WO2025254591A1 - Molécules de liaison à l'antigène anti-ceacam - Google Patents

Molécules de liaison à l'antigène anti-ceacam

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Publication number
WO2025254591A1
WO2025254591A1 PCT/SG2025/050382 SG2025050382W WO2025254591A1 WO 2025254591 A1 WO2025254591 A1 WO 2025254591A1 SG 2025050382 W SG2025050382 W SG 2025050382W WO 2025254591 A1 WO2025254591 A1 WO 2025254591A1
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Prior art keywords
antigen
cancer
cell
binding molecule
seq
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Inventor
Hau Wan LEUNG
Ting Hwee GOH
Boon Hwa Andre Choo
Leonard LEONG
Daniel Tan
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Agency for Science Technology and Research Singapore
Singapore Health Services Pte Ltd
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Agency for Science Technology and Research Singapore
Singapore Health Services Pte Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • 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
    • A61K40/4264—Cancer antigens from embryonic or fetal origin
    • A61K40/4266—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57565—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen [CEA]
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates, in general terms, to antigen-binding molecules.
  • the present disclosure relates to antigen- binding molecules that specifically bind to CEACAM1, CEACAM5 and CEACAM6.
  • Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) belong to the carcinoembryonic antigen (CEA) family.
  • CEACAM1, CEACAM5 and CEACAM6 a e glycosyl phosphatidyl inositol (GPI) anchored cell surface glycoproteins which arc known to be expressed highly in a wide variety of cancers that include gastric, breast, pancreatic, colon and non-small cell lung carcinoma (NSCL).
  • GPI glycosyl phosphatidyl inositol
  • CEACAM1, CEACAM5 and CEACAM6 are promising cancer targets, there are only a limited number of monoclonal antibodies that recognize these targets that are currently in development for cancer treatment in humans. There is therefore a need to develop new antibodies or antibody-drug conjugates against these targets that arc safe and effective as cancer therapeutics.
  • an antigen-binding molecule comprising a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of NYGMN (SEQ ID NO: 1), the VHCDR2 amino acid sequence of WMGWINTNTGEPTYA (SEQ ID NO: 2), and the VHCDR3 amino acid sequence of HYFGLDY (SEQ ID NO: 3), and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the VLCDR2 amino acid sequence of STSNLAS (SEQ ID NO: 5), and the VLCDR3 amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VH VHCDR1 amino acid sequence of NYGMN
  • VHCDR3 amino acid sequence of HYFGLDY SEQ ID NO: 3
  • VL light chain variable region
  • an antigen-binding molecule comprising a heavy chain variable region (VH) comprising the amino acid sequence of NYGMN (SEQ ID NO: 23), the amino acid sequence of WINTNTGEPTYAEEFKG (SEQ ID NO: 24) or WINTNTGEPTYAQGFTG (SEQ ID NO: 25), and the amino acid sequence of HYFGLDY (SEQ ID NO: 26), and a light chain variable region (VL) comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VL light chain variable region
  • an antigen-binding molecule that binds specifically to CEACAM1, CEACAM5 and CEACAM6.
  • Disclosed herein is an isolated polynucleotide comprising a nucleic acid sequence encoding the antigen-binding molecule as defined herein.
  • a construct comprising a polynucleotide as defined herein in operable connection with one or more control sequence.
  • composition comprising an antigen-binding molecule as defined herein and a pharmaceutically acceptable carrier.
  • CAR chimeric antigen receptor
  • Disclosed herein is an isolated polynucleotide encoding a CAR as defined herein.
  • a vector comprising the polynucleotide as defined herein.
  • an engineered cell comprising the vector as defined herein.
  • a method of preparing an engineered immune cell comprising introducing the vector as defined herein into an immune cell.
  • an antigen-binding molecule as defined herein or a composition as defined herein, or an engineered cell as defined herein for use as a medicament Disclosed herein is an antigen-binding molecule as defined herein or a composition as defined herein, or an engineered cell as defined herein for use as a medicament.
  • Disclosed herein is a method of treating or preventing a cancer or an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of an antigen-binding molecule as defined herein, a composition as defined herein or an engineered cell as defined herein to the subject.
  • an antigen-binding molecule as defined herein a composition as defined herein or an engineered cell as defined herein for use in treating or preventing cancer in a subject.
  • an antigen-binding molecule as defined herein a composition as defined herein or an engineered cell as defined herein in the manufacture of a medicament for treating or preventing cancer.
  • a method for detecting cancer in a subject comprising: contacting a sample obtained from the subject with an antigen-binding molecule as defined herein, wherein an increase in the level of binding of the antigen-binding molecule in the sample as compared to a reference is indicative of cancer.
  • a method for identifying a subject susceptible to cancer comprising: contacting a sample obtained from the subject with an antigen-binding molecule as defined herein, wherein an increase in the level of binding of the antigen- binding molecule in the sample as compared to a reference indicates that the subject is susceptible to cancer.
  • kits for use in the method as defined herein comprising an antigenbinding molecule as defined herein, together with instructions for use.
  • FIG. 1 Immunisation with Gefitinib-resistant PC-9 cell line and screening of hybridoma clones
  • A Workflow for semi-cyclic immunisation protocol of Balb/c mice with 3 Gefitinib-resistant PC-9 clones (CL75, CL86 and CL131). After 5 weeks of immunisation, B-cells from the mice were fused with SP2/0 mouse myeloma cells using STEMCELL Technologies ClonaCellTM-HY kit, and mAb-producing hybridoma clones were obtained for initial screening by flow cytometry.
  • B Hybridoma clone, GR 6D11, exhibited strong binding to the 3 immunising cells and was progressed for further characterisation.
  • Figure 2 Protein sequence of GR6D11 variable regions Amino acid sequence of (A) variable heavy chain and (B) variable light chain regions, with the complementaritydetermining regions (CDRs) underlined in red.
  • C The isotype of GR 6D11 mAb was determined to be of a mouse IgG2a, K subtype, as determined by PierceTM Rapid Antibody Isotyping Kit. The putative sequences for the constant heavy and light chain sequences are as shown, with the Uniprot ID referenced.
  • FIG. 3 Antigen for GR6D11 identified as CEACAM1, CEACAM5 & CEACAM6 Immunoprecipitation (IP) using GR6D11 with w'hole cell lysate (WCL) from A549 cells enriched for CEACAM5/6 expression (A549(+)) pulled down two bands of around 150kDa and 50 - 75kDa.
  • Figure 4 Flow cytometry binding across cell lines from various cancer indications and normal cell lines (Table A to K) Flow cytometry binding was tested at 2pg GR 6D11 per 100,000 cells across cell lines spanning various cancer indications and normal cell lines as indicated in their respective table headings. The score is based on a 2% gate set on the secondary antibody only control.
  • GR 6D11 was tested on one sample of neutrophils commercially sourced as indicated, with no binding at the same conditions used for flow cytometry screening of cancer cell lines, demonstrating the specificity of GR 6D11 to cancer-related CEACAM 1/5/6 proteins.
  • FIG. 5 Immunohistology staining of GR 6D11 on human cancer and normal tissues for prevalence and cancer specificity determination
  • a & B Immunohistological staining with GR6D1 1 of tissue microarray (TMA) MNO961 , a multi-organ normal human tissue array, demonstrating low to mid binding in only 6% of samples, mainly in esophageal tissue, and 1 of each in cervical and thymus tissue. The staining has been reviewed by a pathologist and deemed to be intracellular, and not membranous.
  • TMA tissue microarray
  • GR6D11 of tissue microarray (TMA) from multi-organ tumour tissue arrays (MTU481 & MTU951 ), and lung cancer focused arrays (Lc 10012a) has demonstrated high prevalence (> 50%) of GR 6D11 in adenocarcinoma (lung, gastrointestinal and breast cancer), and is also present in other cancers (uterus, bladder, squamous lung, and liver) at a lower level (> 20%).
  • GR 6D11 is again shown to be specific to cancer tissue, with only 2% of cancer adjacent lung normal tissue stained positive.
  • GR 6D11 exhibited cancer cell cytotoxicity as an antibody drug conjugate
  • ADC antibody drug conjugate
  • GR 6D11 was tested for functionality as an antibody drug conjugate (ADC) using an indirect method against the A549(+) cells described previously. Briefly, GR 6D11 was incubated with A549(+) cells at a concentration of 0.2pg/well in a 96-well plate. Anti-mouse secondary antibodies conjugated with different cytotoxins (Saporin: Mslg-ZAP & Monomethyl auristatin E: Mslg-MMAE) was added at 1 : 1 or 1 :4 molar ratio to GR 6D 11 and incubated for 72 hours before read-out of cell growth. GR 6D11 demonstrated potency as an ADC, with up to -60% growth reduction compared to untreated cells.
