WO2025254591A1 - Anti-ceacam antigen-binding molecules - Google Patents

Abstract

The present invention relates, in general terms, to antigen-binding molecules. In particular, the present disclosure relates to antigen-binding molecules that specifically bind to CEACAM1, CEACAM5 and CEACAM6.

Description

ANTI-CEACAM ANTIGEN-BINDING MOLECULES

Technical field

The present invention relates, in general terms, to antigen-binding molecules. In particular, the present disclosure relates to antigen- binding molecules that specifically bind to CEACAM1, CEACAM5 and CEACAM6.

Background

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).

While 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.

It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

Summary

Disclosed herein is 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).

Disclosed herein is 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).

Disclosed herein is 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.

Disclosed herein is a construct comprising a polynucleotide as defined herein in operable connection with one or more control sequence.

Disclosed herein is a host cell that contains the construct as defined herein

Disclosed herein is a composition comprising an antigen-binding molecule as defined herein and a pharmaceutically acceptable carrier.

Disclosed herein is a chimeric antigen receptor (CAR) comprising an antigen- binding molecule as defined herein, a transmembrane domain, one or more co- stimulatory domains and an intracellular signaling domain.

Disclosed herein is an isolated polynucleotide encoding a CAR as defined herein.

Disclosed herein is a vector comprising the polynucleotide as defined herein.

Disclosed herein is an engineered cell comprising the vector as defined herein. Disclosed herein is a method of preparing an engineered immune cell, the method comprising introducing the vector as defined herein into an immune cell.

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.

Disclosed herein is 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.

Disclosed herein is the use of 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.

Disclosed herein is a method for detecting cancer in a subject, the method 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.

Disclosed herein is a method for identifying a subject susceptible to cancer the method 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.

Disclosed herein is a kit for use in the method as defined herein, comprising an antigenbinding molecule as defined herein, together with instructions for use.

Brief description of the drawings

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

Figure 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 Pierce™ Rapid Antibody Isotyping Kit. The putative sequences for the constant heavy and light chain sequences are as shown, with the Uniprot ID referenced.

Figure 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. Cross-probe of the IP done (A) using a commercial anti-CEACAM5 antibody and GR 6D11 and (B) using a commercial anti-CEACAM6 antibody and GR 6D11 demonstrated that GR 6D11 binds both proteins. (C) This was further validated by the overexpression of CEACAM5 and CEACAM6 in the Hl 299 lung cancer cell line (with no endogenous expression), whereby GR 6D11 was shown to bind on the subpopulation with the overexpressed protein. (D) The affinity for GR 6D11 for the CEACAM5 and CEACAM6 protein as measured by Octet® BLI system are 3.78E-10 M and 2.64E-10 M respectively. (E) Cell-based binding EC50 was measured by flow cytometry on two cell lines, PC-9 and A549(+) cells to be 1.37nM and 1.86nM respectively. (F) Cross-reactivity to other members of the human CEACAM family proteins was tested on the Octet® BLI system, and GR 6D11 was found to also bind to CEACAM 1, but not to CEACAM8. (G) From alignment of CEACAM1, 5, 6 and 8, it was postulated that the epitope for GR 6D11 resides in Domain 1 (Ig-like V-type) of CEACAM6 due to higher homology for CEACAM 1, 5 & 6 (binding proteins), but different in CEACAM8 (non-binding). Mutants of CEACAM6 and CEACAM8 was constructed whereby Domain 1 was swapped, and the constructs were expressed in H1299 cells as previously done. The loss of GR 6D11 binding when Domain 1 in CEACAM6 was replaced by the sequence in CEACAM8 (C8Doml-CEACAM6), and the gain of GR6D11 binding with the converse swap of Domain 1 from CEACAM6 into CEACAM8 (C6Doml-CEACAM8) confirmed that GR 6D11 binds at Domain 1 of CEACAM6. (H) 7 amino acid residues were identified that were identical for CEACAM1, 5 & 6, that were different in CEACAM8, and constructs bearing the individual point mutations were made on the CEACAM6 wildtype sequence. Binding of GR 6D11 was completely lost for the A89I CEACAM6 mutant construct, and likely is part of the binding epitope. Commercial anti-CEACAM6 antibody, 9A6, was used as positive control for expression.

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. (L) As CEACAM1 & 6 are known to be expressed in neutrophils, 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. (M) Antigen density was measured using the QIFIKIT® for neutrophils and a variety of cancer cell lines with varying binding intensities. Congruent to the flow cytometry binding data, neutrophils have the lowest number of antigen sites, which is at least half from the lowest binding cancer cell lines, confirming GR 6Dll’s added specificity to cancer- associated CEACAM 1/5/6.

