Therapeutic antibodies

Abstract

LGR5 binding agents, in particular antibodies or fragments thereof that bind to human LGR5, and the use of such binding agents in the treatment of diseases, such as cancer and inflammatory diseases, and in the detection of LGR5.

Description

[Technical field]

[0001] The present invention relates to LGR5 binding agents, in particular antibodies or fragments and derivatives thereof that bind to LGR5, and the use of such binding agents in the treatment of diseases, such as cancer and inflammatory diseases, and in the detection of LGR5. [Background technology]

[0002] The Wnt pathway is one of the core intercellular signaling pathways that orchestrate development in metazoans and regulate stem cell function in adult tissues. Stimulation of the Wnt pathway begins with the engagement of an extracellular ligand, typically a Wnt, by the cognate Wnt receptor on the cell surface and the propagation of the signal within the cell. A series of subsequent molecular interactions culminates in the activation of a context-specific transcriptional program that mediates the biological output.

[0003] The primary role of the Wnt pathway in maintaining self-renewal and proliferation of adult stem cells has been functionally established in a wide range of tissues, including the epithelial stem cell compartment along the gastrointestinal tract, liver, mammary gland, and skin, as well as the mature B-cell compartment. Mutational deregulation of the Wnt pathway resulting in aberrant target gene expression is one of the key drivers of cancer development in these and several other tissues. Oncogenic forms of the Wnt pathway have received considerable attention in colorectal cancer (CRC), where somatic mutations that deregulate pathway activity, most commonly inactivating mutations in the tumor-suppressing adenomatous polyposis coli (APC), are an early and widespread insult in disease pathogenesis. In other epithelial malignancies, a spectrum of cancer-promoting mutations may exist in other Wnt pathway regulatory components; for example, in the development of hepatocellular carcinoma, approximately half of the cases acquire gain-of-function mutations in β-catenin or inactivating mutations in AXIN1. In some epithelial cancers, aberrant pathway activity and deregulated target gene expression are frequent features despite the absence of Wnt pathway mutations and may be the result of convergent regulation by other cell signaling pathways.

[0004] Determining gene expression signatures unique to cancer cells has enabled the identification of prognostic disease markers and provided a basis for determining molecular vulnerabilities that can be exploited therapeutically. One notable study identified a cohort of Wnt target genes in CRC cell lines with somatically inactivated APC. Leucine-rich repeat-containing G protein receptor 5 (LGR5) was identified as a genetic target of oncogenic Wnt pathway activity, and subsequent studies established it as a prototypic stem cell marker in murine and human intestinal epithelium, gastric epithelium, hair follicles, fetal mammary gland, developing kidney nephrons, and regenerating liver.

[0005] LGR5 is a member of the cell surface GPCR-like receptor family that includes LGR4 and LGR6. LGR4 is widely expressed in proliferative cell compartments throughout the body, while LGR6 shows more restricted expression to certain stem cell compartments, including sweat gland, interfollicular epidermal, and nail stem cells. The three LGR family proteins are co-receptors for R-spondin family proteins, and RNF43 or ZNRF3 is the other co-receptor. RNF43 and ZNRF3 are ubiquitin ligases that downregulate cell surface-associated Wnt receptors. Removal of RNF43 / ZNRF3 from Wnt receptors through recruitment to LGR-bound R-spondin acts to increase the steady-state levels of Wnt pathway receptors at the cell surface and the responsiveness of pathway activity to stimulation by Wnt ligands.

[0006] LGR5 has attracted much therapeutic interest due to its overexpression in cancers with oncogenic mutations that deregulate pathway activity. Numerous studies have demonstrated that LGR5 expression is increased in CRC tumors and intestinal adenocarcinomas compared to adjacent normal intestinal tissue, indicating that LGR5 is associated with cells at the invasive front of tumors and metastatic cells. As a prognostic marker in CRC, LGR5 transcript levels in patient samples are associated with shorter overall survival and reduced disease-free survival.

[0007] Functionally, studies of LGR5 sufficiency in CRC cell lines have attributed its role in proliferation, migration, chemosensitivity, colony formation, and in vivo engraftment. Studies using human CRC organoid xenografts have found that LGR5-expressing cell compartments are more proliferative than their non-expressing counterparts.

[0008] Many other malignancies are characterized by increased LGR5 overexpression: basal cell carcinoma, glioblastoma where upregulated LGR5 colocalized with the cancer stem cell marker CD133 predicts poor prognosis, ovarian tumors, ER-negative breast cancer where high LGR5 expression during cancer development correlates with poor prognosis, and B-cell malignancies.

[0009] In summary, although LGR5 may have positive, opposing, or functionally redundant roles in cancer development, overexpression of LGR5 on the cell surface is a hallmark of various cancer types and provides a molecular means to distinguish cancer cells from non-malignant tissues.

[0010] Immunotherapy is a promising class of treatment, part of which relies on the specificity and efficacy of antibody binding for the recognition of cell surface proteins overexpressed on cancer cells. Indeed, antibodies are one of the best-selling classes of drugs today; five of the top ten best-selling drugs are antibodies. The versatility and specificity of antibodies have found widespread use for the detection and specific killing of cancer cells in many cancer types. Therapeutic antibodies include modalities such as antibody-dependent cellular cytotoxicity (ADCC), antibody-drug conjugates (ADC), chimeric antigen receptors (CAR), and bispecific immune cell engagers (e.g., bispecific T cell engagers, BiTEs).

[0011] Despite the clinical promise of antibodies against LGR5, few, if any, have undergone robust validation, hindering efforts to determine relative LGR5 protein levels in normal tissues and disease, study its cell biology, or develop immunotherapies for specific cancer types. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2009 / 047500 [Patent Document 2] US Patent Application Publication No. 2004043401 [Patent Document 3] WO2019200007 issue [Patent Document 4] WO2021108613 No. [Non-patent literature]

[0013] [Non-Patent Document 1] Walsh SJ, Omarjee S, Galloway WRJD, Kwan TTL, Sore HF, Parker JS, Hyvonen M, Carroll JS, Spring DR. A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates. Chem Sci. 2019;10(3):694–700 [Non-Patent Document 2] Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) [Non-Patent Document 3] Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009) [Non-Patent Document 4] Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010)

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[0014] It is an object of the present invention to address the need for antibody-based treatments for use in the treatment of disease, particularly for the treatment of cancer and inflammatory diseases, etc. It is also an object of the present invention to provide antibodies against LGR5 for use as research tools and for use in the treatment and diagnosis of disease. [Means for solving the problem]

[0015] The present invention relates to LGR5 binding agents. In particular, the present invention relates to antibodies that bind to human and cynomolgus monkey (cyno) LGR5, the antibodies binding to an epitope within amino acids 22-37 of LGR5 (SEQ ID NO: 1), and related methods of treating diseases, such as cancer and / or inflammatory diseases, and methods of identifying the presence of LGR5 in biological samples. The present invention is based in part on the work of the inventors, who found that antibodies against a specific epitope within amino acids 22-37 of LGR5 are highly specific, have high affinity, and are rapidly internalized into LGR5-expressing cancer cells. The inventors have shown that the antibodies of the present invention can distinguish cells with high LGR5 expression levels from cells with lower LGR5 expression levels. Thus, the antibodies of the present invention represent excellent research tools and are particularly useful in therapy, such as the treatment of cancer, particularly cancers that express LGR5.

[0016] In one aspect, the invention relates to an antibody or fragment thereof that binds to human LGR5, wherein the antibody binds to an epitope located within amino acids 22-37 of SEQ ID NO:1.

[0017] In one aspect, the present invention relates to an antibody or fragment thereof that binds to an epitope comprising or consisting of amino acids 22 to 37 of SEQ ID NO:1.

[0018] In one embodiment, the antibody is human or humanized, hi one embodiment, the antibody is a murine antibody.

[0019] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, H but having the following CDR1, CDR2, and CDR3: a) CDR1 of SEQ ID NO: 2 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 3 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 4 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 9 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 10 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 14 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 15 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 16 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 20 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 21 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 22 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 26 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 27 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 28 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 32 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 33 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 34 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 38 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 39 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 40 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 44 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 45 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 46 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 50 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 51 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 52 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 56 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 57 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 58 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 62 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 63 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 64 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 68 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 69 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 70 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 74 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 75 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 76 or a sequence having at least 90% homology thereto The present invention relates to an antibody or fragment thereof comprising:

[0020] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, L but having the following CDR1, CDR2, and CDR3: a) CDR1 of SEQ ID NO: 5 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 6 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 7 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 11 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 12 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 13 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 17 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 18 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 19 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 23 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 24 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 25 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 29 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 30 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 31 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 35 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 36 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 37 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 41 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 42 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 43 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 47 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 48 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 49 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 53 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 54 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 55 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 59 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 60 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 61 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 65 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 66 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 67 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 71 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 72 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 73 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 77 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 78 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 79 or a sequence having at least 90% homology thereto The present invention relates to an antibody or fragment thereof comprising:

[0021] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, wherein the antibody is selected from SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104. H The present invention relates to an antibody or fragment thereof comprising the sequence

[0022] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, wherein the antibody is selected from SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105. L The present invention relates to an antibody or fragment thereof comprising the sequence

[0023] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, wherein the antibody: a) V of SEQ ID NO:80 H Sequence and V of SEQ ID NO:81 L array; b) V of SEQ ID NO:82 H Sequence and V of SEQ ID NO:83 L array; c) V of SEQ ID NO:84 H Sequence and V of SEQ ID NO: 85 L array; d) V in SEQ ID NO:86 H Sequence and V of SEQ ID NO:87 L array; e) V in SEQ ID NO:88 H Sequence and V of SEQ ID NO:89 L array; f) V in sequence number 90 H Sequence and V of SEQ ID NO:91 L array; g) V of SEQ ID NO:92 H Sequence and V of SEQ ID NO:93 L array; h) V of SEQ ID NO:94 H Sequence and V of SEQ ID NO: 95 L array; i) V of SEQ ID NO:96 H Sequence and V of SEQ ID NO:97 L array; j) V of SEQ ID NO:98 H Sequence and V of SEQ ID NO: 99L array; k) V of sequence number 100 H Sequence and V of SEQ ID NO: 101 L array l) V of SEQ ID NO:102 H Sequence and V of SEQ ID NO: 103 L A sequence; or m) V of SEQ ID NO: 104 H Sequence and V of SEQ ID NO: 105 L array The present invention relates to an antibody or fragment comprising the following:

[0024] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, wherein the antibody has a V H、 C H、 V L and C L The present invention relates to an antibody or a fragment thereof comprising a combination of SEQ ID NOs: 106 to 156, or a sequence having at least 70%, 80%, or 90% homology thereto, or a sequence of an antibody clone shown in Table 3, or a sequence having at least 70%, 80%, or 90% homology thereto.

[0025] In one embodiment, the antibody or fragment thereof binds to human LGR5. The antibody or fragment may also bind to the corresponding sequence in cynomolgus monkey (cyno) LGR5 (amino acids 22-37).

[0026] In embodiments, the antibody or fragment thereof is a monoclonal antibody.

[0027] In embodiments, the antibody or fragment thereof is a human, humanized, or chimeric antibody.

[0028] In some embodiments, the antibody or fragment thereof is capable of binding to LGR5 with a Kd of less than about 4 nM.

[0029] In embodiments, the fragment comprises a Fab, scFv, or a single domain antibody.

[0030] In some embodiments, the antibody or fragment thereof is conjugated to a toxin, enzyme, radioisotope, label, therapeutic molecule, or other chemical moiety.

[0031] In another aspect, the invention relates to an immunoconjugate comprising an antibody or fragment thereof according to the invention linked to a therapeutic agent.

[0032] The therapeutic agent can be a toxin, enzyme, radioisotope, or other chemical moiety.

[0033] In another aspect, the invention relates to a pharmaceutical composition comprising an antibody or fragment thereof described herein, or an immunoconjugate described herein, and a pharmaceutical carrier.

[0034] In another aspect, the invention relates to a method of treating cancer comprising administering a therapeutically effective amount of an antibody or fragment thereof described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0035] In another aspect, the invention relates to the use of an antibody or fragment thereof described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer.

[0036] In another aspect, the invention relates to an antibody or fragment thereof as described herein, an immunoconjugate as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0037] In another aspect, the invention relates to an antibody or fragment thereof described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein for use in the treatment of cancer.

[0038] In some embodiments, the cancer is an LGR5-positive cancer.

[0039] In embodiments, the cancer overexpresses LGR5.

[0040] In embodiments, the cancer is selected from head or neck cancer, uterine cancer, colorectal cancer, gastric cancer, endometrial cancer, esophageal cancer, leukemia, e.g., acute lymphoblastic leukemia (ALL), liver cancer, e.g., hepatocellular carcinoma, or pancreatic cancer.

[0041] In another aspect, the invention pertains to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof described herein.

[0042] In another aspect, the invention relates to a vector comprising a nucleic acid described herein.

[0043] In another aspect, the invention relates to a host cell comprising a nucleic acid described herein or a vector described herein.

[0044] The host cell can be a bacterial, viral, or mammalian cell.

[0045] In yet another aspect, the invention relates to a method of producing an antibody described herein, comprising culturing a host cell described herein under conditions suitable for expression of a polynucleotide encoding the antibody, and isolating the antibody.

[0046] In another aspect, the invention relates to a method for detecting LGR5 in a biological sample, comprising contacting the biological sample with an antibody described herein under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5.

[0047] In some embodiments, the biological sample may be a cancer sample selected from head or neck cancer, uterine cancer, colorectal cancer, gastric cancer, endometrial cancer, esophageal cancer, leukemia, e.g., acute lymphoblastic leukemia (ALL), liver cancer, e.g., hepatocellular carcinoma, or pancreatic cancer.

[0048] In another aspect, the invention relates to a kit comprising an antibody described herein, an immunoconjugate described herein, optionally with instructions for use, or a pharmaceutical composition described herein.

[0049] In another aspect, the invention relates to a binding agent, e.g., an antibody or fragment thereof that binds to essentially the same epitope as an antibody described herein, or an antibody or fragment thereof that competes with one of the antibodies provided herein for binding to human or cynomolgus monkey LGR5.

[0050] In another aspect, the invention relates to an isolated synthetic or recombinant peptide comprising an epitope, wherein the peptide consists of residues 22-37 of SEQ ID NO:1.

[0051] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an antibody or fragment thereof described herein.

[0052] In another aspect, the invention relates to a CAR comprising SEQ ID NO: 211, or a sequence having at least 70%, 80%, or 90% homology thereto. In one embodiment, the CAR comprises the CDRs of clone 2.4.

[0053] In another aspect, the invention relates to a cell or cell population expressing such a CAR.

[0054] In another aspect, the invention relates to such cells or cell populations for use in adaptive immunotherapy.

[0055] In another aspect, the invention relates to a method for adaptive immunotherapy comprising administering such cells or cell populations.

[0056] In another aspect, the invention relates to an immune cell engager, such as a bispecific T cell engager, or BiTE, or a trispecific killer engager, TriKE, comprising the antibodies or fragments described herein. In one embodiment, the BiTE or TriKE comprises the CDRs of clone 2.4.

[0057] In another aspect, the invention relates to a BiTE comprising SEQ ID NO: 211, or a sequence having at least 70%, 80%, or 90% homology thereto.

[0058] In another aspect, the invention relates to an isolated synthetic or recombinant peptide comprising an epitope, wherein the peptide consists of residues 22-37 of SEQ ID NO:1.

[0059] In another aspect, the invention relates to a method of diagnosing or assessing the progression of cancer, comprising assessing expression of LGR5 and / or assessing protein levels of LGR5.

[0060] The invention is further illustrated in the following non-limiting drawings. [Brief description of the drawings]

