The antibodies, or antigen binding fragments, of the present invention were identified from a functional high-throughput screen of CAR-T cells, wherein the CAR-T cells express a unique chimeric antigen receptor (CARs), each CAR comprising an antibody or antigen binding fragment and/or scFv derived from a diverse scFv library. The antibodies or antigen binding fragments as identified in Table 1 are those exhibiting surprisingly good functional response (i.e. were positive hits) in various assays against cells expressing CLL1 and are therefore deemed to be superior CAR-T cells over that known in the art and provide novel and effective antibodies or antigen binding fragments of CLL1.

The high-throughput screening method employed to identify CLL1 antibodies or antigen binding fragments, and/or bi-specific, tri-specific or multi-specific antigen binding molecules and/or antibodydrug conjugates and/or CAR-T cells derived therefrom is described in the Applicant’s co-pending International application PCT/GB2022/050158 (published as WO2022/157500), the content of which is incorporated by reference.

In embodiments, the antibody or antigen binding fragment can be an isolated antibody having specificity for CLL1 (suitably human CLL1) and can be a full-length antibody or an antibody fragment. The antibody can be polyclonal, monoclonal, recombinant, chimeric, or humanised. Furthermore, the antibody can be of any isotype including without limitation IgA, IgD, IgE, IgG, or IgM. Thus, for example, the antibody can be any IgA such as lgA1 or lgA2, or any IgG such as IgG 1 , lgG2, lgG3, lgG4, or synthetic IgG. The antibody can also be any antibody fragment having specificity for CLL1 , such as F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, dsFv, Fv, scFv-Fc, (scFv)2, a diabody, and a bivalent antibody. The antibody can be any modified or synthetic antibody, including, but not limited to, non-depleting IgG antibodies, T-bodies, or other Fe or Fab variants of antibodies.

In some embodiments, the invention provides an antibody or antigen binding fragment with avidity for CLL1 of about 10 pM or less, 5 pM or less, 2 pM or less, 1 pM or less, 500 nM or less, 400 nM or less, 300 nM or less, or 200 nM or less, or 100 nM or less, or 75 nM or less, or 50 nM or less, or 25 nM or less, or 10 nM or less, or 5 nM or less. Avidity can be measured using art-known techniques, such as ELISA or BIACORE.

The antibody or antigen binding fragment of the invention can be produced by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, the antibody is produced using a mammalian expression system. Alternatively, the antibody or antigen binding fragment of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system. Bacterial expression systems can be used to produce fragments such as a F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, dsFv, Fv, scFv-Fc, (scFv)2, and diabodies. The antibody or antigen binding fragment of the invention can be conjugated to a synthetic molecule. Conjugation of the antibody or antigen binding fragment of the invention to the synthetic molecule may be by any suitable method, for example recombinant engineering. The synthetic molecule can be any molecule such as a drug targeting a tumour or tumour cells. The synthetic molecule can also be a peptide/protein or an antibody or an antigen binding fragment such that the resultant molecule has specificity for more than one antigen, wherein the resulting fusion protein can be produced by conventional recombinant protein expression systems and methods.

In this respect, a further aspect of the invention relates to chimeric antigen receptors (CARs). CARs, are engineered receptors, which confer specificity for a desired antigen onto an immune effector cell, such as a T-cell. CARs may be expressed on the extracellular surface of a cell. Expression may be by any suitable means via retroviral vector expression. The most common form of these molecules are fusions of an antibody or antigen binding fragment, such as an scFv, to the transmembrane and intracellular domains of a native T-cell activation receptor, such as CD3, typically CD3-zeta. Such molecules result in the transmission of a signal, suitable an activation signal, in response to recognition by the scFv of its target. ‘First-generation’ CARs typically have the intracellular domain from the CD3- zeta chain, which is the primary transmitter of signals from endogenous TCRs. ‘Second-generation’ CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, CD137, 41 BB, ICOS) to the intracellular portion of the CAR to provide additional signals to the T cell (see Figure 1). Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells (Maher, et al. (2002) Nat. Biotechnol. 20:70-75; Kowolik, et al. (2006) Cancer Res. 66: 10995-11004). ‘Third-generation’ CARs combine multiple signaling domains, such as CD3z-CD28-41 BB or CD3z-CD28-OX40, to further augment potency (Zhao, et al. (2009) J. Immunol. 183:5563-5574; Pule, et al. (2005) Mol. Ther. 12:933-941 ; Zhong, et al. (2010) Mol. Ther. 18:413-420). Accordingly, in an embodiment of this invention, a CAR is provided that comprises an anti-CLL1 antibody or antigen binding fragment, such as an scFv fragment, as defined in SEQ ID NOS 3 to 12.

