EP3867273A1 - T cell redirecting bispecific antibodies for the treatment of squamous cell cancers - Google Patents

T cell redirecting bispecific antibodies for the treatment of squamous cell cancers

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Publication number
EP3867273A1
EP3867273A1 EP19794927.4A EP19794927A EP3867273A1 EP 3867273 A1 EP3867273 A1 EP 3867273A1 EP 19794927 A EP19794927 A EP 19794927A EP 3867273 A1 EP3867273 A1 EP 3867273A1
Authority
EP
European Patent Office
Prior art keywords
cd3xegfr
cells
antibody
squamous
squamous cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19794927.4A
Other languages
German (de)
French (fr)
Inventor
Amelie Croset
Olivia HALL
Jeremy BERRET
Megane PLUESS
Elodie STAINNACK
Lamine Mbow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ichnos Sciences SA
Original Assignee
Ichnos Sciences SA
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Filing date
Publication date
Application filed by Ichnos Sciences SA filed Critical Ichnos Sciences SA
Publication of EP3867273A1 publication Critical patent/EP3867273A1/en
Withdrawn legal-status Critical Current

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    • 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
    • C07K16/2809—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 against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • 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
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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

Definitions

  • the present invention relates to an antibody or fragment thereof for use in the treatment of a squamous cell carcinoma.
  • the present invention relates to T cell redirecting bispecific antibodies for the treatment of EGFR positive squamous cell cancers.
  • SCCs squamous cell carcinomas
  • cSCC cutaneous squamous cell carcinoma
  • the squamous cell carcinoma of the lung is a histological subtype of non-small cell lung cancer (NSCLC), representing about the 25-30% of NSCLC.
  • NSCLC non-small cell lung cancer
  • the development of the squamous cell carcinoma of the lung is strongly associated with smoking and chemotherapy is considered for primary treatment (Liao et al. Lung Cancer Manag, 2012; 1(4): 293-300).
  • Cancers originating from the squamous epithelium of the upper aerodigestive tract, including lip, oral cavity, tongue, pharynx, larynx and paranasal sinuses, are classified as head and neck squamous cell carcinoma (HNSCC).
  • HNSCC head and neck squamous cell carcinoma
  • EGFR Epidermal Growth Factor Receptor
  • HerBl or HER1 is a transmembrane protein belonging to the ErbB receptor tyrosine kinase family, and often overexpressed in cancer cells, including squamous cell cancer.
  • EGF Epidermal Growth Factor
  • TGFa Transforming Growth Factor a
  • TK tyrosine kinase
  • EGFR plays a crucial role in signaling pathways involved in the modulation of cell growth, proliferation, differentiation and apoptosis; and in cancer cells, its overexpression promotes cancer progression, angiogenesis and metastasis formation.
  • Necitumumab (Portrazza ® ), a fully human IgGl monoclonal antibody, was approved by the FDA in 2015, in combination with gemcitabine and cisplatin for the treatment of patients with metastatic squamous non-small cell lung cancer (Thakur and Wozniak, Lung Cancer: Targets and Therapy, 2017, 8: 13-19).
  • Intrinsic and acquired drug resistance remains one of the main challenges for targeted therapy.
  • SCCs treatments for instance other EGFR targeting molecules, or other strategies, such as immunotherapy needs to be developed.
  • the present invention relates to a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR for use in the treatment of a squamous cell cancer.
  • Squamous cell cancers also known as squamous cell carcinomas (SCCs)
  • SCCs are a class of cancers derived from squamous epithelial cells. This class includes, among others, the squamous cell cancer of the skin, the squamous cell cancer of lung, the squamous cell cancer of the head and neck, which all together have high incidence on the worldwide population.
  • the present invention provides a CD3xEGFR bispecific antibody for the treatment of SCCs, wherein said antibody binds simultaneously the cluster of differentiation 3 (CD3), a T cell co receptor, and EGFR present on the surface of squamous cancer cells.
  • CD3 Cluster of differentiation 3
  • T cell co receptor a T cell co receptor
  • the bispecific antibody of the present invention therefore allows engaging T cells for the redirected lyses of EGFR positive tumor cells.
  • the use of the disclosed bispecific antibody for immunotherapy is advantageous over the use of conventional monoclonal antibodies (mAbs) for EGFR-targeted therapy to bypass resistance mechanisms.
  • mAbs monoclonal antibodies
  • the binding between the disclosed bispecific antibody and EGFR acts as anchorage in the tumor cell to redirect T cells without affecting EGFR function and resulting less affected by EGFR internalization.
  • Kirsten ras (KRAS) oncogene homolog from the mammalian ras gene family is by mutation of the Kirsten ras (KRAS) oncogene homolog from the mammalian ras gene family.
  • KRAS mutations The emergence of KRAS mutations is a frequent driver of acquired resistance to anti-EGFR mAb therapies in colorectal and other cancers. Differently, the bispecific antibody of the present invention has been shown to be effective also on KRAS mutated squamous cancer cells.
  • the present invention discloses a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR for use in the treatment of a squamous cell cancer.
  • the present invention also relates to a method of treating a patient in need thereof by administering a therapeutic effective amount of a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR.
  • a CD3xEGFR bispecific antibody according to the present invention for use as a medicament of an EGFR expressing squamous cell cancer.
  • the antibody according to the present invention binds EGFR on the cell surface of squamous cancer cells and the expression of EGFR on the cell surface of said squamous cancer cells express measured as sABC values is at least about 3000.
  • the antibody according to the present invention binds to EGFR on the cell surface of said squamous cancer cells with an EC 5 o equal to or greater than about 300 pM and equal to or less than about 5500 pM.
  • the antibody according to the present invention induces squamous cancer cell specific killing with an EC 5 o equal to or greater than about 0.1 pM and equal to or less than about 15 pM. More in particular, the antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC 5 o equal to or greater than about 0.01 pM and equal to or less than about 100 pM.
  • the antibody according to the present invention induces squamous cancer cells specific killing, with specific killing percentage equal to or greater than about 80% when the percentage of receptor occupancy is equal to or less than about 10%.
  • the disclosed antibody is administered intravenously at a dose between about 0.0001 mg/kg and about 1 mg/kg body weight, in particular at a dose equal to or greater than about 0.001 mg/kg body weight and equal to or less than about 0.5 mg/kg body weight.
  • said antibody is administered once a week for a number of week comprised between 1 and 3.
  • Co is about 110 ng/mL and/or C ma x is comprised between about 100 and about 200 ng/mL and/or AUC 0-t is about 6400 hr*ng/mL and/or AUCo-m f is comprised between about 7000 and about 13000 hr*ng/mL and/or AUC 0-336 is about 10300 ng*hr/mL and/or T ma x is about 0.25 hr and/or T 1 / 2 is comprised between about 100 and about 150 hr and/or Vz is comprised between about 150 and about 250 ml/kg and/or Vss is comprised between about 100 and about 200 mL/kg and/or CL is comprised between about 0.5 and about 1.5 mL/hr/kg and/or MRT
  • NF is comprised between about 130 and about 170 hr
  • the disclosed antibody is use for the treatment of a squamous cell carcinoma selected from the group comprising squamous cell carcinomas of the skin; squamous cell carcinomas of the head and the neck, comprising squamous cell carcinoma of the larynx, such as squamous cell carcinoma of the epiglottis, squamous cell carcinoma of the supraglottis, squamous cell carcinoma of the glottis, and squamous cell carcinoma of the subglottis, of the oral cavity, such as squamous cell carcinoma of the tongue, of the floor of mouth, of the hard palate, of the buccal mucosa, of the salivary glands and of the alveolar ridges, of the oropharynx, such as squamous cell carcinoma of the lateral pharyngeal walls, of the base of tongue, of the tonsils, and of the soft palate, squamous cell carcinomas of the nasopharyn
  • the CD3xEGFR bispecific antibody of the present invention is selected from the group comprising CD3xEGFR_SFl (SEQ ID NOs: 3, 4 and 5), CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7), CD3xEGFR_SF4 (SEQ ID NOs: 3, 4 and 8), CD3xEGFR_SDl (SEQ ID NOs: 1, 2 and 9) and CD3xEGFR_SD2 (SEQ ID NOs: 10, 9 and 2).
  • the presently disclosed antibodies may be used as a diagnostic tool to quantitatively or qualitatively detect EGFR-expressing squamous cancer cells and/or to characterize said EGFR- expressing squamous cancer cells based on the antibody-EGFR binding properties and/or based on the EGFR-expressing squamous cancer cells killing.
  • the bispecific antibody of the present invention may be used to detect quantitatively or qualitatively and/or to characterize EGFR-expressing squamous cancer cells in a body fluid, a tissue, or an organ. Therefore the presently disclosed antibodies may be provided in a diagnostic kit, which may contain other components that aid EGFR- expressing squamous cancer cells detection and/or characterization.
  • the antibody of the present invention may be used in a method of screening and/or identification and/or classification of SCC patient subpopulations, based on the above said quantitatively or qualitatively EGFR-expressing squamous cancer cells detection and/or characterization, which may be useful to develop personalized treatments.
  • squamous cell carcinoma In the present invention the terms “squamous cell carcinoma”, “epidermal carcinoma”, “squamous cell cancer” and “SSC” are used interchangeably to indicate cancers originating from squamous epithelial cells.
  • Squamous cells are thin, flat cells that look like fish scales, and are found in the tissue that forms the surface of the skin, the lining of the hollow organs of the body, and the lining of the respiratory and digestive tracts.
  • Non limiting examples of squamous cell carcinomas include: skin squamous cell carcinoma or cutaneous squamous cell carcinoma; squamous cell carcinoma of the head and the neck (HNSCC), comprising SCC originating in the larynx (such as epiglottis, supraglottis, glottis, and subglottis SCCs), SCCs originating in the oral cavity or SCCs of the mouth (such as tongue, floor of mouth, hard palate, buccal mucosa, salivary glands and alveolar ridges SCCs), SCCs originating in the oropharynx (such as posterior and lateral pharyngeal walls, base of tongue, tonsils, and soft palate SCCs), SCCs originating in the nasopharynx, nasal cavity, paranasal sinuses, hypopharynx, and salivary glands, squamous cell thyroid carcinoma and squamous cell carcinoma of the eye; SCCs of the es
  • the term “antibody” and the term “immunoglobulin” are used interchangeably.
  • the term “antibody” as referred to herein includes whole antibodies and any antigen binding fragments or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • CL The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR or FW).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino- terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
  • the amino acid sequences of FW1, FW2, FW3, and FW4 all together constitute the "non-CDR region” or "non-extended CDR region” of VH or VL as referred to herein.
  • the term "heavy chain variable framework region” as referred herein may comprise one or more (e.g., one, two, three and/or four) heavy chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)).
  • the heavy chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3.
  • light chain variable framework region may comprise one or more (e.g., one, two, three and/or four) light chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)).
  • the light chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the First component (Clq) of the classical complement system.
  • Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region.
  • Human constant light chains are classified as kappa (CK) and lambda (CX) light chains.
  • Heavy chains are classified as mu (m), delta (d), gamma (y), alpha (a), or epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • isotype as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgGl (IGHG1), lgG2 (IGHG2), lgG3 (IGHG3), lgG4 (IGHG4), IgAl (IGHA1), lgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE).
  • the so-called human immunoglobulin pseudo-gamma IGHGP gene represents an additional human immunoglobulin heavy constant region gene which has been sequenced but does not encode a protein due to an altered switch region (Bensmana M et al., (1988) Nucleic Acids Res. 16(7): 3108).
  • the human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy constant domains (CHI -CH3) and hinge. All open reading frames for its heavy constant domains encode protein domains which align well with all human immunoglobulin constant domains with the predicted structural features.
  • This additional pseudo-gamma isotype is referred herein as IgGP or IGHGP.
  • Other pseudo immunoglobulin genes have been reported such as the human immunoglobulin heavy constant domain epsilon PI and P2 pseudo genes (IGHEP1 and IGHEP2).
  • the IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, lgG2, lgG3 and lgG4. In mice this class comprises subclasses IgGl, lgG2a, lgG2b, lgG2c and lgG3.
  • Antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward ES et al., (1989) Nature, 341 : 544-546) which consists of a single variable, (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vi) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird RE et al, (1988) Science 242: 423-426; Huston JS et al, (1988) Proc.
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • antigen refers to any molecule to which an antibody can specifically bind.
  • antigens include polypeptides, proteins, polysaccharides and lipid molecules.
  • polypeptides include polypeptides, proteins, polysaccharides and lipid molecules.
  • polysaccharides include polypeptides, proteins, polysaccharides and lipid molecules.
  • epitopes can be present.
  • epitopic determinants includes any protein determinant capable of specific binding to/by an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • an antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 mM, for example, ⁇ 1 mM; e.g., ⁇ 100 nM, ⁇ 10 nM or ⁇ 1 nM.
  • Bispecific antibodies are antibodies that can bind two different antigens, or two different epitopes of the same antigen.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, for example, at least 80%, 90%, 95%, or 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • the bispecific antibody provide by the present invention binds to epitopes upon CD3e and EGFR.
  • the cluster of differentiation 3 (CD3) is a T cell co-receptor helps to activate both the cytotoxic T cell (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells).
  • CD3 is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the z-chain (zeta-chain) to generate an activation signal in T lymphocytes.
  • TCR T-cell receptor
  • zeta-chain zeta-chain
  • EGFR Epidermal Growth Factor Receptor
  • FlerBl FlerBl
  • FIERI The Epidermal Growth Factor Receptor
  • the CD3xEGFR bispecific antibody of the present invention is selected from the group CD3xEGFR_SFl (SEQ ID NOs: 3, 4 and 5), CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7), CD3xEGFR_SF4 (SEQ ID NOs: 3, 4 and 8), CD3xEGFR_SDl (SEQ ID NOs: 1, 2 and 9) and CD3xEGFR_SD2 (SEQ ID NOs: 10, 9 and 2).
  • the CD3xEGFR bispecific antibody of the present invention is CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7).
  • the CD3xEGFR bispecific antibody binds EGFR on the cell surface of squamous cancer cells.
  • Method for the quantification of protein expression on the cell surface are known in the art.
  • Non limiting examples include method involving immunohistochemistry and antibody binding capacity assays.
  • the level of expression of EGFR on the surface of squamous cancer cells is quantified by cytometry using a specific Antibody Binding Capacity (sABC) assay.
  • sABC Antibody Binding Capacity
  • the CD3xEGFR bispecific antibody of the present invention binds EGFR on the cell surface squamous cancer cells, wherein the expression of EGFR on the cell surface is at least about 3000 sABC.
  • the expression of EGFR on the surface of said squamous cancer cells is selected from the group comprising at least about 3000 sABC, at least about 5000 sABC at least about 10000 sABC, at least about 50000 sABC, at least about 100000 sABC, at least about 150000 sABC, at least about 200000 sABC, at least about 250000 sABC, at least about 500000 sABC, at least about 1000000 sABC, at least about 1500000 sABC.
  • the present invention also includes sABC values at intervals of 1, 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000 sABC between the above said values.
  • Non limiting examples include ligand binding assays, ELISA binding, Biacore assay, and fluorescence-activated cell sorting (FACS) binding assay.
  • FACS fluorescence-activated cell sorting
  • the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an EC 50 equal to or greater than about 100 pM and equal to or less than about 6000 pM, e.g. equal to or greater than about 300 pM and equal to or less than about 5500 pM.
  • the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an EC 50 selected from the group comprising about about 100 pM, 300 pM, about 500 pM, about 1000 pM, about 1500 pM, about 2000 pM, about 2500 pM, about 3000 pM, about 3500 pM, about 4000 pM, about 4500 pM, about 5000 pM, about 5500 pM, about 6000 pM.
  • the antibody of the present invention also includes EC 5 o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an EC20 equal to or greater than about 50 pM and equal to or less than about 1500 pM.
  • the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an EC20 selected from the group comprising about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1000 pM, about 1100 pM, about 1200 pM, about 1300 pM, about 1400 pM, about 1500 pM.
  • an EC20 selected from the group comprising about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1000 pM, about 1100 pM, about 1200 pM, about 1300 pM, about 1400 pM, about 1500 pM.
  • the antibody of the present invention also includes EC 2 o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an ECso equal to or greater than about 1200 pM and equal to or less than about 21000 pM.
  • the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an ECso selected from the group comprising about 1200 pM, about 3000 pM, about 5000 pM, about 7000 pM, about 10000 pM, about 13000 pM, about 15000 pM, about 17000 pM, about 21000 pM.
  • the antibody of the present invention also includes EC 8 o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 50, 100, 500, 1000, 5000, and 10000 pM between the above said values.
  • the ability of the disclosed bispecific antibody to induce redirected lysis of squamous cancer cells is determined by cytotoxic assay (MTS) to assess specific killing, and by a FACS assay to determine the activation and proliferation of T cells, and the release of cytolytic mediators.
  • MTS cytotoxic assay
  • FACS FACS assay
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an EC 5 o equal to or greater than about 0.05 pM and equal to or less than about 35 pM, in a specific embodiment C 50 equal to or greater than about 0.1 pM and equal to or less than about 15 pM.
  • the antibody of the present invention induces squamous cancer cells specific killing with an EC 50 selected from the group comprising about 0.05 pM, about 0.1 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 50 values at intervals of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an EC 2 o equal to or greater than about 0.01 pM and equal to or less than about 10 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing with an EC 2 o selected from the group comprising about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.5 pM, about 0.7 pM, about about 1 pM, about 3pM, about 5 pM, about 8 pM, about 10 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 2 o values at intervals of 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an ECso equal to or greater than about 0.2 pM and equal to or less than about 140 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing with an ECso selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 25 pM, about 50 pM, about 100 pM, about 120 pM, about 150 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 8 o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing at a dose of at least about 0.0000001 nM, and/or at a dose of at least about 0.000001 nM and/or at a dose of at least about 0.00001 nM, and/or at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM.
  • the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.0000001 nM, at least about 0.000001 nM, at least about 0.00001 nM, at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM.
  • the antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.00000001, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release.
  • FACS assay is used to determine the activation and proliferation of T cells, and the release of cytolytic mediators.
  • the expression of the activation markers CD25 and CD69 is assessed in CD4+ and CD8+ T cells.
  • the expression of the proliferation marker Ki67 is assessed in CD4+ and CD8+ T cells.
  • cytolytic markers produced by CD3+ T cells following the engagement of the disclosed antibody in a RDL assay with squamous cancer cells the expression of CD107a (LAMP-1), a marker of degranulation of cytolytic granules, and granzyme B, a serine protease that mediates apoptosis of targets cells, is assessed in CD4+ and CD8+ T cells.
  • markers useful to assess the effect of the antibody disclosed in the present invention include perforine (pore-forming cytolytic glycoprotein), Fas/FasL (engagement of FasL to Fas induces cell death by caspase activation), Granzyme A and K and IFN-gamma.
  • the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC 5 o equal to or greater than about 0.005 pM and equal to or less than about 150 pM, specifically equal to or greater than about 0.01 pM and equal to or less than about 100 pM, more specifically equal to or greater than about 0.02 pM and equal to or less than about 70 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC 50 selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 30 pM, about 50 pM, about 70 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 50 values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 pM between the above said values.
  • the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell activation with an EC 50 equal to or greater than about 0.01 pM and equal to or less than about 20 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing via T cell activation with an EC 50 selected from the group comprising about 0.01 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 20 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 5 o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10 pM between the above said values.
  • the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell proliferation with an EC 5 o equal to or greater than about 1 pM and equal to or less than about 50 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing via T cell proliferation with an EC 5 o selected from the group comprising about 1 pM, about 5 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 5 o values at intervals of 0.5, 1, 5, 10, 20, 50 pM between the above said values.
  • the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via cell cytolytic granules release with an EC 5 o equal to or greater than about 0.2 pM and equal to or less than about 70 pM.
  • the antibody of the present invention induces squamous cancer cell specific killing via T cell cytolytic granules release with an EC 5 o selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 30 pM, about 50 pM, about 70.
  • the antibody of the present invention also induces squamous cancer cells specific killing with EC 5 o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 pM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell activation at a dose of at least about 0.00001 nM, and/or at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM.
  • the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.00001 nM, at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM.
  • the antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell proliferation at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM.
  • the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM.
  • the antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via cytolytic granules release at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM.
  • the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM.
  • the antibody of the present invention also induces specific killing of squamous cancer cells at closes at intervals of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
  • the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing when the percentage of receptor occupancy is equal to or less than about 10%, or equal to or less than about 8%, or equal to or less of about 5%, or equal to or less of about 2%, or equal to or less of about 1%, or equal to or less of about 0.5%.
  • the percentage of receptor occupancy is equal or less than about 5%.
