 10... -9 M to 10 -14 The antibody or antibody fragment exhibits affinity within the M range for binding to human NKp46. According to certain embodiments, the antibody or antibody fragment is in the form of 10... -9 M to 10 -10The antibody or antibody fragment exhibits affinity within the M range for binding to human NKp46. According to certain embodiments, the antibody or antibody fragment is in the form of 10... -10 M to 10 -11 The antibody or antibody fragment exhibits affinity within the M range for binding to human NKp46. According to certain embodiments, the antibody or antibody fragment is in the form of 10... -11 M to 10 -16 The antibody or antibody fragment exhibits affinity in the M range for binding to human NKp46. According to certain embodiments, the antibody or antibody fragment binds at a concentration higher than 10... -11 M has an affinity for human NKp46. According to some embodiments, the antibody or antibody fragment binds at a concentration higher than 10. -12 M's affinity binds to human NKp46. Each possibility represents an independent implementation of the invention.

[0033] According to some embodiments, the antibody or fragment thereof is at least 5x10 -11 M has an affinity for recognizing the D2 domain of human NKp46 (SEQ ID No: 13). According to certain embodiments, the antibody or a fragment thereof is at least 5 x 10 -13 M has an affinity for recognizing the D2 domain of human NKp46. According to some embodiments, the antibody or a fragment thereof is at least 5 x 10 -14 M has an affinity for recognizing the D2 domain of human NKp46. According to some embodiments, the antibody or a fragment thereof is at least 5 x 10 -15 M has an affinity for recognizing the D2 domain of human NKp46. According to some embodiments, the antibody or antibody fragment is in the form of 10... -13 M to 10 -16 Affinity recognition of the D2 domain of human NKp46 within the M range.

[0034] Analogs and derivatives of the isolated antibodies and fragments are also within the scope of this invention.

[0035] According to some embodiments, the antibody or antibody fragment analog has at least 90% sequence identity with the hypervariable region of the reference antibody sequence.

[0036] According to certain embodiments, the isolated antibody or an analogue or derivative thereof has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with the variable region of the reference antibody sequence. Each possibility represents an independent embodiment of the invention.

[0037] According to certain embodiments, the antibody or antibody fragment according to the invention comprises the heavy chain variable region set forth in SEQ ID No: 7 or an analogue having at least 95% sequence similarity to the sequence.

[0038] According to some embodiments, the antibody or antibody fragment comprises the light chain variable region set forth in SEQ ID No: 8 and / or SEQ ID No: 12 or an analogue having at least 95% sequence similarity to the sequence.

[0039] According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID No: 7 and the light chain comprises SEQ ID No: 8 or SEQ ID No: 12. Analogs of the antibody or fragment having at least 95% sequence similarity to the heavy chain or light chain are also included.

[0040] According to some embodiments, the analog has at least 96, 97, 98, or 99% sequence similarity or identity to the variable region of the antibody light or heavy chain described above. According to some embodiments, the analog comprises no more than one amino acid substitution, deletion, or addition to one or more CDR sequences of the hypervariable region, i.e., any of the CDR sequences set forth in SEQ ID Nos: 1, 2, 3, 4, 5, and 6. Each possibility represents an independent embodiment of the invention. According to some embodiments, the amino acid substitution is a conservative substitution.

[0041] According to some embodiments, the antibody or antibody fragment includes a hypervariable region (HVR) having light and heavy chain regions as defined above, wherein 1, 2, 3, 4, or 5 amino acids are replaced, deleted, and / or added. Each possibility represents an independent embodiment of the invention.

[0042] According to some embodiments, the antibody or antibody fragment comprises an HVR having light chain and heavy chain regions as defined above, wherein one amino acid is substituted. According to a particular embodiment, the antibody or antibody fragment comprises a CDR as defined above, wherein one amino acid is substituted.

[0043] According to a particular embodiment, the antibody is selected from chimeric antibodies and antibody fragments that at least include an antigen-binding portion of the antibody. According to a particular embodiment, the antibody is a chimeric antibody. According to other embodiments, the chimeric antibody includes a human constant region. According to a particular embodiment, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single-chain variable region (scFV), single-chain antibody (scab), "double-chain antibody", and "linear antibody". Each possibility represents an independent embodiment of the invention.

[0044] According to some embodiments, the antibody or antibody fragment comprises a constant region selected from the following: mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3, and human IgG4. Each possibility represents an independent embodiment of the invention.

[0045] According to certain specific implementations, the monoclonal antibody is a chimeric monoclonal antibody.

[0046] According to some implementations, the chimeric antibody contains a constant region derived from humans.

[0047] According to some embodiments, the human constant region of the chimeric antibody is selected from human IgG1, human IgG2, human IgG3, and human IgG4.

[0048] According to some embodiments, the human constant region of the chimeric antibody is selected from human IgG1 and human IgG2.

[0049] This invention also provides a set of six humanized mAbs containing any mAb described herein.

[0050] According to certain embodiments, the humanized antibody or antibody fragment comprises a set of 6 CDRs, wherein: the heavy chain CDR1 sequence comprises the sequence EYSMH (SEQ ID No: 1), the heavy chain CDR2 comprises the sequence GISPNSGGTSYNQKFKG (SEQ ID No: 2), the heavy chain CDR3 comprises the sequence RDFHSSFDY (SEQ ID No: 3), the light chain CDR1 comprises the sequence RASQSISDYLH (SEQ ID No: 4), the light chain CDR2 comprises the sequence YASQSIS (SEQ ID No: 5), and the light chain CDR3 comprises the sequence QNGHSFPLT (SEQ ID No: 6), or analogs thereof comprising no more than 5% amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence.

[0051] According to some embodiments, the humanized antibody or antibody fragment comprises a set of 6 CDR sequences consisting of the following:

[0052] i. Heavy chain CDR1 having the sequence described in SEQ ID No: 1;

[0053] ii. Heavy chain CDR2 having the sequence described in SEQ ID No: 2;

[0054] iii. A heavy chain CDR3 having the sequence described in SEQ ID No: 3;

[0055] iv. A light chain CDR1 having the sequence described in SEQ ID No: 4;

[0056] v. A light chain CDR2 having the sequence set forth in SEQ ID No: 5; and

[0057] vi. A light chain CDR3 having the sequence set forth in SEQ ID No: 6.

[0058] According to some embodiments, the humanized antibody comprises a heavy chain variable region sequence SEQ ID No: 14 or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

[0059] According to some embodiments, the humanized antibody comprises a light chain variable region sequence selected from SEQ ID No: 15 and SEQ ID No: 16, or an analog or derivative thereof having at least 90% sequence identity with the light chain variable region sequence.

[0060] According to some embodiments, the humanized antibody comprises a set of heavy chains and light chains, wherein the set is selected from:

[0061] i.SEQ NOs: 14 and 15; and

[0062] ii.SEQ NOs: 14 and 16.

[0063] According to certain embodiments, a conjugate comprising an antibody or an antibody fragment thereof described herein by the six CDR sequences is provided.

[0064] The antibody or fragment thereof according to the invention may be attached to a radioactive component or an identifiable component.

[0065] According to another aspect of the invention, a polynucleotide sequence encoding an antibody with high affinity and specificity for human NKp46 is provided, as well as a vector and a host cell carrying such polynucleotide sequences.

[0066] According to certain implementations, a multinucleotide sequence encoding the amino acid sequences of the heavy chain variable region and the light chain variable region as defined above is provided.

[0067] According to certain embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope in the human NKp46 protein that binds to the following antibodies: (i) an antibody having the heavy chain variable region of SEQ ID No: 7 and the light chain variable region of SEQ ID No: 8 (referred to herein as clone P4); or (ii) an antibody having the heavy chain variable region of SEQ ID No: 7 and the light chain variable region of SEQ ID No: 8 and / or SEQ ID No: 12 (referred to herein as clone K3).

[0068] According to certain embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising a sequence selected from the sequences described below: (i) SEQ ID No: 7 and SEQ ID No: 8; and (ii) SEQ ID No: 7 and SEQ ID No: 12. Each possibility represents an independent embodiment of the invention.

[0069] According to some other embodiments, the polynucleotide sequence according to the present invention encodes an antibody or antibody fragment or chain thereof comprising a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0070] According to some implementations, the polynucleotide sequence defined above encodes at least one antibody chain or antibody fragment containing at least an antigen-binding portion.

[0071] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody heavy chain variable region comprising the sequence set forth in SEQ ID No: 7, or a variant thereof having at least 90% sequence identity.

[0072] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody light chain variable region comprising the sequence set forth in SEQ ID No: 8, or a variant thereof having at least 90% sequence identity.

[0073] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody light chain variable region comprising the sequence set forth in SEQ ID No: 12 or a variant thereof having at least 90% sequence identity.

[0074] According to some embodiments, the antibody is a monoclonal antibody having two different light chains. According to some embodiments, the antibody is a monoclonal antibody having the light chains described in SEQ ID No: 8 and SEQ ID No: 12.

[0075] According to certain embodiments, the present invention provides a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

[0076] On the other hand, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention. According to some embodiments, the nucleic acid construct is a plasmid.

[0077] According to some embodiments, the plasmid comprises at least one polynucleotide sequence selected from the sequences described in SEQ ID No: 9, SEQ ID No: 10, and SEQ ID No: 11. Each possibility represents an independent embodiment of the invention.

[0078] On the other hand, the present invention provides a cell capable of producing antibodies or antibody fragments comprising a specific CDR sequence and / or a specific heavy chain and light chain variable region as defined above.