  • GR 6D11 functions as an effective CAR T therapeutic in in-vitro and in-vivo lung cancer models
  • VL variable light
  • VH variable heavy
  • the mRNA constructs were transiently transfected into T cells and tested for cell killing potency against PC-9 lung cancer cells. All constructs demonstrated good efficacy in inhibition of cell growth, the construct with the short linker was selected to proceed for further development as lentiviral constructs.
  • C The CAR T cells retained their specificity towards only GR 6D11 binding cells (A549 (+) & PC-9), but spared antigennegative cells (normal - HEK293 and cancer cells - 1GROV-1 & SKOV3).
  • D This cytokine profile data is also congruent with the cell cytotoxicity data in Figure 7B, with the hallmark cytokine typical in CAR T activation released at higher levels in CD28-bearing CAR constructs.
  • E The efficacy of GR 6D11 as a CAR T therapeutic was evaluated in a xenograft lung cancer model with A549(+) target cells.
  • A549(+) was injected subcutaneously to form tumours reaching an average of > 150 mm3 tumour volume before a one-time intravenous treatment with T cells transduced with the CAR(GR6Dl l)-S-28z lentiviral construct.
  • A549(+) cells are negative for CD 19 expression
  • CD19-targeted CAR T bearing constructs with the same signalling domains was used as the control arm.
  • a maximal tumour growth inhibition of up to 69.5% was observed on Day 16 after treatment. Mice from the control arm had to be ethically culled after Day 16 as tumour volumes reach > 2000mm3, but tumour size was monitored for the remaining mice.
  • Figure 8 Characterisation of humanised GR 6D11 antibodies
  • the variable regions of GR 6D11 was humanised by CDR grafting into human germline sequences, and the constant regions replaced by human IgGl and 1g kappa sequences.
  • VH7 framework constructs The three humanised scFv molecules based on the VH7 framework all retained their strong sub-nanomolar binding affinity to CEACAM5 and CEACAM6, while the scFv based on the VH1 framework had a weaker binding at single-digit nanomolar ranges, congruent to what was observed in flow cytometry binding of SNU-16. VH1-VK1 scFv also lost the crossreactivity to CEACAM1 proteins, unlike the VH7 framework constructs.
  • the present disclosure teaches antigen-binding molecules that specifically bind to CEACAM1, CEACAM5 and/or CEACAM6.
  • the antigen-binding molecules may specifically bind to CEACAM1, CEACAM5 and CEACAM6.
  • the antigen-binding molecules specifically binds CEACAM5 and CEACAM6, but not CEACAM1.
  • an antigen-binding molecule comprising a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of NYGMN (SEQ ID NO: 1), the VHCDR2 amino acid sequence of WMGWINTNTGEPTYA (SEQ ID NO: 2), and the VHCDR3 amino acid sequence of HYFGLDY (SEQ ID NO: 3), and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the VLCDR2 amino acid sequence of STSNLAS (SEQ ID NO: 5), and the VLCDR3 amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VH VHCDR1 amino acid sequence of NYGMN
  • VHCDR3 amino acid sequence of HYFGLDY SEQ ID NO: 3
  • VL light chain variable region
  • GR 6D11 is a mouse IgG2a K monoclonal antibody developed against gcfitinib resistant clones of the PC-9 lung adenocarcinoma cell line. It is found to recognise CEACAM5 and CEACAM6 at pico-molar affinities, and also crossreactivity to CEACAM1 at a lower affinity. It binds strongly to gefitinib resistant lung cancer lines, and also to gastric, breast cancer, colorectal, pancreatic and haematological cancer lines on flow cytometry, while retaining negligible binding to normal fibroblast or epithelial cell lines.
  • GR 6D11 showed high selectivity towards tumour samples, with strong staining on gastric, colorectal, breast and lung cancer cores, while no staining was observed on normal tissues or cancer adjacent tissues.
  • GR 6D11 exhibited cytotoxic activity as an antibody drug conjugate (ADC) when indirectly conjugated to saporin or monomethyl auristatin E (MMAE), via a secondary antibody on binding cancer lines in-vitro.
  • ADC antibody drug conjugate
  • MMAE monomethyl auristatin E
  • GR 6D1 1 was further developed as a chimeric antigen receptor (CAR) for T-cells.
  • T-cells bearing the GR 6D11 CAR exhibited expected cytotoxicity and cytokine release in-vitro, and efficacy in in-vivo xenograft models.
  • GR 6D11 has been humanised and grafted onto a human IgGl backbone to achieve similar' pico-molar affinity to CEACAM5 and CEACAM6
  • the antigen-binding molecule binds to CEACAM5 and CEACAM6 with picomolar affinity. In one embodiment, the antigen-binding molecule further cross-reacts with CEACAM1.
  • an antigen-binding molecule comprising a heavy chain variable region (VH) comprising the amino acid sequence of NYGMN (SEQ ID NO: 23), the amino acid sequence of WINTNTGEPTYAEEFKG (SEQ ID NO: 24) or WINTNTGEPTYAQGFTG (SEQ ID NO: 25), and the amino acid sequence of HYFGLDY (SEQ ID NO: 26), and a light chain variable region (VL) comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding molecule may comprise the amino acid sequence of NYGMN (SEQ ID NO: 23), the amino acid sequence of WINTNTGEPTYAEEFKG (SEQ ID NO: 24) or WTNTNTGEPTYAQGFTG (SEQ ID NO: 25), and the amino acid sequence of HYFGLDY (SEQ ID NO: 26) and the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • the antigen-binding molecule comprises a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of NYGM (SEQ ID NO: 18), the VHCDR2 amino acid sequence of W1NTNTGEPTYA (SEQ ID NO: 19), and the VHCDR3 amino acid sequence of HYFGLDY (SEQ ID NO: 20), and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the VLCDR2 amino acid sequence of STSNLAS (SEQ ID NO: 5), and the VLCDR3 amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding molecule comprises a heavy chain variable region (VH) comprising the amino acid sequence of NYGMH (SEQ ID NO: 27), the amino acid sequence of WINTNTGEPTYAEKFQG (SEQ ID NO: 28), and the amino acid sequence of HYFGLDY (SEQ ID NO: 29), and a light chain variable region (VL) comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • VH heavy chain variable region
  • VL light chain variable region comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • the antigen-binding molecule may comprise the amino acid sequence of NYGMH (SEQ ID NO: 27), the amino acid sequence of WINTNTGEPTYAEKFQG (SEQ ID NO: 28), and the amino acid sequence of HYFGLDY (SEQ ID NO: 29) and the amino acid sequence of SASSSVSYMH (SEQ ID NO: 4), the amino acid sequence of STSNLAS (SEQ ID NO: 5), and the amino acid sequence of HQWSSYPWT (SEQ ID NO: 6).
  • the antigen-binding molecules of the present invention may be in isolated, purified, synthetic or recombinant form. Suitable antigen-binding molecules may be selected from antibodies and their antigen-binding fragments, including monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, human antibodies, and antigen-binding fragments of such antibodies.
  • the antigen-binding molecules may be multivalent (e.g., bivalent) or monovalent.
  • the antigen-binding molecules comprise an Fc domain.
  • the antigen-binding molecules lack an Fc domain.
  • the antigen binding molecules are monovalent antigen-binding molecules (e.g., Fab, scFab, Fab’, scFv, one-armed antibodies, etc.).
  • antigen-binding molecule is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigenbinding activity.
  • Representative antigen-binding molecules that are useful in the practice of the present invention include antibodies and their antigen-binding fragments.
  • the term “antigen-binding molecule” includes antibodies and antigen-binding fragments of antibodies.
  • the antigen-binding molecule binds to an epitope that comprises at least A89 of CEACAM1, CEACAM5 and/or CEACAM6. In one embodiment, the antigenbinding molecule binds to an epitope that comprises or consists of TQQA (SEQ ID NO: 30). In one embodiment, the antigen-binding molecule binds to an epitope that comprises or consists of GTQQA (SEQ ID NO: 31). In one embodiment, the antigen-binding molecule binds to an epitope that comprises or consists of TQQAT (SEQ ID NO: 32).
