Antibodies targeting CTLA4 and CD47 and their use

Abstract

Disclosed herein are bispecific antibodies that target both human CTLA4 and CD47. Also provided herein are polynucleotides encoding the bispecific antibodies, pharmaceutical compositions containing the bispecific antibodies, and methods for producing the bispecific antibodies. The medical use of the bispecific antibodies described herein in cancer treatment is also disclosed.

Description

[Technical Field]

[0001] This application claims priority to PCT / CN2023 / 119498, filed on 18 September 2023, which is incorporated fully herein by reference.

[0002] (1. Reference to electronically submitted sequence listings) This application incorporates the sequence listing by reference as an XML file titled "720A001WO02_SL", created on September 18, 2024, and having a size of 66,646 bytes.

[0003] (2. Fields) The present invention relates to molecular biology and cell biology. Provided herein are bispecific antibodies that specifically bind to both human CTLA4 and human CD47, and their use, for example, in the treatment of cancer. [Background technology]

[0004] (3.Background) Immune checkpoint inhibitors (IBTs) represent a promising category of molecules for therapeutic drug development (e.g., those targeting PD-1, Tim-3, and CTLA4). Despite the success of checkpoint inhibitors such as Keytruda® and Opdivo®, current therapeutics targeting CTLA4 or CD47 have achieved limited success (primarily in melanoma) due to their narrow therapeutic scope. Therefore, there is an unmet, urgent need for further cancer therapeutics, particularly those targeting CTLA4 and / or CD47. The compositions and methods provided in this disclosure address this need and offer relevant advantages. [Overview of the Initiative]

[0005] (4. Overview) Provided herein are bispecific antibodies ("bsAb") comprising (i) a light chain variable domain (VL) and a heavy chain variable domain (VH) (wherein the VL / VH pair specifically binds to human CTLA4, and wherein the VL, each comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, each comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively); and (ii) a CD47-binding domain comprising the extracellular domain of SIRPα or a variant thereof. In some embodiments, the VL has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 7, and the VH has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 8. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the CD47-binding domain includes the extracellular domain of human SIRPα variant 2, or a variant thereof. In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 9. In some embodiments, the CD47-binding domain has the amino acid sequence of SEQ ID NO: 9.

[0006] In some embodiments, the bispecific antibodies provided herein include: (1) a first peptide chain (C1) comprising a VL and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising a VH, a heavy chain constant domain 1 (CH1), and a knob-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47-binding domain and a hole-Fc region from the N-terminus to the C-terminus. In some embodiments, the CD47-binding domain and the hole-Fc region are connected by a hinge region. In some embodiments, the hinge region is an IgG1 hinge (SEQ ID NO: 45), an IgG2 hinge (SEQ ID NO: 47), an IgG3 hinge (SEQ ID NO: 48), or an IgG4 hinge (SEQ ID NO: 49); or variants thereof having up to five amino acid mutations. In some embodiments, the hinge region has the amino acid sequence of SEQ ID NO: 46.

[0007] In some embodiments, the bispecific antibodies provided herein include: (1) a first peptide chain (C1) comprising a VL and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising a VH, a heavy chain constant domain 1 (CH1), and a hole-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47-binding domain and a knob-Fc region from the N-terminus to the C-terminus. In some embodiments, the CD47-binding domain and the knob-Fc region are connected by a hinge region. In some embodiments, the hinge region is an IgG1 hinge (SEQ ID NO: 45), an IgG2 hinge (SEQ ID NO: 47), an IgG3 hinge (SEQ ID NO: 48), or an IgG4 hinge (SEQ ID NO: 49); or variants thereof having up to five amino acid mutations. In some embodiments, the hinge region has the amino acid sequence of SEQ ID NO: 46.

[0008] In some embodiments of the bispecific antibodies provided herein, the knob-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including the T366W substitution; and the hole-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including the T366S, L368A, Y407V substitution.

[0009] In some embodiments, the knob-Fc region further includes S354C substitution, and the hole-Fc region further includes Y349C substitution.

[0010] In some embodiments, the knob-Fc region further includes E357K and D399K substitutions, and the hole-region further includes K370E and K409D substitutions.

[0011] In some embodiments of the bispecific antibodies provided herein, (i) the CL region is kappa CL (Cκ; SEQ ID NO: 21) or lambda CL (Cλ; SEQ ID NO: 22), or variants thereof having up to 10 amino acid substitutions; (ii) the CH1 domain is the human IgG1 CH1 domain (SEQ ID NO: 41), or a variant thereof having up to 10 amino acid substitutions; and / or (iii) the knob-Fc region and the hole-Fc region have (1) the amino acid sequences of SEQ ID NOs. 31 and 35, respectively; (2) SEQ ID NOs. 32 and 36, respectively; (3) SEQ ID NOs. 33 and 37, respectively; or (4) the amino acid sequences of SEQ ID NOs. 34 and 38, respectively; or variants thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CH1 domain, knob-Fc region, and hole-Fc region each have the amino acid sequences of (1) SEQ ID NOs. 21, 41, 31, and 35, respectively; (2) SEQ ID NOs. 21, 41, 32, and 36, respectively; (3) SEQ ID NOs. 21, 41, 33, and 37, respectively; or (4) SEQ ID NOs. 21, 41, 34, and 38, respectively.

[0012] In some embodiments of the bispecific antibodies provided herein, C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 51; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 52; and C3 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 53. In some embodiments, C1, C2, and C3 have the amino acid sequences of SEQ ID NO: 51, 52, and 53, respectively.

[0013] In some embodiments, the bispecific antibody: (1) has high avidity for CTLA4 and CD47 double-positive cells; (2) depletes tumor-infiltrating lymphocyte (TIL) regulatory T cells (Treg cells) or Treg cells in the tumor microenvironment (TME); (3) increases cytokine levels in the TME; or (4) enhances T cell proliferation and / or activity against cancer; (5) enhances macrophage-mediated phagocytosis; (6) enhances dendritic cell-mediated antigen presentation; or has any combination of (1) to (6). In some embodiments, the bispecific antibody: (1) has higher affinity for CTLA4 and CD47 double-positive cells than CTLA4 or CD47 single-positive cells; (2) selectively eliminates CTLA4 and CD47-positive cells via antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematotoxicity; or (4) has limited immune-related adverse events (irAE); or has any combination of (1) to (4). In some embodiments, the bispecific antibody has limited mispaired impurities.

[0014] Also provided herein is a composition comprising the bispecific antibody disclosed herein, wherein the purity of the bispecific antibody is at least 95%, and the purity is measured by size exclusion chromatography (SEC) or non-reducing SDS-PAGE.

[0015] In some embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier.

[0016] In some embodiments, provided herein is a polynucleotide encoding a peptide chain of the bispecific antibody disclosed herein. In some embodiments, the polynucleotide can encode all peptide chains of the bispecific antibody. In some embodiments, provided herein are a plurality of polynucleotides disclosed herein that collectively encode all peptide chains of the bispecific antibody.

[0017] In some embodiments, provided herein are vectors comprising polynucleotides disclosed herein.

[0018] In some embodiments, the foregoing provides cells comprising a polynucleotide or a plurality of polynucleotides disclosed herein, or a vector disclosed herein. In some embodiments, the foregoing provides a method for producing bispecific antibodies that specifically bind to human CTLA4 and human CD47, the method comprising culturing the cells disclosed herein under conditions that enable the expression of the bispecific antibodies.

[0019] In some embodiments, provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of a bispecific antibody disclosed herein. In some embodiments, the subject is a human.

[0020] In some embodiments, the use of the bispecific antibodies disclosed herein as pharmaceuticals is provided. In some embodiments, the use of the bispecific antibodies disclosed herein in the treatment of cancer is provided. In some embodiments, the use of the bispecific antibodies disclosed herein for the preparation of pharmaceuticals for the treatment of cancer is provided. [Brief explanation of the drawing]

[0021] (5. Brief explanation of the drawing) [Figure 1] Figures 1A to 1D provide schematic diagrams of bispecific antibodies (bsAbs) provided herein. Figure 1A shows a Fab-Sirpα, knob-into-hole (KIH) shaped bsAb; Figure 1B shows a Sirpα-IgG shaped bsAb; Figure 1C shows an scFv-Sirpα, KIH shaped bsAb; and Figure 1D shows an IgG-Sirpα shaped bsAb.

[0022] [Figure 2] Figure 2 shows flow cytometry results illustrating the binding of HX044 to CD47-positive Jurkat cell lines. Positivity (upper panel) and mean fluorescence intensity (MFI, lower panel) are plotted on the vertical axis. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0023] [Figure 3] Figure 3 shows flow cytometry results illustrating the binding of HX044 to a CHO cell line (CTLA4+CD47-) expressing three hCTLA4 proteins. The binding EC50 was determined according to curve fitting using a four-parameter equation. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0024] [Figure 4] Figure 4 shows flow cytometry results demonstrating the binding of HX044 to CD47+ / CTLA4- HEK293T cells or HEK293T cells transfected with CTLA4 (double-positive for CD47 and CTLA4). "+" indicates membrane CTLA4 expression levels confirmed by flow cytometry. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0025] [Figure 5] Figure 5 provides flow cytometry results showing the binding of HX044 to isolated human peripheral CD4+ lymphocytes, CD8+ lymphocytes, regulatory T cells, and platelets. Ipilimumab, maglorimab (anti-CD47 mAb), and SIRPα-Fc were used as reference antibodies / proteins.

[0026] [Figure 6] Figure 6 provides flow cytometry results showing the binding of HX044 to isolated human erythrocytes, which was undetectable. Ipilimumab, maglorimab, and SIRPα-Fc were used as reference antibodies / proteins.

[0027] [Figure 7] Figure 7 shows flow cytometry results demonstrating the binding of HX044 to human FcRn receptor-positive HEK293T cells. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0028] [Figure 8] Figure 8 shows the results of an ADCC assay demonstrating the ADCC activity of HX044 in CD47+ HEK293T cells with varying levels of CTLA4 expression. "+" indicates membrane CTLA4 expression levels confirmed by flow cytometry. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0029] [Figure 9] Figure 9 shows the results of the in vivo antitumor activity of HX044 in a target gene-humanized syngeneic mouse colon cancer model (MC38-hCD47 model disseminated in hCTLA4×hCD47×hSIRPα HuGEMM mice). Tumor growth was measured twice weekly, and is shown as mean tumor size ± SEM per group. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0030] [Figure 10] Figure 10 shows the results of the in vivo antitumor activity of HX044 in a humanized syngeneic mouse melanoma model (B16F10-hCD47 model seeded on hCTLA4×hCD47×hSIRPα HuGEMM). Tumor growth was measured twice weekly, and is shown as mean tumor size ± SEM per group. Ipilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0031] [Figure 11]Figure 11 provides flow cytometry results showing tumor-infiltrating lymphocytes (TILs) recovered from mice at the end of treatment in a syngeneic mouse model (B16F10-hCD47 model disseminated in hCTLA4×hCD47×hSIRPα mice). Ipilimumab analogs and SIRPα-Fc were used as reference antibodies / proteins.