  • the percentage of receptor occupancy is equal to or less than about 2%.
  • the percentage of receptor occupancy is equal to or less than about 1%.
  • the antibody of the present invention lead to a percentage of specific killing equal to or greater than about 50%, or equal to or greater than about 60%, or equal to or greater than about 70%, or equal to or greater than about 80%, or equal to or greater than about 90%, or equal to 100%.
  • the percentage of specific killing is equal to or greater than about 70%.
  • the percentage of specific killing is equal to or greater than about 80%.
  • the antibody of the present invention induces squamous cancer cells specific killing, with specific killing percentage equal to or greater than about 80% when the percentage of receptor occupancy is equal to or less than about 2%.
  • a method of treating an EGFR expressing squamous cell cancer by administering a therapeutic effective amount of the CD3xEGFR bispecific antibody according to the present invention to a patient in need thereof.
  • administering refers to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent.
  • modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.
  • the antibody or of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. More preferred routes of administration are intravenous or subcutaneous.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody of the invention can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • an effective amount is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • a “patient” for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans.
  • the antibodies of the present invention have both human therapy and veterinary applications.
  • treatment or “treating” in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder.
  • successful administration of an antibody prior to onset of the disease results in treatment of the disease.
  • successful administration of an antibody after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.
  • Treatment and “treating” also encompasses administration of an antibody after the appearance of the disease in order to eradicate the disease.
  • Those "in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
  • the antibody of the present invention can be administered at a single or multiple doses.
  • dose or “dosage” as used in the present invention are interchangeable and indicates an amount of drug substance administered per body weight of a subject or a total dose administered to a subject irrespective to their body weight.
  • the disclosed CD3xEGFR bispecific antibody is administered intravenously at a dose equal to or greater than about 0.0001 mg/kg body weight and equal to or less than about 5 mg/kg body weight.
  • the antibody of the present invention is administered intravenously at a dose equal to or greater than about 0.001 mg/kg body weight and equal to or less than about 0.5 mg/kg body weight.
  • the antibody of the present invention if administered at a dose selected from the group comprising about 0.001 mg/kg body weight, about 0.005 mg/kg body weight, about 0.01 mg/kg body weight, about 0.05 mg/kg body weight.
  • the antibody of the present invention can also be administrated at doses at intervals of 0.0001, 0.001, 0.01, 0.1 and 1 mg/kg body weight between the above said values.
  • the antibody of the present invention is administrated at a dose as specified above in a single intravenous injection or intravenously once a week for at least one week, or for a number of weeks greater than 1 week, e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, in particular for a number of week comprised between 1 and 3.
  • Co is about 110 ng/mL and/or C max is comprised between about 100 and about 200 ng/mL and/or AUCo- t is about 6400 hr*ng/mL and/or AUC 0 -m f is comprised between about 7000 and about 13000 hr*ng/mL and/or AUC O -336 is about 10300 ng*hr/mL and/or T max is about 0.25 hr and/or T1/2 is comprised between about 100 and about 150 hr and/or Vz is comprised between about 150 and about 250 ml/kg and/or Vss is comprised between about 100 and about 200 mL/kg and/or CL is comprised between about 0.5 and about 1.5 mL/hr/kg and/or MRT
  • NF is comprised between about 130 and about 170 hr and/or
  • C 0 represents the initial concentration
  • C max represents the peak plasma concentration of a drug after administration
  • AUC represents the area under the curve, the integral of the concentration-time curve
  • T max represents the time to reach C max
  • T1/2 represents the time required for the concentration of the drug to reach half of its original value
  • Vz represents the volume of distribution during terminal phase after intravenous administration
  • Vss represents the apparent volume of distribution at equilibrium determined after intravenous administration
  • CL represents the clearance, the volume of plasma cleared of the drug per unit time
  • M RTINF represents mean residence time infinity
  • ti ast represents time of last measurable concentration.
  • the present invention also comprises the above said pharmacokinetic parameters at any value comprised between the above said values.
  • Figure 1 Levels of EGFR expression on a panel of seven squamous cancer cell lines. Numbers of EGFR were measured on the surface of the squamous cancer cells SCC-4, NCI-H226, SCC-25, NCI-H1703, SW- 900, NCI-H2286 and NCI-H520 using a specific antigen binding capacity (sABC) method. Error bars represent ⁇ the standard deviation, and LOD the Limit Of Detection.
  • Figure 2. EGFR mRNA expression in different squamous cancer cell lines.
  • A EGFR expression is shown as a relative quantification (delta delta Ct) scaled to beta actin expression and healthy lung or tongue control samples.
  • the bar plot shows the upregulation and downregulation of EGFR expression among different lung and tongue squamous cancers cells.
  • B Correlation between EGFR sABC value (receptor expression at the cell surface; X axis) and EGFR RNA expression (scaled relative quantification; Y axis).
  • C Statistical analysis of correlation which shows a statistically significant correlation (p ⁇ 0.05) and a Pearson coefficient of 0.93 (positive correlation).
  • FIG. 3 Squamous cancer cells do not shed EGFR or release EGF in culture supernatant. Soluble EGFR (A) or EGF (B) were quantified in the culture supernatant of different squamous cancer cells at 24, 48, 72, 96 and 168h by ELISA or Luminex assays respectively. Limit of detection of the Luminex was at 2.6pg/ml.
  • FIG. 4 Detection of CD3xEGFR-SF3 binding by flow cytometry on squamous cancer cell lines.
  • An increasing dose of CD3xEGFR-SF3 or control antibodies were incubated on SCC-4 (A), NCI-H226 (B), SCC-25 (C), NCI-H1703 (D), SW-900 (E), NCI-H2286 (F) and NCI-H520 (G) squamous cancer cell lines and binding was detected with a PE-labelled anti-human IgG.
  • the graphs show the non-linear sigmoidal regression binding curves of the mean fluorescent intensity (MFI) for each treatment. Each data point is the mean ⁇ SEM of duplicates values from 3-4 independent replications.
  • CD3xEGFR-SF3 induces the redirected lysis of squamous cancer cells by CD3+ T cells in an RDL assay.
  • Target cells squamous cancer cells T were incubated with serial dilutions of CD3xEGFR- SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. Specific killing was measured by MTS on squamous cancer cells after removal of the PBMCS.
  • Points represent the mean of (A) 18 donors for NCI-H520, 6 donors for SW-900, 21 donors for NCI-H1703, (B) 9 donors for SCC-25, 18 donors for NCI-H226 and 21 donors for SCC-4. Error bars show the SEM.
  • Squamous cancer cells SCC-4 (A), NCI-H226 (B), SCC-25 (C), NCI-H1703 (D), SW-900 (E) and NCI-H520 (F).
  • CD3xEGFR-SF3 induces CD4+ and CD8+ T cell activation in an RDL assay with squamous cancer cell lines.
  • Target squamous cancer cells T were incubated with serial dilutions of CD3xEGFR- SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours.
  • Percentage of early (CD69) and late (CD25) activation markers were assessed by flow cytometry for both CD4+ and CD8+ T cells. Points represent the means and error bars shows the standard error of the mean.
  • Squamous cancer cells (A) NCI-H520, NCI-H2286, SW-900, (B) NCI-H1703, NCI-H226, SCC- 4.
  • CD3xEGFR-SF3 induces the proliferation of CD4+ and CD8+ T cells in an RDL assay with squamous cancer cells.
  • Target squamous cancer cells T
  • T were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours.
  • E unstimulated human PBMCs
  • Ki67 was assessed by flow cytometry in both CD4+ and CD8+ T cells.
  • Each data point is the mean ⁇ SEM of values from 6 individual donors from 2 independent replications.
  • Squamous cancer cells (A) NCI-FI520, NCI-FI2286, SW-900, (B) NCI-FI1703, NCI-H226, SCC-4.
  • CD3xEGFR-SF3 induces the expression of cytolytic markers by CD4+ T cells in an RDL assay with squamous cancer cells.
  • Target squamous cancer cells T
  • T were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours.
  • E unstimulated human PBMCs
  • the percentages of the cytolytic markers CD107a and Granzyme B (GrB) were assessed by flow cytometry on CD4+ T cells.
  • Each data point is the mean ⁇ SEM of values from 6 individual donors from 2 independent replications.
  • Squamous cancer cells (A) NCI-H520, NCI-H2286, SW-900, (B) NCI- H1703, NCI-H226, SCC-4.
  • CD3xEGFR-SF3 induces the expression of cytolytic markers by CD8+ T cells in an RDL assay with squamous cancer cells.
  • Target squamous cancer cells T
  • T were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours.
  • E unstimulated human PBMCs
  • the percentages of the cytolytic markers CD107a and Granzyme B (GrB)) were assessed by flow cytometry on CD8+ T cells.
  • Each data point is the mean ⁇ SEM of values from 6 individual donors from 2 independent replications.
  • Squamous cancer cells (A) NCI-FI520, NCI-FI2286, SW-900, (B) NCI- H1703, NCI-H226, SCC-4.
  • CD3xEGFR-SF3 induces the redirected lysis of squamous cancer 3D spheroids by CD3+ T cells in an RDL assay.
  • Target 3D spheroids of squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 10:1 for 48 hours. Specific killing was measured by LDH released in the supernatant.
  • Points represent the mean of (A) 9 donors for NCI-FI520, 3 donors for NCI-FI1703, (B) 24 donors for NCI-H226 and 6 donors for SCC-4. Error bars show the standard error of the mean.
  • FIG. 21 Time-course of squamous cancer 3D spheroids during the redirected lysis assay.
  • Target 3D squamous cancer spheroids (T) of NCI-H520 (EGFR negative) and NCI-H226 (EGFR expressing) were plated at low density in poly-FIEMA coated plates.
  • PBMCs Effectors; E
  • E were added at an E:T ratio of 10:1 , and the cells were treated with serial dilutions of CD3xEGFR-SF3 or controls for 48 hours.
  • Images were taken after addition of PBMCs and after 48h of RDL (Post RDL) using an EVOS FL microscope and representative images are shown with a scale bar at lOOpm.
  • FIG. 22 Receptor occupancy of CD3xEGFR-SF3. Receptor occupancy was assessed by flow cytometry on three squamous cancer cell lines: (A) SCC-4, (B) NCI-H226 and (C) NCI-FI1703 using specific Antigen Binding Capacity (sABC) kits. Graphs show the percentage of receptor occupancy bound (EGFR receptors bound by CD3xEGFR-SF3), free (EGFR receptors not bound by CD3xEGFR-SF3) and total (total EGFR receptors at the cells surface). Points represent the mean of two replications and error bars represent the standard error of the mean.
  • sABC Antigen Binding Capacity
  • FIG 23 Detection of CD3xEGFR-SF3 binding by flow cytometry on human primary hepatocytes.
  • a dose response of CD3xEGFR-SF3 or control antibodies were incubated on male (A) and female (B) human primary adult hepatocytes and detected with a PE-labelled anti-human IgG.
  • the graphs show the nonlinear sigmoidal regression binding curves of the mean fluorescent intensity (MFI) for each treatment. Each data point is the mean ⁇ SEM of duplicate values from 3 independent replications.
  • Figure 24 CD3xEGFR-SF3 does not impact the viability of primary human hepatocytes. Primary human hepatocytes were treated with serial dilutions of CD3xEGFR-SF3 or controls for 48h. Viability was measured by MTS. The graphs show the means of cell viability in female and male hepatocytes for each treatment according to the concentration. Each data point is the mean ⁇ SEM of values from 5 independent replications.
  • CD3xEGFR-SF3 induces the redirected lysis of primary human hepatocytes by CD3+ T cells at high doses.
  • Target cells hepatocytes
  • PBMCs effector cells
  • the graphs show the means of specific killing (redirected lysis) in female and male hepatocytes for each treatment according to the concentration.
  • Each data point is the mean ⁇ SEM of values from 6 independent replications.
  • CD3xEGFR-SF3 induces the activation of CD4+ and CD8+ T cells in an RDL assay with hepatocytes at high concentrations.
  • Target cells hepatocytes
  • PBMCs effector cells
  • the graph shows the means T cell activation in female and male hepatocytes for each treatment according to the concentration.
  • Each data point is the mean ⁇ SEM of values from 3 independent replications
  • FIG. 30 Pharmacokinetic profile of CD3XEGFR-SF3 in non xenografted female NOD SCID mice serum.
  • the blood samples for pharmacokinetic assessment were collected at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks).
  • ECL electrochemiluminescence
  • FIG. 31 Tumor grow in female NOD SCID mice xenografted with a mix of hPBMC and NCI-H1703 cells in s.c.
  • a single intravenous injection at a dose of 0.01 mg/kg body was weight performed when tumors reached 100mm3.
  • the tumor size was measured at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks). Tumor growth was
  • FIG. 32 Pharmacokinetic profile of CD3XEGFR-SF3 in female NOD SCID mice serum xenografted with a mix of hPBMC and NCI-H1703 cells in s.c.
  • the blood samples for pharmacokinetic assessment were collected at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks).
  • FIG. 33 Efficacy of CD3XEGFR-SF3 therapeutic treatment in EGFR negative tumor.
  • the expression level of EGFR on NCI-H520 cells was determined by sABC before the graph.
  • CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks at 0.5mg/kg and Vectibix was administrated twice a week for 3 weeks at 20mg/kg.
  • the tumor size quantification was performed by caliper measurement.
  • Tumor volume (mm ) 0.5 c length c 2 3
  • Figure 34 Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 17. The data showed per group the tumor volume of each animal at day 17. Data are extracted from Figure 33.
  • FIG. 35 Efficacy of CD3XEGFR-SF3 therapeutic treatment in NCI-H2286 tumor.
  • the expression level of EGFR on NCI-FI2286 cells was determined by sABC before the graph.
  • CD3XEGFR-SF3 was administered i.v.
  • Tumor volume (mm 3 ) 0.5 x length c width 2 .
  • Figure 36 Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 49 in NCI-H2286 tumor. The data showed per group the tumor volume of each animal at day 49. Data are extracted from Figure 35.
  • FIG. 37 Efficacy of CD3XEGFR-SF3 therapeutic treatment in NCI-H1703 tumor.
  • the expression level of EGFR on NCI-FI1703 cells was determined by sABC before the graph.
  • CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks at different concentration and Vectibix was administrated twice a week for 3 weeks at 20mg/kg.
  • the tumor size quantification was performed by caliper measurement.
  • Figure 38 Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 43. The data showed per group the tumor volume of each animal at day 43. Data are extracted from Figure 37.
  • Example 1 Material and methods EGFR expression in squamous cancer cell lines
  • All cell lines were cultured in the media indicated in Table 1. The cells were passaged twice per week with a subcultivation ratio as per the supplier's recommendation in order to maintain them at an optimal confluency. All cell lines were cultured in a humidified atmosphere of 5% CO2 at 37°C and routinely tested for mycoplasma contamination using the MycoAlert detection kit (Lonza). The cells consistently tested negative for mycoplasma contamination. Cells were harvested for the different assays by trypsinization.
  • Table 1 Culture media for different squamous cell lines. 1 RPMI-1640 medium; 2 DMEM-F12 medium; 3 Fetal Bovine Serum (FBS; Biowest); 4 Penicillin-Streptomycin; 5 Sodium Pyruvate; 6 MEM non Essential Amino Acids; 7 L-Glutamine; 8 HEPES (Gibco); 9 Flydrocortisone (Sigma-Aldrich); “Bovine Serum Albumin (BSA; Roche); “Insulin (Sigma-Aldrich); 12 Transferrrin (Sigma-Aldrich); “Sodium selenite (Sigma-Aldrich); 14 EGF (R&D); “Ethanolamine (Sigma-Aldrich); 16 Phosphorylethanolamine (Sigma- Aldrich); 17 Triiodothyronine (Sigma-Aldrich). All reagents were from Dominique Dutscher unless indicated otherwise.
  • EGFR expression levels were determined by sABC (specific Antibody Binding Capacity) using the QIFIKIT ® (Dako) according to the manufacturer's instructions. Briefly, 100 ⁇ 00 cells were labelled with 10 pg/mL of mouse anti-human EGFR (clone 528, Merck) or mouse lgG2a kappa isotype control (clone eBM2a, eBioscience) and incubated for 20 minutes at 4°C. Cells were washed and incubated along with reference beads with a saturating concentration of anti-mouse IgG-FITC (Dako,) for 20 minutes at 4°C.
  • sABC Specific Antibody Binding Capacity
  • RNA extraction was performed with TRI-Reagent (Zymo Research) and Direct-Zol RNA Miniprep Plus (Zymo Research) according to the manufacturer's instructions. RNA concentrations were measured by Nanodrop and the quality of the RNA was checked using e-gel 2% agarose (ThermoFisher Scientific). cDNA was synthetized from lpg of RNA with SuperScript tm IV Vilo tm Master Mix (ThermoFisher Scientific) according the manufacturer's instructions. Contamination with genomic DNA was excluded using minus RT control provided in the Master Mix kit.
  • Quantitative PCR was performed using the TaqMan ® Gene Expression Assay (ThermoFisher Scientific) for beta actin (reference gene, chosen for its stability among samples compared to other genes tested) and EGFR (target gene) with the TaqMan Fast Advanced MasterMix using a QuantStudio ® 5 Real-Time PCR system (ThermoFisher Scientific).
  • cDNAs from healthy lung cells or tongue tissues (Amsbio) were used as control reference samples.
  • cDNAs were diluted (1/10) and the manufacturer's instructions were followed to perform the quantitative PCR. Relative quantification was calculated in the following manner: (1) Calculation of the average Ct value for the reference gene for each samples.
  • Cells were seeded at 1x10 s cells in T150 flasks. At 24, 48, 72, 96 and 168 hours, lmL of the culture media supernatant was harvested, centrifuged at 350g for 5 minutes in order to remove dead cells, aliquoted in 2ml Eppendorf tube and frozen at -80°C until further analysis.
  • soluble EGFR in culture supernatant was assessed with a commercial ELISA kit (Human EGFR (Full Length) ELISA Kit, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, samples and the diluted kit standards were added to the plate and incubated for 2 hours. Plates were washed, human EGFR detection antibody was added and incubated for 1 hour. Following a washing step, anti-rabbit IgG HRP was added and incubated for 30 minutes. Plates were then washed, stabilized chromogen was added for 30 minutes and the reaction was blocked with stop solution prior to reading with a Synergy HT2-Spectrophotometer (Biotek).
  • a commercial ELISA kit Human EGFR (Full Length) ELISA Kit, ThermoFisher Scientific
  • Luminex quantification ProcartaPlex Human EGF Simplex and ProcartaPlex Human Basic Kit, ThermoFisher Scientific
  • beads, culture supernatants and diluted standards provided by the kit were added to the plate, incubated overnight at 4°C and washed.
  • Detection antibody was added to plate and incubated for 30 minutes at room temperature on a shaker.
  • streptavidin PE was added and incubated for 30 minutes at room temperature on a shaker.
  • the plate was washed, reading buffer was added and incubated at room temperature on a shaker for 5 minutes before reading with the Luminex 200 instrument (Luminex Corporation).
  • FACS simple binding was performed using squamous cancer cells.
  • Cells were incubated with serial dilutions of CD3xEGFR-SF3 (lOpg/ml, 1/3 dilution) and control antibodies were added to the cells and incubated 30min at 4°C.
  • FACS buffer IX PBS + 10% Versene + 2% FBS
  • bound antibodies were detected by the addition of PE-labelled anti-human IgG (Fc-gamma specific).
  • FACS buffer IX PBS + 10% Versene + 2% FBS
  • Fc-gamma specific PE-labelled anti-human IgG
  • PBMCs containing CD3+ T cells were harvested from whole blood filters using a ficoll gradient purification. Briefly, PBS containing Liquemin (Drossapharm) was injected into the filters, collected into 50ml blood separation tubes (SepMate-50; Stemcell) loaded with ficoll, and centrifuged at 1200g for 10 minutes. The PBMCs were harvested, washed three times with PBS and resuspended.
  • effector cells E; 5xl0 4 cells/well
  • target cells T; lxlO 4 cells/well
  • E:T ratio 5:1 E:T ratio 5:1
  • 96-well round bottom plates were coated with 0.3mg/ml of Poly-FIEMA solution (Sigma) and allowed to dry for 72h inside a sterile hood before sterilization with UV light.
  • Squamous cancer cells, NCI-H226 and NCI-FI520 were maintained as described above and seeded at 2xl0 5 cells/ml in T-75cm 2 tissue culture flasks. Cells were harvested using trypsin EDTA and seeded at lxlO 4 cells/well in the Poly- FIEMA coated plates. The plates were then centrifuged for lOmin at 220g and incubated for 24 hours at 37°C, 5% C02, to allow for the formation of 3D spheroids.
