Mutant CD24 protein and use thereof for preventing and treating cancer

Abstract

Provided herein are mutant CD24 proteins, RNAs, compositions thereof, and the use of the proteins and compositions in cancer therapy and as vaccines for cancer prevention.

Description

Technical Field

[0001] The present disclosure relates to CD24 protein and mRNA compositions and the use of such compositions for cancer prevention and treatment. Background Art

[0002] CD24 is a small, highly glycosylated, mucin-like, glycosylphosphatidylinositol (GPI)-linked cell surface protein. CD24 is expressed at higher levels on hematopoietic cells (including B cells, T cells, neutrophils, eosinophils, dendritic cells and macrophages) and non-hematopoietic cells (including neurons, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells and epithelial stem cells). Generally, CD24 tends to be expressed at higher levels in progenitor cells and metabolically active cells, and at lower levels in terminally differentiated cells. The function of CD24 in most cell types is unclear, but a variety of immune functions of CD24 have been reported.

[0003] Although CD24 is present in many normal tissues and cell types, CD24 is overexpressed in nearly 70% of human cancers. High levels of CD24 expression were detected by immunohistochemistry 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 most overexpressed proteins in cancer cells. CD24 expression is upregulated during tumorigenesis, suggesting that it plays a role in tumor progression and metastasis. Overexpression of CD24 in cancer has also been identified as a marker for poor prognosis and a more aggressive course of disease in cancer patients. In breast cancer, CD24 expression is significantly higher in invasive cancers than in benign or precancerous lesions. In non-small cell lung cancer, CD24 expression has been identified as an independent marker for overall survival of patients. In addition, in esophageal squamous cell carcinoma, CD24 overexpression indicates tumor lymph node metastasis, poor tumor grade, and shortened survival time. Similar observations have been found in many other cancers, including colon cancer, hepatocellular carcinoma, glioma, ovarian cancer, and prostate cancer.

[0004] Mature CD24 is a small, highly glycosylated sialoglycoprotein of 31 amino acids with 16 potential O-glycosylation sites and 2 predicted N-glycosylation sites. Glycosylation is one of the most complex post-translational modifications of proteins. It is known that a shift in the normal glycosylation pathway occurs in many cancer cells, resulting in altered glycan expression and leading to high or low glycosylation of many cellular proteins. The altered glycosylation patterns found in cancer cells are the result of many contributing factors, including dysregulation at the transcriptional level, dysregulation of chaperones during glycosylation, and altered glycosidase and glycotransferase activities. Tumor-associated glycan changes include longer or shorter N-glycan branches, higher or lower O-glycan density, the generation of truncated versions of normal counterparts (Tn, sTn, and T antigens), and the generation of abnormal forms of terminal structures (sLea and sLex epitopes) with sialic acid and fucose.

[0005] Although CD24 has been extensively used as a prognostic marker for cancer, it has not been used as a neoantigen that could be a potential target for cancer therapy due to its expression on normal cell types and potential toxicity. There is a need in the art for forms of CD24 that can be used to treat and / or prevent cancer. SUMMARY OF THE INVENTION

[0007] Provided herein are mutant CD24 proteins that mimic one or more CD24 conformations and / or glycoforms that are uniquely present on cancer cells but not on normal cells. The mutant CD24 protein may comprise a mature CD24 polypeptide comprising a sequence shown in one of SEQ ID NOs: 17-19. The mutant CD24 protein may comprise a sequence shown in one of SEQ ID NOs: 14-16. The mutant CD24 protein may further comprise an Fc region of a human immunoglobulin (Ig) fused to the C-terminus of the mutant CD24 protein. Ig may be IgG1, IgG2, IgG3, IgG4, IgA or IgM. Ig may be IgG1, which may have a sequence shown in SEQ ID NO: 24. The mutant CD24 protein may comprise a sequence shown in one of SEQ ID NOs: 1-6.

[0008] The mutant CD24 protein may comprise an N-terminal CD24 signal sequence and a C-terminal CD24 glycosylphosphatidylinositol (GPI) anchor sequence, each of which may be fused to a mature CD24 polypeptide to facilitate proper cellular processing. The CD24 signal sequence may comprise the sequence shown in SEQ ID NO: 42, and the GPI anchor sequence may comprise the sequence shown in SEQ ID NO: 43. The mutant CD24 protein may comprise the sequence shown in one of SEQ ID NOs: 7-13 and 20-23.

[0009] The present invention further provides an RNA encoding a mutant CD24 protein, which may be a messenger RNA (mRNA). The mRNA may comprise a sequence shown in one of SEQ ID NOs: 30-35.

