Novel CD24 bispecific antibodies for treatment of hematopoietic and solid tumors

Abstract

Novel CD24-CD3 bispecific antibodies and methods of their use for treatment of various cancers are described.

Description

NOVEL CD24 BISPECIFIC ANTIBODIES FOR TREATMENT OF HEMATOPOIETIC AND SOLID TUMORSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 676,125 filed on July 26, 2024. The content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] N / AREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The content of the electronic sequence listing (18250600049.xml; Size: 33,225 bytes; and Date of Creation: July 25, 2025) is herein incorporated by reference in its entirety'.BACKGROUND

[0004] With advancements in antibody engineering and recombinant DNA technology, bispecific antibodies (BsAbs) have emerged as a valuable platform in immunotherapy. These BsAbs direct specific immune system effectors to target tumor cells, enhancing their cytotoxicity. Nevertheless, several factors hinder its effectiveness, including short half-life, toxicity, limited potency and persistence, and an immunosuppressive tumor microenvironment (TME). Therefore, strategies for improving the efficacy of BsAb therapies are needed.SUMMARY

[0005] In an aspect, provided herein is a bispecific antibody comprising a CD24 binder; a CD3 binder; and an IgGl Fc heavy chain constant domain (CH); wherein the CD24 binder comprises: a heavy chain variable domain (VH) complementary determining region (CDR) 1 comprising SEQ ID NO: 17; a VH CDR2 comprising SEQ ID NO: 18; a VH CDR3 comprising SEQ ID NO: 19; and a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 20; a VL CDR2 comprising SEQ ID NO: 21; and a VL CDR3 comprising SEQ ID NO: 22; wherein the CD3 binder comprises: a VH CDR1 comprising SEQ ID NO: 9; a VH CDR2 comprising SEQ ID NO: 10; a VH CDR3 comprising SEQ ID NO: 11; a VL CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprisingSEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; and wherein the IgGl Fc CH comprises: a sequence having at least 90% identity to SEQ ID NO: 23, and comprising at least one of D122, N207, and E215; or a sequence having at least 90% identity to SEQ ID NO: 24, and comprising at least one of Al 17; Al 18, and G212.

[0006] The IgGl Fc CH may comprise the sequence having at least 90% identity to SEQ ID NO: 23 and each of D122, N207, and E215; or the sequence having at least 90% identity to SEQ ID NO: 24, and each of Al 17; Al 18, and G212. The IgGl Fc CH may comprise SEQ ID NO: 23 or SEQ ID NO: 24.

[0007] The CD24 binder VH comprises SEQ ID NO: 15 or a sequence having at least 90% identity thereto; wherein the CD24 binder VL comprises SEQ ID NO: 16 or a sequence having at least 90% identity thereto; wherein the CD3 binder VH comprises SEQ ID NO: 7 or a sequence having at least 90% identity thereto; and wherein the CD3 binder VL comprises SEQ ID NO: 8 or a sequence having at least 90% identity thereto.

[0008] The bispecific antibody may further comprise a (G4S)5 linker comprising SEQ ID NO: 27 between the CD3 binder VL and the CD24 binder VH.

[0009] The antibody may comprise: a heavy chain comprising: SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and comprising at least one of D542, N627, and E635; or SEQ ID NO: 4 or a sequence having at least 90% identity thereto, and comprising at least one of A537; A538; and G632; and a light chain comprising SEQ ID NO: 6 or a sequence having at least 90% identity thereto. In embodiments, the heavy chain comprises the sequence having at least 90% identity to SEQ ID NO: 2 and each of D542, N627, and E635. In embodiments, the heavy chain comprises the sequence having at least 90% identity to SEQ ID NO: 4, and each of A537; A538; and G632.

[0010] In another aspect, provided herein is an engineered polynucleotide encoding the bispecific antibody described herein.

[0011] In another aspect, provided herein is a construct comprising the engineered polynucleotide described herein operably linked to a promoter.

[0012] In another aspect, provided herein is a cell comprising the bispecific antibody, the engineered polynucleotide, or the construct described herein. The cell may be an immune cell. The cell may be a T cell, a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell. In embodiments, the cell is a T cell.

[0013] In another aspect, provided herein is a pharmaceutical composition comprising the bispecific antibody, the engineered polynucleotide, the construct, or the cell described herein; and a pharmaceutically acceptable carrier.