[0061] [Figure 1]Validation of novel LGR5-specific antibodies. A. Western blot analysis of HEK293T lysates expressing LGR family transgenes probed with α-LGR5 hybridoma clone 2 (Table 1, antibody 2) and antibodies raised against HA and vinculin. B. Epitope mapping of a-LGR5 hybridoma clone 2 using a fragment of the antigen sequence of 100 amino acids traces the epitope to fragment 1A composed of the N-terminus of human LGR5 (amino acids 22-37). C. Immunofluorescence of HEK293T cells expressing eGFP-fused human LGR4-6 transgenes and cynomolgus monkey LGR5 (green) using FL-α-LGR5 fluorescently labeled in red (Alexa647). D. Flow cytometry analysis of HEK293T cells expressing human LGR4 and LGR5 transgenes using FL-a-LGR5 for detection. E. Preincubation of FL-α-LGR5 with superstoichiometric amounts of fragment 1A peptide abrogates signaling in LGR5-eGFP expressing cells. [Figure 2-1]FIG. 2A shows the specificity of the LGR5 antibodies generated in the study. A. Amino acid sequence of human LGR5 antigen used for immunization and generation of α-LGR5. The sequence is annotated by the fragment used in the RAD display system to localize the a-LGR5 epitope to fragment 1A (see FIG. 1A). Below - annotation of the amino acid sequence of the fragment used for epitope mapping and structural / topological model showing the antigenic region (red) within the extracellular domain of LGR5 (structure coordinates taken from (Peng et al.)). FIG. 2B shows the specificity of the LGR5 antibodies generated in the study. B. Configuration of LGR family transgenic constructs used for antibody validation. All expressed LGR proteins contain a common N-terminal hemagglutinin (HA) tag and a fusion of the vasopressin V2 receptor C-terminal tail followed by eGFP at the C-terminus. FIG. 2C shows the specificity of the LGR5 antibodies generated in the study. C. Western blot analysis of HEK293T lysates expressing human LGR family transgenes probed with antibodies against a-LGR5 hybridoma clones 1, 3, and 4, and HA and vinculin, as indicated. Immunoreactivity was also observed for hybridoma clone 12 (data not shown). No specific immunoreactivity was observed when Western blots were probed for the other 13 hybridoma clones. Figure 2D shows the specificity of the LGR5 antibodies generated in the study. D. Conservation of sequences in α-LGR5 hybridoma clones within the complementarity determining regions (CDRs). Conserved amino acids for clones 2-4 compared to α-LGR5 clone 1 are represented by dashed lines. Amino acid differences are indicated by black circles. Figure 2E shows the specificity of the LGR5 antibodies generated in the study. E. Epitope mapping for a-LGR5 hybridoma clones 1, 3, and 4 as in Figure 1B, using the fragment depicted in Figure 2A. Figure 2F shows the specificity of the LGR5 antibodies generated in the study. F. Sequence alignment of the N-terminal 15 amino acids of human LGR5 corresponding to fragment 1A with the corresponding regions of other LGR family members.Sequence alignment was based on the three invariant cysteine ​​residues boxed. Amino acid differences in the cynomolgus sequence are in red. [Figure 2-2] Figure 2G shows the specificity of the LGR5 antibodies produced in the study. G. Wnt pathway reporter assay (SuperTopFlash assay) for HEK293T cells treated with Wnt3A ligand, R-spondin, and approximately 10-fold molar excess level of either IgG1 or a-LGR5 over R-spondin. ns, no significant difference as determined by two-tailed t-test. Figure 2H shows the specificity of the LGR5 antibodies produced in the study. H. Immunofluorescence using Fl-α-LGR5 for HEK293T cells expressing transgenic LGR4 or LGR5 as in Figure 1C. [Diagram 3] Figure 1: Healthy tissue and cancer survey of LGR5 expression levels. A. Quantification of LGR5 expression levels in normal tissue (FT), 28 ovarian cancer cases (OvC), and 14 peritoneal cancer cases (OmC) from 27 fallopian tube samples from the Cambridge ovarian cancer TMA. Significance levels calculated using a two-tailed t-test comparing ovarian cancer cases (OvC) and peritoneal cancer cases (OmC) with LGR5 protein levels in fallopian tube (FT) samples. B. Quantification of LGR5 expression levels in samples from the Cambridge Brain cancer TMA in five samples each of normal brain tissue (brain), low-grade glioma (LGG), and glioblastoma (GBB). Significance levels calculated using a two-tailed t-test comparing either low-grade glioma (LGG) or glioblastoma (GBM) cases with normal brain tissue. [Figure 4-1]FIG. 4A shows LGR5 expression analysis in cancer. A. Normalized (log2 median centered) LGR5 gene expression levels are shown for each cancer type, ordered by median LGR5 gene expression (black dots). The grey horizontal line shows the median LGR5 expression for all cancer samples. Samples with LGR5 expression lower or higher than the median (grey line) are shown in light or dark red, respectively. Cancer types in which more than 70% of the samples were higher than the median LGR5 expression are defined as "LGR5 high tumors" and printed in bold. FIG. 4B shows LGR5 expression analysis in cancer. B. Comparison of LGR5 gene expression in tumors (red) vs. normal tissues (grey) for LGR5 high tumors, where available. The grey horizontal line shows the median LGR5 expression for all pan-cancer tumors. Significance levels for differences between cancer vs. healthy tissue (Wilcoxon test) are indicated: **p≦0.01, ***p≦0.001, ****p≦0.0001). [Figure 4-2] FIG. 4C shows LGR5 expression analysis in cancer. C. Representative images of fallopian tube tissues (left panel set), ovarian cancer (middle panel set), and peritoneal cancer (right panel set). The arrow in one of the fallopian tube sample sets indicates a very rare example of an epithelial cell containing LGR5-positive intracellular puncta. The white numbers in the insets refer to the relative corresponding LGR5 expression levels consistent with the Cambridge ovarian cancer TMA scoring. FIG. 4D shows LGR5 expression analysis in cancer. D. Relative expression levels of β-catenin in fallopian tube, ovarian cancer, and peritoneal cancer sample sets. Significance levels between fallopian tube (FT) and either ovarian cancer (OvC) or peritoneal cancer (OmC) expression data sets were calculated by two-tailed t-test. [Figure 4-3]FIG. 4E shows LGR5 expression analysis in cancer. E. Representative images of brain tissue (left panel set), GBM (middle panel set), and LGG (right panel set). White numbers in insets refer to LGR5 expression levels based on the criteria used for TMA scoring. FIG. 4F shows LGR5 expression analysis in cancer. F. Quantification of LGR5 protein expression in B cells, CD4+ T cells, and CD8+ T cells from healthy donor PBMC. There was no significant difference in cellular LGR5 expression (mean fluorescence intensity or % LGR5-expressing cells) in the presence or absence of a-LGR5 blocking peptide. [Diagram 5] Characterization of LGR5 expression in NALM6 and LoVo cell lines. A. LGR5 transcript levels in LoVo and SW480 cell lines measured by RT-PCR using TBP as housekeeping gene. B. LGR5 Western blot analysis of lysates from five CRC cell lines. An antibody raised against vinculin was used as a loading control. C. Flow cytometry analysis of LoVo and SW480 cells with antibodies preincubated with Fl-a-LGR5 (red plot) or fragment 1A peptide (black plot). D. Relative LGR5 transcript levels in NALM6, REH, and 697 pre-B-ALL cell lines measured by RT-PCR using TBP as housekeeping gene. E. Western blot analysis of LGR5 protein levels in lysates from pre-B-ALL cell lines. F. LGR5 protein levels in three pre-B-ALL cell lines, 697, REH, and NALM6, analyzed by flow cytometry. [Figure 6] Figure 1 shows the subcellular localization of LGR5. A. Detection of LGR5 in NALM6 and LoVo cell lines using immunofluorescence with Fl-α-LGR5. B. Co-immunofluorescence detection of puncta decorated by either LGR5 or LAMP1 in LoVo cells using antibodies raised against Fl-a-LGR5 or LAMP1. [Figure 7-1]FIG. 7A shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). A. Time course of FL-α-LGR5 internalization by LGR5-eGFP expressing HEK293T cells. Top right panel - magnified image shows association of FL-α-LGR5 with the cell periphery after 5 min, followed by colocalization with internal LGR5-eGFP associated puncta within 30 min (middle right panel). Bottom right panel - no association or internalization of FL-α-LGR5 for cells expressing LGR4-eGFP. Scale bar, 10 μM. FIG. 7B shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). B. Time course of Fl α LGR5v4 (red) internalization by NALM6 cells detected by immunofluorescence. At multiple time points, fixed cells were probed with fluorescent (Alexa488) phalloidin (green) and Hoechst (blue). Bottom panel, Fl-α-LGR5v4 replaced by Fl-α-LGR5v6 (red) as an isotype control. In the bottom right panel, the signal from fluorescent Alexa488 phalloidin is omitted. Scale bar, 10 μM. FIG. 7C shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). C. Time course of Fl-α-LGR5 (red) internalization by LoVo cells. F-actin and nuclei were visualized with Alexa488 phalloidin and Hoechst probes, respectively. For the right two panels, Fl-α-LGR5 was preincubated with fragment 1A. In the rightmost image, the signal from Alexa488 phalloidin was omitted. Scale bar, 10 μM. FIG. 7D shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2).D. Flow cytometric detection of Fl-α-LGR5 (red histogram) or fluorescent isotype control (gray histogram) association with NALM6 cells after 1 h incubation at 4° C. (upper panel) or 37° C. (lower panel). Numbers represent the percentage of cells with detectable fluorescence. FIG. 7E shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). E. Internalization of FL-α-LGR5 by c18 control or c20 CRISPR / Cas9-LGR5 targeted LoVo cell lines monitored and detected by flow cytometry. [Figure 7-2]FIG. 7F shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). F. Flow cytometry analysis of LoVo and NALM6 cells after incubation of Fl-IgG1 (orange) and Fl-α-HER2 (blue) with either Fl-α-LGR5v4 (red) for 60 min. FIG. 7G shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). G. Time course of association of LoVo cells with Fl-α-LGR5v4 and Fl-α-HER2. Percent association was scored as the fraction of cells associated with fluorescent signal from Fl-α-HER2 (light grey bars) or Fl-α-LGR5v4 (dark grey bars) per field at the indicated time points. Data points are the average of six individual scoring experiments, each measuring 80-200 cells for two independent experiments. Error bars indicate SD in the scoring experiments. No significant differences were observed between LoVo cells and Fl-α-LGR5v4 or Fl-α-HER2 association or between any of the time points. Figure 7H shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). H. Time course of percent internalization of Fl-α-LGR5v4 and Fl-α-HER2 by LoVo cells. Internalization data are scored counts of six experiments with 80-200 cells for two independent experiments. Error bars indicate SD. Significant differences in internalization between Fl-α-LGR5v4 or Fl-α-HER2 are at the *p<0.001 significance level as determined by two-tailed t-test at 5-180 min. Figure 7I shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2).I. Colocalization between internalized Fl-α-LGR5v4 puncta and markers of various subcellular components and IQGAP1 in LoVo cells. Colocalization data derived from automated scoring of puncta in images of internalized Fl-α-LGR5v4 and subcellular markers detected by indirect immunofluorescence, composite of at least 200 cells for two individual experiments. Right - Wheel graph showing the fractional association of internalized Fl-α-LGR5v4 with specific subcellular vesicle markers in LoVo cells. Figure 7J shows rapid internalization of cell surface LGR5. Murine clone 2 modified with a fluorescent tag was used (see clone 2 in Tables 1 and 2). J. Colocalization between internalized Fl-α-LGR5 puncta and markers of various subcellular compartments and IQGAP1 in NALM6 cells, scored as above. Right - Wheel graph showing the association of fractions of internalized Fl-α-LGR5v4 with specific intracellular vesicle markers in NALM6 cells. [Figure 8] Figure 1 shows CRISPR / Cas9 targeting of exon 1 of LGR5. Sequencing of exon 1 of the LGR5 locus shows that clone 20 (c20) has an in-frame deletion of 7 amino acids in the coding region of the signal peptide. [Figure 9] Figure 1 shows in vitro killing of LGR5-expressing cell lines by α-LGR5-ADC. NALM6 or REH cell lines were treated with α-LGR5-ADC and cell survival was determined after 72 hours. Cell killing data was fitted to a non-linear EC50 shift model resulting in EC50 values ​​of 4 and 10 nM, respectively. As a control, NALM6 cells were treated with non-cleavable a-LGR5-ADCNC, which did not reduce cell numbers after 72 hours compared to untreated controls. B. LoVo cells were treated with either α-LGR5-ADC or α-LGR5-ADCNC as a non-cell killing control. Modeling of the a-LGR5-ADC-mediated cell killing data resulted in an EC50 of 9 nM. In this figure, murine clone 2 modified with MMAE conjugate is used. Sequences are shown in Tables 1 and 2. [Figure 10] FIG. 1 shows the format and chemistry supporting the IgG and α-LGR5 based ADCs used in the study. (Walsh SJ, Omarjee S, Galloway WRJD, Kwan TTL, Sore HF, Parker JS, Hyvonen M, Carroll JS, Spring DR. A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates. Chem Sci. 2019;10(3):694-700). [Figure 11] Figure 1. In vivo targeting of NALM6 tumors with murine α-LGR5-ADC. A. Experimental design. B. NALM6 tumor burden measured by IVIS imaging over the course of treatment with either a-LGR5-ADC or IgG1-ADC control. *p<0.01, **p<0.001. C. IVIS images of dorsal views of control and a-LGR5-ADC treated mice at the end point of the experiment. D. Spleen mass and absolute number of NALM6 tumor cells in the spleen at the end point of the experiment. E. Relative number of NALM6 cells per gram of blood in mice treated with a-LGR5-ADC or IgG1-ADC control. F. Number of NALM6 cells extracted from femurs of mice from the two treatment groups. (D-F) *p<0.001. In this figure, murine clone 2 modified with MMAE conjugate is used. Sequences are shown in Tables 1 and 2. [Figure 12]Figure 1 shows α-LGR5-ADC targeting of NALM6 tumors. A. H&E staining of tissue sections from IgG1-ADC (top row) and a-LGR5-ADC (bottom row) treated mice. No differences in gross morphology were observed, and there was no difference in the number of NALM6 cells in tissues between experimental groups, except in the spleen, where >20-fold more NALM6 cells were observed (see inset). B. Immunofluorescence of intestinal epithelial sections from IgG1-ADC (top row) and α-LGR5-ADC (bottom row) treated mice using antibodies against Ki67 antigen and β-catenin. No differences in gross morphology of the intestinal epithelial monolayer were observed. C. β-catenin staining at the cell margins adjacent to cell-cell contacts in intestinal epithelial sections from IgG1-ADC (top row) and α-LGR5-ADC treated mice (left graph), and assessment of the average number of Ki67-positive nuclei per confocal section. [Figure 13] Figure 1 shows in vivo targeting of NALM6 tumors with humanized α-LGR5v4-ADC. A. Experimental design. B. NALM6 tumor burden in dorsal view measured by IVIS imaging over the course of treatment with either a-LGR5v4-ADC or a-LGR5v4-ADC control. *p<0.01, **p<0.001. C. IVIS images of mice treated with α-LGR5v4-ADC or α-LGR5v6-ADC control at the experimental endpoint. D. Spleen mass and absolute number of NALM6 cells in spleen at the experimental endpoint. *p<0.001. E. NALM6 cell counts in blood of mice treated with α-LGR5v4-ADC and α-LGR5v6-ADC control. F. NALM6 cell counts in bone marrow of α-LGR5v4-ADC and α-LGR5v6-ADC treated mice at the experimental endpoint. (D–F) *p<0.001. [Figure 14-1]Figure 14A shows that α-LGR5v4 specifically binds to human and cynoLGR5 and α-LGR5v4-ADC targeting NALM6 tumors. A. Western blot analysis of lysates from transgenic murine, human, and cynoLGR5-expressing HEK293T cells using antibodies against HA and humanized antibodies α-LGR5v4 and a-LGR5v6. Note that humanized α-LGR5v6 lost all binding to LGR5. Figure 14B shows that α-LGR5v4 specifically binds to human and cynoLGR5 and α-LGR5v4-ADC targeting NALM6 tumors. B. Indirect immunofluorescence analysis of human and murine LGR family-expressing HEK293T cells using a-LGR5v4 antibody (red), Alexa488-conjugated phalloidin F-actin probe with eGFP fluorescent imaging. In the bottom row of images, LGR5-expressing HEK293T cells were stained with a-LGR5v6. [Figure 14-2]Figure 14C shows that α-LGR5v4 specifically binds to human and cynoLGR5 and α-LGR5v4-ADC targeting NALM6 tumors. C. H&E staining of tissue sections from a-LGR5v6-ADC (top row) and a-LGR5v4-ADC (bottom row) treated mice. No difference in gross morphology was observed, and no difference was detected in the number of NALM6 cells in tissues between experimental groups, except in the spleen, where >20-fold more NALM6 cells were observed (see inset). Figure 14D shows that α-LGR5v4 specifically binds to human and cynoLGR5 and α-LGR5v4-ADC targeting NALM6 tumors. D. Immunofluorescence of intestinal epithelial sections from α-LGR5v6-ADC (top row) and α-LGR5v4-ADC (bottom row) treated mice using antibodies against Ki67 antigen and β-catenin. No differences in gross morphology of the intestinal epithelial monolayer were observed. FIG. 14E shows that α-LGR5v4 specifically binds human and cynoLGR5 and α-LGR5v4-ADC targeting NALM6 tumors. E. β-catenin staining (left graph) at the cell margins adjacent to cell-cell contacts in intestinal epithelial sections from IgG1-ADC (top row) and α-LGR5-ADC treated mice, and assessment of the average number of Ki67 positive nuclei per confocal section. (C-E) Experiments are shown in FIG. 13. [Figure 15-1] Figure 1: CAR cell killing. a-LGR5-CAR cell killing. HEK293 cells were transfected with human (A) and cynomolgus (B) LGR5 to serve as target cells in killing assays. (C) a-LGR5 CAR NK92 cells form efficient synapses with tumor target cells. (D) Tumor cell killing of NALM6, REH, and 647 ALL tumor cell lines at the indicated effector:target ratios at 5 hours. [Figure 15-2]Figure 1: CAR cell killing. a-LGR5-CAR cell killing. HEK293 cells were transfected with human (A) and cynomolgus (B) LGR5 to serve as target cells in killing assays. (C) a-LGR5 CAR NK92 cells form efficient synapses with tumor target cells. (D) Tumor cell killing of NALM6, REH, and 647 ALL tumor cell lines at the indicated effector:target ratios at 5 hours. [Figure 16-1] Figure 16A shows that α-LGR5 bispecific T cell engagers (BiTEs) activate T cells and result in efficient tumor cell destruction. (A) PBMCs were incubated with NALM6 tumor cells in the presence of α-LGR5 scFv control, α-LGR5 LC, or CL BiTEs, respectively. CD4+ and CD8+ T cell activation was determined by flow cytometric analysis of CD69 and CD25 expression after 24 hours. [Figure 16-2] Figure 16B shows that α-LGR5 bispecific T cell engagers (BiTEs) are activated T cells and result in efficient tumor cell destruction. (B) NALM6 tumor cells were co-cultured with cytotoxic CD8+ T cells generated from healthy donor PBMCs in the presence of α-LGR5 scFv control, α-LGR5 LC, or CL BiTEs, respectively. Tumor cell killing was assessed after 5 hours at an effector to target ratio of 5:1. [Figure 17]Analysis of LGR5 expression. A. LGR5 mRNA expression in NALM6 (ALL), LoVo (CRC), and HepG2 (HCC) human cell lines. RNA was extracted from cell lines at multiple time points and subjected to qRT-PCR analysis of LGR5 using Taqman probe. TBP served as a housekeeping gene. B. Western blot analysis of LGR5 protein expression levels in HepG2 cells. HepG2 cells were lysed and loaded with 40 μg protein per well. Membranes were incubated with α-LGR5 antibody (19-24-1, (1:1000), 4° C., overnight) followed by incubation with goat anti-mouse IgG-HRP ((1:15000), RT, 1 h). Actin serves as a loading control. [Figure 18] Figure 1 shows the sensitivity of CRC organoid cell lines expressing different levels of LGR5 to treatment with α-LGR5-ADC.A. Using humanized antibody against LGR5, immunofluorescence imaging of CRC organoid line 1 and 2 was used to quantify the relative level of LGR5 protein.LGR5 protein expression was detectable in CRC organoid line 1, approximately 4-fold higher than CRC organoid line 2.B. The presence of cleaved caspase 3 in CRC organoid line 1 and 2 24 hours after α-LGR5-ADC treatment was used as a proxy for cells undergoing apoptosis in response to treatment with α-LGR5-ADC.CRC organoid line 1 was less sensitive to treatment with α-LGR5-ADC than CRC organoid line 2; the estimated EC50 levels of organoid cell apoptosis were 5nM and 100nM, respectively. [Figure 19]α-LGR5-BiTEs lead to specific activation of human CD4+ and CD8+ T cells in the presence of LGR5+ tumor cells and induce efficient tumor cell destruction in vitro. A. CL(CD3 / LGR5)- and LC(LGR5 / CD3)-BiTE-mediated activation of CD4+ T cells (left panel) and CD8+ T cells (right panel) in healthy donor PBMCs in the presence or absence of NALM6 tumor cells, determined as the percentage cells with combined expression of CD25 and CD69 by flow cytometry after 24 hours. Control (ctrl) with no molecule added, scFv refers to treatment with α-LGR5scFv fragment. Error bars represent SD of three independent activation assays using different healthy donor PBMCs. Significant differences in T cell activation between the addition of scFv and either LC or CL BiTE molecules were determined by two-tailed t-test and indicated at a significance level of **p<0.001. B. α-LGR5-BiTEs result in efficient tumor cell destruction in vitro. Cytotoxic CD8+ T cell killing of NALM6 target cells 5 hours after the absence (ctrl) or addition of scFv or LC or CL BiTEs is shown. Data shown are from three donors and error bars represent SD. Effector to target cell ratios are 10:1 and 5:1, respectively. Significant differences in CD8+ T cell killing were determined by two-tailed t-test with significance levels of *p<0.005 and **p<0.001. [Figure 20]Figure 1: a-LGR5-CAR NK92 cells efficiently kill HEK293 cells overexpressing LGR5 and preferentially kill LGR5HIGH expressing tumor cells. A. a-LGR5-CAR NK92 cells efficiently kill HEK293 cells overexpressing LGR5. Cell killing activity of NK92 cells or NK92 cells expressing a-LGR5-CAR-CD28- or a-LGR5-CAR-4-1BB. hLGR5-eGFP overexpressing HEK293T target cells were incubated with effector NK92 cells at effector to target ratios of 2.5:1 and 10:1, respectively, for 5 or 9 hours. Error bars represent SD of three independent experiments. Significant differences in target cell killing were determined by two-tailed t-test and are indicated as *p<0.05 and **p<0.001. There was no significant difference in target cell killing between the 5:1 or 10:1 cell ratios. B. α-LGR5-CAR NK92 cells preferentially kill LGR5HIGH expressing tumor cells. NALM6 (left panel, LGR5HIGH) and REH (right panel, LGR5LOW) target cell killing activity after 12 hours of incubation of NK92 cells or NK92 cells expressing α-LGR5-CAR-CD28 or α-LGR5-CAR-4-1BB CARs at effector to target ratios of 2.5:1 and 10:1 (top and bottom panels, respectively). Error bars represent SD of three independent experiments. Significant differences in target cell killing were determined by two-tailed t-test with *p<0.05 and **p<0.01. [Figure 21] Figure 1 shows that α-LGR5-CAR NK92 cells efficiently kill HepG2 tumor cells. HepG2 cells were pre-seeded in 96-well plates for 24 hours before assay. NK92 cells and α-LGR5-CAR NK92 cells were added to target cells at a ratio of 5:1. Cell death was assessed using Apotracker Green and monitored for 15 hours using Incucyte SX5 (Sartorius). Error bars represent SEM of 6 different wells. [Figure 22]Figure 1 shows that α-LGR5-CAR NK92 cells kill NALM6 tumors in vivo. 1x10^6 NALM6 cells were injected i,v into NSG mice; on days 5, 7, and 10, mice were injected iv with 10x10^6 parental NK92 cells or α-LGR5-CAR NK92 cells. Mice were harvested on day 12 and Nalm6 cells were counted in the spleen. (Average NALM6 count for NK92 cell treated mice: 20664, average for α-LGR5-CAR NK92 cell treated NSG mice: 11630). One outlier was removed after Grubb's test. [Figure 23] Figure 1 shows that α-LGR5-CAR T cells efficiently kill HepG2 tumor cells. HepG2 cells were pre-seeded in 96-well plates for 24 hours before assay. Non-transduced T cells and a-LGR5-CAR T cells were added to target cells at a ratio of 5:1 (A) or 10:1 (B). Cell death was assessed using Apotracker Green and monitored for 15 hours using Incucyte SX5 (Sartorius). Error bars represent SEM of 6 different wells. [Figure 24] Figure 1 shows that α-LGR5-CAR T cells efficiently kill LoVo tumor cells. LoVo cells were pre-seeded in 96-well plates for 24 hours before assay. Non-transduced and α-LGR5-CAR T cells were added to target cells at a ratio of 5:1 (A) or 10:1 (B). Cell death was assessed using Apotracker Green and monitored for 15 hours using Incucyte SX5 (Sartorius). Error bars represent SEM of 6 different wells. [Diagram 25]α-LGR5-CAR T cells kill NALM6 tumor cells in vivo. 1x10^6 NALM6-LucYFP cells were injected iv into NSG mice. On days 4 and 7, mice received 2.5x10^6 non-transduced T cells or α-LGR5-CAR T cells or 1x10^6 αCD19-CAR T cells, respectively. A. IVIS imaging on day 10. NALM6 cell counts in bone marrow (taken from one tibia) of NSG mice on day 11 treated with non-transduced T cells (negative control), α-LGR5-CAR T cells, or α-CD19-CAR T cells (positive control, baseline). B. Mean NALM6 counts for non-transduced T cell treated NSG mice: 22355, mean for α-LGR5-CAR T cell treated NSG mice: 6296, mean for α-CD19-CAR T cell treated NSG mice: 10. [Figure 26] α-LGR5-CAR-NK92 cells do not kill HEK cells overexpressing human LGR4, LGR6, or mouse Lgr5. α-LGR5-CAR NK92 cells do not target HEK293 cells overexpressing human LGR4, LGR6, but rather weakly target cells overexpressing mouse Lgr5. Cell killing activity of NK92 cells or NK92 cells expressing a-LGR5-CAR-CD28 or a-LGR5-CAR-4-1BB. HEK293T target cells overexpressing the indicated proteins were incubated with effector NK92 cells at effector to target ratios of 2.5:1 and 10:1 for 9 hours. Error bars represent SD of three independent experiments. [Figure 27] Figure 1 shows α-LGR5-CAR-T cell targeting of NALM6 cells. α-LGR5-CAR T cells efficiently kill NALM6 tumor cells. NALM6 tumor target cell killing activity of α-LGR5-41BB CAR-T cells at the indicated effector-to-target ratios was assessed by VITAL assay after 6 hours. Error bars represent SD of three α-LGR5-CAR T cell batches generated from PBMCs of three different healthy donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention is described in detail below. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0063] Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references listed and discussed throughout this application, unless otherwise indicated. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0064] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, and the laboratory procedures and techniques are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0065] The inventors of the present invention have surprisingly identified an antibody that binds to a specific epitope within amino acids 22 to 37 of human LGR5. As demonstrated in the Examples, the antibody is highly specific, has high affinity and is rapidly internalized into cells, making it a highly effective research tool as well as extremely useful in therapy.

[0066] Leucine-rich repeat-containing G protein receptor 5 (LGR5) has been characterized as a stem cell and cancer stem cell marker. Previous analysis of LGR5 transcript levels has shown that high levels of expression distinguish malignant diseases, such as colorectal cancer (CRC) and pre-B acute lymphoblastic leukemia (pre-B ALL), from healthy tissues, suggesting that LGR5 protein expression may provide a molecular tool for prognosis and treatment. The inventors have developed a highly specific, high affinity antibody against the extracellular domain of human LGR5 45 (α-LGR5) that detects high LGR5 protein levels in colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL. In contrast, normal colon and rectal epithelium, liver, ovarian tissue, brain, and immune cell types have low to undetectable levels of LGR5 protein. LGR5 is rapidly internalized from the plasma membrane and transported to intracellular vesicular compartments, including lysosomes.

[0067] We have shown that treatment of high LGR5-expressing CRC and pre-B ALL cancer cell lines with antibody-drug conjugates of a-LGR5 (α-LGR5-ADC) resulted in effective cell killing at nanomolar concentrations. In vivo intervention of pre-B ALL tumors with α-LGR5-ADC resulted in rapid tumor attrition. We have demonstrated the therapeutic utility of humanized α-LGR5 by using α-LGR5 chimeric antigen receptors (CARs) and corresponding scFv fragments for the generation of bispecific T cell engagers (BiTEs). We have demonstrated that α-LGR5-CAR-NK92 cells were effective in killing LGR5-expressing cells, while α-LGR5 / α-CD3 BiTEs induced T cell activation and killing of NALM6 cells by cytotoxic CD8+ T cells. Taken together, the present invention establishes an α-LGR5-based therapeutic modality that effectively distinguishes and targets CRC, HCC, and pre-B ALL tumor cells.

[0068] We also established that LGR5 expression is elevated as a hallmark of CRC, HCC, and pre-B ALL. Importantly, when these tissues are examined for both transcription and LGR5 protein levels, normal tissues have very low to undetectable LGR5 levels, paving the way for therapeutics to target malignancies that overexpress this protein. Indeed, our approach establishes CRC, HCC, and pre-B ALL as high-priority cancer targets for a-LGR5-based therapeutics, and enables future studies to determine other targetable cancer types and to stratify lGR5 overexpression as a prognostic marker. This is particularly pertinent for the assessment of LGR5 protein levels in HCC, where high LGR5 protein expression may be used to further stratify HCC subsets with activating mutations in β-catenin, characterized by low T-cell infiltration and thus referred to as immune deserts. This HCC subset is predicted to be refractory to both checkpoint inhibition and cell therapy.

[0069] Indeed, a recent report from the CheckMate 459 clinical trial (NCT02576509) evaluating nivolumab (a PD1 checkpoint inhibitor) versus sorafenib (a small molecule kinase inhibitor) in HCC failed to meet its endpoint goal of improved overall survival. However, we propose that excluding high LGR5 expressing patient cohorts may prove important in clinical trial outcomes. Thus, stratifying HCC patients with high levels of LGR5 using anti-LGR5 represents an interesting biomarker opportunity to report β-catenin mutant subsets and support the use of therapeutic molecules such as α-LGR5-ADCs where drug efficacy is not dependent on immune infiltration. Thus, the present invention also relates to the use of LGR5 as a prognostic marker and the associated methods described herein.