[0079] According to some embodiments, a cell is provided that comprises at least one polynucleotide sequence disclosed above.

[0080] According to some embodiments, the cells that produce monoclonal antibodies are hybridoma cells.

[0081] According to one aspect, the present invention provides a multispecific binding molecule comprising a binding site of an antibody or antibody fragment described herein.

[0082] According to some embodiments, the multispecific molecule comprises a binding site containing a group of 6 CDRs, wherein: HCCDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0083] According to some embodiments, the multispecific molecule further includes different binding sites for the NKp46 adaptor molecule. According to some embodiments, the multispecific molecule further includes binding sites for NK cell adaptor molecules. According to some embodiments, the multispecific binding molecule further includes binding sites specifically targeting other NK adaptors CD160 or CD16.

[0084] According to some embodiments, the multispecific binding molecule further includes a binding site that specifically targets tumor antigens, viral antigens, bacterial antigens, or fungal antigens.

[0085] According to some embodiments, the multispecific binding molecule further includes a binding site specifically targeting tumor antigens. According to a particular embodiment, the multispecific binding molecule further includes a binding site specifically targeting blood or solid tumors containing, but not limited to, PDL-1, CD38, BCMA, or GPC3.

[0086] According to other embodiments, the multispecific binding molecule further includes a binding site specifically targeting the viral antigen. According to a particular embodiment, the multispecific binding molecule further includes a binding site specifically targeting spike protein 1. According to a particular embodiment, the multispecific binding molecule further includes a binding site specifically targeting ACE2.

[0087] According to some embodiments, the fungal antigen is selected from Epa1, Epa6, and Epa7.

[0088] According to some embodiments, the multispecific binding molecule is fused to or conjugated with a cytokine. According to a particular embodiment, the multispecific binding molecule is fused to or conjugated with a cytokine selected from IL-2, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, TNF-α, GM-CSF, TGF-β, and VEGF. Each possibility represents an independent embodiment of the invention.

[0089] According to some embodiments, the multispecific molecule is a bispecific antibody comprising a binding site containing a set of six CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LCCDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0090] According to some embodiments, the bispecific antibody further includes a binding site that specifically targets tumor antigens, viral antigens, bacterial antigens, or fungal antigens.

[0091] According to some embodiments, the bispecific antibody further includes a binding site specifically targeting the viral antigen. According to some embodiments, the viral antigen is an antigen selected from viruses such as coronavirus, influenza virus, human metapneumovirus (HMPV), human cytomegalovirus (HCMV), Sendai virus, Newcastle disease virus, and poxvirus. Each possibility represents an independent embodiment of the invention.

[0092] According to some implementations, the virus is a mammalian or avian virus.

[0093] According to certain specific implementations, the mammalian virus is a human virus.

[0094] According to some embodiments, the coronavirus is selected from SARS and MERS viruses. According to a particular embodiment, the coronavirus is SARS-CoV-2 (COVID-19).

[0095] According to certain specific implementations, the mammalian influenza virus is selected from human influenza virus and swine influenza virus.

[0096] According to some embodiments, the multispecific molecule is a trispecific antibody that further includes a binding site against the NKp46 adaptor molecule. According to some embodiments, the multispecific molecule is a trispecific antibody that further includes a binding site against the NK adaptor. According to some embodiments, the multispecific molecule is a trispecific antibody that further includes a binding site against CD160 or CD16.

[0097] According to some embodiments, the multispecific molecule is a trispecific antibody that also includes a binding site against a tumor, viral antigen, bacterial antigen, or fungal antigen.

[0098] According to some embodiments, the trispecific antibody is fused with a cytokine. According to a particular embodiment, the trispecific antibody is fused with IL15.

[0099] According to a particular embodiment, the trispecific binding molecule comprises a binding site for the following antibodies or fragments: (i) antibodies or antibody fragments described herein; (ii) antibodies or fragments thereof specifically targeting CD160 or CD16; and (iii) antibodies or fragments thereof specifically targeting tumor antigens.

[0100] According to some embodiments, the multispecific binding molecule is a polypeptide or a polypeptide multimer. According to some embodiments, each polypeptide is independently selected from monospecific, bispecific, and trispecific polypeptides.

[0101] According to some embodiments, the trispecific binding molecule consists of or contains one or more scFv molecules.

[0102] According to another aspect, the present invention provides a pharmaceutical composition comprising at least one antibody or antibody fragment thereof that recognizes the human NKp46 D2 domain with high affinity and specificity as an active ingredient, and optionally at least one pharmaceutically acceptable excipient, diluent, salt or carrier, wherein the at least one antibody or antibody fragment does not block the interaction between NKp46 and its ligand.

[0103] According to some embodiments, the pharmaceutical composition comprises at least one monoclonal antibody, the monoclonal antibody comprising a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0104] According to some embodiments, the pharmaceutical composition comprises an antibody or a fragment thereof containing the heavy chain variable region described in sequence No: 7.

[0105] According to some embodiments, the pharmaceutical composition comprises an antibody or a fragment thereof, the antibody or fragment comprising a light chain variable region having a sequence selected from SEQ ID No: 8, SEQ ID No: 12, and both. Each possibility represents an independent embodiment of the invention.

[0106] According to a particular embodiment, the pharmaceutical composition comprises an antibody or a fragment thereof, the antibody or fragment thereof comprising a heavy chain variable region having the sequence set forth in SEQ ID No: 7 and a light chain variable region having the sequence set forth in SEQ ID No: 8 or SEQ ID No: 12.

[0107] The invention also provides a single-chain variable region (scFv) molecule of the antibody. The scFv molecule contains an antigen-binding site of the antibody expressed in a single polypeptide chain. According to some embodiments, the invention provides an scFv molecule containing heavy and light chain variable regions of an anti-NKp46 antibody. According to some embodiments, the scFv contains a hinge region between the two variable regions.

[0108] According to some embodiments, the scFv includes an NKp46 binding site comprising a set of six CDRs, wherein: HCCDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0109] According to some implementations, the scFv contains the sequence set forth in SEQ ID No: 14 or a variant thereof having at least 90% sequence identity.

[0110] According to some implementations, the scFv contains the sequence set forth in SEQ ID No: 15 or a variant thereof having at least 90% sequence identity.

[0111] According to some implementations, the scFv contains the sequence set forth in SEQ ID No: 16 or a variant thereof having at least 90% sequence identity.

[0112] According to some implementations, the scFv includes the sequences set forth in SEQ ID No: 14 and SEQ ID No: 15.

[0113] According to some implementations, the scFv includes the sequences set forth in SEQ ID No: 14 and SEQ ID No: 16.

[0114] According to another aspect of the invention, a chimeric antigen receptor (CAR) is provided, comprising an extracellular portion (binding domain) capable of binding to NKp46, said CAR comprising an extracellular portion containing any of the antibodies or fragments thereof provided herein.

[0115] According to some embodiments, the CAR includes an NKp46 binding site comprising a set of six CDRs, wherein: HCCDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0116] According to some embodiments, the CAR comprises a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LCCDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6); as well as a transmembrane domain and an intracellular T cell signaling domain.

[0117] According to certain implementations, a lymphocyte engineered to express the CAR described herein is provided.

[0118] According to some embodiments, a T cell engineered to express the CAR described herein is provided, and is referred to as CAR-T. According to some embodiments, an NK cell engineered to express the CAR described herein is provided, and is referred to as CAR-NK.

[0119] According to some embodiments, a population of lymphocytes engineered to express the CAR described herein is provided. According to a particular embodiment, a population of T-cells or NK-cells engineered to express the CAR described herein is provided.

[0120] According to some embodiments, the CAR includes a single-chain variable region (scFv) which contains the heavy and light chain variable regions of the antibody described herein.

[0121] According to some embodiments, the CAR includes at least one protein domain selected from the scFv sequence, CD8 stem domain, CD28™ domain, 41BB domain, and CD3ζ (CD3Z, Zetta) domain. According to some embodiments, the CAR includes the scFv domain.

[0122] According to some embodiments, the CAR comprises an scFv sequence containing the NKp46 binding site of the antibody disclosed herein, and at least one domain selected from the following: CD8 stem domain, CD28™ domain, 41BB domain, and CD3Z domain.

[0123] According to one aspect, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one lymphocyte comprising a CAR described herein.

[0124] According to certain embodiments, the pharmaceutical composition comprises at least one antibody or antibody fragment thereof described herein, CAR or multispecific antibody.

[0125] According to certain embodiments, the pharmaceutical compositions according to the invention are used in cancer immunotherapy or to enhance the immune response against viral infections.

[0126] According to certain embodiments of the invention, the cancer is metastatic cancer. According to certain embodiments, the pharmaceutical composition according to the invention is used to inhibit the formation or distribution of metastatic tumors or reduce the total number of metastatic tumors in a subject.

[0127] According to certain embodiments of the present invention, the cancer is selected from leukemia, lymphoma, melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, kidney cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, kidney cancer, laryngeal cancer, pharyngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial carcinoma, glioblastoma, sarcoma, and myeloid tumors. Each possibility represents an independent embodiment of the present invention.

[0128] In some embodiments, solid tumors are treated with CAR-T or CAR-NK cells. In a particular embodiment, solid tumors are treated with CAR-T cells. In other embodiments, hematological malignancies are treated with CAR-NK or CAR-T cells. In a particular embodiment, hematological malignancies are treated with CAR-NK cells.

[0129] According to some embodiments, the cancer is selected from melanoma, breast cancer, colorectal cancer, kidney cancer, lung cancer, prostate cancer, and brain cancer. Each possibility represents an independent embodiment of the invention.