CARs of this invention can be prepared using standard recombinant protein techniques using sequences of CD3, e.g. CD247 (CD3-zeta) and optionally other costimulatory molecules known in the art. For example, the human CD247 (CD3-zeta) sequence is available under GENBANK accession number NP_932170, the human CD28 sequence is available under GENBANK accession number NP 006130, the human CD8A sequence is available under GENBANK accession number AAH25715, and the human CD137 sequence is available under GENBANK accession number NP_001552.

In particular embodiments, the CAR of the present invention are represented by the SEQ ID NOS: 93 to 112. These CARs include, but not necessarily limited to, either:

1) a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:113), a human CD137 costimulatory domain (SEQ ID NO:114); a human CD8A transmembrane domain (SEQ ID NO:115), a human CD8A hinge domain (SEQ ID NO:116); an antigen binding fragment of the present invention (SEQ ID NOS: 3 to 12) optionally comprising a linker (SEQ ID NO: 117 or

118) between the VH and VL sequences; and a CD8A extracellular signal peptide (SEQ ID NO:

119); or

2) a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:113), a human CD28 costimulatory domain (SEQ ID NO:120); a human CD28 transmembrane domain (SEQ ID NO:121), a human CD28 hinge domain (SEQ ID NO:122); an antigen binding fragment of the present invention (SEQ ID NOS: 3 to 12) optionally comprising a linker (SEQ ID NO: 117 or

118) between the VH and VL sequences; and an CD8A extracellular signal peptide (SEQ ID NO:

119).

In specific embodiments, the CARs of the present invention have sequences defined in SEQ ID NOs: 93 to 102.

In some embodiments, the antibody or antigen binding fragment can be conjugated to a synthetic molecule that can confer specificity for one or more antigens in addition to CLL1 . For example, the antibody of the invention can be engineered (e.g. as a bivalent diabody or a conjugated Fab dimer or trimer) to have specificity for CLL1 and another tumour antigen, e.g., an antigen associated with a disease or disorder as disclosed herein. Alternatively, the antibody can be engineered to have specificity for CLL1 and an antigen that promotes activation or targeting of other cells, such as cytotoxic effector cells or T cells. Accordingly, the invention also includes bispecific, tri-specific and multi-specific molecules such as BiTEs (bi-specific T-cell engagers) and DART™s (dual affinity retargeting reagents).

As is known in the art, a bi-specific T-cell engager (BiTE) refers to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (WO 05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147; Bargou, et al. (2008) Science 321 :974-977). BiTE antibodies have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or H MW- MAA) (Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 11 :22-30). Accordingly, in another embodiment of this invention, an anti-CLL1 antibody or antigen binding fragment (e.g. a scFv) is a component of a bi-specific T-cell engager (BiTE). In particular embodiments, the bi-specific T-cell engager (BiTE) of this invention is composed of an anti-CLL1 antibody or antigen binding fragment and an anti-CD3 antibody fragment fused together by a linker, e.g., a flexible protein linker. See, for example, US 5,929,212. The term ‘BiTE’ as used herein may encompass but is not limited to the bispecific T-cell engager named BiTE® available from Amgen®.

In embodiments, the antibody fragment that specifically binds CD3 comprises:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:123,

(ii) a CDR2 of SEQ ID NO:124, and

(iii) a CDR3 of SEQ ID NO:125; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:126,

(ii) a CDR2 of SEQ ID NO:127, and

(iii) a CDR3 of SEQ ID NO:128.