  • the antigen-binding molecule binds to an epitope that comprises or consists of GTQQAT (SEQ ID NO: 33). In one embodiment, the antigen- binding molecule binds to an epitope that comprises or consists of TQQATP (SEQ ID NO: 34). In one embodiment, the antigen-binding molecule binds to an epitope that comprises or consists of IGTQQA (SEQ ID NO: 35). In one embodiment, the antigen-binding molecule binds to an epitope that comprises or consists of TQQATPG (SEQ ID NO: 36). In one embodiment, the antigenbinding molecule binds to an epitope that comprises or consists of VIGTQQA (SEQ ID NO: 37). In one embodiment, the antigen-binding molecule binds to an epitope that comprises or consists of IGTQQAT (SEQ ID NO: 38).
  • the antigen-binding molecule does not bind to CEACAM8.
  • the antigen-binding molecule as described herein, is conjugated to another molecule or moiety, including functional moieties (e.g., toxins), detectable moieties (e.g., fluorescent molecules, radioisotopes), small molecule drugs and polypeptides.
  • functional moieties e.g., toxins
  • detectable moieties e.g., fluorescent molecules, radioisotopes
  • small molecule drugs e.g., small molecule drugs and polypeptides.
  • antibody is understood to mean any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to, or interacts specifically with, the target antigen.
  • CDR complementarity determining region
  • the term “antibody” includes full-length immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (c.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR, VH or VH) and a heavy chain constant region.
  • the heavy chain constant region typically comprises three domains - CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (which may be abbreviated as LCVR, VL, VK, VK or VL) and a light chain constant region.
  • the light chain constant region will typically comprise one domain (CL1).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that arc more conserved, also referred to as framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL typically comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the antigen-binding molecules described herein may be identical to the FR of germline sequences of the target species (i.e., the species to which the antigen-binding molecules or antigen-binding fragments thereof, as described herein, will be administered). In some embodiments, the FR may be naturally or artificially modified.
  • each of the FR sequences arc identical to FR sequences derived from immunoglobulin molecules of the target species, including to minimize an immune response being raised against the binding molecule upon administration to a subject of the target species
  • the antigen-binding molecule, or antigenbinding fragment thereof may comprise one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in one or more FR from the target species.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins arc called a, 5, s, y, and p, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to a person skilled in the art.
  • the antigen-binding molecule of the present invention has an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.
  • the heavy chain constant region can be a wild-type human Fc region, or a human Fc region that includes one or more amino acid substitutions.
  • the antibodies can have mutations that stabilize the disulphide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of lgG4, as disclosed in the art (e.g., Angal et al., 1993. Mol. Immunol., 30: 105-08). See also, e.g., U.S. 2005/0037000.
  • the heavy chain constant region can also have substitutions that modify the properties of the antigen-binding molecule (e.g., decrease one or more of: Fc receptor binding, antigen-binding molecule glycosylation, deamidation, binding to complement, or methionine oxidation).
  • the antigen-binding molecules may have mutations such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260.
  • the antigen-binding molecule is modified to reduce or eliminate effector function.
  • the heavy chain constant region can be chimeric, e.g., the Fc region can comprise the CHI and CH2 domains of an IgG antibody of the IgG4 isotype, and the CH3 domain from an IgG antibody of the IgGl isotype (see, e.g., U.S. Patent Appl. No. 2012/0100140A1).
  • CDRs complementarity determining regions
  • Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • Each complementarity determining region may comprise amino acid residues from a “complementarity determining region” as defined for example by Kabat (i.e., about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al..
  • a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypcrvariablc loop.
  • the present disclosure extends to antigen binding molecules that bind specifically to native CEAC AM 1/5/6 (i.e., naturally-occurring CEACAM 1/5/6), as well as to variants thereof.
  • Such variants may include CEACAM1/5/6 molecules that differ from a naturally-occurring (wild-type) molecule by one or more amino acid substitutions, deletions and / or insertions.
  • Variant CEACAM1/5/6 molecules of this type may be naturally-occurring or synthetic (e.g., recombinant) forms. It is to be understood, however, that in one embodiment, the antigenbinding molecules described herein bind specifically to a native form of CEACAM 1/5/6, whether of a human or non-human species.
  • the antigen-binding molecule specifically binds to human
  • CEACAM1, CEACAM5 and CEACAM6 may have a sequence with a
  • the CEACAM5 may have a sequence with a
  • the CEACAM6 may have a sequence with a
  • an “antigen-binding site” refers to the site, i.e., one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen.
  • the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • a native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
  • An antigen-binding site of an antigen-binding molecule described herein typically binds specifically to an antigen and more particularly to an epitope of the antigen.
  • antigen-binding fragment As used interchangeably herein to refer to a part of an antigen- binding molecule that participates in antigen-binding. These terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., ph age- anti body libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-graftcd antibodies, one- armed antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMTPs), and shark variable TgNAR domains, are also encompassed within the expression “antigen-binding fragment” as used herein.
  • SMTPs small modular immunopharmaceuticals
  • shark variable TgNAR domains are also encompassed within the expression “antigen-binding fragment” as used herein.
  • an antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) Vn-Cn3; (iv) Vn-Cnl-Cn2; (V) VH-CH1-CH2-CH3, (vi) Vn-Cn2-Cn3; (vii) Vn- C L ; (viii) VL-CH1; (ix) V L -C H 2, (X) V L -C H 3; (xi) V L -C H l-C H 2; (xii) V L -CH1-C H 2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigenbinding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • a multispecific antigen-binding molecule will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antigen-binding molecule format including bispccific antigen-binding molecule formats, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • constant domains or “constant region” as used herein denotes the sum of the domains of an antibody other than the variable region.
  • the constant region is not directly involved in binding of an antigen, but exhibits various immune effector functions.
  • the antigen-binding molecule or antigen-binding fragment thereof is modified for compatibility with the target species.
  • the antigenbinding molecule or antigen-binding fragment thereof is humanized.
  • humanized is meant that the antigen-binding molecule comprises an amino acid sequence that is compatible with humans, such that the amino acid sequence is unlikely to be seen as foreign by the immune system of a human subject.
  • the humanized antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more human immunoglobulin molecules. In some embodiments, all of the framework regions of the humanized antigen-binding molecule will be derived from one or more human immunoglobulin molecules.
  • the humanized antibody may optionally comprise an immunoglobulin heavy chain constant region derived from a human immunoglobulin molecule.
  • the phrase “specifically binds” or “specific binding” refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions.
  • detectable binding agents that are proteins
  • specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies.
  • the specified antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence.
  • Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity to that determinant. This selection may be achieved by subtracting out antigen-binding molecules that cross-react with other molecules.
  • immunoassay formats may be used to select antigen -binding molecules (e.g., immunoglobulins) such that they arc specifically immunoreactive with a particular antigen.
  • antigen -binding molecules e.g., immunoglobulins
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • Methods of determining binding affinity and specificity are also well known in the art (see, for example, Harlow and Lane, supra); Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co. 1976)).
  • Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule e.g., an antigen-binding molecule) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pah e.g., an antigen-binding molecule.
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (k o n and k ou , respectively).
  • equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same.
  • Affinity can be measured by common methods known in the art, including those described herein.
  • a particular method for measuring affinity is biolayer interferometry (BLI).
  • polypeptide polypeptide
  • peptide or protein
  • polypeptide polypeptide
  • peptide or protein
  • the amino acid residues are usually in the natural "L” isomeric form. However, residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • modified antibody includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and arc described, e.g., in U.S. Pat. No. 5,892,019.
  • modified antibody includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen).
  • the antigen-binding fragment may be an antibody or antigen-binding fragment thereof.
  • the antibody or antigen binding fragment thereof may be a full-length antibody, a substantially intact antibody, a Fab fragment, a scFab, a Fab’, a single chain variable fragment (scFv) or a one-armed antibody.
  • the antibody or antigen-binding molecule therefore is humanized.
  • the antigen-binding molecule may comprise: a) a VH region comprising an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 11, 12 or 13; and b) a VL region comprising an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 14.
  • the antigen-binding molecule may comprise a) a heavy chain variable region (VH) as defined herein comprising at least 70% sequence identity to at least one region other than a CDR of the VH amino acid sequence set forth in SEQ ID NO: 11, 12 or 13 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the VH), and b) a light chain variable region (VL) as defined herein comprising at least 70% sequence identity to at least one region other than a CDR of the VL amino acid sequence set forth in SEQ ID NO: 14 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding molecule may comprise: a) a VH as defined herein which is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 11, 12 or 13 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the VH amino acid sequence set forth in SEQ ID NO: 11, 12 or 13 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the VH), and b) a VL as defined herein which is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 14 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the VL amino acid sequence set forth in SEQ ID NO: 14 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the VL).