Figure 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. (C to F) In contrast, immunohistological staining with 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%). Importantly, GR 6D11 is again shown to be specific to cancer tissue, with only 2% of cancer adjacent lung normal tissue stained positive.

Figure 6: GR 6D11 exhibited cancer cell cytotoxicity as an antibody drug conjugate As a proof-of-concept, 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.

Figure 7: GR 6D11 functions as an effective CAR T therapeutic in in-vitro and in-vivo lung cancer models (A) The variable light (VL) and variable heavy (VH) regions for GR 6D11 was cloned into mRNA constructs bearing the C 3L, and CD28 downstream signalling domains for T cell activation with varying linker length. 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. (B) The short linker was tested in two lentiviral constructs bearing the CD3C, and CD28 or the CD3^ and 4- IBB downstream signalling domains, and transduced into T cells to generate stable expressing CAR T cells. For both constructs, a dose-dependent response was observed when PC -9 target cells were treated in-vitro with CAR T effector cells, but the one harbouring the CD28 domain mediated a faster and stronger cytotoxic response than 4- 1BB CARs, especially evident in lower E:T ratios. (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. Briefly, 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. As 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. Of note, complete regression was observed for 2 of 5 mice in the CAR(GR6D11) treatment arm after Day 27, but relapse was observed at the end of the study. The treatment was well tolerated, with no significant drop in body weight throughout the study. (F) Summary of the survival statistics at the end of the study on Day 90 post-treatment, showing statistically significant improvement in survival when the tumour was treated with GR 6D11 CAR T cells.

Figure 8: Characterisation of humanised GR 6D11 antibodies (A) 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. Three sequences for the variable heavy chain region - VH7_Hu, VH7_K & VH7_C, and two sequences for the variable light chain region - VK1 & VK3, were tested and characterised against the mouse parental GR 6D11 antibody. (B) Binding EC50 of the mouse parental GR 6D11, chimeric GR 6D11 (chGR6Dl 1) and 6 combinations of the humanised antibodies by flow cytometry was measured against SNU-16 (antigen-positive) and Hl 299 (antigen-negative). Three of the humanised antibodies, VH7Hu_VKl, VH7C_VK1 & VH7K_VK1, showed comparable binding to SNU-16 as the parental GR 6D11 and chGR6Dll antibodies. The remaining three antibodies, VH7Hu_VK3, VH7C_VK3 & VH7K_VK3, had significantly decreased maximal binding (Bmax), and were not selected to progress for further characterisation. All the tested antibodies retained their antigen specificity and did not show any non-specific binding to antigen-negative H1299 cells. Affinity to (C) CEACAM5 and (D) CEACAM6 proteins were measured by Octet® BLT system for the three selected humanised antibodies and were found to be within 3-fold of chGR6Dll. Hence, all three antibodies are selected to be potential humanised candidates for further pre-clinical development and characterisation. Figure 9: Affinity tuning for hGR6Dll as scFv. (A) The mouse sequence and humanised constructs were also made as his-tagged scFvs to test binding in a more relevant format for CAR constructs. In addition, an additional humanised sequence based on the VH1 framework and shorter CDR definitions (underlined) was designed and tested to obtain potentially lower affinity constructs. The light chain sequence was kept as VK1 version throughout for consistency in comparison. (B) Binding of the scFv protein to Hl 299 cells overexpressing CEACAM6 wild-type and CEACAM6 A89I mutant was evaluated by flow cytometry to ensure epitope specificity was retained for the constructs. Binding of the scFv to the A89I mutant was reduced compared to the CEACAM6 wild-type sequence for all 5 constructs tested, showing that they retained epitope specificity of the parental GR6D11 mAb after humanisation and conversion to the scFv format. Flow cytometry binding to SNU- 16 was evaluated, and the scFv constructs retained strong binding to the gastric cancer cell line, although the new humanised sequence VH1-VK1 showed a significantly lower binding nMFI compared to the other 4 molecules. Affinity to (C) CEACAM5, (D) CEACAM6 and (E) CEACAM1 proteins were measured by Octet® BLI system for the five scFv 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.

Detailed description

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. In another embodiment, the antigen-binding molecules specifically binds CEACAM5 and CEACAM6, but not CEACAM1.

Provided herein is 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).

Without being bound by theory, 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. Importantly, on human tissue arrays, 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. 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.