In other embodiments, the invention provides the antibody or antigen binding fragment of the invention coupled to a synthetic molecule as defined herein to provide a DART™. DART™ refers to an immunoglobulin molecule that includes at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two antigen binding sites, which may recognize the same or different antigens. Each of the polypeptide chains of a DART™ include an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DART™ polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g. the second) DART™ polypeptide chain to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (e.g. the first) of the DART™ polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DART™ polypeptide chain to form an epitope binding site. DART™s may be monospecific, bi-specific, tri- specific, etc., thus being able to simultaneously bind one, two, three or more different antigens (which may be of the same or of different antigens). DART™s may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavelent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of DART™s (i.e., degree of specificity and valency may be combined, for example to produce bispecific antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e. capable of binding four sets of antigens/epitopes), etc. The construction of DART™ molecules is disclosed in WO 2006/113665, WO 2008/157379, and WO 2010/080538. Accordingly, in another embodiment of this invention, an anti-CLL1 antibody or antigen binding fragment is included in a DART™.

In other embodiments, the antibody or antigen binding fragment of the present invention is conjugated, linked or joined to other synthetic molecules including therapeutic agents (or ‘payloads’) such as cytotoxic, cytostatic, or anti-angiogenic agents and radioisotopes, labels or nanoparticles. Such molecules are terms antibody-drug conjugates or ADCs. Examples of ADCs include: Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Trastuzumab emtansine and Trastuzumab emtansine. The invention provides a method of inhibiting cells that express CLL1 (CLL1 cells) by contacting the cells with an antibody, antigen binding fragment or antibody conjugated to a synthetic molecule (e.g. a bispecific T-cell engager such as a BiTE, DART™, or ADC) of the invention. The method can be used to inhibit CLL1 cells in vitro or in a subject (i.e., in vivo). The contacted CLL1 cells can be in, for example, a cell culture or animal model of a disorder associated with aberrant expression or levels of CLL1 . The method is useful, for example, to measure and/or rank (relative to another antibody) the antibody's inhibitory activity for a CLL1 cell type. Inhibiting CLL1 cells can include blocking or reducing the activity or growth of CLL1 cells. Inhibiting can also include the killing of CLL1 cells. Cytotoxicity of an antibody, antibody fragment or antibody conjugated to a synthetic molecule such as a BiTE, DART™, or ADC of the invention can be assessed using any conventional assay including, e.g. a lactate dehydrogenase cytotoxicity assay such as the CYTOTOX 96 non-radioactive cytotoxicity assay commercially available from PROMEGA. Similarly, the invention provides an antibody, antigen binding fragment or antibody conjugated to a synthetic molecule (e.g. a bispecific T-cell engager such as a BiTE, DART™, or ADC) of the invention for use in inhibiting and killing cells expressing CLL1.

The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with aberrant levels of CLL1 or CLL1 cells. As used in the context of the present invention, the term ‘aberrant’ is intended to include increased or decreased CLL1 expression on a cell as compared to expression of CLL1 in normal or healthy cells, or an increase in cells expressing CLL1 . Generally, the method of treatment includes administering a therapeutically effective amount of an isolated antibody, antibody fragment or fusion protein of the invention to the subject. The antibody can be any anti-CLL1 antibody, antigen binding fragment (e.g. chimeric, humanised, synthetic, F(ab)2 , Fv, scFv, F(ab')2, F(ab), VL, VH, dsFv, Fv, or (scFv)2) or antibody conjugated to a synthetic molecule (e.g., a bispecific T-cell engager such as a BiTE, DART™, or ADC) as described herein. Similarly, the invention provides an antibody, antigen binding fragment or antibody conjugated to a synthetic molecule (e.g., a bispecific T-cell engager such as a BiTE, DART™, or ADC) of the invention for use in medicine, and for the treatment of disease and disorders associated with aberrant or elevated CLL1 expression.

Disorders that can be treated include, for example, cancers, such as AML, chronic myeloid (myelogenous) leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monocytic leukemia, acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN); autoimmune diseases such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn’s disease, IBD, IBS, fibromyalga, mastocytosis, and Celiac disease, melanomas, and sarcomas.