[0130] In other embodiments, the cancer is a blood cancer. In some embodiments, the pharmaceutical composition is used in conjunction with human lymphocytes to treat cancer.

[0131] In some embodiments, the human lymphocytes are killer cells selected from T cells, NK cells, and NKT cells. Each possibility represents an independent embodiment of the invention.

[0132] According to some embodiments, the killer cells are autologous or allogeneic.

[0133] According to another aspect, the present invention provides a method for treating cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising at least one antibody or antibody fragment thereof described herein, a CAR or a multispecific antibody.

[0134] The cancer is as described above.

[0135] According to certain embodiments of the present invention, the effective therapeutic dose results in a reduction in tumor size or number of metastatic tumors in the subject.

[0136] According to some embodiments, the method of treating cancer includes administering or performing at least one additional anticancer therapy. According to some embodiments, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

[0137] According to some embodiments, the method of treating cancer includes administering at least one antibody described herein, an antibody fragment thereof, a trispecific binding molecule, a CAR or bispecific antibody, and additional anticancer agents. According to some embodiments, the additional anticancer agents are selected from immunomodulators, activated lymphocytes, kinase inhibitors, and chemotherapy agents.

[0138] According to other embodiments, the immunomodulator is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than human NKp46.

[0139] According to some embodiments, the immunomodulator is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule selected from: human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also known as CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and any other checkpoint molecule or combinations thereof. Each possibility represents an independent embodiment of the invention. According to some embodiments, the additional immunomodulator is an antibody against PD-1. According to some embodiments, the additional immunomodulator is an antibody against CTLA-4.

[0140] According to certain embodiments, the anticancer agent is selected from: cetuximab, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, nitrogen mustard, melphalan, thiotepa, dacarbazine, bleomycin, actinomycin D, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, procainoxine, etoposide, teniposide, and any combination thereof. Each possibility represents an independent embodiment of the invention.

[0141] According to some embodiments, the anticancer agent is an epidermal growth factor receptor (EGFR) inhibitor. According to some embodiments, the EGFR inhibitor is selected from cetuximab. Panitumumab and nexitozumab Each possibility represents an independent implementation of the invention.

[0142] According to certain embodiments of the present invention, the subject is a human subject.

[0143] According to certain embodiments of the invention, the use also includes the use of agents that downregulate the activity or expression of immunosuppressive receptors.

[0144] According to certain embodiments of the present invention, the immunosuppressive receptor is selected from PD-1, TIGIT, PVR, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55 (CD 160), LAIR1, SIGLEC10, and 2B4. Each possibility represents an independent embodiment of the present invention.

[0145] According to some implementations, the method of treating cancer includes enabling the subject to prevent or reduce the formation, growth, or spread of metastatic tumors.

[0146] According to another aspect, the present invention provides a method for treating an infection in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, the pharmaceutical composition comprising at least one antibody, an antibody fragment thereof, a multispecific binding molecule, or a CAR described herein. Each possibility represents an independent embodiment of the invention.

[0147] According to some implementations, the infection is selected from viral infection, bacterial infection, and fungal infection.

[0148] According to some embodiments, the viral infection is an infection with a virus selected from coronaviruses, influenza viruses, human metapneumoviruses (HMPV), human cytomegaloviruses (HCMV), Sendai viruses, Newcastle disease viruses, and poxviruses. Each possibility represents an independent embodiment of the invention.

[0149] According to some implementations, the viral infection is caused by a mammalian or avian virus.

[0150] According to certain specific implementations, the mammalian virus is a human virus.

[0151] According to some implementations, the coronavirus is selected from SARS, MERS, and COVID-19.

[0152] According to certain specific implementations, the mammalian influenza virus is selected from human influenza virus and swine influenza virus.

[0153] According to one aspect, the present invention provides a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of NKp46 in a biological sample of the subject using at least one antibody described herein.

[0154] According to another aspect, the present invention also includes a method for determining or quantifying NKp46 in a sample, the method comprising contacting a biological sample with an antibody or antibody fragment and measuring the level of complex formation, wherein the antibody or antibody fragment comprises a set of six CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0155] According to some embodiments, the antibody comprises a set of heavy chains and light chains, wherein the set is selected from:

[0156] i.SEQ NOs: 14 and 15; and

[0157] ii.SEQ NOs: 14 and 16.

[0158] According to some embodiments, the determination and quantification methods can be performed in vitro or ex vivo, or can be used to diagnose conditions associated with NKp46 expression. The antibodies described in this invention can also be used to formulate screening methods. For example, enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA), as well as methods such as IHC or FACS, can be constructed to measure the levels of secreted or cell-bound peptides using the antibodies and methods known in the art.

[0159] According to some embodiments, the method for detecting or quantifying the presence of NKp46 expressed on cells or secreted into a biological culture medium includes the following steps:

[0160] i. Incubate the sample with an antibody specifically targeting human NKp46 as described herein, or an antibody fragment containing at least an antigen-binding moiety thereof; and

[0161] ii. Use a detectable probe to detect the binding of NKp46.

[0162] According to some embodiments, the method further includes the following steps:

[0163] iii. Compare the amount in (ii) with a standard curve obtained from a reference sample containing a known amount of NKp46; and

[0164] iv. Calculate the amount of NKp46 in the sample from the standard curve.

[0165] According to certain specific implementations, the sample is a bodily fluid.

[0166] According to some embodiments, the method is performed in vitro or ex vivo.

[0167] According to some embodiments, the antibody or antibody fragment for detection or diagnosis comprises a set of 6 CDRs, wherein: heavy chain CDR1 contains the sequence EYSMH (SEQ ID No: 1), heavy chain CDR2 contains the sequence GISPNSGGTSYNQKFKG (SEQ ID No: 2), heavy chain CDR3 contains the sequence RDFHSSFDY (SEQ ID No: 3), light chain CDR1 contains the sequence RASQSISDYLH (SEQ ID No: 4), light chain CDR2 contains the sequence YASQSIS (SEQ ID No: 5), and light chain CDR3 contains the sequence QNGHSFPLT (SEQ ID No: 6).

[0168] A kit for measuring the expression or presence of NKp46 in biological samples is also provided, comprising at least one antibody or antibody fragment according to the present invention. According to some embodiments, the kit comprises an antibody or antibody fragment comprising a set of six CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0169] According to one aspect, the present invention provides a kit for cancer diagnosis or staging, the diagnostic kit comprising an antibody or an antibody fragment thereof disclosed herein.

[0170] Other embodiments and the full scope of applicability of the invention will become apparent from the detailed description given below. However, it should be understood that while the detailed description and specific embodiments indicate preferred embodiments of the invention, they are provided merely as illustrations, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Attached Figure Description

[0171] Figure 1 Identification of the binding sites of anti-NKp46 mAb. The figure illustrates the binding of anti-NKp46 mAb (commercial anti-NKp46 antibodies (9E2), 461-G1, O2, K3, and P4) to the NKp46 D1 domain (D1-Ig), NKp46 D2 domain (D2-Ig), or intact NKp46 protein-Ig (46-Ig).

[0172] Figure 2 Downregulation of NKp46 from the surface of NK cells. Large cultures of activated NK cells were incubated with the indicated anti-NKp46 mAbs (9E2, 461-G1, O2, K3, P4) at 4°C (black bars) or 37°C (red bars) for 8 hours, followed by FACS staining with a secondary antibody (anti-m647 antibody). Solid gray bars represent cells treated at 4°C stained with the secondary antibody alone. Cells treated at 37°C have a similar background (not shown). The figure shows a representative experiment from five experiments performed.

[0173] Figure 3 Antibody dose response. Primary activated large numbers of human NK cells from two donors, NK1 and NK2, were stained with anti-NKp46 mAb (9E2, 461-G1, K3, P4) using FACS (color bar graph). Solid gray bars represent NK cells stained with only the secondary antibody. Full gray bars = secondary antibody only (m-647). Results are presented for different antibody concentrations (0.5 μg, 0.1 μg, 0.05 μg, 0.01 μg, 0.005 μg, 0.001 μg, 0.005 μg, or 0.0001 μg / well) (higher concentrations result in stronger staining).

[0174] Figures 4A-4B Humanized P4 with mutated IgG4 ( Figure 4A ) and K3 ( Figure 4B Affinity (nM) of antibody to NKp46.

[0175] Figures 5A-5DThe binding affinity of NKp46 antibodies P4 and K3, as well as humanized NKp46 antibodies, to the NKp46 D2 domain is shown. The structures of NKp46, the D1 domain, and the D2 domain are presented respectively. Figures 5A-5C The affinity value (Kd(M)) with the D2 domain is shown in... Figure 5D middle. Invention Details

[0177] This invention provides antibodies with high affinity and specificity for human NKp46. It also provides multispecific binding molecules comprising the binding site of the antibody described herein and chimeric antigen receptors (CARs) comprising the binding site of the novel antibody. The antibodies themselves do not affect immune cells carrying NKp46 and can therefore be used as part of a construct or multispecific molecule to cascade said immune cells to specific targets, such as tumor cells or virus-infected cells.