[0032] [Figure 12] Figure 12 shows flow cytometry results illustrating the effect of HX044 treatment on lymphocyte composition in a syngeneic mouse model (hCTLA4×hCD47×hSIRPα HuGemm C57BL / 6J mouse). Ipilimumab analogs and SIRPα-Fc were used as reference antibodies / proteins.

[0033] [Figure 13] Figures 13A and 13B show the results of size exclusion chromatography (SEC) of HX044 (Figure 13A) and the reference bispecific antibody (Figure 13B) at 214 nm and 280 nm.

[0034] [Figure 14] Figures 14A and 14B show the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of HX044 (Figure 14A) and the reference bispecific antibody (Figure 14B). [Modes for carrying out the invention]

[0035] (6. Detailed explanation) Provided herein are bispecific antibodies that specifically bind to both human CTLA4 and human CD47. Pharmaceutical compositions containing therapeutically effective amounts of such antibodies are also disclosed herein. The use of such antibodies and pharmaceutical compositions for the treatment of cancer is also disclosed herein.

[0036] Cytotoxic T lymphocyte-associated protein-4 (CTLA4) is a classic immune checkpoint that suppresses T cell function by blocking the interaction between the costimulatory receptors B7-1 (CD80) / B7-2 (CD86) on antigen-presenting cells (APCs) and the coactivator CD28 on T cells. CTLA4 is involved in tumor infiltration (TIL) T reg In cells, activated CD8 is more prevalent in normal blood compartments and within the tumor microenvironment (TME). + Effector T cells (T eff It is constitutively expressed at a higher level than ). It is also understood that anti-CTLA4 antibodies block CTLA4 from binding to its ligand CD80 / CD86 on APCs, resulting in the release of CD80 / CD86-mediated secondary signals and reactivation of effective T cells. On the other hand, ADCC effect and / or macrophage-mediated tumor-infiltrating regulatory T cells (T reg Depletion of CTLA4, as well as the reconstitution of innate immunity in TMEs through Fcγ receptor (FcγR) engagement, have been shown to contribute to the antitumor activity of CTLA4. However, anti-CTLA4 antibodies have not yet achieved widespread success in cancer treatment due to their narrow therapeutic range, mainly caused by high systemic immune-related adverse effects (irAEs).

[0037] The "don't eat me" receptor CD47 is overexpressed in many human cancers. Along with its ligand on the surface of phagocytic cells, including macrophages and dendritic cells (DCs), CD47 constitutes a major innate and adaptive immune checkpoint protein, as well as a promising immunotherapy target. Targeting of CD47 via anti-CD47 antibodies or CD47 traps (i.e., signal regulatory protein α or SIRPα) has also been widely investigated, but has so far not shown success in clinical practice due to its narrow therapeutic range. For example, due to the broad expression of CD47 on megakaryocytes and erythrocytes, targeting CD47 has been found to be associated with dose-limiting hematological toxicities (DLTs), such as anemia and thrombocytopenia.

[0038] The anti-hCTLA4 / hCD47 bispecific antibody disclosed herein is TIL-T reg They exhibited synergistic activity in promoting the depletion of hCTLA4, superior antitumor activity compared to agents targeting either hCTLA4 or hCD47, and very limited hematological toxicity and immune-related adverse events (irAEs). Having a larger therapeutic range than single-targeting agents, the dual-targeted bsAbs disclosed herein can serve as safer and more effective therapeutic agents for cancer.

[0039] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments described herein, and that the technical terms used herein are for the purpose of describing specific embodiments and are not intended to limit them.

[0040] (6.1 Definition) Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. Overall, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art.

[0041] All publications and patents cited herein are incorporated herein by reference as specifically and individually indicated, and are incorporated herein by reference to disclose and explain the methods and / or materials by which such publications are cited in connection therewith. Any citation of a publication is for its disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention has no prior rights to such publication on the grounds of prior art. Furthermore, the publication date provided may differ from the actual publication date, which may need to be independently verified.

[0042] The term "a" or "an" entity refers to one or more such entities; for example, "an antibody" is understood to mean one or more antibodies.

[0043] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. Accordingly, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (only), and "B" (only). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

[0044] As used herein, the term “approximately” is used to indicate that a value includes variations in inherent errors to the apparatus or method used to determine that value, or variations that exist between the subjects of study. The term “approximately” encompasses the exact numbers listed. In some embodiments, “approximately” means within plus or minus 10% of a given value or range. In some embodiments, “approximately” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to. In some embodiments, “approximately” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to.

[0045] The terms “peptide chain,” “peptide,” “polypeptide,” and “protein,” as used interchangeably herein, and their grammatical equivalents, refer to polymers of amino acids of any length, which may be linear or branched. These may include unnatural or modified amino acids and may be interrupted by non-amino acids. Polypeptides, peptides, polypeptide chains, peptide chains, or proteins may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification.

[0046] As used interchangeably herein, the terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents mean polymers of nucleotides of any length, including DNA and RNA. A nucleotide can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or analogues thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0047] As used herein with respect to a protein or polypeptide having specific sequence features ("reference protein" or "reference polypeptide"), the term "variant" refers to a different protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions compared to the reference protein or reference polypeptide. Changes to the amino acid sequence may be amino acid substitutions. Changes to the amino acid sequence may be conserved amino acid substitutions. Functional fragments or functional variants of a protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0048] As used herein, the term “specifically binds” means that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, rapidly, for a longer duration, with greater affinity, or in some combination of the above, than with alternative substances, including related and unrelated proteins. A binding site (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified, for example, by immunoassays, ELISA, biolayer interference ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific reaction is at least twice the background signal or noise, and may exceed 10 times the background. For considerations regarding antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology, 2nd edition, Raven Press, New York, pp. 332-336. In some embodiments, “specifically binds” means, for example, that the binding site has a K content of approximately 0.1 mM or less. D This means binding to the target molecule. In some embodiments, "specifically binding" means that the polypeptide or molecule has a K content of about 10 μM or less or about 1 μM or less. Dmeans binding to a target. In some embodiments, "specifically binds" means that the polypeptide or molecule binds to a target with a K D of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Due to sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes proteins or targets in multiple species. Similarly, due to homology within specific regions of the polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes multiple proteins or targets.

[0049] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a binding moiety and a target molecule (e.g., an antigen). Binding between a binding moiety and a target molecule is a reversible process, and binding affinity is generally expressed as the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate (k D or k off ) to the association rate (k d or k on ). A lower K a of a binding pair corresponds to a higher affinity. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Specific exemplary embodiments include the following. In some embodiments, the "K D " or "K D value" can be measured by assays known in the art, for example, by a binding assay. K D can be measured in a radiolabeled antigen binding assay (RIA) (Chen et al. (1999) J. Mol Biol 293:865-881). K D or K D values can also be measured, for example, by using biolayer interferometry (BLI) with a Gator system (Probe Life) or an Octet-96 system (Sartorius AG). K D or K D D ​The value can also be measured using a surface plasmon resonance assay with Biacore, for example, using BIAcore(trademark)-2000 or BIAcore(trademark)-3000 (BIAcore, Piscataway, NJ). Binding affinity is the EC2 concentration of the ligand present in a bound state at half the target during the binding assay. 50 It can also be quantified using this method.

[0050] As used herein in relation to cells, the term “CD47-positive” refers to cells having detectable CD47 expression. In some embodiments, cells have detectable CD47 expression on their surface. As used herein in relation to cancer or tumors, the term “CD47-positive” refers to cancer or tumors having cells with detectable CD47 expression. As used herein in relation to cells, the term “CTLA4-positive” refers to cells having detectable CTLA4 expression. In some embodiments, cells have detectable CTLA4 expression on their surface. As used herein in relation to cancer or tumors, the term “CTLA4-positive” refers to cancer or tumors having cells with detectable CTLA4 expression. Those skilled in the art can easily determine whether cancer or tumors have CD47 expression and / or CTLA4 expression using any method known and available in the art, including, for example, immunohistochemistry (IHC), immunocytochemistry (ICC), enzyme-coupled immunosorbent assay (ELISA), flow cytometry (FACS), etc.

[0051] As used herein in relation to two or more polynucleotides or polypeptides, the terms “identical,” “percent “identical,” and their grammatical equivalents refer to two or more sequences or subsequences having the same or a specified percentage of identical nucleotide or amino acid residues when compared and aligned to the greatest extent possible (with gaps introduced if necessary), without considering conservative amino acid substitutions as part of sequence identity. Percential identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variants. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, that is, they have nucleotide or amino acid residue identity of 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, when compared and aligned to the greatest extent possible using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of amino acid sequence having a length of at least about 10 residues, at least about 20 residues, at least about 40–60 residues, at least about 60–80 residues, or any integer value in between. In some embodiments, the identity exists over a region longer than 60–80 residues, e.g., about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding region of the target protein or antibody. In some embodiments, identity exists over a region of nucleotide sequences having a length of at least about 10 bases, at least about 20 bases, at least about 40–60 bases, at least about 60–80 bases, or any integer value in between.In some embodiments, the identity exists over a region longer than 60–80 bases, for example, at least about 80–1000 bases or more, and in some embodiments, the sequence is substantially identical over the entire length of the sequence being compared, for example, the nucleotide sequence encoding the protein of interest.

[0052] As used herein, the term “vector” and its grammatical equivalent refer to a vehicle used to carry genetic material (e.g., polynucleotide sequences) that can be introduced into a host cell and thereafter replicated and / or expressed. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that can be manipulated for stable integration into the chromosomes of the host cell. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, complement nutritional requirement deficiencies, or supply essential nutrients that are not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcriptional enhancers, and transcriptional terminators, which are well known in the art. If two or more polynucleotides are to be co-expressed, both polynucleotides may be inserted, for example, into a single expression vector or into separate expression vectors. In single-vector expression, the coding polynucleotide can be functionally ligated to one common expression regulatory sequence, or to different expression regulatory sequences, such as one inductive promoter and one constitutive promoter. The introduction of the polynucleotide into host cells can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotide is expressed in an amount sufficient to produce the desired product, and further, that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0053] As used herein, the term “encodes” and its grammatical equivalents refer to the inherent property of a particular sequence of nucleotides in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, to function as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, mRNA) or a defined sequence of amino acids, and the biological properties arising therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein. Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0054] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to such an extent that they are no longer in a form found in nature. In some embodiments, isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0055] As used herein in connection with a disease or disorder, or a person having a disease or disorder, the term “treat” and its grammatical equivalents refer to actions, interventions, and / or measures that suppress, eliminate, reduce, and / or improve the symptoms, severity, and / or frequency of such symptoms associated with the disease or disorder being treated. Treatment of osteoporosis can suppress, eliminate, reduce, or improve the symptoms associated with osteoporosis. These symptoms primarily include bone loss, decreased bone density, and an increased risk of fracture, and treatment aims to slow the rate of bone loss, strengthen bone, and reduce the likelihood of fracture.