  • the antigen-binding molecule may comprise: a) a VH region comprising an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 21; and b) a VL region comprising an amino acid sequence having at least 70% (including at least 71 % to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 14.
  • the antigen-binding molecule may comprise a) a heavy chain variable region (VH) as defined herein comprising at least 70% sequence identity to at least one region other than a CDR of the VH amino acid sequence set forth in SEQ ID NO: 21 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the VH), and b) a light chain variable region (VL) as defined herein comprising at least 70% sequence identity to at least one region other than a CDR of the VL amino acid sequence set forth in SEQ ID NO: 14 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding molecule may comprise: a) a VH as defined herein which is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 21 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the VH amino acid sequence set forth in SEQ ID NO: 21 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the VH), and b) a VL as defined herein which is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 14 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the VL amino acid sequence set forth in SEQ ID NO: 14 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the VL).
  • a VH as defined herein which is distinguished from the V
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and 1) or the identical amino acid residue (e.g.
  • Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the antigen-binding molecule as defined herein may comprise one or more conservative amino acid substitutions.
  • a “conservative amino acid substitution” is to be understood as meaning a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as shown in the table below:
  • Conservative amino acid substitution aiso includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants can be screened for their ability to bind specifically to CEACAM1 , CEACAM5 and CEACAM6 using methods known to persons skilled in the art, including those methods described elsewhere herein.
  • the antigen-binding molecule of the present invention is a monovalent antigen-binding molecule.
  • Non-limiting monovalent antigen-binding molecules include: a Fab fragment consisting of VL, VH, CL and Cui domains; a Fab’ fragment consisting of VL, VH, CL and CH1 domains, as well as a portion of a CH2 domain; an Fd fragment consisting of VH and CHI domains; an Fv fragment consisting of VL and VH domains of a single arm of an antibody; a single-chain antibody molecule (e.g., scFab and scFv); a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and a one-armed antibody, such as described in US20080063641 (Genentech) or other monovalent antibody, e.g., such as described in W02007048037 (Amgen).
  • a monovalent antigen-binding molecule comprises an Fv fragment.
  • the Fv fragment is the smallest unit of an immunoglobulin molecule with function in antigenbinding activities.
  • An antigen-binding molecule in scFv (single chain fragment variable) format consists of variable regions of heavy (Vn) and light (VL) chains, which are joined together by a flexible peptide linker that can be easily expressed in functional form in an expression host such as E. coll and mammalian cells, allowing protein engineering to improve the properties of scFv such as increase of affinity and alteration of specificity (Ahmed et al., 2012. Clin Dev Immunol. 2012:980250).
  • Representative examples of linker sequences arc described in Section 4.5 infra. In the scFv construction, the order of the domains can be either Vn-linker-VL or VL-linker-Vn and both orientations can applied.
  • the linker sequences used in scFvs are multimers of the pentapeptide GGGGS (SEQ ID NO: 39) (or G4S or Gly4Ser). Those include the 15-mer (G4S)3 (Huston et al., 1988. Proc Natl Acad Sci USA.
  • sequences with added functionalities e.g., an epitope tag or an encoding sequence containing a Cre-Lox recombination site or sequences improving scFv properties, often in the context of particular antibody sequences.
  • Cloning of the scFv is usually done by a two-step overlapping PCR (also known as Splicing by Overlap Extension or SOE-PCR), as described (Schaefer et al., 2010, supra).
  • the VH and VL domains are first amplified and gel-purified and secondarily assembled in a single step of assembly PCR.
  • the linker is generated either by overlap of the two inner primers or by adding a linker primer whose sequence covers the entire linker or more (three-fragment assembly PCR).
  • Single chain Fv (scFv) antigen-binding molecules may be recombinantly produced for example in E.
  • the monovalent antigen-binding molecule comprises an Fab fragment.
  • the monovalent antigen-binding molecule is a one- armed antibody consisting or consisting essentially of a single antigen-binding fragment (Fab) and a Fc region, wherein the Fc region comprises a first and a second Fc polypeptide, and wherein the first and second Fc polypeptides are present in a complex.
  • Fc-containing monovalent antigen-binding molecules can often lead to undesirable bivalent, homodimer contaminants.
  • Strategies to inhibit formation of homodimers are known including methods that introduce mutations into immunoglobulin constant regions to create altered structures that support unfavorable interactions between polypeptide chains and suppress unwanted Fc homodimer formation.
  • Non-limiting examples of this strategy to promote heterodimerization include the introduction of knobs-into-holes (KIH) structures into the two polypeptides and utilization of the naturally occurring heterodimerization of the CL and CHI domains (see, Kontermann, supra, pp. 1 -28 (2011) Ridgway et al., 1996. Protein Eng. 9(7):617-21; Atwell et al., 1997.
  • KIH knobs-into-holes
  • Modifications in the Fc domain of an antigen- binding molecules may also be desirable to reduce Fc receptor binding and therefore reduce the potential for FcyRIIa-mediated activation of platelets.
  • the so-called ‘LALA’ double mutation (Leu234Ala together with Leu235Ala) in human IgG (including IgGl) is known to significantly impair Fc receptor binding and effector function (Lund el al., 1991, J. Immunol. 147, 2657-2662; Lund et al., 1992, Mol. Immunol. 29:53-59).
  • the antigen- binding molecule e.g., a MAb or an antigen- binding fragment thereof
  • each of the IgGl Fc chains of the antibody carries P329G, L235A, L234A (P329G LALA) mutations or each of the IgG4 Fc chains carries P329G, S228P, L235E mutations, in order to reduce or abolish any undesired cross-linking, platelet activation, or immune effector function (e.g., antibody-dependent cell-meditated cytotoxicity (ADCC), phagocytosis (ADCP) and complement dependent cytotoxicity (CDC)) of the antigen-binding molecule.
  • ADCC antibody-dependent cell-meditated cytotoxicity
  • ADCP phagocytosis
  • CDC complement dependent cytotoxicity
  • each of the IgGl Fc chains of the antigen-binding molecule (or antibody) carries mutations comprising a) S239D, A33OL and I332E or b) F243L, R292P, Y300L, V3O5I and P396L, which enhance immune effector function of the antigen-binding molecule (e.g. ADCC).
  • the present invention contemplates monovalent antigen-binding molecules produced by co-cxprcssion of a light chain, heavy chain and a truncated Fc domain.
  • the heavy chain incorporates hole mutations and P329G LALA mutations
  • the truncated Fc domain incorporates knob mutations and P329G LALA mutations.
  • antigen-binding molecule disclosed herein can be achieved for example in bacterial (e.g., Escherichia colt), yeast, insect or mammalian host cells upon cloning of the protein coding sequences of the constructs in the context of appropriate expression vectors with appropriate translational, transcriptional start sites, and, where appropriate, signal peptide sequences.
  • bacterial e.g., Escherichia colt
  • yeast e.g., Escherichia colt
  • insect or mammalian host cells upon cloning of the protein coding sequences of the constructs in the context of appropriate expression vectors with appropriate translational, transcriptional start sites, and, where appropriate, signal peptide sequences.
  • the antigen-binding molecule is a multivalent antigen-binding molecule, non-limiting examples of which include: immunoglobulins, F(ab’)2, tandem scFv (taFv or scFv2), SCFV-FC, diabody, dAb2/VnH2, minibodies, ZIP miniantibodies, barnase-barstar dimer, knobs-into-holes derivatives, SEED-IgG, heteroFc-scFv, Fab-scFv, Fab)2/sc(Fab)2, scFv-(TNFa)3, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, trimerbody, tribi-minibody, barnase- barstar trimcr, collabody, DNL-F(ab)3, SCFV3-CH!/CL, Fab-scFv2, IgG-scFab, IgG-scFab, I
  • the multivalent antigen-binding molecule is selected from IgG-likc antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech; knobs-into-holes, Genentech; CrossMAbs, Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech; strand exchange engineered domain (SEED) body, EMD Serono; bioIonic, Merus; and Fab-exchanged antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-lg, GSK/Domantis; two-in-one antibody, Genentech; crosslinked MAbs, karmanos cancer center; MAb2, F-star; and Coy X-body, Coy X/Pfizer), IgG fusions (e.g., dual variable domain (DVD)-Ig, Abbott; IgG-likc bispccific antibodies, Eli Lilly; Ts2
  • the antibody is a bispccific or trispccific antibody. In one embodiment, the antibody is a bispecific antibody.