In one embodiment, the antigen-binding molecule binds to CEACAM5 and CEACAM6 with picomolar affinity. In one embodiment, the antigen-binding molecule further cross-reacts with CEACAM1.

In one embodiment, there is provided 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).

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).

In another embodiment, 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).

In another embodiment, 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).

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. In some embodiments, the antigen-binding molecules comprise an Fc domain. In other embodiments, the antigen-binding molecules lack an Fc domain. In some embodiments, the antigen binding molecules are monovalent antigen-binding molecules (e.g., Fab, scFab, Fab’, scFv, one-armed antibodies, etc.).

By “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.

In some embodiments, 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). In one embodiment, 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).

In one embodiment, the antigen-binding molecule does not bind to CEACAM8.

In an embodiment, 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.

The term “antibody”, as used herein, 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. 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). 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. In some embodiments, 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. Whilst it is generally desirable that 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, in some embodiments, 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. Depending on the antibody amino acid sequence of the constant region of its heavy chains, 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.

In one embodiment, 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). In some instances, the antigen-binding molecules may have mutations such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, 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).

As used herein, the term “complementarity determining regions” (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. 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.. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (i.e., about residues 26-32 (LI ), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, 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.

In some embodiments, the antigen-binding molecule specifically binds to human

CEACAM1, CEACAM5 and CEACAM6. The CEACAM1 may have a sequence with a

Uniprot Identification Number of P13688. The CEACAM5 may have a sequence with a

Uniprot Identification Number of P06731. The CEACAM6 may have a sequence with a

Uniprot Identification Number of P40199.

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. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). 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.

The terms “antigen-binding fragment”, “antigen-binding portion”, “antigen-binding domain” and “antigen-binding site” are 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. Such 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. Other 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.

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. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplar}' configurations of 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_H2, (X) V_L-C_H3; (xi) V_L-C_Hl-C_H2; (xii) V_L-CH1-C_H2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the 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. Moreover, 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.

The term “variable region” or “variable domain” 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.

The term “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.

In one embodiment, the antigen-binding molecule or antigen-binding fragment thereof is modified for compatibility with the target species. Thus, in an embodiment, the antigenbinding molecule or antigen-binding fragment thereof is humanized.

By “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. In an embodiment, 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. When using one or more 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. Thus, under designated immunoassay conditions, 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. A variety of immunoassay formats may be used to select antigen -binding molecules (e.g., immunoglobulins) such that they arc specifically immunoreactive with a particular antigen. For example, 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)).

“Affinity” or “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_on and k_ou, respectively). Thus, 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).

The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. 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.

As used herein, the term “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. In addition, the term “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.

In one embodiment, 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).

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). 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).

The term “sequence identity” as used herein 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. Thus, 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:

Amino Acid Subclassification

Conservative amino acid substitution aiso includes groupings based on side chains. For example, 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. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity.

Conservative substitutions arc also shown in the tabic below. 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.

Exemplary and Preferred Amino Acid Substitutions

In one embodiment, 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).

In one embodiment, 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.

In some embodiments, 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. 85(16), 5879-83), the 18-mer GGSSRSSSSGGGGSGGGG (SEQ ID NO: 40) (Andris-Widhopf et al., “Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.” Cold Spring Harbor Protocols, 2011(9)) and the 20-mer (G4S)4 (Schaefer et al., “Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.” In: Antibody Engineering, R. Kontermann and S. Diibel, Springer Verlag, Heidelberg, Germany (2010) pp. 21-44). Many other sequences have been proposed, including 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. coll, insect cells or mammalian host cells upon cloning of the protein coding sequence for the scFv in the context of appropriate expression vectors with appropriate translational, transcriptional start sites and, in the case of mammalian expression, a signal peptide sequence.

In one embodiment, the monovalent antigen-binding molecule comprises an Fab fragment. In an illustrative example of this type, 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.

Recombinant expression of 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. J Mol Biol. 270(l):26- 35; as described in WO 2005/063816). These KTH mutations promote heterodimerization of the knob containing Fc and the hole containing heavy chain, improving the assembly of monovalent antibody and reducing the level of undesired bivalent antibody.

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. For example, 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). For human lgG4, engineering mutations S228P/L235E valiant (SPLE) has previously demonstrated minimal Fc/R bin ing (Newman et al., 2001, Clin. Immunol. 98, 164-174). Mutations in IgGl or IgG4 Fc domains can be combined, for instance combining the LALA mutations in human IgGl with a mutation at P329G or combining the SPLE mutation in human IgG4 with a mutation at P329G, completely abolished FcyR and Clq interactions (Schlothauer el al., 2016, Protein Eng Des. Sei. 29, 457-466).

