Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/71—Decreased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention is in the field of immunotherapy and relates to anti-Nectin-2 antibodies and antibody-conjugates and to therapeutic and diagnostic compositions comprising them, for treating diseases, particularly cancer.
• Cancer immunotherapy is utilized for generating and augmenting an anti-tumor immune response, e.g., by treatment with antibodies specific to antigens on tumor cells, or by specific activation of anti-tumor T cells.
• the ability of recruiting immune cells (e.g., T cells) against tumor cells in a patient provides a therapeutic modality of fighting cancer types and metastasis that are otherwise considered incurable.
• T cell mediated immune responses include multiple sequential steps regulated by a balance between co- stimulatory and co-inhibitory signals that control the magnitude of the immune response.
• the inhibitory signals referred to as immune checkpoints, are crucial for the maintenance of self-tolerance and for the limitation of immune-mediated collateral tissue damage. These inhibitory signals affect the response of T cells and re-shape the immune response.
• Nectin-2 which was also named Poliovirus Receptor-Related Protein-2, Poliovirus Receptor- Like 2, CD 112, or PRR-2, is a single pass transmembrane glycoprotein with two Ig- like C2-type domains and an Ig-like V-type domain. Nectin-2 is involved in mediating cell adhesion to extracellular matrix molecules, serving as one of the plasma membrane components of adherent junctions. It also serves as an entry receptor for certain mutant strains of herpes simplex virus and pseudorabies virus, and it is involved in cell to cell spreading of these viruses. Variations in this gene have been associated with differences in the severity of multiple sclerosis. Importantly, Nectin-2 can also serve as a modulator of T-cell signaling.
• T-cell proliferation and activation can be either a co-stimulator, or a co-inhibitor of T-cell functions, depending on the receptor it binds on these target cells: upon binding to CD226 (DNAM-1), it stimulates T-cell proliferation and cytokine production, including that of IL-2, and IFNy, while upon interaction with PVRIG (CD112R), and/or TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains) it inhibits T-cell proliferation and activation.
• CD226 CD226
• PVRIG CD112R
• TIGIT T-cell immunoreceptor with immunoglobulin and ITIM domains
• Nectin-2 was shown to be overexpressed in various tumors, including breast and ovarian cancers (Oshima et al. Molecular Cancer, 2013, 12:60). The presence of Nectin-2 on tumor cells leads to poor prognosis and reduced activity of T cells (Stamm et al. Oncogene (2016) 37:5269-5280).
• US patent application No. 2017/0037133 discloses inhibitors (e.g., antibodies) against CD112 (Nectin-2, PVRL2), CD155 (PVR), Galectin-9, TIM-3 and/or TIGIT for use in treatment of a blood-borne cancer, in particular acute myeloid leukemia (AML).
• inhibitors e.g., antibodies against CD112 (Nectin-2, PVRL2), CD155 (PVR), Galectin-9, TIM-3 and/or TIGIT for use in treatment of a blood-borne cancer, in particular acute myeloid leukemia (AML).
• AML acute myeloid leukemia
• WO 2020/144697 discloses monoclonal antibodies (mAbs) that recognize human Nectin-2 and block the interactions with TIGIT and CD112R, for use treatment of cancer.
• ADCs Antibody-drug conjugates
• Auristatin a microtubule-destroying drug. It was derived from marine shell-less mollusk Dolabella auricularia called dolastatins. Various derivatives of auristatin have been synthesized, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). MMAE and MMAF were developed by Seattle Genetics and used as payloads for ADCs. MMAF and MMAE have their advantages and disadvantages. MMAE is more membrane-permeable and has a lower IC50 than MMAF. However, MMAF is more hydrophilic and has a lower aggregation tendency to show lower systemic toxicity than MMAE (park et al. Molecules 2019, 24, 2754).
• the present invention provides humanized antibodies that specifically bind Nectin-2.
• the humanized antibodies of the present invention selected from a larger collection of antibody clones, have improved properties compared to other anti-Nectin-2 antibodies.
• the present invention further provides, according to some embodiments, conjugates comprising the antibodies and diagnostic or therapeutic agents.
• the conjugates comprise a cytotoxic moiety that is targeted by said humanized antibodies to tumor cells presenting the Nectin-2 receptor on their surface.
• a large collection of humanized antibodies was produced by combining specific sets of complementarity determining regions (CDR) sequences and human framework (FW) sequences and introducing specific mutations in these sequences to produce antibodies with modified variable regions and improved properties.
• CDR complementarity determining regions
• FW human framework
• the newly designed humanized variable regions described herein preserve the residues critical for the maintenance of the antibody’s conformation and binding affinity, while having lower incidence of potential T cell epitopes, thus minimizing the risk of adverse immune response towards the antibodies.
• the antibodies disclosed herein were designed based on factors including homology, T-cell epitopes, key residues, and predicted structures. Unexpectedly, the humanized antibodies disclosed herein show improved biostability compared with the parental chimeric antibody.
• the humanized antibodies disclosed herein were found to be highly suitable for use as targeted anti-cancer therapy with therapeutic toxins. It is now disclosed that the anti-Nectin-2 monoclonal humanized antibody described herein, conjugated to a cytotoxic moiety, exhibit robust killing of various tumor cell lines.
• the direct targeting of toxins using the antibodies described herein has the potential to increase the anti-tumor efficacy of these toxins, reducing their systemic toxicity, and thus improving the survival of cancer patients.
• FcyRs Human Fc receptors activate Fc-effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP).
• ADCC antibody-dependent cellular cytotoxicity
• ADCP antibody dependent cellular phagocytosis
• Some of the ADCs of the present invention comprise one or more Fc mutations that significantly reduce their binding by FcyR that are expressed on immune cells, thereby increasing their safety and specificity. It is now disclosed that ADCs comprising anti-Nectin-2 monoclonal humanized antibody having specific Fc mutations and linked to toxins are highly effective and safe, suitable for anti-cancer therapy. The inventors of the present invention have shown for the first time that ADCs targeted to Nectin-2 are highly useful for treating cancer, in particular solid tumors.
• the present invention provides a humanized antibody that specifically binds human Nectin-2, or a fragment thereof comprising at least the antigen binding site, wherein the antibody or a fragment thereof comprises a heavy chain (HC) and a light chain (LC), wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
• HC heavy chain
• LC light chain
• the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:
• the humanized antibody or a fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO: 11, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO: 12.