  • bi specific antibodies of the invention are formed using a " protuberance ⁇ into-cavity” strategy, also referred to as "knobs into holes” that serves to engineer an interface between a first and second polypeptide for hetero-oligomerization.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • the "knobs into holes” mutations in the CH3 domain of an Fc sequence has been reported to greatly reduce the formation of homodimers (See, for example. Merchant et al., 1998, Nature Biotechnology, 16:677-681).
  • "Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • a suitably positioned and dimensioned protuberance or cavity exists at the interlace of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.
  • the protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis.
  • knobs into holes see U.S. Patents 5,731,168; 5,807,706; 5,821,333.
  • a general method of preparing a heteromultimer using the "proluberance-inlo-cavity" strategy comprises expressing, in one or separate host cells, a polynucleotide encoding a first polypeptide that has been altered from an original polynucleotide to encode a protuberance, and a second polynucleotide encoding a second polypeptide that has been altered from the original polynucleotide to encode the cavity.
  • the polypeptides are expressed, either in a common host cell with recovery' of the hctcromultimcr from the host cell culture, or in separate host cells, with recovery' and purification, followed by formation of the heteromultimer.
  • the heteromultimer formed is a multimeric antibody, for example a bispecific antibody.
  • an animal e.g. a mouse or rabbit
  • an antigen-binding molecule that specifically binds CEACAM1, CEACAM5 and/or CEACAM6, the method comprising: a) contacting an antibody library with a peptide of any one of SEQ ID NO: 30-38, b) isolating from the animal an antibody that binds specifically to the peptide.
  • the antibody library may, for example, be an antibody phage, yeast or mRNA display library.
  • the antigen- binding molecule is a chimeric molecule that is conjugated to a heterologous moiety.
  • a “chimeric” molecule is one which comprises one or more unrelated types of components or contain two or more chemically distinct regions which can be conjugated to each other, fused, linked, translated, attached via a linker, chemically synthesized, expressed from a nucleic acid sequence, etc.
  • a peptide and a nucleic acid sequence a peptide and a detectable label, unrelated peptide sequences, and the like.
  • the chimeric molecule comprises amino acid sequences of different origin
  • the chimeric molecule includes (1) polypeptide sequences that are not found together in nature (z.e., at least one of the amino acid sequences is heterologous with respect to at least one of its other amino acid sequences), or (2) amino acid sequences that are not naturally adjoined.
  • a “chimeric" antibody” as used herein refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the heterologous moiety is a detectable moiety, a half-life extending moiety or a therapeutic moiety.
  • Detectable moieties contemplated by the present invention include for example any species known in the art that is appropriate for diagnostic detection, including in vitro detection and in vivo imaging.
  • the detectable moiety may be, for example, a fluorophore, a radionuclide reporter, a metal-containing nanoparticle or microparticle, an ultrasound contrast agent (e.g., a nanobubble or microbubble) or an optical imaging dye.
  • This also includes contrast particles visible in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI).
  • Fluorophores can be detected and/or imaged, for example, by fluorescence polarization, fluorescence-activated cell sorting and fluorescence microscopy, which may or may not be in combination with clcctrospray ionization-mass spectrometry (ESLMS) detection, as well as fluorescence emission computed tomography (FLECT) imaging.
  • Radionuclide reporters can be detected and imaged by radionuclide (nuclear) detection, such as, for example, singlephoton emission computed tomography (SPECT), positron emission tomography (PET) or scintigraphic imaging.
  • Metal-containing nanoparticles or microparticles may be detected using optical imaging, including MRI, which is typically used with paramagnetic nanoparticlcs or microparticles, and MPI, which is generally used with supcrparamagnctic particles.
  • Ultrasound contrast agents can be detected using ultrasound imaging including contrast-enhanced ultrasound (CEU).
  • the detectable label may also be an enzyme-substrate label.
  • the enzyme may generally catalyze a chemical alteration of the chromogenic substrate that can be measured using various techniques.
  • the enzyme may catalyze a chemical alteration of the chromogenic substrate that can be measured using the various techniques.
  • the example may catalyze a color change in a substrate, which can be measured spectrophoto metrically.
  • the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, 0-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as unease and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase 0-galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-
  • enzyme- substrate combinations include, for example:
  • Elorseradish peroxidase utilizes hydrogen peroxide to oxidize a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
  • a dye precursor e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)
  • the antigen-binding molecule need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the antigenbinding molecule.
  • the antigen- binding molecule of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, immunohistochemistry and immunoprecipitation assays.
  • the chimeric molecule comprises at least one heterologous moiety that is a “half-life extending moiety”.
  • Half-life extending moieties can comprise, for example, (i) XTEN polypeptides; (ii) Fc; (iii) albumin, (iv) albumin binding polypeptide or fatty acid, (v) the C-terminal peptide (CTP) of the 13 subunit of human chorionic gonadotropin, (vi) PAS; (vii) HAP; (viii) transferrin; (ix) polyethylene glycol (PEG); (x) hydroxyethyl starch (HES), (xi) polysialic acids (PSAs); (xii) a clearance receptor or fragment thereof which blocks binding of the chimeric molecule to a clearance receptor; (xiii) low complexity peptides; (xiv) or any combinations thereof.
  • the half-life extending moiety comprises an Fc region. In other embodiments, the half-life extending moiety comprises two Fc regions fused by a linker.
  • Exemplary heterologous moieties also include, e.g., FcRn binding moieties (e.g., complete Fc regions or portions thereof which bind to FcRn), single chain Fc regions (scFc regions, e.g., as described in U.S. Publ. No. 20080260738, WO 2008/012543 and WO 2008/1439545), or processable scFc regions.
  • a heterologous moiety can include an attachment site for a nonpolypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES),
  • the therapeutic moiety is a toxin.
  • the toxin may, for example, be monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), a mertansine or maytansinoid (such as DM-1), a calicheamicin, a pyrrolobenzodiazepine, saporin, gemcitabine, irinotecan, etoposide, vinblastine, pcmctrcxcd, docetaxel, paclitaxel, platinum agents (for example, cisplatin, oxaliplatin and carboplatin), vinorelbine, capecitabine, mitoxantrone, ixabepilone, eribulin, 5-fluorouracil, trifluridine, tipiracil and a topoisomerase inhibitor (such as SN-38 or deruxtecan).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristat
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the antigen-binding molecule as defined herein.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the chimeric antigen receptor as defined herein.
  • polynucleotide or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxy nucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • Also disclosed herein is a vector that comprises a nucleic acid encoding the antigen-binding molecule as described herein.
  • vector is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or virus, into which a nucleic acid sequence may be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector may be an autonomously replicating vector, i.c., a vector that exists as an cxtrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.
  • construct comprising a polynucleotide as defined herein in operable connection with one or more control sequences.
  • construct refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources.
  • constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined.
  • constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, vims, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked.
  • Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence.
  • Such elements may include control elements or regulatory sequences such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well.
  • the construct may be contained within a vector.
  • the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell.
  • Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.
  • An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell.
  • conventional compositions and methods for preparing and using constructs and host cells arc well known to one skilled in the art, sec for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. 1. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.
  • control element means a nucleic acid sequence (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell.
  • control sequences that arc suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site.
  • Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
  • transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
  • Disclosed herein is a host cell that contains the construct as defined herein.
  • host refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a host cell is any type of cellular system that can be used to generate the antigen binding molecules of the present invention.
  • Host cells include cultured cells, e.g.
  • mammalian cultured cells such as CHO cells, BHK cells, HEK293 cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • a chimeric antigen receptor comprising an antigen-binding molecule as defined herein, a transmembrane domain, one or more co- stimulatory domains and an intracellular signalling domain.
  • the term “chimeric antigen receptor” or “CAR” as used herein refers to an artificial (i.e., man-made) transmembrane protein expressed on a mammalian cell comprising at least an ectodomain, a transmembrane, and an endodomain.
  • the CAR protein includes a “spacer” which covalently links the ectodomain to the transmembrane domain.
  • a spacer is often a polypeptide linking the cctodomain to the transmembrane domain via peptide bonds.
  • the CAR is typically expressed on a mammalian lymphocyte.
  • the CAR is expressed on a mammalian cell such as a T-cell, a tumor infiltrating lymphocyte (TIL) or a CAR NK cell.