[0014] In another aspect, provided herein is a kit comprising at least two bispecific antibodies described herein, engineered polynucleotides encoding said bispecific antibodies, or immune cells comprising said bispecific antibodies. The at least two bispecific antibodies may comprise, a bispecific antibody comprising a heavy chain having at least 90% identity to SEQ ID NO: 2 and each of D542, N627, and E635, and a light chain having at least 90% identity to SEQ ID NO: 6; and a bispecific antibody comprising a heavy chain having at least 90% identity to SEQ ID NO: 4, and each of A537; A538; and G632, and a light chain having at least 90% identity to SEQ ID NO: 6.

[0015] In another aspect, provided herein is a method for treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein. The cancer may comprise at least one of a glioblastoma, a lung carcinoma, a mantle cell lymphoma, and a pancreatic cancer. The cancer may comprise CD24 positive cancer cells. The pharmaceutical composition may comprise a T cell comprising the bispecific antibody described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1B. Recombinant constructs of rCD24 BsAb with decreased (A) or increased (B) cytotoxicity via Fc mutations.

[0017] FIGS. 2A-2F. rCD24 BsAbs binding to CD24 and CD3. Supernatant collected from 293T cells transfected with rCD24 BsAb-expressing plasmids was incubated with human T cells (CD3+) and HBL-2 cells (CD24+), followed by incubation with PE-labeled anti-IgG. (B) demonstrates that rCD24 BsAb-D binds to CD24 in a concentration-dependent manner, and (C) shows that rCD24 BsAb-I binds to CD24 in samples collected over different days. (E) and (F) indicate that both rCD24 BsAbs bind to CD3 in a concentration-dependent manner. (A) and (D) are IgG isotype controls.

[0018] FIGS. 3A-3B. SDS-PAGE of rCD24 BsAb-D (A) and rCD24 BsAb-I (B) under reducing conditions.

[0019] FIGS. 4A-4F. Cytotoxicity assay of rCD24 BsAb-armed activated T cells (ATCs) againstZ138-Nluc. (A-C) Luciferase-activity based-cytotoxicity showing the effects of rCD24-I BsAb- armed ATCs in a time-dependent manner against MCL Z138 cells. (A) 24h, (B) 48h, (C) 72h. (D- F) Luciferase-activity based-cytotoxicity showing the effects of rCD24-D BsAb-armed ATCs in a time-dependent manner against MCL Z138 cells. (D) 24h, (E) 48h, (F) 72h.

[0020] FIGS. 5A-5F. Cytotoxicity assay of rCD24 BsAb-armed ATCs against NALM6-Luc. (A- C) Luciferase-activity based-cytotoxicity showing the effects of rCD24-I BsAb-armed ATCs in a time-dependent manner against acute lymphoblastic leukemia (ALL) NALM6 cells: (A) 24h, (B) 48h, (C) 72h. (D-F) Luciferase-activity based-cytotoxicity showing the effects of rCD24-D BsAb- armed ATCs in a time-dependent manner against ALL NALM6 cells: (D) 24h, (E) 48h, (F) 72h.

[0021] FIGS. 6A-6C. Cytotoxicity assay of rCD24-D BsAb-armed ATCs against A172. (A-C) Cytotoxicity measured in the xCELLigence RTCA MP system showing the effects of rCD24-D BsAb-armed ATCs at different E:T ratios against glioblastoma A172 cells: (A) E:T=1:1, (B) E:T=3:1, (C) E:T=9:1.

[0022] FIGS. 7A-7C. Cytotoxicity assay of rCD24-D BsAb-armed ATCs against T98G. (A-C) Cytotoxicity measured in the xCELLigence RTCA MP system showing the effects of rCD24-D BsAb-armed ATCs at different E:T ratios against glioblastoma T98G cells: (A) E:T=1:1, (B) E:T=3: 1, (C) E:T=9: 1.

[0023] FIGS. 8A-8C. Cytotoxicity assay of rCD24-D BsAb-armed ATCs against U251. (A-C) Cytotoxicity measured in the xCELLigence RTCA MP system showing the effects of rCD24-D BsAb-armed ATCs at different E:T ratios against glioblastoma U251 cells: (A) E:T=1 : 1, (B) E:T=3:1, (C) E:T=9:1.