• the humanized antibody or a fragment thereof comprises a heavy chain and a light chain, wherein the heavy-chain variable region comprises the sequence set forth in SEQ ID NO: 11, and the light-chain variable region comprises the sequence set forth in SEQ ID NO: 12.
• CDR sequences of a given antibody molecule There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as KAB AT, Chothia and IMGT.
• a selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example.
• the CDR sequences of the mAb variable regions are determined using the IMGT method.
• the humanized antibody or a fragment thereof comprises a set of six CDR sequences, wherein heavy-chain CDR1 comprising the sequence SYW, heavy-chain CDR2 comprising the sequence VYPGNSDS (SEQ ID NO: 8), heavy- chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 3), light-chain CDR1 comprising the sequence QNVGIN (SEQ ID NO: 10), light-chain CDR2 comprising the sequence SAS, and light-chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 6).
• the humanized antibody or a fragment thereof comprises a set of six CDR sequences, wherein heavy-chain CDR1 comprising the sequence SYWIH (SEQ ID NO: 1), heavy-chain CDR2 comprising the sequence AVYPGNSDSNYNQKF(KA/QG) (SEQ ID NO: 2), heavy-chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 3), light-chain CDR1 comprising the sequence (K/R)ASQNVGINV(V/A) (SEQ ID NO: 4), light-chain CDR2 comprising the sequence SASYRYS (SEQ ID NO: 5), and light-chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 6).
• heavy-chain CDR1 comprising the sequence SYWIH (SEQ ID NO: 1)
• heavy-chain CDR2 comprising the sequence AVYPGNSDSNYNQKF(KA/QG) (SEQ ID NO: 2)
• heavy-chain CDR3 comprising the sequence LVG
• the heavy-chain CDR2 comprises the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 41) or AVYPGNSDSNYNQKFQG (SEQ ID NO: 42).
• the light-chain CDR1 comprises a sequence selected from the group consisting of KASQNVGINVV (SEQ ID NO: 43), KASQNVGINVA (SEQ ID NO: 44), RASQNVGINVV (SEQ ID NO: 45) and RASQNVGINVA (SEQ ID NO: 46).
• the humanized antibody or a fragment thereof comprises a set of six CDR sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6. According to additional exemplary embodiments, the humanized antibody or a fragment thereof comprises a set of six CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, (SAS), and 6. According to additional embodiments, the humanized antibody or a fragment thereof comprises a set of six CDR sequences set forth in SEQ ID NOs: 1, 41, 3, 43, 5, and 6.
• the humanized antibody or antigen binding fragment thereof comprises a heavy chain variable region, comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs:
• FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27
• FR-H2 is SEQ ID NO: 22
• FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29
• FR-H4 is SEQ ID NO: 24.
• the humanized antibody or antigen binding fragment thereof comprising a light chain variable region, comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs:
• FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39
• FR-E2 is selected from the group consisting of SEQ ID NOs: 31 and 35
• FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40
• FR-L4 is SEQ ID NO: 33.
• the humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs:
• FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27
• FR-H2 is SEQ ID NO: 22
• FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29
• FR-H4 is SEQ ID NO: 24
• the light chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs:
• FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39
• FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35
• FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40
• FR-L4 is SEQ ID NO: 33.
• the humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs:
• FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27
• FR-H2 is SEQ ID NO: 22
• FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29
• FR-H4 is SEQ ID NO: 24
• the light chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NO:
• FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39
• FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35
• FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40
• FR-L4 is SEQ ID NO: 33.
• the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and the light chain variable region comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
• the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and the light chain variable region comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
• the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
• the humanized antibody or fragment thereof is a monoclonal antibody, Fab, F(ab)2, a single-domain antibody, or a single chain variable fragment (scFv).
• the humanized antibody or fragment thereof is an IgG monoclonal antibody.
• the humanized monoclonal antibody comprises a heavy chain constant region selected from IgG4, IgGl, and IgG2.
• the humanized antibody or fragment thereof is an IgG4 subclass.
• the humanized antibody or antigen binding fragment thereof is an IgGl subclass.
• the humanized monoclonal antibody comprises a kappa light chain constant region.
• the humanized monoclonal antibody is IgGl, having a heavy chain comprising an amino acid sequence at least about 90%, 95%, or 98% identical to the sequence set forth in SEQ ID NO: 47. According to certain exemplary embodiments, the humanized monoclonal antibody is IgGl, having a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 47.
• the humanized monoclonal antibody has a kappa light chain comprising an amino acid sequence at least about 90%, 95%, or 98% identical to the sequence set forth in SEQ ID NO: 49. According to certain exemplary embodiments, the humanized monoclonal antibody has a kappa light chain comprising the amino acid sequence set forth in SEQ ID NO: 49.
• the humanized antibody has a mutated Fc domain that prevents FcyR-mediated internalization.
• the humanized antibody comprises a Fc null domain.
• the Fc domain is null for binding to Fey receptors found on immune cells.
• the Fc domain is null for binding to CD64, CD32a, CD32b, CD16a, and/or CD16b.
• the humanized antibody comprises a IgGl Fc domain.
• the IgGl Fc domain is null for binding to Fey receptors.
• the IgGl Fc domain is null for binding to one or more Fey receptors.
• the IgGl Fc domain is null for binding to CD64, CD32a, CD32b, CD16a, and/or CD16b.
• the humanized antibody comprises a human IgG selected from the group consisting of: (i) a human IgGl having the mutation L235S; (ii) a human IgGl having the mutations L235S and E272K; (iii) a human IgGl having the mutation G237I; (iv) a human IgGl having the mutations G237I and E272I; (v) a human IgGl having the mutations G237I and V264R; (vi) a human IgGl having the mutations V215A, E269R and K322A; (vii) a human IgGl having the mutations L234A, L235A and P329G; (viii) a human IgG4 having the mutations S228P, L235P and V264R; (ix) a human IgG2 having the mutation P238H; or (x) a human IgG2 having the mutations P238H; or (x
• the humanized antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 61-70.
• SEQ ID NOs: 61-70 Each possibility represents a separate embodiment of the invention.