[0010] Also provided herein are compositions, which may be pharmaceutical compositions, comprising a mutant CD24 protein or RNA and a physiologically acceptable carrier or excipient.

[0011] Provided herein is a method for treating cancer or cancer prevention in a patient in need thereof, the method comprising administering a mutant CD24 protein, RNA or pharmaceutical composition to the patient. Further provided herein is a mutant CD24 protein, RNA or pharmaceutical composition for treating cancer or cancer prevention in a patient. Also provided is the use of a mutant CD24 protein, RNA or pharmaceutical composition in the preparation of a medicament for treating cancer or cancer prevention in a patient. The cancer may be lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, head and neck cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer or neuroblastoma. The patient may not currently have cancer but may be at high risk of developing cancer, may have fully recovered from cancer but may be at high risk of recurrence, may be genetically susceptible to cancer or may have experienced exposure that increases the patient's risk of cancer. Also provided herein is a method for optimizing a mutant CD24 protein or mRNA composition for treating or preventing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A -B shows a schematic diagram of the CD24 composition disclosed herein. Figure 1A Two schematic diagrams of DNA constructs for expressing CD24 wild-type (SEQ ID NO: 13) and mutant (SEQ ID NO: 9) Fc fusion proteins are shown. The DNA constructs encode WT CD24 or CD24 mutants (T41>A and T51>A) fused to the human heavy chain constant domain (hinge (H)-CH2-CH3). Both proteins lack the polymorphic amino acid at position 57 (based on the full-length sequence of CD24). Figure 1B Two schematic diagrams of WT CD24-Fc and mutant CD24-Fc fusion proteins are shown, showing the Figure 1A Dimeric structures of the two polypeptide components encoded by the DNA constructs shown. T41>A and T51>A mutations of CD24 mutant Fc fusion proteins reveal cancer-specific CD24 conformations.

[0013] Figure 2A -C shows the binding activity of anti-CD24 antibodies to different forms of CD24. Figure 2AThe sequence of the core epitope of the mature cell surface CD24 protein is shown, which has the amino acid sequence of SNSGLAPN (AA42-AA49) (SEQ ID NO: 40) recognized by anti-CD24 antibodies, and flanking amino acids (SEQ ID NO: 41) containing O-glycosylated T41 and T51 (each underlined), which blocks the 6373 epitope. Figure 2B Binding of anti-CD24 antibodies ML5, 6373 and SN3 to 293T cells transfected with wild-type (WT) and mutant CD24 proteins was shown by FACS compared to an isotype control (Iso). Figure 2C Binding of anti-CD24 antibody 6373 to 293T cells expressing wild-type (WT) and mutant CD24 proteins was shown by FACS.

[0014] Figure 3A -B shows the capillary electrophoresis (CE)-SDS analysis of wild-type (WT, SEQ ID NO: 39) and mutant (mut, SEQ ID NO: 3) CD24 fusion proteins expressed in CHO cells. Figure 3A ) and size exclusion-high performance liquid chromatography (SEC-HPLC) ( Figure 3B )analyze.

[0015] Figure 4 Comparison of anti-CD24 (H3L3) and ML5 with WT CD24 fusion protein (hIgG1) and CD24 T41>A,T51>A ELISA data for binding of fc(hlgG1). WT and mutant CD24 fusion proteins were immobilized on the plate, then biotinylated H3L3 and ML5 were added to the plate and detected with avidin-HRP. DETAILED DESCRIPTION OF THE INVENTION

[0017] Targeting cancer-expressed epitopes is a widely adopted approach for treating and preventing cancer. But many such epitopes do not make good drug targets because they are also expressed on normal tissues, which can lead to toxicity issues. An ideal tumor-specific antigen (TSA) would be widely expressed in cancer but rarely or not expressed in normal or essential host organs. Less ideal but equally viable properties of TSAs are those that are expressed but differentially modified in normal tissues compared to cancer tissues: so-called tumor-associated antigens (TAAs). Examples of well-characterized tumor antigens are MAGE-A3, MUC-1, and NY-ESO1.

[0018] TSAs and TAAs can be used to treat or prevent cancer by directly targeting them, such as with tumor-targeting antibodies or T cells that have been genetically engineered to produce artificial T cell receptors for immunotherapy (including CAR-T and TCR therapy). Alternatively, TSAs and TAAs can be used as vaccines, which are administered to people who have cancer or are at risk of developing cancer to elicit an immune response to fight the cancer.