[0070] Thus, the present invention provides antibodies or fragments thereof that bind to human LGR5, where the antibody binds to an epitope located within or including or consisting of amino acids 22-37 of SEQ ID NO: 1, immunoconjugates and pharmaceutical compositions comprising such antibodies, as well as isolated nucleic acid molecules, vectors and host cells for producing such antibodies. Also provided are methods of using the antibodies disclosed herein to detect human LGR5, methods of diagnosing and methods of treating disease, particularly cancer and / or inflammatory disease.

[0071] The antibody of the present invention binds to LGR5 with high affinity and specificity. In addition, the inventors have shown that the antibody of the present invention can be rapidly internalized by cells. Thus, the antibody described herein is capable of binding to the extracellular domain of LGR5 and being internalized. The inventors have also shown that, relative to Trastuzumab, it is extremely rapid internalization (within 5 minutes), whereas only 40% of Teas is internalized after 3 hours. Moreover, when conjugated to a payload, the conjugated antibody mediates effective cell killing at nanomolar concentrations.

[0072] These properties of antibodies mean that they can be exploited therapeutically, for example, to treat cancer and / or inflammatory diseases. For example, antibodies can be used to bring therapeutic molecules, such as drugs, to the vicinity of LGR5. Thus, the antibodies of the present invention can be used to specifically deliver therapeutic molecules to cancers, particularly cancers that express or overexpress LGR5. Our studies show that the specific antibodies of the present invention do not exhibit off-target toxicity, further validating their use in therapy.

[0073] Furthermore, in addition to their use as highly useful research tools, the antibodies of the present invention can be used, for example, as imaging agents in methods for diagnosing cancer and / or inflammatory diseases, or in other biomarker-related methods.

[0074] The antibodies and fragments of the present invention specifically bind to wild-type human LGR5 (UniProt Accession No. O75473), the amino acid sequence of which is shown below (SEQ ID NO: 1).

[0075] [ka]

[0076] Unless otherwise specified, the term LGR5 as used herein refers to human LGR5. The term LGR5 includes variants, isoforms, and species homologs of human LGR5. The antibody binds to the extracellular domain. The antibody that binds to LGR5 is referred to herein as a-LGR5, aLGR5, or α-LGR5 or αLGR5. These terms are used interchangeably.

[0077] The antibody of the present invention binds to LGR5. The term "bind" as used herein means that the antibody binds to an antigen with sufficient affinity so that it is useful as a therapeutic agent in target cells or tissues expressing the antigen. Binding reactions can be demonstrated by standard methods, for example by reference to negative control tests using antibodies of irrelevant specificity.

[0078] The present invention provides antibodies, particularly humanized antibodies, that specifically bind to human LGR5. In other words, binding to the LGR5 antigen is measurably different from non-specific interactions. As shown in the examples, the antibodies of the present invention that bind to humans do not cross-react with mouse LGR5 or human LGR4 or 6. Preferably, the antibodies of the present invention bind to human LGR5 and also bind to cynoLGR5.

[0079] The terms "specific binding," "specifically binds," or "is specific" for a particular polypeptide or epitope on a particular polypeptide target, as used herein, refer to, for example, a binding activity of at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 The specific binding may be exhibited by a molecule having a Kd of 0.7-3 nM or higher. In one embodiment, the antibody or antibody fragment has a Kd of 0.7-3 nM. In one embodiment, the term "specific binding" refers to binding by a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0080] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or an antigen-binding portion thereof, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding properties of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. The term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they retain the antigen-binding ability of the antibody. The term "antibody" as used herein also includes, for example, T cell engagers, such as bispecific T cell engagers (BiTEs). As will be recognized by those skilled in the art, a bispecific T cell engager (BiTE) is a fusion protein that includes two scFvs of different antibodies, where one of the scFvs binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule.

[0081] In one embodiment, a BiTE or CAR described herein comprises an scFv as set forth in SEQ ID NO: 211, or a sequence having at least 70%, 80%, or 90% sequence homology thereto, or a sequence having the CDRs of SEQ ID NO: 211. Thus, in one embodiment, a BiTE comprises the CDRs of clone 2.4, also numbered as SEQ ID NOs: 212, 213, and 214, and the heavy chain CDRs of 215, 216, and 217. In one embodiment, a BiTE or CAR described herein comprises the VH and VL of clone 2.4, or a sequence having at least 90% sequence identity thereto. In several embodiments, an antibody can comprise a monoclonal antibody. The term "monoclonal antibody" refers to an antibody obtained from a single clone of a cell or cell line. The individual antibodies are identical and / or bind to the same epitope. Unlike a polyclonal antibody, which includes different antibodies against different epitopes, each monoclonal antibody in the preparation is directed against a single epitope.

[0082] In a full-length antibody, each heavy chain consists of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is made up of three domains, C H 1. C H 2, and C H Each light chain consists of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region consists of one domain C L It consists of:

[0083] The heavy and light chain variable regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of molecule.

[0084] The term "CDR" refers to the complementarity determining regions in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, named CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0085] The Kabat complementarity determining regions (CDRs) are the basis for sequence diversity and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain).

[0086] The system described by Kabat is used herein. The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These art-recognized terms refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions or antigen-binding portions of an antibody.

[0087] A chimeric antibody is a recombinant protein that contains the variable domains, including the complementarity determining regions (CDRs), of an antibody derived from one species, e.g., a rodent antibody, but the constant domains of the antibody molecule are derived from those of a human antibody. In veterinary applications, the constant domains of the chimeric antibody may be derived from those of another species, e.g., feline or canine.

[0088] A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species, e.g., a rodent antibody, have been transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted with human antibody framework region sequences.

[0089] The term "antigen-binding site" refers to the part of an antibody or antibody fragment which comprises the area that specifically binds to an antigen. An antigen-binding site may be provided by one or more antibody variable domains. Preferably, an antigen-binding site is provided by the associated V H and V L Included within.

[0090] Antibody fragments according to the present invention are functional parts of antibodies, such as F(ab')2, Fab, Fv, sFv, and the like. Thus, the term refers to antigen-binding fragments and is used interchangeably with antigen-binding portion of an antibody. Functional fragments of full-length antibodies retain the target specificity of the full-length antibody. Thus, recombinant functional antibody fragments, such as Fab (fragment, antibody), scFv (single-chain variable fragment) and single domain antibodies (dAbs), have been used to develop therapeutics as an alternative to mAb-based therapeutics. Derivatives of antibodies are also within the scope.

[0091] The scFv fragment (~25 kDa) contains two variable domains, V H and V L In nature, V H and V LThe domains tend to associate and dissociate non-covalently through hydrophobic interactions. However, stable fragments can be engineered by linking the domains with hydrophilic flexible linkers to create single chain Fvs (scFvs). In one embodiment, the scFv comprises SEQ ID NO: 211, or a sequence having at least 70%, 80%, or 90% sequence homology thereto, or a sequence having the CDRs of SEQ ID NO: 211. In one embodiment, the scFv comprises the CDRs of SEQ ID NOs: 212, 213, and 214, and the heavy chain CDRs of 215, 216, and 217.

[0092] The smallest antigen-binding fragment is a single variable fragment, i.e., V H Or V L The binding to the light / heavy chain partners, respectively, is not necessary for target binding. Such fragments are used in single domain antibodies. Thus, single domain antibodies (approximately 12-15 kDa) are H Or V L It consists of or includes any of the domains.

[0093] As used herein, the term "homology" refers to the percentage of amino acid residues in a sequence that are identical to the residues of the reference polypeptide to which it is compared, after aligning the sequences and, in some embodiments, introducing gaps as necessary to achieve the maximum percent homology, without considering any conservative substitutions as part of the sequence identity. Thus, as used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Any N- or C-terminal extensions, tags, or insertions are not interpreted as reducing the identity or homology. Methods and computer programs for alignment are well known. The percent identity or homology between two amino acid sequences can be determined using well-known mathematical algorithms.

[0094] As used herein, when the term homology is used, it is used interchangeably with identity. Thus, when referring to sequence homology, these values ​​also refer to sequence identity, respectively.

[0095] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty equal to 12 and a gap extension penalty equal to 4. The use of GAP is preferred, but other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197), or the TBLASTN program of Altschul et al. (1990), supra, generally using default parameters. In particular, the psi-Blast algorithm (Altschul et al. (1997) Nucl. Acids Res. 25 3389-3402) may also be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments were performed using Snapgene based on the MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithm (Edgar (2004a) Nucleic Acids Res 32: 1792-7; Edgar (2004b) BMC Bioinformatics 5: 113).

[0096] The present invention relates to an antibody or a fragment thereof that binds to an epitope of LGR5 located within amino acids 22 to 37 of human LGR5 (SEQ ID NO: 1).

[0097] Thus, the antibodies of the invention bind to an epitope of LGR5 that includes one or more of residues 22 to 37 of human LGR5 (SEQ ID NO: 1). For example, the antibodies of the invention bind to an epitope of LGR5 that includes one or more of the following residues: 22 , S 23 , S 24 , P 25 , R 26 , S 27 , G 28 , V 29 , L 30 , L 31 , R 32 , G 33 , C 34 , P 35 , T 36 , H 37 The antibody binds to an epitope of LGR5 that includes one or more, for example all, of the following:

[0098] The present invention also relates to an antibody that binds to an epitope consisting of amino acids 22 to 37 of LGR5 (SEQ ID NO: 1).

[0099] Such epitopes are linear epitopes, as described in more detail below.

[0100] The term "epitope" or "antigenic determinant" refers to a site on the surface of an antigen (e.g., LGR5) to which an immunoglobulin, antibody, or antibody fragment specifically binds. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear and conformational epitopes.

[0101] Epitopes in protein antigens can be formed both from contiguous amino acids (usually linear epitopes) or from non-contiguous amino acids adjacent to each other by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically, but not necessarily, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitope a given antibody or antibody fragment binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides are tested for reactivity with a given antibody or antibody fragment.

[0102] In the present invention, epitopes are mapped by peptide mapping. In particular, epitopes are mapped by testing the reactivity of antibodies with overlapping peptide fragments and by performing Western blot analysis.

[0103] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V L In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR3 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. LThe present invention relates to an antibody comprising a CDR3 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0104] In one embodiment, the antibody comprises a V sequence comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79. L It has a CDR3 sequence or a sequence having at least at least 90% or at least 95% homology thereto.

[0105] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V L In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR1 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. LThe present invention relates to an antibody comprising a CDR1 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77. In one embodiment, the homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0106] In one embodiment, the antibody comprises or consists of an amino acid sequence selected from SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77. L It has a CDR1 sequence or a sequence having at least at least 90% or at least 95% homology thereto.

[0107] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V L In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR2 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. LThe present invention relates to an antibody comprising a CDR2 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0108] In one embodiment, the antibody comprises a V sequence comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78. L It has a CDR2 sequence or a sequence having at least at least 90% or at least 95% homology thereto.

[0109] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V H In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR3 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. HThe present invention relates to an antibody comprising a CDR3 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0110] In one embodiment, the antibody comprises a V sequence comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76. H It has a CDR3 sequence or a sequence having at least at least 90% or at least 95% homology thereto.

[0111] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V H In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR1 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. HThe present invention relates to an antibody comprising a CDR1 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more homology to one of SEQ ID NOs: 2, 8, 14, 40, 26, 32, 38, 44, 50, 56, 62, 68, 74. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0112] In one embodiment, the antibody comprises a V H It has a CDR1 sequence or a sequence having at least 90% or at least 95% homology thereto.

[0113] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising any one of the V H In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a CDR2 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. HThe present invention relates to an antibody comprising a CDR2 sequence or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0114] In one embodiment, the antibody comprises a V H It has a CDR2 sequence or a sequence having at least at least 90% or at least 95% homology thereto.

[0115] In one embodiment, the antibody comprises a V H and V L In one aspect, the present invention also includes a combination of CDR1, 2 and 3 sequences of V H and V L The present invention also relates to an antibody having the CDR1, CDR2 and CDR3 combination of

[0116] In one embodiment, the antibody comprises a V H and V L In one embodiment, the antibody comprises a set of CDR1, 2 and 3 sequences having SEQ ID NO:2. H CDR1, V having SEQ ID NO:3 H CDR2, V having SEQ ID NO:4H CDR3, V with SEQ ID NO:5 L CDR1, V with SEQ ID NO:6 L CDR2, and V having SEQ ID NO:7 L In one embodiment, the antibody comprises a V CDR3 having SEQ ID NO:8. H CDR1, V having SEQ ID NO:9 H CDR2, V having SEQ ID NO: 10 H CDR3, V with SEQ ID NO:11 L CDR1, V with SEQ ID NO: 12 L CDR2, and V having SEQ ID NO: 13 L In one embodiment, the antibody comprises a V CDR3 having SEQ ID NO: 14. H CDR1, V with SEQ ID NO: 15 H CDR2, V with SEQ ID NO: 16 H CDR3, V having SEQ ID NO: 17 L CDR1, V with SEQ ID NO: 18 L CDR2, and V having SEQ ID NO: 19 L In one embodiment, the antibody comprises a V CDR3 having SEQ ID NO: 20. H CDR1, V with SEQ ID NO:21 H CDR2, V having SEQ ID NO:22 H CDR3, V with SEQ ID NO:23 L CDR1, V having SEQ ID NO:24 L CDR2, and V having SEQ ID NO: 25 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO:26. H CDR1, V having SEQ ID NO:27 H CDR2, V having SEQ ID NO:28 H CDR3, V having SEQ ID NO:29 L CDR1, V having SEQ ID NO: 30 L CDR2, and V having SEQ ID NO: 31 L In one embodiment, the antibody comprises a V CDR3 having SEQ ID NO: 32. H CDR1, V with SEQ ID NO:33 H CDR2, V having SEQ ID NO:34 H CDR3, V having SEQ ID NO:35 LCDR1, V with SEQ ID NO:36 L CDR2, and V having SEQ ID NO: 37 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO: 38. H CDR1, V with SEQ ID NO:39 H CDR2, V having SEQ ID NO: 40 H CDR3, V having SEQ ID NO:41 L CDR1, V with SEQ ID NO: 42 L CDR2, and V having SEQ ID NO: 43 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO: 44. H CDR1, V having SEQ ID NO:45 H CDR2, V with SEQ ID NO: 46 H CDR3, V having SEQ ID NO:47 L CDR1, V with SEQ ID NO: 48 L CDR2, and V having SEQ ID NO: 49 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO: 50. H CDR1, V with SEQ ID NO:51 H CDR2, V having SEQ ID NO:52 H CDR3, V with SEQ ID NO:53 L CDR1, V with SEQ ID NO:54 L CDR2, and V having SEQ ID NO:55 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO:56. H CDR1, V with SEQ ID NO:57 H CDR2, V with SEQ ID NO:58 H CDR3, V with SEQ ID NO:59 L CDR1, V with SEQ ID NO: 60 L CDR2, and V having SEQ ID NO: 61 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO:62. H CDR1, V with SEQ ID NO:63 H CDR2, V with SEQ ID NO:64 H CDR3, V with SEQ ID NO:65 L CDR1, V with SEQ ID NO: 66 LCDR2, and V having SEQ ID NO:67 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO:68. H CDR1, V with SEQ ID NO:69 H CDR2, V having SEQ ID NO: 70 H CDR3, V with SEQ ID NO:71 L CDR1, V with SEQ ID NO: 72 L CDR2, and V having SEQ ID NO: 73 L In some embodiments, the antibody comprises a V CDR3 having SEQ ID NO: 74. H CDR1, V having SEQ ID NO: 75 H CDR2, V with SEQ ID NO: 76 H CDR3, V having SEQ ID NO:77 L CDR1, V with SEQ ID NO:78 L CDR2, and V having SEQ ID NO: 79 L Includes CDR3.

[0117] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, H but with the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 2 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 3 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 4 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 9 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 10 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 14 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 15 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 16 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 20 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 21 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 22 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 26 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 27 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 28 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 32 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 33 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 34 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 38 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 39 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 40 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 44 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 45 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 46 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 50 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 51 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 52 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 56 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 57 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 58 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 62 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 63 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 64 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 68 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 69 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 70 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 74 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 75 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 76 or a sequence having at least 90% homology thereto The present invention provides an antibody or fragment thereof comprising:

[0118] In one aspect, the present invention provides an antibody or fragment thereof that binds to LGR5, L but with the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 5 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 6 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 7 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 11 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 12 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 13 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 17 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 18 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 19 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 23 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 24 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 25 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 29 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 30 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 31 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 35 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 36 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 37 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 41 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 42 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 43 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 47 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 48 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 49 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 53 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 54 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 55 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 59 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 60 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 61 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 65 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 66 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 67 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 71 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 72 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 73 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 77 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 78 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 79 or a sequence having at least 90% homology thereto The present invention provides an antibody or fragment thereof comprising:

[0119] [Table 1A]

[0120] [Table 1B]

[0121] In Table 1, clones 1, 2, 3, 4 and 12 are murine antibodies, and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 are humanized antibodies based on clone 2.

[0122] In embodiments where the antibody is a humanized antibody, the antibody may comprise the CDR1, CDR2 and CDR3 sequences shown in Table 1 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16, or sequences with at least 90% homology thereto. In one embodiment, the CDR1, CDR2 and CDR3 sequences shown in Table 1 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 contain one, two or three amino acid modifications, e.g., substitutions, deletions or insertions.

[0123] In one embodiment, the antibody provided comprises the CDR1, CDR2 and CDR3 sequences shown in Table 1 for clone 2.4. Clone 2.4 is also referred to herein as LGR5v4, LGR5 variant 4 or human variant 4. See, e.g., Table 5.

[0124] In one embodiment, the present invention relates to an antibody or fragment thereof that binds to human LGR5, wherein the antibody has a V sequence selected from the sequences shown in Table 2 below. H In one aspect, the present invention relates to an antibody or fragment thereof that binds to LGR5, comprising a V selected from SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. H or an antibody comprising a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0125] In one embodiment, the present invention provides an antibody that binds to LGR5, comprising a V L In one aspect, the present invention relates to an antibody that binds to LGR5, comprising a V selected from SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. Lor an antibody comprising a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0126] The present invention also relates to an antibody that binds to LGR5, comprising any one of the V H and V L The present invention also provides an antibody that binds to LGR5, comprising the sequence: a) V of SEQ ID NO:80 H Sequence and V of SEQ ID NO:81 L array; b) V of SEQ ID NO:82 H Sequence and V of SEQ ID NO:83 L array; c) V of SEQ ID NO:84 H Sequence and V of SEQ ID NO: 85 L array; d) V in SEQ ID NO:86 H Sequence and V of SEQ ID NO:87 L array; e) V in SEQ ID NO:88 H Sequence and V of SEQ ID NO:89 L array; f) V in sequence number 90 H Sequence and V of SEQ ID NO:91 L array; g) V of SEQ ID NO:92 H Sequence and V of SEQ ID NO:93 L array; h) V of SEQ ID NO:94 HSequence and V of SEQ ID NO: 95 L array; i) V of SEQ ID NO:96 H Sequence and V of SEQ ID NO:97 L array; j) V of SEQ ID NO:98 H Sequence and V of SEQ ID NO: 99 L array; k) V of sequence number 100 H Sequence and V of SEQ ID NO: 101 L array l) V of SEQ ID NO:102 H Sequence and V of SEQ ID NO: 103 L A sequence; or m) V of SEQ ID NO: 104 H Sequence and V of SEQ ID NO: 105 L array The present invention provides an antibody comprising:

[0127] In one embodiment, the antibody has the V H and V L Contains arrays.

[0128] [Table 2A]

[0129] [Table 2B]

[0130] In Table 2, clones 1, 2, 3, 4 and 12 are murine antibodies and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 are humanized antibodies.

[0131] In some embodiments where the antibody is a humanized antibody, the antibody may have the VV ... H and V LThe sequence may comprise the sequence, or a sequence having at least 90% homology thereto.

[0132] In one embodiment, the V H and V L The sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications, e.g., substitutions, deletions or insertions.

[0133] The antibody may comprise a CH2 domain. The CH2 domain is preferably located N-terminal to the CH3 domain, as in human IgG molecules. The CH2 domain of the antibody is preferably a human IgG1, IgG2, IgG3, or IgG4 CH2 domain, more preferably a human IgG1 CH2 domain. The sequences of human IgG domains are known in the art.

[0134] The antibody may also comprise an immunoglobulin hinge region, or a portion thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate to form a dimer. Preferably, the hinge region or a portion thereof is a human IgG1, IgG2, IgG3 or IgG4 hinge region or a portion thereof. More preferably, the hinge region or a portion thereof is an IgG1 hinge region or a portion thereof.

[0135] The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, and most preferably a human IgG1 CH3 domain.

[0136] The antibodies of the invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. The sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art. The antibodies of the invention may comprise a non-human IgG constant region, for example, a rabbit IgG1 constant region.

[0137] In another embodiment, the antibody comprises or consists of a polypeptide sequence set forth for any one of the antibody clones in Table 3, or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. Thus, the antibody comprises or consists of a polypeptide sequence set forth for any one of the antibody clones in Table 3, or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. H、 C H、 V L and C L or consisting of an amino acid sequence selected from a combination of sequences, or a sequence having at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0138] Thus, the present invention relates to an antibody that binds to LGR5 and: a) V of SEQ ID NO: 106 H Sequence: C of SEQ ID NO: 107 H Sequence: V of SEQ ID NO:108 L and C of SEQ ID NO:109 L array; b) V in SEQ ID NO:110 H Sequence: C of SEQ ID NO:111 H Sequence: V of SEQ ID NO:112 L and C of SEQ ID NO:113 L array; c) V of SEQ ID NO:114 H Sequence: C of SEQ ID NO:115 H Sequence: V of SEQ ID NO:116 L and C of SEQ ID NO:117 L array; d) V of SEQ ID NO:118 H Sequence: C of SEQ ID NO:119 H Sequence: V of SEQ ID NO:120 L and C of SEQ ID NO:121L array; e) V of SEQ ID NO: 122 H Sequence: C of SEQ ID NO:123 H Sequence: V of SEQ ID NO:124 L and C of SEQ ID NO:125 L array; f) V of SEQ ID NO:126 H Sequence: C of SEQ ID NO:127 H Sequence: V of SEQ ID NO:128 L and C of SEQ ID NO:129 L array; g) V of SEQ ID NO:130 H Sequence: C of SEQ ID NO:131 H Sequence: V of SEQ ID NO: 132 L and C of SEQ ID NO:133 L array; h) V of SEQ ID NO: 134 H Sequence: C of SEQ ID NO: 135 H Sequence: V of SEQ ID NO: 136 L and C of SEQ ID NO:137 L array; i) V of SEQ ID NO: 138 H Sequence: C of SEQ ID NO: 139 H Sequence: V of SEQ ID NO:140 L and C of SEQ ID NO:141 L array; j) V of SEQ ID NO:142 H Sequence: C of SEQ ID NO: 143 H Sequence: V of SEQ ID NO: 144 L and C of SEQ ID NO:145 L array; k) V of SEQ ID NO: 146 H Sequence: C of SEQ ID NO:147 H Sequence: V of SEQ ID NO: 148 L and C of SEQ ID NO:149 L array; l) V of SEQ ID NO:150 H Sequence: C of SEQ ID NO:151 H Sequence: V of SEQ ID NO:152 L and C of SEQ ID NO:153 L A sequence; or m) V of SEQ ID NO: 154 HSequence: C of SEQ ID NO: 155 H Sequence: V of SEQ ID NO:156 L and C of SEQ ID NO:157 L array The present invention provides an antibody comprising:

[0139] [Table 3A]

[0140] [Table 3B]

[0141] [Table 3C]

[0142] [Table 3D]

[0143] [Table 3E]

[0144] [Table 3F]

[0145] In Table 3, clones 1, 2, 3, 4 and 12 are murine antibodies and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 are humanized antibodies.