• a conjugate comprising the humanized antibody or fragment thereof described above is provided.
• Antibodies or fragments thereof according to the present invention are attached, according to some embodiments, to a cytotoxic moiety, a radioactive moiety, or an affinity or labeling tag.
• Antibody-drug conjugates according to the present invention comprises the humanized antibodies as described herein, an optional linker, and a toxin.
• the humanized antibody or fragment thereof is conjugated directly or through a linker, to a toxin (pay load).
• the toxin is selected from the group consisting of microtubule inhibitor, DNA synthesis inhibitor, topoisomerase inhibitor and RNA polymerase inhibitor.
• the toxin is a microtubule-destroying drug.
• the toxin is auristatin or a derivative thereof.
• the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).
• the toxin is saporin.
• the toxin is a maytansine derivative.
• the maytansine derivative is DM4 or DM1.
• the toxin is quinoline alkaloid.
• the quinoline alkaloid is SN-38.
• the toxin is a DNA topoisomerase I (TOPI) inhibitor.
• DNA topoisomerase I (TOPI) inhibitor is Exatecan or Exatecan derivative According to certain embodiments, the DNA topoisomerase I (TOPI) inhibitor is DXd.
• the toxin is directly connected to the antibody.
• the antibody and the toxin are connected through a linker or a spacer.
• the toxin is covalently connected to the humanized antibody directly or through a linker or a spacer.
• the linker is cleavable. According to other embodiments, the linker is not cleavable. According to some embodiments, the linker is an enzymatic cleavable linker. According to certain embodiments, the linker is a pH-sensitive linker. According to some embodiments, the linker is a reducible linker (sulfo-SPDB).
• MC Maleimidocaproyl
• MC-VC-PAB Maleimidocaproyl-Valine-Citrulline- p-amino-benzyloxycarbonyl
• SMCC Maleimidomethyl cyclohexane- 1 -carboxylate
• SPDB N-succinimidyl-4-(2- pyridyldithio)butanoate
• polynucleotide sequences encoding the amino acid sequences of heavy chain variable region and light chain variable region described above are provided.
• the polynucleotide sequence encodes a humanized antibody heavy chain variable region
• the polynucleotide comprise a sequence selected from the group consisting of SEQ ID NOs: 51-55 or a variant thereof having at least 80% sequence identity.
• the polynucleotide sequence encodes a humanized antibody light chain variable region
• the polynucleotide comprise a sequence selected from the group consisting of SEQ ID NOs: 56-60 or a variant thereof having at least 80% sequence identity.
• SEQ ID NOs: 56-60 or a variant thereof having at least 80% sequence identity.
• the polynucleotide sequence encodes a humanized antibody heavy chain variable region, the polynucleotide comprise a sequence selected from the group consisting of SEQ ID NOs: 51-55. According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, the polynucleotide comprise a sequence selected from the group consisting of SEQ ID NOs: 56- 60.
• the polynucleotide sequence encodes a humanized antibody heavy chain variable region, said polynucleotide comprise a sequence set forth in SEQ ID NO: 51. According to some embodiments, said polynucleotide sequence encodes a humanized antibody light chain variable region, the polynucleotide comprise a sequence set forth in SEQ ID NO: 56.
• polynucleotide sequences encoding the amino acid sequences of humanized antibody heavy chain and light chain described above are provided.
• the polynucleotide sequence encoding the humanized antibody heavy chain comprises a sequence set forth in SEQ ID NO: 48 or a variant thereof having at least 80% sequence identity.
• the polynucleotide sequence encoding the humanized antibody light chain comprises a sequence set forth in SEQ ID NO: 50 or a variant thereof having at least 80% sequence identity.
• the DNA sequence encoding the amino acid chain of a humanized antibody as described herein comprises a leader sequence.
• the DNA sequence encodes a leader peptide sequence set forth in SEQ ID NO: 71.
• the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one humanized antibody chain or fragment thereof as described herein.
• the nucleic acid construct is a plasmid.
• a cell line comprising the nucleic acids encoding the antibodies of the present invention.
• the cell line is for expression of the humanized antibody or fragment thereof as described herein.
• the cell line is a mammalian cell line such as a Chinese Hamster Ovary (CHO) cell line.
• the present invention provides, according to another aspect, a pharmaceutical composition
• a pharmaceutical composition comprising the humanized antibody or antigen binding fragment described herein or a conjugate comprising the antibody and a pharmaceutically acceptable excipient, carrier, or diluent.
• the pharmaceutical composition is for use in treating cancer.
• Any administration mode may be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral administration modes.
• the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral administration.
• the present invention provides a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of at least one humanized antibody or its conjugate as described herein.
• the cancer is characterized by overexpression of Nectin-2.
• the cancer comprises a solid tumor.
• the cancer is selected from the group consisting of prostate cancer, colorectal cancer, liver cancer, ovarian cancer, endometrial cancer, stomach cancer, thyroid cancer, carcinoid tumor, head and neck cancer, breast cancer, pancreatic cancer, testis cancer, urothelial cancer, cervical cancer, melanoma, lymphoma and lung cancer.
• the cancer is breast cancer. According to some embodiments, the cancer is colorectal adenocarcinoma or lung adenocarcinoma.
• the cancer is a hematological cancer.
• the hematological cancer is selected from leukemia including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphoma, including Hodgkin disease, and non-Hodgkin lymphoma; and multiple myeloma.
• the subject is human.
• the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy.
• the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.
• the method of treating cancer comprises administration of the humanized antibody described herein and an additional anti-cancer agent.
• the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.
• the additional immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human Nectin-2.
• the additional immune-modulator is an antibody against an immune checkpoint molecule.
• the additional immune modulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and any other checkpoint molecule or a combination thereof.
• PD-1 human programmed cell death protein 1
• CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1
• LAG3 lymphocyte activation gene 3
• CD137 also referred to as CD134
• KIR killer cell immunoglobulin-like receptors
• TIGIT TIGIT
• PVR CTLA-4
• NKG2A NKG
• the anti-cancer agent is selected from the group consisting of: erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and any combination thereof.
• erbitux e.g., erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methot
• the method of treating cancer involves preventing or reducing formation, growth or spread of metastases in a subject.