  • target 3D spheroids T were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) isolated as described above at an E:T ratio of 10:1 for 48 hours. Specific killing was measured by LDH released in the supernatant using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega) according to the manufacturer's protocol. Briefly, the supernatant from the spheroids RDL was harvested and mixed with the CytoTox96 solution, incubated for 30 minutes at room temperature and stopped with the kit's Stop solution. Plates were read at 490nm on a Synergy HT2- Spectrophotometer (Biotek).
  • RO Receptor occupancy
  • Cells were plated at lxl0 5 cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. Fluman IgG calibrator beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer. The kit's secondary antibody was added to the wells.
  • EGFRxCD3-SF3 100pg/ml; 1/3
  • antibody controls SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml
  • Cells were plated at lxl0 5 cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. QIFIKIT ® beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer. Mouse anti-human EGFR (Millipore) was diluted at 10pg/ml in FACS Buffer and added to the cells.
  • SF BEAT SP34-IL4 and mouse lgG2a 10pg/ml
  • Cells were plated at lxl0 5 cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. Fluman IgG calibrator beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer.
  • EGFRxCD3-SF3 100pg/ml; 1/3
  • antibody controls SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml
  • EGFRxCD3-SF3 10pg/ml
  • FACS Buffer A saturating dose of EGFRxCD3-SF3 (10pg/ml) was added to the cells and plates were incubated at 4°C for 20 minutes. The plates were then centrifuged at 350g for 3 minutes and washed with FACS Buffer. The kit's secondary antibody was added to the wells. Plates were incubated at 4°C for 20 minutes, centrifuged at 350g for 3 minutes and then washed twice with FACS Buffer. Finally the cells were resuspended in the viability dye 7-AAD (Thermo Fisher Scientific) in FACS buffer, and the beads in FACS buffer before reading on a Cytoflex (Beckman Coulter).
  • 7-AAD Thermo Fisher Scientific
  • FACS binding was performed using primary human hepatocytes cultured as described above. Cells were resuspended in FACS buffer (IX PBS + 10% Versene + 2% FBS), and added to 96-well round bottom plates which were then centrifuged at 350g for 3 minutes. Serial dilutions of CD3xEGFR-SF3 (10pg/ml, 1/3 dilution) and control antibodies were added to the cells and incubated 30 minutes at 4°C. The cells were washed in FACS buffer and stained with anti-human IgG (Fc-gamma specific) PE for 20 minutes 4°C. Cells were washed with FACS buffer and resuspended in FACS buffer containing Sytox green viability dye, for 20 minutes at 4°C and acquired on a CytoFlex (Beckman Coulter).
  • FACS buffer IX PBS + 10% Versene + 2% FBS
  • PBMCs effector cells
  • effector cells E; 8xl0 4 cells/well
  • target cells hepatocytes; 6xl0 4 cells/well
  • E:T ratio 2:1 serial dilutions of CD3xEGFR-SF3
  • the viability of the target cells was assessed at 48h by MTS assay using the CellTiter 96 ® AQ ueous One solution cell proliferation assay (Promega) according to the manufacturer's protocol. Briefly, the supernatant was removed and then the MTS solution was added into the wells.
  • Control cells were lysed with a lysis buffer (Promega). Plates were read at 490nm on a Synergy HT2- Spectrophotometer (Biotek). The plates were considered valid when a sufficient difference between maximum killing (target only that were killed using a Lysis solution) and spontaneous killing (wells with target only) was observed.
  • Luminex data were analyzed using ProcartaPlex Analyst software (eBioscience). The software automatically calculated the cytokines concentrations (in pg/ml), the upper and the lower limit of quantification according to the kit's standard. Cytokine concentrations that were above or below the limit of detection were set to the corresponding limit. Each cytokine concentrations were then normalized to the lower limit of quantification for each specific cytokine.
  • Abs490nm Spontaneous Killing
  • Abs490nm Sample
  • Abs490nm correspond to the OD obtain for a sample
  • Abs490nm Responseaneous Killing
  • Abs490nm Maximum Killing
  • Percentages obtained this way were further analyzed using the dose response analysis method described above.
  • Donor exclusion was performed using JMP software. For the RDL donor exclusion, donors were excluded when the fitting of the dose response curve had an R 2 ⁇ 0.7, or when the no mAb samples had a specific killing higher than 40%.
  • Abs490nm correspond to the OD obtained for a sample
  • Abs490nm correspond to the OD obtained for the mean of lysed cells
  • Abs490nm untreated of cells with media alone. Percentages obtained this way were further analyzed using the dose response analysis method described above.
  • Example 2 Effect of CD3xEGFR-SF3 antibody on squamous cancer cells and on primary human hepatocytes
  • CD3xEGFR-SF3 to induce the redirected lysis (RDL) of EGFR expressing squamous cancer cells by CD3+ T cells
  • RDL redirected lysis
  • a panel of seven different cell lines, derived from lung and tongue squamous cancer patients and expressing different levels of EGFR were assessed.
  • the levels of surface EGFR expression on these squamous cancer cell lines was quantified by flow cytometry using a specific antibody binding capacity (sABC) assay.
  • sABC specific antibody binding capacity
  • Table 2 Characterization of the number of EGFR in different squamous cancer cell lines. EGFR surface expression was measured on squamous cancer cell lines using a sABC method. The table shows the number of individual replications performed (N), average sABC values, the standard deviation and the minimum (min) and maximum (max).
  • EGFR expression was also quantified by measuring relative mRNA levels of EGFR scaled to a housekeeping gene (beta actin) and healthy lung or tongue tissue controls.
  • EGFR mRNA expression levels varied between the cell lines ( Figure 2A) and were significantly correlated with surface EGFR expression when the EGFR negative cell line, NCI-H520 was removed from the analysis to correlate only with cell lines expressing EGFR ( Figure 2B and C).
  • EGF EGF-binding protein
  • CD3xEGFR-SF3 for EGFR
  • EGFR was measured by ELISA and EGF by Luminex in the culture supernatant of NCI-H1703, NCI-H226, SCC-25, SCC-4, NCI-H2286 and NCI-H520 squamous cancer cell lines every 24h over a time-course of 168h. No EGF or EGFR (or levels below 0.4ng/ml) were detected in the culture supernatants of the squamous cancer cell lines at 24, 48, 72, 96 or 168h ( Figure 3A and B).
  • the panel of squamous cancer cells assessed represents a wide range of EGFR surface levels and mRNA expression, and no significant amounts of either EGF or EGFR are released during their culture as adherent monolayers.
  • the SW-900 cells are KRAS mutated, and therefore represent cells that are generally resistant to traditional anti-EGFR therapies such as monoclonal antibodies.
  • a bispecific antibody such as CD3xEGFR-SF3 should overcome this resistance and lead to the lysis of both KRAS wt and KRAS mut cell lines.
  • Table 3 represents the EC20, so and so values extracted from the non-linear sigmoidal regression binding curves of three to four independent replications.
  • CD3xEGFR-SF3 binds all EGFR-expressing squamous cancer cells in a dose- dependent manner but not the EGFR negative cell lines NCI-H520.
  • the EC 50 of the binding does not seem to be linked to the numbers of cell surface receptors, as the SCC-4 cell line with the highest number of EGFRs has a much higher EC 50 than the NCI-FI2286 cell line which has fewer EGFRs (EC50: 0.45pg/ml vs 0.099pg/ml respectively).
  • CD3xEGFR-SF3 Flowever, the maximum binding at 10pg/ml was higher in the cell lines with greater EGFR numbers, therefore showing that more CD3xEGFR-SF3 can bind when more receptors are available.
  • the two cell types with the highest EC50 of CD3xEGFR-SF3 binding to EGFR were SCC-25 and SCC-4 which are both derived from tongue tissues, as compared to the other cell lines which are derived from long tissues, and this may potentially be one of the reasons for this difference.
  • Table 3 EC values of CD3xEGFR-SF3 binding by FACS on squamous cancer cells. Increasing doses ol CD3xEGFR-SF3 and control antibodies were incubated on SCC-4, NCI-H226, SCC-25, NCI-FI1703, SW- 900, NCI-FI2286 and NCI-H520 squamous cancer cell lines and detected with a PE-labelled anti-human IgG (Fc-g). The values represent the mean ⁇ SEM of the EC20, 50, and 80 values extracted from the sigmoidal dose-response binding curves, and n represents the number of independent replications. Redirected lysis assay (RDL)
  • target cancer cells T
  • E effector cells
  • T target cancer cells
  • E effector cells
  • PBMCs PBMCs containing about 55% of CD3+ T cells
  • the redirected lysis of the cancer cells by CD3+ T cells was determined by a cytotoxic assay (MTS) to assess specific killing, and by a FACS assay to determine the activation and proliferation of T cells, and the release of cytolytic mediators.
  • MTS cytotoxic assay
  • CD3xEGFR-SF3 induced redirected cell lysis after 48h in a dose-dependent manner on the EGFR- expressing squamous cancer cells SCC-4, NCI-H226, NCI-FI1703, SCC-25 and SW-900 but not on the EGFR negative NCI-H520 cells (Figure 5).
  • the EC 5 o values were extracted from the sigmoidal dose- response curves from the MTS readout and are all within the low pM range (Table 4).
  • Table 4 EC values of CD3xEGFR-SF3-induced specific redirected lysis on squamous cancer cells.
  • Target cancer cells (T) and effector cells (E; PBMCs) were incubated at an E:T ratio of 5:1 in the presence of increasing concentrations of CD3xEGFR-SF3 or control antibodies and the redirected lysis of the cancer cells was determined by a cytotoxic assay (MTS).
  • MTS cytotoxic assay
  • CD3xEGFR-SF3 The redirected lysis induced by CD3xEGFR-SF3 was statistically significant for all the cell lines tested when compared to the untreated no mAb condition ( Figure 6 and Table 5). Flowever, for the EGFR negative cell line, NCI-FI520, this difference was only significant at InM of CD3xEGFR-SF3 and was not statistically different from the antibody control condition, which shows that CD3xEGFR-SF3 does not induce specific lysis on EGFR negative cell lines.
  • the threshold dose of CD3xEGFR-SF3 which induced specific killing was at 0.000001nM, O.OOOOlnM for SCC-4, O.OOlnM for NCI-H1703, and O.OlnM for NCI-H226.
  • CD3xEGFR-SF3 induced CD4+ and CD8+ T cell activation in a dose- dependent manner on EGFR-expressing squamous cancer cells SCC-4, NCI-H226 ( Figure 8), NCI-FI1703, SW-900 ( Figure 9), and NCI-FI2286 but not on the EGFR negative NCI-H520 cells ( Figure 10).
  • CD3xEGFR-SF3 induced a statistically significant increase in T cell activation on NCI-H520 cells, an increase which is not different from the baseline levels observed with a non-specific antibody control (Figure 10). All statistical analysis for the T cell activation in an RDL assay by CD3xEGFR-SF3 are summarized in Table 6 and Table 7.
  • T cell activation occurs between 0.001 and O.OlnM of CD3xEGFR-SF3, for NCI-H226 T cell activation starts at O.OlnM (the p-value observed at 0.000001nM for CD69+CD4+ T cells is lower than the control value), for SCC-25 T cell activation starts at O.OOOOlnM for CD25+CD4+ T cells, 0.000001nM for CD69+CD4+ T cells, O.OOOlnM for CD25+CD8+ T cells and O.OOOOlnM for CD69+CD8+ T cells, for NCI-H1703 T cell activation starts at O.OlnM, for SW900 T cell activation starts at O.OOlnM, and for NCI-H2286 T cell activation starts at O.OlnM for CD25+CD4+ T cells, O.OOOlnM for CD69+CD4+ T cells, and O.OOlnM for CD8+ T cells.
  • T cell activation marker threshold dose of CD3xEGFR-SF3 may be explained by the time-point (48h), as the peak of activation may occur slightly before or after depending on the cell line and way me not be able to capture the maximum activation of the assay.
  • CD25 and CD69 are known to be expressed sequentially with CD69 being the earliest T cell activation marker, peaking as early as 24h, and CD25 which usually has a peak at around 48h post-activation (Biselli et al. Scand J Immunol 35:439-47 1992), which may explain the differences observed.
  • CD3xEGFR-SF3 induced CD4+ and CD8+ T cell proliferation in a dose-dependent manner on EGFR-expressing squamous cancer cells SCC-4 ( Figure 14), NCI-H226 ( Figure 15), NCI-H1703 ( Figure 16), SW-900 ( Figure 17), and NCI-H2286 ( Figure 18) but not on the EGFR negative NCI-H520 cells ( Figure 19).
  • All statistical analysis for the proliferation of T cells in an RDL assay with CD3xEGFR-SF3 and squamous cancer cells are summarized in Table 8 and Table 9.
  • T cell redirected lysis of cancer cells involves the release of CD107a+ cytolytic granules containing granzyme B, which induces the lysis of target cells (Martinez-Lostao et al. Clin Cancer Res 21(22):5047-56 2015).
  • CD3xEGFR-SF3 induced the production of CD107a and expression of granzyme B in CD4+ and CD8+ T cells in a dose-dependent manner on the EGFR-expressing squamous cancer cells SCC-4 ( Figure 14), NCI-H226 (Figure 15), NCI-H1703 ( Figure 16), SW-900 ( Figure 17), and NCI-H2286 ( Figure 18) but not on the EGFR negative NCI-H520 cells ( Figure 19).
  • the threshold dose of CD3xEGFR- SF3 for any significant increase of CD107a+ in CD4+ and CD8+ T cells in a RDL assay was between 0.1 and O.OlnM for all the cell lines.
  • Squamous cancer cells are known to produce many factors that dampen T cell responses including TGF-beta, IL-10, IDO, PDL1, MMP9 and ROS (Curry et al. Semin Oncol 41(2):217-34 2014). Additionally, a high surface distribution of EGFR may hinder the binding of CD3xEGFR-SF3 to these cells explaining why the efficacy of CD3xEGFR-SF3 is not increased in SCC-4, the cells with the highest number of EGFRs.
  • the cell lines SW-900 and NCI-H2286 had the lowest threshold doses of CD3xEGFR-SF3 for T cell activation, and despite these cells having lower numbers of EGFRs, these are the cells with the highest binding affinity for CD3xEGFR-SF3, which may explain why lower concentrations of CD3xEGFR-SF3 are required to induce the redirected lysis of these cells.
  • CD3xEGFR-SF3 was shown to induce the redirected lysis by activated cytolytic CD4+ and CD8+ T cells of all of the EGFR-expressing human cancer cell lines tested regardless of the KRAS mutational status, but not of an EGFR negative cell line, NCI-H520.
  • an EGFR-expressing squamous cancer cell line, NCI-H226, and an EGFR-negative squamous cancer cell line, NCI-FI520 were cultured as spheroids (targets; T) and used to perform a redirected lysis assay with PBMCs as effectors (E) at an E:T ratio of 10:1 in the presence of a increasing concentrations of CD3xEGFR-SF3 or control antibodies.
  • the redirected lysis of the spheroids by CD3+ T cells was determined by the release of LDH in the culture supernatant as a surrogate measure of cell lysis.
  • CD3xEGFR-SF3 induced cell lysis in a dose-dependent manner on EGFR-expressing squamous cancer cell spheroids but not on spheroids from the EGFR negative NCI- H520 cells after 48h in an RDL assay.
  • the EC 50 value of specific killing is higher in the 3D spheroid cultures than in a 2D monolayer which could be explained by the fact that spheroids have different cell-cell interactions, an outer layer which generally contains proliferating cells and an inner layer with increased necrotic cells, which together reflects a different tumor microenvironment and may affect the T cell tumor penetration and cell lysis potential ( Figure 20 and Table 13).
  • the spheroids can be seen as a mass of cells with scattered PBMCs all around. After 48h of incubation, the PBMCs aggregated towards the center of round bottom in all conditions. Following treatment with CD3xEGFR-SF3 only the spheroids from the EGFR-expressing cell line, NCI-H226, had their structure compromised by the redirected lysis of the cells by the CD3+ T cells as seen the fragmentation of the spheroid.
  • Target cancer spheroids T and effector cells (E; PBMCs) were incubated at an E:T ratio of 10:1 in the presence of increasing doses of CD3xEGFR-SF3 or control antibodies and the redirected lysis of the cancer cells was determined by release of LDH.
  • the EC 50 values ⁇ SEM were extracted from the sigmoidal dose-response curves of specific killing from at least 2 independent replications with n representing the numbers of PBMC donors.
  • EGFR-targeting therapies are of potential off-target effect in EGFR-expressing healthy tissues such as hepatocytes.
  • EGFR expression and CD3xEGFR-SF3 binding to hepatocytes were assessed, and cytotoxicity and redirected lysis assays were performed on a panel of cryopreserved male and female primary human adult hepatocytes (Table 14).
  • EGFR was expressed at a lower level in human primary hepatocytes than in squamous cancer cell lines, with 27925 EGFRs on the surface of male hepatocytes and 25427 EGFRs on the surface of female hepatocytes.
  • Table 14 Results for the different assays on primary human adult male and female hepatocytes.
  • a panel of assays were used to describe the potential effects of CD3xEGFR-SF3 on primary human hepatocytes.
  • the values represent the average surface levels expression of EGFR (sABC), the mean of the EC 5 o values extracted from the sigmoidal dose-response binding curves for FACS binding and redirected lysis assay, as well as the cytotoxic effect.
  • N represents the number of donors assessed for each assay.
  • CD3xEGFR-SF3 binds both male and female human primary hepatocytes in a dose-dependent manner, with an EC 50 of 591.8pM and 414.4pM respectively.
  • CD3xEGFR-SF3 can bind to hepatocytes, we assessed whether this interaction could lead to any potential cytotoxicity.
  • CD3xEGFR-SF3 could induce the redirected lysis of healthy human primary cells expressing EGFR by CD3+ T cells.
  • an RDL assay with human primary hepatocytes as targets and PBMCs containing CD3+ T cells as effector cells was performed. The cells were incubated at an E:T ratio of 1:1 in the presence of serial dilutions of CD3xEGFR-SF3 or control antibodies.
  • the redirected lysis of the hepatocytes by CD3+ T cells was determined by a cytotoxic assay (MTS) to assess specific killing, and by a flow cytometric assay to determine the activation of T cells.
  • MTS cytotoxic assay
  • CD3xEGFR-SF3 induced the redirected cell lysis of human primary hepatocytes in a dose-dependent manner after 48h of RDL (Figure 26).
  • the minimal concentration of CD3xEGFR-SF3 required to induce a significant increase in specific killing was of O.OlnM in both males and females ( Figure 27 and Table 15) compared to O.OOOOlnM in SCC-4 squamous cancer cells (Table 5).
  • CD3+ T cells For the activation of CD3+ T cells following the engagement of CD3xEGFR-SF3 in an RDL assay with human primary male and female hepatocytes, the expression of CD25 and CD69 was assessed in CD4+ and CD8+ T cells after 48h ( Figure 28).
  • CD3xEGFR-SF3 induced CD4+ and CD8+ T cell activation in a dose-dependent manner on human primary hepatocytes and in a similar manner as for the specific killing, the minimal concentration of CD3xEGFR-SF3 required for any significant T cell activation in an RDL assay with both male and female human primary hepatocytes was between O.OlnM (for CD25+) and O.OOlnM (for CD69+) ( Figure 29 and Table 16) compared to O.OOOOlnM or even 0.000001nM for squamous cancer cell lines (Table 6 and Table 7).
  • Primary human adult hepatocytes express lower levels of EGFRs than squamous cancer cells.
  • CD3xEGFR-SF3 due to the efficacy of CD3xEGFR-SF3 to bind to very low numbers of EGFRs and induce the redirected lysis of EGFR-expressing cells by CD3+ T cells, CD3xEGFR-SF3 is capable of inducing the redirected lysis of primary hepatocytes ( Figure 26).
  • the minimal dose required to induce the redirected lysis of primary human hepatocytes is considerably higher than what is required to induce the redirected lysis of squamous cancer cells, despite the EC 50 of binding of CD3xEGFR-SF3 being lower for hepatocytes than squamous cancer cells (503. lpM when both sexes are combined vs. an average of 1611pM for squamous cancer cells).
  • Immortalized squamous cancer cell lines were cultured in the specific media in a humidified atmosphere of 5% C0 2 at 37°C. The cells were passaged 2 to 3 times per week to maintain them at optimal confluency and were routinely tested for mycoplasma contamination using the MycoAlert detection kit. Effector cells: human Peripheral Blood Mononuclear Cells (PBMC)
  • PMBCs Peripheral blood mononuclear cells
  • CD3XEGFR-SF3 was quantified in NOD SCID mice serum by exploratory electrochemiluminescence (ECL) based analytical method using MSD platform.
