EA045813B1 - ANTI-CD24 COMPOSITIONS AND THEIR APPLICATIONS - Google Patents

Description

Field of technology to which the invention relates

The present disclosure relates to aHTu-CD24 antibodies that selectively bind human CD24 expressed in cancer cells, but not human CD24 expressed in non-cancerous cells. The disclosure also relates to the use of such antibodies in the treatment of cancer.

State of the art

CD24 is a small, highly glycosylated cell surface mucin-like protein associated with glycosylphosphatidylinositol (GPI). CD24 is expressed at higher levels on hematopoietic cells, including B cells, T cells, neutrophils, eosinophils, dendritic cells and macrophages, as well as non-hematopoietic cells, including nerve cells, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells glands and epithelial stem cells. In general, CD24 tends to be expressed at higher levels in progenitor cells and metabolically active cells and to a lesser extent in terminally differentiated cells. The function of CD24 in most cell types is unclear, but various immunological functions of CD24 have been reported. Although CD24 is found in many normal tissues and cell types, CD24 is overexpressed in nearly 70% of human cancers. High levels of CD24 expression detected by immunohistochemistry were found in epithelial ovarian cancer (83%), breast cancer (85%), non-small cell lung cancer (45%), prostate cancer (48%) and pancreatic cancer (72%). . CD24 is one of the proteins most highly expressed in cancer cells. CD24 expression is increased during tumorigenesis, suggesting its role in tumor progression and metastasis. Overexpression of CD24 in cancer has also been identified as a marker indicating a poor prognosis and more aggressive disease course for cancer patients. In breast cancer, CD24 expression is significantly higher in invasive carcinoma than in benign or precancerous lesions. In non-small cell lung cancer, CD24 expression has been identified as an independent marker of overall patient survival. In addition, in esophageal squamous cell carcinoma, overexpression of CD24 indicates tumor metastasis to the lymph nodes, high tumor grade, and reduced survival time. Similar observations have been found in many other forms of cancer, including colon cancer, hepatocellular carcinoma, glioma, ovarian cancer, and prostate cancer. Although CD24 has been widely used as a cancer prognosis marker, it has not been used as a neoantigen that could be a potential target for cancer treatment due to its expression on normal cell types and potential toxicity. Mature CD24 is a small, highly glycosylated sialoglycoprotein of 31 amino acids with 16 potential O-glycosylation sites and 2 putative N-glycosylation sites. Glycosylation is one of the most complex post-translational modifications of proteins. It is known that in many cancer cells there is a switch from the normal glycosylation pathway, which leads to changes in glycan expression and leads to hyperglycosylation or hypoglycosylation of many cellular proteins. Altered glycosylation patterns found in cancer cells are the result of many contributing factors, including dysregulation at the transcriptional level, dysregulation of chaperone proteins during glycosylation, and altered glycosidase and glycotransferase activities. Tumor-associated glycan changes include longer or shorter branching of N-glycans, higher or lower density of O-glycans, formation of truncated versions of normal analogues (Tn, sTn and T antigens), and formation of unusual shapes of terminal structures with sialic acid and fucose (sLea and sLex epitopes).

Accordingly, there is a need in the art for improved methods of detecting and treating cancer, particularly methods and compositions capable of distinguishing cancer cells from non-cancerous cells.

Brief description of the invention

Provided herein is an anti-CD24 monoclonal antibody whose binding to CD24 is blocked by glycosylation present in normal cells but not in cancer cells. The antibody can therefore bind a glycan-screened epitope that is not screened on cancer cells but is screened on non-cancerous cells. The antibody can bind a peptide containing the sequence set forth in SEQ ID NO: 48.

In another aspect, the monoclonal antibody can bind cancer cells with minimal or no reactivity to benign tumor cells. In another aspect, the monoclonal antibody can bind tumor cells with minimal or no reactivity to non-tumor cells.

In another aspect, the monoclonal antibody can bind circulating cancer cells with minimal or no reactivity to hematopoietic cells.

In another aspect, a monoclonal antibody cannot bind CD24 on cells lacking cancer-specific glycosylation patterns, but can bind CD24 on cells with cancer-specific glycosylation patterns.

In another aspect, the composition, which may be a pharmaceutical composition, contains a monoclonal antibody or one or more antigen binding fragments thereof.

In another aspect, the composition is used to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC).

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In another aspect, the composition is used to kill cancer cells through antibody-dependent cellular phagocytosis (ADCP).

In another aspect, the composition is used to kill cancer cells through a combination of ADCC and ADCP.

In another aspect, the composition comprises a T cell with a chimeric antigen receptor that can be used to impart cancer cell specificity to the T cells.

In another aspect, the composition contains a 3B6 monoclonal antibody.

In another aspect, the composition comprises a monoclonal antibody comprising the sequences set forth in SEQ ID NOs: 1 and 2.

In another aspect, the composition contains monoclonal antibodies obtained by affinity maturation of the 3B6 monoclonal antibody.

In another aspect, the composition contains a monoclonal antibody containing a heavy chain selected from any of the sequences set forth in SEQ ID NO: 3-10.

In another aspect, the composition comprises a monoclonal antibody comprising a light chain selected from any of the sequences set forth in SEQ ID NOs: 11-16.

In another aspect, the composition contains a monoclonal antibody PP6373 obtained by affinity maturation of the monoclonal antibody 3B6.

In another aspect, the composition comprises a monoclonal antibody comprising the sequences set forth in SEQ ID NOs: 6 and 16.

In another aspect, the composition contains a monoclonal antibody obtained by humanizing the monoclonal antibody PP6373.

In another aspect, the composition comprises a monoclonal antibody comprising a heavy chain selected from any of the sequences set forth in SEQ ID NOs: 29-32.

In another aspect, the composition comprises a monoclonal antibody comprising a light chain selected from any of the sequences set forth in SEQ ID NOs: 33-36.

In another aspect, the pharmaceutical composition contains a monoclonal antibody H2L3 obtained by humanizing the monoclonal antibody PP6373.

In another aspect, the pharmaceutical composition contains a monoclonal antibody H3L3 obtained by humanizing the monoclonal antibody PP6373.

In another aspect, the composition comprises a monoclonal antibody comprising a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 35.

In another aspect, the composition comprises a monoclonal antibody comprising a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 33.

In another aspect, the composition comprises a single chain monoclonal antibody comprising the sequence set forth in SEQ ID NO: 17.

In another aspect, the composition comprises a bispecific antibody comprising a first antibody domain comprising an anti-CD24 antibody or an antigen binding fragment thereof, and a second antibody domain comprising a second antibody or an antigen binding fragment thereof. The bispecific antibody can be used to bridge the gap between cancer cells and immune effector cells in a patient in need of treatment or prevention of cancer.

In another aspect, the second domain of the antibody has a binding specificity that is different from that of the first domain of the antibody.

In another aspect, the second domain of the antibody recruits immune effector T cells to cancer cells.

In another aspect, the second antibody or antigen binding fragment thereof binds CD3.

In another aspect, the second antibody or antigen binding fragment thereof binds a TCR α chain, a TCR β chain, a TCR γ chain, or a TCR δ chain.

In another aspect, the first domain of the antibody includes an antibody comprising the sequence set forth in SEQ ID NO: 17, and the second domain of the antibody contains the sequence set forth in SEQ ID NO: 18.

In another aspect, the first domain of an antibody includes an antibody comprising any of the sequences set forth in SEQ ID NOs: 23-27 and 37-41.

In another aspect, a composition containing a bispecific antibody can be used to treat cancer cells through antibody-dependent cellular cytotoxicity (ADCC).

In another aspect, the composition contains a bispecific antibody with enhanced ADCC activity.

In another aspect, a composition containing a bispecific antibody is used to treat cancer cells through antibody-dependent cellular phagocytosis (ADCP).

In another aspect, a composition comprising a bispecific antibody has enhanced ADCP activity.

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In another aspect, the composition contains a chimeric antigen receptor for use in immunotherapy, wherein said receptor comprises a single chain antibody comprising any of the sequences set forth in SEQ ID NO: 1-36.

In another aspect, a chimeric antigen receptor is used in immunotherapy, wherein said receptor comprises a single chain antibody comprising the sequence set forth in SEQ ID NO: 28.

In another aspect, the pharmaceutical composition is used in conjunction with a second anticancer drug.

Provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient one or more of the antibodies, bispecific antibodies, chimeric antigen receptors or compositions described herein, wherein the cancer is lung cancer, liver cancer, brain cancer brain, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer or neuroblastoma.

Cancer may bind to the anti-CD24 antibody composition described herein.

Further provided herein is a method for diagnosing malignant tissue or metastatic lesions using an anti-CD24 antibody composition. The anti-CD24 antibody composition may bind cancerous tissue or metastatic lesions at levels above a threshold, which may indicate malignant tissue or metastatic lesions.

Also disclosed herein is a method for identifying circulating cancer cells using an anti-CD24 antibody composition. The anti-CD24 antibody composition can bind circulating cancer cells at levels above a threshold, which may indicate circulating cancer cells. Further provided herein is the use of a composition described herein in the manufacture of a medicament for treating a disease or condition described herein.

Description of graphic materials

Fig. 1 is a bar graph of ELISA results showing that binding of the anti-CD24 monoclonal antibody 3B6 is hindered by the presence of a glycan, whereas binding of the commercially available anti-CD24 monoclonal antibody ML5 is not. 3B6 strongly binds CD24, lacking modifications by N-glycan and sialic acid (N-SA-CD24), and CD24, lacking modifications by N-glycan, sialic acid and O-glycan (N-SA-O-CD24), but binds very weakly both CD24 lacking N-glycan modifications (N-CD24) and fully modified (N-glycan + sialic acid + O-glycan modifications) CD24. CD24GST is a negative control CD24-GST fusion protein.