• the present invention provides an antibody-drug conjugates comprising a humanized antibody that specifically binds human Nectin-2 and a toxin, for use in treating cancer.
• the cancer and the toxin are as described above.
• the humanized antibody comprises a mutated Fc domain.
• the present invention further provides, according to an aspect, a method of diagnosing or prognosing cancer in a subject, the method comprises determining the expression level of Nectin-2 in a biological sample of said subject using at least one humanized antibody, fragment or conjugate as described herein.
• the present invention further provides, according to another aspect, a method of determining or quantifying the expression of Nectin-2, the method comprising contacting a biological sample with an antibody or antibody fragment as described herein, and measuring the level of complex formation.
• the method for detecting or quantifying the expression of Nectin-2 comprises the steps of: i. incubating a sample with the antibody specific to Nectin-2 or an antibody fragment thereof comprising at least an antigen-binding portion; ii. detecting the bound Nectin-2 using a detectable probe.
• the method further comprises the steps of: iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of Nectin-2; and iv. calculating the amount of the Nectin-2 in the sample from the standard curve.
• the sample is a body fluid or solid tissue.
• the method is performed in-vitro or ex-vivo.
• a kit for measuring the expression of Nectin-2 in biological sample comprising at least one antibody or antibody fragment as described herein and means for measuring Nectin-2 expression.
• the kit further comprising instruction material directing the use of the kit.
• Figures 1A-1C depict the correlation of Nectin-2 mRNA levels (high or low as indicated) with survival probability of Low-grade glioma (Figure 1A), Kidney Renal Clear Cell Carcinoma ( Figure IB) and lung adenocarcinoma ( Figure 1C) patients.
• Data sets were obtained from the TCGA site and analyzed using oncolnc.org site (https://doi.org/10.7717/peerj-cs.67).
• N depicts number of patients included at the analysis.
• FIG 2 is a schematic illustration of receptors expressed on immune cells and their respective affinities to Nectin-2 (CD 112), which is expressed by tumors or on antigen presenting cells (APCs).
• TIGIT is a co-inhibitory receptor on many immune cells (e.g., T and NK cells);
• DNAM-1 also termed CD2266 is an activating receptor on many immune cells (e.g., T cells), and CD112R (also termed PVRIG) and TIGIT are co-inhibitory receptors on lymphoid immune cells (e.g., T and NK cells);
• Nectin-2 acts as an inhibitory ligand for immune cells, mainly via its high-affinity binding to CD112R (rectangle).
• Figure 3 is a graph showing percent of tumors positive for Nectin-2 expression. Data obtained from proteinatlas.com using the HPA012759 mAb (anti Nectin-2; Sigma- Aldrich®). In 17/20 indications moderate-high membranous expression of Nectin-2 is seen.
• Figures 4A-4C depict improved on-cell binding of the humanized variants of the parental (chimeric) anti-Nectin2 mAb.
• Nectin-2 expressing cells were analyzed by FACS analysis for binding of serially diluted mAb humanized variants. EC-50 values were calculated for each variant and are reported relative to the EC-50 value of the chimeric Ab (HOKO), which was set as 1.
• Figure 4A fold change EC-50 binding to 293T cells expressing human Nectin-2.
• Figure 4B - fold change EC-50 binding to Vero cells derived from African green monkey (AFG, Chlorocebus) and naturally expressing Nectin-2 (XP_007995342.1).
• Figure 4C results of CD112R blocking by the different humanized mAb variants, as assessed by FACS analysis of CD112R-Fc binding to CH0(K)l cells overexpressing human Nectin-2, in presence of the anti- Nectin-2 humanized clones in concentrations from 66-0.8 InM. Variants maintaining >70% blocking capacity at the lowest dose were considered superior.
• Figures 5A-5C depict the killing effect of humanized anti-Nectin-2 ADC variants.
• ADC was based on Saporin (ZAP) and the A549 target cells (lung adenocarcinoma) were confirmed to express Nectin-2.
• Figure 5A shows the superior killing effect of the lead clone H3K3, compared to other humanized variants tested at two concentrations.
• Figure 5B depicts comparable levels of ADC activity between the humanized H3K3 and H4K2 variants.
• Figure 5C - comparable levels of ADC activity between the chimeric mAb (HOKO), and the humanized H3K3 variant at three concentrations.
• H3K3 was also tested when grafted on IgG2-P238H, which has an FcgR-null Fc, and which had no effect on the potency of this variant. All killing results are significant (p ⁇ 0.001, two-way t-test), unless indicated by NS (not significant).
• Figures 6A-6C demonstrate that humanized H3K3-FcgR nu11 anti-Nectin-2 ADCs, conjugated to various toxins, lead to robust killing of various solid tumor cell lines. The Figures show significant killing activity of the H3K3-FcgR nu11 ADCs, at different ADC concentrations and across different cancer targets after 72 hours of incubation.
• Figure 6A depicts killing of RKO cells (colorectal adenocarcinoma).
• Figure 6B depicts killing of MDA-MB-231 cells (triple negative breast cancer).
• Figure 6C depicts killing of A549 cells (lung adenocarcinoma). Killing of target cells was significant (p ⁇ 0.001), unless indicated by NS (not significant). While all ADCs had a toxic effect, its magnitude varied pending the conjugated toxin and nature of target cells. Still, the ADCs containing either MMAE or MMAF were superior to all others across all target cells.
• Figure 7 demonstrates that humanized H3K3-FcgR nuU anti-Nectin-2 ADCs are not internalized via interaction with the high affinity FcgR - CD64.
• S228P h!gG4
• hlgGl Fc both conjugated to DM4
• Figure 8 demonstrates that humanized H3K3-FcgR nu11 anti-Nectin-2 ADCs lead to robust killing of a hematological cell line.
• H3K3-FcgR nu11 Abs with the indicated linker-payload combinations, were incubated at concentration from 9-1 pg/ml, for 72 hours with HL-60 cells (an AML model). All linker-payload combinations, except DM1, resulted in >90% killing at the high dose and significant killing at all doses tested (p ⁇ 0.001).
• Figures 9A-9B depict in-vivo efficacy of the humanized H3K3-FcgR nu11 anti-Nectin-2 ADCs against the hematological tumor cells line, HL-60 implanted s.c. to Nude female mice.