  • ECL electrochemiluminescence
  • CD3XEGFR-SF3 was captured using EGFR-His and plate bound CD3XEGFR-SF3 was detected using biotin conjugated Affini-Pure Goat Anti-Fluman IgG, Fey fragment specific antibody followed by Streptavidin-Sulpho tag.
  • ECL response was measured following the addition of read buffer using MSD plate reader.
  • CD3XEGFR-SF3 showed slow clearance and limited volume of distribution.
  • Immortalized squamous cancer cell lines were cultured in the specific media in a humidified atmosphere of 5% C0 2 at 37°C. The cells were passaged 2 to 3 times per week to maintain them at optimal confluency and were routinely tested for mycoplasma contamination using the MycoAlert detection kit. Effector cells: human Peripheral Blood Mononuclear Cells (PBMC)
  • PMBCs Peripheral blood mononuclear cells
  • CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks and Vectibix was administrated twice a week for 3 weeks.
  • CD3xEGFR-SF3 efficacy was tested in two different EGFR expressing tumors: NCI-FI2286 and NCI- FI1703.
  • NCI-FI2286 tumors Figure 35, Figure 36 and Table 20
  • CD3-EGFR-SF3 treatment induced a slight tumor volume reduction compared to the control (around 200mm3 mean differences at day 49). Flowever, no complete tumor regression was observed in animals treated with CD3xEGFR-SF3.
  • CD3xEGFR-SF3 showed a really good efficacy at really low doses compared to Vectibix.
  • 54% of animal treated with CD3xEGFR-SF3 at 0.05mg/kg showed complete tumor regression, 40% in the group CD3xEGFR-SF3 at O.Olmg/kg, 30% in the group CD3xEGFR-SF3 at 0.005mg/kg and 10% in the group CD3xEGFR-SF3 at O.OOlmg/kg (Table 21).

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Abstract

The present invention relates to an antibody or fragment thereof for use in the treatment of a squamous cell carcinoma. In particular the present invention relates to T cell redirecting bispecific antibodies for the treatment of EGFR positive squamous cell cancers.

Description

T cell redirecting bispecific antibodies for the treatment of squamous cell cancers
TECHNICAL FIELD
The present invention relates to an antibody or fragment thereof for use in the treatment of a squamous cell carcinoma. In particular the present invention relates to T cell redirecting bispecific antibodies for the treatment of EGFR positive squamous cell cancers.
BACKGROUND
Cancers originating from squamous epithelial cells (cells forming the surface of the skin, the lining of the hollow organs of the body and the lining of the respiratory and digestive tracts) are known as squamous cell carcinomas (SCCs) or epidermoid carcinoma. A common squamous cell carcinoma is the one developed from squamous cells of the skin, known as cutaneous squamous cell carcinoma (cSCC); this type of cancer is often associated with the exposure to ultraviolet radiation from the sun and represents the 20-50% of the skin cancers. Typically surgery enables cSSC eradication, but in a subset of patients the likelihood of recurrence, metastasis, and death can still be high (Que et al. JAAD, 2018; 78(2): 237-347). The squamous cell carcinoma of the lung is a histological subtype of non-small cell lung cancer (NSCLC), representing about the 25-30% of NSCLC. The development of the squamous cell carcinoma of the lung is strongly associated with smoking and chemotherapy is considered for primary treatment (Liao et al. Lung Cancer Manag, 2012; 1(4): 293-300). Cancers originating from the squamous epithelium of the upper aerodigestive tract, including lip, oral cavity, tongue, pharynx, larynx and paranasal sinuses, are classified as head and neck squamous cell carcinoma (HNSCC). The possibility to develop these types of cancer increases with certain environmental and lifestyle risk factors like tobacco and alcohol consumption. The treatment of HNSCC often requires multimodality therapy that nevertheless results in low survival rate (Kozakiewcz et al. Oncology Letters, 2018, 15: 7497-7505).
Conventional cancer treatments, such as surgery, radiotherapy, chemotherapy are for instance only effective in 50% of HNSCC patients (Boeckx et al. The Oncologist, 2013; 18:850-864). The unsatisfactory outcome of the conventional treatments coupled with their toxicity highlights the need for new therapeutic approaches. Progress in genetic and molecular biology have led to the development of targeted cancer therapies to replace (or to be combined with) the conventional ones.
An example of relevant target for anti-cancer therapies is the Epidermal Growth Factor Receptor (EGFR) (Seshacharyulu et al. Expert Opin Ther Targets, 2012; 16(1): 15-31). EGFR, also known as HerBl or HER1, is a transmembrane protein belonging to the ErbB receptor tyrosine kinase family, and often overexpressed in cancer cells, including squamous cell cancer. When activated by its ligands, the Epidermal Growth Factor (EGF) or the Transforming Growth Factor a (TGFa), EGFR undergoes homodimerization or heterodimerization with other members of its family. This allows the activation of its tyrosine kinase (TK) domain followed by autophosphorylation that triggers the activation of downstream signaling. In particular, EGFR plays a crucial role in signaling pathways involved in the modulation of cell growth, proliferation, differentiation and apoptosis; and in cancer cells, its overexpression promotes cancer progression, angiogenesis and metastasis formation.
Two main approaches have been developed for EGFR targeted therapy. One takes advantage of small molecules tyrosine kinase inhibitors, which target EGFR intracellular TK domain to inhibit its autophosphorylation and the consequent signal transmission. The other approach exploits anti-EGFR monoclonal antibodies that bind to EGFR extracellular domain preventing EGFR interaction with its ligands and the consequent receptor activation. Monoclonal antibodies targeting EGFR have been demonstrated to be efficacious in the treatment of squamous cell cancers. Cetixumab (Erbitux®), for instance, a human-murine chimeric anti-EGFR antibody, was approved by the FDA in 2006 for the treatment of locally advanced HNSCC in combination with radiotherapy. Clinical studies have also reported promising results for the treatment of FINSCC by the use of the humanized monoclonal antibody Nimotuzumab (BIOMAb-EGFR®), in combination with chemotherapy and radiotherapy (Kozakiewcz et al. Oncology Letters, 2018, 15: 7497-7505). Necitumumab (Portrazza®), a fully human IgGl monoclonal antibody, was approved by the FDA in 2015, in combination with gemcitabine and cisplatin for the treatment of patients with metastatic squamous non-small cell lung cancer (Thakur and Wozniak, Lung Cancer: Targets and Therapy, 2017, 8: 13-19).
Despite EGFR targeting agents providing an alternative therapeutic path for the cure of SCCs, not all the patients respond to the treatments; the failure to respond to a small-molecule or to a monoclonal antibody is known as primary resistance. Other patients initially benefit of therapeutic treatment but over the time they acquire resistance. Resistance mechanisms can develop at the level of EGFR or its downstream effectors, or by the activation of alternative parallel signaling pathways (Chong and Janne, Nature Medicine, 2013, 19(11); Boeckx et al. The Oncologist, 2013; 18:850-864).
Intrinsic and acquired drug resistance remains one of the main challenges for targeted therapy. To overcome that and to offer patients with a higher level of personalized care, other SCCs treatments, for instance other EGFR targeting molecules, or other strategies, such as immunotherapy needs to be developed. SUMMARY
The present invention relates to a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR for use in the treatment of a squamous cell cancer. Squamous cell cancers, also known as squamous cell carcinomas (SCCs), are a class of cancers derived from squamous epithelial cells. This class includes, among others, the squamous cell cancer of the skin, the squamous cell cancer of lung, the squamous cell cancer of the head and neck, which all together have high incidence on the worldwide population. Among the current treatments for SCCs, there are monoclonal antibody-based therapies, such as Epidermal Growth Factor Receptor (EGFR) targeted therapies, where monoclonal antibodies are used to bind EGFR and hamper cancer cells proliferation. Despite the promising results, such treatments are not always effective, depending on the patient and on therapy resistance development. Curing SCCs therefore remains a major challenge which requires the development of new therapies. The present invention provides a CD3xEGFR bispecific antibody for the treatment of SCCs, wherein said antibody binds simultaneously the cluster of differentiation 3 (CD3), a T cell co receptor, and EGFR present on the surface of squamous cancer cells. The bispecific antibody of the present invention therefore allows engaging T cells for the redirected lyses of EGFR positive tumor cells. The use of the disclosed bispecific antibody for immunotherapy is advantageous over the use of conventional monoclonal antibodies (mAbs) for EGFR-targeted therapy to bypass resistance mechanisms. Also the binding between the disclosed bispecific antibody and EGFR acts as anchorage in the tumor cell to redirect T cells without affecting EGFR function and resulting less affected by EGFR internalization. Additionally, one well elucidated mechanism by which cancer cells can become resistant to anti-EGFR mAb therapy, is by mutation of the Kirsten ras (KRAS) oncogene homolog from the mammalian ras gene family. The emergence of KRAS mutations is a frequent driver of acquired resistance to anti-EGFR mAb therapies in colorectal and other cancers. Differently, the bispecific antibody of the present invention has been shown to be effective also on KRAS mutated squamous cancer cells.
In particular the present invention discloses a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR for use in the treatment of a squamous cell cancer.
The present invention also relates to a method of treating a patient in need thereof by administering a therapeutic effective amount of a CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR.
In accordance with another aspect of the present invention there is provided a CD3xEGFR bispecific antibody according to the present invention for use as a medicament of an EGFR expressing squamous cell cancer. In one embodiment, the antibody according to the present invention binds EGFR on the cell surface of squamous cancer cells and the expression of EGFR on the cell surface of said squamous cancer cells express measured as sABC values is at least about 3000.
In another embodiment, the antibody according to the present invention binds to EGFR on the cell surface of said squamous cancer cells with an EC5o equal to or greater than about 300 pM and equal to or less than about 5500 pM.
In a further embodiment, the antibody according to the present invention induces squamous cancer cell specific killing with an EC5o equal to or greater than about 0.1 pM and equal to or less than about 15 pM. More in particular, the antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC5o equal to or greater than about 0.01 pM and equal to or less than about 100 pM.
In another embodiment, the antibody according to the present invention induces squamous cancer cells specific killing, with specific killing percentage equal to or greater than about 80% when the percentage of receptor occupancy is equal to or less than about 10%.
According to one aspect of the present invention, the disclosed antibody is administered intravenously at a dose between about 0.0001 mg/kg and about 1 mg/kg body weight, in particular at a dose equal to or greater than about 0.001 mg/kg body weight and equal to or less than about 0.5 mg/kg body weight. According to a particular aspect, said antibody is administered once a week for a number of week comprised between 1 and 3. In a more particular aspect of the present invention, when the disclosed antibody is administered as a single intravenous injection at a dose of about 0.01 mg/kg body weight , Co is about 110 ng/mL and/or Cmax is comprised between about 100 and about 200 ng/mL and/or AUC0-t is about 6400 hr*ng/mL and/or AUCo-mf is comprised between about 7000 and about 13000 hr*ng/mL and/or AUC0-336 is about 10300 ng*hr/mL and/or Tmax is about 0.25 hr and/or T1/2 is comprised between about 100 and about 150 hr and/or Vz is comprised between about 150 and about 250 ml/kg and/or Vss is comprised between about 100 and about 200 mL/kg and/or CL is comprised between about 0.5 and about 1.5 mL/hr/kg and/or MRT|NF is comprised between about 130 and about 170 hr and/or t|ast is about 330 hr.
In one embodiment of the present invention, the disclosed antibody is use for the treatment of a squamous cell carcinoma selected from the group comprising squamous cell carcinomas of the skin; squamous cell carcinomas of the head and the neck, comprising squamous cell carcinoma of the larynx, such as squamous cell carcinoma of the epiglottis, squamous cell carcinoma of the supraglottis, squamous cell carcinoma of the glottis, and squamous cell carcinoma of the subglottis, of the oral cavity, such as squamous cell carcinoma of the tongue, of the floor of mouth, of the hard palate, of the buccal mucosa, of the salivary glands and of the alveolar ridges, of the oropharynx, such as squamous cell carcinoma of the lateral pharyngeal walls, of the base of tongue, of the tonsils, and of the soft palate, squamous cell carcinomas of the nasopharynx, squamous cell carcinomas of the nasal cavity, squamous cell carcinomas of the paranasal sinuses, squamous cell carcinomas of the hypopharynx, squamous cell carcinomas salivary glands, squamous cell thyroid carcinoma and squamous cell carcinoma of the eye; squamous cell carcinomas of the esophagus; squamous cell carcinomas of the lung; squamous cell carcinomas of the bladder; squamous cell carcinomas of the cervix; vaginal squamous cell carcinoma; vulvar squamous cell carcinoma; squamous cell carcinomas of the penis; squamous cell carcinomas of the anus; squamous cell carcinomas of the prostate; early forms of squamous cell cancer such as Bowen's disease and Erythroplasia of Queyrat; and KRAS mutated squamous cell cancer.
In a further aspect, the CD3xEGFR bispecific antibody of the present invention is selected from the group comprising CD3xEGFR_SFl (SEQ ID NOs: 3, 4 and 5), CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7), CD3xEGFR_SF4 (SEQ ID NOs: 3, 4 and 8), CD3xEGFR_SDl (SEQ ID NOs: 1, 2 and 9) and CD3xEGFR_SD2 (SEQ ID NOs: 10, 9 and 2).
In addition, the presently disclosed antibodies may be used as a diagnostic tool to quantitatively or qualitatively detect EGFR-expressing squamous cancer cells and/or to characterize said EGFR- expressing squamous cancer cells based on the antibody-EGFR binding properties and/or based on the EGFR-expressing squamous cancer cells killing. For example, the bispecific antibody of the present invention may be used to detect quantitatively or qualitatively and/or to characterize EGFR-expressing squamous cancer cells in a body fluid, a tissue, or an organ. Therefore the presently disclosed antibodies may be provided in a diagnostic kit, which may contain other components that aid EGFR- expressing squamous cancer cells detection and/or characterization.
Additionally, the antibody of the present invention may be used in a method of screening and/or identification and/or classification of SCC patient subpopulations, based on the above said quantitatively or qualitatively EGFR-expressing squamous cancer cells detection and/or characterization, which may be useful to develop personalized treatments.
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry, laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.
In the present invention the terms "squamous cell carcinoma", "epidermal carcinoma", "squamous cell cancer" and "SSC" are used interchangeably to indicate cancers originating from squamous epithelial cells. Squamous cells are thin, flat cells that look like fish scales, and are found in the tissue that forms the surface of the skin, the lining of the hollow organs of the body, and the lining of the respiratory and digestive tracts. Non limiting examples of squamous cell carcinomas include: skin squamous cell carcinoma or cutaneous squamous cell carcinoma; squamous cell carcinoma of the head and the neck (HNSCC), comprising SCC originating in the larynx (such as epiglottis, supraglottis, glottis, and subglottis SCCs), SCCs originating in the oral cavity or SCCs of the mouth (such as tongue, floor of mouth, hard palate, buccal mucosa, salivary glands and alveolar ridges SCCs), SCCs originating in the oropharynx (such as posterior and lateral pharyngeal walls, base of tongue, tonsils, and soft palate SCCs), SCCs originating in the nasopharynx, nasal cavity, paranasal sinuses, hypopharynx, and salivary glands, squamous cell thyroid carcinoma and squamous cell carcinoma of the eye; SCCs of the esophagus; SCCs of the lung; SCCs of the bladder; SCCs of the cervix; vaginal squamous cell carcinoma; vulvar squamous cell carcinoma; SCCs of the penis; SCCs of the anus; SCCs of the prostate; early forms of SCCs such as Bowen's disease and Erythroplasia of Queyrat; and KRAS mutant SCCs.
In the present invention, the term "antibody" and the term "immunoglobulin" are used interchangeably. The term "antibody” as referred to herein includes whole antibodies and any antigen binding fragments or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR or FW). Each VH and VL is composed of three CDRs and four FWs, arranged from amino- terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino acid sequences of FW1, FW2, FW3, and FW4 all together constitute the "non-CDR region" or "non-extended CDR region" of VH or VL as referred to herein. The term "heavy chain variable framework region" as referred herein may comprise one or more (e.g., one, two, three and/or four) heavy chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)). Preferably the heavy chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3. The term "light chain variable framework region" as referred herein may comprise one or more (e.g., one, two, three and/or four) light chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)). Preferably the light chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the First component (Clq) of the classical complement system.
Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (CK) and lambda (CX) light chains. Heavy chains are classified as mu (m), delta (d), gamma (y), alpha (a), or epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Thus, "isotype" as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl (IGHG1), lgG2 (IGHG2), lgG3 (IGHG3), lgG4 (IGHG4), IgAl (IGHA1), lgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP gene represents an additional human immunoglobulin heavy constant region gene which has been sequenced but does not encode a protein due to an altered switch region (Bensmana M et al., (1988) Nucleic Acids Res. 16(7): 3108). In spite of having an altered switch region, the human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy constant domains (CHI -CH3) and hinge. All open reading frames for its heavy constant domains encode protein domains which align well with all human immunoglobulin constant domains with the predicted structural features. This additional pseudo-gamma isotype is referred herein as IgGP or IGHGP. Other pseudo immunoglobulin genes have been reported such as the human immunoglobulin heavy constant domain epsilon PI and P2 pseudo genes (IGHEP1 and IGHEP2). The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, lgG2, lgG3 and lgG4. In mice this class comprises subclasses IgGl, lgG2a, lgG2b, lgG2c and lgG3.
Antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward ES et al., (1989) Nature, 341 : 544-546) which consists of a single variable, (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vi) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird RE et al, (1988) Science 242: 423-426; Huston JS et al, (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83), (vii) bispecific single chain Fv dimers (PCT/US92/09965), (viii) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson I & Hollinger P (2000) Methods Enzymol. 326: 461-79; WO94/13804; Holliger P et al, (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-48) and (ix) scFv genetically fused to the same or a different antibody (Coloma MJ & Morrison SL (1997) Nature Biotechnology, 15(2): 159-163).
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
The term "antigen" as used herein, refers to any molecule to which an antibody can specifically bind. Examples of antigens include polypeptides, proteins, polysaccharides and lipid molecules. In the antigen one or more epitopes can be present.
The term "epitope" includes any protein determinant capable of specific binding to/by an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For instance, an antibody is said to specifically bind an antigen when the dissociation constant is < 1 mM, for example, < 1 mM; e.g., < 100 nM, < 10 nM or < 1 nM.
Bispecific antibodies are antibodies that can bind two different antigens, or two different epitopes of the same antigen.
As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, for example, at least 80%, 90%, 95%, or 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
In one embodiment, the bispecific antibody provide by the present invention binds to epitopes upon CD3e and EGFR. The cluster of differentiation 3 (CD3) is a T cell co-receptor helps to activate both the cytotoxic T cell (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells). CD3 is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the z-chain (zeta-chain) to generate an activation signal in T lymphocytes. The Epidermal Growth Factor Receptor (EGFR), also known as FlerBl or FIERI, is a transmembrane protein that belongs to the ErbB family of receptor tyrosine kinase, and it is often overexpressed in cancer cells, including in squamous cell carcinomas.
In a particular embodiment the CD3xEGFR bispecific antibody of the present invention is selected from the group CD3xEGFR_SFl (SEQ ID NOs: 3, 4 and 5), CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7), CD3xEGFR_SF4 (SEQ ID NOs: 3, 4 and 8), CD3xEGFR_SDl (SEQ ID NOs: 1, 2 and 9) and CD3xEGFR_SD2 (SEQ ID NOs: 10, 9 and 2). In a preferred embodiment the CD3xEGFR bispecific antibody of the present invention is CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7).
In one embodiment of the present invention the CD3xEGFR bispecific antibody binds EGFR on the cell surface of squamous cancer cells.
Method for the quantification of protein expression on the cell surface, such as receptor, are known in the art. Non limiting examples include method involving immunohistochemistry and antibody binding capacity assays. In a particular embodiment of the present invention the level of expression of EGFR on the surface of squamous cancer cells is quantified by cytometry using a specific Antibody Binding Capacity (sABC) assay. In a more particular embodiment, the CD3xEGFR bispecific antibody of the present invention binds EGFR on the cell surface squamous cancer cells, wherein the expression of EGFR on the cell surface is at least about 3000 sABC. More specifically, the expression of EGFR on the surface of said squamous cancer cells is selected from the group comprising at least about 3000 sABC, at least about 5000 sABC at least about 10000 sABC, at least about 50000 sABC, at least about 100000 sABC, at least about 150000 sABC, at least about 200000 sABC, at least about 250000 sABC, at least about 500000 sABC, at least about 1000000 sABC, at least about 1500000 sABC. The present invention also includes sABC values at intervals of 1, 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000 sABC between the above said values.