[0178] When used herein, the term "NKp46" refers to natural killer protein 46, also known as natural cytotoxicity trigger receptor 1 (NCR1) or CD335. NKp46 has two Ig-like extracellular domains (D1 and D2), followed by a stem region of ~40 residues, a class I transmembrane domain, and a short cytoplasmic tail region. NKp46 is an important NK cell activation receptor involved in the elimination of HCV and other virally infected cells and has been shown to regulate the interaction of NK cells with other immune cells, including T cells and dendritic cells (DCs). The exemplary NKp46 described according to the present invention is illustrated in UniPort and GenBank symbol or accession number UniProtKB-O76036 (NCTR1_HUMAN) and Gene ID: 9437. NKp46 has two Ig-like extracellular domains (D1 and D2), followed by a stem region of ~40 residues, a class I transmembrane domain, and a short cytoplasmic tail region. The D2 domain (or NKp46D2) contains 134 amino acid residues (corresponding to residues 121-254 of the full-length protein of isoform a). An example of a human D2 domain sequence can be found in SEQ ID No: 13.

[0179] The antibody or a fragment thereof according to the present invention binds to an epitope in NKp46. Specifically, the antibody binds to an epitope within the D2 domain of the NKp46 protein.

[0180] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments long enough to exhibit the desired biological activity, namely binding to human NKp46.

[0181] When used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen molecule that specifically reacts with a particular antibody. Using methods known in the art, peptide sequences derived from epitopes can be used alone or in combination with a vector moiety to immunize animals and generate additional polyclonal or monoclonal antibodies. Isolated peptides derived from epitopes can be used in diagnostic methods to detect antibodies.

[0182] It should be noted that antibody affinity can be quantified using known methods, such as surface plasmon resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M., Surface plasmon resonance imaging for affinity-based biosensors, Biosens Bioelectron. 2010, 25:957-66), and can be calculated using, for example, the dissociation constant Kd, such that a lower Kd reflects a higher affinity. Unless otherwise specified, any numerical value used in this disclosure for “affinity” is a dissociation constant.

[0183] When used herein, the term "antibody" can refer to any polypeptide or polypeptide complex that includes one or more immunoglobulin-like antigen-binding domains. The term "antibody" can include complete antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), multispecific antibodies and their proteolytic fragments such as Fab or F(ab')2 fragments, single-chain antibodies and their complexes.

[0184] Immunoglobulins consist of two heavy chains and two light chains linked together by disulfide bonds, with each light chain connected to the corresponding heavy chain in a "Y" configuration via disulfide bonds. Antibodies undergo proteolytic digestion into Fv (variable fragment) and Fc (crystallizable fragment) domains. The antigen-binding domain (Fab) includes regions where the polypeptide sequence varies. The term F(ab')2 represents the two Fab' arms linked together by disulfide bonds. Each heavy chain has a variable domain (V) at one end. H ), followed by multiple constant structure domains (C H Each light chain has a variable structural domain (V) at one end. L ) and has a constant structural domain (C) at its other end. LThe variable domain of the light chain is aligned with the variable domain of the heavy chain, and the constant domain of the light chain is aligned with the first constant domain (CH1) of the heavy chain. Each pair of variable domains of the light and heavy chains forms an antigen-binding site. The domains on the light and heavy chains have the same overall structure, and each domain contains four relatively conserved frame regions connected by three hypervariable domains called complementarity-determining regions (CDRs 1-3). These domains contribute to the specificity and affinity of the antigen-binding site.

[0185] The identification or determination of CDRs derived from a given heavy or light chain variable sequence is typically performed using one of several methods known in the art. For example, such determination is performed according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970; 132:211–50) and IMGT (Lefranc MP et al., Dev Comp Immunol, 2003, 27:55-77).

[0186] When the term "CDR having a certain sequence" or similar term is used, it includes the option that the CDR contains the specified sequence and the option that the CDR is composed of the specified sequence.

[0187] Antigen specificity of an antibody is based on the hypervariable region (HVR), which is a unique CDR sequence formed by the light and heavy chains together to form the antigen-binding site.

[0188] The heavy chain isoform (γ, α, δ, ε, or μ) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain has two isoforms (κ or λ). Both isoforms are present in all antibody classes.

[0189] The present invention also provides chimeric antibodies comprising a human-derived constant region and humanized antibodies specifically targeting human NKp46. Advantageously, the humanized antibodies avoid the risk of adverse immune responses to said antibodies and are therefore safe for in vivo use in humans.

[0190] antibody fragments

[0191] An "antibody fragment" contains only a portion of a complete antibody, typically including the antigen-binding site of the complete antibody, and thus retains the ability to bind antigens. Examples of antibody fragments covered by this definition include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having a single arm of antibody with VL and VH domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 1989, 341, 544-546); (vii) isolated CDR regions; (viii) F(ab')2 fragments, which are bivalent fragments comprising two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science). (x) "Double-chain antibodies" having two antigen-binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (see, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "Linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., Protein Eng., 1995, 8, 1057-1062; and U.S. Patent No. 5,641,870.

[0192] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are produced by the proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, the antibody fragments can be isolated from an antibody phage library. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for the production of antibody fragments will be readily apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv).

[0193] Single-chain antibodies can be single-chain complex polypeptides with antigen-binding ability and containing amino acid sequences homologous to or similar to the variable regions of immunoglobulin light and heavy chains, i.e., linked V... H -V L Or a single-chain Fv (scFv). The technology used to produce single-chain antibodies (US Patent No. 4,946,778) can be adapted to produce single-chain antibodies against NKp46.

[0194] When used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possible presence of naturally occurring mutations, albeit in small amounts. Monoclonal antibodies are highly specific and target a single antigen. Furthermore, unlike polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. mAbs can be obtained by methods known to those skilled in the art. For example, the monoclonal antibody used according to the invention can be prepared by a hybridoma method first described by Kohler et al., Nature 1975,256,495, or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J.Mol.Biol., 1991, 222: 581-597.

[0195] The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of functional variable heavy chain (VH) and variable light chain (VL) genes, as well as the design and cloning of synthetic DNA sequences optimized for expression in recombinant bacteria, are described in Fields et al., 2013, 8(6):1125-48.

[0196] The mAbs of this invention can be any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained through in vivo production, wherein cells derived from an individual hybridoma are intraperitoneally injected into primiparously immunized Balb / c mice to produce ascites containing a high concentration of the desired mAb. mAbs can be purified from these ascites or from culture supernatants using methods known to those skilled in the art.

[0197] Anti-idiotype antibodies that have specific immunoreactivity with the hypervariable region of the antibody of the present invention have also been taken into consideration.

[0198] This invention provides a monoclonal antibody or antibody fragment comprising an antigen-binding domain (ABD), said antigen-binding domain comprising three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity to the ABD of a monoclonal mouse antibody comprising the following chains: (i) a variable heavy chain comprising the amino acid sequence SEQ ID NO: 7 and a variable light chain comprising the amino acid sequences SEQ ID No: 8 and / or SEQ ID No: 12. Such an antibody may have an ABD domain having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or similarity, or 100% sequence identity, with respect to the corresponding ABD of antibody clone K3 or clone P4.

[0199] Sequence identity is the number of precisely matching amino acids or nucleotides between two different sequences. Sequence similarity allows for the identification of conserved substitutions of amino acids as identical amino acids. The polynucleotide sequences described herein can be codon-optimized for expression in specific cells, such as human cells. Codon optimization does not alter the coding amino acid sequence of the antibody chain but can, for example, improve expression in cells.

[0200] This invention also provides conserved amino acid variants of the antibody molecules described according to the invention. The variants described according to the invention can also be prepared to retain the overall molecular structure of the encoded protein. Given the properties of the individual amino acids constituting the disclosed protein products, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, or “conserved substitutions,” can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. When used herein, the term “antibody analog” refers to an antibody derived from another antibody through one or more conserved amino acid substitutions.

[0201] When used herein, the term "antibody variant" refers to any molecule containing the antibody of the present invention. For example, a fusion protein in which the antibody or its antigen-binding fragment is linked to another chemical entity is considered an antibody variant.

[0202] Analogs and variants of the antibody sequence are also within the scope of this application. These include, but are not limited to, conserved and non-conserved substitutions, insertions, and deletions of amino acids within the sequence. Such modifications and the resulting antibody analogs or variants are within the scope of this invention, provided they provide or even enhance the binding of the antibody to human NKp46.

[0203] Conservative substitutions of amino acids known to those skilled in the art are within the scope of this invention. Conservative amino acid substitutions include replacing one amino acid with another amino acid having the same type of functional group or side chain (e.g., aliphatic, aromatic, positively charged, negatively charged). These substitutions may enhance oral bioavailability, penetration and targeting to specific cell populations, immunogenicity, etc. Those skilled in the art will recognize that alterations to the sequence of a peptide, polypeptide, or protein, the addition or deletion of a single amino acid or a small proportion of amino acids in the coding sequence, where the alteration results in the substitution of a chemically similar amino acid, are “variants of conserved modification.” Tables providing conserved substitutions of functionally similar amino acids are well known in the art. For example, according to a table known in the art, the following six groups each contain amino acids that are conserved substitutions for each other:

[0204] 1) Alanine (A), Serine (S), Threonine (T);

[0205] 2) Aspartic acid (D), glutamic acid (E);

[0206] 3) Asparagine (N), glutamine (Q);

[0207] 4) Arginine (R), Lysine (K);

[0208] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0209] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0210] It should be emphasized that the variant strand sequence is determined by sequencing methods using specific primers. Using different sequencing methods for the same sequence, due to technical issues and different primers, may produce slightly different sequences, especially at the ends of the sequence.