  • TIL tumor infiltrating lymphocyte
  • a CAR expressed on an NK cell is referred to herein as a "CAR NK-cell” or "CAR-NK”.
  • a CAR expressed on a T-cell is referred to herein as a “CAR T- cell” or “CAR-T.”
  • the CAR-T is a T helper cell, a cytotoxic T-cell, a natural killer T-cell, a memory T-cell, a regulatory T-cell, or a gamma delta T-cell.
  • a CAR-T with antigen binding specificity to the patient's tumor is typically engineered to express on a native T-cell obtained from the patient.
  • the engineered T-cell expressing the CAR is then infused back into the patient.
  • the CAR- T is thus often an autologous CAR-T although allogeneic CAR-T are included within the scope of the invention.
  • the ectodomain of a CAR comprises an antigen binding region, such as an antibody or antigen binding fragment thereof (e.g., scFv), that specifically binds under physiological conditions with a target antigen, such as a tumor specific antigen.
  • a biochemical chain of events i.e., signal transduction results in modulation of the immunological activity of the CAR-T.
  • CD3-z CD3-zeta chain
  • IAM immunoreceptor tyrosinebased activation motif
  • the transmembrane domain is a transmembrane domain selected from the group consisting of a T cell receptor a chain, a T cell receptor chain, a CD3 zeta chain, a CD28, a CD3c, a CD45, a CD4, a CD5, a CD8, a CD9, a CD16, a CD22, a CD33, a CD37, aCD64, a CD80, a CD86, a CD134, a CD137, an ICOS, a CD154 a KIR2D, a NKG2D and a GITR.
  • the co-stiinulatory domain is a co- stimulatory domain selected from the group consisting of a CD27, CD28, CD40, CD40L, a 4-1BB, a GITR, an ICOS-1, a CD27, an OX-40, Toll-like receptor (TLR), DAP10, DAP12 or 2B4.
  • a co- stimulatory domain selected from the group consisting of a CD27, CD28, CD40, CD40L, a 4-1BB, a GITR, an ICOS-1, a CD27, an OX-40, Toll-like receptor (TLR), DAP10, DAP12 or 2B4.
  • the activating domain comprises a CD3 zeta activating domain.
  • the CAR may comprise an scFv (VL-linker-VH or VH-linker-VL).
  • the scFv may be linked to a transmembrane domain, a co-stimulatory domain (such as CD28) and an activating domain (such as CD3 zeta activating domain).
  • the linker may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 22.
  • Also provided herein is a vector comprising the polynucleotide as defined herein.
  • an engineered cell comprising the vector as defined herein.
  • Also provided herein is a method of preparing an engineered immune cell, the method comprising introducing the vector as defined herein into an immune cell.
  • composition comprising an antigen-binding molecule as defined herein or a chimeric molecule as defined herein.
  • pharmaceutically acceptable carrier a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.c., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (c.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated.
  • the pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., dispersions or suspensions, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Suitable pharmaceutical compositions may be administered intravenously, subcutaneously or intramuscularly.
  • the compositions are in the form of injectable or infusible solutions.
  • a preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the pharmaceutical composition is administered by intravenous infusion or injection.
  • the pharmaceutical composition is administered by intramuscular or subcutaneous injection.
  • parenteral administration and “administered parenterally” 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.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline.
  • Intravenous vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • an agent of the present disclosure may be conjugated to a vehicle for cellular delivery.
  • the agent may be encapsulated in a suitable vehicle to either aid in the delivery of the agent to target cells, to increase the stability of the agent, or to minimize potential toxicity of the agent.
  • a variety of vehicles arc suitable for delivering an agent of the present disclosure.
  • suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • An antigen-binding molecule of the present disclosure can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of modified polypeptide or antigen in the patient. Alternatively, the antigenbinding molecule can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the antigen-binding molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 0.01 to 40 mg/kg, e.g., 0.01 to 0.1 mg/kg, e.g., about 0.1 to 1 mg/kg, about 1 to 5 mg/kg, about 5 to 25 mg/kg, about 10 to 40 mg/kg.
  • the dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.
  • dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • an antigen-binding molecule as defined herein a chimeric molecule as define herein, an engineered cell as defined herein, or a pharmaceutical composition as defined herein for use as a medicament.
  • Disclosed herein is a method for reducing or inhibiting proliferation and/or viability of a cancer cell, the method comprising contacting the cancer cell with a therapeutically effective amount of an antigen-binding molecule as defined herein, a chimeric molecule as defined herein, an engineered cell as defined herein or a pharmaceutical composition as defined herein.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth.
  • cancer refers to non-metastatic and metastatic cancers, including early stage and late stage cancers.
  • non-metastatic is meant a cancer that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site.
  • metal cancer refers to cancer that has spread or is capable of spreading from one part of the body to another.
  • a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer.
  • a metastatic cancer is usually a stage IV cancer.
  • cancer includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and/or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astro
  • the cancer cell is a solid or haematological cancer cell.
  • solid cancer may refer to one or more of breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and/or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiformc, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary
  • haematological cancer may refer to one or more of leukemia, lymphoma. Chronic Myeloproliferative Disorders, Langerhans Cell Histiocytosis, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplasia Syndromes, Myelodysplastic/Myeloproliferative Neoplasms or a combination thereof.
  • leukemia is any one or more of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Hairy Cell Leukemia (HCL) or a combination thereof.
  • ALL Acute Lymphoblastic Leukemia
  • AML Acute Myeloid Leukemia
  • CLL Chronic Lymphocytic Leukemia
  • CML Chronic Myelogenous Leukemia
  • HCL Hairy Cell Leukemia
  • lymphoma is any one or more of AIDS-Related Lymphoma, Cutaneous T- Cell Lymphoma, Hodgkin Lymphoma, Mycosis Fungoides, Non-Hodgkin Lymphoma, Primary Central Nervous System Lymphoma, Sezary Syndrome, T-Cell Lymphoma, Cutaneous, Waldenstrom Macroglobulincmia, B cell lymphoma or a combination thereof.
  • the cancer is a metastatic cancer.
  • the cancer may be a refractory or a relapsed cancer.
  • the cancer is selected from the group consisting of gefitinib-resistant lung cancer, osimertinib-resistant lung cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, stomach (or gastric) cancer, small intestine cancer, oesophageal cancer, colorectal cancer, haematological cancer, squamous cell lung cancer, cervical cancer, endometrium cancer or liver cancer.
  • Disclosed herein is a method of treating or preventing a cancer or an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of an antigen-binding molecule as defined herein, a chimeric molecule as defined herein, an engineered cell as defined herein, or a pharmaceutical composition as defined herein to the subject.
  • a method of treating a disease or condition associated with an undesired expression of CEACAM 1/5/6 in a subject wherein the method comprises administering a therapeutically effective amount of an antigen-binding molecule as defined herein, a chimeric molecule as defined herein, an engineered cell as defined herein, or a pharmaceutical composition as defined herein to the subject.
  • treating may refer to (1) delaying the appearance of one or more symptoms of the condition; (2) inhibiting the development of the condition or one or more symptoms of the condition; (3) relieving the condition, i.e., causing regression of the condition or at least one or more symptoms of the condition; and/or (4) causing a decrease in the severity of the condition or of one or more symptoms of the condition.
  • vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such as from the genus Macaco, (e.g., cynomolgus monkeys such as Macaco fascicularis, and/or rhesus monkeys (Macaco mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpan
  • primates e.g., humans, monkeys and apes
  • species of monkeys such as from the genus Macaco, (e.g., cynomolgus monkeys such as Macaco fascicularis,
  • the cancer is positive for or overexpresses CEACAM1, CEACAM5 and/or CEACAM6.
  • the cancer may be positive for or overexpresses CEACAM 1, CEACAM5 or CEACAM6.
  • the cancer may be positive for or overexpresses CEACAM1 and CEACAM5, CEACAM1 and CEACAM6, or CEACAM5 and CEACAM6.
  • the cancer may be positive for or overexpresses CEACAM 1 , CEAC AM5 and CEACAM6.
  • the methods as disclosed herein may comprise the administration of a “therapeutically effective amount” of an agent (c.g., an antigen-binding molecule, a chimeric molecule, a polynucleotide, a construct, a vector, an engineered cell or a pharmaceutical composition) to a subject.