Fig. 2A,B - binding assays show that 3B6 binds neuroblastoma and medulloblastoma cell lines. Fig. 2A - normalized affinity graphs of monoclonal anti-CD24 antibodies ML5, 3B6 and SN3 and control antibodies tested against 6 neuroblastoma cell lines, IMR32, SK-N-SH, SH-SY5Y, SK-N-BE(2), SK-N -AS and SK-N-BE(2)C. Although 3B6 has some affinity for all neuroblastoma cell lines except SK-N-AS, the affinity of 3B6 was significantly lower compared to the commercially available anti-CD24 antibodies ML5 (BD Bioscience Cat#555426) and SN3 (Thermo Fisher Cat#MA5-11833 ). Fig. 2B is a fluorescent image of the results of treatment of 4 medulloblastomas with 3B6. 3B6 associated 3 out of 4 tumors.

Fig. 3 is a graph of a competitive ELISA comparing the ability of 3B6 variants to block the binding of 3B6 to the CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

Fig. 4 is a graph of a competitive ELISA comparing 3B6 variants for their ability to block the binding of 3B6 to the CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

Fig. 5 is a graph of a competitive ELISA comparing 3B6 variants for their ability to block the binding of 3B6 to the CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

Fig. 6 is a bar graph of ELISA results showing the relative affinity of affinity matured chimeric anti-CD24 antibodies to CD24 expressed by CHO (Chinese hamster ovary) cells. Twelve clones showed increased affinity and different specificity for fully glycosylated CD24, N-CD24, SA-CD24 and N-SA-CD24 compared to 3B6 (PP6226).

Fig. 7 is a titration analysis of various affinity matured chimeric anti-CD24 antibodies tested against the lung cancer cell line NCI-H727 (left panel) and the neuroblastoma cell line IMR32 (right panel). The maximum antibody concentration tested was 5 μg/mL with a titration factor of 2x to a minimum concentration of 0.01 μg/mL. An unstained (0 μg/mL) negative control is also shown.

Fig. 8 - Quantitative comparison of binding between different CD24 glycoforms and anti-CD24 antibodies: parent PP6229 versus affinity matured PP6373.

CD24 with the Fc removed (crystallizing fragment) was applied to an ELISA plate and then

- 3 045813 were treated with either buffer (CD24), NanA (SA-), or NanA+N-glycanase (SA-N-) before adding given doses of PP6626 (left panel) or PP6373 (right panel).

The maximum concentration tested was 7812.50 ng/mL with a titration factor of 5x to a minimum concentration of 0.02 ng/mL.

Fig. 9 - Mapping of 3B6 binding site using peptide inhibition assay. Of the five overlapping CD24 peptides tested, only one (peptide 4) contains an antigenic epitope.

Fig. 10 - Mapping of PP6373 binding site using peptide inhibition assay. Of the five CD24 overlapping peptides tested, only one (SNSGLAPNT (SEQ ID NO: 46)) contains an antigenic epitope.

Fig. 11 - Mapping of the PP6373 epitope with truncated peptides from the antigenic epitope sequence of peptide 4. The data indicate that the optimal epitope is contained in the sequence SNSGLAPN (SEQ ID NO: 48).

Fig. 12 is a graph showing that PP6373 reduces tumor growth in vivo in a mouse model. Nude mice bearing a palpable lung cancer xenograft received either control human IgG or PP6373 at two time points indicated by arrows, after which tumor growth was measured weekly.

Fig. 13 is a graph showing PP6373-induced cellular cytotoxicity (ADCC) against the human cancer cell line H727. H727 cells were co-incubated with PBL effector cells with PP6373 and human IgG FC at 5 μg/ml, which induced ADCC.

Fig. 14 is a graph showing that PP6373 without core fucosylation (d6873) induces higher ADCC against the human cancer cell line H727 than PP6373. H727 cells were co-incubated with PBL effector cells with d6373, PP6373 and human FC IgG at 5 μg/ml to induce ADCC.

Fig. 15 are flow cytometry plots showing that the combination of PP6373-groove and OKT3-overhang shows higher bispecificity than PP6373-overhang and OKT3-hole. Jurkat cells were stained with tissue culture supernatants of 293T cells transfected with PP6373, OKT3, PP6373-overhang and OKT3-indentation, or PP6373-indentation and OKT3-overhang, followed by incubation with biotinylated SA-N-CD24 protein. The PE-streptavidin (phycoerythrin-streptavidin) signal was measured by flow cytometry. Three independent experiments were carried out.

Fig. 16 - Flow cytometry plots showing that PP6373-OCTZ induces higher bispecific activity than OKTZ-PP6373. Jurkat cells were stained with tissue culture supernatants of 293T cells transfected with empty plasmid (negative control), PP6373OKT3, or OKTZ-PP6373, followed by incubation with biotinylated SA-N-CD24 protein. The PE-streptavidin signal was measured by flow cytometry. Three independent experiments were carried out.

Fig. 17 is a flow cytometry graph showing that the bispecific antibody PP6373OKT3 has antitumor activity. H727 lung cancer cells and activated human T cells were incubated at a ratio of 1:5 with tissue culture supernatants of untreated 293T cells or transfected with empty plasmid (untransfected), PP6373, OKT3, PP6373OKT3 for 12 hours. Cytokines (IFNr, TNF, IL10, IL6, IL4 and IL2) in tissue culture media were measured using flow cytometry. Three independent experiments were carried out.

Fig. 18 is a flow cytometry graph showing that the bispecific antibody PP6373OKT3 induced cytotoxicity of tumor cells by T cells. H727 lung cancer cells and activated human T cells were incubated in a 1:5 ratio with tissue culture supernatants of untreated 293T cells or transfected with an empty plasmid (untransfected), PP6373, OKT3, PP6373-OCT3 for 12 hours. Lung cancer cells and human T cells cells were collected and stained with anti-human CD45 and Aqua Live/Dead reagent. Tumor cell counts were plotted as double negative for anti-CD45 and Aqua. Three independent experiments were carried out.

Fig. 19 - analysis of high bispecific activity induced by FIT-Ig using flow cytometry. Jurkat cells were stained with negative control (untransfected 293T supernatant) or FIT-Ig, followed by incubation with biotinylated SA-N-CD24 protein. The PE-steptavidin signal was measured by flow cytometry. Three independent experiments were carried out.

Fig. 20 - Flow cytometry analysis shows that FIT-Ig has higher antitumor activity than PP6373-OCTZ and OKTZ-PP6373. H727 lung cancer cells and activated human T cells were incubated at a ratio of 1:5 with negative control (untransfected 293T supernatant), PP6373-OCT3, OKT3-PP6373, or FIT-Ig for 12 hours. Cytokines (IFNr, TNF, IL10, IL6, IL4 and IL2) in tissue culture media were measured using flow cytometry. Three independent experiments were carried out.

Fig. 21 - Flow cytometry analysis shows that FIT-Ig induces tumor cell cytotoxicity by T cells. H727 lung cancer cells and activated human T cells

- 4 045813 was incubated 1:5 with negative control (untransfected supernatant 293T), PP6373-OKT3, OKTZ-PP6373 or FIT-Ig for 12 hours. Lung cancer cells and human T cells were collected and stained with anti-human CD45 and Aqua Live reagent /Dead. Tumor cell counts were plotted as double negative for anti-CD45 and Aqua. Three independent experiments were carried out.

Fig. 22 - Flow cytometry analysis shows that FIT-Ig has higher thermal stability than PP6373-OCTZ and OKTZ-PP6373. All bispecific antibodies PP6373OKT3, OKTZ-PP6373 and FIT-Ig were incubated at the indicated temperature for 20 min, and the supernatants after centrifugation at 14000 g for 5 min were used to stain Jurkat cells. Biotinylated SA-N-CD24 protein was then incubated with Jurkat cells, and the PE-steptavidin signal was measured by flow cytometry.

Fig. 23 is a diagram of a CarT construct containing anti-CD24-scFv.

Fig. 24 is a graph of CD24 CART-induced cytotoxicity for the A549 lung cancer cell line.

Fig. 25 is a graph of CART activation by a tumor cell line as evidenced by IFNy production.

Fig. 26 is a bar graph of the antitumor activity of CD24 CART against various tumor types. The E/T ratio for the data presented is 5.

Fig. 27 is a strip diagram of the three-dimensional structural alignment of chimeric PP6373 (FR: white, CDR: light gray) and huVHv1VLv1 (FR: gray, CDR: dark gray).

Fig. 28 is a graph of the relative potency of various antibody pairs for CD24-GST expression and binding.

Fig. 29 is a graph of H2L3 and H3L3 binding to human cancer cell lines NCI-H727 (top) and IMR32 (bottom). Data shown are average fluorescence intensities using a wide range of antibodies.

Fig. 30 - Cell death plots show that at low concentration, HL33 is more effective than PP6373 in ADCC. The lung cancer cell line A549 was used as a target, and human PBL was used as effectors. The dose of antibody used was 3 μg/ml (top panel) or 9 μg/ml (lower panel).

Fig. 31 Cell death plots show that H3L3 confers strong ADCC activity against multiple tumor cell lines, including the lung cancer cell lines A549 and NCI-H727 and the neuroblastoma cell line IMR-32. Human PBMCs (peripheral blood mononuclear cells) were used as effector cells.

Fig. 32 Cell death plots show that H3L3 confers strong ADCC activity in multiple tumor cell lines, including the lung cancer cell lines A549 and NCI-H727 and the neuroblastoma cell line IMR-32. NK (natural killer) cells purified from human PBMCs are used as effector cells.