• Figure 9 A compares the effect of treatment with different combinations of linker pay loads (LP). Two of the LP combinations had a significant effect: DM4 resulted in significant tumor growth inhibition (TGI, p ⁇ 0.05), while complete tumor regression was seen for MMAE.
• TGI tumor growth inhibition
• MMAE The effect of the MMAE -based ADC is shown in Figure 9B without including the other treatments.
• Figure 10 depicts in-vivo efficacy of the humanized H3K3-FcgR nu11 anti-Nectin-2 ADC (1107- MC-VC-MMAE) against the solid (colorectal adenocarcinoma) tumor cell line, RKO. An empty vehicle was used as control.
• FIG 11 depicts in-vitro killing of MDA-MB-231 (triple negative breast cancer or TNBC) cells by the humanized H3K3-FcgR nu11 anti-Nectin-2 mAb (NTX1107) linked to Dxd, compared to Trodelvy (Sacituzumab govitecan), an approved ADC drug for TNBC.
• Both ADCs use the same class of cytotoxic payload (i.e., topoisomerase I (TOPI) inhibitors).
• TOPI topoisomerase I
• MDA- MB-231 cells were incubated in presence of the indicated concentration of these ADCs. Killing was evaluated after 120 hours. Robust killing of target cells was induced by both ADCs with significant superiority of Trodelvy at both concentrations (***p ⁇ 0.0005).
• Figure 12 depicts in-vivo efficacy of humanized H3K3-FcgR nu11 anti-Nectin-2 ADC (NTX1107), compared to Trodelvy, against MDA-MB-231 s.c. tumor model in Nude female mice.
• NTX1107-Dxd was able to significantly inhibit tumor growth, resulting in tumor stasis, while Trodelvy had no effect on tumor growth at the same treatment regimen.
• Figure 13 compares the effect of in-vivo treatment with H3K3-FcgR null -Dxd (NTX1107-Dxd) to that of H3K3-FcgR nu11 MMAE (NTX1107-MMAE) against MDA-MB-231 s.c. tumor model in Nude female mice. Complete tumor regression was seen for H NTX1107-MMAE with 7/7 mice having no measurable tumors at the end of the study. Tumor regression was seen for NTX1 107-Dxd with 4/7 mice having no measurable tumors at the end of the study.
• the present invention provides humanized monoclonal antibodies that recognize Nectin- 2.
• the antibodies of the invention are almost fully humanized, thus avoiding the risk of adverse immune response towards the antibodies and are therefore likely to be safe for use in humans.
• the antibodies of the invention are characterized by having unique CDR sequences and novel humanized framework sequences and design.
• the present invention further provides in some embodiments antibody-drug conjugates, or ADCs, comprising the humanized antibodies described herein, which are useful in treating cancer.
• ADCs antibody-drug conjugates
• certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details.
• the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.”
• the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.
• the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
• Nectin-2 or “Nectin Cell Adhesion Molecule 2”, as used herein refers to a human plasma membrane glycoprotein, also known as CD 112, and PVRL2.
• the Nectin-2 protein is a single-pass type I membrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. This protein is one of the plasma membrane components of adherent junctions. It also serves as an entry for certain mutant strains of herpes simplex virus and pseudorabies virus, and it is involved in cell to cell spreading of these viruses.
• Nectin-2 is set forth in SwissPort, UniPort and GenBank symbols or accession numbers: Gene ID: 5819, Q92692, 168093, NP_001036189.1, NP_002847.1, and # Q92692.
• the present invention provides a humanized antibody that specifically bind Nectin-2, or a fragment thereof comprising at least the antigen binding site, wherein the antibody or a fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
• the present invention provides a humanized antibody that specifically binds Nectin-2, or a fragment thereof comprising at least the antigen binding site, wherein the antibody, or a fragment thereof, comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising:
• FR-H1 frame work heavy chain No. 1
• FR-H2 is SEQ ID NO: 22
• FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29
• FR-H4 is SEQ ID NO: 24
• the light chain variable region comprising:
• FR-L1 frame work light chain No. 1
• FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35
• FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40
• FR-L4 is SEQ ID NO: 33.
• FR-H1 is the sequence before CDR1 in the heavy variable chain
• FR-H2 is the sequence between CDR1 and CDR2
• FR-H3 is the sequence between CDR2 and CDR3
• FR-H4 is the sequence after CDR3.
• FR-L1 is the sequence before CDR1 in the light variable chain
• FR-L2 is the sequence between CDR1 and CDR2
• FL-H3 is the sequence between CDR2 and CDR3
• FR-L4 is the sequence after CDR3.
• the present invention provides a humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences, the CDRs comprising the sequences SYW, SEQ ID NO: 8, and SEQ ID NO: 3; and ii. a set of four heavy chain framework sequences, wherein: FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29;
• FR-H4 is SEQ ID NO: 24; and the light chain variable region comprising: i. a set of three CDR sequences, the CDRs comprising the sequences set forth in SEQ ID NO: 10, SAS, and SEQ ID NO: 6; and ii. a set of four light chain framework sequences, wherein: FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33.
• the humanized antibody or fragment thereof comprises a heavy chain variable region comprising a set of three CDR sequences, the CDRs comprising the sequences SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and a light chain variable region comprising a set of three CDR sequences, the CDRs comprising the sequences SEQ ID NO: 10, SAS, and SEQ ID NO: 6.
• an antibody includes, but is not limited to, full- length, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and/or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity).
• immunoglobulin classes and/or isotypes e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM
• biologically relevant (antigen-binding) fragments or specific binding portions thereof including but not limited to Fab, F(ab')2, Fv, and scFv (single
• a monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts.
• a polyclonal antibody is a preparation that includes different antibodies of varying sequences that generally are directed against two or more different determinants (epitopes).
• the monoclonal antibody can comprise a human IgGl constant region.
• the monoclonal antibody can comprise a human IgG4 constant region.
• antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
• Fab fragment antigen binding
• F(ab')2 fragments fragment antigen binding
• Fab' fragments fragment antigen binding
• Fv fragments fragment antigen binding
• rlgG fragment antigen binding fragments
• single chain antibody fragments including single chain variable fragments (sFv or scFv) fragments.
• single domain antibodies e.g., sdAb, sdFv, nanobody
• the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
• antibody should be understood to encompass functional antibody fragments thereof.