[0056] As used herein, the term “administer” and its grammatical equivalents refer to the act of delivering or causing to be delivered a therapeutic agent or pharmaceutical composition into the body of a subject by means of a method described herein or otherwise known in the art. A therapeutic agent may be a compound, polypeptide, antibody, cell, or population of cells. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms such as creams, jellies, powders, and patches; oral dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

[0057] As used herein, the terms “effective dose,” “therapeutic effective dose,” and their grammatical equivalents refer to the amount of a drug administered to a subject, either as a single dose or as part of a series of doses, alone or as part of a pharmaceutical composition, that, when administered to the subject, can produce any detectable positive effect against any symptom, aspect, or characteristic of a disease, disorder, or illness. The therapeutic effective dose can be determined by measuring the relevant physiological effect. The exact required dose will vary from subject to subject, depending on the subject’s age, weight, and overall condition, the severity of the condition being treated, and the clinician’s judgment. The appropriate “effective dose” in individual cases can be determined by those skilled in the art using routine experiments.

[0058] As used herein, the term “mispairing impurities” and its grammatical equivalents refer to unintended antibody species resulting from improper association or pairing of immunoglobulin chains during manufacturing, particularly in recombinant or artificial antibody systems. These impurities arise when heavy and light chains fail to pair correctly, leading to the formation of heavy chain homodimers, light chain homodimers, or heavy chains paired without their corresponding light chains. For illustrative purposes, in a bispecific antibody described herein, comprising three peptide chains: (1) a first peptide chain (C1) which is an IgG light chain containing anti-CTLA4 VL; (2) a second peptide chain (C2) which is an IgG heavy chain containing anti-CTLA4 VL and a “knob” Fc region; and (3) a third peptide chain (C3) which contains a “hole” Fc region fused to a CD47 binding domain, mispairing impurities may occur when C2 and C3 pair without the accompanying light chain, thereby resulting in a light-chain-deficient heterodimer, or when C1, C2, and C3 each form homodimers. The presence of such mispairing impurities may adversely affect the structural integrity, specificity, efficacy, and safety of the antibody product.

[0059] As used herein, the term “subject” refers to any animal (e.g., mammal) that is to be a recipient of a particular treatment, including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. Examples of mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects in need of treatment may be those who have a disease, are at risk of developing a disease, or are suspected of having a disease. Subjects with a disease can be identified by routine medical examinations, such as physical examination, clinical examination, organ function tests, CT scans, or ultrasound. Subjects suspected of having any of such diseases may exhibit one or more symptoms of the disease. Subjects at risk of disease may have one or more risk factors for that disease. Subjects may be human. Subjects may have a particular disease or illness.

[0060] Scope: Through this disclosure, various aspects of the present invention can be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, a range description should be considered to specifically disclose all possible subranges, as well as the individual numbers within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0061] Exemplary genes and polypeptides are described herein with reference to their GenBank numbers, GI numbers, and / or sequence numbers. Those skilled in the art will understand that homologous sequences can be readily identified by referencing sequence sources, including, but not limited to, GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0062] (6.2 Bispecific antibodies targeting human CTLA4 and human CD47) Provided herein are bispecific antibodies that specifically bind to both human CTLA4 and human CD47. In some embodiments, the bispecific antibodies provided herein are monoclonal antibodies. In some embodiments, the bispecific antibodies provided herein are isolated. In some embodiments, the bispecific antibodies provided herein are substantially pure.

[0063] As used herein and as understood in the art, “antibody” is an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein) through at least one antigen-binding fragment, usually located within the variable region of the immunoglobulin molecule. “Antibodies” can be of many different types and structures. For example, an antibody can be a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, a monospecific antibody, a monovalent antibody, or any other modified immunoglobulin molecule containing an antigen-binding site. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the uniqueness of their heavy chain constant domains, which are called alpha, delta, epsilon, gamma, and mu, respectively. Unless otherwise expressly indicated, the term “antibody” as used herein includes the “antigen-binding fragment” of an intact antibody. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenicity-determining variable region of the intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, heavy-chain antibody molecules (e.g., scFv), heavy-chain antibodies (HCAb), light-chain antibodies (LCAb), disulfide-bound scFv (dsscFv), diabodies, triabodies, tetrabodies, minibodies, bivariate-domain antibodies (DVD), single-variate-domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), and single-variate-domains of heavy-chain antibodies (VHH).

[0064] As used herein and as understood in the art, a “bispecific” antibody is an artificial hybrid antibody having two different antigen-binding fragments. The two different antigen-binding fragments specifically bind to two different target antigens. A bispecific antibody can be formed from antibody fragments.

[0065] The structure of immunoglobulins is well-characterized (see, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989))). Typically, immunoglobulins consist of two pairs of polypeptide chains: one pair of light (L; low molecular weight) chains and one pair of heavy (H; high molecular weight) chains, all four of which are interconnected by disulfide bonds.

[0066] Each light chain of an immunoglobulin typically contains a variable light chain region ("VL region") and a constant light chain region ("CL region"). Based on the amino acid sequence of the CL region, there are two distinct types of light chains, called kappa (κ) or lambda (λ). The amino acid sequences of the CL region are well known in the art.

[0067] Each heavy chain typically contains a variable heavy chain region ("VH region") and a constant heavy chain region ("CH region"). Based on their amino acid sequences, the VH region can be of five distinct types: alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). When combined with a light chain, these different types of heavy chains give rise to five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM. There are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the CH regions of different antibody classes are well known in the art.

[0068] The CH region of immunoglobulins contains multiple domains. For example, the CH region of an IgG antibody consists of three domains: heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and heavy chain constant domain 3 (CH3). The highly flexible region between the CH1 and CH2 domains is called the "hinge region." The disulfide bond within the hinge region is the part of the interaction between the two heavy chains in the immunoglobulin. The "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region. In the IgG, IgA, and IgD isotypes, the Fc region consists of the CH2 and CH3 domains; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4). The amino acid sequences of the Fc regions of human IgG, IgA, IgD, IgM, and IgE, as well as the subtypes IgG1, IgG2, IgG3, and IgG4, are known to those skilled in the art. The natural Fc region can be modified. Modifications of the Fc region are described further below. In some embodiments, the bispecific antibodies provided herein may contain paired Fc domains that, instead of forming homodimers, have paired different modifications that promote their mutual association.

[0069] Unless otherwise specified or contextually inconsistent, references to amino acid positions within the constant region are based on EU numbering (Edelman et al., PNAS. 1969; 63:78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991 NIH Publication No. 91-3242). A list of exemplary amino acid sequences of the constant domains / regions of human IgG antibodies is provided below. Some exemplary variants are included, and more variants are disclosed in the sections below.

[0070] Table 1. Natural human IgG constant region / domain [Table 1] TIFF2026532634000002.tif178170

[0071] The term "variable region" typically refers to the portion of the light or heavy chain of an immunoglobulin located at the amino terminus of the light or heavy chain, and is used in relation to the binding and specificity of each particular antibody to its particular antigen. The variable region of the light chain is called the "light chain variable region" or "VL region," and it contains at least one, usually one, "light chain variable domain" or "VL." The variable region of the heavy chain is called the "heavy chain variable region" or "VH region," and it contains at least one, usually one, "heavy chain variable domain" or "VH." The sequences of variable domains differ significantly between different antibodies. A "VL-VH pair" can be related to each other and form a binding site that specifically binds to a target antigen or epitope.

[0072] The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs) (or hypervariable regions where the morphology of sequencely and / or structurally defined loops can be hypervariable). Sequence variability is concentrated in the CDRs, while the less variable portions within the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily involved in antibody-antigen interactions. Each VH and VL typically consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, and also J Mol Biol 1987;196:901-17).

[0073] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. The CDR region is well known to those skilled in the art and is defined by various methods / systems. These systems and / or definitions have been developed and refined over decades and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the most hypervariable region within the antibody variable (V) domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). Software programs for antibody sequence analysis and CDR determination (e.g., abYsis) are available and known to those skilled in the art.

[0074] As used herein, the term “humanized antibody” refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human sequences. Typically, a humanized antibody is a human immunoglobulin. In some cases, the variable region residues of a human immunoglobulin are replaced with corresponding residues in an antibody derived from a non-human species. In some cases, the residues of the CDR are replaced with residues derived from a CDR of a non-human species (e.g., mouse, rat, hamster, camel, rabbit, goat, shark, llama) that have the desired specificity, affinity, and / or binding ability. Humanized antibodies can be further modified by substitution of any additional residues in the variable region and / or within the replaced non-human residues to improve and optimize the antibody's specificity, affinity, and / or binding ability. As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to a human-produced antibody prepared using any technique known in the art.

[0075] The bispecific antibodies provided herein include VL and VH pairs that specifically bind to the human CTLA4 and human CD47 binding domains, including the extracellular domain (ECD) of SIRPα. Exemplary VL / VH pairs that specifically bind to human CTLA4 are provided below, as are exemplary SIRPα ECDs. In addition to the specific CTLA4-targeted VL / VH pairs and CD47 binding domains exemplified below, variants of these VL / VH pairs and CD47 binding domains that retain their binding to CTLA4 and CD47, respectively, are expressly assumed herein.

[0076] Table 3A: Exemplary VL / VH and CDR of anti-CTLA4 antibodies [Table 2]

[0077] Table 3B: SIRPα domain [Table 3]

[0078] In some embodiments, provided herein are bispecific antibodies comprising (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) (wherein the VL1 / VH1 pair specifically binds to human CTLA4, and wherein the VL1 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 1, 2, and 3 according to the Kabat definition, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs. 4, 5, and 6 according to the Kabat definition); and (ii) a CD47-binding domain comprising the extracellular domain of SIRPα or a variant thereof.

[0079] In some embodiments of the bispecific antibodies disclosed herein, the VL has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. A bispecific antibody may have a VL having at least 85% sequence identity with SEQ ID NO: 7. A bispecific antibody may have a VL having at least 90% sequence identity with SEQ ID NO: 7. A bispecific antibody may have a VL having at least 95% sequence identity with SEQ ID NO: 7. A bispecific antibody may have a VL having at least 98% sequence identity with SEQ ID NO: 7. A bispecific antibody may have a VL having at least 99% sequence identity with SEQ ID NO: 7. A bispecific antibody may have a VL having the amino acid sequence of SEQ ID NO: 7.