Method for the investigation of protein-protein interaction, such as antibody-target binding, are known in the art. Non limiting examples include ligand binding assays, ELISA binding, Biacore assay, and fluorescence-activated cell sorting (FACS) binding assay. In one embodiment, the binding of the CD3xEGFR bispecific antibody according to the present invention to EGFR-expressing squamous cancer cells is assessed by FACS binding assay.
In one specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an EC50 equal to or greater than about 100 pM and equal to or less than about 6000 pM, e.g. equal to or greater than about 300 pM and equal to or less than about 5500 pM. In a more particular aspect, the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an EC50 selected from the group comprising about about 100 pM, 300 pM, about 500 pM, about 1000 pM, about 1500 pM, about 2000 pM, about 2500 pM, about 3000 pM, about 3500 pM, about 4000 pM, about 4500 pM, about 5000 pM, about 5500 pM, about 6000 pM. The antibody of the present invention also includes EC5o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 pM between the above said values.
In another specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an EC20 equal to or greater than about 50 pM and equal to or less than about 1500 pM. In a more particular aspect, the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an EC20 selected from the group comprising about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1000 pM, about 1100 pM, about 1200 pM, about 1300 pM, about 1400 pM, about 1500 pM. The antibody of the present invention also includes EC2o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 pM between the above said values.
In a further specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody binds to EGFR on the cell surface of squamous cancer cells with an ECso equal to or greater than about 1200 pM and equal to or less than about 21000 pM. In a more particular aspect, the antibody of the present inventions binds to EGFR on the cell surface of squamous cancer cells with an ECso selected from the group comprising about 1200 pM, about 3000 pM, about 5000 pM, about 7000 pM, about 10000 pM, about 13000 pM, about 15000 pM, about 17000 pM, about 21000 pM. The antibody of the present invention also includes EC8o values of the binding of disclosed antibody to EGFR on the cell surface of squamous cancer cells at intervals of 1, 5, 10, 50, 100, 500, 1000, 5000, and 10000 pM between the above said values.
In one embodiment of the present invention, the ability of the disclosed bispecific antibody to induce redirected lysis of squamous cancer cells is determined by cytotoxic assay (MTS) to assess specific killing, and by a FACS assay to determine the activation and proliferation of T cells, and the release of cytolytic mediators.
In a more specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an EC5o equal to or greater than about 0.05 pM and equal to or less than about 35 pM, in a specific embodiment C50 equal to or greater than about 0.1 pM and equal to or less than about 15 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cells specific killing with an EC50 selected from the group comprising about 0.05 pM, about 0.1 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC50 values at intervals of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20 pM between the above said values.
In another specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an EC2o equal to or greater than about 0.01 pM and equal to or less than about 10 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing with an EC2o selected from the group comprising about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.5 pM, about 0.7 pM, about about 1 pM, about 3pM, about 5 pM, about 8 pM, about 10 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC2o values at intervals of 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 pM between the above said values. In a further specific embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing with an ECso equal to or greater than about 0.2 pM and equal to or less than about 140 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing with an ECso selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 25 pM, about 50 pM, about 100 pM, about 120 pM, about 150 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC8o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 pM between the above said values.
In accordance with one aspect of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing at a dose of at least about 0.0000001 nM, and/or at a dose of at least about 0.000001 nM and/or at a dose of at least about 0.00001 nM, and/or at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM. In a more particular aspect, the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.0000001 nM, at least about 0.000001 nM, at least about 0.00001 nM, at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM. The antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.00000001, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
In one embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release.
In one aspect of the present invention, FACS assay is used to determine the activation and proliferation of T cells, and the release of cytolytic mediators. In a specific aspect of the present invention, to measure the activation of CD3+ T cells following the binding of the disclosed antibody in a RDL assay with squamous cancer cells, the expression of the activation markers CD25 and CD69 is assessed in CD4+ and CD8+ T cells. In another specific aspect of the present invention, to measure the proliferation of CD3+ T cells following the engagement of the disclosed antibody in a RDL assay with squamous cancer cells, the expression of the proliferation marker Ki67 is assessed in CD4+ and CD8+ T cells. In a further specific aspect of the present invention, to measure cytolytic markers produced by CD3+ T cells following the engagement of the disclosed antibody in a RDL assay with squamous cancer cells, the expression of CD107a (LAMP-1), a marker of degranulation of cytolytic granules, and granzyme B, a serine protease that mediates apoptosis of targets cells, is assessed in CD4+ and CD8+ T cells. Non limiting examples of other markers useful to assess the effect of the antibody disclosed in the present invention include perforine (pore-forming cytolytic glycoprotein), Fas/FasL (engagement of FasL to Fas induces cell death by caspase activation), Granzyme A and K and IFN-gamma.
In a particular aspect, the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC5o equal to or greater than about 0.005 pM and equal to or less than about 150 pM, specifically equal to or greater than about 0.01 pM and equal to or less than about 100 pM, more specifically equal to or greater than about 0.02 pM and equal to or less than about 70 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC50 selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 30 pM, about 50 pM, about 70 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC50 values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 pM between the above said values.
In a more particular aspect, the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell activation with an EC50 equal to or greater than about 0.01 pM and equal to or less than about 20 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing via T cell activation with an EC50 selected from the group comprising about 0.01 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 20 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC5o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10 pM between the above said values.
In another particular aspect, the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via T cell proliferation with an EC5o equal to or greater than about 1 pM and equal to or less than about 50 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing via T cell proliferation with an EC5o selected from the group comprising about 1 pM, about 5 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM. The antibody of the present invention also induces squamous cancer cells specific killing with EC5o values at intervals of 0.5, 1, 5, 10, 20, 50 pM between the above said values.
In a further particular aspect, the CD3xEGFR bispecific antibody of the present invention induces squamous cancer cell specific killing via cell cytolytic granules release with an EC5o equal to or greater than about 0.2 pM and equal to or less than about 70 pM. In a more particular aspect, the antibody of the present invention induces squamous cancer cell specific killing via T cell cytolytic granules release with an EC5o selected from the group comprising about 0.2 pM, about 0.5 pM, about 1 pM, about 5 pM, about 10 pM, about 30 pM, about 50 pM, about 70. The antibody of the present invention also induces squamous cancer cells specific killing with EC5o values at intervals of 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 pM between the above said values.
According to one aspect of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell activation at a dose of at least about 0.00001 nM, and/or at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM. In a more particular aspect, the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.00001 nM, at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM. The antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
According to another aspect of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via T cell proliferation at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM. In a more particular aspect, the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM. The antibody of the present invention also induces specific killing of squamous cancer cells at doses at intervals of 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
According to a further aspect of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing via cytolytic granules release at a dose of at least about 0.0001 nM, and/or at a dose of at least about 0.001 nM, and/or at a dose of at least about 0.01 nM, and/or at a dose of at least about 0.1 nM, and or at a dose at least about 1 nM, and/or at a dose at least about 10 nM. In a more particular aspect, the dose of the CD3xEGFR bispecific antibody of the present invention that induces specific killing of squamous cancer cells is selected from the group comprising at least about 0.0001 nM, at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least about 1 nM, at least about 10 nM. The antibody of the present invention also induces specific killing of squamous cancer cells at closes at intervals of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM between the above said values.
In one embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody induces squamous cancer cells specific killing when the percentage of receptor occupancy is equal to or less than about 10%, or equal to or less than about 8%, or equal to or less of about 5%, or equal to or less of about 2%, or equal to or less of about 1%, or equal to or less of about 0.5%. In a preferred embodiment the percentage of receptor occupancy is equal or less than about 5%. In an even more preferred embodiment the percentage of receptor occupancy is equal to or less than about 2%. In another preferred embodiment the percentage of receptor occupancy is equal to or less than about 1%. In a particular aspect of the present invention with the above specified receptor occupancy percentages, the antibody of the present invention lead to a percentage of specific killing equal to or greater than about 50%, or equal to or greater than about 60%, or equal to or greater than about 70%, or equal to or greater than about 80%, or equal to or greater than about 90%, or equal to 100%. In a preferred embodiment the percentage of specific killing is equal to or greater than about 70%. In an even more preferred embodiment the percentage of specific killing is equal to or greater than about 80%. In a particularly preferred embodiment, the antibody of the present invention induces squamous cancer cells specific killing, with specific killing percentage equal to or greater than about 80% when the percentage of receptor occupancy is equal to or less than about 2%.
In accordance with another aspect of the present invention there is provided a method of treating an EGFR expressing squamous cell cancer by administering a therapeutic effective amount of the CD3xEGFR bispecific antibody according to the present invention to a patient in need thereof.
The term "administering," as used herein, refers to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.
The antibody or of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. More preferred routes of administration are intravenous or subcutaneous. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the invention can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
By "effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
A "patient” for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans. Thus the antibodies of the present invention have both human therapy and veterinary applications. The term "treatment" or "treating" in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder. Thus, for example, successful administration of an antibody prior to onset of the disease results in treatment of the disease. As another example, successful administration of an antibody after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.
"Treatment" and "treating" also encompasses administration of an antibody after the appearance of the disease in order to eradicate the disease. Successful administration of an antibody after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease, comprises treatment of the disease. Those "in need of treatment" include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
The antibody of the present invention can be administered at a single or multiple doses. The term "dose" or "dosage" as used in the present invention are interchangeable and indicates an amount of drug substance administered per body weight of a subject or a total dose administered to a subject irrespective to their body weight.
In one embodiment of the present invention, the disclosed CD3xEGFR bispecific antibody is administered intravenously at a dose equal to or greater than about 0.0001 mg/kg body weight and equal to or less than about 5 mg/kg body weight. In particular, the antibody of the present invention is administered intravenously at a dose equal to or greater than about 0.001 mg/kg body weight and equal to or less than about 0.5 mg/kg body weight. Preferably, the antibody of the present invention if administered at a dose selected from the group comprising about 0.001 mg/kg body weight, about 0.005 mg/kg body weight, about 0.01 mg/kg body weight, about 0.05 mg/kg body weight. The antibody of the present invention can also be administrated at doses at intervals of 0.0001, 0.001, 0.01, 0.1 and 1 mg/kg body weight between the above said values.
In a more particular embodiment, the antibody of the present invention is administrated at a dose as specified above in a single intravenous injection or intravenously once a week for at least one week, or for a number of weeks greater than 1 week, e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, in particular for a number of week comprised between 1 and 3.
In a more particular embodiment, when the antibody according to the present invention is administered as a single intravenous injection at a dose of about 0.01 mg/kg body weight, , Co is about 110 ng/mL and/or Cmax is comprised between about 100 and about 200 ng/mL and/or AUCo-t is about 6400 hr*ng/mL and/or AUC0-mf is comprised between about 7000 and about 13000 hr*ng/mL and/or AUCO-336 is about 10300 ng*hr/mL and/or Tmax is about 0.25 hr and/or T1/2 is comprised between about 100 and about 150 hr and/or Vz is comprised between about 150 and about 250 ml/kg and/or Vss is comprised between about 100 and about 200 mL/kg and/or CL is comprised between about 0.5 and about 1.5 mL/hr/kg and/or MRT|NF is comprised between about 130 and about 170 hr and/or tiast is about 330 hr. Wherein C0 represents the initial concentration; Cmax represents the peak plasma concentration of a drug after administration; AUC represents the area under the curve, the integral of the concentration-time curve; Tmax represents the time to reach Cmax; T1/2 represents the time required for the concentration of the drug to reach half of its original value; Vz represents the volume of distribution during terminal phase after intravenous administration; Vss represents the apparent volume of distribution at equilibrium determined after intravenous administration; CL represents the clearance, the volume of plasma cleared of the drug per unit time and M RTINF represents mean residence time infinity; and tiast represents time of last measurable concentration. The present invention also comprises the above said pharmacokinetic parameters at any value comprised between the above said values.
Figure 1. Levels of EGFR expression on a panel of seven squamous cancer cell lines. Numbers of EGFR were measured on the surface of the squamous cancer cells SCC-4, NCI-H226, SCC-25, NCI-H1703, SW- 900, NCI-H2286 and NCI-H520 using a specific antigen binding capacity (sABC) method. Error bars represent ± the standard deviation, and LOD the Limit Of Detection. Figure 2. EGFR mRNA expression in different squamous cancer cell lines. (A) EGFR expression is shown as a relative quantification (delta delta Ct) scaled to beta actin expression and healthy lung or tongue control samples. The bar plot shows the upregulation and downregulation of EGFR expression among different lung and tongue squamous cancers cells. (B) Correlation between EGFR sABC value (receptor expression at the cell surface; X axis) and EGFR RNA expression (scaled relative quantification; Y axis). (C) Statistical analysis of correlation which shows a statistically significant correlation (p<0.05) and a Pearson coefficient of 0.93 (positive correlation).
Figure 3. Squamous cancer cells do not shed EGFR or release EGF in culture supernatant. Soluble EGFR (A) or EGF (B) were quantified in the culture supernatant of different squamous cancer cells at 24, 48, 72, 96 and 168h by ELISA or Luminex assays respectively. Limit of detection of the Luminex was at 2.6pg/ml.
Figure 4. Detection of CD3xEGFR-SF3 binding by flow cytometry on squamous cancer cell lines. An increasing dose of CD3xEGFR-SF3 or control antibodies were incubated on SCC-4 (A), NCI-H226 (B), SCC-25 (C), NCI-H1703 (D), SW-900 (E), NCI-H2286 (F) and NCI-H520 (G) squamous cancer cell lines and binding was detected with a PE-labelled anti-human IgG. The graphs show the non-linear sigmoidal regression binding curves of the mean fluorescent intensity (MFI) for each treatment. Each data point is the mean ± SEM of duplicates values from 3-4 independent replications.
Figure 5. CD3xEGFR-SF3 induces the redirected lysis of squamous cancer cells by CD3+ T cells in an RDL assay. Target cells squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR- SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. Specific killing was measured by MTS on squamous cancer cells after removal of the PBMCS. Points represent the mean of (A) 18 donors for NCI-H520, 6 donors for SW-900, 21 donors for NCI-H1703, (B) 9 donors for SCC-25, 18 donors for NCI-H226 and 21 donors for SCC-4. Error bars show the SEM.
Figure 6. Statistical comparison of the specific killing in an RDL assay with squamous cancer cell lines between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 5 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area). Squamous cancer cells: SCC-4 (A), NCI-H226 (B), SCC-25 (C), NCI-H1703 (D), SW-900 (E) and NCI-H520 (F).
Figure 7. CD3xEGFR-SF3 induces CD4+ and CD8+ T cell activation in an RDL assay with squamous cancer cell lines. Target squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR- SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. Percentage of early (CD69) and late (CD25) activation markers were assessed by flow cytometry for both CD4+ and CD8+ T cells. Points represent the means and error bars shows the standard error of the mean. Squamous cancer cells: (A) NCI-H520, NCI-H2286, SW-900, (B) NCI-H1703, NCI-H226, SCC- 4.
Figure 8. Statistical comparison of the T cell activation in an RDL assay with SCC-4 (A, B) and NCI- H226 (C, D) between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 7 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 9. Statistical comparison of the T cell activation in an RDL assay with NCI-H1703 (A, B) and SW-900 (C, D) between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 7 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 10. Statistical comparison of the T cell activation in an RDL assay with NCI-H2286 (A, B) and NCI-H520 (C,D) between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 7 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 11. CD3xEGFR-SF3 induces the proliferation of CD4+ and CD8+ T cells in an RDL assay with squamous cancer cells. Target squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. The percentage of the proliferation marker Ki67 was assessed by flow cytometry in both CD4+ and CD8+ T cells. Each data point is the mean ± SEM of values from 6 individual donors from 2 independent replications. Squamous cancer cells: (A) NCI-FI520, NCI-FI2286, SW-900, (B) NCI-FI1703, NCI-H226, SCC-4.
Figure 12. CD3xEGFR-SF3 induces the expression of cytolytic markers by CD4+ T cells in an RDL assay with squamous cancer cells. Target squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. The percentages of the cytolytic markers CD107a and Granzyme B (GrB) were assessed by flow cytometry on CD4+ T cells. Each data point is the mean ± SEM of values from 6 individual donors from 2 independent replications. Squamous cancer cells: (A) NCI-H520, NCI-H2286, SW-900, (B) NCI- H1703, NCI-H226, SCC-4.
Figure 13. CD3xEGFR-SF3 induces the expression of cytolytic markers by CD8+ T cells in an RDL assay with squamous cancer cells. Target squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 5:1 for 48 hours. The percentages of the cytolytic markers CD107a and Granzyme B (GrB)) were assessed by flow cytometry on CD8+ T cells. Each data point is the mean ± SEM of values from 6 individual donors from 2 independent replications. Squamous cancer cells: (A) NCI-FI520, NCI-FI2286, SW-900, (B) NCI- H1703, NCI-H226, SCC-4.
Figure 14. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with SCC-4 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 15. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with NCI-H226 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 16. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with NCI-H1703 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 17. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with SW-900 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 18. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with NCI-H2286 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 19. Statistical comparison of the release of cytolytic markers (A, B) and proliferation (C) in an RDL assay with NCI-H520 between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 11 to Figure 13 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 20. CD3xEGFR-SF3 induces the redirected lysis of squamous cancer 3D spheroids by CD3+ T cells in an RDL assay. Target 3D spheroids of squamous cancer cells (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) at an E:T ratio of 10:1 for 48 hours. Specific killing was measured by LDH released in the supernatant. Points represent the mean of (A) 9 donors for NCI-FI520, 3 donors for NCI-FI1703, (B) 24 donors for NCI-H226 and 6 donors for SCC-4. Error bars show the standard error of the mean.
Figure 21. Time-course of squamous cancer 3D spheroids during the redirected lysis assay. Target 3D squamous cancer spheroids (T) of NCI-H520 (EGFR negative) and NCI-H226 (EGFR expressing) were plated at low density in poly-FIEMA coated plates. Following 24 h of spheroid formation, PBMCs (Effectors; E) were added at an E:T ratio of 10:1 , and the cells were treated with serial dilutions of CD3xEGFR-SF3 or controls for 48 hours. Images were taken after addition of PBMCs and after 48h of RDL (Post RDL) using an EVOS FL microscope and representative images are shown with a scale bar at lOOpm.
Figure 22. Receptor occupancy of CD3xEGFR-SF3. Receptor occupancy was assessed by flow cytometry on three squamous cancer cell lines: (A) SCC-4, (B) NCI-H226 and (C) NCI-FI1703 using specific Antigen Binding Capacity (sABC) kits. Graphs show the percentage of receptor occupancy bound (EGFR receptors bound by CD3xEGFR-SF3), free (EGFR receptors not bound by CD3xEGFR-SF3) and total (total EGFR receptors at the cells surface). Points represent the mean of two replications and error bars represent the standard error of the mean.
Figure 23. Detection of CD3xEGFR-SF3 binding by flow cytometry on human primary hepatocytes. A dose response of CD3xEGFR-SF3 or control antibodies were incubated on male (A) and female (B) human primary adult hepatocytes and detected with a PE-labelled anti-human IgG. The graphs show the nonlinear sigmoidal regression binding curves of the mean fluorescent intensity (MFI) for each treatment. Each data point is the mean ± SEM of duplicate values from 3 independent replications. Figure 24. CD3xEGFR-SF3 does not impact the viability of primary human hepatocytes. Primary human hepatocytes were treated with serial dilutions of CD3xEGFR-SF3 or controls for 48h. Viability was measured by MTS. The graphs show the means of cell viability in female and male hepatocytes for each treatment according to the concentration. Each data point is the mean ± SEM of values from 5 independent replications.
Figure 25. Statistical comparison of the cell viability of hepatocytes between CD3xEGFR-SF3 and no mAb condition in an RDL assay. Data from Figure 24 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 26. CD3xEGFR-SF3 induces the redirected lysis of primary human hepatocytes by CD3+ T cells at high doses. Target cells (hepatocytes) and effector cells (PBMCs) were incubated at an E:T ratio of 2:1 in the presence of increasing doses of CD3xEGFR-SF3 or control antibodies and the redirected lysis of the hepatocytes was determined by a cytotoxic assay (MTS). The graphs show the means of specific killing (redirected lysis) in female and male hepatocytes for each treatment according to the concentration. Each data point is the mean ± SEM of values from 6 independent replications.