[0019] The identification of new TSAs and TAAs is a limiting factor in the development of new or more effective cancer therapies, particularly for those cancers for which no tumor antigens currently exist. CD24 is a good cancer target for the following reasons: it is widely overexpressed in more than 70% of all human cancers and is differentially glycosylated in cancer, it appears to be oncogenic and is associated with poor prognosis and significantly shorter patient survival in various cancers, and it is a marker for cancer stem cells that can cause recurrence and metastasis by generating new tumors. The inventors herein disclose compositions of mutant CD24 proteins that mimic the conformation and / or glycosylation pattern of CD24 in cancer and their use as therapeutic and prophylactic vaccines for cancer treatment.

[0020] 1. Definition

[0021] The terms used herein are used only for the purpose of describing specific embodiments and are not intended to be limiting. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The word "about" associated with a numerical value represents a reasonable approximation of the value. In some cases, "about" can be interpreted as being within as much as 10% of the specific value associated with it. For example, the phrase "about 100" will cover any value between 90 and 110.

[0022] For the recitation of numerical ranges herein, each intervening number with equal precision therebetween is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0023] As used herein, "cancer" refers to a neoplasm or tumor caused by abnormal, uncontrolled growth of cells. As used herein, cancer explicitly includes leukemias and lymphomas. The term refers to a disease involving cells that have the potential to metastasize to distant sites. The patient can be a human.

[0024] As used herein, "mutant" means any polypeptide containing at least one amino acid sequence change compared to the amino acid sequence of the corresponding wild-type polypeptide. As used herein, "amino acid sequence change" can be, for example, a replacement, deletion or insertion of one or more amino acids. A mutant can also refer to a protein having an amino acid sequence substantially identical to a reference protein, the reference protein having an amino acid sequence that retains at least one biological activity. A mutant can be a derivative, analog or homolog of a polypeptide. A mutant can also be a soluble portion of a polypeptide. Conservative replacement of amino acids, i.e., replacing amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor changes. As understood in the art, these minor changes can be identified in part by considering the hydropathic index of amino acids. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). In the context of mRNA vaccines, a mutant also means a change in the nucleotide sequence in a changed region or non-coding region, regardless of whether the nucleotide is replaced.

[0025] "Treatment" or "treating" may mean inhibiting a disease, which may involve administering a composition of the disclosure to an animal after the disease has clinically appeared.

[0026] "Prevention" may mean preventing a disease, which may be by administering a composition of the present disclosure to an animal prior to the onset of the disease.

[0027] 2. CD24 protein composition

[0028] Provided herein are mutant CD24 proteins and compositions thereof. Natural, mature CD24 is a glycosylphosphatidylinositol (GPI) anchored cell surface protein, which has 31 amino acids (AA) after cleavage of the signal peptide (AA1-26) and the GPI anchor signal sequence (AA60-80) after translation from the full-length CD24 protein. The CD24 protein can be a full-length CD24 protein and include one or more of the signal peptide and the GPI anchor signal sequence.

[0029] Two alleles of mature CD24 exist in humans, which have the WT CD24 sequence SEQ ID NO: 37 or 38, respectively. The polymorphism is located at amino acid 57 and is a valine (CD24 v , mature sequence SETTTGTSSNSSQSTSNSGLAPNPTNATTKV (SEQ ID NO: 44)) or alanine (CD24 A, mature sequence SETTTGTSSNSSQSTSNSGLAPNPTNATTKA (SEQ ID NO: 45)). Anti-CD24 antibodies that specifically bind to cancer cells and tissues recognize a core epitope of the mature cell surface protein, which has the amino acid sequence SNSGLAPN (SEQ ID NO: 40) (AA42-AA49 of the full-length CD24 sequence).

[0030] Because the sequence is unchanged in cancer cells, the inventors realized that the epitope must be masked by the amino acid-anchored glycan moiety surrounding the core epitope in normal cells. Various combinations of mutations can be introduced into the CD24 coding region of the mature protein. The mutant CD24 protein can have a mature sequence shown in one of SEQ ID NOs: 14-16.

[0031] SETTTGTSSNSSQSASNSGLAPNPTNATTK(SEQ ID NO:14)

[0032] SETTTGTSSNSSQSTSNSGLAPNPANATTK(SEQ ID NO:15)

[0033] SETTTGTSSNSSQSASNSGLAPNPANATTK(SEQ ID NO:16)

[0034] The CD24 protein may have mutations designed to mimic the cancer-specific CD24 conformation and may have a mature sequence as shown in one of SEQ ID NOs: 17-19, wherein X represents any amino acid except S, T or N, which can anchor a glycan.