In one embodiment, the antigen- binding molecule (e.g., a MAb or an antigen- binding fragment thereof), in which 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.

In one embodiment, 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).

In one embodiment, the present invention contemplates monovalent antigen-binding molecules produced by co-cxprcssion of a light chain, heavy chain and a truncated Fc domain. Suitably, the heavy chain incorporates hole mutations and P329G LALA mutations, while the truncated Fc domain incorporates knob mutations and P329G LALA mutations.

Expression of the 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.

In one embodiment, 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-scFv, scFv-IgG, scFv₂-Fc, F(ab')2-scFv2, scDB-Fc, SCDF-CH3, Db-Fc, scFv2-H/L, DVD-Tg, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, dAb-Fc-dAb, tetrabody, streptabody (scFv- streptavidin)4, (scFv-p53)4, [sc(Fv)2]2; tandem diabody (tandab) and combinations thereof.

In one embodiment, 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; Ts2Ab, Medimmune/AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idee; TvAb, Roche) Fc fusions (e.g., scFv/Fc fusions, Academic Institution; SCORPION, Emergent BioSolutions/Trubion, ZymoGenetics/BMS; dual affinity retargeting technology (Fc- DART), MacroGenics; dual (ScFv)2-Fab, National Research Center for Antibody Medicine) Fab fusions (e.g., F(ab)2, Medarex/AMGEN; dual-action or Bis-Fab, Genentech; Dock-and- Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (e.g., bispccific T cell engagers (BiTEs), Micromct; tandem diabodies (Tandab), Affimed; DARTs, MacroGenics; Single-chain diabody, Academic; TCR-like antibodies, AIT, Receptor Logics; human serum albumin scFv fusion, Merrimack; and COMBODIES, Epigen Biotech), IgG/non-IgG fusions (e.g., immunocytokins, EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantigen fusion protein, Active Biotech; and immune mobilizing mTCR Against Cancer, InunTAC) and oligoclonal antibodies (e.g., Symphogen and Merus).

In one embodiment, the antibody is a bispccific or trispccific antibody. In one embodiment, the antibody is a bispecific antibody.

In one embodiment, 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). Where 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. For further description of 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. In some embodiments, the heteromultimer formed is a multimeric antibody, for example a bispecific antibody.

Disclosed herein is a method for preparing an antigen-binding molecule that specifically binds CEACAM1, CEACAM5 and/or CEACAM6, the method comprising: a) immunizing an animal (e.g. a mouse or rabbit) with a peptide of any one of SEQ ID NO: 30-38, b) isolating from the animal a B-cell that binds specifically to the peptide, and c) determining the amino acid sequence of the antibody that is expressed by the B-cell.

Disclosed herein is a method for preparing 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.

In one embodiment, the antigen- binding molecule is a chimeric molecule that is conjugated to a heterologous moiety.

As used herein, 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. For example, a peptide and a nucleic acid sequence, a peptide and a detectable label, unrelated peptide sequences, and the like. In embodiments in which 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. For example, 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.

In one embodiment, 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. For example, the enzyme may catalyze a chemical alteration of the chromogenic substrate that can be measured using the various techniques. For example, the example may catalyze a color change in a substrate, which can be measured spectrophoto metrically. Alternatively, 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. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), 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.

Examples of enzyme- substrate combinations include, for example:

1) Elorseradish peroxidase (HRPO) utilizes hydrogen peroxide to oxidize a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));

2) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and

3) 0-D-galactosidase (0-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-P-D- galactosidasc) or Anorogenic substrate 4-mcthylumbcllifcryl-|3-D-galactosidasc. In another embodiment of the invention, 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.

In one embodiment, 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. In some embodiments, 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. In some embodiments, a heterologous moiety can include an attachment site for a nonpolypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES),

In one embodiment, 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).

Disclosed herein is an isolated polynucleotide comprising a nucleic acid sequence encoding the antigen-binding molecule as defined herein. In one embodiment, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the chimeric antigen receptor as defined herein.

The term “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.

By “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. Accordingly, 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. Alternatively, 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.

Disclosed herein is a construct comprising a polynucleotide as defined herein in operable connection with one or more control sequences. The term “construct” refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, 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. Representative 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. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, 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. For the practice of the present invention, 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.

By “control element”, “control sequence”, "regulatory sequence" and the like, as used herein, mean a nucleic acid sequence (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The 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.