Fig. 33 - Flow cytometry analysis shows that antibodies recognizing the glycan-screened epitope do not recognize B cells, red blood cells and interact poorly with neutrophils.

Detailed Description of the Invention

Targeting epitopes expressed by cancer is a widely used approach for cancer treatment. However, many such epitopes are not good drug targets because they are also expressed in normal tissues, which can lead to toxicity problems. An ideal tumor specific antigen (TSA) should have high expression in the tumor but minimal or no expression in important host organs. Attributes of less ideal but equally functional TSAs are those that are expressed but differentially modified in normal and cancer tissues, the so-called tumor antigens (TAAs). Examples of well-characterized tumor antigens are MAGE-A3, MUC-1, and NY-ESO 1.

The identification of new TSAs and TAAs is a limiting factor in the development of new or more effective cancer treatments, especially those cancers for which no tumor antigens currently exist. CD24 is a good target for cancer for the following reasons: it is overexpressed in more than 70% of all human cancers and is differentially glycosylated in cancers, it appears to be an oncogene and is associated with poor prognosis in various cancers and significantly shorter lifespan patient survival and is a marker of cancer stem cells that can cause relapse and metastasis, giving rise to new tumors. The inventors discovered anti-CD24 antibodies whose binding to CD24 is blocked by glycosylation, which occurs in normal cells but not in cancer cells. As a result, the antibodies bind to cancer cells and cancer tissues, but with minimal reactivity to many normal tissues and hematopoietic cells. Antibodies and antigen-binding fragments thereof are provided herein. The antibody may be a monoclonal antibody, a human antibody, a chimeric antibody, or a human antibody.

- 5 045813 modified antibody. The antibody may be monospecific, bispecific, trispecific or multispecific. The antigen-binding antibody fragment can immunospecifically bind CD24, and in particular human CD24, preferably expressed on the surface of a living cell at endogenous or transfected concentrations. The antigen binding fragment can bind CD24. The antibody may be labeled with a detectable label or may include a conjugated toxin, drug, receptor, enzyme, or receptor ligand.

In addition to directly targeting tumors, the immune system has the ability to recognize and eliminate cancer in experimental model systems and in patients. As a result, cancer immunotherapy is becoming one of the most promising areas of cancer therapy. Active cancer immunotherapy includes agents that enhance natural immune responses (including antibodies against PD-1, PD-L1, or CTLA-4); bispecific molecules, such as antibodies, that form a bridge between cancer and immune effector T cells; or adoptive cell transfer (ACT) using ex vivo stimulated tumor infiltrating lymphocytes (TILs), activated natural killer (NK) cells, or genetically engineered T cells (chimeric antigen receptor (CAR) and T cell receptor modified T cells (TCR)). Many of these technologies require a tumor targeting component for specificity and effectiveness.

1. Definitions.

The terminology used herein is intended to describe specific embodiments only and should not be interpreted in a limiting manner. As used in the specification and the accompanying claims, the singular number also denotes the plural unless the context clearly dictates otherwise. The word about (approximately, approximately) in combination with a numerical value denotes a reasonable approximation to that value. In some cases, about can be interpreted as being within 10% of the specific meaning with which it is used. For example, the phrase about 100 could cover any value from 90 to 110. When listing numeric ranges, this document explicitly relies on treating each intermediate number within the range with the same degree of precision. For example, for the range 6-9, the numbers 7 and 8 are counted in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3 are explicitly counted. 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

Treat or cure, in relation to protecting an animal from a disease, means preventing, suppressing, repressing or completely eliminating a disease. Prevention of disease includes administering the composition as disclosed to the animal prior to the onset of disease. Suppression of the disease involves administering the composition as disclosed to the animal after the induction of the disease, but before its clinical manifestation. Disease suppression involves administering the composition to the animal upon clinical manifestation of the disease.

As used herein, the term antibody is intended to refer to an immunoglobulin molecule that has a variable region antigen recognition site. The term variable region is intended to distinguish such an immunoglobulin domain from domains that are common among antibodies (such as the Fc domain of an antibody). The variable region includes a hypervariable region, the residues of which are responsible for antigen binding. The hypervariable region contains amino acid residues of the complementarity determining region or CDR (that is, generally, residues at approximately positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region and residues approximately at positions 27-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region) and/or hypervariable loop residues (i.e. residues 26-32 (L1), 50-52 (L2) and 9196 (L3) in the light chain variable region and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region). Framework region residues or FRs are those variable region residues that differ from the hypervariable region residues as defined herein. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelid antibodies, single chain antibodies, disulfide-linked Fv (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id and anti-anti-Id antibodies to the antibodies of the invention). In particular, such antibodies include immunoglobulin molecules of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), any class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

As used herein, the term antigen-binding antibody fragment refers to one or more portions of an antibody that contain the antibody CDR and, optionally, framework residues that constitute the antigen recognition site of the antibody variable region and exhibit immunospecific antigen-binding ability. Such fragments include Fab', F(ab') ₂ , Fv, single chain fragment (ScFv) and mutants thereof, naturally occurring variants and fusion proteins comprising an antibody variable region antigen recognition site and a heterologous protein (eg, toxin, antigen recognition site for another antigen, enzyme, receptor or receptor ligand, etc.). As used herein, the term fragment refers to a peptide or polypeptide containing an amino acid sequence consisting of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues

- 6 045813 residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues , at least 200 contiguous amino acid residues or at least 250 contiguous amino acid residues.

Human, chimeric or humanized antibodies are particularly preferred for in vivo use in humans, however murine antibodies or antibodies of other species can be used successfully for many applications (eg, in vitro or in situ detection assays, in vivo acute use, etc. .).

A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Chimeric antibodies containing one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91/09967; and US Pat. Nos. 5,225,539, 5,530,101 and 5,585,089, each of which is incorporated herein in its entirety), veneering or variable domain resurfacing (EP 592,106; EP 519,596, each of which is incorporated herein) by reference) and chain shuffling (US Patent No. 5,565,332, the contents of which are incorporated herein by reference).

As used herein, the term humanized antibody refers to an immunoglobulin containing a human framework region and one or more CDRs from a non-human (usually murine or rat) immunoglobulin. The non-human immunoglobulin from which the CDRs are taken is called the donor, and the human immunoglobulin from which the framework is taken is called the acceptor. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, that is, at least about 85-90% identical, preferably about 95% or more. Therefore, all portions of the humanized immunoglobulin, with the possible exception of the CDRs, are substantially identical to the corresponding portions of the natural human immunoglobulin sequences. A humanized antibody is an antibody comprising an immunoglobulin with a humanized light chain and a humanized heavy chain. For example, a humanized antibody will not include a typical chimeric antibody because, for example, the entire variable region of the chimeric antibody is non-human. A donor antibody may be referred to as humanized during the humanization process because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody from which the CDRs are taken. In general, humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable region residues from the recipient are replaced by hypervariable region residues from a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity and binding capacity. In some cases, framework region (FR) residues of human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further improve the characteristics of the antibodies. In general, a humanized antibody will comprise substantially the entire sequence of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions are non-human immunoglobulin sequences, and all or substantially all of the FRs are human immunoglobulin sequences. The humanized antibody may also optionally include at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin, that immunospecifically binds to an FcyRIIB polypeptide that has been altered by introducing substitutions, deletions, or additions of amino acid residues (i.e., mutations).

2. Compositions of anti-CD24 antibodies.

This document describes an anti-CD24 antibody that can specifically target a cancer-specific glycoform of CD24. The Ahtu-CD24 antibody can be used to develop cancer treatments, including, but not limited to: antibody-drug conjugates, ADCC-enhanced therapeutic antibodies, bispecific antibodies, CAR-T therapies and TCR therapies. In particular, an anti-CD24 antibody or antigen-binding fragment thereof may bind to a glycan-screened epitope that is not screened on cancer cells but is screened on non-cancerous cells. In particular, an anti-CD24 antibody or an antigen-binding fragment thereof may

- 7 045813 can bind to the CD24 peptide containing the amino acid sequence SNSGLAPN (SEQ ID NO: 48).

The Ahtu-CD24 antibody may be 3B6, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 1 and a light chain variable region containing the sequence set forth in SEQ ID NO: 2. Ahtu-CD24 antibody or its antigen binding fragment may be an affinity matured version of 3B6 and may include a heavy chain variable region comprising any of the sequences set forth in SEQ ID NO: 3-10 and a light chain variable region containing any of the sequences set forth in SEQ ID NO: 11-16. The Ahtu-CD24 antibody or antigen binding fragment thereof may be PP6373, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 6 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 16. For for therapeutic use in humans, the anti-CD24 antibody or antigen binding fragment thereof may be a humanized version of PP6373 and may comprise a heavy chain variable region comprising any of the sequences set forth in SEQ ID NOs: 29-32 and a light chain variable region comprising any of the sequences specified in SEQ ID NO: 33-36. In particular, the humanized anti-CD24 antibody or antigen binding fragment thereof may be H2L3, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 30 : 35; or may be H3L3, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 31 and a light chain variable region containing the sequence set forth in SEQ ID NO: 35.

3. Antibody-drug conjugate compositions.

A tumor-targeted antibody can be used to prevent or limit the growth of tumors by directly influencing tumor biology. For example, a humanized monoclonal anti-VEGF antibody (bevacizumab; Avastin) blocks tumor growth by preventing VEGF-induced tumor vascularization. Other tumor-targeting antibodies are used to inhibit the growth of tumor cells or kill cancer cells by modifying the antibody itself. For example, tumor-targeting immunoconjugates consist of an antibody and an effector moiety linked together by covalent cross-links or genetic fusion. The effector moiety may be a cytotoxic drug (antibody-drug conjugate), a protein toxin (immunotoxin), or a radionuclide (radioimmunoconjugate). An example of an antibody-drug conjugate is brentuximab vedotin (ADCETRIS®, Seattle Genetics), which consists of the chimeric monoclonal antibody brentuximab (cAC10, which targets the cell membrane protein CD30) linked to three to five units of the antimitotic agent monomethyl auristatin E (MMAE, which is reflected by the expression vedotin in the name of the drug).