[0146] In embodiments in which the antibody is a humanized antibody, the antibody may comprise a sequence as shown in Table 3 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16, or a sequence having at least 90% homology thereto.

[0147] In some embodiments, the invention provides an antibody that is a variant of any of the above antibodies or fragments thereof having one or more amino acid modifications, such as substitutions, deletions, additions, insertions, or other modifications, and that retains the biological function of the antibody. Thus, variant antibodies may be sequence engineered. Modifications include at least one substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence compared to the native antibody or polypeptide. Amino acid substitutions may be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, e.g., a leucine for a serine, i.e., a conservative amino acid exchange. Insertions or deletions may optionally range from about 1 to 5 amino acids. Permitted variations may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. Variants of antibodies described herein have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the non-variant molecule, preferably at least 95%, 96%, 97%, 98% or 99% sequence homology.

[0148] In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an 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. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the functions described in (c)-(l) above) using the functional assays described herein.

[0149] In some embodiments, the invention provides antibodies that are variants of an antibody selected from the antibodies shown in Table 3, which comprise one or more sequence modifications and have one or more improved properties, such as binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility, compared to an unmodified antibody. The sequence modifications may be in CDR1, CDR2, and / or CDR3, or in one or more framework regions.

[0150] The antibodies of the invention may comprise a human IgG Fc that has effector functions.

[0151] Fc receptors (FcR) are important immunoregulatory receptors that link antibody-mediated (humoral) immune responses with cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRIla, FcyRIIb and FcyRIIc) and CD16 (FcyRIIIa and FcyRIIIb). FcyRI is classified as a high affinity receptor (Kd in the nanomolar range), whereas FcyRI I and FcyRIII are low to moderate affinity (Kd in the micromolar range).

[0152] In antibody-dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) bind to the Fc region of IgG, which itself binds to target cells. Upon binding, signaling pathways are triggered that result in the secretion of various substances, such as lytic enzymes, perforin, granzymes, and tumor necrosis factor, which mediate the destruction of the target cells. The level of ADCC effector function varies with IgG subtypes. This depends on the allotype and the specific FcvR, but in brief, ADCC effector function is high for human IgG1 and IgG3 and low for IgG2 and IgG4. For IgG subtype variations in effector function, see below, ranked in order of decreasing potency.

[0153] FcyRs bind IgG asymmetrically across the hinge and upper CH2 regions. Since the binding sites were known, engineering efforts have been made to modulate IgG effector functions.

[0154] Antibodies of the invention may have an Fc with effector function, enhanced effector function, or reduced effector function.

[0155] The potency of an antibody can be increased by enhancing its ability to mediate cytotoxic functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Several mutations in the Fc domain have been identified that directly or indirectly enhance Fc receptor binding and significantly enhance cytotoxicity: S239D / A330L / I332E ("3M"), F243L, or G236A. Alternatively, enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, where FcyR interacts with carbohydrates on the CH2 domain, and the glycan composition has a substantial effect on effector function activity. Defucosylated (non-fucosylated) antibodies show greatly enhanced ADCC activity through increased binding to FcyRIIIa.

[0156] Although activation of ADCC and CDC may be desirable for some therapeutic antibodies, in some embodiments, antibodies that do not activate effector functions are preferred. Due to the lack of effector functions, IgG4 antibodies are the preferred IgG subclass for receptor blockade without cell depletion. However, IgG4 molecules can exchange half molecules in a dynamic process called Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation has been shown to prevent this recombination process, allowing the design of IgG4 antibodies with reduced propensity for Fab-arm exchange. Fc engineering approaches have been used to determine the key interaction sites of the IgG1 Fc domain with the Fey receptor and C1q, and then mutate these positions to reduce or eliminate binding. Through alanine scanning, the binding site of C1q to the region encompassing the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of the antibody or fragment of the present invention may comprise one or more mutations that reduce or abolish the binding of the CH2 domain to one or more Fey receptors, such as FcyRI, FcyRIla, FcyRIIb, FcyRIII, and / or complement. The CH2 domain of a human IgG domain normally binds to Fey receptors and complement, and a reduction in binding to Fey receptors is expected to reduce antibody-dependent cellular cytotoxicity (ADCC), and a reduction in binding to complement is expected to reduce the complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations that reduce or abolish the binding of the CH2 domain to one or more Fey receptors and / or complement are known in the art. The antibody molecule of the present invention may comprise an Fc with modifications that almost completely abolish FcyR and C1q binding: K322A / L234A / L235A or L234F / L235E / P331S ("TM"). The antibody molecules of the invention may comprise a CH2 domain containing alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be reduced by mutation of Pro329 (position according to EU numbering) to, for example, either P329A or P329G.Antibody molecules of the invention may comprise a CH2 domain comprising alanine residues at EU position 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) and an alanine (LALA-PA) or glycine (LALA-PG) at EU position 329 (position 114 according to the IMGT numbering). Additionally or alternatively, antibody molecules of the invention may comprise an alanine, glutamine or glycine at EU position 297 (position 84.4 according to the IMGT numbering).

[0157] Altered glycosylation at asparagine 297 of the Fc domain, known to be necessary for optimal FcR interaction, can confer loss of binding to FcR; loss of binding to FcR has been observed with N297 point mutation. The antibody molecule of the present invention can comprise an Fc with N297A, N297G or N297Q mutation. The antibody molecule of the present invention with a defucosylated Fc domain can be obtained by enzymatic deglycosylation, by recombinant expression in the presence of glycosylation inhibitors, or after expression of the Fc domain in bacteria.

[0158] IgG naturally persists for long periods in serum due to FcRn-mediated recycling, with a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependent interactions of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant conferred an approximately two-fold increase in IgG half-life (when assessed in rhesus monkeys), whereas the M252Y / S254T / T256E variant (YTE") conferred an approximately four-fold increase in IgG half-life (when assessed in cynomolgus monkeys). Prolonged half-life may allow the possibility of reducing dosing frequency while maintaining or improving efficacy.

[0159] Antibodies of the present invention preferably have a Kd value of less than about 4 nM, less than about 3 nM, less than about 3 nM, less than about 2 nM, or less than about 1 nM, as shown in the Examples.

[0160] The term "Kd" refers to the "equilibrium dissociation constant" and refers to the value obtained in a titration measurement at equilibrium or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). "Ka" refers to the affinity constant. The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffer at equilibrium. Other experimental approaches and instruments can be used, such as the BIAcore® (Biomolecular Interaction Analysis) assay.

[0161] In embodiments, antibodies according to the invention have Kd values ​​in the nanomolar range.

[0162] The present invention provides antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, that are capable of competing with an antibody of the invention described herein (e.g., a set of HCDRs and LCDRs defined by the Kabat nomenclature, and / or the VH and / or VL amino acid sequence of any one of the clones in Tables 1-3) for binding to an epitope within an isolated peptide comprising or consisting of residues 22-37 (SEQ ID NO:1).

[0163] Competitive assays include immunoassays, such as ELISA, HTRF; flow cytometry; fluorescent immunoassays based on Fluorescence Micro-Volume Assay Technology (FMAT) assays, Mirrorball, high content imaging, radioligand binding assays, biolayer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays, as well as cell-based and cell-free binding assays.

[0164] An antibody that binds to the same or overlapping epitope as a reference antibody refers to an antibody that blocks the reference antibody from binding to its binding partner (e.g., an antigen or "target") by 50% or more in a competitive assay, and / or conversely, the reference antibody blocks binding of the antibody to its binding partner by 50% or more in a competitive assay. Such antibodies are said to compete for binding to the epitope of interest.

[0165] The present invention further provides isolated nucleic acid encoding an antibody of the present invention. The nucleic acid may comprise DNA and / or RNA.

[0166] In one aspect, the present invention thus relates to the clones shown in Table 4, H、 C H、 V L and C L The present invention also relates to a nucleic acid sequence comprising or consisting of a sequence selected from a combination of sequences. The nucleic acid sequences of Table 4 encode the antibodies shown in Table 3.

[0167] Thus, the present invention provides a nucleic acid sequence which binds to LGR5 and has the following nucleic acid sequence: a) V of SEQ ID NO: 158 H Sequence: C of SEQ ID NO:159 H Sequence: V of SEQ ID NO: 160 L and C of SEQ ID NO:161 L array; b) V of SEQ ID NO: 162 H Sequence: C of SEQ ID NO: 163 H Sequence: V of SEQ ID NO: 164 L and C of SEQ ID NO: 165 L array; c) V of SEQ ID NO: 166 H Sequence: C of SEQ ID NO: 167 H Sequence: V of SEQ ID NO: 168 L and C of SEQ ID NO: 169 L array; d) V of SEQ ID NO:170 H Sequence: C of SEQ ID NO:171 H Sequence: V of SEQ ID NO:172L and C of SEQ ID NO:173 L array; e) V of SEQ ID NO: 174 H Sequence: C of SEQ ID NO:175 H Sequence: V of SEQ ID NO: 176 L and C of SEQ ID NO:177 L array; f) V of SEQ ID NO:178 H Sequence: C of SEQ ID NO: 179 H Sequence: V of SEQ ID NO: 180 L and C of SEQ ID NO:181 L array; g) V of SEQ ID NO: 182 H Sequence: C of SEQ ID NO: 183 H Sequence: V of SEQ ID NO: 184 L and C of SEQ ID NO: 185 L array; h) V of SEQ ID NO: 186 H Sequence: C of SEQ ID NO:187 H Sequence: V of SEQ ID NO: 188 L and C of SEQ ID NO:189 L array; i) V of SEQ ID NO:190 H Sequence: C of SEQ ID NO:191 H Sequence: V of SEQ ID NO: 192 L and C of SEQ ID NO:193 L array; j) V of SEQ ID NO:194 H Sequence: C of SEQ ID NO: 195 H Sequence: V of SEQ ID NO: 196 L and C of SEQ ID NO: 197 L array; k) V of sequence number 198 H Sequence: C of SEQ ID NO: 199 H Sequence: V of SEQ ID NO: 200 L and C of SEQ ID NO:201 L array; l) V of SEQ ID NO:202 H Sequence: C of SEQ ID NO:203 H Sequence: V of SEQ ID NO:204 L and C of SEQ ID NO: 205 L A sequence; or m) V of SEQ ID NO: 206 H Sequence: C of SEQ ID NO:207 H Sequence: V of SEQ ID NO:208 L and C of SEQ ID NO:209 L array The present invention provides an antibody or fragment thereof comprising:

[0168] In one embodiment, the nucleic acid is SEQ ID NO:218.

[0169] [Table 4A]

[0170] [Table 4B]

[0171] [Table 4C]

[0172] [Table 4D]

[0173] [Table 4E]

[0174] [Table 4F]

[0175] [Table 4G]

[0176] [Table 4H]

[0177] [Table 4I]

[0178] [Table 4J]

[0179] [Table 4K]

[0180] [Table 4L]

[0181] [Table 4M]

[0182] In one embodiment, the nucleic acid sequence has at least 60%, 70%, 80%, 90%, 95% or more sequence identity to one of the sequences selected from Table 4. In one embodiment, the sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

[0183] Nucleic acids according to the invention may comprise DNA or RNA and may be wholly or partially synthetically or recombinantly produced. References to nucleotide sequences herein include DNA molecules having the specified sequence and, unless specifically required otherwise, include RNA molecules having the specified sequence in which U is substituted for T.

[0184] Furthermore, the present invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, a vector, a transcription or expression cassette.

[0185] The present invention also relates to an isolated recombinant host cell comprising one or more of the nucleic acid constructs described above. The host cell may be bacterial, viral, mammalian, or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.

[0186] The invention also provides a method of producing an antibody described herein, comprising culturing a host cell under conditions suitable for expression of a polynucleotide encoding the antibody, and isolating the antibody.

[0187] In one embodiment, the antibodies of the invention are conjugated to a toxin, enzyme, radioisotope, label, therapeutic agent or other chemical moiety.

[0188] In some embodiments, the antibody is conjugated (or labeled) to a detectable or functional label. The label can be any molecule that produces or can be induced to produce a signal, including, but not limited to, a fluorophore, a fluorescent substance, a radioactive label, an enzyme, a chemiluminescent substance, a nuclear magnetic resonance active label, or a photosensitizer. Thus, binding can be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzymatic activity, or light absorption.

[0189] The invention provides immunoconjugates comprising an antibody of the invention conjugated to at least one therapeutic and / or diagnostic agent.

[0190] In some embodiments, the therapeutic agent may be a toxin, an enzyme, a radioisotope, or other chemical moiety, hi one embodiment, the therapeutic agent comprises a toxin, such as a cytotoxic radionuclide, a chemical toxin, or a protein toxin.

[0191] The present invention thus relates to an antibody drug conjugate (ADC) comprising (i) an antibody or antigen-binding fragment thereof as described herein, (ii) a payload, such as a toxin having cytostatic activity capable of killing a cell, and (iii) a payload-antibody linker moiety covalently attached to the antibody or antigen-binding fragment thereof. Linker groups for attaching biologically active moieties are well known in the art.

[0192] The payload can be a cytotoxin. In another embodiment, the payload can be an immunomodulatory molecule, such as a TLR8 or TLR7 agonist, or a STING agonist (Gingrich J. How the Next Generation Antibody Drug Conjugates Expands Beyond Cytotoxic Payloads for Cancer Therapy-J. ADC. April 7, 2020).

[0193] Examples of toxins that can be conjugated to antibodies are well known in the art and include, for example, auristatins, such as monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF), or duocarmycins, calicheamicins, pyrrolobenzodiazepines, and SN-38. In some embodiments, the immunoconjugate comprises an antibody linked to MMAE. MMAE is a synthetic antimitotic anti-neoplastic agent. MMAE is 100-1000 times more potent than doxorubicin, but its toxicity is such that it cannot be used as a drug by itself. However, it has been used as part of an antibody drug conjugate (ADC) in which MMAE is linked to an antibody that recognizes a specific marker expressed in cancer cells, directing MMAE to cancer cells.

[0194] The structure linking the antibody to MMAE may include a linking group (maleimide (aml) and caproic acid (cap)), a spacer (para-aminobenzoic acid), and a cathepsin-cleavable linker (amino acids valine (Val) and citrulline (Cit)). When the ADC binds to a targeted cancer cell antigen and enters the cancer cell, the structure connecting MMAE to the antibody may be cleaved by cathepsin, after which the ADC releases toxic MMAE and activates a potent anti-mitotic mechanism. The ADC reduces the deleterious systemic effects of highly potent cytotoxic agents.

[0195] MMAF is a synthetic anti-neoplastic agent used as a toxic moiety in some experimental anti-cancer ADCs. The structure linking the antibody to MMAF can include maleimide and caproic acid.

[0196] Monomethylauristatin E (MMAE, desmethylauristatin E) is a synthetic antimitotic antineoplastic agent. The lUPAC name for MMAE is (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamide)butanamide.

[0197] Monomethyl auristatin E or MMAE is 100-1000 times more potent than doxorubicin, but its toxicity is such that it cannot be used as a drug by itself. However, it has been used as part of an antibody drug conjugate or ADC, in which MMAE is linked to a monoclonal antibody (mAb) that recognizes a specific marker expressed in cancer cells to direct MMAE to cancer cells. Because MMAE is toxic, it has only been used as a therapeutic agent when conjugated to a monoclonal antibody (mAb) to target MMAE to cancer cells. In the international generic name for the MMAE-mAb conjugate, the name "vedotin" refers to the linking structure for MMAE plus the antibody. The structure linking the targeting mAb to MMAE may include a linking group (maleimide (mal) and caproic acid (cap)), a spacer (para-aminobenzoic acid), and a cathepsin-cleavable linker (amino acids valine (Val) and citrulline (Cit)).

[0198] The tether connecting MMAE to the monoclonal antibody is stable in extracellular fluids, but is cleaved by cathepsins when the antibody-drug conjugate binds to the targeted cancer cell antigen and enters the cancer cell, after which the ADC releases the toxic MMAE and activates a potent antimitotic mechanism. The antibody-drug conjugate enhances the antitumor effect of the antibody and reduces the deleterious systemic effects of highly potent cytotoxic agents.

[0199] Monomethylauristatin F (MMAF, desmethylauristatin F) is a synthetic antineoplastic agent. The lUPAC name for MMAF is (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2- ((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropionic acid.

[0200] MMAF is a toxic payload used in some experimental anti-cancer antibody drug conjugates, such as borsetuzumab mafodotin and SGN-CD19A. In the international generic name of MMAF antibody conjugates, the name mafodotin refers to its binding structure to MMAF plus antibody. The binding group can be composed of maleimide and caproic acid.

[0201] Auristatins and their use as components of ADCs are reviewed by Maderna and Leverett in "Recent Advances in the Development of New Auristatins: Structural Modifications and Application in Antibody Drug Conjugates"; Mol. Pharmaceutics, 2015, 12 (6), pp 1798-1812. Mendelsohn et al., "Investigation of Hydrophilic Auristatin Derivatives for Use in Antibody Drug Conjugates". Bioconjugate Chem., Article ASAP DOI: 10.1021 / acs.bioconjchem.6b00530, Publication Date (Web): January 6, 2017, describe derivatives of the natural product dolastatin 10 that contain pyridine and other basic amines, which were tested to assess whether the more hydrophilic auristatin derivatives would be sufficiently potent for use in ADCs. The pyridine derivative, monomethylauristatin PYE, showed the greatest efficacy when tested in vivo.

[0202] MMAE may be conjugated to the antigen-binding moiety via a valine-citrulline (vc) linker (vc-MMAE). MMAF is conjugated to the antigen-binding moiety via a maleimidocaproyl linker (mc-MMAF) using HiPEG™ technology (WO 2009 / 047500). Thus, in one embodiment, D is MMAE or a derivative thereof conjugated to the antigen-binding moiety via a valine-citrulline (vc) linker (vc-MMAE). In another embodiment, D is MMAF or a derivative thereof conjugated to the antigen-binding moiety via a maleimidocaproyl linker (mc-MMAF). In another embodiment, LD is vedotin or mafodotin.

[0203] The immunoconjugates, compositions, and methods of the invention may feature an auristatin that is either monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF), or a derivative thereof.

[0204] Immunoconjugates, in which an antibody is conjugated to a toxin, can be used to mediate specific cell killing of LGR5-expressing cancer cells without systemic toxicity.

[0205] The diagnostic agent may be a detectable or functional label as described above.

[0206] In another aspect of the invention, there is provided a pharmaceutical composition comprising an antibody according to the invention, or an immunoconjugate according to the invention, and optionally a pharma- ceutically acceptable carrier.

[0207] The antibodies, immunoconjugates, or pharmaceutical compositions of the invention may be administered by any convenient route, including, but not limited to, orally, topically, parenterally, sublingually, rectally, vaginally, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneally, intravenously, subcutaneously, intracerebral, transdermal, transmucosal, by inhalation, or topically, particularly to the ear, nose, eye, or skin, or by inhalation.

[0208] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0209] The pharma- ceutically acceptable carrier or vehicle may be particulate, such that the composition is in, for example, tablet or powder form. The term "carrier" refers to a diluent, adjuvant, or excipient with which the drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers may be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents may be used. In one embodiment, when administered to animals, the antibody or composition of the present invention and the pharma- ceutically acceptable carrier are sterile. When the drug antibody conjugate of the present invention is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0210] The pharmaceutical compositions of the invention may be in the form of a liquid, such as a solution, emulsion, or suspension. The liquid may be useful for delivery by injection, infusion (e.g., IV infusion), or subcutaneously.

[0211] When intended for oral administration, the compositions are preferably in solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included in the forms considered herein as either solid or liquid.

[0212] As a solid composition for oral administration, the composition can be formulated into powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or similar form. Such solid composition typically contains one or more inert diluents. In addition, one or more of the following may be present: binder, such as carboxymethylcellulose, ethylcellulose, crystalline cellulose or gelatin; excipient, such as starch, lactose or dextrin; disintegrating agent, such as alginic acid, sodium alginate, corn starch and similar; lubricant, such as magnesium stearate; lubricant, such as colloidal silicon dioxide; sweetener, such as sucrose or saccharin; flavoring, such as peppermint, methyl salicylate or orange flavor; and coloring. When the composition is in the form of capsule (e.g., gelatin capsule), it can contain, in addition to the above-mentioned type of materials, liquid carrier, such as polyethylene glycol, cyclodextrin or fatty oil.

[0213] The composition may be in liquid form, for example, elixir, syrup, solution, emulsion, or suspension. The liquid may be useful for oral administration or delivery by injection. When intended for oral administration, the composition may contain one or more of sweeteners, preservatives, dyes / colorants, and flavor enhancers. In the composition administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may also be included.

[0214] The compositions may be in the form of one or more dosage units.

[0215] In certain embodiments, it may be desirable to administer the compositions locally to the area in need of treatment or by intravenous injection or infusion.

[0216] The amount of the antibody of the present invention that is effective / active in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be optionally used to help identify optimal dosage ranges. The exact dose to be used in the composition also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. Factors such as age, body weight, sex, diet, administration time, excretion rate, host condition, drug combination, reaction sensitivity, and severity of the disease should be taken into consideration.

[0217] Typically, the amount is at least about 0.01% of the antibody of the invention by weight of the composition. When intended for oral administration, this amount may vary from about 0.1% to about 80% of the composition by weight. A preferred oral composition may contain about 4% to about 50% of the antibody of the invention by weight of the composition.

[0218] Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by mass of the antibody of the present invention.

[0219] For administration by injection, the composition may typically comprise about 0.1 mg / kg to about 250 mg / kg of animal body weight, preferably about 0.1 mg / kg to about 20 mg / kg of animal body weight, and more preferably about 1 mg / kg to about 10 mg / kg of animal body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The administration schedule may vary, for example, from once a week to once every 2, 3, or 4 weeks.

[0220] The present invention provides methods of treating or diagnosing a disease, e.g., cancer, in a subject, e.g., a mammal (e.g., a human patient), comprising administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition of the present invention. The present invention provides methods of diagnosing a disease, e.g., cancer, e.g., by assessing LGR5 expression in a target tissue. The methods are described in detail herein.

[0221] As used herein, "treat", "treating" or "treatment" means to inhibit or alleviate a disease or disorder. For example, treatment can include postponing the onset of symptoms associated with a disease or disorder and / or reducing the severity of such symptoms that occur or are expected to occur with the disease. The term includes ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the term indicates that a beneficial result is conferred on at least a portion of the subject, e.g., human patient, being treated. Many medical treatments are effective for some, but not necessarily all, of the patients who receive the treatment.

[0222] The term "subject" or "patient" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, such as, but not limited to, a human or a non-human mammal, such as, but not limited to, a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0223] As used herein, the term "effective amount" means an amount of an antibody that, when administered to a cell, tissue, or subject, alone or in combination with an additional therapeutic agent, is effective to achieve a desired therapeutic or prophylactic effect under the conditions of administration.

[0224] The present invention also relates to an antibody, an immunoconjugate, or a pharmaceutical composition of the invention for use in the treatment or prevention of a disease.