[0024] FIGS. 9A-9C. Cytotoxicity assay of rCD24-D BsAb-armed ATCs against A549. (A-C) Cytotoxicity measured in the xCELLigence RTCA MP system showing the effects of rCD24-D BsAb-armed ATCs at different E:T ratios against lung cancer A549 cells: (A) E:T=1:1, (B) E:T=3: 1, (C) E:T=9: 1.

[0025] FIGS. 10A-10C. Cytotoxicity assay of rCD24-D BsAb-armed ATCs against PANC-1. (A- C) Cytotoxicity measured in the xCELLigence RTCA MP system showing the effects of rCD24- D BsAb-armed ATCs at different E:T ratios against pancreatic cancer PANC-1 cells: (A) E:T=1: 1, (B) E:T=3:1, (C) E:T=9: 1.DETAILED DESCRIPTION

[0026] CD24, a glycosylphosphatidylinositol-anchored membrane protein, is highly glycosylated and overexpressed in many human carcinomas, correlating with poor prognosis. By interacting with the inhibitory receptor Siglec-10 on macrophages, CD24 transmits a “don’t eat me” signal, preventing cancer cell phagocytosis and presenting a strategic vulnerability for cancer treatment. Our innovative design includes two recombinant CD3 x CD24 BsAbs: one with decreased antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), and the other with increased ADCP, ADCC, and CDC via Fc mutations. Our research demonstrates that these unique BsAbs bind to both the CD3 receptor (activating cytotoxic T lymphocytes) and CD24 on tumor cells, thereby activating T cells and promoting tumor cell destruction. The CD3 x CD24 BsAbs exhibit potent cytotoxicity across various tumor types, including both liquid and solid tumors. This innovative strategy highlights the potential of recombinant bispecific antibodies by combining targeting of CD3 and CD24 with modulated ADCP, ADCC, and CDC activities. This approach offers a novel pathway for developing BsAb therapies for diverse tumors, significantly expanding commercial opportunities upon successful implementation.

[0027] In a first aspect, provided herein is a bispecific antibody or antigen binding fragment thereof comprising a CD24 binder, a CD3 binder, and an IgGl Fc heavy chain constant domain (CH); wherein the CD24 binder comprises a heavy chain variable domain (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 17; a VH CDR2 comprising SEQ ID NO: 18; a VH CDR3 comprising SEQ ID NO: 19; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 20; a VL CDR2 comprising SEQ ID NO: 21; and a VL CDR3 comprising SEQ ID NO: 22; and wherein the CD3 binder comprises a VH CDR1 comprising SEQ ID NO: 9; a VH CDR2 comprising SEQ ID NO: 10; a VH CDR3 comprising SEQ ID NO: 11; a VL CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; and wherein the IgGl Fc CH comprises: a sequence having at least 90% identity to SEQ ID NO: 23, and comprising at least one of D122, N207, and E215; or a sequence having at least 90% identity to SEQ ID NO: 24, and comprising at least one of Al 17; Al 18, and G212.

[0028] In embodiments, the IgGl Fc CH comprises the sequence having at least 90% identity to SEQ ID NO: 23 and each of D122, N207, and E215. In embodiments the IgGl Fc, CH comprises the sequence having at least 90% identity to SEQ ID NO: 24, and each of Al 17; Al 18, and G212.

[0029] The VH of the CD24 binder may comprise SEQ ID NO: 15 or a sequence having at least 90% identity thereto. The VL of the CD24 binder may comprise SEQ ID NO: 16 or a sequence having at least 90% identity thereto. The VH of the CD3 binder VH may comprise SEQ ID NO: 7 or a sequence having at least 90% identity thereto. The VL of the CD3 binder may comprise SEQ ID NO: 8 or a sequence having at least 90% identity thereto.

[0030] The bispecific antibody may further comprise a (G4S)5 linker comprising SEQ ID NO: 27 between the CD3 binder VL and the CD24 binder VH.

[0031] The bispecific antibody may comprise a heavy chain comprising SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and comprising at least one of D542, N627, and E635, or SEQ ID NO: 4 or a sequence having at least 90% identity thereto, and comprising at least one of A537; A538; and G632; and a light chain comprising SEQ ID NO: 6 or a sequence having at least 90% identity thereto. In embodiments, the heavy chain comprises a sequence having at least 90% identity to SEQ ID NO: 2 and each of D542, N627, and E635. In embodiments, the heavy chain comprises a sequence having at least 90% identity to SEQ ID NO: 4 and each of A537; A538; and G632.