• the term also encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
• the antibody can comprise a human IgGl constant region.
• the antibody can comprise a human IgG4 constant region.
• CDR identification or determination from a given heavy or light chain variable sequence is typically made using one of few methods known in the art. For example, such determination is made according to the Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).
• CDR having a sequence includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.
• the antigen specificity of an antibody is based on the hyper variable region (HVR), namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding site.
• HVR hyper variable region
• antibody fragments refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
• antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; singlechain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments.
• the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
• a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework region (FR) amino acid residues are derived from human FRs.
• a humanized antibody optionally may 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 variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
• some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
• a non-human antibody e.g., the antibody from which the CDR residues are derived
• the amino acid residues in the Fc domain can be substituted to be null, meaning the Fc domain does not bind Fc receptors or can bind with such low affinity and/or avidity as to not cause any Fc receptor signaling as a result of binding.
• the Fc domain can be null for binding to Fey receptors.
• Fey receptors for which the Fc domain can be null for binding can be, but not limited to, FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), FcyRIIIA (CD16a) F158 variant, FcyRIIIA (CD16a) V158 variant, or FcyRIIIB (CD 16b).
• the Fc domain may have one or more, two or more, three or more, or four or more amino acid substitutions that decrease binding of the Fc domain to an Fc receptor.
• the humanized antibody has a mutated Fc domain that prevents FcyR-mediated internalization.
• the humanized antibody comprises a Fc null domain.
• the Fc domain is null for binding to a Fey receptors.
• an "Fc null” refers to a domain that exhibits weak to no binding to one or more of the Fey receptors.
• the present invention provides a conjugate comprising the humanized antibody as described herein fused to a toxin.
• the conjugate comprises an antibody or fragment thereof comprising a heavy chain and a light chain
• the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16
• the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
• the toxin is selected from the group consisting of microtubule inhibitor, DNA synthesis inhibitor, topoisomerase inhibitor, and RNA polymerase inhibitor.
• the toxin is selected from the group consisting of microtubule inhibitor, DNA synthesis inhibitor, topoisomerase inhibitor, and RNA polymerase inhibitor.
• the toxin is a microtubule-destroying drug.
• the toxin is auristatin or a derivative thereof.
• the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).
• the toxin is saponin.
• the toxin is a maytansine derivative.
• the maytansine derivative is DM4 or DM1.
• the toxin is quinoline alkaloid.
• the quinoline alkaloid is SN-38.
• the toxin is selected from the group consisting of MMAE, MMAF, Saporin, DM4, DM1, SN-38, Calicheamicin, DXd, PBD, Duocarmycin, Sandramycin, alpha- Amanitin, Chaetocin, Daunorubicin, 17-AAG, Agrochelin A, Doxorubicin, Methotrexate, Colchicine, Cordycepin, Hygrolidin, Herboxidiene, Ferulenol, Curvulin, Englerin A, Taltobulin, Triptolide, Cryptophycin, and Nemorubicin.
• MMAE MMAE
• MMAF Saporin
• DM4 DM1, SN-38
• Calicheamicin DXd
• PBD Duocarmycin
• Sandramycin alpha- Amanitin
• Chaetocin Daunorubicin
• 17-AAG 17-AAG
• Agrochelin A Doxorubicin
• the toxin is a DNA topoisomerase I (TOPI) inhibitor.
• the DNA topoisomerase I (TOPI) inhibitor is DXd.
• toxins names are used herein as known in the art.
• suitable payloads include: - DM1 - a N 2 '-deacetyl-N 2 '-(3-mercapto-l-oxopropyl)-maytansine- a tubulin inhibitor.
• SN-38 - a potent DNA topoisomerase I inhibitor, a member of the class of pyranoindolizinoquinolines that is (4S)-pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione bearing two additional ethyl substituents at positions 4 and 11 as well as two additional hydroxy substituents at positions 4 and 9.
• MMAF - a monomethyl auristatin F - a tubulin inhibitor, having the formula C39H65N5O8.
• MMEA - a monomethyl auristatin E- a tubulin inhibitor, having the formula C39H67N5O7.
• the antibody is directly linked to the toxin.
• the antibody and the toxin are linked through a linker.
• the humanized described herein is covalently linked to the toxin.
• the linker is cleavable. According to additional embodiments, the linker is not cleavable.
• the linker is cleaved in response to changes in pH or redox potential. According to some embodiments, the linker is cleaved when contacted with lysosomal enzymes.
• the present invention provides, according to another aspect, a pharmaceutical composition
• a pharmaceutical composition comprising the humanized antibody or antigen binding fragment described herein or a conjugate comprising the antibody and a pharmaceutically acceptable excipient, carrier, or diluent.
• the pharmaceutical composition according to the invention is for use in treating cancer characterized by overexpression of Nectin-2.
• Nectin-2 overexpression related cancer types can be identified using known data bases such as The Cancer Genome Atlas (TCGA).
• the cancer treatable with a composition according to the present invention is selected from the group consisting of adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary kidney carcinoma (KIRP), lung adenocarcinoma (LU AD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC), cutaneous mela
• ACC ad
• the term “individual,” “patient,” or “subject” refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating.
• the individual is a mammal.
• the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak.
• the individual is a human.
• an “effective amount” refers to the amount of a therapeutic that causes a biological effect when administered to a mammal.
• Biological effects include, but are not limited to, inhibition or blockade of a receptor ligand interaction (e.g., PVR-TIGIT, PD-1- PD-L1/PD-L-2), inhibition of a signaling pathway, reduced tumor growth, reduced tumor metastasis, or prolonged survival of an animal bearing a tumor.
• a “therapeutic amount” is the concertation of a drug calculated to exert a therapeutic effect.
• a therapeutic amount encompasses the range of dosages capable of inducing a therapeutic response in a population of individuals.
• the mammal can be a human individual.
• the human individual can be afflicted with or suspected or being afflicted with a tumor.
• checkpoint inhibitor refers a drug that inhibits a biological molecule (“checkpoint molecule”) produced by an organism that negatively regulates the anti- tumor/cancer activity of T cells in the organism.
• Checkpoint molecules include without limitation PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, and CD112R.
• the molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known.
• excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof.
• PBS phosphate buffered saline
• dextrose glycerol
• ethanol ethanol
• suitable carriers are well known to those skilled in the art.
• the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.
• treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
• cancer and “tumor” relate to the physiological condition in mammals characterized by deregulated cell growth.
• Cancer is a class of diseases in which a group of cells display uncontrolled growth or unwanted growth. Cancer cells can also spread to other locations, which can lead to the formation of metastases. Spreading of cancer cells in the body can, for example, occur via lymph or blood. Uncontrolled growth, intrusion, and metastasis formation are also termed malignant properties of cancers. These malignant properties differentiate cancers from benign tumors, which typically do not invade or metastasize.
• the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.
• the anti-cancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof.
• the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea.
• the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine.
• the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan.
• the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine.
• the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin.
• the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.
• the present invention provides, according to another aspect, a method of treating a cancer in an individual afflicted with a cancer comprising administering to the individual a therapeutically effective amount of the humanized antibody or antigen binding fragment thereof or the pharmaceutical composition, and an inhibitor of PD-1, PD-L1, CTLA-4 or CD112R signaling.
• the cancer comprises a solid tumor.
• the cancer is selected from the group consisting of lung cancer, colon cancer, glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer.
• the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1.
• the antibody or fragment thereof that binds to PD-1 is Pembrolizumab, Nivolumab, AMP-514, Tislelizumab, Spartalizumab, or a PD-1 binding fragment thereof.
• the inhibitor of PD-1 signaling is an antibody that specifically binds PD- L-l or PD-L-2.
• the antibody that specifically binds PD-L1 or PD-L2 comprises Durvalumab, Atezolizumab, Avelumab, BMS-936559, or FAZ053, or a PD-L1 or PD-L2 binding fragment thereof.
• the inhibitor of PD-1 signaling comprises an Fc-fusion protein that binds PD-1, PD-E1, or PD-E2.
• the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.
• the inhibitor of PD-1 signaling comprises a small molecule inhibitor of PD-1, PD-E1, or PD-E2.
• the small molecule inhibitor of PD-1, PD-E1, or PD-E2 signaling comprises on or more of: N- ⁇ 2-[( ⁇ 2-methoxy-6-[(2-methyl[l,l’-biphenyl]-3- yl)methoxy]pyridin-3-yl]methyl)amino]ethyl ⁇ acetamide (BMS 202); (2-((3- cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5- methylbenzyl)-D-serine hydrochloride; (2R,4R)-l-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3- (2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxy
• compositions for treating a cancer in an individual afflicted with cancer comprising admixing the humanized antibody or antigen binding fragment thereof and a pharmaceutically acceptable excipient, carrier, or diluent.
• the cancer comprises a solid tumor.
• the cancer is selected from the group consisting of colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, glioblastoma, cervical cancer, prostate cancer, and lung cancer.
• Also described herein is a method of producing the humanized antibody or antigen binding fragment thereof comprising incubating the cell line described herein in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen binding fragment thereof.
• the additional anti-cancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof.
• bevacizumab carboplatin
• cyclophosphamide doxorubicin hydrochloride
• gemcitabine hydrochloride gemcitabine hydrochloride
• topotecan hydrochloride thiotepa
• Example 1 High expression of Nectin-2 mRNA correlates with poor survival probability of various cancer patients.
• Nectin-2 binds and affects immune cells through specific receptors.
• TIGIT relates to a co-inhibitory receptor on immune cells such as T and NK cells
• DNAM-1 also termed CD2266
• CD112R also termed PVRIG
• PVRIG relates to a co-inhibitory receptor on lymphoid immune cells (e.g., T and NK cells)
• Nectin-2 (CD112) servs as an inhibitory ligand for immune cells, mainly via its binding to CD112R.
• humanized anti-Nectin- 2 mAbs block Nectin-2 interactions with its high affinity receptor CD112R and may target cancer cells for specific toxin delivery and killing.
• the database Proteinatlas.com was searched for all the aliases of Nectin-2 (NECTIN2, CD112, HVEB, PRR2, PVRL2, PVRR2). Under the pathology rubric, data using three different mAbs was found. HPA0127569 mAb has the highest validation score (Enhanced) as it was validated by orthogonal method. Thus, the expression data across different tumors was selected for this clone only, and is depicted in Figure 3. The graph is showing percent of tumors positive for Nectin-2 expression. In 17/20 indications membranous expression of Nectin-2 is seen at Moderate-High levels.
• Murine anti-human Nectin-2 clone 2.11 disclosed in Patent application publication No. WO2020144697, was selected as the lead mAb for humanization. Based on structural analysis, a large preliminary set of sequence segments were identified that were used to create the humanized variants. These segments were selected and analyzed using iTopeTM technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008) and using the TCEDTM of known antibody sequence-related T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II, or that scored significant hits against the TCEDTM, were discarded.
• Table 1 Improved characteristics of the humanized anti-Nectin-2 mAbs. Illustration of predicted MHCII epitopes (iTope score) of the parental (VO) and humanized heavy (VH1-5) and light (Vkl-5) variable chains used to generate humanized variants for lead drug selection.
• the parental heavy chain (VHO) has 8 predicted high affinity motifs and the parental light chain (VkO) has 4 such motifs.
• VHO parental heavy chain
• VkO parental light chain
• the number of predicted high affinity MHCII epitopes for the heavy chain was reduced to 3 (VH2, VH3) or even to 1 (VH4, VH5) and for the light chain the predicted high affinity MHCII motifs were reduced to 2 (VK3-5).
• a protein’s denaturation profile provides information about its thermal stability and represents a structural ‘fingerprint’ for assessing structural and formulation buffer modifications.
• a widely used measure of the thermal structural stability of a protein is the temperature at which it unfolds from the native state to a denatured state. For many proteins, this unfolding process occurs over a narrow temperature range and the mid-point of this transition is termed ‘melting temperature’ or ‘Tm’.
• Tm melting temperature
• UNcle measures the fluorescence of Sypro Orange (which binds to exposed hydrophobic regions of proteins) as the protein undergoes conformational changes. Increased Tm is a desired characteristic in Ab lead selection as it predicts a more stable Ab.