[0225] In another aspect, the invention relates to the antibody, immunoconjugate or pharmaceutical composition of the invention for use in the treatment or prevention of cancer.

[0226] In another aspect, the invention relates to the use of an antibody, immunoconjugate or pharmaceutical composition of the invention in the treatment or prevention of a disease.

[0227] In another aspect, the invention relates to the use of an antibody, immunoconjugate or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of cancer.

[0228] In an embodiment of the present invention, the antibody may be used as part of a CAR-T cell to treat cancer. The CAR-T cell may comprise a chimeric antigen receptor (CAR) comprising the antibody of the present invention, i.e., an antibody that binds to LGR5 or a fragment thereof.

[0229] Adoptive cell therapy (ACT) has attracted much attention as a technique for the treatment of cancer. One therapeutic approach of ACT involves genetically engineering T cells to express chimeric antigen receptors (CARs) on the surface of the T cells to allow targeting of specific tumors. When CARs are expressed in T cells, CAR-modified T cells (CAR-T or CAR-T cells) acquire properties including antigen-specific recognition, activation, and proliferation, and the cells thus act as "living drugs". Thus, the purpose of expressing CARs in T cells is to redirect the immune reactivity of the cells to a selected target. Furthermore, CARs with different strengths and signaling can also modulate T cell expansion and change the strength of T cell activation.

[0230] CARs are synthetic receptors that typically consist of a targeting / binding moiety associated with one or more signaling domains in a single fusion molecule. The binding moieties of CARs typically consist of paired antibody light and heavy chain variable domains (VCH) fused to a single polypeptide chain via a short flexible linker. L and V H) The scFv is capable of binding to a specific target of interest with the same specificity and similar affinity as the full-length antibody from which it is derived. In addition to the extracellular antigen-binding domain, the CAR also contains a transmembrane domain and signaling molecules such as a costimulatory endodomain and a CD3 zeta chain.

[0231] CARs combine antigen specificity and T cell activation properties in a single fusion molecule. First generation CARs typically contained the cytoplasmic region of CD3 zeta or the Fc receptor gamma chain as their signaling domain. First generation CARs have been tested in Phase I clinical trials in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, where they induced moderate responses (reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009). Second generation CARs, containing both CD28 and CD3 zeta signaling domains, provide dual signaling to direct combined activation and costimulatory signals. Third generation CARs have three or more signaling domains and are more complex.

[0232] The term "chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to an engineered receptor that grafts antigen specificity onto a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof), thus combining the antigen-binding properties of the antigen-binding domain with the lytic ability and self-renewal of the T cell. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. The term "antigen-binding domain" or "antigen-specific targeting domain" as used herein refers to the region of the CAR that targets and binds a specific antigen, as explained above. When the CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain).

[0233] One of skill in the art will be aware that a CAR may contain additional elements.

[0234] One of skill in the art will also know that such elements of a CAR (other than the antigen-specific targeting domain described herein) are well known in the art. Thus, the present invention is not limited to specific domains of a CAR in addition to the antigen-specific targeting domain described herein.

[0235] As mentioned above, first generation CARs have been tested in various Phase I clinical trials in patients with cancer. Second and third generation CARs have also been described as more complex, with three or more signaling domains (Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009; reviewed in Sterner, RC, Sterner, RM CAR-T Cell therapy: current limitations and potential strategies. Blood Cancer J. 11, 69, 2021). CARs are also described in U.S. Patent Application Publication No. 2004043401, WO2019200007, and WO2021108613, all of which are incorporated herein by reference.

[0236] For example, the CAR of the present invention may be a molecule of the formula: LGR5-binding scFv - a transmembrane domain - an intracellular signaling domain. Exemplary domains, transmembrane domains and intracellular signaling domains are described below and are known in the art.

[0237] Thus, the present invention also provides a chimeric antigen receptor comprising the antibody of the present invention or a fragment thereof, such as an scFv. In one embodiment, the CAR may be labeled. In one embodiment, the CAR comprises an scFv as shown in SEQ ID NO: 211, or a sequence having 70%, 80 or 90% sequence identity thereto, as encoded by SEQ ID NO: 218, or a sequence having 70%, 80 or 90% sequence identity thereto. In one embodiment, the CAR comprises an scFv comprising the CDRs of clone 2.4 numbered herein as SEQ ID NOs: 212, 213 and 214, and the heavy chain CDRs of 215, 216 and 217. In one embodiment, the CAR comprises an scFv comprising the VH and VL of clone 2.4, or a sequence having at least 90% sequence identity thereto.

[0238] The invention also relates to cells expressing the CAR of the invention, e.g., immune cells, selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), TCR expressing cells, dendritic cells, or NK-T cells, and regulatory T cells. The cells can be autologous or allogeneic T cells.

[0239] The present invention also relates to a cell or cell population comprising the CAR described herein.The present invention also relates to a cell population comprising the CAR described herein for use in adaptive immunotherapy, for example, for treating cancer.The present invention also relates to a method of treating cancer, for example, adoptive adaptive immunotherapy, comprising administering a cell population comprising the CAR described herein.

[0240] The present invention also relates to a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a subject, comprising administering to the subject an effective amount of a cell or cell population comprising a CAR described herein.

[0241] The invention also relates to a method of producing a cell population ex vivo for use in adaptive immunotherapy, comprising transforming the cells with a nucleic acid encoding a CAR, such as SEQ ID NO: 218, or a sequence having 70%, 80, or 90% sequence identity thereto.

[0242] The present invention also provides a method of generating a population expressing a CAR, comprising: (i) contacting a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface; (ii) contacting a cell (e.g., T cell) population with a nucleic acid molecule of SEQ ID NO: 218, or a sequence having 70%, 80, or 90% sequence identity thereto, thereby providing a cell (e.g., T cell) population comprising the nucleic acid molecule; and (iii) harvesting the cell population (e.g., T cells) for storage (e.g., re-formulating the cell population in cryopreservation medium) or administration. The present invention also relates to a method comprising the steps of:

[0243] As will be appreciated by those of skill in the art, the term "chimeric antigen receptor" or "CAR" refers to a recombinant molecule that contains an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain.

[0244] The extracellular domain comprises a ligand specific for a target tumor antigen, such as an antibody or fragment thereof that binds to LGR5 as described herein.

[0245] The extracellular domain is tethered to a transmembrane region, which may be selected from, for example, the transmembrane region of the alpha, beta or zeta chain of the T cell receptor, PD-1, 4-1BB, OX40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

[0246] The intracellular (cytoplasmic) domain of the CAR may provide activation of at least one of the normal effector functions of an immune cell. Thus, the CAR of the present invention may further comprise an intracellular signaling domain. An "intracellular signaling domain," "cytoplasmic domain," or "endodomain" is a domain that transmits an activation signal to a T cell and directs the cell to carry out its specialized function.

[0247] "Intracellular signaling domain," as that term is used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell or a CAR-expressing NK cell. For example, examples of immune effector function in a CART cell or a CAR-expressing NK cell include cytolytic activity and helper activity, including secretion of cytokines.

[0248] In one embodiment, the intracellular signaling domain can include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include domains derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain can include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include domains derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can include a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can include a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0249] The intracellular signaling domain contains a signaling domain, such as CD28, OX40, and / or CD3 zeta. The intracellular signaling domain can include a costimulatory domain.Costimulatory domains include CD28, CD8, OX40, 4-1BB, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM1l, B7-H3, CDS, ICAM-l, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-l, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, signaling domain of a ligand that specifically binds CD83, or any combination thereof.

[0250] In one embodiment of the invention, the antibodies may be used as part of a bispecific T cell engager (BiTE) to treat cancer. The BiTE may comprise an antibody of the invention, i.e., an antibody or fragment thereof that binds to LGR5.

[0251] Thus, the present invention also provides BiTEs comprising the antibodies of the present invention.

[0252] As will be appreciated by those skilled in the art, the term "bispecific T cell engager" or "BiTE" refers to a fusion protein comprising two scFvs of different antibodies, where one of the scFvs binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (e.g., an antibody or fragment thereof that binds to LGR5). The CD3 molecule non-covalently associates with the T cell receptor (TCR) and is involved in antigen-specific signaling, which activates T cells and initiates redirection of tumor cell lysis by the T cell.

[0253] In one embodiment, the BiTE comprises an scFv as set forth in SEQ ID NO:211, or a sequence having 70%, 80, or 90% sequence identity thereto, or one or more CDRs of SEQ ID NO:211.

[0254] The cancer may be a solid or non-solid tumor. For example, the cancer may be selected from head or neck cancer, uterine cancer, colorectal cancer, gastric cancer, endometrial cancer, esophageal cancer, leukemia, such as acute lymphoblastic leukemia (ALL), liver cancer, such as hepatocellular carcinoma, or pancreatic cancer.

[0255] In one embodiment, the tumor is a solid tumor.Solid tumors that can be treated include, for example, colorectal cancer.Some examples of such tumors include epidermoid cysts, squamous cell tumors, such as head and neck tumors, colorectal tumors.

[0256] In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia.

[0257] In one embodiment, the cancer is selected from colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL.

[0258] In one embodiment, the cancer is identified as an LGR5 positive cancer. The term "LGR5 positive cancer" as used herein means a cancer whose cells express LGR5. The antibodies and pharmaceutical compositions of the present invention are particularly useful for treating cancers that express abnormally high levels of LGR5, such as cancers that overexpress LGR5. The term "overexpress" as used herein means that the cells express more LGR5 than observed in normal non-cancerous cells. Cancer cells may express 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% more LGR5 than observed in normal non-cancerous cells. Those skilled in the art are well aware of methods to assess whether a cancer is LGR5 positive and / or overexpresses LGR5. Methods include, for example, using fluorescent in situ hybridization, immunohistochemistry approaches, flow cytometry, RT-PCR.

[0259] The inventors have found that by utilizing the antibodies of the present invention, a toxic payload in the form of an antibody drug conjugate (ADC) can be delivered to LGR5-expressing cancer cells to kill those cells without exhibiting systemic toxicity.

[0260] In one embodiment, the cancer is locally advanced, unresectable, metastatic, or recurrent cancer.

[0261] In one embodiment, the cancer has progressed following another treatment, for example chemotherapy.

[0262] The antibodies, immunoconjugates or pharmaceutical compositions of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agents. A therapeutic agent is a compound or molecule that is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, proapoptotic agents, antiangiogenic agents, boron compounds, photoactive agents, or dyes and radioisotopes. Antibody molecules may be full-length antibodies or fragments thereof (e.g., Fab, F(ab')2, Fv, single chain Fv fragments (scFv), or single domain antibodies, e.g., V H domain) or antibody mimetic proteins.

[0263] In one embodiment, the treatment is used in combination with an existing treatment or therapeutic agent, for example an existing anti-cancer treatment. Thus, in another aspect, the invention also relates to a combination therapy comprising the administration of an antibody, immunoconjugate, or pharmaceutical composition of the invention and an anti-cancer treatment. The anti-cancer treatment may comprise a therapeutic agent or a radiotherapeutic agent, including gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anti-cancer therapy, or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, anti-neoplastic agents, immunogenic agents, attenuated cancer-like cells, tumor antigens, antigen-presenting cells, such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biomolecules (e.g., agents that bind to tumor-specific antigens, including components of signal transduction pathways, such as regulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or immunosuppressants, and cells transfected with genes encoding immune stimulating cytokines (e.g., GM-CSF), chemotherapy. In one embodiment, the antibody is used in combination with surgery.

[0264] In certain embodiments of the invention, the composition is administered simultaneously with a chemotherapeutic agent or radiation therapy. In another particular embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the composition of the invention, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably several months (e.g., up to 3 months) before or after administration of the composition of the invention.

[0265] In some embodiments, an antibody, immunoconjugate or pharmaceutical composition of the invention may be administered with two or more therapeutic agents.

[0266] The antibody, immunoconjugate or pharmaceutical composition of the invention may be administered at the same time or at different times as other therapeutics or therapeutic compounds or treatments, eg, simultaneously, separately or sequentially.

[0267] In another aspect, the invention relates to an antibody, an immunoconjugate or a pharmaceutical composition of the invention for use in the treatment or prevention of an inflammatory disease.

[0268] In another aspect, the invention relates to the use of an antibody, immunoconjugate or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of an inflammatory disease.

[0269] In another aspect, the invention relates to a method of treating an inflammatory disease, comprising administering a therapeutically effective amount of an antibody described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0270] As will be appreciated by those skilled in the art, the term "inflammatory disease" refers to a disease in which tissue is inflamed, for example due to increased recruitment of leukocytes to the tissue, which can cause swelling, pain, and loss of function. Inflammatory diseases include, for example, arthritis, asthma, Crohn's disease, colitis, dermatitis, and irritable bowel syndrome. In particular, chronic inflammation is often a precursor to cancer. Inflammatory diseases also include autoimmune diseases and other inflammatory disorders, such as myositis, ankylosing spondylitis, and vasculitis.

[0271] In some embodiments, the inflammatory disease is an inflammatory disease that expresses abnormally high levels of LGR5, for example, an inflammatory disease that overexpresses LGR5. The term "overexpress" as used herein means that the cells express more LGR5 than observed in normal cells that do not have inflammatory disease. The cells may express 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% more LGR5 than observed in normal cells. Those skilled in the art will be familiar with the methods to assess whether an inflammatory disease overexpresses LGR5. Methods include, for example, fluorescent in situ hybridization, immunohistochemistry approaches, flow cytometry, RT-PCR.

[0272] Without wishing to be bound by theory, the inventors hypothesize that chronic inflammation in tissues leads to upregulation of stem cell pathways and overexpression of LGR5. Thus, by targeting LGR5 using the antibodies of the present invention, the inventors can target cells affected by inflammatory diseases.

[0273] The inventors have found that by utilizing the antibodies of the present invention, a toxic drug payload in the form of an antibody drug conjugate (ADC) can be delivered to LGR5-expressing cells to kill those cells without systemic toxicity.

[0274] In another aspect, the invention provides a kit comprising an antibody, immunoconjugate or pharmaceutical composition described herein.

[0275] In another aspect, the invention provides kits for detecting LGR5 to treat or prevent disease and / or to diagnose, prognose, or monitor disease comprising an antibody or immunoconjugate of the invention. Such kits may contain other components, packaging, instructions, or materials to aid in the detection of LGR5 protein. The kits may include a labeled antibody of the invention as described above, and one or more compounds for detecting the label.

[0276] In another aspect, the invention provides an antibody of the invention packaged in lyophilized form or packaged in an aqueous medium.

[0277] The invention also relates to the antibodies herein described with reference to the figures and examples.

[0278] In another embodiment, the antibodies of the invention are used for non-therapeutic purposes, such as in diagnostic tests and assays. A method for detecting the presence of LGR5 in a biological sample comprises contacting the sample with an antibody according to the invention under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5.

[0279] The present inventors have demonstrated that the antibody of the present invention binds to cancer cells expressing LGR5 with high affinity and specificity, and therefore can be used in a method for detecting cancer in a sample from a subject.

[0280] Thus, the invention provides a method for detecting cancer in a biological sample from a subject, comprising contacting the sample with an antibody according to the invention under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5. The method is particularly useful for detecting cancers that are positive for or overexpress LGR5.

[0281] Thus, the invention provides a method for detecting an inflammatory disease in a biological sample from a subject, comprising contacting the sample with an antibody according to the invention under conditions that permit binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5. The method is particularly useful for detecting inflammatory diseases that overexpress LGR5.

[0282] The methods may be performed in vivo, in vitro or ex vivo.

[0283] The modification of antibodies for diagnostic purposes is well known in the art.For example, antibodies may be modified with ligand groups, such as biotin, or detectable marker groups, such as fluorescent groups, radioisotopes, or enzymes.The compounds of the present invention can be labeled using conventional techniques.Suitable detectable labels include, but are not limited to, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands with specific binding partners.

[0284] Biological samples can include, for example, tissue samples, blood and blood components (e.g., serum), mucus, saliva, urine, vomit, stool, sweat, semen, vaginal fluid, tears, pus, sputum, or pleural fluid.Preferably, the biological sample is a blood sample or a tissue sample.As will be appreciated by those skilled in the art, the method of detecting cancer can therefore be an in vitro method of detecting cancer in a subject, which is performed on a sample provided by the subject.

[0285] Also provided is a method for detecting cancer in a biological sample from a subject, comprising contacting the sample with an antibody according to the invention under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5. The method is particularly useful for detecting cancer that is LGR5 positive or overexpresses LGR5. If a complex is formed between the antibody and LGR5, this may indicate the presence of cancer in the subject. In such a case, the method may further comprise administering a cancer treatment to the subject. In some embodiments, the treatment may comprise administering an antibody, immunoconjugate or pharmaceutical composition of the invention, particularly an immunoconjugate or pharmaceutical composition, where the antibody is conjugated to a therapeutic agent, such as a toxin, enzyme, radioisotope or other chemical moiety (as described above).

[0286] Also provided is a method for detecting an inflammatory disease in a biological sample from a subject, comprising contacting the sample with an antibody according to the invention under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5. The method is particularly useful for detecting inflammatory diseases that overexpress LGR5. If a complex is formed between the antibody and LGR5, this indicates that an inflammatory disease may be present in the subject. In such a case, the method may further comprise administering a treatment to the subject. In some embodiments, the treatment may comprise administering an antibody, immunoconjugate or pharmaceutical composition of the invention, particularly an immunoconjugate or pharmaceutical composition, where the antibody is conjugated to a therapeutic agent, such as a toxin, enzyme, radioisotope or other chemical moiety (as described above).

[0287] In a preferred embodiment, the present invention relates to an in vitro method for detecting an epitope of the present invention in a sample, comprising incubating an antigen binding protein of the present invention with a sample of interest and determining binding of the antigen binding protein to an epitope of the present invention present in the sample, wherein binding of the antigen binding protein indicates the presence of the epitope of the present invention in the sample. Methods for detecting binding of an antigen binding protein to its target antigen are known in the art and include ELISA, ICC, IHC, immunofluorescence, Western blot, IP, SPR and flow cytometry. The sample of interest may be a sample obtained from an individual. The individual may be a human. Samples include, but are not limited to, tissues such as brain tissue, cerebrospinal fluid (CSF), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, semen, follicular fluid, semen, amniotic fluid, milk, whole blood, plasma, serum, blood derived cells, urine, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0288] After incubation, binding of the antigen-binding protein to the antigen, e.g., antibody-antigen binding, is detected using an appropriate detection system. Detection methods may be direct or indirect and may generate fluorescent or chromogenic signals. Direct detection involves the use of a primary antibody directly conjugated to a label. Indirect detection methods use a labeled secondary antibody raised against the primary antigen-binding protein, e.g., antibody, host species. Indirect methods may include an amplification step to increase signal intensity. Labels commonly used for visualization (i.e., detection) of antigen-binding protein-antigen (e.g., antibody-epitope) interactions include fluorophores and enzymes that convert soluble material into insoluble chromogenic end products.

[0289] The term "detect" as used herein is used in its broadest sense to include both qualitative and quantitative measurements of a target molecule. Detection includes identifying the mere presence of a target molecule in a sample as well as determining whether the target molecule is present in a sample at detectable levels. Detection may be direct or indirect.

[0290] Suitable detectable labels that can be conjugated to antigen binding proteins, such as antibodies, are known in the art and include radioisotopes, such as iodine-125, iodine-131, yttrium-90, indium-111, and technetium-99; fluorescent dyes, such as fluorescein, rhodamine, phycoerythrin, Texas Red, and cyanine dye derivatives, such as Cy7, Alexa750, and Alexa Fluor 647; chromogenic dyes, such as diaminobenzidine; latex beads; enzyme labels, such as horseradish peroxidase; phosphorescent or laser / fluorescent dyes that have spectrally resolved absorption or emission characteristics; electrochemiluminescent labels that can be detected via electrical stimulation in the appropriate chemical environment, such as SULFO-TAG; and chemical moieties that can be detected via binding to a specific cognate detectable moiety, such as labeled avidin or streptavidin, such as biotin.

[0291] In another aspect, the present invention relates to an isolated synthetic or recombinant peptide comprising an epitope, the peptide consisting of residues 22-37 of SEQ ID NO: 1, which may be used in immunization methods to identify and select antibodies. Thus, immunization methods using fragments are also contemplated.

[0292] Furthermore, the present invention relates to the use of LGR5 expression as a prognostic marker.

[0293] Thus, the present invention relates to methods of diagnosing or assessing the progression of cancer comprising assessing LGR5 expression and / or LGR5 protein levels. Assessing expression includes measuring expression using conventional methods. Protein levels can be assessed using the antibodies of the present invention.

[0294] In one embodiment, the method may further comprise comparing the expression level to a threshold level.

[0295] In one embodiment, the threshold level is the expression level in normal tissue.

[0296] In one embodiment, the method may further comprise selecting a treatment if the expression level is above a threshold.

[0297] In one embodiment, the method may further comprise administering said treatment.

[0298] In one embodiment, the treatment is an antibody or antibody fragment described herein.

[0299] In one embodiment, the cancer is CRC, HCC, and pre-B ALL.

[0300] In one embodiment, the cancer is HCC, and the method may further comprise assessing the presence of a mutation in β-catenin.

[0301] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The foregoing disclosure provides a general description encompassed within the scope of the present invention, including how to make and use the invention and the best mode thereof, but the following examples are provided to further enable those skilled in the art to practice the invention and provide a complete description of the requirements thereof. However, those skilled in the art will recognize that the details of these examples should not be read as limiting the invention, the scope of which should be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0302] All documents referred to in this application are incorporated herein by reference in their entirety, including references to gene accession numbers and references to patent publications.

[0303] "And / or," as used herein, should be taken as a specific disclosure of each of the two specified features or components, with or without each other. For example, "A and / or B" should be taken as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein. Unless the context indicates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0304] The invention is further illustrated in the following non-limiting examples. EXAMPLES

[0305] The inventors undertook significant experiments to develop the antibodies of the present invention, and in the process established antibodies with high binding affinity and specificity that can be used both as research tools and for therapeutic purposes.

[0306] Materials and Methods Plasmid constructs The plasmid for hLGR5-eGFP expression plasmid was previously described. All other LGR transgenes used in the study were constructed by directly replacing the LGR5 coding sequence with PCR amplicons from the corresponding LGR family coding sequences, either by Gibson assembly (New England Biolabs) or by restriction enzyme cloning. The origins of the LGR family coding sequences were as follows: hLGR4 (HG15689), Sino Biological; mLGR4 (MR219497) Origene; mLGR5 (MR219702) Origene; and hLGR6 (LGR6_OHu16329D) GenScript.

[0307] Sequencing of antibody hybridoma clones and generation of plasmids encoding the heavy and light chains and scFv fragments of humanized a-LGR5 antibodies (α-LGR5v4 and α-LGR5v6) were obtained from Absolute Antibody.

[0308] Preparation of proteins from bacterial expression The coding sequence of the N-terminal 100 amino acids of human LGR5 lacking the signal peptide was inserted into the pGEX-4T1 bacterial expression vector and expressed in E. coli XL1 blue. The expressed GST fusion protein was absorbed onto glutathione-Sepharose 4B (Sigma-Aldrich), after which the column was washed extensively with phosphate-buffered saline (PBS) and eluted with 20 mM glutathione. The eluate was digested overnight with 2U thrombin protease (Sigma), and the LGR5 N-terminal fragment was separated in PBS on a Superdex 75 10 / 300 gel filtration column in the AKTA pure system.