[0211] When used herein, the terms "molecule having an antigen-binding portion of an antibody" and "antigen-binding fragment" are intended to include not only any isotype and produced by any animal cell line or microorganism, but also its antigen-binding reactive portion, including, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, variable portions of their heavy and / or light chains, Fab microantibodies (see, for example, WO 93 / 15210, U.S. Patent Application 08 / 256,790, WO 96 / 13583, U.S. Patent Application 08 / 817,788, WO 96 / 37621, U.S. Patent Application 08 / 999,554), and single-chain antibodies incorporating such reactive portions, as well as any other type of molecule in which such antibody reactive portions have been physically inserted. Such molecules can be provided by any known technology, including but not limited to enzyme cleavage, peptide synthesis, or recombinant technology.

[0212] The antibodies described herein specifically include “chimeric” antibodies, wherein a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such “chimeric” antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Furthermore, complementarity-determining region (CDR) transplantation can be performed to alter certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR transplantation are disclosed in US Patent 5,225,539.

[0213] Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those with a variable region derived from mouse mAbs and a constant region of human immunoglobulins. Antibodies with a variable region framework residue that is essentially derived from human antibodies (called receptor antibodies) and a CDR that is essentially derived from mouse antibodies (called donor antibodies) are also known as humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in applications and to increase yield in production, for example, in cases where mouse mAbs have higher yields from hybridoma production but higher immunogenicity in humans, human / mouse chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (e.g., PCT patent applications WO 86 / 01533, WO97 / 02671, WO 90 / 07861, WO 92 / 22653 and U.S. patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

[0214] According to some implementations, the antibody is a monoclonal antibody.

[0215] According to certain specific implementations, the monoclonal antibody is a chimeric monoclonal antibody.

[0216] According to some implementations, the chimeric antibody contains a constant region derived from humans.

[0217] According to some embodiments, the human constant region of the chimeric antibody is selected from human IgG1, human IgG2, human IgG3, and human IgG4.

[0218] According to some embodiments, the human constant region of the chimeric antibody is selected from human IgG1 and human IgG2.

[0219] According to a specific embodiment, a chimeric monoclonal antibody recognizing human NKp46 is provided, comprising a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0220] According to some implementations, the antibody is a humanized antibody.

[0221] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR, and all or substantially all of the FR amino acid residues are derived from human FR. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a humanized variant of a non-human antibody, which typically has reduced immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. According to some embodiments, certain FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or enhance antibody specificity or affinity.

[0222] "Human antibody" is an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced from a human or human cell source or from a non-human source using a human antibody library or other sequences encoding human antibodies (including human antibody libraries). The term does not include humanized forms of non-human antibodies containing non-human antigen-binding regions (e.g., non-human antibodies where all or substantially all CDRs are non-human).

[0223] Pharmacology

[0224] In pharmaceutical manufacturing and drug formulations, the active pharmaceutical agent is preferably used in conjunction with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The carrier must be pharmaceutically acceptable in a manner compatible with other components of the dosage form and not unduly harmful to the recipient. The active pharmaceutical agent is provided in an amount sufficient to effectively achieve the desired pharmacological effect as described above and in an amount suitable for achieving the desired exposure.

[0225] Typically, the antibodies of the present invention, and fragments comprising the antigen-binding portion of the antibody or comprising another polypeptide including peptide mimics, are suspended in sterile saline solution for therapeutic use. The pharmaceutical composition may optionally be formulated to control the release of the active ingredient (a molecule comprising the antigen-binding portion of the antibody) or to prolong its presence in a patient system. A large number of suitable drug delivery systems are known, including, for example, implantable drug delivery systems, hydrogels, hydroxymethyl cellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled-release formulations can be prepared by using polymers to compound or adsorb the molecules according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of stearic acid dimer and polyanhydride copolymers of sebacic acid. The rate of release of the molecules, i.e., antibodies or antibody fragments, according to the present invention from such matrices depends on the molecular weight of the molecules, the amount of the molecules within the matrices, and the size of the dispersed particles.

[0226] The pharmaceutical compositions of the present invention can be administered by any suitable means, such as intravenous, intratumoral, oral, intranasal, subcutaneous, intramuscular, intra-arterial, intra-articular, intralesional, or parenteral. Typically, intravenous (iv) administration is used for antibody delivery.

[0227] It will be apparent to those skilled in the art that the therapeutically effective amount of the molecule according to the invention will depend in particular on the dosing schedule, the unit dose of the molecule, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health, the therapeutic activity of the molecule, its persistence in the bloodstream, and the judgment of the attending physician.

[0228] When used herein, the term "therapeuticly effective amount" refers to the amount of a drug that is effective in treating a disease or condition in mammals. In the case of cancer, a therapeutically effective amount of said drug may reduce the number of cancer cells, decrease tumor size, inhibit (i.e., to some extent slow down and preferably stop) the infiltration of cancer cells into surrounding organs, inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate one or more symptoms associated with said condition to some extent. Where said drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it may have cytoseptic and / or cytotoxic properties. For cancer therapies, in vivo efficacy can be determined, for example, by assessing length of survival, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0229] Cancers suitable for treatment by this invention include, but are not limited to: leukemia or lymphoid malignancies, epithelial carcinoma, lymphoma, blastoma, and sarcoma. 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), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney 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 (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, etc.). The cancers include HL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, giant mass NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with nevus hamartoma, edema (e.g., edema associated with brain tumors) and Meigs syndrome. According to some embodiments, the cancers are selected from breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid tumors, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. Cancerous conditions suitable for treatment by the present invention include metastatic cancers.

[0230] According to a specific implementation, the cancer is selected from NK cell leukemia and large granular lymphocytic (LGL) leukemia.

[0231] According to other embodiments, the pharmaceutical composition according to the invention is used to treat cancers characterized by overexpression of NKp46.

[0232] According to other implementations, "therapeutic effective amount" refers to the amount of drug that is effective in treating viral diseases or conditions in mammals.

[0233] It is known that the molecules of the present invention, as active ingredients, are dissolved, dispersed, or mixed in pharmaceutically acceptable excipients compatible with said active ingredients. Suitable excipients are, for example, water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and combinations thereof. Other suitable carriers are known to those skilled in the art. Furthermore, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, etc.

[0234] The pharmaceutical composition according to the present invention can be administered together with an antitumor composition.

[0235] When used herein, the term "treatment" refers to both therapeutic treatment and preventative measures. Subjects requiring treatment include those who already have the condition and those who will take precautions to prevent the condition.

[0236] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, leukemia, epithelial carcinoma, lymphoma, blastoma, and sarcoma. More specific examples of cancer include leukemia, lymphoma, melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, bone marrow cancer, ovarian cancer, uterine cancer, sarcoma, bile duct cancer, or endometrial cancer.

[0237] According to some embodiments, the method of treating cancer includes administering the pharmaceutical composition as part of a treatment regimen that includes administering at least one additional anticancer agent.

[0238] According to some embodiments, the anticancer agent is selected from antimetabolites, mitotic inhibitors, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics, and combinations thereof. Each possibility represents an independent embodiment of the invention.

[0239] According to some embodiments, the antimetabolite is selected from cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from topotecan and irinotecan. According to some embodiments, the alkylating agent is selected from busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, nitrogen mustard, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from bleomycin, actinomycin D, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and procainoxine. According to some embodiments, the topoisomerase II inhibitor is selected from etoposide and teniposide. Each possibility represents an independent embodiment of the invention.

[0240] According to certain specific embodiments, the additional anticancer agent is selected from bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each possibility represents an independent embodiment of the invention.

[0241] The monoclonal antibody described in this invention can be used as part of a combination therapy in conjunction with at least one anticancer agent. According to some embodiments, the additional anticancer agent is an immunomodulator, activated lymphocytes, a kinase inhibitor, or a chemotherapy agent.

[0242] According to some embodiments, the anticancer agent is an immunomodulatory agent that acts as an agonist or antagonist, such as an antibody targeting immune checkpoint molecules.

[0243] Checkpoint immunotherapy blockade has proven to be an exciting new frontier in cancer treatment. Immune checkpoint pathways consist of a series of co-stimulatory and inhibitory molecules that work together to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors utilize certain checkpoint pathways to evade the immune system. Therefore, inhibiting these pathways has become a promising anti-cancer therapeutic strategy.

[0244] The anti-cytotoxic T-lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapy agent to show benefit in treating cancer patients. This antibody interferes with inhibitory signaling during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks negative immune regulatory signaling by the PD-1 receptor expressed by T cells. Another anti-PD-1 agent was submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Active research is currently exploring many other immune checkpoints, including: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also known as CD134), and killer cell immunoglobulin-like receptor (KIR).

[0245] According to certain specific embodiments, the immunomodulator is selected from: antibodies inhibiting CTLA-4, antibodies against human programmed cell death protein 1 (PD-1), PD-L1, and PD-L2, activated cytotoxic lymphocytes, lymphocyte activators, antibodies against CEACAM, antibodies against TIGIT, and RAF / MEK pathway inhibitors. Each possibility represents an independent embodiment of the invention. According to certain specific embodiments, the additional immunomodulator is selected from mAbs against PD-1, mAbs against PD-L1, mAbs against PD-L2, mAbs against CEACAM1, mAbs against CTLA-4, mAbs against TIGIT, PVR, interleukin-2 (IL-2), or lymphokine-activated killer (LAK) cells.

[0246] According to other embodiments, the additional anticancer agent is a chemotherapeutic agent. Chemotherapy agents that can be administered together with or separately from the antibody according to the invention may include any such agents known in the art that exhibit anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle toxins derived from periwinkle: vincristine, vinblastine, vinorelbine (paclitaxel), paclitaxel, docetaxel; alkylating agents: nitrogen mustard, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercapto-6-mercapto-6-carboxylic acid. Purines; 5-fluorouracil, cytarabine, gemcitabine; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazine; antibiotics: doxorubicin (doxorubicin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprorelin, flutamide, and medroxyprogesterone acetate.