[0035] SETTTGTSSNSSQSXSNSGLAPNPTNATTK(SEQ ID NO:17)

[0036] SETTTGTSSNSSQSTSNSGLAPNPXNATTK(SEQ ID NO:18)

[0037] SETTTGTSSNSSQSXSNSGLAPNPXNATTK(SEQ ID NO:19)

[0038] The mutant CD24 protein may also have a mutation that avoids an immune response to the polymorphic V or A at position 57 of the AA of the full-length CD24 protein sequence. In one example, the CD24 protein (which may be based on the WT or mutant CD24 protein sequence disclosed above) does not include the polymorphic AA at position 57. The full-length CD24 mutant sequence may have a sequence as shown in one of SEQ ID NOs: 7-12, containing a signal sequence and a GPI anchor sequence (underlined at the N-terminus and C-terminus of SEQ ID NO: 7, respectively), and incorporating the mutations described for the mature CD24 protein sequences SEQ ID NOs: 14-19, respectively (the mutations are shown in bold).

[0039] MGRAMVARLGLGLLLLALLLPTQIYS SETTTGTSSNSSQSASNSGLAPNPTNATTK AGGALQSTASLFVVSLSLLHLYS (SEQ ID NO:7) (GPI anchor, missing A / V polymorphism at AA57)

[0040] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPANATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 8) (GPI anchor, deletion of the A / V polymorphism at AA57)

[0041] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSASNSGLAPNPANATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO:9) (GPI anchor, missing A / V polymorphism at AA57)

[0042] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSXSNSGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 10) (GPI anchor, missing A / V polymorphism at AA57)

[0043] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPXNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 11) (GPI anchor, missing A / V polymorphism at AA57)

[0044] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSXSNSGLAPNPXNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 12) (GPI anchor, missing A / V polymorphism at AA57)

[0045] The full length WT CD24 protein sequence with the polymorphic AA at position 57 removed may have the following sequence:

[0046] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 13) (GPI anchor, missing A / V polymorphism at AA57)

[0047] In another example, the full-length mutant CD24 protein may comprise a mutation at T41 and / or in the flanking amino acids in addition to lacking the polymorphic AA at position 57. The full-length mutant CD24 protein may comprise a sequence as set forth in one of SEQ ID NOs: 20-23 (mutations are shown in bold).

[0048] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQATSNSGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO:20) (GPI anchor, missing A / V polymorphism at AA57)

[0049] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTANSGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO:21) (GPI anchor, missing A / V polymorphism at AA57)

[0050] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQAAANSGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 22) (GPI anchor, missing A / V polymorphism at AA57)

[0051] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNAGLAPNPTNATTKAGGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 23) (GPI anchor, missing A / V polymorphism at AA57)

[0052] 3. CD24 fusion protein composition

[0053] The CD24 protein described herein can be fused to another protein at the N-terminus or the C-terminus. The other protein can be a part of a mammalian Ig protein, and the mammal can be a human or a mouse. The part can include the Fc region of the Ig protein. The Fc region can include the hinge region and the CH2 and CH3 domains of the Ig protein. The Ig protein can be human IgG1, IgG2, IgG3, IgG4 or IgA. The Ig protein can also be IgM, and the Fc portion can include the hinge region and the CH3 and CH4 domains of IgM. In one example, the Fc region is a human IgG1 comprising the amino acid sequence shown in SEQ ID NO:24.

[0054] In another example, the CD24 protein comprises a mutant mature CD24 protein sequence disclosed herein fused to the Fc region of an Ig protein, and the Fc region of the Ig protein may be the Fc region of human IgG1, in particular the Fc region shown in SEQ ID NO: 24. The mature CD24 fusion protein may have a sequence shown in one of SEQ ID NOs 1-6, which includes a C-terminal IgG1 Fc region and incorporates mature CD24 mutant proteins of SEQ ID NOs: 14-19, respectively.

[0055] SETTTGTSSNSSQSASNSGLAPNPTNATTKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0056] SETTTGTSSNSSQSTSNSGLAPNPANATTKDKTHTCPCPPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2)

[0057] SETTTGTSSNSSQSASNSGLAPNPANATTKDKTHTCPCPPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)

[0058] SETTTGTSSNSSQSXSNSGLAPNPTNATTKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)

[0059] SETTTGTSSNSSQSTSNSGLAPNPXNATTKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:5)

[0060] SETTTGTSSNSSQSXSNSGLAPNPXNATTKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)

[0061] The CD24 protein can also be fused to a protein tag at the N-terminus or C-terminus, and the protein tag can include GST, His or FLAG or albumin. Methods for preparing fusion proteins and purifying fusion proteins are well known in the art.