  • an agent c.g., an antigen-binding molecule, a chimeric molecule, a polynucleotide, a construct, a vector, an engineered cell or a pharmaceutical composition
  • therapeutically effective amount includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine
  • an antigen-binding molecule as defined herein a chimeric molecule as defined herein, an engineered cell as defined herein, or a pharmaceutical composition as defined herein, in the manufacture of a medicament for treating or preventing cancer.
  • a method for detecting cancer in a subject comprising: contacting a sample obtained from the subject with an antigen-binding molecule as defined herein, wherein an increase in the level of binding of the antigen-binding molecule in the sample as compared to a reference is indicative of cancer.
  • a method for identifying a subject susceptible to cancer comprising: contacting a sample obtained from the subject with an antigen-binding molecule as defined herein, wherein an increase in the level of binding of the antigen-binding molecule in the sample as compared to a reference indicates that the subject is susceptible to cancer.
  • the reference may be the level of binding of the antigen-binding molecule in a sample from a subject of the same species without cancer, or an average level of binding of the antigenbinding molecule in samples from a population of subjects of the same species (c.g., of varying ages, ethnic backgrounds and genders) without cancer.
  • the reference may also be the level of binding of the antigen-binding molecule in a sample from the same subject before the suspected onset of cancer, before the start of a treatment regimen, or at a different time-point during the course of cancer or during the course of treatment for the cancer.
  • the reference values can be stored in a database and used as a reference in subsequent analyses.
  • the terms “increased” and “increase” are used herein to mean an increase by a statistically significant amount.
  • the terms “increased” and “increase” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • the terms “decreased” and “decrease” are used herein to mean a decrease by a statistically significant amount.
  • the terms “decreased” and “decrease” can mean a decrease of at least 10% as compared to a reference level, for example a decrease of at least about 20%>, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to and including a 100% decrease or any decrease between 10-100% as compared to a reference level.
  • the antigen-binding molecule comprises a detectable label.
  • kits for use in the method as defined herein comprising an antigenbinding molecule as defined herein, together with instructions for use.
  • PC -9, H1299 and SNU-16 were cultured in RPMI (Invitrogen, USA) supplemented with 10% foetal bovine serum (HyClone GE Healthscience, South America).
  • PC-9 cultures were exposed to increasing concentration of gefitinib (Selleckchem, USA), starting from 2nM and gradually increased with each subsequent passage to a final concentration of 6.4pM.
  • GR clones, CL75, CL86 and CL131 were maintained in 6.4pM gefitinib thereafter.
  • A549 and A549(+) were cultured in DMEM (Invitrogen, USA) supplemented with 10% foetal bovine serum.
  • A549(+) were derived from A549 parental cells by magnetic bead separation with a mouse anti-CEACAM6 antibody and CELLectionTM Pan Mouse IgG Kit.
  • H1299 is a cell line with no endogenous expression of the antigen for GR 6D11. Plasmids containing the CEACAM5 or CEAC AM6 open reading frame cDNA, or CEACAM6 mutant constructs, was transfected into the cells using LipofectamineTM (Invitrogen, USA), and selected for stable expression by antibiotic selection.
  • Cells were harvested as single cell suspensions using trypsin. 1E5 cells were used per sample, and incubated with lOOpl of mAb culture supernatant for 30 min. Cells were then washed with 1% bovine serum albumin in PBS, and further incubated with lOOul of goat anti-mouse antibody fluorescein isothiocyanate (FITC)-conjugated (1 :500, DAKO, Denmark) for 15 min at 4°C in the dark. Cells were again washed and resuspended in 200pL of 1% BSA/PBS for analysis on MACSQuantX (Milteny Biotec, Germany).
  • FITC goat anti-mouse antibody fluorescein isothiocyanate
  • Membrane proteins were extracted from cell pellets using the Membrane Protein Extraction Kit (BioVision, USA). Briefly, cell pellets of 5E7 cells were resuspended in ImL of Homogenize Buffer and cell membranes broken in a dounce homogenizer. This as transferred into an Eppendorf tube and centrifuged at 700 x g for 10 min at 4°C to remove cell debris. The supernatant was transferred to a new' Eppendorf tube and centrifuged at 12,000 x g for 30 min at 4°C to pellet the membrane.
  • the membrane was finally resuspended in 500pl of lx Cell Lysis Buffer (Cell Signaling Technology, USA) containing protease inhibitors (Pierce ThcrmoScicntific, USA).
  • the membrane protein solution was clarified with by centrifugation at 15,000 x g for 5 min at 4°C, to remove any insoluble proteins. Protein was quantified using the Pierce 660nM Protein Assay Reagent.
  • IP Immunoprecipitation
  • Cell membrane protein extracts, or IP products were denatured by in protein loading dye containing SDS at a final concentration of 1%, and heated at 95°C for 5 min.
  • the sample was then loaded into pre-cast gradient gel (NuPAGE 4-12% gradient gel, Invitrogen), and separated by SDS-PAGE running MOPS Running Buffer (NuPAGE Invitrogen, USA).
  • the resolved proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (BioRad, USA) in a transfer buffer containing 20% methanol, 10% Tris-Glycinc in DI water at constant voltage of 110V for 90 min.
  • PVDF polyvinylidene fluoride
  • the membrane was then blocked with 5% milk prepared in PBS/0.1% Tween-20 (PBS-T) for 30 min at room temperature.
  • the membrane was then washed in PBS-T, followed by overnight incubation of GR 6D11 at 2pg/mL in 2.5% milk at 4°C. Subsequently, the membrane was washed in PBS-T, before incubation with goat anti-mouse secondary antibodies horseradish peroxidase-conjugated (1: 10000, Dako) for 1 hour at room temperature. After a final wash with PBS-T, the binding of HRP-conjugatcd secondary antibodies was visualized by ECL detection (GE Healthcare, Sweden).
  • Antibody affinity to antigen proteins was measured by Octet® Bio-Layer Interferometry (BLI).
  • Avi-tag biotinylated CEACAM1, 5, 6 and 8 proteins (ACROBiosystems, USA) were immobilized onto Octet® SA biosensors, and dipped into antibody solutions of varying concentrations to obtain the association and dissociation curves.
  • Binding kinetic parameters, KD, kon and koff were calculated by the Octet® Data Analysis Version 7.1 , based on a 1 : 1 model.
  • Antibody affinity to cells was measured by flow cytometry based EC50. Briefly, cells were harvested as single cell suspensions using trypsin. 1E5 cells were used per sample, and incubated with lOOpl of antibody ranging from lOpg/mL to 0.0195pg/mL for 30 min at 4°C. Cells were then washed with 1% bovine serum albumin in PBS, and further incubated with lOOpl of goat anti-mouse antibody fluorescein isothiocyanate (FlTC)-conjugated for 15 min at 4°C in the dark.
  • FlTC goat anti-mouse antibody fluorescein isothiocyanate
  • TMA slides containing FFPE tissues were first heated in an oven at 60°C for 30 min to remove any solvents. The slides were then dewaxed and re -hydrated through sequential immersion in Histoclear (2x), 100% ethanol (2x), 95% ethanol, 70% ethanol, and finally in DI water.
  • Heat-induced epitope retrieval was done in a solution containing lOmM Tris Base, ImM EDTA, 0.05% Tween 20 at pH 9.0, and heated at 95°C for 20 min.
  • the container with the antigen retrieval solution and slides was then removed and allowed to cool to room temperature for an additional 20 min.
  • the slides were then washed in DI water. Endogenous peroxidase activity was then blocked by incubation of the slides with 3% H2O2 in PBS for 30 min at room temperature.
  • the slides were washed in DI water, followed by a blocking step with 10% normal goat serum in PBS for 30 min.
  • the slides were then incubated with GR 6D1 1 at 0.5pg/mL in blocking solution overnight at 4°C.
  • the slides were then washed an incubated with a polymer-based anti-mouse secondary antibody conjugated with HRP (DAKO, USA) for 30 min at room temperature, and developed with the recommended DAB chromogen substrate solution for 2 min, and counterstained with Gill’ s Hematoxylin solution.
  • the stained slides were subsequently dehydrated through immersion in 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol (2x) and Histoclear (2x), before mounting with a glass cover slip.
  • the slides were then imaged with the Zeiss AxioScan Digital Slide Scanner. Images were scored using the ImmunoMembrane ImageJ plugin.