[0080] In some embodiments of the bispecific antibodies disclosed herein, VH has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. A bispecific antibody may have VH having at least 85% sequence identity with SEQ ID NO: 8. A bispecific antibody may have VH having at least 90% sequence identity with SEQ ID NO: 8. A bispecific antibody may have VH having at least 95% sequence identity with SEQ ID NO: 8. A bispecific antibody may have VH having at least 98% sequence identity with SEQ ID NO: 8. A bispecific antibody may have VH having at least 99% sequence identity with SEQ ID NO: 8. A bispecific antibody may have VH having the amino acid sequence of SEQ ID NO: 8.

[0081] In some embodiments of the bispecific antibodies disclosed herein, VL and VH, which specifically bind to human CTLA4, have the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0082] In some embodiments of the bispecific antibodies disclosed herein, the CD47-binding domain comprises the extracellular domain of human SIRPα. Human SIRPα is an immunomodulatory receptor that binds to CD47 and transmits a "don't eat me" signal, thereby helping to prevent phagocytosis by macrophages. SIRPα contains an extracellular domain having three immunoglobulin superfamily (IgSF) domains, including an NH2-terminal ligand-binding V-domain. The intracellular domain of SIRPα contains both the ITIM and ITSM motifs, which are essential for the receptor's inhibitory activity. For illustrative purposes, full-length human SIRPα may be a 504-amino acid protein (Uniprot accession number P78324-1) containing an extracellular domain (amino acids 31-373), a transmembrane domain (amino acids 374-394), and a cytoplasmic domain (amino acids 395-504). SIRPα has several variants, primarily variant 1 (Genbank: AAH33092.1; SEQ ID NO: 12) and variant 2 (Genbank: AAH26692.1; SEQ ID NO: 10). Although similar in some respects, the two variants of human SIRPα differ by 13 amino acids, all located in their extracellular domain. Currently, there are no approved therapeutic products containing either variant 1 or variant 2 of human SIRPα, although several research therapies targeting the CD47-SIRPα pathway are in clinical trials. Due to the delicate balance and complexity of cancer immunology, the choice between variant 1 and variant 2 in therapeutic design is highly unpredictable. Variations in factors such as binding affinity to CD47, the degree of immune cell activation, and the risk of off-target effects or toxicity can tilt the therapeutic balance towards undesirable immunosuppression or hyperactivation. As will be discussed in detail below, bispecific antibodies containing human SIRPα ECD variant 2 strike a good balance, achieving synergistic antitumor immunity with the anti-CTLA4 domain while minimizing off-target effects.

[0083] In some embodiments, the CD47-binding domain comprises human SIRPα or a variant thereof that retains its binding to CD47. In some embodiments, the CD47-binding domain comprises human SIRPα. In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the CD47-binding domain has the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the CD47-binding domain has the amino acid sequence of SEQ ID NO: 12.

[0084] In some embodiments, the CD47-binding domain comprises the extracellular domain of human SIRPα or a variant thereof that retains its binding to CD47. In some embodiments, the CD47-binding domain comprises the extracellular domain of human SIRPα. In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 31-373 of SEQ ID NO: 10. In some embodiments, the CD47-binding domain has the amino acid sequence of amino acids 31-373 of SEQ ID NO: 12. In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 31-373 of SEQ ID NO: 12.

[0085] In a preferred embodiment, the CD47-binding domain comprises a fragment of the extracellular domain of SIRPα that retains its binding to CD47 (e.g., SEQ ID NO: 9). In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence that is at least 85% sequence-identical to SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence that is at least 90% sequence-identical to SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence that is at least 95% sequence-identical to SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence that is at least 98% sequence-identical to SEQ ID NO: 9. In some embodiments, the CD47-binding domain has the amino acid sequence of SEQ ID NO: 9.

[0086] In some embodiments, the CD47-binding domain includes a fragment of the extracellular domain of SIRPα that retains its binding to CD47 (e.g., SEQ ID NO: 11). In some embodiments, the CD47-binding domain has an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence that is at least 85% sequence-identical to SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence that is at least 90% sequence-identical to SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence that is at least 95% sequence-identical to SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence that is at least 98% sequence-identical to SEQ ID NO: 11. In some embodiments, the CD47-binding domain has the amino acid sequence of SEQ ID NO: 11.

[0087] Those skilled in the art can select known amino acid sequence variants of SIRPa by referring to the literature, for example, the literature of LEE et al., The Journal of Immunology, 179, 7741-7750, 2007, each of which is fully incorporated by citation, and the literature of HATHERLEY et al., The Journal of Biological Chemistry, 282:19, pp. 14567-14575, 2007. More information on human SIRPa can be found in public databases under the following IDs: UniProtKB / Swiss-Prot: P78324; HGNC: 9662; MIM:602461; VeuPathDB: HostDB:ENSG00000198053; and neXtProt: NX_P78324. Three alternatively spliced ​​transcript variants (Uniprot NOs: P78324-1, P78324-2, and P78324-4) encoding different isoforms have been described for the human SIRPa gene.

[0088] In some embodiments, the bispecific antibodies provided herein have a “knob-into-hole” or “KIH” structure (e.g., Figures 1A and 1C). The “KIH” model promotes the formation of heterodimers of the modified bispecific antibodies instead of heavy-chain homodimers.

[0089] Modifications that promote the association of a pair of Fc domains in bispecific antibodies include so-called "knob-into-hole" modifications, which involve a "knob" modification in one Fc domain and a "hole" modification in the other Fc domain. The knob-into-hole technique is described, for example, in US No. 5,731,168; US No. 7,695,936; Ridgway et al., Prot. Eng. 9, 617-621 (1996); and Carter, J Immunol. Meth. 248, 7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of the first Fc ("knob-Fc") and a corresponding cavity ("hole") at the interface of the second Fc ("hole-Fc"), so that the projection can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusions are constructed by replacing smaller amino acid side chains (e.g., tyrosine or tryptophan) originating from the interface of the first polypeptide with larger side chains. Complementary cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine).

[0090] Therefore, the "knob-Fc region" and the "hole-Fc region" are designed to form a heterodimer pair. The knob-Fc region refers to an Fc region in which an amino acid in the CH3 domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a projection within the CH3 domain that can be placed in the cavity within the CH3 domain of the hole-Fc region. In this hole-Fc region, an amino acid residue in the CH3 domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain in which the projection within the CH3 domain of the first subunit can be placed. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusions and cavities can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis.

[0091] In some embodiments, the threonine residue at position 366 of the knob-Fc region is replaced with a tryptophan residue (T366W), the tyrosine residue at position 407 of the whole-Fc region is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A). In some embodiments, the knob-Fc region further comprises either the serine residue at position 354 being replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 being replaced with a cysteine ​​residue (E356C), and the whole-Fc region further comprises the tyrosine residue at position 349 being replaced with a cysteine ​​residue (Y349C). In some embodiments, the whole-Fc region further comprises a serine residue at position 354 replaced by a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced by a cysteine ​​residue (E356C), and the knob-Fc region further comprises a tyrosine residue at position 349 replaced by a cysteine ​​residue (Y349C). In some embodiments, the knob-Fc region contains the amino acid substitutions S354C and T366W, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V. In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C and T366W, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, and Y407V. All amino acid residues are numbered according to the EU index.

[0092] To further promote heterodimerization while suppressing homodimerization, three negatively charged residues in the CH3 domain of one chain can be paired with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are E356-K439, E357-K370, and D399-K409, and vice versa. In some embodiments, two of the following three mutations: E356K, E357K, and D399K are introduced into the knob-Fc region, and two of the following three mutations: K370E, K409D, and K439E are introduced into the hole-Fc region. In some embodiments, two of the following three mutations: E356K, E357K, and D399K are introduced into the whole-Fc region, and two of the following three mutations: K370E, K409D, and K439E are introduced into the knob-Fc region.

[0093] Therefore, in some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, E357K, and D399K, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, K370E, and K409D. In some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, K370E, and K409D, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, E357K, and D399K.

[0094] In some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, E356K, and D399K, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, K439E, and K409D. In some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, K439E, and K409D, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, E356K, and D399K.

[0095] In some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, E356K, and E357K, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, K439E, and K370E. In some embodiments, the knob-Fc region contains the amino acid substitutions S354C, T366W, K439E, and K370E, and the whole-Fc region contains the amino acid substitutions Y349C, T366S, L368A, Y407V, E356K, and E357K.

[0096] In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, E357K, and D399K, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, K370E, and K409D. In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, K370E, and K409D, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, E357K, and D399K.

[0097] In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, E356K, and D399K, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, K439E, and K409D. In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, K439E, and K409D, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, E356K, and D399K. All amino acid residues are numbered according to the EU index.

[0098] In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, E356K, and E357K, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, K439E, and K370E. In some embodiments, the knob-Fc region contains the amino acid substitutions Y349C, T366W, K439E, and K370E, and the whole-Fc region contains the amino acid substitutions S354C, T366S, L368A, Y407V, E356K, and E357K. All amino acid residues are numbered according to the EU index.

[0099] In some embodiments, the herein provides a bispecific antibody comprising (i) a light chain variable domain (VL) and a heavy chain variable domain (VH) (where the VL / VH pair specifically binds to human CTLA4) and (ii) a CD47-binding domain comprising the extracellular domain of SIRPα or a variant thereof. As shown in Figure 1A, in some embodiments, the bispecific antibody provided herein may have three peptide chains: (1) a first peptide chain (C1) comprising the VL and the light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising the VH, the heavy chain constant domain 1 (CH1), and the knob-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising the CD47-binding domain and the hole-Fc region from the N-terminus to the C-terminus.

[0100] As those skilled in the art will understand, in the above-described bispecific antibody having a KIH configuration, the knob-Fc and hole-Fc regions can be interchanged. That is, in the C2 and C3 pairs of a bispecific antibody employing a KIH design, C2 may contain a knob-Fc region and C3 may contain a hole-Fc region; or, in some embodiments, C2 may contain a hole-Fc region and C3 may contain a knob-Fc region. Thus, in some embodiments, the bispecific antibody provided herein may have three peptide chains: (1) a first peptide chain (C1) comprising a VL and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising a VH, a heavy chain constant domain 1 (CH1), and a hole-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47-binding domain and a knob-Fc region from the N-terminus to the C-terminus.