Disclosed herein is a host cell that contains the construct as defined herein.

The terms “host”, “host cell”, “host cell line” and “host cell culture” are used interchangeably and refer 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.

Provided herein is a chimeric antigen receptor (CAR) 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. Optionally, 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. In some embodiments, the CAR is expressed on a mammalian cell such as a T-cell, a tumor infiltrating lymphocyte (TIL) or a CAR NK cell. 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.” In some embodiments 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. When used clinically in, c.g. adoptive cell transfer, 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. Upon specific binding a biochemical chain of events (i.e., signal transduction) results in modulation of the immunological activity of the CAR-T. Thus, for example, upon specific binding by the antigen binding region of the CAR-T to its target antigen can lead to changes in the immunological activity of the T-cell activity as reflected by changes in cytotoxicity, proliferation or cytokine production. Signal transduction upon CAR-T activation is achieved in some embodiments by the CD3-zeta chain (“CD3-z”) which is involved in signal transduction in native mammalian T-cells. CAR-Ts can further comprises multiple signaling domains such as CD28, 41BB or 0X40, to further modulate immunomodulatory response of the T-cell. CD3-z comprises a conserved motif known as an immunoreceptor tyrosinebased activation motif (IT AM) which is involved in T-cell receptor signal transduction.

In one embodiment, 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. In one embodiment, 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.

In one embodiment, 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.

Provided herein is an isolated polynucleotide encoding a CAR as defined herein.

Also provided herein is a vector comprising the polynucleotide as defined herein.

Also provided herein is 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.

Disclosed herein is a pharmaceutical composition comprising an antigen-binding molecule as defined herein or a chimeric molecule as defined herein.

By “pharmaceutically acceptable carrier” is meant 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. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered intravenously, subcutaneously or intramuscularly. In some embodiments, the compositions are in the form of injectable or infusible solutions. A preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In specific embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In other embodiments, the pharmaceutical composition is administered by intramuscular or subcutaneous injection.

The phrases “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. Examples of 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. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral 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.

More particularly, pharmaceutical 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. In such cases, 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. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin and/or by the maintenance of the required particle size. In specific embodiments, an agent of the present disclosure may be conjugated to a vehicle for cellular delivery. In these embodiments, 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. As will be appreciated by a skilled artisan, a variety of vehicles arc suitable for delivering an agent of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating agents of the present disclosure into delivery vehicles are known in the art. Although various embodiments are presented below, it will be appreciated that other methods known in the art to incorporate an antigen-binding molecule, as described herein, into a delivery vehicle are contemplated.

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.

It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein 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.

Dosages and therapeutic regimens of the antigen-binding molecule can be determined by a skilled artisan. In certain embodiments, 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.

It is to be noted that 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.

Disclosed herein is 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.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. By “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. The term "metastatic cancer" refers to cancer that has spread or is capable of spreading from one part of the body to another. Generally, 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, on the other hand, is usually a stage IV cancer.

The term "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, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof.

In one embodiment, the cancer cell is a solid or haematological cancer cell.

The term “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 adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer, sarcomas or a combination thereof.

The term “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. In some embodiments, 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. In some embodiments, 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.

In one embodiment, the cancer is a metastatic cancer. The cancer may be a refractory or a relapsed cancer.

In one embodiment, 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. Disclosed herein is 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.

The term “treating” as used herein 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.

The terms “subject”, “patient”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable 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 chimpanzees (Pan troglodytes , rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In one embodiment, the subject is a human subject.

In one embodiment, 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. Alternatively, the cancer may be positive for or overexpresses CEACAM1 and CEACAM5, CEACAM1 and CEACAM6, or CEACAM5 and CEACAM6. Alternatively, 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. As used herein the term “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 experimentation.

Disclosed herein is the use 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, in the manufacture of a medicament for treating or preventing cancer.

Disclosed herein is a method for detecting cancer in a subject, the method 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.

Disclosed herein is a method for identifying a subject susceptible to cancer, the method 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. For monitoring cancer progression, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, the antigen-binding molecule comprises a detectable label.

Disclosed herein is a kit for use in the method as defined herein, comprising an antigenbinding molecule as defined herein, together with instructions for use.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary' skill in the art to which this invention belongs.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

Materials and Methods

Culture of cancer cell lines, and generation ofGefitinib resistant & protein over-expression lines

PC -9, H1299 and SNU-16 were cultured in RPMI (Invitrogen, USA) supplemented with 10% foetal bovine serum (HyClone GE Healthscience, South America).