[0062] 4. Mutant CD24 mRNA Composition

[0063] Further provided herein is an RNA encoding mutant CD24, which may be a messenger RNA (mRNA). The mature mRNA may include a 5' untranslated region (UTR), a coding sequence (CDS), a 3'UTR, and a polyadenosine tail (poly A tail). Various 5'UTRs and 3'UTRs are known in the art and may be incorporated into the mRNA sequence to achieve protein expression.

[0064] In one example, the mRNA encodes a CD24 protein disclosed herein, which may be a GPI-anchored CD24 protein. The 5'UTR of the mRNA may include a nucleotide sequence selected from the group consisting of SEQ ID NO: 25-29, which is an endogenous 5'UTR from transcript variants 1, 2, 3, 4, and 7 encoding human CD24. The coding sequence of the mutant CD24 may include a nucleotide sequence selected from the group consisting of SEQ ID NO: 30-35, which encodes proteins in SEQ ID NO: 7-12, respectively. In one example, the 3'UTR of the mRNA includes a nucleotide sequence shown in SEQ ID NO: 36, which is an endogenous 3'UTR from a transcript encoding human CD24 shared between variants 1, 2, 3, 4, and 7. The poly A tail may be any poly A tail known in the art.

[0065] 5. Treatment Methods

[0066] The CD24 proteins, fusion proteins and mRNA compositions described herein can be used as vaccines to treat or prevent cancer or another abnormal proliferative disease. Provided herein are methods of such use in patients in need thereof, comprising administering to the patient a CD24 protein, fusion protein or mRNA composition or a pharmaceutical composition comprising the above. Such molecules and pharmaceutical compositions can also be used to prepare a medicament for treating or preventing cancer or another abnormal proliferative disease.

[0067] The cancer or other abnormal proliferative disease may be one or more of (but not limited to) the following: carcinoma, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer and skin cancer; including squamous cell carcinoma; hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and early Myelocytic leukemia; Tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; Other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; Central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma and schwannoma; Tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma. Cancers caused by apoptosis aberrations that can also be treated by the methods and compositions of the present invention are also contemplated. Such cancers may include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, as well as precancerous lesions (e.g., familial adenomatous polyposis) and myelodysplastic syndrome. In specific embodiments, malignancies or abnormal proliferative changes (e.g., metaplasia and dysplasia) or hyperproliferative disorders are treated or prevented by the methods and compositions of the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. The cancer may also be a sarcoma, melanoma, or leukemia.

[0068] An mRNA vaccine encoding a GPI-anchored CD24 protein as described herein will result in expression of the encoded mutant CD24 protein on the surface of endogenous cells, which may be particularly useful for eliciting an immune response against the protein that mimics the conformation seen on cancer cells, thereby resulting in a robust anti-tumor and durable immune response.

[0069] Cancer vaccines comprising CD24 proteins, fusion proteins or mRNA molecules described herein can be used in combination with one or more other anti-tumor therapies, including but not limited to current standard and experimental chemotherapy, hormone therapy, biological therapy, immunotherapy, radiotherapy or surgery. In some embodiments, the vaccine 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 diseases, infectious diseases or poisoning. Such agents include, for example, any of the above-mentioned biological response modifiers, cytotoxins, antimetabolites, alkylating agents, antibiotics or antimitotic agents and immunotherapeutics.

[0070] Vaccines can be used in combination with one or more anti-tumor immunotherapies. Anti-tumor immunotherapy can involve one or more molecules that destroy or enhance alternative immunomodulatory pathways (e.g., TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, PD-1, CTLA-4, TIGIT, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27, or LAG3) or modulate effector molecules (e.g., cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-β, IFNg, Flt3, BLys) and chemokines (e.g., CCL21)) to enhance immunomodulatory effects. In one example, the vaccine is combined with anti-PD-1 or anti-CTLA-4 antibodies.

[0071] 6. Production

[0072] CD24 protein or fusion protein vaccine can be prepared using eukaryotic expression system. The expression system may need to be expressed from a vector in mammalian cells, such as Chinese hamster ovary (CHO) cells. The system can also be a viral vector, such as a replication-deficient retroviral vector that can be used to infect eukaryotic cells. The vaccine can also be produced by a stable cell line that expresses antibodies from a portion of the vector or a vector that has been integrated into the cell genome.