[0032] In embodiments, the CD24 binder VH and the IgGl Fc of the bispecific antibody comprises at least one amino acid substitution mutation selected from a position corresponding to amino acids L234, L235, and P329 relative to SEQ ID NO: 26. The amino acid substitution mutations may be L234A, L235A, and P329G.

[0033] In embodiments, the CD24 binder VH and the IgGl Fc of the bispecific antibody comprises at least one amino acid substitution mutation selected from a position corresponding to amino acids S239, S324, and 1332 relative to SEQ ID NO: 25. The amino acid substitution mutations may be S239D, S324N, and I332E.

[0034] The terms “antibody” and “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain. Antibodies include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g. ability to bind to the protein of interest or variant) are retained.

[0035] The term antibody includes “antibody fragments” or “antibody-derived fragments” and“antigen binding fragments” which comprise an antigen binding domain and displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Fragments may comprise a heavy chain variable region (VH domain) and light chain variable region (VL). Fragments may comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.

[0036] The term "bispecific" means that the binding protein is able to specifically bind to at least two distinct moieties (e.g., antigen binding sites). Typically, a bispecific molecule comprises two different binding sites, each of which is specific for a different moiety (e.g., antigen). A bispecific molecule may be capable of simultaneously binding two moieties, particularly two moieties expressed on two distinct cells (e.g., a tumor cell and a T cell). The bispecific antibodies described herein are capable of binding a target antigen, specifically CD24, and a T-cell antigen, specifically CD3. Particularly, the bispecific antibodies are capable of binding to the surface of tumor cells and T cells simultaneously, allowing for activation of the T cells and the targeting killing of tumor cells bound to the bispecific antibody.

[0037] The terms “complementarity determining region” and “CDR” refer to part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B-cells and T-cells respectively, where these molecules bind to their specific antigen. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen binding site. Since the antigen binding sites are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen binding site that can collectively come into contact with the antigen. A single whole antibody molecule has two antigen binding sites and thereforecontains twelve CDRs. For further example, sixty CDRs can be found on a pentameric IgM molecule.

[0038] Within the variable domain, CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, and all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions. The tertiary structure of an antibody is important to analyze and design new antibodies.

[0039] The human VH complex is composed of approximately 100 gene segments per haploid genome, including at least 51 functional genes, as judged by successful rearrangement in cloned cDNA. On the basis of nucleic acid sequence homology, the VH genes have been grouped into 6- 7 families (VH 1-7). Among the seven families, the VH3 family is the largest.

[0040] Antibodies can be genetically engineered from the CDRs, VH, VL, and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0041] One may wish to engraft one or more CDRs from the monoclonal antibodies described herein into alternate scaffolds. For example, standard molecular biological techniques can be used to transfer the DNA sequences encoding the antibody's CDR(s) to (1) full IgG scaffold of human or other species; (2) a scFv scaffold of human or other species, or (3) other specialty vectors. If the CDR(s) have been transferred to a new scaffold all of the previous modifications described can also be performed. For example, one could consult Biotechnol Genet Eng Rev, 2013, 29: 175-86 for a review of useful methods.

[0042] The antibodies or antibody fragments can be wholly or partially synthetically produced. Thus, the antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants. Thus, the antibody molecules can be produced in vitro or in vivo. The antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.

[0043] An antibody fragment crystallizable region (Fc region) is the tail region of an antibody thatinteracts with cell surface receptors called Fc receptors and some proteins of the complement system. This region allows antibodies to activate the immune system, for example, through binding to Fc receptors.

[0044] Furthermore, the antibody or antibody fragment can further comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0045] Antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). Monoclonal antibodies can be produced by immunizing inbred mice with a peptide antigen. The mice may be immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response. The mice may receive an initial immunization on day 0 and be rested for about 3 to about 30 weeks. Immunized mice may be given one or more booster immunizations of by the intravenous (IV) or subcutaneous (SC) route. Lymphocytes, from antibody positive mice may be obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells may be produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas. The antibody producing cells and fusion partner cells may be fused in polyethylene glycol at concentrations from about 30% to about 50%. Fused hybridoma cells may be selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids may be collected from growth-positive wells and screened for antibody production by an immunoassay such as an Enzyme Linked Immunosorbent Assay. Hybridoma cells from antibody positive wells may be cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques or by limiting dilution in which hybridomas are diluted in suitable growth media and re-plated such that ~1 hybridoma cell is pipetted into a single well of a 96-well plate where it grows over the course of 10 days as a single clone, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.