The Ahtu-CD24 antibody or antigen-binding fragment thereof may be included in antibody-drug conjugates, immunotoxins or radioimmunoconjugates. The anti-CD24 targeting component of such compositions may allow specific delivery of the conjugate to cancer cells and tissues while limiting exposure to normal cells and tissues and thereby preventing off-target toxicity.

4. Compositions of ADCC antibodies.

An Ahtu-CD24 antibody or an antigen-binding fragment thereof, or an antibody composition containing one of the above, can be used to promote the death of cancer cells through at least one of the following mechanisms: antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCC is an immune defense mechanism by which a specific set of immune cells (effector cells) in the body actively interact with and lyse a target cell (such as a pathogen). ADCC has been identified as an important cell-mediated innate immune response, functions as the body's first line of defense against pathogens, and acts to limit and control infections. The ADCC process is designed to kill the antibody-coated target cell through a non-phagocytic process and is characterized by either the targeted release of cytotoxic vacuoles or the expression of molecules that induce cell death. ADCC is usually initiated when specific antibodies (mainly IgG classes) of the host recognize and bind membrane surface antigens of target cells and simultaneously interact with Fc receptors (FcRs) on the surface of effector cells. The most common effector cells that mediate ADCC are natural killer (NK) cells, although monocytes, macrophages, neutrophils, eosinophils, and dendritic cells are also capable of mediating the ADCC response. Although ADCC is a fairly rapid response, the effectiveness varies depending on several parameters, such as the density of antigen on the surface of target cells and the affinity of the antigen-antibody interaction, as well as the characteristics of Fc fragments, which determine the interactions of antibodies with various members of the Fc receptor family.

- 8 045813

The binding of an antibody to specific cell surface receptors on target cells, a process called opsonization, is a key event in the ADCC process. The process of opsonization attracts phagocytes to the target cell and can initiate phagocytosis. Binding of the antibody Fc region to the FcR on phagocytes also promotes the formation of C3b, a cleavage product of complement component 3, which is an essential protein that initiates the engulfment of antibody-opsonized target cells. Antibody-mediated phagocytosis is also often called antibody-dependent cell-mediated phagocytosis (ADCP). However, in the case of ADCC, killing the pathogen does not require it to be phagocytosed. As noted above, FcR on the surface of cytotoxic effector cells is key to the induction of ADCC. In humans, the most important classes of FcRs capable of causing ADCC are FcyRI (CD64), FcyRIIa and FcyRIIc (CD32), and FcyRIIIa (CD16). However, the FcyRIIb receptor suppresses the ADCC response. Thus, the balance between activating and inhibitory signals from FcyRs is an important factor determining the magnitude of the ADCC response. Upon target recognition, specialized intracellular vacuoles (also called secretory lysosomes) are released by cytotoxic effector cells in a calcium-dependent polarized exocytotic process. Perforin, cytolysin and granzyme B are the key components that are released from the vacuoles. Perforin penetrates and forms pores in the target cell membrane. This process requires calcium. Granzyme B causes DNA fragmentation in the target cell. An example of a therapeutic antibody that works via ADCC is trastuzumab (Herceptin, Genentech). Trastuzumab targets HER2, which is expressed at abnormally high levels in a wide range of breast cancers, often referred to as HER2-positive breast cancer, and suppresses the growth of HER2-positive breast cancer by inducing ADCC in the host. Antibodies used to induce ADCC usually require some modification to enhance their ADCC activity. There are a number of technologies available to do this, which typically involve designing the antibody so that the oligosaccharides in the Fc region of the antibody do not contain fucose sugar units, which improves binding to the FcyIIIa receptor. Afucosylated antibodies exhibit increased antibody-dependent cellular cytotoxicity (ADCC). For example, Biowa's Potelligent® technology uses a FUT8 knockout CHO cell line to produce 100% afucosylated antibodies. FUT8 is the only gene encoding an α1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to GlcNAc via a complex-type α1,6 oligosaccharide linkage. Probiogen has developed a CHO line that is designed to produce lower levels of fucosylated glycans on MAbs, although not by knocking out FUT. The Probiogen system introduces a bacterial enzyme that redirects the de-novo fucose synthesis pathway to a nucleotide sugar that cannot be metabolized by the cell. As an alternative approach, Seattle Genetics has a proprietary feeding system that produces lower levels of fucosylated glycans on MAbs produced in CHO (and possibly other) cell lines. Xencor has developed XmAb Fc domain technology designed to improve the immune system's elimination of tumor and other pathological cells. This Fc domain has two amino acid substitutions, resulting in a 40-fold increase in affinity for FcyRIIIa. It also increases affinity for FcyRIIa with the potential to recruit other effector cells such as macrophages, which play a role in immunity by engulfing and digesting foreign material. An anti-CD24 antibody or an antigen-binding fragment thereof may be included in antibodies that kill cancer through ADCC. The anti-CD24 targeting component of such compositions can provide specific targeting to cancer cells for ADCC-mediated destruction while sparing normal cells and tissues. The ADCC activity of an anti-CD24 antibody or an antigen-binding fragment thereof may be enhanced by one or more of the modifications described herein.

5. Compositions of bispecific antibodies.

Further provided herein is a bispecific antibody that comprises a first antibody domain comprising a first antibody or an antigen binding fragment thereof linked to a second antibody or an antigen binding fragment thereof. The first domain of the antibody may comprise an anti-CD24 antibody or an antigen binding fragment thereof described herein, and the second antibody or an antigen binding fragment thereof may bind to other immunostimulatory molecules. In a specific embodiment, the second domain of the antibody includes an anti-CD3 antibody or an antigen binding fragment thereof. In this case, the bispecific antibody can specifically target tumor cells expressing the cancer-specific glycoform of CD24 while simultaneously binding CD3 on cytotoxic T cells, thereby attracting the T cells to the tumor site, whereby the T cells will infiltrate the tumor and cause cytotoxicity in the tumor . Other examples of antibody partners for use in a bispecific antibody to attract cytotoxic T cells or other effector cells to a tumor site are known in the art.

The second antibody or antigen-binding fragment thereof may target a complementary antitumor pathway or mechanism. The second domain of the antibody may include a cancer immunotherapy antibody or an antigen binding fragment thereof that enhances natural immune responses.

- 9 045813 you. Examples of such antibodies for cancer immunotherapy include anti-PD-1, anti-B7-H1, anti-B7-H3, anti-B7-H4, anti-LIGHT, anti-LAG3, anti-TIM3, anti-TIM4, anti-CD40 , anti-OX40, anti-GITR, anti-BTLA, anti-CD27, anti-ICOS or anti-4-1BB antibodies. Such antibodies can be used to treat cancer. The second antibody or antigen-binding fragment thereof may bind the TCR α chain, the TCR β chain, the TCR γ chain, or the TCR 5 chain.

The bispecific antibody may contain the sequences set forth in SEQ ID NOs: 17 and 18, or any of the sequences set forth in SEQ ID NOs: 23-27 and 37-41. There are many different bispecific antibody technologies known in the art. Most require that the antibodies of the two components be in a single-chain format so that the two parts can be expressed in a single construct. The preferred method is to express the antibody as a single chain variable fragment (scFv). Non-limiting examples of bispecific antibody technologies include BiTE (which stands for Bi-specific T-cell Engager), DART (which stands for Dual-Affinity Re-Targeting), Fab tandem immunoglobulin (FIT- Ig) and protrusions-to-recessions type. Such bispecific antibodies containing an anti-CD24 antibody or an antigen-binding fragment thereof are particularly contemplated herein.

6. CAR-T therapeutic compositions.

Chimeric antigen receptor (CAR) T-cell therapy, or CAR-T therapy, is a type of cell treatment in which a cancer patient's T cells are genetically modified ex vivo to express the CAR protein so they can attack cancer cells . In particular, T cells are obtained from the patient's blood, which in particular may be the patient's own blood (autologous), and are transfected with a gene construct that expresses the recombinant CAR receptor. Large numbers of CAR-T cells are then grown in the laboratory and injected back into the patient, where they can target and destroy the patient's cancer cells. The T cells may also be allogeneic from a suitable donor or from a universal or off-the-shelf T cell line in which one or more of the TCR gene loci and the HLA class I loci of the allogeneic T cells are disrupted and the resulting T cells are unable to recognize allogeneic antigens.