[0309] A bacterial expression plasmid for RAD display of the LGR5 N-terminal fragment was generated by Gibson assembly, expressed in bacteria, and purified by heat denaturation and Ni-Sepharose affinity chromatography as previously described (Rossmann et al.).

[0310] mammalian cell lines Cell lines were purchased from the European Collection of Cell Cultures and were authenticated by short tandem repeat (STR) DNA profiling. Upon receipt, cell lines were frozen and individual aliquots were cultured for analysis, typically <10 passages. Cells were grown in a humidified incubator at 37°C with 5% CO2 and were mycoplasma-test negative (MycoProbe® mycoplasma detection kit, R&D systems). HEK293T cells and colorectal cancer cell lines LoVo, SW480, HT29, HCT116, CaCo and DLD1 were maintained in DMEM supplemented with 10% heat-inactivated FCS (Gibco) and 100 U / ml penicillin / streptomycin (Gibco). The pre-B ALL cell lines NALM6, REH, 697, RS4-11, HAL-01, NALM16, SupB15 KOPN8, and MHH-CALL2 were maintained in RPMI-1640 (Gibco) medium supplemented with 10% heat-inactivated FCS (Gibco) and 100 U / ml penicillin / streptomycin (Gibco).

[0311] Cell assays and manipulations Indirect immunofluorescence, Western blot analysis, flow cytometry and TopFlash assay were previously described. For immunofluorescence, detection was performed by confocal spinning disk microscopy using an Andor Dragonfly 500 (Oxford Instruments). Images were processed using Imaris software (Bitplane / Oxford Instruments). Flow cytometry was performed on a BD LSR Fortessa or BD LSR Symphony cell analyzer using BD FACSDiva software (BD Biosciences Inc.). Quantitative real-time PCR (qRT-PCR) was previously described and used a Taqman probe specific for human LGR5 (Life Technology, Hs00969422_m1) and TBP (Life Technology, Hs00427620_m1) as a control housekeeping gene. Data sets from TopFlash assay and qRT-PCR experiments were graphed using the Prism software package and statistical analysis was performed using a two-tailed Student's t-test.

[0312] For overexpression of LGR family proteins, HEK 293T cells were transfected with the corresponding plasmids using lipofectamine 2000 (Life Technologies) according to the manufacturer's recommendations. Cells were transfected overnight and allowed to recover in culture medium for an additional 16 h before immunofluorescence, Western blot, or flow cytometry.

[0313] LGR5v4 and a-LGR5v6 were generated by transfecting 15 μg of each encoding plasmid (Absolute Antibody) into HEK293T 4×T175 flasks. Antibody-containing media was collected 2- and 4 days after transfection and subjected to protein G purification. Fluorescent forms of a-LGR5 (Fl-a-LGR5), α-LGR5v4 (Fl-α-LGR5v4) and α-LGR5v6 (Fl-α-LGR5v6) were generated using Dylight TM650 antibody labeling kit (Thermo Fisher Scientific). Fluorescent trastuzumab (Fl-α-HER2) was generated using Dylight TM550 antibody labeling kit, respectively. For some controls for flow cytometry and immunofluorescence, Fl-α-LGR5 was preincubated with superstoichiometric amounts of RAD-displayed Frag1A or Frag1B or Frag1A peptides (Cambridge Peptides) at a molar ratio of 10:1.

[0314] Antibody internalization assays were performed by incubating cells with 20 μg / ml Fl-α-LGR5 or Fl-a-HER2 for various time points, followed by fixation with 4% paraformaldehyde and immunofluorescence. Images from z-stacks of cells were analyzed for Fl-a-LGR5v4 and for fluorescent signals from antibodies against various cell markers, fluorescent phalloidin to visualize cortical actin, and Hoechst 33342 to visualize nuclei. Images were processed and analyzed using Arivis Vision 4D software. Fluorescence properties: Segmentation of images in 3D to delineate whole cells, nuclei, and puncta represented by cortical F-actin was performed using Blob Finder. Arivis Vision 4D software was then used to classify puncta associated with the cell membrane or within the cells and to determine the degree of colocalization between LGR5 and compartment-specific markers - colocalization is defined as >50% overlap with LGR5 puncta.

[0315] For internalization kinetics experiments, images were analyzed using Arivis Vision 4D software in combination with deep learning segmentation using Cellpose. Two regions were defined: the cell outer membrane and the cytoplasm. Segmented dot signals corresponding to Fl-a-LGR5 or Fl-a-LGR5v4, as well as segment-like signals corresponding to Fl-α-HER2, were classified according to their location within these two regions. Ratiometric analysis was performed to quantify the internalization of both markers at various time points.

[0316] TCGA data mining for LGR5 expression in cancer Publicly available gene expression data (RNAseq V2) from The Cancer Genome Atlas (TCGA; https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga) were downloaded using Firebrowse (http: / / firebrowse.org / ). Gene-level read counts were quantile normalized using Voom, and (log2 median-centered) LGR5 gene expression was determined for each sample. Tumor subtypes in which >70% of samples had higher than the pan-cancer median LGR5 expression were triaged as "LGR5 high tumors."

[0317] Generation of a-LGR5 antibodies Fusions were generated from splenocytes of NMRI mice or SPRD rats (Taconic) immunized twice, 14 days apart, with 30 mg of LGR5 N-terminal fragment glutaraldehyde conjugated to diphtheria toxoid, SC. ​​Antigen was administered with GERBU Pa adjuvant according to the manufacturer's recommendations. Four days prior to fusion, animals were boosted with 15 mg of antigen IV, administered with adrenaline.

[0318] Fusions and screening were performed essentially as previously described with the murine SP2 myeloma cell line as the fusion partner.

[0319] Purification of the immunoglobulin fraction was performed by absorbing 1 L of hybridoma supernatant onto a 3 ml packed volume of Protein G fast flow Sepharose equilibrated in PBS. After extensive washing of the column with PBS, the immunoglobulin fraction was eluted with 100 mM glycine, pH 2.7, and immediately neutralized with 200 mM Tris pH 8.0.

[0320] Western blotting Western blots of HEK293t, LoVo and NALM6 lysates were performed on 40 μg of protein. Western blots of RAD-displayed peptides were performed on 2.5, 12 and 60 ng of purified protein.

[0321] Wnt signaling reporter assay TopFlash assays were performed as previously reported (de la Roche et al.).

[0322] DNA sequencing of the CDR3 region of anti-LGR5 mouse monoclonal antibody This was outsourced to Absolute Antibody.

[0323] Humanization of anti-LGR5 mouse monoclonal antibody This was outsourced to Absolute Antibody.

[0324] cell culture All cells were grown in a humidified incubator at 37°C and 5% CO2. Colorectal cancer cell lines, LoVo, SW480, HT29, HCT116, CaCo and DLD1, were maintained in DMEM medium supplemented with 10% heat-inactivated FCS (Gibco) and 100 U / ml penicillin / streptomycin (Gibco). HEK 293T were maintained in DMEM medium supplemented with 10% heat-inactivated FCS (Gibco) without 100 U / ml penicillin / streptomycin (Gibco). The Colo205 colorectal cancer cell line and the pre-B-ALL cell lines, NALM6, REH and 697, were maintained resuspended in RPMI-1640 (Gibco) medium supplemented with 10% heat-inactivated FCS (Gibco) and 100 U / ml penicillin / streptomycin (Gibco).

[0325] Quantification of LGR5 expression at the mRNA level Cells harvested for RNA extraction were washed twice in ice-cold PBS and flash frozen as dry pellets for storage in a -80°C freezer. Total RNA was extracted using a Single cell RNA purification kit (Geneflow) or PureLink RNA mini kit (Life Tech) according to the number of cells obtained, following the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed using a One-Step qRT-PCR kit (Thermo Fisher SuperScript III Platinum) with a Taqman probe specific for human LGR5 (Life Technology, Hs00969422_m1) and TBP (Life Technology, Hs00427620_m1) as a control housekeeping gene. Each experiment included a no-template water control to ensure a clean reaction background. A no-RT control was performed replacing SuperScript Platinum III RT with Platinum Taq DNA polymerase (Life Technology, Cat. No. 10966018) to control for the non-specificity of RT amplifying genomic DNA. Expression levels of gene transcripts were calculated by the ΔCt method: the cycle threshold (Ct) of the gene of interest was subtracted from that of the housekeeping gene and transformed by a factor 2^(-ΔCt) to give the fold expression relative to the housekeeping gene.

[0326] Patient samples and immunofluorescence detection of LGR5 and β-catenin All human tissue biopsies used in the study were fixed in paraformaldehyde, paraffin embedded, probed with antibodies against α-LGR5 and β-catenin, and visualized with fluorescent secondary antibodies against mouse labeled with Alexa488 and rabbit labeled with Alexa555. All immunostained samples were imaged using a PhenoImager HT™ Automated Quantitative Pathology Imaging System (Akoya Biosciences). Scoring of all human biopsies for co-expression of LGR5 and β-catenin was performed by an individual blinded to the origin of the samples and graded from no expression - 0 to high expression - 3 for all sample sets. The Prism software package was used to plot LGR5 or β-catenin expression levels for all biopsy sample sets and to determine statistical differences using a two-tailed Student's t-test. Unless otherwise stated, all relevant legal and ethical guidelines of Addenbrooke's Hospital (Cambridge, UK) were adhered to regarding the collection of samples and provision of the present studies. Informed consent for research applications was obtained from all subjects.

[0327] LGR5 expression in individual colorectal cancer cases and adjacent healthy tissue was determined on biopsies provided by Dr Olivier Giger (OG; (IRAS: 162057). In these tissue samples, areas were annotated from consecutive H&E sections as normal, dysplastic, or invasive tissue. LGR5 and β-catenin protein levels were further determined in CRC by immunofluorescence using the Bern CRC sample set provided by Dr Inti Zlobec. The Bern CRC sample set is a duplicated highly annotated tumor microarray (TMA) consisting of 160 individual cases with determined phenotypic characteristics - sex, age, tumor stage, treatment intervention, and MSI status. Biopsies used for the construction of the TMA were collected under ethics 2020-00498 approved by the Ethics Committee of the Canton of Bern, Switzerland. All relevant guidelines of the National Institute of Medical Sciences (NIST, Bern, Switzerland) were followed for the construction of the TMA.

[0328] The Cambridge HCC TMA consists of 104 human liver samples and was collected with informed consent by Drs Sarah Aitken and Matthew Hoare at Addenbrooke's Hospital, Cambridge, UK, following procedures approved by the East of England Local Research Ethics Committee (16 / NI / 0196 and 20 / EE / 0109). Liver samples classified as healthy were obtained from resections from two women with hepatic inflammatory adenoma and two women with focal nodular hyperplasia, or a man with HNF1a-inactivating adenoma. All healthy liver tissue biopsies were obtained from patients aged between 25 and 36 years.

[0329] High-grade ovarian serous cystadenoma (HGSOC) samples constituting the Cambridge Ovarian Cancer TMA were provided by Prof James Brenton. Tumor samples were obtained from patients enrolled in the Cambridge Translational Cancer Research Ovarian Study 04 (CTCROV04, abbreviated OV04) study, approved by the Institutional Review Board (REC08 / H0306 / 61). Samples were processed according to a standardized operating protocol outlined in the OV04 study design. Tissue quality was assessed using hematoxylin and eosin (H&E) sections and high purity areas were selected for tissue microarray (TMA) generation (using 0.1 cm cores). The TMA consisted of healthy fallopian tubes (FT; 27 samples), 28 ovarian cancer cases (OvC) and 14 peritoneal cancer cases (OmC).

[0330] The Cambridge Brain Cancer TMA consisted of five healthy brain tissue samples, five low-grade gliomas, and five glioblastomas collected in accordance with approved local research ethics (LREC 18 / EE / 0172) via the ICARUS biorepository, Addenbrooke's Hospital.

[0331] Sections of PDAC and healthy pancreas were provided by Dr Eva Serrao and obtained from the Cambridge University Hospital Human Tissue bank, Cambridge, UK, following procedures approved by the Cambridge South Research Ethics Committee (REC18 / EE / 0227).

[0332] Primary hematological malignancy samples used in this study were provided by the Blood Cancer UK Childhood Leukaemia Cell Bank (LREC 16 / SW / 0219) and Cambridge Blood and Stem Cell Biobank (LREC 18 / EE / 0199) and the Blood Cancer UK Biobank (REC16 / SW / 0219) following procedures approved by the South West-Central Bristol Research Ethics Committee.

[0333] Buffy coats from healthy donors were obtained from NHS Blood and Transplant (Cambridge) under appropriate ethics (Research into Altered Lymphocyte Function in Health and Disease, REC reference number: 17 / YH / 0304). PBMCs were isolated using SepMate PBMC isolation tubes (Stemcell Technologies) and B cells and CD8+ T cells were isolated from these using human CD19 microbeads (Miltenyi Biotec) or human CD8+ isolation kits (Miltenyi Biotec).

[0334] Generation of ADCs and in vitro killing assays All antibodies and IgG1 (Sigma-Aldrich) were conjugated to MMAE through a divinylpyrimidine bridge linker inserted into the heavy-light chain disulfide linkage for a precise drug to antibody ratio of 4. For in vitro killing assays, LoVo target cells were seeded in opaque 96-well plates overnight, allowed to settle, and then treated for 3 days with ADCs bearing cleavable or non-cleavable control linkers at doses of 30, 10, 3, 1, 0.3, and 0.1 nM. NALM6 and REH target cells were seeded in opaque 96-well plates and directly treated for 3 days with ADCs bearing cleavable or non-cleavable control linkers at doses of 30, 10, 3, 1, 0.3, and 0.1 nM. On day 3, cell viability was assessed by CellTiter-Glo 2.0 cell viability assay (Promega) according to the manufacturer's instructions. Bioluminescence was measured using a CLARIOStar (BMG Labtech).

[0335] The efficacy of α-LGR5-ADC in targeting CRC was tested in CRC organoid models from the de la Roche Institute Biobank. For the killing assay, organoid models 1 and 2 were treated with a range of concentrations of α-LGR5-ADC from 1 to 100 nM. Organoid killing was assessed after 24 hours by the percentage of cleaved caspase 3 (CC3) expressing cells.

[0336] Flow cytometry analysis of LGR5 internalization. Cells were blocked with human TruStain fcX (Biolegend) for 15 min and then incubated with Fl-labeled a-LGR5 mAb at a ratio of 10:1 (peptide:mAb) in the presence or absence of blocking with epitope peptide (synthesized by Cambridge Peptides). Incubations were performed at 37°C (surface and internalized LGR5) or 4°C (surface LGR5). For cell surface staining, cells were washed twice with ice-cold PBS and incubated with Fixable Viability dye eFlour780 (eBioscience) for 10 min at room temperature protected from light. Cells were then washed once with FACS buffer consisting of PBS (Gibco), 3% FCS (Biosera), 0.05% sodium azide (Sigma), and 2 mM EDTA (Sigma). Cells were then incubated with appropriate dilutions of fluorophore-conjugated antibodies in the presence of human TruStain fcX (Biolegend) for 30 min in the dark at 4° C. Cells were washed twice with FACS buffer and subjected to flow cytometric analysis on a BD Fortessa or Symphony cell analyzer, and data were analyzed by FlowJo software (Tree Star Inc., version 10.4).

[0337] Immunofluorescence (IF) microscopy Regarding overexpression of different LGRs from different species (human, mouse, and macaque), HEK 293T cells were cultured on glass coverslips, grown in culture overnight, and then transfected with various expression vectors (Figure 2B) using lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Transfected cells were further incubated overnight to allow transgene expression before being subjected to IF staining and microscopy.

[0338] LoVo cells were seeded on glass coverslips and grown in culture for 2 days before staining for IF microscopy. NALM6 cells were washed twice with RPMI medium without FCS, resuspended, seeded on glass slides, and placed in a 37°C incubator for 8 min to allow firm attachment before further IF staining and microscopy.

[0339] To visualize internalization, NALM6 cells were incubated with Fl-conjugated anti-LGR5 mAbs or Fl-conjugated anti-LGR5 mAbs blocked by epitope peptide as a negative control in complete medium for 5, 15, 30, and 60 min in a 37°C incubator. Then, NALM6 cells were washed twice with RPMI medium without FCS, resuspended, plated on glass slides, and placed in a 37°C incubator for 8 min. For LoVo, cells were plated on cover slips and grown in culture for 2 days, and incubated with Fl-conjugated anti-LGR5 mAbs or Fl-conjugated anti-LGR5 mAbs blocked by epitope peptide as a negative control in complete medium for 5, 15, 30, and 60 min in a 37°C incubator for further IF staining and microscopy.

[0340] Cells were washed twice with PBS and then fixed with 4% PFA (CN Technical Services) for 10 min at room temperature. Slides were washed five times with PBS and blocked with blocking buffer containing 1% bovine serum albumin (BSA, Sigma) and 0.1% TritonX-100 (Alfa Aesar) in PBS for 30 min at room temperature. Blocking buffer was aspirated and slides were stained with primary Abs in blocking buffer for 1 h at room temperature and then washed five times with blocking buffer. Secondary antibodies (anti-mouse Alexa488 (Thermo) and Alexa647 AffiniPure donkey anti-human IgG (H+L) (Jackson ImmunoResearch)) with or without Fl-conjugated phalloidin (Alexa488 or Alexa555, Thermo) in blocking buffer were added to the slides for 30 min at room temperature. After incubation, slides were washed 5 times with blocking buffer and stained with Hoechst33342 (Invitrogen) prepared in blocking buffer for 5 minutes at room temperature protected from light. Slides were washed 5 times with blocking buffer and mounted with ProLong Diamond Antifade Mountant (Fisher). Excess mounting fluid was wiped off and slides were placed at room temperature protected from light overnight before imaging. Confocal spinning disk microscopy was performed on an Andor Dragonfly 500 (Oxford Instruments). Images were processed using Imaris software (Bitplane / Oxford Instruments).

[0341] ADC Generation All antibodies and IgG1 were conjugated to MMAE using previously reported protocols (Walsh et al.).

[0342] In vitro cytotoxicity of ADCs against CRC and pre-B ALL cell lines LoVo target cells were seeded in opaque 96-well plates overnight and allowed to settle, followed by treatment with ADCs with cleavable or non-cleavable control linkers at doses of 30, 10, 3, 1, 0.3, and 0.1 nM for 3 days. NALM6 and REH cells were seeded in opaque 96-well plates and directly treated with ADCs with cleavable or non-cleavable control linkers at doses of 30, 10, 3, 1, 0.3, and 0.1 nM for 3 days. On day 3, cell viability was assessed by CellTiter-Glo® 2.0 Cell Viability Assay (Promega) according to the manufacturer's instructions. Bioluminescence was measured by CLARIOStar (BMG Labtech).

[0343] In vivo therapeutic efficacy of murine and humanized ADCs against human NALM6 pre-B ALL cells in an immune-deficient NSG mouse model NSG mice (NOD scid gamma; strain NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) were purchased from Charles River UK Ltd (Margate, UK) and housed under specific pathogen-free conditions in accordance with UK Home Office regulations at the CRUK Cambridge Laboratory, University of Cambridge. NALM6 cells were transduced with a stably expressing LucEYFP reporter.

[0344] Immunodeficient NSG mice were engrafted by intravenous (iv) injection with NALM6 cells, monitored for weight loss, and imaged using IVIS imaging at 2-3 day intervals (Figure 11A and Figure 13A). For IVIS imaging, mice were administered D-luciferin ip at a dose of 150 mg / kg 10 min prior to imaging with IVIS2000 under general anesthesia with isoflurane. Mice were randomized into control and treatment groups according to bioluminescence signals detected 5 days after engraftment. Treatment with 5 mg / kg of mIgG-ADC or α-LGR5-ADC was performed by iv injection every other day for 4 times starting on day 6. Treatment with 5 mg / kg of α-LGR5v6-ADC (control) or α-LGR5v4-ADC was performed by iv injection on days 6 and 7. At the experimental endpoint on day 20, all animals were euthanized in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Spleen, blood, heart, kidneys, lungs, liver, small intestine, femur and tibia were collected at the experimental endpoint for histology and / or FACS.

[0345] Single-cell suspensions prepared from spleen, blood, and femoral and tibial bone marrow were stained with eflour780 fixable live / dead, followed by staining with a fluorescently conjugated antibody against human CD19 to identify NALM6 cells (CD19+ EYFP+) by FACS analysis. The absolute number of NAML6 cells was quantified by using AccuCheck Counting Beads (Thermo).

[0346] The whole heart, kidney, lung, liver, small intestine, and part of the spleen were fixed with paraformaldehyde, dehydrated with 70% ethanol, and embedded in paraffin blocks. Tissue sections were stained with hematoxylin and eosin (H&E) to evaluate the general cellular structure of different tissues. Small intestine sections were stained with fluorescently conjugated antibodies against Ki67 and β-catenin.

[0347] Engineering of LoVo and NALM6 cell lines The LGR5 and LGR4 genes were targeted by CRISPR-Cas9 using the following guide RNA (gRNA) targeting the first exon of both genes: LGR5-5'-GCTGCTGCAGCTGGCGACCGG-3'; (SEQ ID NO: 219), LGR4-5'-GCGCGGCGCCGCCTCTCTGCG-3' (SEQ ID NO: 220).

[0348] The DNA sequence corresponding to the gRNA was incorporated into pSpCas9(BB)-2A-GFP (Addgene plasmid 48138). LoVo and NALM6 cell lines were transfected with the pSpCas9(BB)-2A-GFP plasmid carrying the gRNA, and the next day, single GFP-expressing cells were seeded into wells of a 96-well plate. Clonal targeted cell lines were confirmed by PCR amplification and sequencing of the targeted region.

[0349] The luciferase transgene was introduced into LoVo and NALM6 cell lines by transduction with the lentiviral vector lenti-luc.

[0350] Patient samples CRC Patient Sections - CRC tumor samples were obtained ethically. Tumor sections were evaluated by a histopathologist (OTG) to identify areas of normal colonic epithelium, dysplastic epithelial growths, and cancer.

[0351] For the Bern TMA-studies, all relevant guidelines of the Institute of Pathology, University of Bern, Canton Bern, Switzerland were followed. Informed consent was obtained from all subjects or, if the subject was under 18 years of age, from a parent and / or legal guardian. During the construction of the ngTMA, similar tissue types, i.e. normal epithelium, tumor epithelium, or tumor stroma, were pooled onto the same TMA recipient block, and thus prior knowledge of the expected content of the tissue punch was available.

[0352] Determination of binding affinity Binding affinity was determined by biolayer interferometry using the Octet platform. For svFv-α-LGR5, Kd values ​​were derived for scFv-α-LGR5 absorbed to Ni-NTA biosensor from the change in interferometry upon addition of the α-LGR5 epitope peptide N-SSPRSGVLLRGCPTHCHC-C (SEQ ID NO: 210).

[0353] Preparation of scFv-α-LGR5 An expression plasmid containing the 6xHis-tagged scFv-α-LGR5 transgene was transfected into HEK293T cells, and the protein was purified from the conditioned medium using Ni-NTA-Sepharose. The purified protein was dialyzed in phosphate-buffered saline (pH 7.2).

[0354] Cambridge Liver Cancer TMA Ethics for liver and liver cancer samples will follow Rec-20 / NI / 0109 to MH.