Figure 27. Statistical comparison of the specific killing of hepatocytes by CD3+ T cells between CD3xEGFR-SF3 and no mAb condition in an RDL assay. Data from Figure 26 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 28. CD3xEGFR-SF3 induces the activation of CD4+ and CD8+ T cells in an RDL assay with hepatocytes at high concentrations. Target cells (hepatocytes) and effector cells (PBMCs) were incubated at an E:T ratio of 2:1 in the presence of increasing doses of CD3xEGFR-SF3 or control antibodies and the activation of CD4+ and CD8+ T cell was measured by the expression of CD25 and CD69 at the cell surface by flow cytometry. The graph shows the means T cell activation in female and male hepatocytes for each treatment according to the concentration. Each data point is the mean ± SEM of values from 3 independent replications
Figure 29. (A-D) Statistical comparison of the T cell activation in an RDL assay with primary human hepatocytes between treatment with CD3xEGFR-SF3 and no mAb. Data from Figure 28 were analyzed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. Significant differences of the mean are shown as the needles bars outside of the decision limits (95% Cl interval for each treatment; gray area).
Figure 30. Pharmacokinetic profile of CD3XEGFR-SF3 in non xenografted female NOD SCID mice serum. The pharmacokinetics of CD3XEGFR-SF3 was evaluated in non xenografted female NOD SCID mice (n=27 total mice, N=3/time points) following a single intravenous injection at a dose of 0.01 mg/kg body weight. The blood samples for pharmacokinetic assessment were collected at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks). The concentrations of CD3XEGFR-SF3 in these serum samples were quantified using electrochemiluminescence (ECL) based analytical method using MSD platform (N=l experiment).
Figure 31.Tumor grow in female NOD SCID mice xenografted with a mix of hPBMC and NCI-H1703 cells in s.c. A single intravenous injection at a dose of 0.01 mg/kg body was weight performed when tumors reached 100mm3. The tumor size was measured at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks). Tumor growth was
3 determined by external caliper measurements. The graphs show the mean tumor size (in mm ) of 3 animals per time point. 2 PBMC donors were included in this study (N=l experiment).
Figure 32. Pharmacokinetic profile of CD3XEGFR-SF3 in female NOD SCID mice serum xenografted with a mix of hPBMC and NCI-H1703 cells in s.c. The pharmacokinetics of CD3XEGFR-SF3 was evaluated in female NOD SCID mice xenografted with a mix of hPBMC and NCI-FI1703 cells (E:T ratio 1:1) in s.c. (n=27 total mice, N=3/time points, 2 PBMC donors) following a single intravenous injection at a dose of 0.01 mg/kg body weight when tumors reached 100mm3. The blood samples for pharmacokinetic assessment were collected at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks). The concentrations of CD3XEGFR-SF3 in these serum samples were quantified using electrochemiluminescence (ECL) based analytical method using MSD platform (N=l experiment).
Figure 33. Efficacy of CD3XEGFR-SF3 therapeutic treatment in EGFR negative tumor. The expression level of EGFR on NCI-H520 cells was determined by sABC before the graph. A mix of tumor cells (target cells, T) and PBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was
3
administered intravenously (i.v.) when tumor reached 100m in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks at 0.5mg/kg and Vectibix was administrated twice a week for 3 weeks at 20mg/kg. The tumor size quantification was performed by caliper measurement. The
3
tumor volumes were calculated using the following formula: Tumor volume (mm ) = 0.5 c length c 2 3
width . The graphs show the mean tumor size (in mm ) ± SEM. 3 PBMC donors were included. N=1 experiment.
Figure 34. Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 17. The data showed per group the tumor volume of each animal at day 17. Data are extracted from Figure 33.
Figure 35. Efficacy of CD3XEGFR-SF3 therapeutic treatment in NCI-H2286 tumor. The expression level of EGFR on NCI-FI2286 cells was determined by sABC before the graph. A mix of tumor cells (target cells, T) and PBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was administered intravenously (i.v.) when tumor reached 100m3 in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks at 0.05mg/kg or O.Olmg/kg and Vectibix was administrated twice a week for 3 weeks at 20mg/kg. The tumor size quantification was performed by caliper measurement. The tumor volumes were calculated using the following formula: Tumor volume (mm3) = 0.5 x length c width2. The graphs show the mean tumor size (in mm3) ± SEM. 3 PBMC donors were included. N=1 experiment.
Figure 36. Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 49 in NCI-H2286 tumor. The data showed per group the tumor volume of each animal at day 49. Data are extracted from Figure 35.
Figure 37. Efficacy of CD3XEGFR-SF3 therapeutic treatment in NCI-H1703 tumor. The expression level of EGFR on NCI-FI1703 cells was determined by sABC before the graph. A mix of tumor cells (target cells, T) and PBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was administered intravenously (i.v.) when tumor reached 100m3 in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks at different concentration and Vectibix was administrated twice a week for 3 weeks at 20mg/kg. The tumor size quantification was performed by caliper measurement. The tumor volumes were calculated using the following formula: Tumor volume (mm3) = 0.5 x length c width2. The graphs show the mean tumor size (in mm3) ± SEM. 2-5 PBMC donors were included. N=2 independent experiments.
Figure 38. Tumor volume comparison between CD3XEGFR-SF3 and Vectibix in therapeutic treatment vs control group at day 43. The data showed per group the tumor volume of each animal at day 43. Data are extracted from Figure 37. Example 1: Material and methods EGFR expression in squamous cancer cell lines
Cell culture conditions
All cell lines were cultured in the media indicated in Table 1. The cells were passaged twice per week with a subcultivation ratio as per the supplier's recommendation in order to maintain them at an optimal confluency. All cell lines were cultured in a humidified atmosphere of 5% CO2 at 37°C and routinely tested for mycoplasma contamination using the MycoAlert detection kit (Lonza). The cells consistently tested negative for mycoplasma contamination. Cells were harvested for the different assays by trypsinization.
Table 1: Culture media for different squamous cell lines. 1 RPMI-1640 medium; 2 DMEM-F12 medium; 3 Fetal Bovine Serum (FBS; Biowest); 4 Penicillin-Streptomycin; 5 Sodium Pyruvate; 6 MEM non Essential Amino Acids; 7 L-Glutamine; 8 HEPES (Gibco); 9 Flydrocortisone (Sigma-Aldrich); “Bovine Serum Albumin (BSA; Roche); “Insulin (Sigma-Aldrich); 12Transferrrin (Sigma-Aldrich); “Sodium selenite (Sigma-Aldrich); 14EGF (R&D); “Ethanolamine (Sigma-Aldrich); 16Phosphorylethanolamine (Sigma- Aldrich); 17Triiodothyronine (Sigma-Aldrich). All reagents were from Dominique Dutscher unless indicated otherwise.
Characterization of EGFR expression on squamous cancer cell lines
Expression levels of EGFR were determined by sABC (specific Antibody Binding Capacity) using the QIFIKIT® (Dako) according to the manufacturer's instructions. Briefly, 100Ό00 cells were labelled with 10 pg/mL of mouse anti-human EGFR (clone 528, Merck) or mouse lgG2a kappa isotype control (clone eBM2a, eBioscience) and incubated for 20 minutes at 4°C. Cells were washed and incubated along with reference beads with a saturating concentration of anti-mouse IgG-FITC (Dako,) for 20 minutes at 4°C. Cells were washed prior to addition of the viability dye 7-AAD (eBioscience) and analysed by flow cytometry on a CytoFLEX (Beckman Coulter). Data were analyzed with FlowJo software (Tree Star). The limit of detection of the assay was set as the mean sABC values of known EGFR negative cell lines + 3.3 standard deviations.
EGFR mRNA quantification in squamous cancer cell lines
Squamous cancer cells were harvested, washed twice with PBS, centrifuged at 500g for 5 minutes, and all the supernatant was removed. Cells pellets were frozen at -20°C until RNA extraction. RNA extraction was performed with TRI-Reagent (Zymo Research) and Direct-Zol RNA Miniprep Plus (Zymo Research) according to the manufacturer's instructions. RNA concentrations were measured by Nanodrop and the quality of the RNA was checked using e-gel 2% agarose (ThermoFisher Scientific). cDNA was synthetized from lpg of RNA with SuperScripttm IV Vilotm Master Mix (ThermoFisher Scientific) according the manufacturer's instructions. Contamination with genomic DNA was excluded using minus RT control provided in the Master Mix kit.
Quantitative PCR was performed using the TaqMan® Gene Expression Assay (ThermoFisher Scientific) for beta actin (reference gene, chosen for its stability among samples compared to other genes tested) and EGFR (target gene) with the TaqMan Fast Advanced MasterMix using a QuantStudio® 5 Real-Time PCR system (ThermoFisher Scientific). cDNAs from healthy lung cells or tongue tissues (Amsbio) were used as control reference samples. cDNAs were diluted (1/10) and the manufacturer's instructions were followed to perform the quantitative PCR. Relative quantification was calculated in the following manner: (1) Calculation of the average Ct value for the reference gene for each samples. (2) Calculation of relative quantification (RQ) based on the following formula for each samples: 2(ct a'/erage reference gene Ct target gene) (3) Calculation of the scaled RQ to obtain the normalization to the reference sample: RQsampies/RQreference sample- (4) The scaled RQ is used to show the difference in expression for a specific gene. The Log2(Scaled RQ) should be used for statistical analysis. Detection of EGFR and EGF in culture supernatant
Cells were seeded at 1x10s cells in T150 flasks. At 24, 48, 72, 96 and 168 hours, lmL of the culture media supernatant was harvested, centrifuged at 350g for 5 minutes in order to remove dead cells, aliquoted in 2ml Eppendorf tube and frozen at -80°C until further analysis.
The presence of soluble EGFR in culture supernatant was assessed with a commercial ELISA kit (Human EGFR (Full Length) ELISA Kit, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, samples and the diluted kit standards were added to the plate and incubated for 2 hours. Plates were washed, human EGFR detection antibody was added and incubated for 1 hour. Following a washing step, anti-rabbit IgG HRP was added and incubated for 30 minutes. Plates were then washed, stabilized chromogen was added for 30 minutes and the reaction was blocked with stop solution prior to reading with a Synergy HT2-Spectrophotometer (Biotek).
The presence of soluble EGF culture in supernatant was assessed by Luminex quantification (ProcartaPlex Human EGF Simplex and ProcartaPlex Human Basic Kit, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, beads, culture supernatants and diluted standards provided by the kit, were added to the plate, incubated overnight at 4°C and washed. Detection antibody was added to plate and incubated for 30 minutes at room temperature on a shaker. The plate was washed, streptavidin PE was added and incubated for 30 minutes at room temperature on a shaker. The plate was washed, reading buffer was added and incubated at room temperature on a shaker for 5 minutes before reading with the Luminex 200 instrument (Luminex Corporation).
EGFR FACS binding
FACS simple binding was performed using squamous cancer cells. Cells were incubated with serial dilutions of CD3xEGFR-SF3 (lOpg/ml, 1/3 dilution) and control antibodies were added to the cells and incubated 30min at 4°C. After two washes with FACS buffer (IX PBS + 10% Versene + 2% FBS) bound antibodies were detected by the addition of PE-labelled anti-human IgG (Fc-gamma specific). After incubation for 20min at 4°C, cells were washed twice with FACS buffer and resuspended in FACS buffer containing Sytox green viability dye, for 20min at 4°C and acquired on a CytoFlex (Beckman Coulter).
Redirected lysis assay (RDL)
Prior to each assay, the squamous cancer cells were assessed for specific Antibody Binding Capacity (sABC; QIFIKIT®) to verify the surface EGFR expression. If any significant shifts in the population were detected, the cells were discarded. PBMCs containing CD3+ T cells (effector cells) were harvested from whole blood filters using a ficoll gradient purification. Briefly, PBS containing Liquemin (Drossapharm) was injected into the filters, collected into 50ml blood separation tubes (SepMate-50; Stemcell) loaded with ficoll, and centrifuged at 1200g for 10 minutes. The PBMCs were harvested, washed three times with PBS and resuspended.
For the redirected lysis assay, effector cells (E; 5xl04 cells/well) and target cells (T; lxlO4 cells/well) (E:T ratio 5:1) were plated in 96-well flat bottom plates incubated for 48h at 37°C in the presence of serial dilutions of CD3xEGFR-SF3 (IOhM, 1/10 dilution) and controls. The viability of the target cells was assessed at 48h by MTS assay using the CellTiter 96® AQueous One solution cell proliferation assay (Promega) according to the manufacturer's protocol. Briefly, positive control wells (maximum killing) were lysed with lysis buffer (Promega) for 15 minutes, the plates were washed 3 times and then the MTS solution was added into the wells. Plates were read at 490nm on a Synergy HT2- Spectrophotometer. The plates were considered valid when a sufficient difference between the maximum killing (target cells that were killed using a lysis solution) and spontaneous killing (wells with target cells only) was observed.
Analysis of the expression of T cell activation and proliferation markers at the surface of CD4+ and CD8+ T cells and cytolytic markers was performed after 48 hours of RDL. The culture supernatant containing PBMCs was transferred into a new U-bottom plate. Plates were centrifuged for 3 minutes at 350 g and cells were stained with live/dead yellow for 30 minutes at 4°C, washed with FACS buffer (IX PBS + 10% Versene + 2% FBS) and stained with anti-human CD4-PE-eFluor 610 (clone RPA-T4), CD8a-Alexa Fluor 700 (clone RPA-T8), CD25-APC-eF780 (clone BC96), CD69 PE-Cyanine7 (clone FN50), CD107a (clone eBioFI4A3), Granzyme B-PE (clone QA16A02), Perforine-FITC (clone Delta G9), and Ki67- APC (clone 20Rajl) (all from Thermo Fischer Scientific or BioLegend) for 20 minutes at 4°C. Cells were washed, resuspended in FACS buffer and analysed by flow cytometry on a CytoFLEX (Beckman Coulter).
Preparation of spheroids
96-well round bottom plates were coated with 0.3mg/ml of Poly-FIEMA solution (Sigma) and allowed to dry for 72h inside a sterile hood before sterilization with UV light. Squamous cancer cells, NCI-H226 and NCI-FI520, were maintained as described above and seeded at 2xl05cells/ml in T-75cm2 tissue culture flasks. Cells were harvested using trypsin EDTA and seeded at lxlO4 cells/well in the Poly- FIEMA coated plates. The plates were then centrifuged for lOmin at 220g and incubated for 24 hours at 37°C, 5% C02, to allow for the formation of 3D spheroids.
For the RDL assay, target 3D spheroids (T) were incubated with serial dilutions of CD3xEGFR-SF3 or controls in the presence of unstimulated human PBMCs (E) isolated as described above at an E:T ratio of 10:1 for 48 hours. Specific killing was measured by LDH released in the supernatant using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega) according to the manufacturer's protocol. Briefly, the supernatant from the spheroids RDL was harvested and mixed with the CytoTox96 solution, incubated for 30 minutes at room temperature and stopped with the kit's Stop solution. Plates were read at 490nm on a Synergy HT2- Spectrophotometer (Biotek).
Images were taken using an EVOS FL microscope and analyzed using ImageJ software (NIH).
Receptor occupancy assay
Receptor occupancy (RO) was assessed by flow cytometry using specific Antigen Binding Capacity kits (QIFIKIT®; Dako and Fluman IgG Calibrator; BioCytex). We used three different protocols to measure the bound, free and total RO. All assays were performed in FACS buffer (IX PBS + 10% Versene + 2% FBS).
Bou nd receptor occupancy
Cells were plated at lxl05cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. Fluman IgG calibrator beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer. The kit's secondary antibody was added to the wells. Plates were incubated at 4°C for 20 minutes, centrifuged at 350g for 3 minutes and then washed twice with FACS Buffer. Finally the cells were resuspended in the viability dye 7-AAD (Thermo Fisher Scientific) in FACS buffer, and the beads in FACS buffer alone before reading on a Cytoflex (Beckman Coulter).
Free receptor occupancy
Cells were plated at lxl05cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. QIFIKIT® beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer. Mouse anti-human EGFR (Millipore) was diluted at 10pg/ml in FACS Buffer and added to the cells. Plate were then incubated at 4°C for 20minutes, centrifuged at 350g for 3 minutes and the cells were washed with FACS Buffer. The kit's secondary antibody was added to the wells. Plates were incubated at 4°C for 20 minutes, centrifuged at 350g for 3 minutes and then washed twice with FACS Buffer. Finally the cells were resuspended in the viability dye 7-AAD (Thermo Fisher Scientific) in FACS buffer, and the beads in FACS buffer only before reading on a Cytoflex (Beckman Coulter). Total receptor occupancy
Cells were plated at lxl05cells/well in a 96 well round bottom plate and centrifuged at 350g for 3 minutes. A serial dilution of EGFRxCD3-SF3 (100pg/ml; 1/3) and antibody controls (SF BEAT SP34-IL4 and mouse lgG2a, 10pg/ml) were added to the wells, cells were resuspended, and plates were incubated at 4°C for 20 minutes. Fluman IgG calibrator beads were added, plates were centrifuged at 350g for 3 minutes and washed with FACS Buffer. A saturating dose of EGFRxCD3-SF3 (10pg/ml) was added to the cells and plates were incubated at 4°C for 20 minutes. The plates were then centrifuged at 350g for 3 minutes and washed with FACS Buffer. The kit's secondary antibody was added to the wells. Plates were incubated at 4°C for 20 minutes, centrifuged at 350g for 3 minutes and then washed twice with FACS Buffer. Finally the cells were resuspended in the viability dye 7-AAD (Thermo Fisher Scientific) in FACS buffer, and the beads in FACS buffer before reading on a Cytoflex (Beckman Coulter).
Effects of CD3xEGFR-SF3 on primary human hepatocytes
Cryopreserved primary human adult hepatocytes
A panel of male and female cryopreserved primary human adult hepatocytes (Primacyt) from different donors were thawed according to the supplier's instructions. Cells were counted and 6xl04cells/well were plated in flat bottom 96-well plates for 24h before being used in assays.
Simple FACS binding
FACS binding was performed using primary human hepatocytes cultured as described above. Cells were resuspended in FACS buffer (IX PBS + 10% Versene + 2% FBS), and added to 96-well round bottom plates which were then centrifuged at 350g for 3 minutes. Serial dilutions of CD3xEGFR-SF3 (10pg/ml, 1/3 dilution) and control antibodies were added to the cells and incubated 30 minutes at 4°C. The cells were washed in FACS buffer and stained with anti-human IgG (Fc-gamma specific) PE for 20 minutes 4°C. Cells were washed with FACS buffer and resuspended in FACS buffer containing Sytox green viability dye, for 20 minutes at 4°C and acquired on a CytoFlex (Beckman Coulter).
Cytotoxicity measurement
After overnight culture in maintenance media, cells were treated with serial dilutions of CD3xEGFR- SF3 (starting at lOnM) or control antibodies. The viability of the cells was assessed after 48h by MTS assay using the CellTiter 96® AQueous One solution cell proliferation assay (Promega) according to the manufacturer's protocol. Plates were read at 490nm on a Synergy HT2- Spectrophotometer (Biotek).
Redirected lysis assay
PBMCs (effector cells) were harvested as described above. For the redirected lysis, effector cells (E; 8xl04 cells/well) were added to the target cells (hepatocytes; 6xl04 cells/well) (E:T ratio 2:1) and incubated for 48h at 37°C in the presence of serial dilutions of CD3xEGFR-SF3 (starting at lOnM) and controls. The viability of the target cells was assessed at 48h by MTS assay using the CellTiter 96® AQueous One solution cell proliferation assay (Promega) according to the manufacturer's protocol. Briefly, the supernatant was removed and then the MTS solution was added into the wells. Control cells were lysed with a lysis buffer (Promega). Plates were read at 490nm on a Synergy HT2- Spectrophotometer (Biotek). The plates were considered valid when a sufficient difference between maximum killing (target only that were killed using a Lysis solution) and spontaneous killing (wells with target only) was observed.
Data and statistical analysis
Dose response analysis
Data were plotted and analyzed using Prism (GraphPad). Data were first transformed using X = Log(X). Using transformed data, a 4 parameters logistic regression (4PL) fitting was applied resulting in a sigmoidal dose-response curve (Hillslope fixed to 1). ECF values (F = 20, 50 and 80) corresponding to the percentage F of the response variables of the tested sample were obtained according to the curve fitting.
Flow cytometry data
Data were analyzed using FlowJo (BD) and either mean fluorescence intensity (MFI) or percentage of specific cells population were extracted. Data were then processed for each experiment.
Lu minex Data
Luminex data were analyzed using ProcartaPlex Analyst software (eBioscience). The software automatically calculated the cytokines concentrations (in pg/ml), the upper and the lower limit of quantification according to the kit's standard. Cytokine concentrations that were above or below the limit of detection were set to the corresponding limit. Each cytokine concentrations were then normalized to the lower limit of quantification for each specific cytokine.