[0247] According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxin, antibiotics, L-asparaginase, topoisomerase inhibitors, interferon, platinum coordination complexes, anthrone-substituted ureas, methylhydrazine derivatives, adrenocorticotropic hormone inhibitors, adrenocorticotropic steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from 5-fluorouracil (5-FU), folate (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. One or more chemotherapeutic agents may be used.

[0248] According to some embodiments, the pharmaceutical composition according to the present invention is used to treat cancer or to treat an infection. According to some embodiments, the infection is a viral infection, a bacterial infection, or a fungal infection.

[0249] According to certain embodiments, a pharmaceutical composition comprising at least one antibody or fragment thereof as described in the present invention and a pharmaceutical composition comprising an immunomodulator or kinase inhibitor are used for treating cancer by separate administration.

[0250] According to another aspect, the present invention provides a method for treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a monoclonal antibody or antibody fragment, CAR molecule or multispecific binding molecule according to the present invention.

[0251] When used herein, the term "effective amount" refers to a sufficient amount of the monoclonal antibody or antibody fragment that has the expected therapeutic effect when administered to a subject. The effective amount required to achieve the desired therapeutic outcome may depend on a variety of factors, including, for example, the specific type of tumor and the severity of the patient's condition, and whether the combination is further co-administered with radiation. In the context of this invention, the effective amount (dosage) of the active agent should be sufficient to achieve a beneficial therapeutic response in the subject over time, including, but not limited to, inhibition of tumor growth, reduction of tumor growth rate, prevention of tumor and metastatic tumor growth, and improved survival.

[0252] The toxicity and therapeutic efficacy of the compositions described herein can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the IC50 (concentration providing 50% inhibition) and maximum tolerated dose of the subject compound. Data obtained from these cell culture assays and animal studies can be used to formulate various dosages for human use. The dosage may vary, in particular, depending on the dosage form used, the chosen dosing regimen, the composition of the agent used for treatment, the route of administration, and other relevant factors. The precise dosage form, route of administration, and dosage can be selected by individual physicians based on the patient's condition. Depending on the severity and responsiveness of the condition being treated, administration may also be a single dose of a sustained-release composition, and the treatment process may last from days to weeks or until a cure or reduction of the disease state is achieved. The amount of composition to be administered, of course, depends on the subject being treated, the severity of the disease, the route of administration, the prescribing physician's judgment, and all other relevant factors.

[0253] The term “administer” refers to the administration of a substance, compound, or agent to a subject, which can be performed using one of a variety of different methods known to those skilled in the art. For example, a compound or agent can be administered enterically or parenterally. Enterally refers to administration via the gastrointestinal tract, including oral, sublingual, or rectal administration. Parenterally administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal (by absorption, e.g., via a skin catheter). A compound or agent can also be suitably introduced via refillable or biodegradable polymer devices or other devices such as patches and pumps, or by providing a dosage form that delivers an extended, slow, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods. In some embodiments, administration includes both direct administration (including self-administration) and indirect administration (including the act of prescribing). For example, when used herein, a physician instructing a patient to self-administer the drug or to have another person administer the drug, and / or a physician prescribing the drug to a patient, is administering the drug to the patient.

[0254] Antibodies are typically administered at doses ranging from about 0.1 to about 20 mg / kg of patient body weight, typically from about 0.5 to about 10 mg / kg, and typically from about 1 to about 5 mg / kg. In this regard, antibodies with a circulating half-life of at least 12 hours, preferably at least 4 days, and more preferably up to 21 days are preferred. Chimeric antibodies are expected to have a circulating half-life of up to 14-21 days. In some cases, it may be advantageous to administer a large loading dose during the treatment period, followed by periodic (e.g., weekly) maintenance doses. Antibodies can also be delivered via sustained-release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0255] The antibodies of this invention can be used in CAR-based adoptive immunotherapy, which utilizes engineered CAR-containing lymphocytes to treat cancer. CAR-T systems are described herein as non-limiting examples.

[0256] The T-cell therapy mentioned above utilizes chimeric antigen receptors (CARs) (i.e., CAR-T cell therapy) in the treatment of cancer or tumors. CAR-T cell therapy is a type of cellular immunotherapy that involves administering genetically engineered T cells to a cancer patient. These T cells act on tumor cells and induce apoptosis in the tumor cells. The genetically engineered T cells are prepared by expressing CARs on T cells using gene transfer technology. These CARs contain variable regions (VL and VH) of antibodies combined with fragments of intracellular domains, such as the CD3ζ chain sequence. CAR is a general term for a chimeric protein in which the light and heavy chains of the variable regions of a monoclonal antibody specifically targeting a tumor antigen are linked together and then attached to a T-cell receptor (TCR) chain at the C-terminus.

[0257] According to some embodiments, the CAR includes at least one protein domain selected from the CD8 stem domain, CD28™ domain, 41BB domain, and CD3ζ domain.

[0258] According to some embodiments, the CAR includes co-stimulatory domains derived from 4-1BB (or 41BB or CD137), ICOS, OX40, CD27, KIR2DS2, MYD88–CD40, or CD28. In some embodiments, the CAR includes signal transduction domains of CD3ζ, 41BB, and CD28.

[0259] According to some embodiments, the CAR includes a transmembrane domain (TM) selected from CD28TM, DAP12TM, CD8TM, CD3ζTM, DAP10TM, and ICOSTM.

[0260] According to some embodiments, the CAR includes a hinge region sequence. According to some embodiments, the hinge region sequence is derived from the CD8, CD28, or IgG4 hinge.

[0261] According to some embodiments, a chimeric antigen receptor (CAR) is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL) as described in the present invention. According to some embodiments, a genetically modified lymphocyte expressing a CAR on its surface is provided. According to certain specific embodiments, a genetically modified T cell (CAR-T cell) expressing a CAR on its surface is provided.

[0262] According to some embodiments, the CAR includes an NKp46 binding site, the binding site comprising a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HCCDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6), or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment.

[0263] According to certain embodiments, the analogue or derivative has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the antibody or fragment sequence.

[0264] According to some embodiments, the CAR includes an NKp46 binding site, the binding site comprising: three complementary determinant regions (CDRs) including the heavy chain (HC) variable region of SEQ ID No: 7, and three CDRs including the light chain (LC) variable region of SEQ ID No: 8 or SEQ ID No: 12.

[0265] According to some embodiments, the CAR includes an NKp46 binding site, the binding site comprising a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HCCDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

[0266] According to one aspect, the present invention provides a cell comprising the CAR described herein. According to some embodiments, the cell expresses or is capable of expressing the CAR of the present invention. According to some embodiments, the cell is a lymphocyte. According to some embodiments, the cell is selected from T cells and natural killer (NK) cells.

[0267] According to some embodiments, the cells, such as T-cells, contain nucleic acid molecules encoding the CAR of the present invention. According to other embodiments, the cells, such as T-cells, contain nucleic acid constructs containing nucleic acid molecules encoding the CAR of the present invention. According to another embodiment, the present invention provides a vector containing a nucleic acid construct or molecule encoding the CAR of the present invention. According to these embodiments, the T-cells are capable of expressing or expressing the CAR of the present invention.

[0268] According to some embodiments, lymphocytes engineered to express the CAR described herein are provided. According to some embodiments, T cells engineered to express the CAR described herein are provided.

[0269] According to other embodiments, an NK cell engineered to express the CAR described herein is provided.

[0270] The CAR of the present invention comprises a transmembrane domain (TM domain), a co-stimulatory domain, and an activation domain. According to some embodiments, the TM domain is a TM domain selected from the receptors of CD4, CD3ζ, CD28, and CD8, or an analogue having at least 85% amino acid identity with the original sequence; and / or the co-stimulatory domain is selected from the protein co-stimulatory domains selected from CD28, 4-1BB, OX40, iCOS, CD27, CD80, and CD70, their analogues having at least 85% amino acid identity with the original sequence, and any combination thereof; and / or the activation domain is selected from FcRγ and CD3ζ activation domains. According to some embodiments, the CAR comprises a leader peptide.

[0271] According to some embodiments, the present invention provides a cell composition comprising a plurality of cells of the present invention, such as CAR display cells.

[0272] According to another aspect, the present invention provides a multispecific binding molecule comprising the binding site of the antibody or antibody fragment described herein.

[0273] According to some embodiments, the bonding portion includes a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LCCDR3 is QNGHSFPLT (SEQ ID No: 6).

[0274] According to some embodiments, the multispecific antibody is a bispecific antibody.

[0275] According to some embodiments, the multispecific antibody is a trispecific antibody.

[0276] According to some embodiments, the multispecific antibody further comprises a binding site against an NK cell adaptor molecule. When used herein, the term "adaptor" refers to a molecule that can activate or enhance the activity of immune cells. According to some embodiments, the multispecific antibody further comprises a binding site specifically against CD160. According to some embodiments, the multispecific antibody further comprises a binding site specifically against CD16.

[0277] CD160 is a natural killer cell receptor and a member of the immunoglobulin superfamily. It is a 27 kDa glycoprotein whose expression is closely associated with peripheral blood NK cells and CD8 T lymphocytes with cytolytic effector activity. Synonyms for CD160 include BY55, NK1, and NK28. An exemplary sequence of CD160 can be found in UniProt registry number O95971.