CAR protein constructs have modular structures typically containing the following core components: an antibody-derived extracellular single chain variable fragment (scFv) attached to a hinge/spacer peptide and a transmembrane domain, which is further linked to the intracellular T cell signaling domains of the T cell receptor. ScFv is a targeting element and is expressed on the surface of CAR-T cells to impart antigen specificity. The spacer connects the extracellular targeting element to the transmembrane domain and influences CAR function and scFv flexibility. The transmembrane domain crosses the cell membrane, anchors CAR to the cell surface, and connects the extracellular domain to the intracellular signaling domain, thereby affecting cell surface expression of CAR. The costimulatory domain is derived from the intracellular signaling domains of costimulatory proteins such as CD28 and 4-1BB, which enhance cytokine production. The CD3 zeta domain is derived from the intracellular signaling portion of the T cell receptor, which mediates downstream signaling during T cell activation. Examples of CAR-T therapeutics include those that target B cell surface antigens CD19 (such as JCAR017 and JCAR014 [Juno Therapeutics]), CTL019 (tisagenlecleucel-T (Kymriah™) [Novartis]), and CTE-C19 (axicabtagene ciloleucel (Yescarta®) [Kite Pharma]) and CD22 (JCAR014 [Juno Therapeutics]). Other examples of CAR-T therapeutics include those that target Ll-CAM (JCAR023 [Juno Therapeutics]), ROR-1 (JCAR024 [Juno Therapeutics]), and MUC16 (JCAR020 [Juno Therapeutics]). The ScFv portion of CAR is a critical component that provides specificity for cancer cells while preventing activity against normal cells that is associated with off-target toxicity. Therefore, the scFv portion is typically derived from a portion of the antibody that recognizes a target protein that is specifically expressed on cancer cells but is much less commonly, or ideally not, expressed at all on other cells and tissues. Accordingly, a scFv fragment derived from any of the anti-CD24 antibodies described herein can be used as a cancer-targeting component of a recombinant CAR protein. In particular, the scFv protein may contain the sequence set forth in SEQ ID NO: 28.

CAR T cells have demonstrated impressive effects against hematological tumors such as acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia, adult myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL) and multiple myeloma (MM). However, to date, CAR T therapy has shown only limited effect against solid tumors. Due to the characteristic expression pattern of CD24 in tumors and normal tissues, data obtained using CD24 CAR-T have demonstrated that the types of cancer that can be targeted

- 10 045813 include, but are not limited to, brain tumors, head and neck cancer, sarcoma, lung cancer, gastrointestinal cancer, breast cancer, testicular cancer, prostate cancer, pancreatic cancer, cervical cancer, cancer ovarian, liver cancer or hematological malignancies.

7. TCR therapeutic compositions.

Similar to CAR-T therapy, genetically engineered T-cell receptor (TCR) therapy is a type of cell-based treatment in which a cancer patient's T cells are genetically modified ex vivo to express a modified TCR in order to improve the T-cell receptor's ability to recognize and attack specific cell antigens when the cells are reintroduced to the patient. However, unlike CAR T cells, which recognize proteins expressed on the surface, T cell immunotherapy using gene-edited TCRs has been targeted primarily at solid tumors. TCRs can recognize tumor-specific proteins within cells. When tumor-specific proteins are broken down into fragments, they are presented on the cell surface with another protein called the major histocompatibility complex, or MHC. TCRs are designed to recognize a tumor-specific protein fragment/MHC combination. Examples of TCR-modified T cell targets include Mage-Az targets such as KITE-718 (Kite Pharma), Wilms tumor antigen 1 (WT-1) such as JTCR016 (Juno Therapeutics) and NY-ESO 1.

The TCR is a heterodimer consisting of two subunits, TCRa and TCRe. Each subunit contains a constant region, which is adjacent to the T cell membrane and anchors the receptor to the cell membrane, as well as a hypervariable region, which is involved in antigen recognition. Accordingly, a scFv fragment derived from any of the anti-CD24 antibodies described herein can be used as a cancer-targeting component of a recombinant TCR protein. In particular, the scFv protein may contain the sequence set forth in SEQ ID NO: 28.

8. Peptide compositions.

An anti-CD24 antibody or antigen binding fragment thereof described herein can bind a glycan-screened epitope that is not screened on cancer cells but is screened on non-cancerous cells. In particular, an anti-CD24 antibody or an antigen-binding fragment thereof can bind a CD24 peptide containing the amino acid sequence SNSGLAPN (SEQ ID NO: 48). Accordingly, peptides containing the sequence set forth in SEQ ID NO: 48 can be used to neutralize anti-CD24 antibodies that bind to epitopes containing the core sequence set forth in SEQ ID NO: 48. This can be used in assays based on anti-drug antibodies to detect neutralizing antibodies. Peptides containing the sequence set forth in SEQ ID NO: 48 can be used to inhibit the potential adverse effects associated with antibodies that bind to epitopes constituting the core sequence set forth in SEQ ID NO: 48. The peptide can be modified to improve stability for use in vivo using methods known in the art, including, but not limited to, the use of D-amino acids, the replacement of O with S in one or more peptide bonds, the addition of a fusion sequence to improve solubility or half-life (for example, fusion with albumin ). In yet another embodiment, a molecule comprising the sequence set forth in SEQ ID NO: 48 can be used as a vaccine for the treatment and prevention of cancer.

9. Methods of treatment.

Anti-CD24 antibody compositions or cell therapeutics including such antibody compositions described herein can be used to treat or prevent cancer or other abnormal proliferative disease. Provided herein is a method of such administration to a patient in need thereof, which may include administering to the patient an anti-CD24 antibody or an antigen binding fragment thereof or a pharmaceutical composition containing the foregoing. Such molecules and pharmaceutical compositions may also be used in the manufacture of a medicament for the treatment or prevention of cancer or other abnormal proliferative disease. As used herein, the term cancer refers to a growth or tumor resulting from abnormal, uncontrolled cell growth. As used herein, the term cancer clearly includes leukemia and lymphomas. The term refers to a disease involving cells that can metastasize to distant sites. The patient may be human. Cancer or other abnormal proliferative disease may be (but is not limited to) one or more of the following: carcinoma, including carcinoma of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid glands and skin; including squamous cell carcinoma; hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkett's lymphoma; hematopoietic tumors of myeloid origin, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin

- 11 045813 nia, including fibrosarcoma and rhabdomyosarcoma; other tumors including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoactantoma, seminoma, follicular thyroid cancer and teratocarcinoma. It is also contemplated that cancers caused by aberrations of apoptosis can also be treated with the methods and compositions of the present invention. Such cancers may include, but are not limited to, follicular lymphomas, p53 mutation carcinomas, hormone-dependent breast, prostate and ovarian tumors, as well as precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific embodiments, malignant or dysproliferative changes (such as metaplasias and dysplasias) or hyperproliferative disorders are treated or prevented by the methods and compositions of the present invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. Cancer can also be sarcoma, melanoma or leukemia.

The Ahtu-CD24 antibody or antigen-binding fragment thereof may be used in combination with one or more other anticancer therapies, including, but not limited to, current standard and experimental chemotherapy, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery. In some embodiments, an anti-CD24 antibody or antigen-binding fragment thereof can be administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies, or other agents known to those skilled in the art for the treatment and/or prevention of cancer, autoimmune disease, infectious illness or intoxication. Such agents include, for example, any of the biological response modifiers discussed above, cytotoxins, antimetabolites, alkylating agents, antibiotics or antimitotic agents, and immunotherapies.

The Ahtu-CD24 antibody or antigen-binding fragment thereof can be used in combination with one or more antitumor immunotherapies. Antitumor immunotherapies may include molecules that disrupt or enhance alternative immunomodulatory pathways (such as TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, PD-1, B7-H3, B7-H4, LIGHT , BTLA, ICOS, CD27, or LAG3) or modulate the activity of effector molecules such as cytokines (eg, IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-beta, IFNg , Flt3, BLys) and chemokines (for example, CCL21), in order to enhance immunomodulatory effects. Specific embodiments include a bispecific antibody comprising an anti-CD24 antibody or binding fragment thereof and anti-PD-1 (pembrolizumab (Keytruda®) or nivolumab (Opdivo®)), anti-B7-H1 (atezolizumab (Tecentriq®) or durvalumab) , anti-B7-H3, anti-B7-H4, anti-LIGHT, anti-LAG3, antiTIM3, anti-TIM4, anti-CD40, anti-OX40, anti-GITR, anti-BTLA, anti-CD27, anti-ICOS or anti-41BB. In yet another embodiment, an anti-CD24 antibody or antigen binding fragment thereof can be administered in combination with molecules that activate various stages or aspects of the immune response to achieve a broader immune response. In a more preferred embodiment, an anti-CD24 antibody or antigen binding fragment thereof can be combined with anti-PD-1 or anti-4-1BB antibodies without increasing autoimmune side effects.

10. Receipt.

Ahtu-CD24 antibody or antigen binding fragment thereof can be produced using a eukaryotic expression system. The expression system may include expression from a vector in mammalian cells, such as Chinese hamster ovary (CHO) cells. The system may also be a viral vector, such as a replication-defective retroviral vector, which can be used to infect eukaryotic cells. The Ahtu-CD24 antibody or antigen binding fragment thereof can also be produced in a stable cell line that expresses the antibody from a vector or portion of a vector integrated into the cellular genome. A stable cell line can express an antibody from an integrated replication-defective retroviral vector. The expression system may be GPExTM.

The Ahtu-CD24 antibody or antigen binding fragment thereof can be purified using, for example, chromatographic techniques such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, size exclusion chromatography and hydroxyapatite chromatography. In some embodiments, the fusion proteins can be designed to contain an additional domain containing an amino acid sequence that allows the polypeptides to be captured by an affinity matrix. For example, antibodies described herein containing the Fc region of an immunoglobulin domain can be isolated from cell culture supernatant or cytoplasmic extract using a protein A column. Additionally, a tag such as c-myc, hemagglutinin, can be used to facilitate purification of the polypeptide , polyhistidine or Flag™ (Kodak). Such marks can be inserted anywhere along the

- 12 045813 lipeptide, including the carboxyl or amine terminus. Other fusions that may be useful include enzymes that aid in polypeptide detection, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. Vaccines

Provided herein is a method of treating cancer or providing prevention of the cancer described herein in a patient. Using this method, a patient can be vaccinated against cancer. The method may include administering a composition comprising the sequence set forth in SEQ ID NO: 48 to a patient in need thereof. The composition can also be administered to a patient in need of treatment of side effects associated with therapy, including the use of anti-CD24 antibodies or cells expressing CD24 binding receptors. The composition can also be used in the manufacture of a medicament for treating cancer or providing cancer prevention.