The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with elevated levels of CLL1 on a cell, or by elevated levels of CLL1 -cells by adoptive transfer of the recombinant host cells, e.g. T-cells described herein, which express an antibody or antigen binding fragment conjugated to a synthetic molecule of the invention as a CAR that selectively binds CLL1 . Similarly, the invention provides recombinant cells expressing an antibody or antigen binding fragment conjugated to a synthetic molecule (e.g. CAR) of the invention for use in medicine.

Recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T- cells, e.g. effector memory T-cells from the subject to be treated. The recombinant T-cells are transferred, typically by infusion, to the patient. The transferred T-cells of the invention can then mount an immune response against CLL1 expressing cells (CLL1 -cells) in the patient.

The adoptive transfer method can be used, for example, to treat subjects that have or are suspected to have cancers, such as AML, chronic myeloid (myelogenous) leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monocytic leukemia, acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN); autoimmune diseases such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn’s disease, IBD, IBS, fibromyalga, mastocytosis, and Celiac disease, melanomas, and sarcomas. In embodiments, and suitable for use in treatment, the invention also provides a pharmaceutical composition containing an antibody or antigen binding fragment or antibody conjugated to a synthetic molecule (e.g., a CAR, a bispecific T-cell engager such as a BiTE, DART™, or ADC) as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared from any of the antibody, antigen binding fragment (e.g. chimeric, humanised, synthetic, F(ab)2 , Fv, scFv, F(ab')2 , F(ab), VL, VH, dsFv, Fv, or (scFv)2) or antibody conjugated to a synthetic molecule (e.g., a CAR, a bispecific T-cell engager such as a BiTE, DART™, or ADC) as described herein.

The composition of the invention can include a carrier for the antibody or antigen binding fragment, or antibody conjugated to a synthetic molecule, desirably a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. The term ‘pharmaceutically acceptable carrier’, as used herein, means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances, which are suitable for administration into a human or veterinary patient (e.g. a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term ‘carrier’ denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The pharmaceutically acceptable carrier can be co-mingled with one or more of the active components, e.g., a hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy. ‘Pharmaceutically acceptable’ materials typically are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset.

The pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The pharmaceutical compositions also optionally can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

The pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well-known in the pharmaceutical arts. Such methods include the step of bringing the antibody of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

High throughput screening to identify CARs/antibodies

The present invention benefits from the high-throughout screening methodology as described in the copending International application no. PCT/GB2022/050158, the content of which is incorporated herein by reference.

The high-throughout screening method allows resources to be focussed primarily on the diversity of the CAR library and identification of the CARs that are able to demonstrate promising activity in a functional assay relevant to a clinical context.

The invention is described in greater detail by the following non-limiting examples.

EXAMPLES

Example 1 :

A synthetic human scFv phage library was panned against recombinant human CLL1 protein (R&D Biosystems™). To this end, the library was grown to log phase, and then rescued with M13KO7 helper phage (Antibody Design Lab™, PH010L) before being amplified overnight at 32°C in a shaker. The phage library was subsequently precipitated with PEG/NaCI, re-suspended in PBS and stored at -80°C. Protein G coated magnetic beads were coated with hCLL1-Fc or Fc recombinant protein in PBS and subsequently blocked in PBS+ BSA. Phage particles were incubated for 30 minutes with negative magnetic particles. Subsequently, magnetic particles were pelleted and the supernatant was incubated for 1 h with hCLL1 coated magnetic beads under rotation. After 1 h incubation, unbound and non- specifically bound phages were washed away by rinsing the beads with PBST. Bound phages were eluted by 100 mM triethylamine (TEA), and the eluate was neutralized by 1 M Tris-HCI (pH 7.4). The eluate was then used to infect exponentially growing E. co// TGI cells. The panning was repeated for an additional two to four cycles.

Example 2:

After the last panning step, scFv sequences were PCR amplified from the eluted phages or the isolated plasmids using Q5 DNA polymerase (NEB) and scFv-specific forward and reverse primers. Resulting amplicon was cleaned by PCR clean-up and 1 pg PCR product were digested with restriction enzymes for 2h at 37°C. In parallel, a CAR scaffold library, consisting of pooled plasmids containing different CAR scaffolds with variation in hinge domain, transmembrane domain and intracellular signalling domains, was digested using Esp3l . After 2h incubation the CAR scaffold library and the scFv amplicons were cleaned by PCR clean-up columns and ligated using T4 DNA ligase (Thermo Fisher™) for 12h over-night. The resulting CAR-library was electroporated into electrocompetent bacteria and grown overnight at 30°C, followed by plasmid maxi-prep isolation. The CAR sequences from the resulting plasmid library were briefly PCR amplified and sequenced on an Oxford Nanopore Technology™ MinlON™, using the amplicon sequencing kit.