  • Cells were seeded into a black coated 96 well plate (Grenier Bio-one, UK) at a range of density from 1000-5000 cells per well (depending on the cell type used). The plate was then incubated for 24 hours at 37oC in humidified air with 5% CO2. After 24 hours, antibody and the anti-mouse secondary conjugated to MMAE (Moradec LLC, USA) or saponin (Advanced Targeting Systems, USA) to was added to each well and the plate was again placed at 37oC in humidified air with 5% CO2. 4 days after addition of mAb or buffer, lOOpL of CTG substrate (Promega, Wisconsin, USA) was added to each well. The plate was then left in the dark for 10 minutes, with vigorous shaking. The cell growth of the samples was then quantified using Tecan I-control (Tecan, Switzerland).
  • the different CAR constructs was cloned into pcDNA3.1(+) plasmids.
  • In vitro transcription of linearised templates was performed using the HiScribeTM T7 ARC A mRNA kit according to manufacturer’s instructions (NEB, USA) to generate CAR mRNA and stored at -80°C.
  • Nucleofection of T cells was performed on a 4D-NucleofectorTM System using the P3 Primary Cell 4D-NucleofectorTM Kit according to manufacturer’s protocol (Lonza, Switzerland), and the transfected T cells used immediately for functional assays.
  • Shortlisted CAR constructs were synthesised into third-generation lentiviral CAR vectors by Vector Builder, USA.
  • T cells were cultured in TexMACSTM Medium (Miltenyi Biotec, Germany) supplemented with IL-7 (20 U/mL), IL-15 (10 U/mL) and IL-21 (0.04 U/mL), and activated with anti-CD3/CD28 Dynabeads for 3 days before transduction.
  • Lentiviral CAR vectors were then added to T cells at a multiplication of infection (MOI) of 10, before undergoing two rounds of spinoculation at 1200 x g, 37°C for 2 hours.
  • CAR-transduced T cells were harvested and maintained in standard culture.
  • CAR T cells were co-incubated with target cells at E:T of 10:1 for 6 hours at 37°C, 5% CO2.
  • Cell supernatants were collected and the cytokine analysed using the MACPlex Cytokine 12 kit (Miltenyi Biotec) according to manufacturer’s protocol on the MACSQuant Analyzer X.
  • NIKO mice NOD-scid IL-2RY-Knock-Out mice were used for the in vivo studies to assess the efficacy of GR6D11 CAR T cells.
  • NIKO mice were generated by knocking-out IL-2R gamma chain with CRISPR/cas9 technology in NOD-scid mice (A*STAR, Singapore).
  • tumour growth inhibition ratio was calculated using the following equation:
  • GR 6D11 is a mouse lgG2a K monoclonal antibody that recognizes CEACAM1, CEACAM5 and CEACAM6 at the conserved Ig-like V-type domain (Domain 1) on human cells, with the protein sequence encoding the variable heavy and light antibody chains defined in the supplementary document.
  • GR 6D1 1 is highly selective to the tumour antigen target and does not bind to normal cells, both in flow cytometry assays, or formalin fixed paraffin embedded (FFPE) tissue arrays.
  • FFPE formalin fixed paraffin embedded
  • GR 6D11 demonstrates strong binding for several cancer indications in both flow cytometry and FFPE samples.
  • flow cytometry GR 6D11 showed strong binding to a subset of clones that has acquired drug resistance to gefitinib, as compared to low binding on the parental gefinitib sensitive parental lines. This makes it useful not only as a novel general cancer biomarker, but potentially a means of diagnosis for acquisition of drug resistance, and identifying the mechanism of resistance.
  • GR 6D11 On FFPE tissue arrays, GR 6D11 exhibited positive reactivity to 6% lung adenocarcinoma tissues (63 out of 99), 30% lung squamous cell carcinoma tissues (52 out of 170), 41% other adenocarcinoma tissues, such as gastric and colorectal (7 out of 17), 30% other squamous cell carcinoma tissues, such as oesophagus and uterus (3 out of 10), and 78% invasive ductal/lobular carcinoma (7 out of 9), 0% cancer adjacent and normal tissues (0 out of 147).
  • A549 cells were sorted into two populations: CEACAM6(+) and CEACAM6(-).
  • GR 6D1 1 can potentially be used as an antibody drug conjugate. Indirect conjugation of GR 6D11 with the ribosome toxin, saporin (mAb-ZAP), demonstrated killing of about 50% after 72-hour treatment on A549-CEACAM6(+) cells.
  • GR 6D11 can also be potentially used as a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the VH and VL domain sequences for GR 6D11 were used to generate the single-chain variable fragment (scFv) antigen-recognition domain for a 2nd-generation CAR, using the CD3g domain for stimulation, and either CD28 or 4-1BB for co-stimulation.
  • GR 6D11 CARs were transfected into T-cell using cither mRNA or 3rd-gcncration lentiviral vectors.
  • GR 6D11 CAR T-cells with variant spacer lengths were co-incubated with target cell tumour cells, with all constructs demonstrating efficient cytotoxicity kinetics, suggesting good immune synapse formation.
  • Dose-response titrations revealed the sensitivity of GR 6D11 CAR T-cell, which were capable of depleting target PC-9 cells within 48 hours at an effector- to-target ratio of 2:1 minimally.
  • GR 6D11 CAR T-cells also secrete inflammatory cytokines in response to the recognition of target cells.
  • This inflammatory cytokine secretion profile of GR 6D11 CAR T-cells supports their anti-tumour functionality.
  • GR 6D11 CAR T-cells controlled the tumour growth of A549- CEACAM6(+) tumour-bearing mice.
  • GR 6D11 CAR T-cells reduced the growth rate of tumour xenografts and was capable in some cases of mediating long-term tumour control.
  • GR 6D1 1 CAR T-cells prolonged mouse survival, with mice treated with GR 6D11 CAR T-cells surviving for a median of 61 day compared with 19 days for mice in the control group.
  • the sequence for the mouse GR6D1 1 antibody has also been humanised, and 4 variable heavy chain sequences, and 2 variable light chain sequences were identified that will be used for development in the final therapeutic format.
  • VH7 Hu humanised VH
  • CDRs underlined VH7 Hu
  • VH7 K humanised VH
  • CDRs underlined CDRs underlined
  • VH7 C humanised VH (CDRs underlined)
  • VK1 humanised VL (CDRs underlined)
  • VK3 humanised VL
  • CDRs underlined VK3
  • PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16

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Abstract

La présente invention concerne, d'une manière générale, des molécules de liaison à l'antigène. En particulier, la présente invention concerne des molécules de liaison à l'antigène qui se lient spécifiquement à CEACAM1, CEACAM5 et CEACAM6.
PCT/SG2025/050382 2024-06-05 2025-06-05 Molécules de liaison à l'antigène anti-ceacam Pending WO2025254591A1 (fr)

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US20210095024A1 (en) * 2019-09-27 2021-04-01 Janssen Biotech, Inc. Anti-ceacam antibodies and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210095024A1 (en) * 2019-09-27 2021-04-01 Janssen Biotech, Inc. Anti-ceacam antibodies and uses thereof

Non-Patent Citations (3)

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Title
CHAOGU ZHENG, JING FENG, DI LU, PING WANG, SHU XING, JEAN-LUC COLL, DONGLING YANG, XIYUN YAN: "A Novel Anti-CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity", PLOS ONE, vol. 6, no. 6, pages e21146, XP055145536, DOI: 10.1371/journal.pone.0021146 *
HOLLANDSWORTH HANNAH M., AMIRFAKHRI SIAMAK, FILEMONI FILEMONI, SCHMITT VERENA, WENNEMUTH GUNTHER, SCHMIDT ALEXEJ, HOFFMAN ROBERT M: "Anti-carcinoembryonic antigen-related cell adhesion molecule antibody for fluorescence visualization of primary colon cancer and metastases in patient-derived orthotopic xenograft mouse models", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 11, no. 4, 28 January 2020 (2020-01-28), United States , pages 429 - 439, XP055854596, ISSN: 1949-2553, DOI: 10.18632/oncotarget.27446 *
JANCEWICZ IGA, ŚMIECH MAGDALENA, WINIARSKA MAGDALENA, ZAGOZDZON RADOSLAW, WISNIEWSKI PAWEL: "New CEACAM-targeting 2A3 single-domain antibody-based chimeric antigen receptor T-cells produce anticancer effects in vitro and in vivo", CANCER IMMUNOLOGY, IMMUNOTHERAPY, SPRINGER, BERLIN, DE, vol. 73, no. 2, Berlin, DE , XP093293134, ISSN: 1432-0851, DOI: 10.1007/s00262-023-03602-4 *

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