[0278] According to other embodiments, the multispecific antibody is fused with or conjugated to cytokines.

[0279] According to some embodiments, the multispecific antibody further includes a binding site that specifically targets an antigen located at a cancer site.

[0280] According to other embodiments, the multispecific antibody further includes a binding site specifically targeting viral antigens. According to other embodiments, the multispecific antibody further includes a binding site specifically targeting fungal antigens. According to other embodiments, the multispecific antibody further includes a binding site specifically targeting bacterial antigens.

[0281] According to some embodiments, the multispecific binding molecule is a polypeptide or a polypeptide multimer. According to some embodiments, each polypeptide is independently selected from monospecific, bispecific, and trispecific polypeptides.

[0282] According to some embodiments, the multispecific binding molecule consists of or contains one or more scFv molecules.

[0283] For a binding molecule with at least three specificities, it is sufficient for each binding specificity to be represented by exactly one binding site. Therefore, the three-specific binding molecule according to the invention can be a trivalent molecule. The three binding specificities can also be achieved by different numbers of binding sites. It may be preferred that one binding specificity is represented by more binding sites than the other two binding specificities, or that two binding specificities are represented by more binding sites than the third binding specificity.

[0284] According to certain embodiments, a set of six CDRs is a preferred embodiment for a given binding site. As is known in the art, a set of six CDRs comprises first and second groups, each consisting of three CDRs. These two groups of three CDRs can be located on the same polypeptide chain or on different polypeptide chains, resulting in different molecular structures. Furthermore, it is envisioned that in a given binding molecule according to the invention, some binding sites result in all six CDRs constituting the given binding site being located on a single polypeptide chain, while other binding sites result in the first and second groups of three CDRs constituting the given binding site being located on different polypeptides. The different polypeptides can differ only in form and be identical in other respects, particularly in terms of amino acid sequence. Alternatively, the different polypeptides can differ from each other in terms of amino acid sequence.

[0285] The term "about" means the acceptable range of error that should be assumed for a particular value, such as at most 5% or 10%.

[0286] diagnosis

[0287] The present invention also discloses methods for diagnosing and prognosing cancer.

[0288] According to one aspect, the present invention provides a method for diagnosing and / or prognosing a cancer or infectious disease in a subject, the method comprising the step of determining the expression level of NKp46 in a biological sample of the subject using at least one antibody described herein.

[0289] The term "biological sample" encompasses a wide variety of sample types obtained from a living organism that can be used for diagnostic or monitoring assays. The term covers biologically derived blood and other fluid samples, solid tissue samples such as biopsy samples, or tissue cultures or cells derived from them and their progeny. Furthermore, the term may cover circulating tumor or other cells. The term specifically covers clinical samples and also includes cells in cell cultures, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, and biological fluids and tissue samples, including aqueous humor and vitreous fluid for ocular samples. The term also covers samples that have been manipulated in any way after acquisition, such as samples treated with reagents, solubilized, or enriched with certain components.

[0290] The expression level of NKp46 can be determined using a labeled anti-NKp46 antibody as described herein. This expression can be determined, for example, by ELISA.

[0291] The method of the present invention may further include the step of comparing the expression level with a control level.

[0292] The following embodiments are presented to illustrate certain implementations of the invention more fully. They should in no way be construed as limiting the scope of the invention. Example

[0293] The invention will now be described in a non-limiting manner with reference to the following embodiments, which, together with the description above, illustrate the invention.

[0294] Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbiological, immunological, and recombinant DNA technologies. Such techniques are well known in the art. For the reader's convenience, other general references relating to well-known procedures are provided throughout this document.

[0295] Example 1. Generation and selection of Nkp46 antibodies

[0296] Immunogen (Nkp46-Fc) expression was performed in mammalian HEK 293T cells. An NKp46-Fc fusion protein, consisting of the extracellular domain of the target (NKp46) protein and a fragment of human IgG1 Fc, was generated and purified.

[0297] For immunization, BALB / c mice were injected with 50 μg of immunogen in Freund's complete adjuvant (CFA), followed by 50 μg of immunogen in Freund's incomplete adjuvant (IFA) on day 14 post-immunization. Serum anti-NKp46-Fc antibody titers were then analyzed by ELISA. Mice with the highest titers were boosted with 50 μg of immunogen in PBS. Three days later, spleens were harvested from immunized mice, and after erythrocyte lysis, spleen cells were fused with the SP2 / 0 cell line. Potential hybridoma cells were seeded in 20% RPMI 1640 medium containing hypoxanthine, aminopterin, and thymidine (HAT), and stable hybridoma cell lines were selected. The above procedure was repeated five times, and anti-NKp46-Fc antibody secretion was screened from a total of 4600 wells by ELISA. The positivity of 84 cell lines from the supernatants of these wells, which showed positive binding assays on NKp46-Fc-coated ELISA plates, was then retested. Cross-reactivity assays were performed in parallel on unrelated Fc fusion proteins. This yielded five hybridoma cell lines that secreted antibodies that specifically recognize the extracellular domain of NKp46. Of these antibodies, four were further selected for their IgG isotypes (but not IgM isotypes). Subclones of all these candidates that showed specific signals in ELISA were then tested for their ability to recognize native human NKp46 protein on transfected cell lines, as described below.

[0298] First, binding was studied on mouse thymoma BW transfected cells expressing NKp46 (BW NKp46). As controls, we used a commercially available anti-NKp46 mAb (designated 9E2) and a previously developed anti-NKp46 mAb in our lab, 461-G1 (Arnon et al., 2004; Mandelboim et al., 2001). All antibodies tested interacted specifically with BW NKp46 but not with parental BW cells (data not shown). To confirm that the antibodies recognize NKp46 naturally expressed by human NK cells, we stained a large number of primary human NK cells activated by IL-2 (activated NK cells). The activated NK cells used throughout the experiment were isolated from PBMCs. Our purification protocol achieved approximately 97% purity, and we verified their identity as CD56+CD3- cells (data not shown). Indeed, all antibodies positively stained the activated NK cells. Similar results were obtained using PBMCs derived from several donors (data not shown).

[0299] At this point, four different stable clonal cell lines were selected for large-scale production and purification. Large-scale Ab production was then carried out, and all monoclonal antibodies were purified from serum-free medium in amounts of a few milligrams using a GE AKTA Prime Plus liquid chromatography system and a HiTrap Protein G column.

[0300] To identify the binding site of anti-NKp46 mAb, Ig-D1, Ig-D2, and Ig-NKp46 were plated at a density of 0.1 μg / well. The plates were incubated overnight at 4°C. The next day, blocking was performed (blocking buffer was PBS x 1, 0.05% Tween-20, and 5% BSA), followed by the addition of 0.1 μg / well mAb and 10 μl / well SN antibody (both diluted to a final volume of 100 μl in blocking buffer). The plates were incubated overnight at 4°C. The next day, biotinylated anti-mouse antibody (diluted in blocking buffer) was added, followed by streptavidin-POD (diluted in blocking buffer), and the samples were detected at OD 650 nm using TMB. Figure 1 As shown, clones K3 and P4 antibodies bind to the NKp46 D2 domain, while commercially available anti-NKp46 antibody clones 02 and 461-G1 (published NKp46 antibodies) bind to the NKp46 D1 domain.

[0301] Next, we examined whether any anti-NKp46 mAb could block the interaction between NKp46 and its ligand. For this purpose, BJAB, MCF7, and C1R tumor cells expressing unknown NKp46 ligands were used. NKp46-Ig was incubated on ice, alone or with various different anti-NKp46 mAbs. The incubated NKp46-Ig fusion protein was then used for FACS staining of the tumor cells. None of the anti-NKp46 mAbs blocked the binding of NKp46-Ig to the cells.

[0302] Example 2. Study on the downregulation of NKp46 from the surface of NK cells

[0303] The effect of anti-NKp46 mAb on the reduction of NKp46 expression on the surface of NK cells was tested. Activated NK cells were incubated with anti-NKp46 mAb at 4°C or 37°C for 8 hours. The cells were then stained with FACS using a conjugated anti-mouse secondary antibody. Only one mAb, O2, resulted in a decrease in NKp46 levels. Figure 2 Other antibodies do not induce downregulation of NKp46 derived from the surface of NK cells. These antibodies specifically bind to NKp46 and do not interfere with the binding of NKp46 to its homologous ligands. To confirm these findings, dose-response FACS staining was performed on two types of activated primary NK cells using these antibodies. Figure 3As can be recognized, the decrease in K3 and P4 antibody concentrations corresponds to a decrease in the intensity of fluorescence signals on the cell surface. Therefore, the K3 and P4 antibodies exhibit a strong dose response and are suitable for generating bispecific or trispecific antibodies that bridge NK cells and tumor cells.

[0304] Example 3. Humanized anti-NKp46 antibody, activity and affinity

[0305] Humanization of the parental heavy and light chain sequences of the anti-NKp46 antibody clones P4 and K3 was designed using Macromoltek software. The sequences were confirmed by structural biology and molecular modeling experts. The resulting humanized sequences are elucidated in SEQ ID No: 14 (humanization of the heavy chains of K3 and P4), SEQ ID No: 15 (humanization of the light chain of P4), and SEQ ID No: 16 (humanization of the light chain of K3). Four humanized clones were prepared: B341001, B341002, B341003, and B341004.

[0306] The binding of a humanized antibody carrying mutated IgG4 to cells expressing NKp46 was examined. The experiment was performed as follows:

[0307] 1. Test the coating in three matrices with three different concentrations relative to three different buffer solutions to identify the optimal signal.