Example 3:

In order to assess if the CAR library was functionally expressed and recognizing CLL1 , the CAR library against CLL1 was used to produce lentiviral vector particles. The lentiviral library was titrated and MOI (Multiplicity of Infection) determined on Jurkat cell line. Subsequently, the library was transduced into human T cells or Jurkat cell line, to reach a transduction efficiency of around 20%. The resulting CAR- T cell library was expanded for six more days after transduction and assessed for CLLI binding. CLL1- CAR-T cell library cells were stained with CLL1-Fc fusion protein or a negative control protein (recombinant Fc, R&D systems). In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibody to detect transduced cells. The expression of CARs on the cell surface was assessed.

Example 4:

A CAR-T cell library was produced by activation of Peripheral Blood Mononuclear Cells (PBMCs) with TransAct™ (Miltenyi™) in the presence of human IL-2 (100 lll/ml) and transduced at day two with the lentiviral CLL1 CAR library. The next day, cells were washed and further expanded until day 8 of the process. Transduction was assessed by flow cytometry and transduced cells were enriched through CD34 microbeads (Miltenyi™). Purity was assessed by flow cytometry. The CAR-T-cell library was cocultured with CLL1-positive cells (HL60) or no cells. After 6h activation, CAR-T cells were prepared for single cell sequencing analysis. CAR-T cell libraries from different donors were prepared for 10x genomics single cell gene expression analysis (1 Ox genomics 3’ sequencing kit V3 or Single Cell 5’ Kit v2) and sequenced on a NovaSeq™ 6000 (Illumina™). Cell ranger™ software (10x Genomics™) was used for downstream processing and alignment of reads. In order to deconvolute the single cell sequencing data and identify the CAR sequence and cell barcode identity, CAR sequences were amplified using long PCR with a forward read 1 primer and CAR-specific reverse primer. After 10 cycles of PCR, the product was cleaned using SPRI bead clean-up and a second, nested PCR was performed. PCR products were barcoded and an Oxford Nanopore library was prepared using the Oxford Nanopore ligation sequencing kit. Libraries were then sequenced on a MinlON® flow cell and CAR sequences and 10x cell barcodes were determined. Example 5:

Single CAR constructs were selected based on best activation propensity from the single cell gene expression analysis, and CAR sequences were synthetized by Integrated DNA technologies (IDT). CAR sequences were cloned into a lentiviral expression construct, encoding a 2A peptide and a truncated CD34 reporter. Lentiviral vectors were produced with the individual CAR constructs and titrated on Jurkat cell line. PBCMs were activated on day 0 with TransAct™ (Miltenyi™) and transduced with equal MOI (Multiplicity of Infection) of lentiviral vectortwo days after activation, in the presence of recombinant human IL2 (Miltenyi™). On day 4, cells were washed, counted and seeded for expansion until day 8. Cell expansion was quantified using LUNA-FL cell counter and live/dead fluorescence dye. Average cell expansion between donors was calculated as the fold change of total cell count between day 4 and day 8 (Figure 3). Cells were subsequently used for functional assays. The affinity/avidity with which the different CARs bind to CLL1 was assessed. Cells transduced with the different CLL1-CAR constructs were stained with CLL1-Fc fusion protein (R&D systems). In a second step, cells were stained with PE- conjugated anti-Fc and an APC-conjugated anti-CD34 antibodies. Median fluorescence intensity of CLL1-Fc staining is shown for CD34+ CAR-T cells (Figures 2A and 2B).

The labels on the x axis indicate different CAR constructs, as indicated below.

CAR 11 and CAR 12 comprise respective antibody/antigen-binding fragments sequences defined as Antibody No. 11 and Antibody No. 12 (Table 2).