[0046] The terms “specifically binds”, or “binding specificity” refer to the ability of the antibody to form one or more noncovalent bonds with an epitope or antigen via the antibody variable domains. Specificity can be characterized by an antibody-antigen affinity, e.g. as characterized bya dissociation constant (KD) of <100 nM, 10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nanomolar (nM) (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M).

[0047] The terms “protein” or “polypeptide” or “peptide” are used interchangeably to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids. A protein typically comprises a polymer of naturally or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0048] The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3 -Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelicacid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'- Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine.

[0049] In a second aspect, provided herein is an engineered polynucleotide comprising any of the bispecific antibodies described herein.

[0050] The terms “engineered polynucleotide”, “recombinant polynucleotide”, “genetically engineered polynucleotide”, and “genetically modified polynucleotide” refer to any manipulation of a polynucleotide that results in a detectable change in a naturally occurring polynucleotide, wherein the manipulation includes, but is not limited to, changes in the sequence of the polynucleotide or inclusion of non-naturally occurring nucleotides or nucleosides.

[0051] “Percentage of sequence identity", “percent similarity”, or “percent identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0052] The term “substantial identity" or “substantial similarity” of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 75% sequence identity. Alternatively, percent identity can be any integer from 75% to 100%. Embodiments described herein have at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0053] In a third aspect, provided herein is a construct comprising any of the engineered polynucleotides described herein. As used herein, the term “construct” refers to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic). For example, theconstructs may comprise a portion of the coding region of a transgene of interest operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) is from the genome of a different species, or (3) is synthetic. The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0054] In a fourth aspect, provided herein is a cell comprising any of the bispecific antibodies, engineered polynucleotides, or constructs described herein. A “cell” or “engineered cell” is a cell in which the engineered polynucleotide is expressed or a cell comprising the bispecific antibody. The cell may be a mammalian cell. The cell may be a human cell. In exemplary embodiments, the cell is an immune cell. The cell may comprise a T cell, a natural killer (NK) cell, a B cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell. Accordingly, the cell comprising the CAR protein and / or the engineered polynucleotide may be a CAR-T cell, CAR- NK cell, CAR-B cell, a CAR-macrophage, or a CAR-iNKT cell. In exemplary embodiments, the cell is a T cell.

[0055] In a fifth aspect, provided herein is a pharmaceutical composition comprising any of the bispecific antibodies, engineered polynucleotides, constructs, or cells described herein, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Examples of compositions appropriate for such therapeutic applications include preparations for parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronarial, intramyocardial, intraperitoneal, intravenous or intraarterial (e.g., injectable), or intratracheal administration, such as sterile suspensions, emulsions, and aerosols. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically acceptable excipients, diluents, or carriers such assterile water, physiological saline, glucose or the like.

[0056] In a sixth aspect, provided herein is a kit comprising at least two of the bispecific antibodies, engineered polynucleotides, constructs, or cells described herein. At least one bispecific antibody may comprise at least one amino acid substitution mutation selected from a position corresponding to amino acids L234, L235, and P329 relative to SEQ ID NO: 26. The amino acid substitutions may comprise each of L234, L235, and P329 relative to SEQ ID NO: 26. At least one bispecific antibody may comprise at least one amino acid substitution mutation selected from a position corresponding to amino acids S239, S324, and 1332 relative to SEQ ID NO: 25. The amino acid substitutions may comprise each of S239, S324, and 1332 relative to SEQ ID NO: 25.

[0057] As used herein the term "wild type" refers to the typical form of an organism, strain, gene, protein or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a gene or protein molecule having a polynucleotide or amino acid sequence that differs from a reference polynucleotide or protein molecule. A variant or mutant protein may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant protein may one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference polypeptide.

[0058] In a seventh aspect, provided herein is a method for treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions, bispecific antibodies, engineered polynucleotides, constructs, or cells described herein. In embodiments, the cancer is a hematopoietic cancer. In embodiments, the cancer comprises a solid tumor. In embodiments, the cancer comprises at least one of a glioblastoma, a lung carcinoma, a mantle cell lymphoma, and a pancreatic cancer. The cancer may comprise CD24 positive cancer cells.