[0073] Vaccines comprising Ig Fc fusion proteins can be purified using, for example, chromatographic methods, such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, the fusion protein can be engineered to contain an additional domain containing an amino acid sequence that allows the polypeptide to be captured on an affinity matrix. For example, antibodies comprising an Fc region of an immunoglobulin domain as described herein can be isolated from cell culture supernatants or cytoplasmic extracts using a protein A column. In addition, tags such as c-myc, hemagglutinin, polyhistidine, or Flag can be used to separate the Fc region of the immunoglobulin domain from the cell culture supernatant or cytoplasmic extract. TM (Kodak) can be used to aid in the purification of polypeptides. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino termini. Other useful fusions include enzymes that aid in the detection of polypeptides, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides.

[0074] CD24 mRNA compositions can be prepared by in vitro chemical synthesis. Large-scale production of mRNA vaccines typically involves 1 or 2 in vitro reactions followed by a purification platform with multiple steps that may include DNA enzyme digestion, precipitation, chromatography, or tangential flow filtration. The manufacturing process can be grouped into upstream processing, which includes enzymatic production of mRNA, and downstream processing, which includes unit operations required to purify the mRNA product. These are complementary to the LNP formulation and fill-to-finish steps.

[0075] 7. Pharmaceutical Compositions

[0076] Provided herein are pharmaceutical compositions comprising a CD24 protein, fusion protein or mRNA (which may be present in a therapeutically effective amount) and a physiologically acceptable carrier or excipient. The pharmaceutical composition may comprise a prophylactically or therapeutically effective amount of a CD24 protein, fusion protein or mRNA and a pharmaceutically acceptable carrier.

[0077] The term "pharmaceutically acceptable" may mean approved by a regulatory agency of a federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly for humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition 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 excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0078] Typically, the ingredients of the pharmaceutical composition can be provided separately or mixed in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a sachet indicating the amount of active agent. When the pharmaceutical composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the pharmaceutical composition by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0079] The pharmaceutical composition can be formulated as a neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0080] The pharmaceutical composition may include one or more or all of histidine buffer, sucrose, and polysorbate 80 (PS80). In one example, the pharmaceutical composition includes about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM histidine buffer. In particular, the histidine buffer concentration may be 20 mM. The pharmaceutical composition may include about 6%, 7%, 8%, 9%, or 10% w / v sucrose. In one example, the pharmaceutical composition includes 8% sucrose. The pharmaceutical composition may include about 0.01%, 0.02%, or 0.03% PS80. In one example, the PS80 concentration is 0.02%. In one example, the pharmaceutical composition includes 20 mM histidine buffer, 8% sucrose, and 0.02% w / v PS80. The pharmaceutical composition may have a pH of about 5, 5.5, or 6.0. In one example, the pH is 5.5. Prior to administration to a subject, the pharmaceutical composition may be diluted in 0.9% sodium chloride or 5% dextrose solution.

[0081] 8. Application method

[0082] Methods of administering the composition and pharmaceutical compositions thereof include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In specific embodiments, the composition is administered intramuscularly, intravenously, or subcutaneously. The composition can be administered by any convenient route, for example, by infusion or bolus, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with one or more other bioactive agents. Administration can be systemic or local.

[0083] The CD24 protein or fusion protein can be present in the pharmaceutical composition at about 10, 15, 20, 25 or 30 mg / mL and can be administered at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 mg / kg. If the subject suffers an adverse event associated with the administration of the antibody composition, the subsequent dose can be adjusted downward from the previous dose. If the antibody composition does not have a strong enough effect on the cancer, the subsequent dose can be adjusted upward from the previous dose.

[0084] mRNA encoding CD24 can be delivered using different strategies, including: i) direct injection of naked mRNA; ii) conjugation with lipid-based carriers, polymers or peptides; and iii) via transfection of dendritic cells (DCs). Direct injection of mRNA is a more cost-effective delivery alternative for DC vaccines. In vivo delivery of naked, complexed or encapsulated mRNA can be successfully performed through a variety of administration routes, such as intradermal, intramuscular, intranasal, intratumoral, intranodal or even intravenous. Using this method, a dose consisting of only tens or hundreds of micrograms of mRNA (10-250 μg) is administered to each patient to trigger an immune response. The first clinical trial evaluating direct injection used naked mRNA in patients with melanoma. This approach is feasible and safe, but no clinical effectiveness has been observed. Self-adjuvants Vaccine is a technology developed by CureVac that uses a mixture of protamine complexes and naked mRNA to enhance the immune stimulatory effect of the vaccine. This technology has been successfully used in Phase I and Phase I / II clinical trials for liver, prostate, lung and melanoma cancers. Novel delivery methods using liposomes and LNPs have been widely used in recent clinical trials. Recent results show that both technologies can be successfully applied to treat melanoma, lymphoma and solid tumors. Example

[0085] The present disclosure has several aspects, illustrated by the following non-limiting examples.