Table 2: Comparative example antibodies from US2020/0115457A1 and US2013/0295118A1 respectively

As is evident from Figures 2A and 2B, each of the CARs corresponding to CAR SEQ ID NOs 93 to 102, comprising the scFv SEQ ID NOs 3 to 12 (and the associated CDRs as detailed in Table 1) showed enhanced activity against CLL1 + cells than the prior art CARs. This demonstrates the surprisingly beneficial effect of the antibody/antigen binding fragments of the present invention, and the utility of such antibody/antigen binding fragments in CARs, other bi-specific, tri-specific and multi-specific molecules (such as BiTEs and DART™s), and ADCs. Example 6:

To further evaluate the activity of different anti-CLL1 CARs, CLL1-CAR T-cells were evaluated for cellular toxicity and activity against cancer cells expressing CLL1 , or controls lacking expression of CLL1.

The ability of the different CARs to elicit functional responses was explored by incubating the CLL1- CAR T-cells for 24 hours at 1 :1 ratio with CLL1 -expressing cancer cell line (HL60). After 24h co-culture, cells were stained with anti-CD3 Pacific Blue and anti-CLL1 APC in order to separate T cells and cancer cells. Cells were acquired and counted using flow cytometry and cell killing was calculated compared to HL60 cells cultured in the absence of CAR-T cells (Figure 5).

CAR-T cell activation was assessed via CD69 upregulation on CD3+, CD34+ CAR-T cells as an indicator for T-cell activation (Figure 4).

CAR-T potency was assessed by collecting supernatant of CAR-T cells stimulated with HL60 cells or non-stimulated controls. After 24h of co-culture supernatant was collected and assessed for cytokine production via IFNg ELISA (Figure 6). Left bar on each CAR-T number indicate IFNg values of CAR-T cells stimulated with HL60 cells. Right bar indicate non-stimulated controls.

As is evident from Figure 4, each of the CARs corresponding to CAR SEQ ID NOs 93 to 102, comprising the scFvs SEQ ID NOs 3 to 12 (and the associated CDRs as detailed in Table 1) showed enhanced activity via CD69 upregulation against CLL1 + cells than the prior art CARs. This demonstrates the surprisingly beneficial effect of the antibody/antigen binding fragments of the present invention, and the utility of such antibody/antigen binding fragments in CARs, other bi-specific, tri-specific and multispecific molecules (such as BiTEs and DART™s), and ADCs.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the invention. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention.

Sequence listing:

Claims

1. An antibody, or antigen binding fragment, which binds to CLL1 selected from the group consisting of:

Antibody 5, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:27,

(ii) a CDR2 of SEQ ID NO:37, and

(iii) a CDR3 of SEQ ID NO:47; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:67,

(ii) a CDR2 of SEQ ID NO:77, and

(iii) a CDR3 of SEQ ID NO:87; and,

Antibody 1 , comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:23,

(ii) a CDR2 of SEQ ID NO:33, and

(iii) a CDR3 of SEQ ID NO:43; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:63,

(ii) a CDR2 of SEQ ID NO:73, and

(iii) a CDR3 of SEQ ID NO:83; and,

Antibody 2, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:24,

(ii) a CDR2 of SEQ ID NO:34, and

(iii) a CDR3 of SEQ ID NO:44; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:64,

(ii) a CDR2 of SEQ ID NO:74, and

(iii) a CDR3 of SEQ ID NO:84; and,

Antibody 3, comprising;

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:25,

(ii) a CDR2 of SEQ ID NO:35, and

(iii) a CDR3 of SEQ ID NO:45; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:65,

(ii) a CDR2 of SEQ ID NO:75, and

(iii) a CDR3 of SEQ ID NO:85; and, Antibody 4, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:26,

(ii) a CDR2 of SEQ ID NO:36, and

(iii) a CDR3 of SEQ ID NO:46; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:66,

(ii) a CDR2 of SEQ ID NO:76, and

(iii) a CDR3 of SEQ ID NO:86; and,

Antibody 6, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:28,

(ii) a CDR2 of SEQ ID NO:38, and

(iii) a CDR3 of SEQ ID NO:48; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:68,