To obtain PC-9 clones with acquired gefitinib resistance, 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 CELLection™ 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.

Generation of GR mAb panel Immunisation of PC-9 GR lines, CL75, CL86 and CL131 , was done with 5E6 cells resuspended 1: 1 with Fraund’s complete adjuvant. Immunisation was done once per week for the first immunisation in week 1-3 with only a single line each, and a mixed suspension of all three lines for the subsequent immunisations in week 4-5. After 5 weeks, mice were sacrificed, and the B-cclls collected for fusion with SP2/0 mouse myeloma lines using STEMCELL Technologies ClonaCell™-HY kit as per manufacturer’s instructions. Single hybridoma clones were picked into 96-wells, and the culture supernatant collected for screening by flow cytometry.

Flow cytometry

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). For interrogation of intercellular binding, cells were fixed with 4% PFA/PBS (Affymetrix, USA) at room temperature for 10 min, washed in PBS, and pcrmcabiliscd with 0.1% triton/PBS for 5 min at room temperature, before proceeding with incubation with mAb supematant/primary antibody. For staining with propidium iodide, PI was added to a final concentration of 5pg/mL for 5 min just prior to analysis by the flow cytometer.

Western blot and immunoprecipitation

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.

Immunoprecipitation (IP) was conducted using the automated Phynexus MEA system (Phyncxus Inc., USA). GR 6D11 was captured onto Protein G PhyTip columns containing 5ul of resin bed. The column was then washed with PBS to remove unbound proteins, and cell membrane protein extract was introduced to bind to GR 6D11 that has been captured on the column. The column was then washed with Wash Buffer 11 ( 140mM NaCl, pH7.4) before elution at low pH with Elution Buffer (200 mM NaH2PO4/140 mM NaCl pH 2.5) and neutralized immediately with 1 M Tris-Cl pH 9.0. The IP product is then subjected to analysis by Western blot.

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). After gel electrophoresis, 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.

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 measurements

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. Cells were again washed and resuspended in 200pL of 1% BSA/PBS for analysis on MACSQuantX (Miltenyi Biotec, Germany). Mean fluorescence index was plotted against antibody concentration, and binding curve was fitted to a four-parameter logistic equation to obtain the EC50 and maximal signal, Bmax using GraphPad Prism 10.

Immunohistochemistry staining

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.

Indirect antibody drug conjugate, assay

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).

Generation of CAR T cell lines

For the transient mRNA nucleofection of T-cells, 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. To prepare T cells for transduction, 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.

In vitro assays for CAR T functionality characterisation

For measurements of cell cytotoxicity from CAR T treatment, real-time cell growth for adherent target cell lines was monitored on the xCELLigence RCPA MP Instrument (ACEA Biosciences). Target cells were seeded into E- Plates and allowed to expand for 24 hours before transfected or transduced CAR T cells were spiked in at various effector-to-target (E:T) ratios. Cell indexes were normalised to the spike-in timepoint, and growth curve tracked for 72 hours post-treatment.

For measurements of cytokine release profiles, 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.

In vivo studies for CAR T functionality assessment

NIKO (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).

A549(+) cells were introduced to NIKO mice via subcutaneous injection into the right flank under general anaesthetic to form tumour xenografts. Mice tumour volumes were measured twice a week. When mean tumour volumes reached 100mm3, 5 million CAR T cells were administered via tail vein injection. Mice were culled when their tumour volume exceeded 2000 mm3, when the tumour site was severely ulcerated, or when severe, persistent weight loss was observed. The tumour growth inhibition ratio (TGI%) was calculated using the following equation:

Where: C = Mean control tumour volume T = Mean treated tumour volume

Animals were handled according to A*STAR (BRC) IACUC Protocol No.: 151001, in accordance with the National Advisory Committee for Laboratory Animal Research (N ACLAR) guidelines.

EXAMPLE 1

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.

Conversely, GR 6D11 demonstrates strong binding for several cancer indications in both flow cytometry and FFPE samples. In 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.

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(-). It was found in literature that A549-CEACAM6(+) cells are more aggressive and form bigger tumours in-vivo (Hong KP, Shin MH, Yoon S, et al. Biomaterials. 2015;67:32-41). Hence, the A549-CEACAM6(+) cells were used in subsequent experiments. 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. In addition, indirect conjugation of GR 6D11 with the anti-mitotic toxin, MMAE, demonstrated killing of about 30% after 72-hour treatment on A549-CEACAM6(+) cells. This would be further improved by changing the concentration, linker and type of toxin used for conjugation.