Percentage of s killing formula in a RDL
Abs490nm ( Spontaneous Killing)- Abs490nm ( Sample )
% Specific killing (Sample) Abs490nm ( Spontaneous Killing)- Abs490nm ( Maximum killing) x 100
Where Abs490nm (Sample) correspond to the OD obtain for a sample, Abs490nm (Spontaneous Killing) correspond to the OD obtained for the mean of target only wells and Abs490nm (Maximum Killing) corresponds to the mean OD obtained for the lysed target cells only. Percentages obtained this way were further analyzed using the dose response analysis method described above. Donor exclusion
Donor exclusion was performed using JMP software. For the RDL donor exclusion, donors were excluded when the fitting of the dose response curve had an R2 < 0.7, or when the no mAb samples had a specific killing higher than 40%.
Percentage of cell viability calculation for hepatocyte cytotoxicity
% Cell viability (Sample) 100
Where Abs490nm (Sample) correspond to the OD obtained for a sample, Abs490nm (Lysed cells) correspond to the OD obtained for the mean of lysed cells, and Abs490nm (untreated) of cells with media alone. Percentages obtained this way were further analyzed using the dose response analysis method described above.
Statistical analysis
Statistical comparisons were performed using JMP. Fit Least Square nested models were performed to compare the effect of the treatment and the concentration of the treatment. After the model fitting, if the treatment concentration had an effect on the response variable, a Dunnett's comparison was performed using the no mAb condition as control. These comparisons were performed after donor exclusion and for each time point (if applicable) separately.
Correlation analysis were perform using a Bivariate Fit model. On this model, a linear fit was applied, and if this linear fit was correlated, the model showed a significant correlation between the variables. The correlation was expressed as the Pearson coefficient (where 1 is a positive correlation, 0 no correlation and -1 a negative correlation).
Example 2: Effect of CD3xEGFR-SF3 antibody on squamous cancer cells and on primary human hepatocytes
Quantification of EGFR expression in squamous cancer cell lines
To determine the in vitro efficacy of CD3xEGFR-SF3 to induce the redirected lysis (RDL) of EGFR expressing squamous cancer cells by CD3+ T cells, a panel of seven different cell lines, derived from lung and tongue squamous cancer patients and expressing different levels of EGFR were assessed. For this, the levels of surface EGFR expression on these squamous cancer cell lines was quantified by flow cytometry using a specific antibody binding capacity (sABC) assay. Surface EGFR expression was observed at different levels in all the cell lines tested except in NCI-H520 cells which have an EGFR level below the limit of detection, thus allowing for a wide range of EGFR expression levels and an EGFR negative cell line (Figure 1 and Table 2). The variability in EGFR levels observed can be explained by slight shifts in EGFR expression levels depending on the passage number of the cell lines. Therefore, cells were only used at low passage numbers for the assays and with homogenous EGFR levels.
Table 2: Characterization of the number of EGFR in different squamous cancer cell lines. EGFR surface expression was measured on squamous cancer cell lines using a sABC method. The table shows the number of individual replications performed (N), average sABC values, the standard deviation and the minimum (min) and maximum (max).
To confirm the characterization of these cell lines, EGFR expression was also quantified by measuring relative mRNA levels of EGFR scaled to a housekeeping gene (beta actin) and healthy lung or tongue tissue controls. EGFR mRNA expression levels varied between the cell lines (Figure 2A) and were significantly correlated with surface EGFR expression when the EGFR negative cell line, NCI-H520 was removed from the analysis to correlate only with cell lines expressing EGFR (Figure 2B and C).
If any EGF is produced by the squamous cancer cells, or if any EGFRs are shed, this could potentially interfere with our different assays by competing either for binding to EGFR (for EGF) or CD3xEGFR-SF3 (for EGFR). Therefore, EGFR was measured by ELISA and EGF by Luminex in the culture supernatant of NCI-H1703, NCI-H226, SCC-25, SCC-4, NCI-H2286 and NCI-H520 squamous cancer cell lines every 24h over a time-course of 168h. No EGF or EGFR (or levels below 0.4ng/ml) were detected in the culture supernatants of the squamous cancer cell lines at 24, 48, 72, 96 or 168h (Figure 3A and B).
In summary, the panel of squamous cancer cells assessed represents a wide range of EGFR surface levels and mRNA expression, and no significant amounts of either EGF or EGFR are released during their culture as adherent monolayers. Moreover, the SW-900 cells are KRAS mutated, and therefore represent cells that are generally resistant to traditional anti-EGFR therapies such as monoclonal antibodies. A bispecific antibody such as CD3xEGFR-SF3 should overcome this resistance and lead to the lysis of both KRASwt and KRASmut cell lines.
Assessment of CD3xEGFR-SF3 binding to EGFR-expressing squamous cancer cell lines To assess the binding of CD3xEGFR-SF3 to EGFR-expressing squamous cancer cell lines, a FACS binding assay was performed. For this, increasing concentrations of CD3xEGFR-SF3, or control antibodies were incubated on the following squamous cancer cell line SCC-4 (Figure 4A), NCI-H226 (Figure 4B), SCC-25 (Figure 4C), NCI-H1703 (Figure 4D), SW-900 (Figure 4E), NCI-H2286 (Figure 4F) and NCI-H520 (Figure 4G) and binding was detected using a PE-labelled anti-human IgG. Table 3 represents the EC20, so and so values extracted from the non-linear sigmoidal regression binding curves of three to four independent replications. CD3xEGFR-SF3 binds all EGFR-expressing squamous cancer cells in a dose- dependent manner but not the EGFR negative cell lines NCI-H520. The EC50 of the binding does not seem to be linked to the numbers of cell surface receptors, as the SCC-4 cell line with the highest number of EGFRs has a much higher EC50 than the NCI-FI2286 cell line which has fewer EGFRs (EC50: 0.45pg/ml vs 0.099pg/ml respectively). Flowever, the maximum binding at 10pg/ml was higher in the cell lines with greater EGFR numbers, therefore showing that more CD3xEGFR-SF3 can bind when more receptors are available. The two cell types with the highest EC50 of CD3xEGFR-SF3 binding to EGFR were SCC-25 and SCC-4 which are both derived from tongue tissues, as compared to the other cell lines which are derived from long tissues, and this may potentially be one of the reasons for this difference.
Table 3: EC values of CD3xEGFR-SF3 binding by FACS on squamous cancer cells. Increasing doses ol CD3xEGFR-SF3 and control antibodies were incubated on SCC-4, NCI-H226, SCC-25, NCI-FI1703, SW- 900, NCI-FI2286 and NCI-H520 squamous cancer cell lines and detected with a PE-labelled anti-human IgG (Fc-g). The values represent the mean ± SEM of the EC20, 50, and 80 values extracted from the sigmoidal dose-response binding curves, and n represents the number of independent replications. Redirected lysis assay (RDL)
To assess the ability of CD3xEGFR-SF3 to induce the redirected lysis of various EGFR-expressing human squamous cancer cell lines by CD3+ T cells, target cancer cells (T) and effector cells (E; PBMCs containing about 55% of CD3+ T cells) were incubated at an E:T ratio of 5:1 in the presence of increasing doses of CD3xEGFR-SF3 or controls. The redirected lysis of the cancer cells by CD3+ T cells was determined by a cytotoxic assay (MTS) to assess specific killing, and by a FACS assay to determine the activation and proliferation of T cells, and the release of cytolytic mediators.
CD3xEGFR-SF3 induced redirected cell lysis after 48h in a dose-dependent manner on the EGFR- expressing squamous cancer cells SCC-4, NCI-H226, NCI-FI1703, SCC-25 and SW-900 but not on the EGFR negative NCI-H520 cells (Figure 5). The EC5o values were extracted from the sigmoidal dose- response curves from the MTS readout and are all within the low pM range (Table 4).
Table 4. EC values of CD3xEGFR-SF3-induced specific redirected lysis on squamous cancer cells.
Target cancer cells (T) and effector cells (E; PBMCs) were incubated at an E:T ratio of 5:1 in the presence of increasing concentrations of CD3xEGFR-SF3 or control antibodies and the redirected lysis of the cancer cells was determined by a cytotoxic assay (MTS). The EC2o, so and so values ± SEM were extracted from the sigmoidal dose-response curves of specific killing from at least 3 independent replications with n representing the numbers of PBMC donors.
The redirected lysis induced by CD3xEGFR-SF3 was statistically significant for all the cell lines tested when compared to the untreated no mAb condition (Figure 6 and Table 5). Flowever, for the EGFR negative cell line, NCI-FI520, this difference was only significant at InM of CD3xEGFR-SF3 and was not statistically different from the antibody control condition, which shows that CD3xEGFR-SF3 does not induce specific lysis on EGFR negative cell lines. For SCC-25 and SW-900 the threshold dose of CD3xEGFR-SF3 which induced specific killing was at 0.000001nM, O.OOOOlnM for SCC-4, O.OOlnM for NCI-H1703, and O.OlnM for NCI-H226. The highest tested dose of CD3xEGFR-SF3 (lOnM), induced different percentages of (maximum) specific killing depending on the cell line, with 62.30 ± 2.00% for SCC-4, 60.4 ± 3.61% for NCI-H226, 89.48 ± 1.56% for SCC-25, 82.99 ± 2.07% for NCI-H1703, and 91.87 ± 1.32% for SW-900.
Table 5. Statistical analysis summary of the percent specific killing as compared to no mAb. The concentrations within the treatments were analysed by fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05). To measure the activation of CD3+ T cells following the binding of CD3xEGFR-SF3 in an RDL assay with squamous cancer cells, the expression of the activation markers CD25 and CD69 was assessed in CD4+ and CD8+ T cells at 48h (Figure 7). CD3xEGFR-SF3 induced CD4+ and CD8+ T cell activation in a dose- dependent manner on EGFR-expressing squamous cancer cells SCC-4, NCI-H226 (Figure 8), NCI-FI1703, SW-900 (Figure 9), and NCI-FI2286 but not on the EGFR negative NCI-H520 cells (Figure 10). At high doses, CD3xEGFR-SF3 induced a statistically significant increase in T cell activation on NCI-H520 cells, an increase which is not different from the baseline levels observed with a non-specific antibody control (Figure 10). All statistical analysis for the T cell activation in an RDL assay by CD3xEGFR-SF3 are summarized in Table 6 and Table 7.
For SCC-4 cells, T cell activation occurs between 0.001 and O.OlnM of CD3xEGFR-SF3, for NCI-H226 T cell activation starts at O.OlnM (the p-value observed at 0.000001nM for CD69+CD4+ T cells is lower than the control value), for SCC-25 T cell activation starts at O.OOOOlnM for CD25+CD4+ T cells, 0.000001nM for CD69+CD4+ T cells, O.OOOlnM for CD25+CD8+ T cells and O.OOOOlnM for CD69+CD8+ T cells, for NCI-H1703 T cell activation starts at O.OlnM, for SW900 T cell activation starts at O.OOlnM, and for NCI-H2286 T cell activation starts at O.OlnM for CD25+CD4+ T cells, O.OOOlnM for CD69+CD4+ T cells, and O.OOlnM for CD8+ T cells. The differences in T cell activation marker threshold dose of CD3xEGFR-SF3 may be explained by the time-point (48h), as the peak of activation may occur slightly before or after depending on the cell line and way me not be able to capture the maximum activation of the assay. Moreover, CD25 and CD69 are known to be expressed sequentially with CD69 being the earliest T cell activation marker, peaking as early as 24h, and CD25 which usually has a peak at around 48h post-activation (Biselli et al. Scand J Immunol 35:439-47 1992), which may explain the differences observed.
Table 6. Statistical analysis summary of the percent CD4+ T cell activation. The percent of CD25+ or CD69+ CD4+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0,05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05)..
Table 7. Statistical analysis summary of the percent CD8+ T cell activation. The percent of CD25+ or CD69+ CD8+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05).
When T cells become activated, the cells undergo proliferation to ensure a robust immune response. To measure the proliferation of CD3+ T cells following the engagement of CD3xEGFR-SF3 in an RDL assay with squamous cancer cells, expression of the proliferation marker Ki67 was assessed in CD4+ and CD8+ T cells at 48h (Figure 11). CD3xEGFR-SF3 induced CD4+ and CD8+ T cell proliferation in a dose-dependent manner on EGFR-expressing squamous cancer cells SCC-4 (Figure 14), NCI-H226 (Figure 15), NCI-H1703 (Figure 16), SW-900 (Figure 17), and NCI-H2286 (Figure 18) but not on the EGFR negative NCI-H520 cells (Figure 19). RDL assays with SCC-4, NCI-H226, SW-900 and NCI-H2286 led to significant proliferation of CD4+ T cells at O.OlnM, and with NCI-H1703 at O.lnM of CD3xEGFR-SF3. For the proliferation of CD8+ T cells, RDL assays with SCC-4 led to significant proliferation at O.lnM and with NCI-H226, NCI-H1703 and SW-900 at O.OlnM of CD3xEGFR-SF3. All statistical analysis for the proliferation of T cells in an RDL assay with CD3xEGFR-SF3 and squamous cancer cells are summarized in Table 8 and Table 9. In summary, addition of O.lnM of CD3xEGFR-SF3 in an RDL assay with EGFR- expressing squamous cancer cell lines and PBMCs led to efficient CD4+ and CD8+ T cell proliferation.
Table 8. Statistical analysis summary of the percent of proliferation in CD4+ T cells in an RDL assay with CD3xEGFR-SF3. The percent of KΪ67+ CD4+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05).
Table 9. Statistical analysis summary of the percent of proliferation in CD8+ T cells in an RDL assay with CD3xEGFR-SF3. The percent of KΪ67+ CD8+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05).
The main mechanism of T cell redirected lysis of cancer cells involves the release of CD107a+ cytolytic granules containing granzyme B, which induces the lysis of target cells (Martinez-Lostao et al. Clin Cancer Res 21(22):5047-56 2015). For the analysis of cytolytic markers produced by CD3+ T cells following the engagement of CD3xEGFR-SF3 in an RDL assay with squamous cancer cells, expression of CD107a (LAMP-1), a marker of degranulation of cytolytic granules, and granzyme B, a serine protease that mediates apoptosis of targets cells, was assessed in CD4+ (Figure 12) and CD8+ (Figure 13) T cells at 48h. CD3xEGFR-SF3 induced the production of CD107a and expression of granzyme B in CD4+ and CD8+ T cells in a dose-dependent manner on the EGFR-expressing squamous cancer cells SCC-4 (Figure 14), NCI-H226 (Figure 15), NCI-H1703 (Figure 16), SW-900 (Figure 17), and NCI-H2286 (Figure 18) but not on the EGFR negative NCI-H520 cells (Figure 19). The threshold dose of CD3xEGFR- SF3 for any significant increase of CD107a+ in CD4+ and CD8+ T cells in a RDL assay was between 0.1 and O.OlnM for all the cell lines. For the expression of granzyme B by CD4+ and CD8+ T cells in an RDL assay with CD3xEGFR-SF3, the threshold dose varied between the cell lines with O.OlnM for SCC-4, NCI-H1703 and NCI-H2286, O.OOl-O.OOOlnM for NCI-H226 and SW-900. Statistical analysis summarized in Table 10 and Table 11.
Table 10. Statistical analysis summary of the percent of cytolytic markers in CD4+ T cells in an RDL assay with CD3xEGFR-SF3. The percent of CD107a+ or Granzyme B+ CD4+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05).
Table 11. Statistical analysis summary of the percent of cytolytic markers in CD8+ T cells in an RDL assay with CD3xEGFR-SF3. The percent of CD107a+ or Granzyme B+ CD8+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. The table shows the p-value for each cell line. * statistically significant difference (p<0.05).
The EC5o (pM) values of CD3xEGFR-SF3 induced T cell activation, release of cytolytic granules and proliferation on squamous cancer cells are given in Table 12.
Table 12. EC5o values of CD3xEGFR-SF3 induced T cell activation, release of cytolytic granules and proliferation on squamous cancer cells. Target cancer cells (T) and effector cells (E; PBMCs) were incubated at an E:T ratio of 5:1 in the presence of increasing doses of CD3xEGFR-SF3 or controls and the expression of activation markers (CD25 and CD69), cytolytic markers (CD107a and GrB) as well as the proliferation (Ki67) were assessed by flow cytometry. The EC50 values (pM) were extracted from the sigmoidal dose-response curves from two independent replications. NA signifies a value that could not be calculated due to a lack of sigmoidal curve.
In all the assays RDL assays with squamous cancer cells and CD3xEGFR-SF3, differences between the cell lines were observed for the different readouts that did not correlate with the number of EGFRs at the surface of the cells. The slight variability in the outcome of these RDL assays with CD3xEGFR-SF3 and squamous cancer cell lines may be due to the differences observed in the binding of CD3xEGFR- SF3 to the cell lines (Table 3), and to differences in the tumor microenvironment. Indeed, the different tumor cell lines may secrete various factors that may impact the T cells activated by CD3xEGFR-SF3 and suppress their cytolytic function. Squamous cancer cells are known to produce many factors that dampen T cell responses including TGF-beta, IL-10, IDO, PDL1, MMP9 and ROS (Curry et al. Semin Oncol 41(2):217-34 2014). Additionally, a high surface distribution of EGFR may hinder the binding of CD3xEGFR-SF3 to these cells explaining why the efficacy of CD3xEGFR-SF3 is not increased in SCC-4, the cells with the highest number of EGFRs. For many of the T cell parameters assessed, the cell lines SW-900 and NCI-H2286 had the lowest threshold doses of CD3xEGFR-SF3 for T cell activation, and despite these cells having lower numbers of EGFRs, these are the cells with the highest binding affinity for CD3xEGFR-SF3, which may explain why lower concentrations of CD3xEGFR-SF3 are required to induce the redirected lysis of these cells. Despite these slight differences between the different squamous cancer cell lines, CD3xEGFR-SF3 was shown to induce the redirected lysis by activated cytolytic CD4+ and CD8+ T cells of all of the EGFR-expressing human cancer cell lines tested regardless of the KRAS mutational status, but not of an EGFR negative cell line, NCI-H520.
Redirected lysis of spheroids
The redirected lysis of target cancer cells by CD3xEGFR-SF3 was assessed in squamous cancer cell lines in a 2D monolayer RDL assay with PBMCS (Figure 5 to Figure 19). Flowever, in vivo, the tumors are more heterogeneous and complex than in a 2D monolayer culture and to better represent this, a 3D spheroid model was assessed. For this, an EGFR-expressing squamous cancer cell line, NCI-H226, and an EGFR-negative squamous cancer cell line, NCI-FI520, were cultured as spheroids (targets; T) and used to perform a redirected lysis assay with PBMCs as effectors (E) at an E:T ratio of 10:1 in the presence of a increasing concentrations of CD3xEGFR-SF3 or control antibodies. The redirected lysis of the spheroids by CD3+ T cells was determined by the release of LDH in the culture supernatant as a surrogate measure of cell lysis. CD3xEGFR-SF3 induced cell lysis in a dose-dependent manner on EGFR-expressing squamous cancer cell spheroids but not on spheroids from the EGFR negative NCI- H520 cells after 48h in an RDL assay. The EC50 value of specific killing is higher in the 3D spheroid cultures than in a 2D monolayer which could be explained by the fact that spheroids have different cell-cell interactions, an outer layer which generally contains proliferating cells and an inner layer with increased necrotic cells, which together reflects a different tumor microenvironment and may affect the T cell tumor penetration and cell lysis potential (Figure 20 and Table 13). As depicted in the microscopy images of the RDL (Figure 21), in the untreated condition after addition of the PBMCs, the spheroids can be seen as a mass of cells with scattered PBMCs all around. After 48h of incubation, the PBMCs aggregated towards the center of round bottom in all conditions. Following treatment with CD3xEGFR-SF3 only the spheroids from the EGFR-expressing cell line, NCI-H226, had their structure compromised by the redirected lysis of the cells by the CD3+ T cells as seen the fragmentation of the spheroid. When the tumor cells and PBMCs were labelled with fluorescent cell tracking dyes, it was possible to observe the aggregation of PBMCs (which is characteristics of T cell activation) and their penetration inside the spheroid structure. As observed previously in 2D monolayers, CD3xEGFR-SF3 had no effect on the spheroids from the EGFR negative cell line NCI-H520.
spheroids. Target cancer spheroids (T) and effector cells (E; PBMCs) were incubated at an E:T ratio of 10:1 in the presence of increasing doses of CD3xEGFR-SF3 or control antibodies and the redirected lysis of the cancer cells was determined by release of LDH. The EC50 values ± SEM were extracted from the sigmoidal dose-response curves of specific killing from at least 2 independent replications with n representing the numbers of PBMC donors.