[0086] Example 1

[0087] Generation of monoclonal antibodies against hypoglycosylated CD24

[0088] On normal cells, the mature extracellular CD24 protein is highly glycosylated, while cancer cells have altered glycan expression, resulting in either hyper- or hypo-glycosylation of many cellular proteins. It has been demonstrated that both CD24 amino acids T41 and T51 are fully O-glycosylated in normal cells, which prevents the binding of the cancer-specific anti-CD24 mAb, 6373. However, at least one of the T41 and T51 residues is not glycosylated in cancer cells, which allows 6373 to bind. Therefore, 6373 is a cancer-specific anti-CD24 antibody.

[0089] To test whether mutant CD24 mimics hypoglycosylated CD24 on cancer cells, 293T cells were transfected with WT and mutant CD24 proteins with SEQ ID NOs: 7, 8, 13, and 20-23, and the conformation of CD24 was assessed using antibodies that bind to all (ML5), sialylated (SN3), and hypoglycosylated (6373) forms of CD24. As shown in Figure 2, cells expressing WT CD24 bind ML5 and SN3, but not 6373. Mutations in T41 (T41>A, SEQ ID NO: 7) and T51 (T51>A, SEQ ID NO: 8) confer reactivity to 6373. Because the mutated residues are not required for 6373 binding, and the replacement glycosylation site exposes the 6373 epitope, these data indicate that the 6373 epitope is shielded by glycans on T41 and T51. Cells transfected with mutations at both residues also showed enhanced 6373 binding. Other mutations, including 40S>A (SEQ ID NO:20), 44S>A (SEQ ID NO:23), did not confer binding to 6373. These data suggest that CD24 on cancer cells differs from CD24 on normal cells in the glycans surrounding the 6373 core epitope, and that mutations at T41 and T51 of the CD24 core recapitulate the CD24 conformation in cancer cells.

[0090] Example 2

[0091] Fusion proteins that mimic cancer-specific CD24 conformations

[0092] To generate a fusion protein that mimics the cancer CD24-specific conformation, a fusion protein was generated that contained the CD24 extracellular domain with T>A mutations at residues T41 and T51 (mature CD24 protein) fused to the Fc region containing the hinge region and the CH2 and CH3 domains of human IgG1 (CD24 T41>A,T51>A Fc) (SEQ ID NO: 3), or wild-type CD24 extracellular protein fused to the Fc region (SEQ ID NO: 39). These proteins were expressed in 293T cells and purified by affinity chromatography (Figure 3).

[0093] To test whether the mutant fusion proteins mimic CD24 cancer conformations, the binding of humanized antibodies derived from 6373, H3L3, and ML5 (which recognize all CD24 molecules regardless of its glycosylation state) to CD24 was measured by ELISA. T41>A,T51> A Fc binding. Figure 4 As shown, CD24 T41>A,T51>A Fc showed slightly stronger binding to H3L3 than ML5.

[0094] Example 3

[0095] Induction of cancer-specific anti-CD24 antibodies using CD24 vaccine

[0096] The main obstacle to an effective cancer vaccine is immune tolerance to CD24. In order to reproduce immune tolerance, CD24 knock-in mice were generated. These mice were vaccinated with a vaccine comprising a mutant CD24 having one of SEQ ID NOs: 1-12, 14-23, and 30-35. After two immunizations, mouse sera were collected and tested for binding to the vaccine and binding to normal and cancer cells using flow cytometry or immunohistochemistry. As described in Example 4, the vaccine with the strongest antibody response will be further tested for the treatment and prevention of cancer.

[0097] Example 4

[0098] Cancer treatment or prevention

[0099] As a first test of CD24 vaccine prevention, human CD24 knock-in mice will be immunized two or more times and challenged with a syngeneic tumor cell line expressing human CD24. Mice that received mock immunization will serve as controls. The vaccine's preventive activity is evaluated based on the growth kinetics of tumors expressing human CD24.

[0100] In order to test the effect of the vaccine on mice with genetic susceptibility to cancer, transgenic mice expressing oncogenes in a tissue-specific manner are vaccinated before tumorigenesis begins. One such model is the TRAMP mouse, which expresses the SV40 large T antigen (oncogene) under the control of the probasin promoter. Previous studies by Wang et al. have shown that CD24 plays a key role in the carcinogenic process of TRAMP mice (Nat Commun 6, 5909 (2015), doi.org / 10.1038 / ncomms6909). TRAMP mice express SV40 T antigen during puberty, which initiates the tumorigenesis process in the prostate. Therefore, vaccination begins before puberty in order to test the preventive activity of the CD24 vaccine. The same cancer model can also be used to test the therapeutic effect of the vaccine, the only difference being that the vaccine will be administered after tumor formation.