GR 6D11 can also be potentially used as a chimeric antigen receptor (CAR). 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. GR 6D11 CAR T-cells with either CD28 or 4-1BB co-stimulation secreted elevated levels of GM-CSF, IL-2, TFN-y, and TNF-a after co-incubation with PC-9 or A549- CEACAM6(+) cells. This inflammatory cytokine secretion profile of GR 6D11 CAR T-cells supports their anti-tumour functionality.

In a xenograft murine model, 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. Furthermore, 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.

Amino acid sequences of exemplary antiuen-bindinu molecules

VH CDR1

NYGMN (SEQ ID NO: 1)

VH CDR2

WMGWINTNTGEPTYA (SEQ ID NO: 2)

VH CDR3

HYFGLDY (SEQ ID NO: 3)

VL CDR1

SASSSVSYMH (SEQ ID NO: 4)

VL CDR2

STSNLAS (SEQ ID NO: 5)

VL CDR3

HQWSSYPWT (SEQ ID NO: 6)

VH (GR D611) (CDRs underlined)

OIOLVOSGPALKKPGETVKISCKASGYTFTNYGMNWVKOAPGKGLKWMGWINT NTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARHYFGLDYWGO GTTLTVSS (SEQ ID NO: 7)

VL (GR D611) (CDRs underlined)

OIVLTOSPA1MSASLGEE1TLTCSASSSVSYMHWYOQKSGTSPKLLIYSTSNLASGV PSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYPWTFGGGTKLEIK (SEQ ID NO: 8) Mouse IgG2a Fc (Uniprot accession P01863)

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAV

LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPA

PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ

TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV

RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPV

LDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ

ID NO: 9)

Mouse Ig Kappa Fc (Uniprot accession P01837)

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW

TDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID

NO: 10)

VH7 Hu (humanised VH) (CDRs underlined)

OVOLVOSGSALKKPGGSVKVSCKASGYTFTNYGMNWVROAPGOGLEWMGWIN

TNTGEPTYAEEFKGRFVFSLETSVS1AYLO1NNLKAEDTAVYFCARHYFGLDYWG

QGTTLTVSS (SEQ ID NO: 11)

VH7 K (humanised VH) (CDRs underlined)

OIOLVOSGSELKKPGASVKVSCKASGYTFTNYGMNWVROAPGOGLEWMGWINT

NTGEPTYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARHYFGLDYWGO

GTLVTVSS (SEQ ID NO: 12)

VH7 C (humanised VH) (CDRs underlined)

OVOLVOSGSELKKPGASVKVSCKASGYTFTNYGMNWVROAPGOGLEWMGWINT

NTGEPTYAOGFTGRFVFSLDTSVSTAYLOISSLKAEDTAVYYCARHYFGLDYWGO

GTLVTVSS (SEQ ID NO: 13)

VK1 (humanised VL) (CDRs underlined)

DIOLTOSPSSLSASVGDRVT1TCSASSSVSYMHWYOOKPGKAPKLL1YSTSNLASG

VPSRFSGSGSGTDYTLTISSLOPEDFATYYCHOWSSYPWTFGOGTKVEIK (SEQ ID

NO: 14) VK3 (humanised VL) (CDRs underlined)

EIVLTOSPATLSLSPGERATLSCSASSSVSYMHWYOQKPGOAPRLLIYSTSNLASGI

PARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSYPWTFGOGTKVEIK (SEQ ID NO: 15)

Human IgGl Fc (Uniprot accession P01857)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTY1CNVNHKPSNTKVDKKVEPKSCDKTHTCPPC

PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT1SKA

KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16)

Human Ig Kappa Fc (Uniprot accession P01834)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17)

VH1 CDR1

NYGM (SEQ ID NO: 18)

VH1 CDR2

W1NTNTGEPTYA (SEQ ID NO: 19)

VH1 CDR3

HYFGLDY (SEQ ID NO: 20)

VH1

EVOLVOSGAEVKKPGATVKISCKVSGYTFTNYGMHWVOOAPGKGLEWMGWINT

NTGEPTYAEKFOGRVTITLDTSTDTAYMELSSLRSEDTAVYYCARHYFGLDYWGO

GTTLTVSS (SEQ ID NO: 21) scFV linker GGGGSGGGGSGGGGS (SEQ ID NO: 22)

Claims

1. 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 SASSS VSYMH (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).

2. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises a) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11, 12 or 13; and b) a VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 14.