[0101] In some embodiments of the bispecific antibodies disclosed herein, the Fc region (either a knob-Fc region or a hole-Fc region) may include a hinge region. In some embodiments, the hinge region is the IgG1 hinge (SEQ ID NO: 45), the IgG2 hinge (SEQ ID NO: 47), the IgG3 hinge (SEQ ID NO: 48), or the IgG4 hinge (SEQ ID NO: 49); or variants thereof having up to five amino acid mutations. In some embodiments, the hinge region is the IgG1 hinge (SEQ ID NO: 45) or a variant thereof having up to five amino acid mutations. In some embodiments, the hinge region has the amino acid sequence of SEQ ID NO: 46. In some embodiments, the hinge region is the IgG2 hinge (SEQ ID NO: 47) or a variant thereof having up to five amino acid mutations. In some embodiments, the hinge region is the IgG3 hinge (SEQ ID NO: 48) or a variant thereof having up to five amino acid mutations. In some embodiments, the hinge region is the IgG4 hinge (SEQ ID NO: 49) or a variant thereof having up to five amino acid mutations.

[0102] In some embodiments, the CD47-binding domain and the Fc region are connected by a flexible linker, such as a GS linker. In some embodiments, the CD47-binding domain and the Fc region are directly connected, i.e., the C-terminus of the CD47-binding domain is directly connected to the N-terminus of the Fc region (usually the hinge of the Fc region) without any additional linkers.

[0103] The inclusion of flexible linkers is a common choice in the design of therapeutic molecules, as it can increase flexibility by introducing a spatial separation between the SIRPα domain and the Fc region, allowing each domain to function independently. Increased flexibility can improve the binding efficiency of each domain to its respective target (CD47 or immune cells) and can also help achieve simultaneous binding to CD47 and CTLA4, especially when these two targets are located at varying distances on different cells (e.g., tumor cells and T cells). Flexible linkers can also minimize steric collisions, particularly in crowded TMEs, thereby improving the accessibility of both the SIRPα domain and the CTLA4 targeting domain, and increasing the overall potency of the bispecific molecule. Furthermore, GS linkers are commonly used in protein engineering to promote better protein folding and stability during expression, thereby achieving higher production yields of bispecific molecules in recombinant systems, reducing aggregation, and improving their manufacturability.

[0104] Here, it was an unexpected discovery by the inventors that a bispecific antibody lacking a flexible linker between the CD47-binding domain and the Fc region maintained a similar binding affinity to CD47+ / CTLA4+ cells compared to a counterpart molecule with a flexible linker, despite having reduced affinity to CD47+ / CTLA4- cells. While not constrained by theory, it is possible that the increased rigidity optimally positioned the two binding factors to engage both antigens simultaneously. This spatial constraint enhances the molecular selectivity, ensuring that it effectively binds and activates only tumor cells or immune cells co-expressing both CD47 and CTLA4, such as Tregs within the tumor microenvironment (TME). Furthermore, it was also unexpectedly discovered that the bispecific antibody lacking a flexible linker between the CD47-binding domain and the Fc region actually exhibits superior productivity, achieving higher expression levels and fewer mispairing impurities. Therefore, in some preferred embodiments of the bispecific antibodies disclosed herein, the CD47-binding domain (e.g., ECD of human Sirpα variant 2) and the Fc region are directly linked without a linker.

[0105] The bispecific antibodies provided herein in KIH form comprise a CL region, a CH1 domain, and two Fc regions (a knob-Fc and a hole-Fc, each containing a hinge, a CH2 domain, and a CH3 domain, respectively). The amino acid sequences of the CH1, CL, and Fc regions of the bispecific antibodies disclosed herein may be derived from any suitable source, e.g., the constant region of an antibody, e.g., IgG1, IgG2, IgG3, or IgG4. The amino acid sequences of the heavy and light chain constant regions of antibodies, e.g., those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference, are well known in the art.

[0106] In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL region, and Fc region (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein may contain one or more amino acid substitutions that differ from wild-type immunoglobulin. Such substitutions are known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0107] In some embodiments, the CH1 domain of the bispecific antibodies provided herein can be selected from the group consisting of a human IgG1 CH1 domain (SEQ ID NO: 41), a human IgG2 CH1 domain (SEQ ID NO: 42), a human IgG3 CH1 domain (SEQ ID NO: 43), or a human IgG4 CH1 domain (SEQ ID NO: 44); or variants thereof having up to 10 amino acid substitutions. The CH1 domain may be a human IgG1 CH1 domain (SEQ ID NO: 41) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be a human IgG2 CH1 domain (SEQ ID NO: 42) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be a human IgG3 CH1 domain (SEQ ID NO: 43) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be a human IgG4 CH1 domain (SEQ ID NO: 44) or a variant thereof having up to 10 amino acid substitutions.

[0108] In some embodiments, the CL region of the bispecific antibody provided herein may be kappa CL (Cκ; SEQ ID NO: 21). In some embodiments, the CL region of the bispecific antibody provided herein may be lambda CL (Cλ; SEQ ID NO: 22).

[0109] In some embodiments, the Fc region of the bispecific antibodies provided herein may be a variant of the human IgG1 Fc region. In some embodiments, the knob-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including the T366W substitution; and the hole-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including the Y407V substitution. In some embodiments, the hole-Fc region may further have T366S and L368A substitutions. In some embodiments, the knob-Fc and hole-Fc regions may further include S354C and Y349C substitutions, respectively. In some embodiments, the knob-Fc and hole-Fc regions may further include Y349C and S354C substitutions, respectively. In some embodiments, two of the following three mutations: E356K, E357K, and D399K are introduced into the knob-Fc region, and two of the following three mutations: K370E, K409D, and K439E are introduced into the whole-Fc region. In some embodiments, E357K and D399K are introduced into the knob-Fc region, and K370E and K409D are introduced into the whole-Fc region. In some embodiments, two of the following three mutations: E356K, E357K, and D399K are introduced into the whole-Fc region, and two of the following three mutations: K370E, K409D, and K439E are introduced into the knob-Fc region. In some embodiments, E357K and D399K are introduced into the whole-Fc region, and K370E and K409D are introduced into the knob-Fc region. All amino acid residues are numbered according to the EU index.

[0110] In some embodiments, the knob-Fc region and the whole-Fc region may have the amino acid sequences of (1) SEQ ID NOs. 31 and 35, respectively; (2) SEQ ID NOs. 32 and 36, respectively; (3) SEQ ID NOs. 33 and 37, respectively; or (4) SEQ ID NOs. 34 and 38, respectively. The knob-Fc region and the whole-Fc region may have the amino acid sequences of SEQ ID NOs. 31 and 35, respectively. The knob-Fc region and the whole-Fc region may have the amino acid sequences of SEQ ID NOs. 32 and 36, respectively. The knob-Fc region and the whole-Fc region may have the amino acid sequences of SEQ ID NOs. 33 and 37, respectively. The knob-Fc region and the whole-Fc region may have the amino acid sequences of SEQ ID NOs. 34 and 38, respectively.

[0111] Table 4: Exemplary Fc arrays in the KIH model (hinge removed) [Table 4]

[0112] In some embodiments of the bispecific antibodies provided herein, (i) the CL region is kappa CL (Cκ; SEQ ID NO: 21) or lambda CL (Cλ; SEQ ID NO: 22), or variants thereof having up to 10 amino acid substitutions; (ii) the CH1 domain is the human IgG1 CH1 domain (SEQ ID NO: 41), or a variant thereof having up to 10 amino acid substitutions; and / or (iii) the knob-Fc region and the hole-Fc region have the amino acid sequences of (1) SEQ ID NOs. 31 and 35, respectively; (2) SEQ ID NOs. 32 and 36, respectively; (3) SEQ ID NOs. 33 and 37, respectively; or (4) SEQ ID NOs. 34 and 38, respectively; or variants thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CH1 domain, knob-Fc region, and hole-Fc region have the amino acid sequences of SEQ ID NOs. 21, 41, 31, and 35, respectively. In some embodiments, the CL region, CH1 domain, knob-Fc region, and whole-Fc region have the amino acid sequences of SEQ ID NOs. 21, 41, 32, and 36, respectively. In some embodiments, the CL region, CH1 domain, knob-Fc region, and whole-Fc region have the amino acid sequences of SEQ ID NOs. 21, 41, 33, and 37, respectively. In some embodiments, the CL region, CH1 domain, knob-Fc region, and whole-Fc region have the amino acid sequences of SEQ ID NOs. 21, 41, 34, and 38, respectively.

[0113] Table 5A provides a diagram of three peptide chains of an exemplary bispecific antibody in KIH format.

[0114] Table 5A: Peptide chains of exemplary bispecific antibodies (KIH) [Table 5] Note: The VL1 / VH1 pair specifically binds to CTLA4 (e.g., SEQ ID NOs. 7 and 8); SIRPα specifically binds to CD47 (e.g., SEQ ID NOs. 9 or 11); Cκ refers to kappa CL. (H): Hinge region. Table 5B: Sequences of exemplary bispecific antibodies (KIH) [Table 6]

[0115] In some embodiments, provided herein are bispecific antibodies that specifically bind to human CTLA4 and human CD47, wherein the bispecific antibody comprises a first peptide chain (C1), a second peptide chain (C2), and a third peptide chain (C3), where C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 51; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 52; and C3 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence-identical to SEQ ID NO: 53. In some embodiments, C1, C2, and C3 each have the amino acid sequences of SEQ ID NOs: 51, 52, and 53, respectively.

[0116] This disclosure further envisions further variants and equivalents that are substantially homologous to the bispecific antibodies described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, but not limited to, it is desirable to modulate the biological properties of the antibody, including specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the antibody, for example, by changing the number or location of glycosylation sites or by altering membrane anchoring properties.

[0117] Antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of the antibody of this embodiment are also provided herein. Functional variants of the heavy chain, light chain, VL, VH, or CDR used in relation to the antibody still enable the antibody to retain at least a substantial proportion (at least about 90%, 95%, or more) of the functional characteristics of the “reference” and / or “parent” antibody, including affinity and / or specificity / selectivity, Fc inactivity, and PK parameters such as half-life, Tmax, and Cmax. Such functional variants typically have heavy and light chains of substantial sequence identity to the parent antibody and / or substantially similar lengths. Exemplary variants include those that differ from the parent antibody sequence's heavy and / or light chain, VH and / or VL, and / or CDR region, primarily through conservative substitutions, where, for example, 10 of the substitutions in the variant, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1, may be conservative amino acid residue substitutions.

[0118] In some embodiments, variants of the bispecific antibody disclosed herein can retain their ability to bind to CTLA4 and CD47 to the same degree, the same degree, or a higher degree than the parent bispecific antibody. In some embodiments, the variant can have an amino acid sequence that is identical to the parent antibody by at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, a variant of the bispecific antibody disclosed herein comprises the amino acid sequence of the parent bispecific antibody disclosed herein having one or more conserved amino acid substitutions. Conserved amino acid substitutions include amino acid substitutions that are known in the art and have specific physical and / or chemical properties, in which one amino acid has the same or similar chemical or physical properties as another amino acid.