[0059] In embodiments, the pharmaceutical composition comprises a T cell comprising the bispecific antibody comprising at least one amino acid substitution mutation selected from a position corresponding to amino acids L234, L235, and P329 relative to SEQ ID NO: 26 of the heavy chain. The amino acid substitutions may comprise each of L234, L235, and P329 relative to SEQ ID NO: 26. This pharmaceutical composition may be administered when a decrease antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity(ADCC), and complement-dependent cytotoxicity (CDC) is desired.

[0060] In embodiments, the pharmaceutical composition comprises a T cell comprising the bispecific antibody comprising at least one amino acid substitution mutation selected from a position corresponding to amino acids S239, S324, and 1332 relative to SEQ ID NO: 25. The amino acid substitutions may comprise each of S239, S324, and 1332 relative to SEQ ID NO: 25. This pharmaceutical composition may be administered when an increase in antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC) is desired.

[0061] The method may further comprise administering to the subject an additional cancer treatment. Additional cancer treatments include, but are not limited to chemotherapy, radiation, bone marrow transplant, surgery and immunotherapy.

[0062] As used herein, the term “administering” an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0063] The term “subject” or “patient” are used herein interchangeably to refer to a mammal to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. The subject may be a human. The subject may be a mammal in need of treatment for a cancer.

[0064] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The amount of the pharmaceutical composition that is therapeutically effective may vary depending on the particular pathogen or the condition of the subject. Appropriate dosages may be determined, for example, by extrapolation from cell culture assays, animal studies, or human clinical trials taking into accountbody weight of the patient, absorption rate, half-life, disease severity and the like. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0065] Miscellaneous

[0066] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0067] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0068] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0069] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspectof a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0070] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0071] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0072] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents citedherein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0073] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0074] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0075] The present disclosure will be more fully understood upon consideration of the following non-limiting examples.

[0076] EXAMPLES

[0077] Example 1

[0078] Background

[0079] Bispecific antibody (BsAb) therapy has shown significant potential in cancer treatment. However, its efficacy is often hindered by several factors, including short half-life, toxicity, limited potency and persistence, and a suppressive tumor microenvironment (TME). To address these challenges, we have designed and constructed two bispecific antibody constructs by utilizing the sequences of anti-CD3 and anti-CD24 antibodies. These BsAbs possess dual variable domains that bind to CD3 and CD24.

[0080] The BsAb heavy chain was constructed by sequentially linking the CD3 variable light chain - (G4S)6 linker - CD3 variable heavy chain - (G4S)5 linker (with a G to T substitution in repeat3) - CD24 heavy chain variable sequence - human IgGl Fc. The BsAb light chain was constructed by linking the CD24 variable light chain to the human constant region of the kappa light chain.

[0081] In summary, the BsAbs designs are as follows:

[0082] rCD24 BsAb-D: This unique BsAb design specifically targets CD24 tumors and includes three amino acid replacements in the Fc CH2 region to decrease antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC): L234A, L235A, and P329G.

[0083] rCD24 BsAb-I: This unique BsAb design also targets CD24 tumors but features three amino acid replacements in the Fc CH2 region to increase ADCP, ADCC, and CDC: S239D, S324N, and I332E.

[0084] The L234A, L235A, and P329G mutations are introduced to ensure that the antibody’s therapeutic activity is driven exclusively by its bispecific binding to tumor antigen CD24 and CD3 on T-cells, rather than non-specific immune activation. By reducing off-target effects and minimizing toxicities, such as cytokine release syndrome (CRS), these mutations enhance the precision, safety, and efficacy of cancer therapies, directing the immune response specifically toward tumor cells.

[0085] Results

[0086] We generated BsAb cells from 293T cells and purified them from cell-free supernatants using Protein A beads. After the expression of BsAbs by 293T cells, their binding to CD3 and CD24 was confirmed (FIG. 3). To further verify BsAb expression, non-reducing SDS-PAGE gel analysis was conducted, demonstrating the correct formation of both heavy and light chains (FIG.4). Next, we evaluated the cytotoxic activity of T cells armed with our BsAbs. Tumor target cells, specifically luciferase-stably expressed target cells (Z138, NALM6), were co-cultured with rCD24 BsAb-armed T cells or unarmed T cells for 24 to 72 hours. Cytotoxicity was measured using bioluminescence assays, which showed sustained cytotoxic activity in BsAb-armed T cells (FIGs. 5-6). Additionally, as depicted in FIGs. 6-11, rCD24 BsAb-D armed T cells mediated significant cytotoxicity against solid tumor cell lines (A172, T98G, U251, A549, PANC-1) in a dosedependent manner, compared to unarmed T cells.