3. The antigen-binding molecule of claim 1 or 2, wherein the antigen-binding molecule is an antibody or antigen-binding fragment thereof.

4. The antigen -binding molecule of claim 3, wherein the antibody or antigen binding fragment thereof is 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.

5. The antigen-binding molecule of any one of claims 1 to 4, wherein the antigenbinding molecule binds specifically to CEACAM1, CEACAM5 and CEACAM6.

6. The antigen-binding molecule of any one of claims 1 to 5, wherein the antigenbinding molecule comprises is conjugated to a radioisotope or cytotoxin.

7. The antigen-binding molecule of claim 6, wherein the cytotoxin is selected from the group consisting of 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, pemetrexed, 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).

8. The antigen -binding molecule of any one of claims 1 to 7, wherein the antigenbinding molecule selectively binds to a cancer that is positive for CEACAM1, CEACAM5 and/or CEACAM6.

9. The antigen -binding molecule of any one of claims 1 to 8, wherein the antigenbinding molecule selectively binds to a gefitinib-resistant lung cancer cell, an osimertinib-resistant lung cancer cell, a non-small cell lung cancer cell, a breast cancer cell, pancreatic cancer cell, stomach (or gastric) cancer cell, small intestine cancer cell, esophageal cancer cell, a colorectal cancer cell, haematological cancer cell, squamous cell lung cancer, cervival cancer, endometrium cancer or liver cancer.

10. 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 W1NTNTGEPTYAEEFKG (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).

11. An antigen-binding molecule that binds specifically to CEACAM1, CEACAM5 and CEACAM6.

12. The antigen-binding molecule of claim 11, wherein the antigen-binding molecule docs not bind to CEACAM8.

13. The antigen-binding molecule of claim 1 1 or 12, wherein the antigen-binding molecule binds to an epitope that comprises at least A89 of CEACAM1, CEACAM5 and/or CEACAM6.

14. The antigen-binding molecule of any one of claims 11 to 13, wherein the antigenbinding molecule binds specifically to an amino acid sequence of SEQ ID NO: 30.

15. An isolated polynucleotide comprising a nucleic acid sequence encoding the antigenbinding molecule according to any one of claims 1 to 14.

16. A construct comprising a polynucleotide of claim 15 in operable connection with one or more control sequence.

17. A host cell that contains the construct of claim 16.

18. A composition comprising an antigen-binding molecule of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of claims 1 to 14, a transmembrane domain, one or more costimulatory domains and an intracellular signaling domain.

20. An isolated polynucleotide encoding a CAR of claim 19.

21. A vector comprising the polynucleotide of claim 20.

22. The vector of claim 21, wherein the vector is a viral vector.

23. An engineered cell comprising the vector of claim 21 or 22.

24. The engineered cell of claim 23, wherein the cell is a T cell, an NK T cell or an NK cell.

25. A method of preparing an engineered immune cell, the method comprising introducing the vector of claim 21 or 22 into an immune cell.

26. An antigen-binding molecule of any one of claims 1 to 14 or a composition of claim 18 or an engineered cell of claim 23 or 24 for use as a medicament.

27. 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 of any one of claim 1 to 14, a composition of claim 18 or an engineered cell of claim 23 or 24 to the subject.

28. The method of claim 27, wherein the cancer is positive for CEACAM1, CEACAM5 and/or CEACAM6.

29. The method of claim 28, wherein 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, squamous cell lung cancer, cervival cancer, endometrium cancer or liver cancer.

30. An antigen-binding molecule of any one of claims 1 to 14, a composition of claim 18 or an engineered cell of claim 23 or 24 for use in treating or preventing cancer in a subject.

31. Use of an antigen-binding molecule of any one of claims 1 to 14, a composition of claim 18 or an engineered cell of claim 23 or 24 in the manufacture of a medicament for treating or preventing cancer.

32. A method for detecting cancer in a subject, the method comprising: contacting a sample obtained from the subject with an antigen-binding molecule of any one of claims 1 to 14, 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.

33. A method for identifying a subject susceptible to cancer, the method comprising: contacting a sample obtained from the subject with an antigen-binding molecule of any one of claims 1 to 14, wherein an increase in the level of binding of the antigenbinding molecule in the sample as compared to a reference indicates that the subject is susceptible to cancer.

34. The method according to claim 32 or 33, wherein the antigen-binding molecule comprises a detectable label.

35. A kit for use in the method of any one of claims 32 to 34, comprising an antigenbinding molecule of any one of claims 1 to 14, together with instructions for use.