[0119] In some embodiments, the bispecific antibody variants disclosed herein include an amino acid sequence of a parent antibody having one or more non-conservative amino acid substitutions. In some embodiments, the bispecific antibody variants disclosed herein include an amino acid sequence of a parent-bound antibody having one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In some embodiments, one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions can enhance the biological activity of the variant such that the biological activity of the functional variant is increased compared to that of the parent antibody.

[0120] In some embodiments, the variant may have one, two, three, four, or five amino acid substitutions in the binding CDR (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3).

[0121] In some embodiments, the bispecific antibodies provided herein include modifications to their Fc region. In some embodiments, the modified antibody (e.g., the modified Fc region) provides altered effector function, which in turn affects the antibody's biological profile. For example, in some embodiments, deletion or inactivation of the constant region (by point mutation or other means) reduces the Fc receptor binding of the modified antibody in circulation. In some embodiments, constant region modification reduces the immunogenicity of the antibody. In some embodiments, constant region modification increases the serum half-life of the antibody. In some embodiments, constant region modification decreases the serum half-life of the antibody. In some embodiments, constant region modification reduces or eliminates the ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region by corresponding IgG1 or IgG4 residues reduce the effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, constant region modification increases or enhances the ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate a disulfide bond or oligosaccharide moiety. In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide attachment sites for one or more cytotoxins, oligosaccharides, or carbohydrates.

[0122] In some embodiments, the variant may include the addition of amino acid residues at the amino-terminus and / or carboxyl-terminus of the antibody. The length of the additional amino acid residues may range from 1 to 100 or more residues. In some embodiments, the variant may include an N-terminal methionyl residue. In some embodiments, the variant may be modified to be detectable and may include a detectable label and / or protein (e.g., a fluorescent tag or enzyme).

[0123] The variant antibodies described herein can be prepared using methods known in the art, including, but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0124] In some embodiments, the bispecific antibodies disclosed herein may be chemically modified naturally or by intervention. In some embodiments, the bispecific antibodies are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linkage to cellular ligands or other proteins. Any of the numerous chemical modifications can be carried out by known techniques. The bispecific antibodies provided herein may include one or more analogues of amino acids (e.g., including non-natural amino acids) and other modifications known in the art.

[0125] The bispecific antibodies of this disclosure can be analyzed for their physical, chemical, and / or biological properties by various methods known in the art. In some embodiments, the bispecific antibodies provided herein are tested for their ability to bind to human CTLA4 and CD47. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the antibodies can be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency.

[0126] In some embodiments, the bispecific antibodies disclosed herein can be conjugated to detectable substances or molecules that enable the drug to be used for detection. Examples of detectable substances include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and flavin; fluorescent substances such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansilchloride, cyanine (Cy3), and phycoerythrin; bioluminescent substances such as luciferase; radioactive materials; positron-emitting metals; and magnetic metal ions.

[0127] The anti-CTLA4 / CD47 bispecific antibodies disclosed herein can be immobilized on a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized bispecific antibodies are used in immunoassays. In some embodiments, the immobilized bispecific antibodies are used in purification.

[0128] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein can achieve various technological advantages compared to drugs that target only CTLA4 (e.g., ipilimumab) or drugs that target only CD47 (e.g., Sirpα). The anti-CTLA4 / CD47 bispecific antibodies provided herein can achieve various technological advantages compared to counterpart molecules having a more flexible structure by including, for example, a counterpart molecule having a different CD47 binding domain (e.g., a different Sirpα variant) or an additional flexible linker. For illustrative purposes, in some embodiments, the anti-CTLA4 / CD47 bispecific antibody provided herein (e.g., HX044) has greater selectivity for CTLA4 and CD47 bipositive cells. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody provided herein (e.g., HX044) has greater stability. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein has fewer mispairing impurities (e.g., homodimers and / or inaccurate heterodimers). In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein can achieve greater purity. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein has greater expression efficiency.

[0129] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) have limited mispairing impurities.

[0130] In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) selectively remove CTLA4 and CD47-positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Hematological toxicity is adverse effects on the blood and bone marrow, including a decrease in red blood cells, white blood cells, and / or platelets. Because the bispecific antibodies disclosed herein (e.g., HX044) specifically target Treg cells within the TME and avoid nonspecific binding to peripheral cells, in some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have low hematological toxicity. In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have improved safety (e.g., lower hematological toxicity) compared to antibodies that target only CD47 (e.g., Sirpα). Immune-related adverse events (irAEs) are inflammatory toxicity that occur when immune checkpoint inhibitors—e.g., anti-CTLA-4, anti-PD-1, and anti-PD-L1 therapies—disrupt immune self-tolerance and enhance T-cell activity not only against tumor cells but also against normal tissues. These events result from an overactivated immune response that leads to autoimmune-like effects across various organ systems, including the skin, gastrointestinal tract, liver, endocrine glands, lungs, and nervous system. Clinically, irAEs can range from mild to severe and may manifest as dermatitis, colitis, hepatitis, endocrine disorders, pneumonia, or neurological disorders. Due to their low affinity for CTLA4+ / CD47- cells, the bispecific antibodies disclosed herein (e.g., HX044) have a low risk of inducing irAEs in some embodiments. In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a low risk of inducing cytokine release syndrome (CRS). In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a lower risk of inducing irAEs than single-target immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4).In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a lower risk of inducing CRS than single-target immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4).

[0131] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody provided herein (e.g., HX044) (1) has high binding affinity to CTLA4 and CD47 bipositive cells; (2) depletes tumor-infiltrating lymphocytes (TILs) regulatory T cells (Treg cells) or Treg cells in the tumor microenvironment (TME); (3) increases cytokine levels in the TME; (4) enhances T cell proliferation and / or activity against cancer; and (5) CTLA4 or CD4 (1) has a higher affinity for CTLA4 and CD47 double-positive cells than for single-positive cells; (2) selectively removes CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematological toxicity; (4) has limited immune-related adverse events (irAEs); (5) enhances macrophage-mediated phagocytosis; (6) enhances dendritic cell-mediated antigen presentation; or (7) has limited mispairing impurities; or any combination of (1) to (11).

[0132] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) (1) have high binding affinity to CTLA4 and CD47 bipositive cells; (2) deplete tumor-infiltrating lymphocytes (TILs) regulatory T cells (Treg cells) or Treg cells in the tumor microenvironment (TME); (3) increase cytokine levels in the TME; (4) enhance T cell proliferation and / or activity against cancer; (5) enhance macrophage-mediated phagocytosis; (6) enhance dendritic cell-mediated antigen presentation; or any combination of (1) to (6).

[0133] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) have (1) a higher affinity for CTLA4 and CD47 bispecific cells than for CTLA4 or CD47 monopositive cells; (2) selective removal of CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) limited hematological toxicity; (4) limited immune-related adverse events (irAEs); or any combination of (1) to (4).

[0134] The technical effects of the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein can be measured by any assay disclosed herein or otherwise known in the art. For example, the affinity of HX044 or any other reference antibody (e.g., the reference bsAb disclosed in the Experiments section below) for cells expressing only CD47, only CTLA4, or both CD47 and CTLA4 can be measured, for example, by ELISA or FACS. The inventors intend to confirm that HX044 has a lower affinity than the reference bsAb for cells expressing CD47 but not CTLA4, but at least the same or higher affinity than the reference bsAb for cells expressing both CD47 and CTLA4.

[0135] The improved safety of HX044 can be measured by FACS analysis showing its effects on RBC, HGB, and platelet count in a syngeneic mouse model (hCTLA4×hCD47×hSIRPα HuGemm C57BL / 6J mouse) (see the exemplary studies described in the Experiments section below). We intend to confirm that HX044 has a milder effect on RBC / PLT depletion and a reduced CTLA-4-related irAE compared to the reference bsAb.

[0136] (6.3 Polynucleotides, vectors, and cells) Provided herein are polynucleotides encoding at least one light chain or one heavy chain of an anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the polynucleotides provided herein encode one polypeptide, e.g., the light chain or heavy chain of a bispecific antibody. In some embodiments, the polynucleotides provided herein encode multiple polypeptides. In some embodiments, the polynucleotides provided herein can, for example, encode the light chain and heavy chain of a bispecific antibody provided herein, respectively. The cistron can be separated, for example, by an internal ribosome entry site (IRES) or a 2A element. An IRES, as understood in the art, refers to a nucleotide sequence in an expression cassette that, when transcribed to mRNA, can directly recruit ribosomes without prior scanning of the untranslated region of the mRNA by ribosomes. A 2A element, as understood in the art, encodes a self-cleaving short 2A peptide (about 20 amino acids) that provides a mechanism for subsequent separation of the polypeptide of interest produced in equimolar amounts. A family of self-cleaving 2A peptides has been described in the Art (see, for example, Kim, JH et al. (2011) PLoS ONE 6:el8556). Those skilled in the art will understand that other linkers recognized in the Art may be suitable for use in the constructs of the Disclosure (e.g., those encoded by the nucleic acids of the Disclosure). Similarly, those skilled in the art will understand that other polycistronic constructs may be suitable for use provided herein.

[0137] In some embodiments, provided herein are polynucleotides encoding peptide chains C1, C2, C3, or any combination thereof of an anti-CTLA4 / CD47 bispecific antibody having a KIH structure disclosed herein. In some embodiments, provided herein are polynucleotides encoding C1, C2, C3, or any combination thereof of a bispecific antibody named HX044. In some embodiments, provided herein are a plurality of polynucleotides collectively encoding C1, C2, and C3 of a bispecific antibody named HX044.

[0138] The term “polynucleotide encoding a polypeptide” encompasses polynucleotides containing only the coding sequence of a polypeptide and polynucleotides containing further coding and / or non-coding sequences. The polynucleotides of this disclosure may be in the form of RNA or DNA. The DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. Single-stranded DNA may be a coding strand or a non-coding (antisense) strand. The polynucleotides of this disclosure may be mRNA.

[0139] This disclosure also provides variants of the polynucleotides described herein, wherein the variants have nucleotide sequences that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the polynucleotide sequence encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. As used herein, the phrase "polynucleotide having a nucleotide sequence at least about 95% identical to the polynucleotide sequence" means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. That is, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur individually between nucleotides in the reference sequence, or as one or more consecutive groups within the reference sequence, at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions.