[0087] Table 1. Informal Sequence Listing

Claims

CLAIMSWhat is claimed is:

1. A bispecific antibody comprising a CD24 binder; a CD3 binder; and an IgGl Fc heavy chain constant domain (CH); wherein the CD24 binder comprises: a heavy chain variable domain (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 17; a VH CDR2 comprising SEQ ID NO: 18; a VH CDR3 comprising SEQ ID NO: 19; and a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 20; a VL CDR2 comprising SEQ ID NO: 21; and a VL CDR3 comprising SEQ ID NO: 22; wherein the CD3 binder comprises: a VH CDR1 comprising SEQ ID NO: 9; a VH CDR2 comprising SEQ ID NO: 10; a VH CDR3 comprising SEQ ID NO: 11; a VL CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; and wherein the IgGl Fc CH comprises: a sequence having at least 90% identity to SEQ ID NO: 23, and comprising at least one of DI 22, N207, and E215; or a sequence having at least 90% identity to SEQ ID NO: 24, and comprising at least one of Al 17; Al 18, and G212.

2. The bispecific antibody of claim 1, wherein the IgGl Fc CH comprises the sequence having at least 90% identity to SEQ ID NO: 23 and each of D122, N207, and E215; or the sequence having at least 90% identity to SEQ ID NO: 24, and each of Al 17; Al 18, and G212.

3. The bispecific antibody of claim 2, wherein the IgGl Fc CH comprises SEQ ID NO: 23 or SEQ ID NO: 24.

4. The bispecific antibody of any one of claims 1-3, wherein the CD24 binder VH comprises SEQ ID NO: 15 or a sequence having at least 90% identity thereto; wherein the CD24 binder VL comprises SEQ ID NO: 16 or a sequence having at least 90% identity thereto; wherein the CD3 binder VH comprises SEQ ID NO: 7 or a sequence having at least 90% identity thereto; and wherein the CD3 binder VL comprises SEQ ID NO: 8 or a sequence having at least 90% identity thereto.

5. The bispecific antibody of any one of claims 1-4, further comprising a (G4S)5 linker comprising SEQ ID NO: 27 between the CD3 binder VL and the CD24 binder VH.

6. The bispecific antibody of any one of claims 1-5, wherein the antibody comprises: a heavy chain comprising:SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and comprising at least one of D542, N627, and E635; orSEQ ID NO: 4 or a sequence having at least 90% identity thereto, and comprising at least one of A537; A538; and G632; and a light chain comprising SEQ ID NO: 6 or a sequence having at least 90% identity thereto.

7. The bispecific antibody of claim 6, wherein the heavy chain comprises the sequence having at least 90% identity to SEQ ID NO: 2 and each of D542, N627, and E635.

8. The bispecific antibody of claim 6, wherein the heavy chain comprises the sequence having at least 90% identity to SEQ ID NO: 4, and each of A537; A538; and G632.

9. An engineered polynucleotide encoding the bispecific antibody of any one of claims 1-8.

10. A construct comprising the engineered polynucleotide of claim 9 operably linked to a promoter.

11. A cell comprising the bispecific antibody of any one of claims 1-8, the engineered polynucleotide of claim 9, or the construct of claim 10.

12. The cell of claim 11, wherein the cell is an immune cell.

13. The cell of claim 12, wherein the cell is a T cell, a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell.

14. The cell of claim 13, wherein the cell is a T cell.

15. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1- 8, the engineered polynucleotide of claim 9, the construct of claim 10, or the cell of any one of claims 11-14; and a pharmaceutically acceptable carrier.

16. A kit comprising the bispecific antibody of claim 7 and the bispecific antibody of claim 8, engineered polynucleotides encoding said bispecific antibodies, or immune cells comprising said bispecific antibodies.

17. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 15.

18. The method of claim 17, wherein the cancer comprises at least one of a glioblastoma, a lung carcinoma, a mantle cell lymphoma, and a pancreatic cancer.

19. The method of claim 17 or 18, wherein the cancer comprises CD24 positive cancer cells.

20. The method of any one of claims 17-19, wherein the pharmaceutical composition comprises a T cell comprising the bispecific antibody of claim 8.

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