[0355] BiTE production, T cell activation and killing assays a-LGR5 fused to scFv antibody fragment of CD3ε scFv Expression plasmids containing the coding regions of the transgenic BiTE forms of LGR5 in both orientations were constructed: LGR5scFv at the N-terminus, LGR5 scFv - CD3ε scFv (LC BiTE), or CD3ε at the N-terminus scFv (CL BiTE). CL and LC BITEs were engineered to contain an N-terminal signal sequence and a FLAG epitope tag. Both BiTEs were purified from conditioned medium of transfected HEK293T cells using HiTrap Protein L column (Cytiva) chromatography.

[0356] T cell activation assays were performed using 10 6 10 PBMCs and 10 6The incubation was initiated by adding 100% NALM6 cells to the mixture. After 24 h incubation, cells were stained with eflour780 fixable live / dead dye followed by fluorescently conjugated antibodies against CD4, CD8, CD25 and CD69. Flow cytometry was used to assess the expression of T cell activation markers CD25 and CD69 on CD4+ and CD8+ T cells.

[0357] BiTE-mediated killing assays were performed using cytotoxic CD8+ T cells. For this, CD8+ T cells were isolated from PBMCs as described and stimulated with 25 μL / mL ImmunoCult™ human CD3 / CD28 / CD2 T cell activator (STEMCELL) for 72 h. CD8+ T cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with 100 U / ml human IL-2 (Miltenyi Biotec) and 100 U / ml penicillin / streptomycin (Gibco). Cells were restimulated at a concentration of 1×106 cells / mL with 1 μg / mL plate-bound anti-CD3ε antibody (clone UCHT1, Biolegend) for 48 h on days 8-10. BiTE-mediated killing assays were performed on days 14-16 of culture. NALM6 target cells were labeled with CellVue membrane dye (CellVue Claret Far Red Fluorescent Cell Linker Kit protocol; Sigma-Aldrich) at 6.25 × 10 4 Pieces or 1.25 x 10 5 Cytotoxic CD8+ T cells were used at the indicated effector-to-target ratios. After 6 h of coculture at 37°C, cells were labeled with eFluor780 fixable live / dead dye (ThermoFisher) and NALM6 cells were analyzed for viability by flow cytometry.

[0358] Generation of α-LGR5 CAR-NK92 and CAR-T cells The α-CD19-CAR lentiviral plasmid was a gift from Dr John James (University of Warwick) and contained the coding region of the α-CD19 scFv domain (clone FM363) fused to the CD8 stalk and transmembrane domains, 4-1BB or CD28 intracellular co-stimulatory domains, respectively, and mScarlet. The humanized a-LGR5 scFv fragment (Absolute Antibody) was used to swap the α-CD19 scFv domain in the α-CD19-CAR lentiviral plasmid (pHR-SIN) using restriction enzyme digestion and Gibson assembly. The pHR-SIN backbone was cut with BamHI and MluI and the following PCR primers were used:

[0359] [Table 5]

[0360] Lentivirus was produced using HEK293T cells. For transfection, 1.5 mL of OptiMEM (Gibco, Cat. No. 31985070) was mixed with 30 μL of 1 mM HEPES (made in-house) and 42 μL of TransIT-293 transfection reagent (Geneflow, Cat. No. E7-0026) and incubated for 5 min at RT. In parallel, 6 μg of the desired lentiviral plasmid was mixed with 4 μg of lentiviral packaging plasmid pCMV8.91 and 4 μg of lentiviral envelope plasmid pMDG. OptiMEM / TransIT solution was then added slowly to mix with the lentiviral plasmid. After 15 min incubation at RT, 13 ml of pre-warmed complete DMEM was added and the mixture was added to the HEK293T cells.

[0361] After 48 and 72 hours, respectively, viral supernatants were collected and centrifuged at 250g for 5 minutes, then filtered through 0.45mm filters into 30ml conical ultracentrifuge tubes. The viral supernatants were ultracentrifuged at 25000g for 90 minutes at 4°C using an SW-28 rotor (Beckmann). After centrifugation, the supernatants were discarded and the pellets were gently resuspended in 200-300μl of PBS containing 1% heat-inactivated FCS by pipetting several times without creating air bubbles above the pellet or removing it. The resuspended concentrated virus was stored in aliquots at -80°C.

[0362] NK92 cells were transduced at an MOI of 10, where virus stock was mixed with 6 μg / ml protamine sulfate and transfected with 0.2 × 10 cells that had been pre-washed the day before. 6 NK92 cells seeded at 100x were added to the culture. Spinfection was performed at 1800 rpm for 15 min at 32°C. mScarlet expression was assessed 48-72 hours later and NK92 cells were sorted for mScarlet. >95% Scarlet positive NK92 cells were used for all in vitro and in vivo assays.

[0363] CD8+ T cells were isolated from PBMCs as described above and stimulated with 25 μL / mL ImmunoCult™ human CD3 / CD28 / CD2 T cell activator (STEMCELL). CD8+ T cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with 100 U / ml human IL-2 (Miltenyi Biotec) and 100 U / ml penicillin / streptomycin (Gibco). After 24 hours, CD8 T cells were transduced at an MOI of 5 and used between 15 and 20 days post-stimulation. α-LGR5-CAR+ CD8 T cells were sorted for in vitro killing assays and purity was typically >90%.

[0364] a-LGR5 CAR-NK and CAR-T cell killing assays NALM6, REH or HEK293T cells overexpressing hLGR5-eGFP, mLGR5-eGFP or cLGR5-eGFP were used as target cells in the VITAL killing assay and preloaded with CellVue membrane dye (CellVue Claret Far Red Fluorescent Cell Linker Kit protocol; Sigma-Aldrich). a-LGR5 CAR-NK and CAR-T cells or respective non-transduced control cells were added at the indicated effector-to-target ratios. Percent target cell killing at the indicated time points was assessed by flow cytometry.

[0365] LoVo and HepG2 cells were used as target cells in incucyte killing assays, where tumor cells were seeded in 96-well plates 24 hours prior to the assay. a-LGR5 CAR-NK and CAR-T cells or respective non-transduced control cells were added at the indicated effector-to-target ratios. Cell death was assessed using Apotracker Green (Biolegend, 1:200) and monitored over 15 hours using an Incucyte SX5 (Sartorius).

[0366] result Generation and validation of antibodies against LGR5 For monoclonal antibody production, we immunized mice with the N-terminal 100 amino acids of the human LGR5 extracellular domain (Figure 2A). To mount an effective immune response, the antigen needed to be conjugated to diphtheria toxin. The fusion reaction resulted in the generation of 18 hybridoma clones. To investigate the function of the clones, we generated transgenic versions of human and murine LGR family members: all human and murine LGR4-6 transgenes contain a common N-terminal influenza hemagglutinin (HA) epitope tag and a C-terminal extension consisting of a fusion fused to the vasopressin V2 receptor C-terminal tail for enhanced protein stabilization, which is then fused to eGFP (Figure 2B). We also generated a human LGR5 version with Gly1Ser and Val8Ala substitutions to match the corresponding cynomolgus monkey LGR5 N-terminus (cynoLGR5). The N-terminal 100 amino acid residues of cynoLGR5 are otherwise identical to the human protein. Western blot analysis of lysates from HEK293T cells expressing various LGR transgenes demonstrated specific immunoreactivity of hybridoma clones 1, 2, 3, and 4 against human and cynomolgus monkey LGR5 (Figure 1A, Figure 2C). None of the hybridoma clones were reactive against murine LGR5 protein, nor against human or murine LGR4 and LGR6 proteins. Moreover, we did not observe any specific immunoreactivity of the hybridoma clones against HEK293T cells, since these cells do not express endogenous LGR5 in the absence of pathway activity.

[0367] All a-LGR5 antibodies bind to a common epitope in the LGR5 N-terminus Upon sequencing, we found that the complementarity determining regions (CDRs) of the light and heavy chains of the four a-LGR5 antibodies were highly conserved, exhibiting only four variable amino acid positions (Figure 2D). To determine whether the a-LGR5 clones bind to a common epitope, we designed four overlapping fragments (fragments 1-4) of approximately 35 amino acids in length of the 100 amino acid antigen whose sequences are unique to the human but not to the murine LGR5 protein (Figure 2A). We expressed the individual fragments as RAD display fusion constructs and purified them from expressing bacteria using standard protocols. Western blot analysis showed that all four a-LGR5 clones specifically bound to fragment 1 (Figure 1B; Figure 2E). We further narrowed the epitope to fragment 1A, obtaining a 15 amino acid sequence at the N-terminus of LGR5 protein bound by all four a-LGR5 clones (Figure 1B, Figure 2E). None of the clones bound to the adjacent LGR5 fragment 1B, which contains five overlapping amino acid residues. Notably, the sequence of the fragment 1A epitope is substantially divergent from the corresponding regions in human LGR4 / 6 and mouse LGR4 / 5, by two residues in the corresponding cynoLGR5 sequence (Figure 2F), explaining why all of the hybridoma clones are specific for human and cynomolgus LGR5.

[0368] The binding affinity between a-LGR5 clones and RAD-fragment 1 fusions was determined by biolayer interferometry using the Octet platform. Due to the bi-dentate binding of the two arms of the antibody and the high binding affinity exhibited by the LGR5 antibody, inclusion of 10 μM of fragment 1 peptide was necessary to accurately determine the Kd value. We observed high affinity binding between captured fragment 1A and the original murine clones 1 and 2, with Kd values ​​of 0.76 and 1.1 nM, respectively (Table 5). No detectable binding was observed between all LGR5 antibodies and captured fragment 1B.

[0369] [Table 6]

[0370] Humanized variant 4 corresponds to clone 2.4 as per Tables 1 and 2. a-LGR5 humanization is a direct transfer of murine clone 2 variable domain sequences onto a human IgG scaffold. The variable domain protein sequences of this humanized clone are identical to murine clone 2.

[0371] Humanized variant 6 (a-LGR5v6) is also an antibody generated by the inventors, which lost all reactivity and did not bind to either the human or cynomolgus LGR5 proteins (FIG. 14A) or to the LGR5 epitope. This variant was used as a negative control in the experiment.

[0372] Clones 1, 2, 3 and 4 in Table 5 are murine antibodies 1, 2, 3 and 4 as in Tables 1 and 2.

[0373] Because the a-LGR5 epitope on LGR5 is close to the binding site of its R-spondin family ligand, we used TopFlash assay to determine whether antibody binding interferes with the ability of R-spondin1 to enhance Wnt pathway activity. We treated HEK293T cells expressing LGR5- eGFP or eGFP control with Wnt and R-spondin1 in the presence of 10-fold molar excess of murine IgG1 or α-LGR5 (relative to R-spondin1) (Figure 2G). No significant difference was observed in the enhancement of Wnt / R-spondin1 activity between IgG1 or α-LGR5 treated cells, indicating that antibody binding to LGR5 does not interfere with R-spondin1 binding.

[0374] In summary, the a-LGR5 antibody binds with high affinity to the N-terminus of human and cynomolgus LGR5 at a site that does not interfere with the binding of the R-spondin1 ligand.

[0375] The α-LGR5 antibody recognizes cells expressing human LGR5, but not other LGR family members. To determine the specificity of the antibody in detecting cellular LGR5 expression, we overexpressed the LGR transgene in HEK293T cells and probed expression by immunofluorescence with murine a-LGR5 clone 2 (α-LGR5) conjugated to the Alexa647 fluorophore (Fl-a-LGR5). HEK293T cells do not express endogenous LGR5 at steady state, and thus we observed no fluorescent signal from Fl-a-LGR5 in the absence of transgenic expression. We observed complete colocalization of Fl-a-LGR5 with overexpressed human LGR5-GFP, and the signal from the antibody was abrogated by preincubation with the fragment 1A peptide (Figure 1C). Fl-a-LGR5 was able to detect the overexpressed cynoLGR5 transgene (Fig. 1C ), but no signal was observed in HEK293T cells overexpressing human LGR4 or LGR6 transgenes or murine LGR (Fig. 1C ; Fig. 2H ).

[0376] To determine whether fluorescent a-LGR5 specifically detects native LGR5 expressed on the cell surface, we performed flow cytometry analysis of live HEK293T cells overexpressing human, murine, and cynoLGR forms. Cells expressing either human or cynoLGR5-eGFP were detected by fluorescent a-LGR5 antibody. In contrast, HEK293T cells overexpressing either human LGR4-eGFP, LGR6-eGFP, or murine LGR were not detected by flow cytometry using Fl-a-LGR5 (Figure 1D). Importantly, the fluorescent signal observed in LGR5-eGFP expressing cells was attenuated by preincubation with fragment 1A or a superstoichiometric level (10:1; Figure 1E) of a blocking peptide based on this sequence.

[0377] Investigating LGR5 expression levels in healthy tissues and cancer Previous studies have quantified LGR5 transcript levels in multiple cancer types and established that LGR5 mRNA expression is increased in some cancers compared to matched normal tissues. This study raises the intriguing possibility of using LGR5-based therapeutic strategies to identify and target cancer cells. To investigate this in more detail, we performed a comprehensive survey of LGR5 transcript levels for 33 cancer types using datasets extracted from the TCGA database. Read counts were quantile normalized to genome for direct comparison in median expression levels of extracted LGR5 data determined across cancer subtypes, sample sets, and the entire dataset (Figure 4A). We triaged 12 cancer types as "high LGR5 expressers" with more than 70% of the constituent cases having LGR5 expression levels higher than the median. Brain cancer, ovarian cancer, and uterine carcinosarcoma were excluded from this analysis due to the lack of availability of normal tissue samples. For these high LGR5 expressing cancers, we compared LGR5 expression to matched healthy tissues. In all cases, median LGR5 mRNA expression was higher in cancers, except for adrenal cancer, compared with matched healthy tissues. Highly significant increases in LGR5 expression were observed in head and neck, endometrial, gastric, colon, and rectal cancers (Figure 4B).

[0378] We also analyzed human expression data, see Hung-Chang Chen et al., LGR5 targeting molecules as therapeutic agents for multiple cancer types Preprint, BioRxiv, https: / / doi.org / 10.1101 / 2022.09.01.506182.

[0379] Transcriptional data ranked ovarian cancer as the highest LGR5-expressing cancer. However, the lack of transcriptome data for fallopian tubes in the TCGA database, a putative tissue source for ovarian cancer, prevented us from determining whether this represents malignancy-specific LGR5 overexpression. Therefore, we probed 24 fallopian tube biopsies with a-LGR5 and b-catenin, along with a TMA containing 28 ovarian and 14 peritoneal cancer cases. b-catenin was expressed in the cortex of all epithelial cells; however, in all cancer cases, we did not detect discernible LGR5 protein levels in b-catenin-positive fallopian tube epithelium, except in very rare cases where individual cells expressing LGR5 were localized in intracellular spots (<0.1% of cells; Figure 4C). When comparing LGR5 expression between fallopian tube samples and ovarian and peritoneal cancer cases, we did not observe a significant increase in protein levels (Figure 3A), nor did we observe significant differences in b-catenin protein expression (Figure 4D).

[0380] We next used a-LGR5 to determine whether LGR5 is upregulated in brain cancers (glioblastoma and low-grade glioma; LGG) using TMAs containing malignant and normal resected brain biopsies. No LGR5 expression was detected in healthy brain tissue and in the cancer cases we examined, and no significant upregulation of LGR5 protein levels was observed compared to resected non-malignant brain controls (Figure 3B; Figure 4H).

[0381] Finally, we investigated LGR5 protein expression in immune cells from healthy donors. Flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) with Fl-a-LGR5 did not detect any B cells, CD4+ T cells, or CD8+ T cells expressing LGR5 protein (Figure 4F). Consistent with previous studies, we determined whether LGR5 transcript levels were upregulated in tumor cells from acute lymphoblastic leukemia (ALL) patients compared to non-malignant controls. For further human expression data and our analysis, see Hung-Chang Chen et al., LGR5 targeting molecules as therapeutic agents for multiple cancer types Preprint, BioRxiv, https: / / doi.org / 10.1101 / 2022.09.01.506182.

[0382] The resulting transcript and protein datasets established that (i) LGR5 overexpression is unique to many cancer types, and (ii) a substantial therapeutic window of LGR5 expression exists between normal tissues and cancer. Collectively, the data support therapeutic intervention in CRC, HCC, and acute lymphoblastic leukemia through strategies that target cellular LGR5 overexpression.

[0383] α-LGR5 detects endogenous expression of LGR5 Based on our LGR5 expression data from CRC and ALL tumors, we sought to determine whether endogenous cellular LGR5 expression could be detected in cell line models of the disease. The LoVo colon cancer cell line was previously shown to express sufficient levels of LGR5 for antibody detection, and indeed we found that, among a panel of five colorectal cancer cell lines, LoVo cells expressed at least 10-fold higher LGR5 transcripts compared to the SW480 cell line (Figure 5A). We were able to detect high LGR5 protein levels in lysates from LoVo, HCT116, and DLD1 cells, but low levels of protein were detectable in lysates from SW480 and CaCo cells (Figure 5B).

[0384] Cell surface-expressed LGR5 was detected in LoVo cells using Fl-α-LGR5, but could be abrogated by preincubating the antibody with the fragment 1A peptide (Figure 5C). LGR5 was not detected by flow cytometry in SW480 cells.

[0385] We further investigated LGR5 protein expression in pre-B-ALL cell lines and identified that NALM6 cells expressed the highest transcript levels of LGR5, followed by REH cells with intermediate levels, and 697 cells with the lowest levels (Figure 5D). The LGR5 transcript levels in pre-B-ALL cell lines are consistent with the relative levels of LGR5 protein detected by Western blot (Figure 5E) and flow cytometry (Figure 5F).

[0386] LGR5 associates with unique subcellular compartments Endogenous LGR5 expression in NALM6 and LoVo cells allowed us to determine its cellular localization using Fl-α-LGR5. Consistent with the localization pattern of overexpressed transgenic LGR5-eGFP, the majority of endogenous LGR5 localizes to internal puncta (Figure 6A). Previous studies using transgenic LGR5 overexpression in HEK293T determined that LGR5 puncta are associated with LAMP1-positive compartments, and we tested whether the endogenous protein behaved accordingly in LoVo cells. In both instances, antibodies against LAMP1 and LGR5 proteins detected puncta; however, these are by no means coincident, indicating that they are localized to distinct cellular compartments (Figure 6B).

[0387] Rapid internalization of α-LGR5 antibodies by LGR5-overexpressing cell lines We next sought to determine whether they could target exogenous cargo, such as FL-α-LGR5, to LGR5-modified intracellular puncta. Treatment of HEK293T cells overexpressing LGR5-eGFP with FL-α-LGR5 at 37° C. resulted in specific internalization of the antibody (FIG. 7A). The kinetics of internalization was rapid; within 5 min, Fl-a-LGR5 was internalized and localized to puncta close to the cell surface, which eventually became fully associated with intracellular LGR5-eGFP puncta over the course of 45 min. HEK293T cells transfected with LGR4-eGFP were unable to bind or internalize FL-α-LGR5 (FIG. 7A).

[0388] To determine whether endogenous LGR5 expression could direct Fl-α-LGR5 internalization, we repeated the internalization assay using NALM6 cells. Similar to HEK293T cells overexpressing hLGR5, NALM6 cells rapidly internalized Fl-α-LGR5 within 15 min, but did not internalize when the antibody was preincubated with the fragment 1A peptide (Figure 7B).

[0389] To distinguish between cell surface binding and internalization of Fl-α-LGR5 in NALM6 cells, we performed the assay at 4° C. and 37° C., respectively. We detected antibody binding to only a small percentage (approximately 1%) of NALM6 cells, but the cells uniformly internalized Fl-α-LGR5 after 30 min of incubation (FIG. 7C).

[0390] LoVo cells also rapidly internalized Fl-α-LGR5 within 5 min unless the antibody was preincubated with the fragment 1A peptide (FIG. 7D).

[0391] To investigate the specific LGR5 internalization by Fl-α-LGR5 uptake in LoVo cells, we generated control lines in which they inactivated LGR5 by targeting the first exon of the LGR5 gene using CRISPR / Cas9. Interestingly, some of the targeted clonal lines had lesions in both LGR6 alleles, encoding proteins lacking 7 amino acids of the signal sequence (Supplementary, Fig. 5). The mutant proteins were expressed at comparable levels in the targeted cell lines compared to the parental lines (Fig. 5E); however, flow cytometry analysis using Fl-α-LGR5 to probe cell surface LGR5 did not detect the mutant proteins in the LGR5 targeted cell lines (Fig. 5F). The data suggest that mutant LGR5 proteins lacking the complete signal peptide are not translated in the endoplasmic reticulum for incorporation into the secretory pathway and for cell surface expression.

[0392] Taken together, the internalization data indicate that α-LGR5 binds to and is rapidly internalized by cells with low and transient cell surface LGR5 levels.

[0393] Validation of α-LGR5 antibody drug conjugates We next engineered a form of α-LGR5 fused to the microtubule toxin MMAE through a divinylpyrimidine disulfide bridge linker inserted into the heavy-light chain disulfide bond for precise 4:1 stoichiometry (Figure 10). Two forms of α-LGR5-MMAE conjugates were generated for in vitro testing: a sulfatase-cleavable form (α-LGR5-ADC; (Walsh et al.; Bargh et al.)) and a non-cleavable form (α-LGR5-ADCNC; Figure 10). We also generated a control IgG1 conjugated to MMAE via a cleavable linker. Importantly, α-LGR5-ADC demonstrated similar epitope binding affinity to the parent antibody (Table 5).

[0394] Single dose treatment of NALM6 cells with α-LGR5-ADC showed effective cell killing over 3 days with an EC50 of 4 nM (Figure 9A). α-LGR5-ADC was slightly less effective against the REHpre-B-All cell line, which expresses lower LGR5 levels, with an EC50 of 10 nM. We found that treatment of NALM6 cells with α-LGR5-ADCNC was ineffective, consistent with previous studies that found that a non-cleavable form of an ADC based on LGR5 antibodies was ineffective in cell killing. In vitro killing assays using LoVo cells showed similar results, showing effective cell killing with an EC50 value of 9 nM.

[0395] Targeting NALM6 tumors in vivo To test the in vivo efficacy of α-LGR5-ADC, we implanted NALM6 cells constitutively expressing a luciferase transgene into NSG mice. Five days after implantation, IVIS imaging was performed to stratify the mice into two groups with identical total tumor burdens. On days 6, 8, 10, and 12 after implantation, mice were treated with 5 mg / kg α-LGR5-ADC via tail vein injection (Figure 11A). A control cohort of mice received 5 mg / kg IgG1-ADC control injections on these days. Tumor burden was monitored at 2-3 day intervals by IVIS imaging. NALM6 tumors treated with IgG1-ADC grew at a logarithmic rate, but we observed tumor regression within 4 of the first 6 days of α-LGR5-ADC treatment (Figure 11A). Tumor regression continued throughout the course of treatment, with tumor growth resuming on day 16 after implantation, with a latency of 4 days. At the endpoint of the experiment on day 20, the a-LGR5-ADC-treated group had less than 0.5% of the tumor burden of control mice (Figure 11A, Figure 11B). We also observed a significant reduction in spleen mass (approximately 2-fold reduction) and residual NALM6 cells (approximately 100-fold reduction; (Figure 11C) as well as the absolute number of NALM6 cells in the blood (100-fold reduction) and bone marrow (50-fold reduction) of the α-LGR5-ADC-treated mice (Figure 11D, Figure 11E). No tissue toxicity was observed with α-LGR5-ADC treatment, and proliferation of stem cells in the intestinal crypts was unaffected (Figure 12).