11. Pharmaceutical compositions.

Provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the anti-CD24 antibodies, cell therapeutics or peptide compositions described above and a physiologically acceptable carrier or excipient. The pharmaceutical composition may contain a prophylactically or therapeutically effective amount of an anti-CD24 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In a specific embodiment, the term pharmaceutically acceptable means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more particularly, in humans. The term carrier refers to the diluent, adjuvant (for example, Freund's adjuvant (complete and incomplete), excipient or carrier medium with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including petroleum, animal , vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene , glycol, water, ethanol, etc. The pharmaceutical composition, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

Typically, the ingredients of a pharmaceutical composition may be supplied either separately or admixed in unit dosage form, for example as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. If the pharmaceutical composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile water or pharmaceutical grade saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration. The pharmaceutical composition can be obtained in neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and salts formed with cations such as those derived from sodium hydroxides, potassium, ammonium, calcium, iron, isopropylamine, triethylamine, 2ethylaminoethanol, histidine, procaine, etc.

12. Methods of administration.

Methods of administration of the formulations and their pharmaceutical compositions include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and mucosal (eg, intranasal and oral routes). In a specific embodiment, the composition is administered intramuscularly, intravenously or subcutaneously. The composition can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (for example, oral mucosa, rectal mucosa, etc.) and can be administered in conjunction with one or more other biologically active substances. Administration may be systemic or local.

Examples

The disclosure of the invention has many aspects, illustrated by the following non-limiting examples.

Example 1. Synthesis of monoclonal antibodies to hypoglycosylated CD24.

Overexpression of NEU1 and CD24 in tumors suggests glycosidase dysregulation. On

- 13 045813 glycosidase dysregulation suggests that CD24, like MUC1, may be hypoglycosylated in tumors. Binding of the antibody, 3B6, to CD24 is hampered by sialic acid glycans (Fig. 1). Compared to the commercially available anti-CD24 antibody, ML5 (BD bioscience), 3B6 binds strongly to N-SA-CD24 and N-SA-O-CD24, but only weakly N-CD24 or fully glycosylated CD24, as detected by ELISA . This indicated that the epitopes that 3B6 binds are indeed a protein backbone, and that 3B6 binding is prevented by glycosylation of the epitope. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IFA) staining results show that 3B6 binds multiple cancer cell lines, including neuroblastoma and medulloblastoma (Fig. 2A-B). 3B6 binds the neuroblastoma cell lines IMR32, SK-N-SH, SH-SY5Y, SK-N-BE(2), and SK-N-BE(2)C, but not SK-N-AS (Fig. 2A). 3B6 also bound 3 of 4 patient-derived medulloblastoma tumors as assessed by IFA staining (Fig. 2B). These data indicate that 3B6 is capable of binding cancer cell lines and tumors.

Example 2: Affinity maturation.

The binding affinity of 3B6 to CD24 was significantly lower compared to the commercial antibodies ML5 (BD Bioscience) and SN3 (Thermo Fisher). To increase the affinity and specificity of binding of 3B6 to its antigen, affinity maturation of 3B6 was performed. First, the heavy (IgH) and light (IgL) chains of the 3B6 antibody were cloned and the Ig variable region sequence was identified as follows: 3B6 IgH (SEQ ID NO: 1, CDRs in underlined and bold) EVKFEESGGGLVQPGGSIKLS CAASGVTFSEAWMDWVRO SPEKGLEWVAEI

RDKTKNYVTYYAESVKGRFTISRDDSKSRVYLOMNNLRTEDTGIYYCTGA

MDYWGOGTSVTVSS

3B6 IgL (SEQ ID NO: 2, CDRs in underlined and bold) DIVMTOTPLSLSVTIGOPASISCKSSOSLLYSNGKTYLNWLOORPGOSPKRLI

YQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCLQGTSYPWTFGG

GTKLEIK

The VH and VL fragments of the original 3B6 antibody were converted to scFv format and cloned into a phage display vector. ScFv was displayed monovalently on the phage, allowing the selection of phage clones with higher affinity. To test the display level of scFv, scFv was fused to the Flag-6xHis detection tag. Phage ELISA was performed to test the binding of the parent antibody to the antigen in a phage display format. The binding signal from the phage supernatant was significant and the project moved on to library construction.

Three rounds of selection and screening were conducted. Reducing the concentrations of CD24-GST antigen and biotinylated CD24-GST was used in screening to select clones with higher binding. Forty-eight clones from each CDR mutagenesis library were selected, cultured, analyzed for binding, and sequenced. After confirmation of the sequences of the affinity matured scFv clones, the affinity matured scFv clones were reformatted into full length antibody genes and transiently expressed in mammalian cells. All affinity matured antibodies were prepared in small batches of 0.01 L. The parent antibody was also generated for direct comparison. Plasmids for the indicated heavy and light chains (Table 1) were transfected into suspension HEK293 cells using chemically defined media in the absence of serum to obtain antibodies. Five days after transfection, conditioned media were collected and clarified. Whole antibodies in conditioned media were purified using MabSelect SuRe Protein A media (GE Healthcare).

- 14 045813

Table 1

Antibodies produced in HEK293 cells by transient transfection and purified with IgG1

Parental With Affinity Ripe, Panel 1 With Affinity Ripe, Panel 2 PP6226 - H4040+L4040 anti- CD24 PP6228 - H4041+L4040 P3050.N1.A4 PP6368 - H4069+L4069 P3050.ComFl.All PP6230- H4042+L4040 P3050.N2.A7 PP6369 - H4070+L4069 P3050.ComFl.H4 PP6231 - H4043+L4040 P3050.N2.B11 PP6370 - H4071+L4069 P3050.ComFl.2F4 PP6232- H4040+L4041 P3050.L3.B9 P6371 - H4072+L4070 P3050.ComFl.2F5 PP6233- H4040+L4042 P3050.L3.C7 PP6372 - H4073+L4071 P3050.ComFl.C9 PP6234- H4040+L4043 P3050.L3.D8 PP6373 - H4069+L4071 P3050.ComFl.2Hl PP6235- H4041+L4042 P3050.H1.A4.L3.C7 PP6387 - H4071+L4071 P3050.ComF2.Bl PP6236- H4043+L4042 P3050.H2.B11.L3.C7 PP6388- H4072+L4069 P3050.ComF2.A5 Table: List of transient transfections and purifications performed to obtain IgG. H40xx means heavy chain design and L40xx means light chain design. P3050.xx denotes the original clone obtained by phage panning. All IgGs were well expressed. PP numbers are serial codes used to distinguish the proteins produced.

The purified affinity matured antibodies and the parent antibody were assessed for their affinity for the antigen using a competitive ELISA. Antibody PP6226 (parental variable regions 3B6) was applied to the plates at a concentration of 2 μg/ml. Affinity-matured antibodies were first incubated with CD24-GST, then plate incubated, followed by antibody incubation for secondary detection. As shown in FIG. 3-5, 16 antibodies were obtained with varying abilities to compete with their parental clones. The amino acid sequences of the heavy and light chains of these antibodies are SEQ ID NO: 3-10 (heavy chain) and SEQ ID NO: 11-16 (light chain). To determine whether affinity matured clones had stronger binding to CD24 and whether the interactions were glycan regulated, CD24 was treated with either N-glycanase (NCD24), NanA sialidase (SA-CD24), or both enzymes (N-SA- CD24). The 16 clones described in FIG. 3-5 were tested using ELISA. As shown in FIG. 6, despite significant affinity for CD24-GST, PP6231 and PP6230 cannot bind CD24 expressed by mammalian cells, regardless of glycosylation. On the other hand, most other clones retained preferential binding to CD24 that were treated with sialidase and/N-glycanase. However, because the relative influence of sialidase and N-glycanase on antibody binding varies significantly among clones, each clone must be tested individually to determine their sensitivity to glycan screening.

Six clones with strong SA-N-CD24 binding but that exhibit minimal binding to CD24 were selected and tested for binding to two cancer cell lines, the lung cancer cell line H727 and the neuroblastoma cell line IMR32. As shown in FIG. 7, despite similarity in their binding to SA-N-CD24, 6 clones showed significantly different binding to cancer cells. Importantly, PP6373 exhibited significantly stronger binding to both cancer cell lines tested. Therefore, this clone was selected for further study. The heavy chain sequence of PP6373 is listed in SEQ ID NO: 6 and the light chain sequence is listed in SEQ ID NO: 16. Compared to the parent sequence, the heavy chain has three mutations in CDR2, while the light chain has one mutation in CDR3 of the light chain. As shown in FIG. 8, these mutations not only increase binding to SA-N-CD24 by nearly 100-fold, but also make the interaction more tightly regulated through desialylation. It is also noteworthy that PP6373 acquired the ability to bind to CD24 even without deglycosylation at a level of 1/1000 that of SA-N-CD24. However, since CHO cells are known to be incompletely glycosylated, it is likely that binding reflects greater sensitivity of the antibody to detect the minor glycoform in recombinant CD24 derived from CHO cells.

Example 3. Antigenic epitope recognized by 3B6 and PP6373.

To determine the antigenic epitope recognized by 3B6 and the affinity matured clone PP6373

- 15 045813, overlapping peptides covering the mature amino acid sequence of CD24 (SEQ ID NO: 42) were synthesized and pre-incubated with the 3B6 antibody before adding 3B6 to plates pre-coated with NO' CD24 protein (CD24Fc, pre-treated sequentially with N- glycosidase, NanA and O-glycosidase). As shown in FIG. 9, of the 5 peptides tested (SEQ ID NO: 43-47), only peptide 4 (SEQ ID NO: 46) showed significant blocking of the 3B6-CD24 interaction, suggesting that the CD24 binding epitope is contained within this sequence. To confirm that PP6373 recognizes the same epitope, five peptides were titrated over a wide range of doses. As shown in FIG. 10, only peptide 4 showed dose-dependent inhibition of PP6373 binding to SA-N-CD24.