[0140] Polynucleotide variants may contain modifications in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain modifications that result in silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants include silent substitutions that do not result in a change in the amino acid sequence of the polypeptide (due to degeneracy of the genetic code). Polynucleotide variants may be produced for various reasons, for example, to optimize codon expression for a particular host (e.g., to change codons in human mRNA to those preferred by a bacterial host such as Escherichia coli (E. coli)). In some embodiments, polynucleotide variants contain at least one silent mutation in the non-coding or coding region of the sequence.

[0141] In some embodiments, polynucleotide variants are produced to modulate or modify the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant results in increased expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, the polynucleotide variant results in decreased expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0142] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) fused to a polynucleotide (e.g., a leader sequence that functions as a secretion sequence to control polypeptide transport) that assists in the expression and secretion of the polypeptide from the host cell within the same reading frame. The polypeptide may have a leader sequence that is cleaved by the host cell to form a “mature” form of the polypeptide.

[0143] In some embodiments, the polynucleotide includes the coding sequence of a polypeptide (e.g., an antibody) fused to the marker or tag sequence within the same reading frame. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS-tag) that enables efficient purification of the polypeptide fused to the marker. In some embodiments, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence is a hemagglutinin (HA) tag derived from influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG® tag. In some embodiments, the marker can be used in conjunction with other markers or tags.

[0144] In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

[0145] In some embodiments, what is provided herein is also a vector containing the polynucleotides disclosed herein. The term “vector” as used herein, and its grammatical equivalents, refers to a vehicle used to carry genetic material (e.g., a polynucleotide sequence) which can be introduced into a host cell where it can be replicated and / or expressed. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes that can contain functionally selectable sequences or markers for stable integration into the chromosomes of a host cell. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply essential nutrients not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcriptional enhancers, transcriptional terminators, and the like, which are well known in the art. If two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or into separate expression vectors. For single-vector expression, the encoding polynucleotide can be functionally ligated to a single common expression regulatory sequence, or to different expression regulatory sequences, such as one inductive promoter and one constitutive promoter. The introduction of the polynucleotide into host cells can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotide is expressed in an amount sufficient to produce the desired product, and further understood that the expression level can be optimized using methods well known in the art to obtain sufficient expression.

[0146] In some embodiments, the vectors provided herein may be expression vectors. In some embodiments, the vectors provided herein include a polynucleotide encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, provided herein is a recombinant expression vector that can be used to amplify and express a polynucleotide encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. For example, a recombinant expression vector may be a replicable DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein, functionally linked to a suitable transcription and / or translation regulatory element derived from a mammalian, microorganism, virus, or insect gene. In some embodiments, a viral vector is used. DNA regions are "functionally linked" if they are functionally related to each other. For example, a promoter is functionally linked to a coding sequence if it controls the transcription of the sequence; or a ribosome-binding site is functionally linked to a coding sequence if it is positioned to enable translation. In some embodiments, the structural elements intended for use in a particular expression system include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In some embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0147] Examples of vectors include plasmids, autonomous replication sequences, and transposable elements. Useful expression vectors for bacterial hosts include known bacterial plasmids, e.g., E. coli plasmids including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, e.g., M13 and other filamentous single-stranded DNA phages. Further exemplary vectors, but not limited to, include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of animal viruses useful as vectors, but not limited to, include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Exemplary transposon systems such as Sleeping Beauty and PiggyBac, which can be stably integrated into the genome, can be used (e.g., Ivics et al., Cell, 91(4): 501-510 (1997); Cadinanos et al. (2007) Nucleic Acids Research. 35(12): e87).

[0148] In some embodiments, the vector is an episomal vector or a vector maintained outside of chromosomes. As used herein, the term “episomal” means a vector that can replicate without being incorporated into the host’s chromosomal DNA and without being gradually lost from a dividing host cell, and also means that the vector replicates outside of chromosomes or as an episome. The vector is modified to possess a DNA replication origin or “ori” derived from a lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a sequence encoding a lymphotropic herpesvirus or gamma herpesvirus replication origin corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), herpesvirus thymiri (HS), or Marek’s disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Host cells typically contain viral replication transactivator proteins that activate replication.

[0149] The "expression regulatory sequences," "regulatory elements," or "regulatory sequences" present in an expression vector are the untranslated regions of the vector—the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Schein-Dalgarno sequence or Kozak sequence), introns, polyadenylated sequences, and 5' and 3' untranslated regions—which interact with host cell proteins to perform transcription and translation. Such elements can vary considerably in length and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0150] Examples of ubiquitous expression regulatory sequences that can be used in this disclosure include the cytomegalovirus (CMV) very early promoter, the virulence salivirus 40 (SV40) promoter (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Roussarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the vaccinia virus-derived H5, P7.5, and P11 promoters, the elongation factor 1-α (EF1a) promoter, the early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), and glyceraldehyde. Examples include, but are not limited to, 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa β, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0151] Examples of inductive promoters / systems include, but are not limited to, steroid-inductive promoters, e.g., promoters of genes encoding glucocorticoid or estrogen receptors (induced by treatment with the corresponding hormones), metallothionein promoters (induced by treatment with various heavy metals), MX-1 promoters (induced by interferon), the "GeneSwitch" mifepristone controllable system (Sirin et al., 2003, Gene, 323:67), cumate-inductive gene switches (WO 2002 / 088346), and tetracycline-dependent regulatory systems. The bispecific antibodies described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise indicated, the invention will be carried out using conventional techniques in the relevant fields within the scope of the art, including molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and other fields within the scope of the art. These techniques are described and adequately explained in the references cited herein. For example, Maniatis et al. (1982), *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001), *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons (1987 and annually updated editions);Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated editions), Gait (ed.) (1984); Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991); Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (ed.) (1999); Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995); Antibody Engineering, 2nd edition, Oxford University Press; See Lo (ed.) (2006), Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press; these are incorporated herein in full by citation.

[0152] This disclosure also provides cells comprising polynucleotides disclosed herein that encode at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the cells provided herein comprise polynucleotides encoding C1, C2, and C3 of the anti-CTLA4 / CD47 bispecific antibody disclosed herein having a KIH structure. In some embodiments, the cells provided herein comprise a plurality of polynucleotides that collectively encode C1, C2, and C3 of the anti-CTLA4 / CD47 bispecific antibody disclosed herein having a KIH structure.

[0153] Cells containing the vectors disclosed herein are also conceivable. In some embodiments, provided herein are host cells containing a vector comprising a polynucleotide disclosed herein. In some embodiments, the host cells provided herein contain a vector or a plurality of vectors comprising a polynucleotide collectively encoding the polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the host cells provided herein produce the anti-CTLA4 / CD47 bispecific antibody described herein.

[0154] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human fetal kidneys) cell lines, and their variants. Mammalian expression vectors may include a replication origin, suitable promoters and enhancers linked to the gene to be expressed, and non-transcription elements such as other 5' or 3' flanking non-transcription sequences, as well as 5' or 3' untranslated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art.

[0155] (6.4 Production Method) Also provided herein are methods for producing the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the bispecific antibody disclosed herein consists of a plurality of polypeptide chains that can be produced individually or together. In some embodiments, the method provided herein produces at least one polypeptide chain of the bispecific antibody disclosed herein. In some embodiments, the method provided herein produces all polypeptide chains of the bispecific antibody disclosed herein.

[0156] The bispecific antibodies or polypeptides described herein can be produced and isolated using methods known in the art. Polypeptides can be synthesized whole or partially using chemical methods (see, for example, Caruthers (1980), Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995), Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be carried out using various solid-phase techniques (see, for example, Roberge, Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)), and automated synthesis can be achieved using, for example, an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combinatorial methods. Synthetic residues and polypeptides can be synthesized using various procedures and methods known in the art (see, for example, Organic Synthesis Collective Volumes, Gilman et al. (ed.), John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Peptide sequence mutations, derivatives, substitutions, and modifications can also be performed using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)), and other techniques can be applied to cloned DNA to produce the inventive peptide sequence, variants, fusions, and chimeras, as well as their mutations, derivatives, substitutions, and modifications.

[0157] Various host expression vector systems can be used to recombinantly express one or more of the bispecific antibodies or their polypeptide chains described herein. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein production, including antibody production, are well known in the art. Such host expression systems are media that can produce and subsequently purify the coding sequences of the bispecific antibodies described herein, but are also cells that can express the bispecific antibodies described herein in situ when transformed or transfected with an appropriate polynucleotide coding sequence.These include microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence of the compounds described herein (e.g., Escherichia coli and B. subtilis); and yeast transformed with recombinant yeast expression vectors containing the coding sequence of the compounds described herein (e.g., Saccharomyces pichia). This includes, but is not limited to, COS, CHO, BHK, 293, 293T, 3T3 cells, lymphoid cells (see U.S. Patent No. 5,807,715), and Per C.6 cells (human retinal cells developed by Crucell) that possess a recombinant expression construct containing a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0158] In bacterial systems, many expression vectors can be advantageously selected depending on the intended use of the expressed protein. For example, when large quantities of such proteins are to be produced, a vector directing high levels of easily purified protein product expression may be desirable for the preparation of the bispecific antibody pharmaceutical compositions described herein. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983), EMBO J. 2: 1791-1794); and the pIN vector (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), J. Biol. Chem. 24:5503-5509). Polypeptides can also be expressed as fusion proteins with glutathione S-transferase (GST) using pGEX vectors. Generally, such proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. To allow the cloned target gene product to be released from the GST region, the pGEX vector is designed to contain a thrombin or factor X protease cleavage site.

[0159] Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Several virus-based expression systems can be used in mammalian host cells. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human fetal kidney), and their variants. Mammalian expression vectors may include a replication origin, a suitable promoter and enhancer linked to the gene to be expressed, and other non-transcription elements such as other 5' or 3' adjacent non-transcription sequences, as well as 5' or 3' untranslated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art. The cutworm (Autographa californica) nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes.

[0160] Furthermore, a host cell line can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important to the protein's function. For example, in one embodiment, the antibody described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor) that requires proteolytic cleavage by innate or recombinant cellular mechanisms to form a distinct polypeptide of the bispecific antibody described herein. Thus, this disclosure encompasses modifying a nucleic acid sequence to encode a polyprotein precursor molecule containing the polypeptide of the bispecific antibody described herein, wherein the nucleic acid sequence includes a coding sequence that can direct post-translational cleavage of the polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the polypeptide of the bispecific antibody described herein. Posttranslational cleavage of precursor molecules containing polypeptides of the compounds described herein can occur in vivo (i.e., in a host cell, by a native or recombinant cell line / mechanism, e.g., by furin cleavage at an appropriate site) or in vitro (e.g., incubation of the polypeptide chain in a composition containing a protease or peptidase of known activity and / or in a composition containing conditions or reagents known to promote the desired proteolytic activity). Purification and modification of recombinant proteins are well known in the art to the extent that the design of polyprotein precursors can include many embodiments that are readily understood by those skilled in the art. Any known protease or peptidase known in the art can be used for the modification of the precursor molecules described herein.