Receptor occupancy assay
To assess the receptor occupancy of CD3xEGFR-SF3 on the EGFR at the surface of squamous cancer cells, several assays were performed to measure the total of EGFR at the cell surface (RO total), the percentage of EGFRs bound by CD3xEGFR-SF3 (RO bound), and the percentage of free receptors not bound by CD3xEGFR-SF3 (RO free) (Figure 22). The total amount of EGFR at the cell surface was set to 100% for each cell lines. The percentage of EGFRs bound by CD3xEGFR-SF3 increased in a dose dependent manner and reached 100% for each cell lines at the highest concentration of 100pg/ml. Based on the concentrations of CD3xEGFR-SF3 that induced 80% of specific killing for each cell lines in an RDL assay, we extrapolated the percentage of receptor occupancy (bound receptors) that this would represent for each cell line tested. The receptor occupancy by CD3xEGFR-SF3 leading to 80% of specific killing for SCC-4 was of 2.07%, of 1.23% for NCI-H226, and of 1.28% for NCI-FI1703. Taken together these results indicate that a very low percentage (1-2%) of receptor occupancy by CD3xEGFR- SF3 is necessary to lead to at least 80% of specific killing of target squamous cancer cells in an RDL assay.
Effects of CD3xEGFR-SF3 on primary human hepatocytes
A common concern for EGFR-targeting therapies is of potential off-target effect in EGFR-expressing healthy tissues such as hepatocytes. To assess whether CD3xEGFR-SF3 may impact hepatocytes, EGFR expression and CD3xEGFR-SF3 binding to hepatocytes were assessed, and cytotoxicity and redirected lysis assays were performed on a panel of cryopreserved male and female primary human adult hepatocytes (Table 14). EGFR was expressed at a lower level in human primary hepatocytes than in squamous cancer cell lines, with 27925 EGFRs on the surface of male hepatocytes and 25427 EGFRs on the surface of female hepatocytes.
Table 14. Results for the different assays on primary human adult male and female hepatocytes. A panel of assays were used to describe the potential effects of CD3xEGFR-SF3 on primary human hepatocytes. The values represent the average surface levels expression of EGFR (sABC), the mean of the EC5o values extracted from the sigmoidal dose-response binding curves for FACS binding and redirected lysis assay, as well as the cytotoxic effect. N represents the number of donors assessed for each assay.
To assess the binding of CD3xEGFR-SF3 to EGFR-expressing primary human male and female adult hepatocytes, a FACS binding assay was performed using an increasing dose of CD3xEGFR-SF3 and control antibodies, and detected with a PE-labelled anti-human IgG (Fc-g) (Figure 23). CD3xEGFR-SF3 binds both male and female human primary hepatocytes in a dose-dependent manner, with an EC50 of 591.8pM and 414.4pM respectively. As CD3xEGFR-SF3 can bind to hepatocytes, we assessed whether this interaction could lead to any potential cytotoxicity. For this, male and female human primary hepatocytes were treated with an increasing dose of CD3xEGFR-SF3 and control antibody for 48h and the viability was assessed with an MTS assay. The viability of primary human hepatocytes was not impacted by CD3xEGFR-SF3, and no effect of the sex of the donor was observed (Figure 24 and Figure 25).
To assess whether CD3xEGFR-SF3 could induce the redirected lysis of healthy human primary cells expressing EGFR by CD3+ T cells, an RDL assay with human primary hepatocytes as targets and PBMCs containing CD3+ T cells as effector cells was performed. The cells were incubated at an E:T ratio of 1:1 in the presence of serial dilutions of CD3xEGFR-SF3 or control antibodies. The redirected lysis of the hepatocytes by CD3+ T cells was determined by a cytotoxic assay (MTS) to assess specific killing, and by a flow cytometric assay to determine the activation of T cells.
CD3xEGFR-SF3 induced the redirected cell lysis of human primary hepatocytes in a dose-dependent manner after 48h of RDL (Figure 26). However, unlike with squamous cancer cell lines, the minimal concentration of CD3xEGFR-SF3 required to induce a significant increase in specific killing (about 10% of specific killing) was of O.OlnM in both males and females (Figure 27 and Table 15) compared to O.OOOOlnM in SCC-4 squamous cancer cells (Table 5).
Table 15. Statistical analysis summary of the percent specific killing of primary human hepatocytes in an RDL assay. The percent of specific killing was analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0.05) to compare the means against the no mAb control. * statistically significant difference (p<0.05).
For the activation of CD3+ T cells following the engagement of CD3xEGFR-SF3 in an RDL assay with human primary male and female hepatocytes, the expression of CD25 and CD69 was assessed in CD4+ and CD8+ T cells after 48h (Figure 28). CD3xEGFR-SF3 induced CD4+ and CD8+ T cell activation in a dose-dependent manner on human primary hepatocytes, and in a similar manner as for the specific killing, the minimal concentration of CD3xEGFR-SF3 required for any significant T cell activation in an RDL assay with both male and female human primary hepatocytes was between O.OlnM (for CD25+) and O.OOlnM (for CD69+) (Figure 29 and Table 16) compared to O.OOOOlnM or even 0.000001nM for squamous cancer cell lines (Table 6 and Table 7). Primary human adult hepatocytes express lower levels of EGFRs than squamous cancer cells. However, due to the efficacy of CD3xEGFR-SF3 to bind to very low numbers of EGFRs and induce the redirected lysis of EGFR-expressing cells by CD3+ T cells, CD3xEGFR-SF3 is capable of inducing the redirected lysis of primary hepatocytes (Figure 26). However, the minimal dose required to induce the redirected lysis of primary human hepatocytes is considerably higher than what is required to induce the redirected lysis of squamous cancer cells, despite the EC50 of binding of CD3xEGFR-SF3 being lower for hepatocytes than squamous cancer cells (503. lpM when both sexes are combined vs. an average of 1611pM for squamous cancer cells). Taken together these data suggest that the potential therapeutic window of CD3xEGFR-SF3 dosage should not impact non-tumoral cells expressing EGFR such as hepatocytes.
Table 16. Statistical analysis summary of the percent CD4+ and CD8+ T cell activation. The percent of CD25+ or CD69+ CD4+ and CD8+ T cells were analyzed by concentrations within the treatment in a fit least square model followed by a Dunnett's comparison (a=0,05) to compare the means against the no mAb control. The table shows the p-value for each cell population. * statistically significant difference (p<0.05).
Example 3: Pharmacokinetic profile of CD3XEGFR-SF3 in NOD SCID mice serum
Material and methods
Cell line culture conditions
Immortalized squamous cancer cell lines were cultured in the specific media in a humidified atmosphere of 5% C02 at 37°C. The cells were passaged 2 to 3 times per week to maintain them at optimal confluency and were routinely tested for mycoplasma contamination using the MycoAlert detection kit. Effector cells: human Peripheral Blood Mononuclear Cells (PBMC)
Peripheral blood mononuclear cells (PMBCs) were harvested from blood filters obtained from La Chaux-de-Fonds Transfusion Center. For blood filter processing, 40 ml of PBS supplemented with 1% Liquemine® (Drossapharm) was injected into the blood filter and the solution containing the PBMCs was collected in 50ml blood separation tubes (Chemie Brunschwig) previously loaded with ficoll (GE Healthcare). The ficoll tubes were centrifuged for 20 min at 800g at room temperature (RT) without the brake and the PBMC "buffy coat" ring was harvested and transferred into 50 ml falcon tubes containing 30 ml of PBS. The PBMCs were washed three times in PBS before being processed.
Animal husbandry
In vivo experiments were performed in female 6-7-week-old immunodeficient NOD.CB17/AlhnRj- Prkdcscid/Rj (NOD/SCID) mice characterized by T cell, B cell, and natural killer cell deficiency (Envigo/Janvier labs). The mice were maintained under standardized environmental conditions in rodent micro-isolator cages (20 ± 1°C room temperature, 50 ± 10% relative humidity, 12 hours light dark cycle). Mice received irradiated food and bedding and 0.22 pm-filtered drinking water. All experiments were performed according to the Swiss Animal Protection Law with previous authorization from the cantonal and federal veterinary authorities.
Xenograft experiments
A mix of tumor cells (target cells, T) and hPBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was administered intravenously (i.v.) when tumor reached 100m3 in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks and Vectibix was administrated twice a week for 3 weeks. The tumor size quantification was performed by caliper measurement. The tumor volumes were calculated using the following formula: Tumor volume (mm3) = 0.5 c length c width2.
Statistical treatment
Data were analyzed using Graphpad Prism 5 software; the data are presented as the mean ± SEM. Statistical analysis was performed by ANOVA one way and a Dunnett's multiple comparisons test. P<0.05 was considered as statistically significant.
Results and conclusions
The pharmacokinetics of CD3XEGFR-SF3 was evaluated in two independent studies. The first one in non xenografted female NOD SCID mice (n=27 total mice, N=3/time points following a single intravenous injection at a dose of 0.01 mg/kg body weight. The second one in female NOD SCID mice xenografted with a mix of hPBMC and NCI-H1703 cells (E:T ratio 1:1) in S.C. (n=27 total mice, N=3/time points, 2 PBMC donors) following a single intravenous injection at a dose of 0.01 mg/kg body weight when tumors reached 100mm3. In both studies, the blood samples for pharmacokinetic assessment were collected at different time points of 0, 0.25, 1, 24, 48, 72, 96, 168 and 336 hours post dose over a period of 14 days (two weeks). The concentrations of CD3XEGFR-SF3 was quantified in NOD SCID mice serum by exploratory electrochemiluminescence (ECL) based analytical method using MSD platform. In this method, CD3XEGFR-SF3 was captured using EGFR-His and plate bound CD3XEGFR-SF3 was detected using biotin conjugated Affini-Pure Goat Anti-Fluman IgG, Fey fragment specific antibody followed by Streptavidin-Sulpho tag. The ECL response was measured following the addition of read buffer using MSD plate reader.
In non xenografted female NOD SCID mice, quantifiable levels were observed until the last sampling point (2 weeks). The Co/CmaX and AUC are as anticipated at the tested dose level of 10pg/kg. The ti/2 is around 5 days (Figure 30 and Table 17). In female NOD SCID mice xenografted with a mix of hPBMC and NCI-H1703 cells in s.c., tumors are sensitive to CD3XEGFR-SF3 treatment (Figure 31). Quantifiable levels were also observed until the last sampling point (2 weeks), there is a standard PK profile in both donors, PK parameters are in line with historical data, the ti/2 is around 5 days (Figure 32 and Table 18).
The serum concentration profile appeared to follow a bi-exponential disposition with an initial distribution phase followed by a longer terminal elimination phase. CD3XEGFR-SF3 showed slow clearance and limited volume of distribution.
Table 17. Mean pharmacokinetic parameters of CD3XEGFR-SF3 in non xenografted female NOD SCID mice serum. Cmax: The peak plasma concentration of a drug after administration. AUC: area under the curve, the integral of the concentration-time curve. TmaX: Time to reach CmaX. T1/2, the time required for the concentration of the drug to reach half of its original value. Vz: Volume of distribution during terminal phase after intravenous administration. Vss: Apparent volume of distribution at equilibrium determined after intravenous administration. CL: clearance, the volume of plasma cleared of the drug per unit time. MRT|NF: mean residence time infinity
Table 18. Mean pharmacokinetic parameters of CD3XEGFR-SF3 in female NOD SCID mice serum xenografted with a mix of hPBMC and NCI-H1703 cells in s.c. Cmax: The peak plasma concentration of a drug after administration. AUC: area under the curve, the integral of the concentration-time curve. Tmax: Time to reach Cmax. Tl/2, the time required for the concentration of the drug to reach half of its original value. Vz: Volume of distribution during terminal phase after intravenous administration. Vss: Apparent volume of distribution at equilibrium determined after intravenous administration. CL: clearance, the volume of plasma cleared of the drug per unit time. MRT|NF: mean residence time infinity.
Example 4: Efficacy of CD3XEGFR-SF3 therapeutic treatment in NOD SCID xenografted mouse model
Material and methods Cell line culture conditions
Immortalized squamous cancer cell lines were cultured in the specific media in a humidified atmosphere of 5% C02 at 37°C. The cells were passaged 2 to 3 times per week to maintain them at optimal confluency and were routinely tested for mycoplasma contamination using the MycoAlert detection kit. Effector cells: human Peripheral Blood Mononuclear Cells (PBMC)
Peripheral blood mononuclear cells (PMBCs) were harvested from blood filters obtained from La Chaux-de-Fonds Transfusion Center. For blood filter processing, 40 ml of PBS supplemented with 1% Liquemine® (Drossapharm) was injected into the blood filter and the solution containing the PBMCs was collected in 50ml blood separation tubes (Chemie Brunschwig) previously loaded with ficoll (GE Healthcare). The ficoll tubes were centrifuged for 20 min at 800g at room temperature (RT) without the brake and the PBMC "buffy coat" ring was harvested and transferred into 50 ml falcon tubes containing 30 ml of PBS. The PBMCs were washed three times in PBS before being processed. Animal husbandry
In vivo experiments were performed in female 6-7-week-old immunodeficient NOD.CB17/AlhnRj- Prkdcscid/Rj (NOD/SCID) mice characterized by T cell, B cell, and natural killer cell deficiency (Envigo/Janvier labs). The mice were maintained under standardized environmental conditions in rodent micro-isolator cages (20 ± 1°C room temperature, 50 ± 10% relative humidity, 12 hours light dark cycle). Mice received irradiated food and bedding and 0.22 pm-filtered drinking water. All experiments were performed according to the Swiss Animal Protection Law with previous authorization from the cantonal and federal veterinary authorities.
Xenograft experiments
A mix of tumor cells (target cells, T) and hPBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was administered intravenously (i.v.) when tumor reached 100m3 in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks and Vectibix was administrated twice a week for 3 weeks. The tumor size quantification was performed by caliper measurement. The tumor volumes were calculated using the following formula: Tumor volume (mm3) = 0.5 c length c width2.
Statistical treatment
Data were analyzed using Graphpad Prism 5 software; the data are presented as the mean ± SEM. Statistical analysis was performed by ANOVA one way and a Dunnett's multiple comparisons test. P<0.05 was considered as statistically significant.
Results and conclusions
The efficacy of CD3xEGFR-SF3 in vivo was tested on three different squamous cancer tumors. All experiments were performed in 6-7-week-old female NOD SCID mice. A mix of tumor cells (target cells, T) and hPBMCs (effector cells, E) were injected s.c. at an E:T ratio of 1:1 into the right flank area of NOD/SCID mice (n = 4-5 per group per PBMC donor). Protocol was therapeutic, the treatment was administered intravenously (i.v.) when tumor reached 100m3 in average. CD3XEGFR-SF3 was administered i.v. once a week for 3 weeks and Vectibix was administrated twice a week for 3 weeks.
In EGFR negative tumors, NCI-H520 (Figure 33) CD3xEGFR-SF3, injected at high dose 0.5mg/kg, did not display any efficacy. As showed in Figure 34 and Table 19, at day 17 after xenograft, no significant difference was observed between CD3xEGFR-SF3 and the control group (P valueVectibix = 0.8578 ; P valuecD3xEGFR SF3 = 0.5740).
Table 19. Statistical analysis of Figure 34 (Dunnett's multiple comparisons test at day 17).
CD3xEGFR-SF3 efficacy was tested in two different EGFR expressing tumors: NCI-FI2286 and NCI- FI1703. In NCI-FI2286 tumors (Figure 35, Figure 36 and Table 20), CD3-EGFR-SF3 treatment induced a slight tumor volume reduction compared to the control (around 200mm3 mean differences at day 49). Flowever, no complete tumor regression was observed in animals treated with CD3xEGFR-SF3.
In NCI-FI1703 tumors (Figure 37, Figure 38 and Table 21), CD3xEGFR-SF3 showed a really good efficacy at really low doses compared to Vectibix. At day 43 after xenograft, 54% of animal treated with CD3xEGFR-SF3 at 0.05mg/kg showed complete tumor regression, 40% in the group CD3xEGFR-SF3 at O.Olmg/kg, 30% in the group CD3xEGFR-SF3 at 0.005mg/kg and 10% in the group CD3xEGFR-SF3 at O.OOlmg/kg (Table 21).
Table 21. Additional analysis of Figure 38 (percent of animals with a complete tumor regression at day 43). The difference of CD3xEGFR-SF3 efficacy between NCI-FI2286 and NCI-FI1703 is still unexplained. It may be due to differences in the tumor micro environment or tumor architecture. Distribution and half-life of hPBMC within the two different tumor types have yet to be characterized. CD3xEGFR-SF3 efficacy in additional tumor types will be also performed.

Claims

1. A CD3xEGFR bispecific antibody which binds to epitopes upon CD3e and EGFR for use in the treatment of a squamous cell cancer.
2. The antibody of claim 1 wherein said antibody binds to EGFR on the cell surface of squamous cancer cells and wherein the expression of EGFR on the cell surface of said squamous cancer cells measured as sABC values is at least about 3000.
3. The antibody of any one of the preceding claims wherein said antibody binds to EGFR on the cell surface of said squamous cancer cells with an EC50 equal to or greater than about 300 pM and equal to or less than about 5500 pM.
4. The antibody of any one of the preceding claims wherein said antibody induces squamous cancer cells specific killing with an EC5o equal to or greater than about 0.1 pM and equal to or less than about 15 pM.
5. The antibody of any one of the preceding claims wherein said antibody induces squamous cancer cells specific killing via T cell activation and/or T cell proliferation and/or T cell cytolytic granules release with an EC5o equal to or greater than about 0.01 pM and equal to or less than about 100 pM.
6. The antibody of anyone of the preceding claims wherein said antibody induces squamous cancer cells specific killing, with specific killing percentage equal to or greater than about 80% when the percentage of receptor occupancy is equal to or less than about 10%.
7. The antibody of anyone of the preceding claims, wherein said antibody is administered intravenously at a dose equal to or greater than about 0.001 mg/kg body weight and equal to or less than about 0.5 mg/kg body weight.
8. The antibody of claim 8, wherein said antibody is administered once a week for a number of week comprised between 1 and 3.
9. The antibody of anyone of the preceding claims wherein, when said antibody is administered as a single intravenous injection at a dose of about 0.01 mg/kg body weight, C0 is about 110 ng/mL and/or Cmax is comprised between about 100 and about 200 ng/mL and/or AUCo-t is about 6400 hr*ng/mL and/or AUC0-mf is comprised between about 7000 and about 13000 hr*ng/mL and/or AUCo is about 10300 ng*hr/mL and/or Tmax is about 0.25 hr and/or T is comprised between about 100 and about 150 hr and/or Vz is comprised between about 150 and about 250 ml/kg and/or Vss is comprised between about 100 and about 200 mL/kg and/or CL is comprised between about 0.5 and about 1.5 mL/hr/kg and/or M RTINF is comprised between about 130 and about 170 hr and/or tiast is about 330 hr.
10. The CD3xEGFR bispecific antibody of anyone of the preceding claims wherein said squamous cancer is selected from the group comprising squamous cell carcinomas of the skin; squamous cell carcinomas of the head and the neck, comprising squamous cell carcinoma of the larynx, such as squamous cell carcinoma of the epiglottis, squamous cell carcinoma of the supraglottis, squamous cell carcinoma of the glottis, and squamous cell carcinoma of the subglottis, of the oral cavity, such as squamous cell carcinoma of the tongue, of the floor of mouth, of the hard palate, of the buccal mucosa, salivary glands and of the alveolar ridges, of the oropharynx, such as squamous cell carcinoma of the lateral pharyngeal walls, of the base of tongue, of the tonsils, and of the soft palate, squamous cell carcinomas of the nasopharynx, squamous cell carcinomas of the nasal cavity, squamous cell carcinomas of the paranasal sinuses, squamous cell carcinomas of the hypopharynx, squamous cell carcinomas salivary glands, squamous cell thyroid carcinoma and squamous cell carcinoma of the eye; squamous cell carcinomas of the esophagus; squamous cell carcinomas of the lung; squamous cell carcinomas of the bladder; squamous cell carcinomas of the cervix; vaginal squamous cell carcinoma; vulvar squamous cell carcinoma; squamous cell carcinomas of the penis; squamous cell carcinomas of the anus; squamous cell carcinomas of the prostate; early forms of squamous cell cancer such as Bowen's disease and Erythroplasia of Queyrat; and KRAS mutated squamous cell cancer.
11. The CD3xEGFR bispecific antibody of any one of the proceedings claims selected from the group comprising CD3xEGFR_SFl (SEQ ID NOs: 3, 4 and 5), CD3xEGFR_SF3 (SEQ ID NOs: 6, 2 and 7), CD3xEGFR_SF4 (SEQ ID NOs: 3, 4 and 8), CD3xEGFR_SDl (SEQ ID NOs: 1, 2 and 9) and CD3xEGFR_SD2 (SEQ ID NOs: 10, 9 and 2).
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