• Purified lead humanized antibody variants in duplicates, were diluted to a final test concentration of 0.5 mg/ml in PBS into which Sypro Orange (at 160x Stock solution) was added to a final concentration of 20x solution. 9 pL of each sample mixture was loaded in duplicate into UNi microcuvettes. Samples were subjected to a thermal ramp from 15 - 95 °C, with a ramp rate of 0.3 °C/minute and excitation at 473 nm. Full emission spectra were collected from 250-720 nm, and the area under the curve between 510 - 680 nm was used to calculate the inflection points of the transition curves (Tonset and Tm). As seen in Table 2, for all of the antibodies tested, Tml and Tonset are higher for the humanized variants compared with the chimeric antibody (VH0/VK0).
• the humanized anti-Nectin-2 mAbs binding to human Nectin-2 expressed by 293T cells, (protein id: Q92692) and Chlorocebus (African green monkey, AFG) Vero cells were assessed and EC50 values were established.
• AFG expresses Nectin-2 protein (XP_007995342.1) with 97% similarity to human Nectin-2.
• Figures 4A and 4B depict fold change of EC50 values to human and AFG cells expressed Nectin-2, respectively. Both cell lines were plated at 0.5xl0 5 cells per well.
• the chimeric (HOKO) and humanized mAbs were added at concentration range of 20-0.001 nM.
• anti-human- APC Ab was used at 1:200 dilution (Jackson Immunoresearch AB_2340526). Cells were analyzed by FACS and EC50 was calculated and compared to that of the chimeric Ab (fold improve). This analysis revealed improved binding of the humanized variants, and similar degree of cross-reactivity to monkey Nectin-2, comparing to the chimeric mAh ( Figures 4A vs. 4B). To evaluate the blocking capacity of the humanized Abs, human Nectin-2 was overexpressed in Chinese hamster ovary (CHO) cells.
• Figure 4C shows data generated when 10 5 of CHO-hNectin-2/well were incubated with 30nM of human CD112R-mIgG2a in presence of the indicated humanized Abs, in concentrations ranging from 66-0.81nM. Bound CD112R was detected by using amIgG2a-647 (Jackson Immunoresearch Cat 115-607-186) at 1:200 dilution followed by FACS analysis. As can be seen, all humanized variants maintained full blocking capacity.
• FIG. 5 depicts the results of the initial screen which included top humanized clones and A549 (lung adenocarcinoma) cells as targets. 2xl0 3 cells per well were plated and allowed to adhere over 4-6 hours period. The ADCs were added, and the cells were incubated with the ADCs.
• H3K3-FcgR nu11 mAbs were generated by mutating key residues in the hinge region of human IgGl as indicated in Table 3.
• the presented linker payload combinations were chosen according to the desired release mechanism and were generated according to the standard protocol by Abzena LTD. Briefly, the mAbs were reduced and incubated with an excess of the linker-payload to obtain the desired drug antibody ratio (DAR) of 4 for all the linker-payloads except for SN-38, which had a target DAR of 8. The final products were then purified, and the DARs were established by LC/MS method. Selected tumor cell lines, representing various solid tumors, were used to assess potency of the various linker-payload combinations in-vitro. The indicated target cells were plated at 2xl0 3 cells per well and allowed to adhere over 4-6 hours period.
• DAR drug antibody ratio
• the ADCs were added at concentrations of 4-0.16
• CHO cells overexpressing the high affinity Fc receptor hCD64 (CHO- hCD64) were generated. These cells do not express human Nectin-2 and thus no target- specific killing is expected.
• Parental CHO cells and CHO-hCD64 were plated at 2xl0 3 cells per well and allowed to adhere over 4-6 hours.
• the different Fc variant ADCs were added at 12pg/ml. After incubation of 72 hours, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo® 2.0 Cell Viability Assay - (Promega G9242).
• HL-60 cells (AML model) were plated at 2xl0 3 cells per well and the ADCs were added at concentrations of 9-lpg/ml, using 3-fold dilutions. The cells were incubated with the ADCs for 72 hours. Then, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo® 2.0 Cell Viability Assay - (Promega G9242) as described above. As seen in Figure 8, except for the DM1 pay load (indicated in the graph), all other compounds led to significant killing of the targets in all concentrations tested and all exceeded 90% killing at the highest concentration (>60nM). Based on these results the SMCC-DM1 linker payload was excluded from future experiments.
• PBS vehicle
• H3K3-FcgR null -MMAE H3K3-FcgR null -MMAF
• H3K3-FcgR null -DM4 H3K3-FcgR null -SN- 38. All treatments were at 5 mg/kg
• H3K3-FcgR null -MMAF and the SN-38 variants did not attenuate tumor growth.
• H3K3-FcgR null -DM4 led to significant tumor growth inhibition (TGI) of 49%, while H3K3-FcgR null -MMAE led to significant tumor regression.
• Figure 9B shows the mean effect of H3K3-FcgR null -MMAE.
• PBS Vehicle
• H3K3-FcgR null -MMAE led to significant tumor regression, as can be seen in Figure 10, representing >80% TGI compared to vehicle treated group.
• the cells were incubated with the ADCs for 120 hours, after which tumor cell killing was evaluated using CellTiter-Glo® 2.0 Cell Viability Assay - (Promega G9242), following a standard protocol. Robust killing of MDA-MB-231 cells was induced by both ADCs with significant superiority of Trodelvy at both concentrations (***p ⁇ 0.0005).
• PBS vehicle
• NTX1107-Dxd NTX1107-Dxd
• Trodelvy 5 mg/kg
• NTX1107-Dxd was able to significantly inhibit tumor growth (*p ⁇ 0.02, ** ⁇ 0.005.***p ⁇ 0.0005).
• Anti-hNectin-2 H3K3-FcgR nul1 can regress aggressive MDA-MB-231 tumors in vivo when conjugated to tubulin or TOPI targeting agents
• NTX1107-Dxd For the animals treated with NTX1107-Dxd, four had no tumors, while for the remaining three the average tumor volume was 210mm 3 , indicating tumor stasis.
• Table 5 Framework (Non-CDR) sequences of the humanized heavy chain variable regions.
• Table 6 Framework (Non-CDR) sequences of the humanized light variable regions.
• SEQ ID NO: 65 Human IgGl (G237I / V264R) (including VH3)

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