[0101] Once therapeutic and preventive efficacy is established, human CD24 knock-in mice will be used to evaluate the toxicity of the vaccine. In one set of experiments, mice will be vaccinated with increasing doses of the vaccine, once every two weeks for 10 weeks. Mice will be monitored for potential adverse reactions, including weight loss, blood chemistry changes, complete blood counts, and flow cytometric analysis of leukocyte subsets. Organs will be examined by histopathological methods. Autoantibodies against blood cells expressing CD24 (including B cells, macrophages, neutrophils, and dendritic cells) can be used as markers of autoimmunity.

[0102] Once safety is established in animal models, the vaccine's safety and clinical activity, including therapeutic and preventive activity, will be evaluated in human cancer patients.

Claims

1. A mutant CD24 protein comprising at least one mutation that mimics a cancer-specific CD24 conformation.

2. The mutant CD24 protein of claim 1, wherein the mutant CD24 protein comprises a mature CD24 polypeptide, and the mature CD24 polypeptide comprises a sequence shown in any one of SEQ ID NOs: 17-19.

3. The mutant CD24 protein of claim 2, wherein the mature CD24 polypeptide comprises a sequence as shown in any one of SEQ ID NOs: 14-16.

4. The mutant CD24 protein according to claim 2 or 3, further comprising an Fc region of a human immunoglobulin (Ig) fused to the C-terminus of the mutant CD24 protein.

5. The mutant CD24 protein of claim 4, wherein the Ig is IgG1, IgG2, IgG3, IgG4, IgA or IgM. The mutant CD24 protein of claim 5 , wherein the Ig is IgG1.

7. The mutant CD24 protein of claim 6, wherein the Fc region has the sequence shown in SEQ ID NO:

24.

8. The mutant CD24 protein of claim 7, wherein the mutant CD24 protein comprises a sequence shown in any one of SEQ ID NOs: 1-6.

9. The mutant CD24 protein of claim 2 or 3, further comprising an N-terminal CD24 signal sequence and a C-terminal CD24 glycosylphosphatidylinositol (GPI) anchor sequence, each fused to the mature CD24 polypeptide.

10. The mutant CD24 protein of claim 9, wherein the CD24 signal sequence comprises SEQ ID NO:42, and the GPI anchor sequence comprises the sequence shown in SEQ ID NO:

43.

11. The mutant CD24 protein of claim 10, wherein the mutant CD24 protein comprises a sequence as shown in any one of SEQ ID NOs: 7-12.

12. RNA encoding the mutant CD24 protein according to any one of claims 1 to 11.

13. The RNA of claim 12, wherein the RNA is messenger RNA (mRNA).

14. The mRNA of claim 13, wherein the mRNA encodes a mutant CD24 protein comprising a sequence shown in one of SEQ ID NOs: 1-12.

15. The mRNA of claim 14, wherein the mRNA comprises a sequence as shown in one of SEQ ID NOs: 30-35.

16. A composition comprising the mutant CD24 protein or RNA according to any one of claims 1 to 15, and a physiologically acceptable carrier or excipient.

17. A method for treating or preventing cancer in a patient in need thereof, the method comprising administering to the patient a mutant CD24 protein, RNA or composition according to any one of claims 1 to 16.

18. The method of claim 17, wherein the cancer is lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, head and neck cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer, or neuroblastoma.

19. The method of claim 17 or 18, wherein the method is cancer prevention.

20. The method of claim 19, wherein the patient does not currently have cancer but is at high risk of developing cancer.

21. The method of claim 19, wherein the patient has fully recovered from cancer but is at high risk of recurrence.

22. The method of claim 19, wherein the patient is genetically susceptible to cancer.

23. The method of claim 19, wherein the patient has experienced an exposure that increases the patient's risk of cancer.

24. Use of the mutant CD24 protein, RNA or composition of any one of claims 1-16 in the preparation of a medicament for treating or preventing cancer in a patient.

25. The use of claim 24, wherein the cancer is lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, head and neck cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer or neuroblastoma.

26. The use of claim 24 or 25, wherein the patient does not currently have cancer but is at high risk of developing cancer; the patient has fully recovered from cancer but is at high risk of recurrence; the patient is genetically susceptible to cancer; and / or the patient has experienced an exposure that increases the patient's risk of cancer.

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