[0308] 2. Coat the ELISA plate with optimal signal conditions, using standard sealing and cleaning procedures.

[0309] 3. The first antibody (MAb with humanized variable region + mutated IgG4) was applied at concentrations with 10 dilutions.

[0310] 4. The second antibody used to detect binding is an anti-Huκ antibody (TMB substrate) containing HRP.

[0311] 5. Measure the absorbance at 450 nm.

[0312] The antigen is Acro Biosystems' human NKp46 expressed in HEK293 cells. Figures 4A-4B As shown, both humanized clones have strong affinity for NKp46 (20.5 pM and 27 pM).

[0313] The affinity of the humanized antibody for the NKp46 D2 domain was further examined. Figure 5D As shown, the humanized antibody exhibits an exceptionally high affinity for the D2 domain of NKp46.

[0314] Example 4. Bispecific and trispecific antibodies

[0315] Binding molecules with two or three specificities were prepared. The bispecific binding molecules comprise the antibody binding site disclosed herein and a binding site specifically targeting a tumor ligand. The trispecific binding molecules comprise the antibody binding site described herein, a binding site targeting CD160, and a binding site targeting a tumor ligand. First, for the trispecific antibodies, blocking assays against NKp46 and CD160 were performed to confirm that the antibodies do not block the binding of the receptors to their natural ligands. Redirection assays using antibody clones K3 and P4 and the anti-CD160 antibody were used to demonstrate that antibody binding does not impair NK cell function.

[0316] In vivo assays were performed using SCID / Beige mice that do not express T, B, or NK cells because the tumors used for testing were of human origin. Tumor establishment in mice expressing the selected tumor antigen was achieved by subcutaneous injection of progressively increasing amounts of cancer cells (2 x 10⁻⁶). 5 5x10 5 1x10 6 5x10 6 The mice were assessed for the presence of palpable tumors every two days, and once observed, tumor volume was measured daily using digital calipers. Depending on the tumor type and injection volume, tumors typically appeared within 10–14 days. Once a palpable tumor was observed, the mice were monitored for two weeks (tumor volume, mouse weight, and overall appearance). The humane endpoint was set at a tumor volume of 1 cm. 3 Or, mice were allowed to lose 20% of their initial body weight. Two weeks later, the mice were sacrificed, the tumors were removed, and the mice were weighed. The ideal working number of cells was defined as the minimum number of cells that maintained a palpable tumor throughout the entire study period, as mentioned above. Six mice were tested per group for each cell line.

[0317] Dose-response experiments were used to determine the safe amount of bispecific or trispecific antibodies required to reduce tumor size. Once the ideal working number of cells was determined according to the above criteria, the antibodies and single antibodies (K3, P4, anti-CD160 antibody, and anti-tumor antigen antibody) were injected into SCID / Beige mice with tumors at gradually increasing concentrations.

[0318] SCID / Being mice were initially injected with the determined number of cancer cells assessed in the previous section. Once a palpable tumor was observed, its volume was determined by measurement using digital calipers. Subsequently, the bispecific or trispecific and single antibody were injected intraperitoneally at gradually increasing doses (30 μg and 60 μg). Human NK cells were then injected into all mouse groups except the PBS-injected group.

[0319] The foregoing description of specific embodiments sufficiently reveals the general nature of the invention, enabling others to readily modify and / or adapt these specific embodiments to suit a variety of different applications by applying current knowledge, without excessive experimentation and without departing from the general concepts. Therefore, such adaptations and modifications should and are intended to be understood within the meaning and scope of equivalence to the disclosed embodiments. It should be understood that phrases or terms used herein are for descriptive purposes and not for limitation. sequence list <110> Jerusalem Hebrew University Isem Research and Development Co., Ltd. Rijeka Medical University <120> Antibodies and their constructs targeting NKp46 for the treatment of cancer and infections. <130> Yissum / 0168 PCT <150> US 63 / 005457 <151> 2020-04-06 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Glu Tyr Ser Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Mus musculus <400> 2 Gly Ile Ser Pro Asn Ser Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3 Arg Asp Phe His Ser Ser Phe Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Mus musculus <400> 4 Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Asn Gly His Ser Phe Pro Leu Thr 1 5 <210> 7 <211> 118 <212> PRT <213> Mus musculus <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Phe 35 40 45 Gly Gly Ile Ser Pro Asn Ser Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Phe His Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Mus musculus <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Phe Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 9 <211> 354 <212> DNA <213> Mus musculus <400> 9 gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctgggacttc agtgaagata 60 tcctgcaaga cttctggata cacattcact gaatacagca tgcactgggt gaagcagagc 120 catggaaaga gccttgagtg gtttggaggt attagtccta acagtggtgg taccagctac 180 aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240 atggagctcc gcagcctgac atctgaggat tctgcagtct attactgtgc acgaagggac 300 ttccatagta gctttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354 <210> 10 <211> 321 <212> DNA <213> Mus musculus <400> 10 gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60 ctctcctgca gggccagcca gagtattagc gactacttac attggtttca acaaaaatca 120 catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180 aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240 gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccgctcac gttcggtcct 300 gggaccaagc tggagctgaa a 321 <210> 11 <211> 321 <212> DNA <213> Mus musculus <400> 11 gatattgtcc taactcagtc tccagccacc ctgtctgtga ctccaggaaa tagcgtcagt 60 ctttcctgca gggccagcca aagtattagc gactacttac attggtttca acaaaaatca 120 catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180 aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240 gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccgctcac gttcggtcct 300 gggaccaagc tggagctgaa a 321 <210> 12 <211> 107 <212> PRT <213> Mus musculus <400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asn Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Phe Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 13 <211> 134 <212> PRT <213> Homo sapiens <400> 13 Tyr Asp Thr Pro Thr Leu Ser Val His Pro Gly Pro Glu Val Ile Ser 1 5 10 15 Gly Glu Lys Val Thr Phe Tyr Cys Arg Leu Asp Thr Ala Thr Ser Met 20 25 30 Phe Leu Leu Leu Lys Glu Gly Arg Ser Ser His Val Gln Arg Gly Tyr 35 40 45 Gly Lys Val Gln Ala Glu Phe Pro Leu Gly Pro Val Thr Thr Ala His 50 55 60 Arg Gly Thr Tyr Arg Cys Phe Gly Ser Tyr Asn Asn His Ala Trp Ser 65 70 75 80 Phe Pro Ser Glu Pro Val Lys Leu Leu Val Thr Gly Asp Ile Glu Asn 85 90 95 Thr Ser Leu Ala Pro Glu Asp Pro Thr Phe Pro Ala Asp Thr Trp Gly 100 105 110 Thr Tyr Leu Leu Thr Thr Glu Thr Gly Leu Gln Lys Asp His Ala Leu 115 120 125 Trp Asp His Thr Ala Gln 130 <210> 14 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Humanized antibody <400> 14 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Phe 35 40 45 Gly Gly Ile Ser Pro Asn Ser Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Phe His Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 15 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Humanized antibody <400> 15 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Phe Gln Gln Lys Pro His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 16 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Humanized antibody <400> 16 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Phe Gln Gln Lys Pro His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe 115

Claims

1. An antibody that binds to NKp46, or an antibody fragment thereof comprising at least an antigen-binding moiety, wherein the antibody or antibody fragment comprises a set of 6 CDRs, wherein: HC CDR1 is EYSMH (SEQ ID No: 1); HC CDR2 is GISPNSGGTSYNQKFKG (SEQ ID No: 2); HC CDR3 is RDFHSSFDY (SEQ ID No: 3); LC CDR1 is RASQSISDYLH (SEQ ID No: 4); LC CDR2 is YASQSIS (SEQ ID No: 5); and LC CDR3 is QNGHSFPLT (SEQ ID No: 6).

2. The antibody or antibody fragment according to claim 1, comprising the heavy chain variable region described in SEQ ID No:

7.

3. The antibody or antibody fragment according to claim 1 or 2, comprising the light chain variable sequence set forth in SEQ ID No:

8.

4. The antibody or antibody fragment according to any one of claims 1 to 3, comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence shown in SEQ ID No: 7 and the light chain comprises the sequence shown in SEQ ID No:

8.

5. The antibody fragment according to any one of claims 1 to 4, wherein the antibody fragment is a single-chain Fv (scFv).

6. The antibody or antibody fragment according to any one of claims 1 to 5, wherein the antibody is in the form of 10 -11 M to 10 -16 M's affinity binds to human NKp46.

7. The antibody or antibody fragment according to any one of claims 1 to 6, wherein the antibody is a humanized antibody.

8. The antibody or antibody fragment of claim 7, wherein the humanized antibody comprises the heavy chain described in SEQ ID No: 14 and a light chain selected from SEQ ID Nos: 15 and 16.

9. A polynucleotide or combination of polynucleotides encoding a heavy and light chain variable region of an antibody or antibody fragment according to any one of claims 1 to 7.

10. The polynucleotide or combination of polynucleotides of claim 9, encoding an antibody heavy chain variable region comprising the sequence of SEQ ID No: 9 or a variant thereof having at least 85% identity with said sequence; and an antibody light chain variable region comprising the sequence of SEQ ID No: 10 or a variant thereof having at least 85% identity with said sequence.

11. A plasmid comprising a polynucleotide or combination of polynucleotides according to any one of claims 9 or 10.

12. A cell comprising a polynucleotide or a combination of polynucleotides according to any one of claims 9 or 10.

13. A cell capable of producing an antibody according to any one of claims 1 to 7.

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