To determine the minimal binding site of PP6373, peptide 4 was trimmed one amino acid at a time and its inhibition of PP6373 binding to SA-N-CD24 was compared. As shown in FIG. 11, while deletion of 3 amino acids from the C-terminus eliminates inhibition, deletion of one or two amino acids significantly improves inhibition (left panel). In addition, deletion of any amino acid from the N-terminus of peptide 4 also abolished inhibition (right panel). These data identify SNSGLAPN (SEQ ID NO: 48) as the optimal epitope recognized by PP6373. The identification of the antigenic epitope allows the generation of additional antibodies with similar properties. In one embodiment, new antibodies can be generated using a synthetic peptide containing the sequence SNSGLAPN (SEQ ID NO: 48). The peptide may be linked to another immunogenic carrier protein or used in conjunction with adjuvants. In another embodiment, the peptide can be used to identify other anti-CD24 mAbs that recognize the same epitope to generate cancer-specific antibodies for the diagnosis and treatment of cancer. In yet another embodiment, the antigenic peptide can be used to neutralize or inhibit potential adverse effects associated with antibodies that bind to epitopes containing the core sequence of SEQ ID NO: 48. The peptide can be modified for better stability when used in vivo using methods known in the art. in the art, including, but not limited to, the use of D-amino acids, replacement of O with S in one or more peptide bonds, addition of a fusion sequence to improve solubility or half-life (eg, albumin fusion). In yet another embodiment, a molecule comprising the amino acid sequence of SEQ ID NO: 48 can be used as a vaccine for the treatment and prevention of cancer.

Example 4 Antigenic Epitope Expression in Normal versus Malignant Tissues.

To determine whether the epitope recognized by PP6373 is presented preferentially in cancer tissues compared to normal tissues, tissue binding was analyzed by immunofluorescence using biotinylated PP6373. Data on normal tissues are summarized in Table. 2, and data on cancer tissues are in table. 3. In addition, the binding of the antibody to normal benign and malignant brain cancer was assessed. The data is presented in table. 4.

Table 2 Immunofluorescent staining of PP6373 showed minimal binding to normal tissues

Organ +/- Staining pattern Normal stomach - Normal duodenum - Normal small intestine - Normal colon - Normal parotid salivary gland - Normal thyroid gland - Normal pancreas + Weakly - cell surface, intracellular? Normal prostate - Normal aorta - Normal testicle - Normal greater omentum -

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Normal mammary gland - Normal lymph node - Normal skin - Normal medulla oblongata Normal spleen - Few positive, cell surface? Normal uterus - Normal vagina - Normal bladder - Normal nerve -

Table 3

Reactivity of PP6373 to malignant tissues

Organ Percent positive Staining pattern Malignant colon 0/1 Malignant esophagus 0/1 Malignant stomach 0/2 Malignant ovary 16/25 cell surface Malignant soft tissue 0/1 Malignant kidney 1/1 surface weak Malignant liver 14/19 cell surface Malignant mammary gland 12/20 cell surface Malignant skin 1/1 cell surface Malignant testicle 1/1 intracellular/cell surface Malignant lung 11/39 cell surface

Table 4

Binding of PP6373 to normal benign and malignant brain tumors

Pathology cell surface inside the cell negative Astrocytoma 2/24 (8%) 17/24 (71%) 5/24 (21%) Glioblastoma 3/8 (38%) 2/8 (25%) 5/8 (37%) Oligodendroglioma 4/8 (50%) 3/8 (38%) 1/8 (12%) Ependymoma 5/8 (63%) 0/8 (0%) 3/8 (37%) Medulloblastoma 7/10 (70%) 0/10(0%) 3/10(30%) Benign meningioma 0/22 (0%) 15/22 (68%) 7/22 (32%) Normal CNS tissue 0/16(0%) 0/16(0%) 16/16(100%)

As shown in table. 2, with the exception of the pancreas and possibly the spleen, PP6373 did not stain normal tissue. It should be noted that most staining in the pancreas is intracellular. In the spleen, cell staining was rare. On the contrary, as shown in table. 3, most cancers tested showed strong binding to PP6373. As shown in table. 4, while normal CNS tissues lack CD24, benign meningioma exhibits intracellular staining but does not exhibit cell surface staining. It is important to note that malignant brain tumors, including astrocytoma, glioblastoma, and oligodendroglioma, exhibit 8–70% cell surface staining, and some cancer tissues exhibit intracellular staining.

In one embodiment, PP6373 can be used to distinguish a malignant brain tumor from normal or benign brain tissue. In another embodiment, PP6367 can be used to identify cancerous tissue in solid organs such as liver, lung, breast and ovary.

Example 5 PP6373 slows the growth of lung cancer in vivo.

To test whether PP6373 could slow tumor growth in vivo, nude mice were infected with the human lung cancer cell line H727 subcutaneously. Once the tumor became palpable, tumor-bearing mice received two injections of PP6373 at a dose of 5 mg/kg (14 and 21 days after H727 inoculation). As shown in FIG. 12, compared with the IgG control, the tumor treated with PP6373 grew significantly more slowly. These data demonstrate that unmodified PP6373 is capable of exerting antitumor activity in vivo.

Consistent with the delay in tumor development in vivo, in vitro studies demonstrate that

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PP6367 mediates potent antibody-dependent cellular cytotoxicity as shown in FIG. 13.

Because ADCC is affected by glycosylation, especially fucosylation, antibody engineering was used to generate PP6373 without fucosylation of the FC core (d6373). As shown in FIG. 14, fucosylation increases the ADCC activity of PP6373. The data presented show that PP6373 can be used to treat cancer. In one embodiment, PP6373 WT IgG1 can be used as therapeutic antibodies against cancer for administration to cancer patients. In another embodiment, the antibody can be glycoengineered either chemically or produced in a cell line lacking fucosyltransferase.

Example 6. Bispecific antibodies based on the sequence of PP6373 and OKT3.

For the use of anti-CD24 antibodies, bispecific antibodies have been produced that bind both CD24 and CD3. In one embodiment, anti-CD24 and anti-CD3 (OKT3) antibodies are converted to single-chain antibodies with reactivity to CD24 and CD3, respectively, and linked by the flexible linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 49). The sequence of the single chain antibody PP6373 is shown in SEQ ID NO: 17, and the single chain sequence of OKT3 is shown in SEQ ID NO: 18.

In one embodiment, a bispecific antibody is created using knob and trench technology, in which two partners of the bispecific molecule have complementary mutations in the Fc region to create a knob and trench to facilitate the formation of bispecific heterodimers. The sequences of the PP6373 and OKT3 knob and valley variants are listed in SEQ ID NO: 1922. To evaluate the bispecificity of the various knob and valley configurations, an assay was developed consisting of staining Jurkat cells with the cotransfection product of various products produced by the knob-valley technology. Briefly, CD3+ Jurkat cells were first stained with tissue culture supernatants from transfected 293T cells. After washing away unbound antibodies, cells were incubated with biotinylated SA-N-CD24. The amount of SA-N-CD24 on Jurkat cells was determined using PE-streptavidin. As shown in FIG. 15, the combination of PP6373-groove and OKTZ-overhang results in the highest CD24 binding to Jurkat cells, indicating that the pair of PP6373-groove and OKTZ-overhang is the most suitable for the protrusion-tooth strategy.

In another embodiment, a bispecific antibody is generated through a tandem repeat of two single chain binding motifs. And this time we also compared the activity of two configurations with different binding motifs in opposite orders, PP6373-OKT3 and OKT3PP6373, as indicated in SEQ ID NO: 23 and 24, respectively. As shown in FIG. 16, a design with a single chain of PP6373 at the N-terminus (PP6373-OCT3; SEQ ID NO: 23) shows higher bispecific activity. To determine whether the bispecific antibody had antitumor activity, the lung cancer cell line H727 was co-incubated with T cells that had been activated with anti-CD3 and anti-CD28 for 2 days. First, they tested whether a cancer cell could specifically trigger the production of cytokines. As shown in FIG. 17, significant cytokines were induced by the bispecific antibody, but not by OKT3-Fc PP6373-Fc. More importantly, the bispecific antibody does not induce cytokine production unless both T cells and tumor cells are present together. These data demonstrate that bispecific antibodies trigger T cell activation, involving both T cells and tumor cells.

Simultaneously with the cytokine release assay, tumor cell cytotoxicity was also assessed using microparticle-based dye-labeled live tumor cell counting by flow cytometry. As shown in FIG. 18, bispecific antibodies cause tumor cell loss if and only if T cells are present.

In yet another embodiment, the bispecific antibody can be produced using FIT-Ig technology. Briefly, a bispecific antibody is generated by coexpression of three constructs encoding VL6373-CL-VHokt3-CH1-FC (SEQ ID NO: 25), VH6373-CH1 (SEQ ID NO: 26) and VLokt3-CL (SEQ ID NO: 27), respectively. As shown in FIG. 19, FIT-Ig antibody showed good bispecific binding activities for both OKT3 and SA-N-CD24. In addition to binding, this bispecific antibody was also found to induce significant cytokine response (FIG. 20) and cytotoxicity toward tumor cells (FIG. 21). In addition, this bispecific antibody (FIT-Ig) showed higher thermostability compared to the previous bispecific antibodies PP6373-OCT3 and OKT3-PP6373 (Fig. 22).