[0396] Targeting NALM6 tumors with a humanized ADC form of α-LGR5 To generate an ADC product for potential clinical use, we humanized the a-LGR5 antibody to generate 16 variants based on a human IgG scaffold. We proceeded with one of the humanized clones, variant 4 (a-LGR5v4), which showed specific immunoreactivity to expressed human or cynoLGR5 (Figure 14A) and high affinity binding Kd-2nM to the LGR5 epitope comparable to the parental murine antibody (Table 5). As a negative control for the experiment, we used humanized variant 6 (a-LGR5v6), which lost all reactivity and did not bind to either the human or cynoLGR5 protein (Figure 14A) or the LGR5 epitope (Table 5). Moreover, we observed essentially the same specificity as α-LGR5 when using a-LGR5v4 as an immunofluorescence probe; it detected overexpressed transgenic human and cynomolgus monkey LGR5 proteins, but not any of the other LGR proteins (Figure 14B).

[0397] We generated ADC versions of α-LGR5v4 and α-LGR5v6 control for treatment of NALM6 tumor-bearing mice with a two-dose treatment regimen on days 6 and 8 after implantation. Consistent with previous in vivo studies, we observed a significant reduction in tumor growth after a four-day latency period from the first treatment date, and the reduction in tumor growth persisted for at least two days after the last α-LGR5v4 treatment (Figure 13A). Total NALM tumor burden, measured by IVIS imaging, was approximately half of the control value at the end of the experiment (Figure 13A, Figure 13B). Similar to treatment with α-LGR5-ADC, we observed a reduction in spleen mass (by 3-fold) and a reduction in residual NALM6 cells (by approximately 50-fold), as well as a reduction in NALM6 cells in the blood by approximately 10-fold. Interestingly, this treatment regimen was observed to maintain NALM6 tumor burden in the bone marrow, but there was no significant difference in total tumor cell number. No tissue toxicity was observed and stem cell proliferation in the intestinal crypts was unaffected by α-LGR5-ADC treatment (FIG. 14).

[0398] Taken together, the in vivo data obtained by the inventors indicate that α-LGR5 ADCs are effective in suppressing tumor growth, but require persistent treatment for sustained responses.

[0399] Binding affinity constant of scFv-α-LGR5 We next sought to develop LGR5 antibody fragments to determine whether such fragments exhibited binding activity comparable to the a-LGR5 clone. We prepared single chain variable fragments (scFvs) by transfecting an expression plasmid containing a 6xHis-tagged scFv-α-LGR5 transgene into HEK293T cells and purifying the protein from the conditioned medium. Binding affinity was determined by biolayer interferometry on an Octet platform. We observed extremely high affinity binding between scFv-α-LGR5 and the peptide, with a Kd value of 770 pM (table 5).

[0400] Generation of CARs and BiTEs The inventors next used the antibodies of the invention to develop chimeric antigen receptors (CARs) and bispecific T cell engagers (BiTEs).

[0401] A humanized scFv fragment of the LGR5 antibody (scFv-α-LGR5) was engineered for use as a bispecific T cell engager (BiTE). α-LGR5-BiTEs consist of a polypeptide chain of scFv-α-LGR5 fused to an scFv fragment of an antibody that binds to the CD3ε subunit of the T cell receptor (TCR). The two BiTE formats, either scFv-α-LGR5 or scFv-α-CD3ε as the N-terminal part, are compatible with either bacterial expression or expression in mammalian cell lines, which are purified and used as BiTEs.

[0402] scFv-α-LGR5 was engineered for use as a chimeric antigen receptor (CAR), which was expressed from lentivirally transduced cells as a single polypeptide chain consisting of scFv-αLGR5 fused to the CD8 alpha stalk and transmembrane domains, then fused to either the CD28 or 4-1BB costimulatory domains, and terminating in the CD3 zeta signaling domain.

[0403] The α-LGR5-CAR is compatible for expression in T cells, NK cells, and other immune cells.

[0404] As shown in Figure 15, a-LGR5-CAR NK cells are efficient cell killers with favorable killing kinetics. Second generation CAR constructs were generated using humanized a-LGR5 scFv. CAR constructs were stably introduced into NK92 cells by lentiviral delivery. (A,B) HEK293 cells were transfected with human (A) and cynomolgus (B) LGR5 as target cells in the killing assay. a-LGR5 CAR NK92 cells were co-cultured with modified HEK target cells at the indicated effector-to-target ratios for 5 and 9 hours, respectively. (C) a-LGR5 CAR NK92 cells were incubated with NALM6 target cells labeled with blue cell dye for 50 minutes, and immunological synapse formation was assessed by confocal microscopy, showing that a-LGR5 CAR NK92 cells formed effective synapses with tumor target cells. (D) a-LGR5 CAR NK92 cells were incubated with acute lymphoblastic leukemia (ALL) human tumor cell lines at the indicated effector to target ratios and specific tumor cell killing was assessed after 12 hours.

[0405] As shown in FIG. 16, α-LGR5 bispecific T cell engagers (BiTEs) are activated T cells and result in efficient tumor cell destruction. BiTE constructs were generated in both orientations (LC and CL, respectively) using humanized α-LGR5 scFv and humanized α-CD3 scFv. (A) PBMCs were isolated from healthy donors and incubated with NALM6 tumor cells in the presence of α-LGR5 scFv control, α-LGR5 LC or CL BiTEs, respectively. CD4+ and CD8+ T cell activation was determined by flow cytometric analysis of CD69 and CD25 expression after 24 hours. (B) NALM6 tumor cells were co-cultured with cytotoxic CD8+ T cells generated from healthy donor PBMCs in the presence of α-LGR5 scFv control, α-LGR5 LC or CL BiTEs, respectively. Tumor cell killing was assessed after 5 hours at an effector to target ratio of 5:1.

[0406] The sequences used are shown below.

[0407] α-LGR5 scFv fragment used in experiments related to CAR and BiTE constructs, see Figures 15-27.

[0408] Human variant 4 (clone 2.4 shown in Tables 1 and 2) is used to generate scFv fragments and thus are incorporated into the CAR and BiTE constructs. Thus, the CARs and BiTEs referred to in Figures 17-27 use human variant 4 (clone 2.4, see also Tables 2 and 3).

[0409] Peptide: SEQ ID NO: 211

[0410] [ka]

[0411] The CDRs of the light and heavy chains are underlined and are as follows (see also sequences in Table 2): RASQDISNRLN CDR1 Light Chain SEQ ID NO:212 YRSRRHT CDR2 Light Chain SEQ ID NO:213 QQGNSLPPT CDR3 Light Chain SEQ ID NO:214 NYWMQ CDR1 Heavy Chain SEQ ID NO:215 EIDPSDSYTNYNQKFQG CDR2 Heavy Chain SEQ ID NO:216 SLSGYVDY CDR3 Heavy Chain SEQ ID NO:217 Nucleic acid: SEQ ID NO:218

[0412] [ka]

[0413] summary Our survey of human cancers revealed that pre-B-ALL, CRC and HCC are indicator diseases for our portfolio of a-LGR5 therapeutics (Figure 3).

[0414] Human pre-B-ALL cells (NALM6), CRC cells (LoVo), and HCC cells (HepG2) express high levels of LGR5 transcripts and protein (FIG. 5, FIG. 17).

[0415] The a-LGR5 ADCs demonstrate high efficacy in targeting NALM, REH and LoVo cells in vitro (Figure 9), and importantly, preferentially kill LGR5HIGH patient-derived CRC organoids (Figure 18). Murine and humanized α-LGR5 ADCs demonstrate preclinical efficacy in the NSG model of ALL (NALM6) (Figure 11).

[0416] a-LGR5 BiTE efficiently activates human CD4+ and CD8+ T cells and, importantly, efficiently leads to specific tumor cell death (NALM6) when incubated in the presence of tumor and human CD8+ cytotoxic T cells (Figure 19).

[0417] a-LGR5-CAR NK92 cells efficiently and specifically target HEK293T cells overexpressing human and cynomolgus LGR5, but not HEK293T cells overexpressing human LGR4, human LGR6, and to a lesser extent cells expressing mouse Lgr5 (Figure 20 and Figure 26). a-LGR5-CAR NK92 cells also specifically target LGR5HIGH NALM6 cells in vitro and to a much lesser extent LGR5LOW REH cells (Figure 20). Taken together, these experiments demonstrate the essential specificity and favorable therapeutic window of a-LGR5-CAR by not targeting LGR5 low expressing cells (potentially stem cells). Furthermore, a-LGR5-CAR NK92 cells effectively kill HepG2 cells (Figure 21) and show moderate preclinical efficacy in vivo in the NSG model of ALL (NALM6) (Figure 27).

[0418] α-LGR5-CAR T cells efficiently kill NALM6 (Figure 23), HepG2 (Figure 24), and LoVo tumor cells (PP Figure 13) in vitro. Surprisingly, our α-LGR5-CAR T cells show excellent preclinical efficacy in vivo in the NSG model of ALL (NALM6) (Figure 25).

[0419] Consideration The present inventors have extensively tested and characterized novel antibodies against the extracellular domain of LGR5 with the aim of establishing it as a research tool and to determine its potential therapeutic applications. α-LGR5 is distinct from previously reported anti-LGR5 therapeutic antibodies due to its target epitope on the LGR5 protein, its unique variable heavy and light chains, and in particular its high binding affinity to its epitope.

[0420] LGR5 has been established as a marker of stem cells in many epithelial tissues through extensive expression analysis in genetically engineered mouse models (GEMMs). Moreover, it has received considerable attention as a marker of certain malignancies through functional studies in GEMMs as well as detailed transcriptional analysis of cancer patient datasets.

[0421] The inventors have shown that the developed α-LGR5 antibody is a sensitive and specific tool for determining the cellular levels and localization of LGR5 protein in healthy and malignant tissues. Using transcriptomic analysis of LGR5 expression, the inventors have characterized LGR5 protein expression in a wide range of clinical samples from various cancers and matched healthy tissues. Thereby, the inventors have established elevated LGR5 expression as a hallmark of various cancers, including CRC, HCC and pre-B-ALL. Importantly, examination of tissues at both the transcriptional and LGR5 protein levels shows that normal tissues generally have very low LGR5 levels, paving the way for therapeutic targeting of malignancies that overexpress the protein.

[0422] The inventors observed differences between the results from their transcript analysis and previous transcript analysis related to LGR5 protein levels in brain and ovarian cancer; although both malignancies consistently score high on LGR5 transcript levels, the inventors were only able to detect very low LGR5 protein levels in very few cells, less than 10% of the tumors tested. This difference may be due in part to the different case samples used in creating the dataset, but it highlights the need to establish LGR5 protein levels as a prognostic indicator to target cancers based on LGR5 overexpression.

[0423] The antibody developed by the present inventors specifically recognizes native LGR5 on the cell membrane and faithfully detects LGR5 expression under various fixation conditions, making it an excellent research tool, and we will use a-LGR5 to characterize the subcellular localization of endogenous LGR5.

[0424] All detectable LGR5 protein in NALM6 and LoVo cells, as well as colon epithelium, CRC tumors, HCC, and some ovarian cancer cases examined, are localized to intracellular specks. The accessibility of LGR5-positive intracellular specks through rapid internalization of Fl-a-LGR5 supports dynamic shuttling between a highly transient population of LGR5 on the cell surface and LGR5-associated vesicular compartments. Indeed, this hypothesis is supported by our comparative studies of LoVo cells and LGR5 targeting, in which LGR5 protein lacking a signal peptide did not traffic to the plasma membrane along the secretory pathway; these cells do not internalize LGR5.

[0425] Overexpression of LGR5 protein by some cancers and the highly dynamic nature of its internalization make LGR5 an ideal cellular target for ADC-based therapeutics. Using a recently developed protocol to link MMAE payloads to a-LGR5, the inventors have produced ADCs that show little change in Kd value for epitope binding and no loss of binding affinity. In vitro validation showed low nanomolar EC50 values ​​comparable to the Kd value of epitope binding in targeting LGR5-expressing NALM6 and LoVo cells.

[0426] In vivo experiments targeting NALM6 tumors with a-LGR5-ADC are consistent with previous studies targeting LoVo tumors; after a 2-day refractory period, we observed tumor attrition that persisted over the course of treatment. Upon removal of α-LGR5-ADC, tumor growth was restored, suggesting that NALM6 cells may reversibly localize to less drug-accessible pockets during the course of treatment. This idea was again reinforced by clinical trials using a humanized version of a-LGR5-ADC. Four days of treatment effectively targeted NALM6 tumors, but tumor growth eventually recovered. In this example, bone marrow was one of the compartments examined that contained significant levels of NALM6 cells that were not eliminated by the low-dose targeting regimen.

[0427] Importantly, no detectable off-target toxicity of α-LGR5-ADC was observed in treated mice, paving the way for future in vivo clinical trials with increased treatment doses of α-LGR5-ADC that may provide increased efficacy in targeting potential tumor cell cavities.

[0428] The present inventors have developed and validated novel antibodies and antibody fragments against human and cynoLGR5. The antibodies are highly specific, have high affinity, and are rapidly taken up by LGR5-expressing cancer cells. They represent an excellent research tool, but also show great efficacy when used as ADC in vivo in models of ALL, and therefore represent a therapeutic tool that can be used to treat cancer, especially cancers in which LGR5 is overexpressed. For example, the present inventors have designed and developed chimeric antigen receptors (CARs) and bispecific T cell engagers (BiTEs) using the antibodies of the present invention that can be used, for example, in the treatment of cancer.

[0429] (References) TIFF2025508984000030.tif102164

Claims

1. An antibody or fragment thereof that binds to LGR5, wherein the antibody binds to an epitope located within amino acids 22-37 of SEQ ID NO:

1.

2. The antibody or fragment thereof according to claim 1, wherein the antibody binds to an epitope consisting of amino acids 22 to 37 of SEQ ID NO:

1.

3. Antibody V H but with the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 50 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 51 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 52 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 2 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 3 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 4 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 9 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 10 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 14 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 15 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 16 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 20 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 21 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 22 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 26 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 27 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 28 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 32 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 33 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 34 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 38 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 39 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 40 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 44 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 45 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 46 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 56 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 57 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 58 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 62 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 63 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 64 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 68 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 69 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 70 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 74 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 75 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 76 or a sequence having at least 90% homology thereto The antibody or fragment thereof according to claim 1 or 2, comprising:

4. Antibody V L but with the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 53 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 54 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 55 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 5 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 6 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 7 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 11 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 12 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 13 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 17 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 18 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 19 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 23 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 24 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 25 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 29 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 30 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 31 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 35 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 36 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 37 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 41 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 42 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 43 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 47 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 48 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 49 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 59 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 60 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 61 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 65 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 66 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 67 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 71 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 72 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 73 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 77 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 78 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 79 or a sequence having at least 90% homology thereto The antibody or fragment thereof according to claim 1 or 2, comprising:

5. V selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104 H 3. The antibody or fragment thereof of claim 1 or 2, comprising the sequence:

6. V selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105 L 3. The antibody or fragment thereof of claim 1 or 2, comprising the sequence:

7. i) V of SEQ ID NO: 96 H Sequence and V of SEQ ID NO: 97 L array; b) V in SEQ ID NO: 80 H Sequence and V of SEQ ID NO: 81 L array; c) V of SEQ ID NO: 82 H Sequence and V of SEQ ID NO: 83 L array; d) V of SEQ ID NO: 84 H Sequence and V of SEQ ID NO: 85 L array; e) V of SEQ ID NO: 86 H Sequence and V of SEQ ID NO: 87 L array; f) V in SEQ ID NO: 88 H Sequence and V of SEQ ID NO: 89 L array; g) V in SEQ ID NO: 90 H Sequence and V of SEQ ID NO: 91 L array; h) V of SEQ ID NO: 92 H Sequence and V of SEQ ID NO: 93 L array; i) V of SEQ ID NO: 94 H Sequence and V of SEQ ID NO: 95 L array; j) V in SEQ ID NO: 98 H Sequence and V of SEQ ID NO: 99 L array; k) V of sequence number 100 H Sequence and V of SEQ ID NO: 101 L array l) V of SEQ ID NO: 102 H Sequence and V of SEQ ID NO: 103 L an array; or m) V of SEQ ID NO: 104 H Sequence and V of SEQ ID NO: 105 L array The antibody or fragment thereof according to claim 1 or 2, comprising:

8. 3. The antibody or fragment thereof according to claim 1 or 2, comprising the sequence of an antibody clone shown in Table 3, or a sequence having at least 70%, 80%, or 90% homology thereto.

9. 3. The antibody or fragment thereof according to claim 1 or 2, wherein the fragment is an scFv comprising SEQ ID NO: 211, or an array having at least 70%, 80% or 90% homology thereto.

10. 3. The antibody or fragment thereof of claim 1 or 2, wherein the antibody binds to human LGR5, optionally the antibody is a monoclonal antibody, and optionally the antibody is a human, humanized, or chimeric antibody.

11. The antibody or fragment thereof of claim 1 or 2, wherein the antibody is conjugated to a toxin, enzyme, radioisotope, label, therapeutic agent or other chemical moiety, optionally wherein the antibody is capable of binding to LGR5 with a Kd of less than approximately 4 nM, and optionally wherein the fragment comprises a Fab, scFv, or single domain antibody.

12. 10. An immunoconjugate comprising the antibody or fragment thereof of claim 1 linked to a therapeutic agent, optionally wherein the therapeutic agent is a toxin, enzyme, radioisotope, or other chemical moiety.

13. A pharmaceutical composition comprising the antibody of claim 1, the immunoconjugate of claim 12, and a pharmaceutical carrier.

14. 14. The antibody or fragment thereof of claim 1, the immunoconjugate of claim 12, or the pharmaceutical composition of claim 13 for use as a medicament.

15. 14. The antibody or fragment thereof of claim 1, the immunoconjugate of claim 12, or the pharmaceutical composition of claim 13 for use in the treatment or diagnosis of cancer or an inflammatory disease.

16. 16. The antibody or fragment thereof for use according to claim 15, wherein the cancer is an LGR5-positive cancer, optionally wherein the cancer overexpresses LGR5.

17. 16. The antibody or fragment thereof for use according to claim 15, wherein the cancer is selected from head or neck cancer, uterine cancer, colorectal cancer, gastric cancer, endometrial cancer, esophageal cancer, leukemia, such as acute lymphoblastic leukemia (ALL), liver cancer, such as hepatocellular carcinoma, or pancreatic cancer.

18. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof of claim 1.

19. A vector comprising the nucleic acid of claim 18.

20. 20. A host cell comprising the nucleic acid of claim 18 or the vector of claim 19.

21. A method for detecting LGR5 in a biological sample, comprising contacting the biological sample with an antibody described in claim 1 or 2 under conditions that allow binding of the antibody to LGR5, and detecting whether a complex is formed between the antibody and LGR5, optionally wherein the biological sample is a cancer sample selected from head or neck cancer, uterine cancer, colorectal cancer, gastric cancer, endometrial cancer, esophageal cancer, leukemia, e.g., acute lymphoblastic leukemia (ALL), liver cancer, e.g., hepatocellular carcinoma, or pancreatic cancer.

22. An antibody or a fragment thereof that binds to LGR5, wherein the VH of the antibody has the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 50 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 51 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 52 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 2 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 3 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 4 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 9 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 10 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 14 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 15 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 16 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 20 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 21 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 22 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 26 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 27 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 28 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 32 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 33 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 34 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 38 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 39 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 40 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 44 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 45 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 46 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 56 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 57 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 58 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 62 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 63 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 64 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 68 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 69 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 70 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 74 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 75 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 76 or a sequence having at least 90% homology thereto An antibody or fragment thereof comprising:

23. The VL of the antibody has the following CDR1, CDR2 and CDR3: a) CDR1 of SEQ ID NO: 53 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 54 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 55 or a sequence having at least 90% homology thereto; or b) CDR1 of SEQ ID NO: 5 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 6 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 7 or a sequence having at least 90% homology thereto; or c) CDR1 of SEQ ID NO: 11 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 12 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 13 or a sequence having at least 90% homology thereto; or d) CDR1 of SEQ ID NO: 17 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 18 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 19 or a sequence having at least 90% homology thereto; or e) CDR1 of SEQ ID NO: 23 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 24 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 25 or a sequence having at least 90% homology thereto; or f) CDR1 of SEQ ID NO: 29 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 30 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 31 or a sequence having at least 90% homology thereto; or g) CDR1 of SEQ ID NO: 35 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 36 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 37 or a sequence having at least 90% homology thereto; or h) CDR1 of SEQ ID NO: 41 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 42 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 43 or a sequence having at least 90% homology thereto; or i) CDR1 of SEQ ID NO: 47 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 48 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 49 or a sequence having at least 90% homology thereto; or j) CDR1 of SEQ ID NO: 59 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 60 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 61 or a sequence having at least 90% homology thereto; or k) CDR1 of SEQ ID NO: 65 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 66 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 67 or a sequence having at least 90% homology thereto; or l) CDR1 of SEQ ID NO: 71 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 72 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 73 or a sequence having at least 90% homology thereto; or m) CDR1 of SEQ ID NO: 77 or a sequence having at least 90% homology thereto, CDR2 of SEQ ID NO: 78 or a sequence having at least 90% homology thereto, CDR3 of SEQ ID NO: 79 or a sequence having at least 90% homology thereto The antibody or fragment thereof according to claim 22, which binds to LGR5 and comprises:

24. The antibody or fragment thereof that binds to LGR5 according to claim 22, comprising a VH sequence selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, and 104.

25. The antibody or fragment thereof that binds to LGR5 according to claim 22, comprising a VL sequence selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, and 105.

26. 23. A chimeric antigen receptor (CAR) comprising the antibody or fragment of claim 1 or 22, optionally comprising SEQ ID NO: 211, or a sequence having at least 70%, 80%, or 90% homology thereto.

27. 27. A cell or population of cells expressing the CAR of claim 26, optionally wherein the cell is an immune cell, optionally wherein the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), a TCR-expressing cell, a dendritic cell, or an NK-T cell, and a regulatory T cell, and optionally wherein the cell is an autologous T cell or an allogeneic T cell.

28. 28. The cell or cell population of claim 27 for use in adaptive immunotherapy.

29. 23. An immune cell engager such as BiTE or TriKE comprising the antibody or fragment of claim 1 or 22, optionally comprising SEQ ID NO: 211 or a sequence having at least 70%, 80%, or 90% homology thereto.

30. 23. A method for producing an antibody, comprising culturing a host cell according to claim 1 or 22 under conditions suitable for expression of a polynucleotide encoding the antibody, and isolating the antibody.

31. An isolated synthetic or recombinant peptide comprising an epitope, wherein the peptide consists of residues 22 to 37 of SEQ ID NO:

1.

32. 23. An in vivo or ex vivo method for diagnosing or assessing the progression of cancer, comprising a step of assessing LGR5 expression and / or LGR5 protein levels, optionally further comprising a step of comparing the expression level with a threshold level, optionally the threshold level being the expression level in normal tissue, optionally further comprising a step of selecting a treatment if the expression level is above the threshold, optionally wherein the protein level is assessed using an antibody or antibody fragment described in claim 1 or 22.

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