(ii) a CDR2 of SEQ ID NO:78, and

(iii) a CDR3 of SEQ ID NO:88; and,

Antibody 7, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:29,

(ii) a CDR2 of SEQ ID NO:39, and

(iii) a CDR3 of SEQ ID NO:49; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:69,

(ii) a CDR2 of SEQ ID NO:79, and

(iii) a CDR3 of SEQ ID NO:89; and,

Antibody 8, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NQ:30,

(ii) a CDR2 of SEQ ID NQ:40, and

(iii) a CDR3 of SEQ ID NQ:50; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NQ:70,

(ii) a CDR2 of SEQ ID NQ:80, and

(iii) a CDR3 of SEQ ID NQ:90; and, Antibody 9, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:31 ,

(ii) a CDR2 of SEQ ID NO:41 , and

(iii) a CDR3 of SEQ ID NO:51 ; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:71 ,

(ii) a CDR2 of SEQ ID NO:81 , and

(iii) a CDR3 of SEQ ID NO:91 ; and,

Antibody 10, comprising:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:32,

(ii) a CDR2 of SEQ ID NO:42, and

(iii) a CDR3 of SEQ ID NO:52; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:72,

(ii) a CDR2 of SEQ ID NO:82, and

(iii) a CDR3 of SEQ ID NO:92. The antibody, or antigen binding fragment, of claim 1 , is comprised in a single chain variable fragment (scFv). The antibody, or antigen binding fragment, of claim 2, wherein the scFv is selected from the group consisting of: SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5; SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8, SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11 ; and SEQ ID NO. 12. The antibody, or antigen binding fragment, of any one of claims 1 to 3, wherein the antibody is conjugated to a synthetic molecule. The antibody, or antigen binding fragment, of claim 4, wherein the antibody is a chimeric antigen receptor and the synthetic molecule comprises a transmembrane region and an intracellular T- cell receptor signaling domain. The antibody, or antigen binding fragment, of claim 5, wherein the transmembrane region is from CD8A or CD28 and/or the intracellular T-cell receptor signaling domain is from CD247 (CD3-zeta). The antibody, or antigen binding fragment, of claim 6, further comprising an intracellular domain of a costimulatory protein receptor. The antibody, or antigen binding fragment, of claim 4, wherein the antibody is a bi-specific T- cell engager (BiTE) and the synthetic molecule comprises an antigen binding domain which binds to a T-cell antigen. The antibody, or antigen binding fragment, of claim 8, wherein the antigen binding domain comprises an antibody fragment that specifically binds CD3. The antibody, or antigen binding fragment, of claim 4, wherein the antibody is an antigen-drug conjugate and the synthetic molecules is a cytotoxic drug or a therapeutic radioisotope. A vector encoding the antibody, or antigen binding fragment, of any one of claims 1 to 10. A pharmaceutical composition comprising the antibody or antigen binding fragment, of any one of claims 1 to 10 and a pharmaceutically acceptable carrier. A chimeric antigen receptor comprising: a) The antibody or antigen binding fragment as claimed in any one of claims 1 to 10; b) A transmembrane region; and c) An intracellular signaling domain. The chimeric antigen receptor of Claim 13, wherein the transmembrane region is from CD8A or CD28 and/or the intracellular signaling domain is from CD247 (CD3-zeta). The chimeric antigen receptor of claim 13, further comprising an intracellular signaling domain of a costimulatory protein receptor. The chimeric antigen receptor of any one of claims 13 to 15, wherein the CAR comprises: a. a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:113), a human CD137 co-stimulatory domain (SEQ ID NO:114); a human CD8A transmembrane domain (SEQ ID NO:115), a human CD8A hinge domain (SEQ ID NO:116); antibody or antigen binding fragment as claimed in any one of claims 1 to 12; and an CD8A extracellular signal peptide (SEQ ID NO:119); or b. a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO: 113), a human CD28 costimulatory domain (SEQ ID NQ:120); a human CD28 transmembrane domain (SEQ ID NO:121), a human CD28 hinge domain (SEQ ID NO:122); antibody or antigen binding fragment as claimed in any one of claims 1 to 12; and an CD8A extracellular signal peptide (SEQ ID NO:119). The chimeric antigen receptor of any one of claims 13 to 16, wherein the CAR has a sequence selected from SEQ ID NO: 93; SEQ ID NO. 94; SEQ ID NO. 95; SEQ ID NO. 96; SEQ ID NO. 97; SEQ ID NO. 98; SEQ ID NO. 99, SEQ ID NO. 100; SEQ ID NO. 101 ; and SEQ ID NO. 102. A recombinant T cell comprising the chimeric antigen receptor of any one of claims 13 to 17. A bispecific T-cell engager comprising: a) The antibody or antigen binding fragment as claimed in any one of claims 1 to 10; b) An antigen binding domain which binds to a T-cell antigen; and c) A linker joining (a) and (b). The bi-specific T-cell engager of claim 19, wherein the antigen binding domain comprises an antigen binding fragment that specifically binds CD3. The bi-specific T-cell engager of claim 20, wherein the antibody fragment that specifically binds CD3 comprises:

(a) a heavy chain variable region comprising,

(i) a CDR1 of SEQ ID NO:123,

(ii) a CDR2 of SEQ ID NO:124, and

(iii) a CDR3 of SEQ ID NO:125; and

(b) a light chain variable region comprising,

(i) a CDR1 of SEQ ID NO:126,

(ii) a CDR2 of SEQ ID NO:127, and

(iii) a CDR3 of SEQ ID NO:128; An antibody-drug conjugate comprising: a) The antibody or antigen binding fragment as claimed in any one of claims 1 to 10; b) A therapeutic drug substance; c) A linker joining (a) and (b). The antibody-drug conjugate of claim 22, wherein the synthetic molecule is a cytotoxic drug. An antibody, or antigen binding fragment, of any one of claims 1 to 10, a pharmaceutical composition of claim 12, a chimeric antigen receptor of any one of claims 13 to 17, a recombinant T-cell of claim 18, a bi-specific T-cell engager of any one of claims 19 to 21 or the antibody-drug conjugate of any one of claims 22 to 23 for use as a medicament. An antibody, or antigen binding fragment, of any one of claims 1 to 10, a pharmaceutical composition of claim 12, a chimeric antigen receptor of any one of claims 13 to 17, a recombinant T-cell of claim 18, a bi-specific T-cell engager of any one of claims 19 to 21 or the antibody-drug conjugate of any one of claims 22 to 23 for use in killing or inhibiting the growth of cells expressing CLL1 . An antibody, or antigen binding fragment, of any one of claims 1 to 10, a pharmaceutical composition of claim 12, a chimeric antigen receptor of any one of claims 13 to 17, a recombinant T-cell of claim 18, a bi-specific T-cell engager of any one of claims 19 to 21 or the antibody-drug conjugate of any one of claims 22 to 23 for use in medicine. An antibody, or antigen binding fragment, of any one of claims 1 to 10, a pharmaceutical composition of claim 12, a chimeric antigen receptor of any one of claims 13 to 17, a recombinant T-cell of claim 18, a bi-specific T-cell engager of any one of claims 19 to 21 or the antibody-drug conjugate of any one of claims 22 to 23 for use in the treatment of a disease or disorder wherein the disease or disorder is selected from the group consisting of: cancers, such as AML, chronic myeloid (myelogenous) leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monocytic leukemia, acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN); autoimmune diseases such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn’s disease, IBD, IBS, fibromyalga, mastocytosis, and Celiac disease, melanomas, and sarcomas. A method of treating a disease or condition associated with the expression of CLL1 , said method comprising administering to a patient in need thereof an effective amount of an antibody, or antigen binding fragment, of any one of claims 1 to 10, a pharmaceutical composition of claim 12, a chimeric antigen receptor of any one of claims 13 to 17, a recombinant T-cell of claim 18, a bi-specific T-cell engager of any one of claims 19 to 21 or the antibody-drug conjugate of any one of claims 22 to 23 to a patient in need thereof. The method of treatment of Claim 28, wherein the disease or condition is selected from the group consisting of: cancers, such as AML, chronic myeloid (myelogenous) leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monocytic leukemia, acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN); autoimmune diseases such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn’s disease, IBD, IBS, fibromyalga, mastocytosis, and Celiac disease, melanomas, and sarcomas.