Example 7 Use of PP6373 in chimeric antigen receptor (CAR) modified T cells (CAR-T) for the treatment of cancer.

Ahtu-CD24 antibodies react with a wide range of cancer cells and can be used to produce a chimeric antigen receptor that imparts anticancer activity to T cells. In one embodiment, a single chain Fv sequence of PP6373 (SEQ ID NO: 28) or another single chain anti-CD24 mAb (alphaCD24SC) is inserted into a CAR-T vector known in the art, as shown in FIG. 23. The construct is then inserted into gene vectors known in the art, including vectors derived from retrovirus, lentivirus, adeno-associated virus, or adenoviral vectors.

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To test CAR activity, PBMCs from a healthy donor were enriched for T cells using a human pan-T cell isolation kit (Miltenyl Biotec) (day 0). Human pan T cells were stimulated with anti-CD3 and anti-CD28 for 24 h and cultured with IL-2 for 2 days. Activated T cells were mock treated (control T) or infected with lentivirus carrying CD24-CAR (day 2). To test CAR-T antitumor activity, control T cells or CD24 CAR-T cells were cocultured with CellTrace Violet-labeled tumor cells (Thermo Fisher) overnight. Tumor cell lysis was measured by Fixable Viability Dye eFluor™ 660 staining (eBioscience) and calculated using the formula:

Lysis% = (dead% - autolysis%)/(1-autolysis%).

As shown in FIG. 24, over a wide range of effector/target (E/T) ratios, CD24 CAR-T exhibits potent cytotoxicity compared with the A549 lung cancer cell line.

To test whether CAR-T is activated by cancer cells, 4x10 ⁴ CAR-T or control T cells were incubated with A549 tumor cells overnight and IFNy was measured in the supernatants. As shown in FIG. 25, CAR-T, but not control T cells, produced IFNy in response to stimulation by A549 tumor cells.

Because CD24 is widely expressed among many cancer types. As shown in FIG. 26, CD24 CAR-T exhibits broad cytotoxicity against many types of cancer, including lung cancer, breast cancer, prostate cancer, cervical cancer, neuroblastoma, and glioma.

Taken together, the data presented demonstrate that CD24 CAR-T based on the antibody disclosed herein has great potential in the treatment of cancer. Types of cancer that can be targeted include, but are not limited to, brain tumors, head and neck cancer, sarcoma, lung cancer, gastrointestinal cancer, breast cancer, testicular cancer, prostate cancer, pancreatic cancer, liver cancer or hematological malignancies.

Example 8: Humanization of PP6373 for the treatment of cancer.

A homology model of Fv PP6373 was constructed using the pdb structure of 4PB0 as a model structure. Both VH and VL share more than 90% homology with those of 4PB0. When querying the human Ig database, the human germline V region sequence of IGHV3-73*01 and the J region sequence of IGHJ4*01 were identified as suitable structures and used as the human acceptor framework for the heavy chain CDR regions (Onc-1 VH ). The human germline V region IGKV2-29*02 and the J region sequence IGKJ4*01 were used as the human acceptor framework for the light chain CDR regions (Onc-1 VL). Four VH sequences and four VL sequences were designed (SEQ ID NO: 29-36). New products improve humanization rates from 73% to over 83% in VH and from 80% to over 83% in VL. The structural alignment of mouse Fv PP6373 and the humanized version of PP6373 Fv (huVHv1VLv1; SEQ ID NOs: 29 and 33) demonstrated a high degree of similarity (Fig. 27). To select the best working combination of HuVH and HuVL for CD24 binding, different combinations were co-transfected into 293 cells for 72 hours. Two ELISAs were then performed with expression medium. ELISA 1: A 96-well plate was coated with purified goat anti-human polyclonal IgG (GAH) and after blocking, expression medium or purified control IgG was added and goat anti-human IgG-HRP antibody was used as the detection antibody. ELISA 2: A 96-well plate was coated with CD24-GST protein and after blocking, expression medium or purified control IgG was added and goat anti-human IgG-HRP was used as the detection antibody. If binding of the chimeric antibody PP6373 is considered to be 100% in both ELISAs, different combinations of VH and VL exhibiting different degrees of binding will be compared with those of the chimeric antibody and ranked by relative binding (candidates selected after pre-screening will be compared again after purification) . Data from the first round of prescreening are summarized in FIG. 28, and the data from this experiment suggest that: a) L3 showed high binding capacity per unit protein, making L3 a candidate; and b) H1L3 (SEQ ID NOs: 29 and 35), H2L3 (SEQ ID NOs: 30 and 35), H3L3 (SEQ ID NOs: 31 and 35) and H4L3 (SEQ ID nos: 32 and 35) - four humanization candidates for PP6373.

To test whether the H2L3 and H3L3 antibody candidates retained their ability to bind tumor cells, the humanized antibodies were biotinylated along with PP6373. As shown in FIG. 29, although PP6373 had the best binding to the two human cancer cells tested, both H2L3 and H3L3 exhibited strong binding in the nanomolar IC50 range.

ADCC assays were performed using either PBL (FIG. 30, 31) or purified NK cells (FIG. 32) from PBL as effectors and A549 cells as target cells. Surprisingly, although H2L3 and H3L3 bind poorly to tumor cells (Fig. 29), they are more potent effectors in ADCC when using low concentrations of antibodies (Fig. 30). As expected, defucosylated PP6373 (d6373) is more active in ADCC (Figs. 31, 32).

Taken together, the data presented here demonstrate that humanized PP6373 clones exhibit significant binding to human cancer cells and an unexpectedly high a.c.

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Claims (13)

activity of ADCC. In one embodiment, the antibodies can be used to treat cancer. In another embodiment, the humanized antibody can be used as a key component of a bispecific antibody. To investigate this activity, two constructs containing H3 and L3 were generated to generate bispecific antibodies based on FIT-Ig technology. The sequences of the humanized FIT-Ig antibodies are listed in SEQ ID NO: 37 and 38 and are used in conjunction with SEQ ID NO: 27. In addition, some mutations were also introduced to optimize the humanized FIT-Ig sequences and they were listed in SEQ ID NO: 39-41. Specifically: all three sequences contain a signal sequence at the N-terminus for protein purification and synthesis; in SEQ ID NO: 39: a mutation (D to A substitution) was introduced into the Fc region to prevent ADCC; in SEQ ID NO: 27 there is one additional R residue between VLOKT3 and CL, which was caused by the restriction enzyme site during construction, and in SEQ ID NO: 41 the extra R residue was removed. In yet another embodiment, humanized antibodies can be used as a key component of CAR-T for cancer therapy using methods known in the art. Example 9: Ahtu-CD24 antibodies that bind glycan-screened epitopes do not bind to normal cells with high CD24 expression. A key requirement of antibody-based immunotherapy is minimal reactivity in normal tissues. Because CD24 is abundantly expressed on hematopoietic cells, especially granulocytes, B cells, some erythrocytes and some monocytes, PP6373 and its two humanized clones, H2L3 and H3L3, were compared with a conventional anti-CD24 mAb, ML5. As shown in FIG. 33, while ML5 exhibits strong binding to cells that typically express high levels of CD24, H2L3 and H3L3 do not bind to B cells and red blood cells and bind poorly to granulocytes. This result demonstrates minimal binding to other cell types such as macrophages and to the non-B lymphocyte fraction. CLAIM 1. An Ahtu-CD24 antibody for the treatment of cancer, comprising: (a) a heavy chain variable region from the sequence set forth in SEQ ID NO: 6 and a light chain variable region from the sequence set forth in SEQ ID NO: 16, or (b) a heavy chain variable region containing the sequence shown in SEQ ID NO: 31 or 30, and a light chain variable region containing the sequence shown in SEQ ID NO: 35. 2. Ahtu-CD24 antibody according to claim 1, wherein the antibody is single chain. 3. Ahtu-CD24 antibody according to claim 1 or 2, containing a heavy chain variable region containing the sequence specified in SEQ ID NO: 30, and a light chain variable region containing the sequence specified in SEQ ID NO: 35. 4. Ahtu-CD24 antibody according to claim 1 or 2, containing a heavy chain variable region containing the sequence specified in SEQ ID NO: 31, and a light chain variable region containing the sequence specified in SEQ ID NO: 35. 5. Ahtu-CD24 antibody according to any one of claims 1-4, further comprising a human immunoglobulin constant region (Fc). 6. A bispecific antibody for treating cancer, comprising a first antibody domain comprising an anti-CD24 antibody according to any one of claims 1 to 5, and a second antibody domain comprising a second antibody or an antigen binding fragment thereof. 7. The bispecific antibody of claim 6, wherein the second domain of the antibody recruits immune effector T cells to cancer cells for cancer immunotherapy and/or binds CD3, TCR α chain (T cell receptor), TCR β chain, γ chain TCR or TCR δ chain. 8. The bispecific antibody of claim 7, wherein the second antibody or antigen binding fragment thereof binds CD3. 9. The bispecific antibody of claim 8, comprising a second antibody domain comprising the sequence set forth in SEQ ID NO: 18. 10. A chimeric antigen receptor containing a single chain antibody according to any one of claims 1 to 5. 11. An antibody-drug conjugate (ADC) comprising an anti-CD24 antibody according to any one of claims 1 to 5, associated with an effector moiety selected from the group consisting of a cytotoxic drug, a protein toxin and a radionuclide. 12. A composition for the treatment of cancer comprising an anti-CD24 antibody as defined in any one of claims 1 to 5, a bispecific antibody as defined in any one of claims 6 to 9, a chimeric antigen receptor as defined in claim 10, or an antibody-drug conjugate as claimed in claim 12. .11 and a second anti-cancer agent. 13. The composition of claim 12, wherein the second anticancer agent comprises an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a LAG3. -