[0161] Different host cells possess characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. A suitable cell line or host system can be selected to ensure the precise modification and processing of expressed foreign proteins. For this purpose, eukaryotic host cells possessing cellular machinery for proper processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, and T47D, CRL7030, as well as Hs578Bst.

[0162] For long-term high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the compounds described herein can be artificially created. Rather than using expression vectors containing viral replication origins, host cells can be transformed with DNA and selection markers controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introduction of the foreign DNA, the artificially created cells can be grown in enriched medium for 1-2 days, and then transferred to a selection medium. The selection markers in the recombinant plasmid confer resistance to selection, allowing the cells to stably incorporate the plasmid into their chromosomes, grow, and form growth foci, which can then be cloned and expanded into a cell line. This method can be advantageously used to artificially create cell lines that express the compounds described herein. Such artificially created cell lines may be particularly useful in screening and evaluation of compounds that directly or indirectly interact with the compounds described herein.

[0163] Several select systems can be used, including, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al. (1977), Cell 11: 223-232), hypoxanthine guanine phosphoribosyltransferase (Szybalska et al. (1992), Bioessays 14: 495-500), and adenine phosphoribosyltransferase (Lowy et al. (1980), Cell 22: 817-823) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance is conferred by the following genes: dhfr (Wigler et al. (1980) PNAS 77:3567-3570; O'Hare et al. (1981) PNAS, 78: 1527-1531), which confers resistance to methotrexate; gpt (Mulligan et al. (1981) PNAS, 78: 2072-2076), which confers resistance to mycophenolate; neo (Tolstoshev (1993), Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993), Science 260:926-932; and Morgan et al. (1993), Ann. Rev. Biochem. 62: 191-217), and can also be used as a basis for selecting hygro (Santerre et al. (1984) Gene 30: 147-156) to confer resistance to hygromycin.The commonly known methods in the field of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler's Literature, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Chapters 12 and 13 of Current Protocols in Human Genetics, Dracopoli et al. (eds.), 1994, John Wiley & Sons, NY.

[0164] The expression levels of the bispecific antibodies or their polypeptide chains described herein can be increased by vector amplification (see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987) for a review). If the vector-based markers described herein are amplified, an increase in the level of the inhibitor present in the host cell culture increases the number of copies of the marker gene. Since the amplified region is related to the nucleotide sequence of the target protein, the production of the target protein also increases (see Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0165] Host cells can be co-transfected with multiple expression vectors, each encoding a polypeptide chain of one of the bispecific antibodies described herein. The vectors may contain identical selection markers that enable equivalent expression of all polypeptides. Alternatively, a single vector encoding two or more polypeptides can be used. The polypeptide coding sequences of the compounds described herein may include cDNA or genomic DNA.

[0166] Once a bispecific antibody or polypeptide described herein has been recombinantly expressed, it can be purified by any method known in the Art for the purification of polypeptides, polyproteins, or antibodies (similar to antibody purification schemes based on antigen selectivity), for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens (optionally, after selection of protein A if the compound contains an Fc domain (or a portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of polypeptides or antibodies.

[0167] Provided herein are methods for producing the anti-CTLA4 / CD47 bispecific antibody or the bispecific antibody polypeptide chain described herein, comprising obtaining the cells described herein and expressing the polynucleotide described herein in said cells. In some embodiments, the methods provided herein include culturing the cells under conditions that enable the expression of the bispecific antibody. In some embodiments, the methods further include isolating and purifying the bispecific antibody or polypeptide chain described herein.

[0168] The bispecific antibodies described herein can be tested for binding to human CTLA4 and / or CD47, for example, by standard ELISA. Briefly, a microtiter plate is coated with purified antigen and then blocked with bovine serum albumin. Diluted antibody is added to each well and incubated. The plate is washed and incubated with a secondary reagent conjugated with horseradish peroxidase (HRP) (for example, in the case of human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent). After washing, the plate can be color-developed and analyzed by spectrophotometer. The antibodies can be further tested by flow cytometry for binding to cell lines expressing human CTLA4 and / or CD47, rather than to control cell lines that do not express the target antigen. Briefly, antibody binding can be evaluated by incubation of CHO cells expressing CTLA4 and / or CD47 with the bispecific antibodies provided herein. The cells can be washed, and binding can be detected with an anti-human IgG antibody. Flow cytometry analysis can be performed using FACS can flow cytometry (Becton Dickinson, San Jose, CA).

[0169] The anti-CTLA4 / CD47 bispecific antibodies provided herein can be further tested for reactivity with target antigens by Western blotting. Other methods known in the art for analyzing the binding affinity, cross-reactivity, and binding reaction rate of the various anti-CTLA4 / CD47 bispecific antibodies described herein include, for example, biolayer interferometry (BLI) using a Gator system (Probe Life) or an Octet-96 system (Sartorius AG), or BIACORE® surface plasmon resonance (SPR) analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0170] In practice of the present invention, unless otherwise indicated, conventional techniques in molecular biology, cell biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of the skills of those skilled in the art are utilized. These techniques are described and fully explained in the references cited herein. For example, Maniatis et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated editions); Current Protocols in Immunology, John Wiley & Sons Sons (1987 and annually updated editions), Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press;See Birren et al. (eds.) (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995), Antibody Engineering, 2nd edition, Oxford University Press; Lo (ed.) (2006), Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press; each of these is incorporated herein by reference in its entirety.

[0171] (6.5 Composition) Provided herein are compositions comprising an anti-CTLA4 / CD47 bispecific antibody disclosed herein, wherein the purity of the bispecific antibody is at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, wherein the purity is measured by size exclusion chromatography (SEC) or non-reducible SDS-PAGE. In some embodiments, the purity of the bispecific antibody in the composition is at least 95%. In some embodiments, the purity of the bispecific antibody in the composition is at least 96%. In some embodiments, the purity of the bispecific antibody in the composition is at least 97%. In some embodiments, the purity of the bispecific antibody in the composition is at least 98%. In some embodiments, the purity of the bispecific antibody in the composition is at least 99%. In some embodiments, the purity of the bispecific antibody in the composition is in the range of 95% to 99%, 95% to 98%, 95% to 97%, 96% to 99%, 96% to 98%, 96% to 97%, or 95% to 96%. In some embodiments, the purity of the bispecific antibody in the composition is in the range of 95% to 99%. In some embodiments, the purity of the bispecific antibody in the composition is in the range of 95% to 98%. In some embodiments, the purity of the bispecific antibody in the composition is in the range of 95% to 97%. In some embodiments, the compositions provided herein have mispairing impurities of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the compositions provided herein have mispairing impurities of less than 10%. In some embodiments, the compositions provided herein have mispairing impurities of less than 8%. In some embodiments, the compositions provided herein have mispairing impurities of less than 5%. In some embodiments, the compositions provided herein have less than 3% mispairing impurities. In some embodiments, the compositions provided herein have less than 2% mispairing impurities.In some embodiments, the compositions provided herein have less than 1% mispairing impurities. In some embodiments, the compositions provided herein do not have detectable mispairing impurities when measured by non-reducing SDS-PAGE. Mispairing impurities include, for example, homodimers and / or inaccurate heterodimers. In some embodiments, the compositions provided herein do not have detectable homodimers when measured by non-reducing SDS-PAGE. In some embodiments, the compositions provided herein do not have detectable inaccurate heterodimers when measured by non-reducing SDS-PAGE.

[0172] Provided herein are pharmaceutical compositions comprising the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in the treatment of inflammatory or autoimmune diseases.

[0173] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refer to a material suitable for drug administration to an individual together with an activator without causing undesirable biological effects or interacting in an adverse manner with any of the other components of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions disclosed herein may include one or more of buffer systems, preservatives, tonicity modifiers, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0174] In some embodiments, the pharmaceutical compositions provided herein contain the anti-CTLA4 / CD47 bispecific antibody provided herein. The anti-CTLA4 / CD47 bispecific antibody can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein contain 1 to 1000 mg / mL of the anti-CTLA4 / CD47 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions contain 10 to 500 mg / mL, 10 to 400 mg / mL, 10 to 300 mg / mL, 10 to 200 mg / mL, 10 to 100 mg / mL, 20 to 100 mg / mL, or 50 to 100 mg / mL of the anti-CTLA4 / CD47 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions provided herein contain anti-CTLA4 / CD47 bispecific antibodies provided herein in doses of approximately 10 mg / mL, approximately 20 mg / mL, approximately 30 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 150 mg / mL, approximately 180 mg / mL, approximately 200 mg / mL, approximately 300 mg / mL, approximately 500 mg / mL, approximately 800 mg / mL, or approximately 1000 mg / mL. The dosage can be easily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in the dosage.

[0175] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., anti-CTLA4 / CD47 bispecific antibody) can be coated in the material to protect it from the action of acids and other natural conditions that may inactivate it.

[0176] Provided herein are pharmaceutical compositions or formulations that improve the stability of anti-CTLA4 / CD47 bispecific antibodies to enable their long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise (a) the anti-CTLA4 / CD47 bispecific antibody disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a filler; and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years, or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0177] A buffer useful in a pharmaceutical composition or formulation disclosed herein may be a weak acid or base used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) with isotonic agents and potentially pH adjustment with acids or bases known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained at about 2 to about 10, or about 4 to about 8.

[0178] Stabilizers are added to pharmaceutical products to stabilize their products. Such agents can stabilize proteins in various ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine; carbohydrates such as glucose, sucrose, trehalose, raftnose, or maltose; polyols such as glycerol, mannitol, sorbitol; cyclodextrins or destran of any type and molecular weight; or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the antibody in the lyophilized preparation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0179] Fillers can be added to pharmaceutical compositions or formulations to add volume and mass to the product, thereby facilitating its accurate measurement and handling. Common fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0180] A surfactant is an amphiphilic substance having hydrophilic and hydrophobic groups. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0181] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersants, emulsions, and multiphase materials. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

[0182] In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which a physician or patient adds a solvent and / or diluent before use.

[0183] The pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0184] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the required particle size in the case of dispersants, and by the use of surfactants.

[0185] These compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the sterilization procedure described above and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbate. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, extension of absorption in injectable pharmaceutical forms can be achieved by the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0186] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. In some embodiments, provided herein are pharmaceutical compositions comprising anti-CTLA4 / CD47 bispecific antibodies or cells provided herein, wherein the composition is suitable for topical administration.

[0187] Pharmaceutical compositions or formulations must typically be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents, such as lecithin; in the case of dispersants, by maintaining the required particle size; and by the use of surfactants. Often, compositions may contain isotonic agents, such as sugars, polyhydric alcohols like mannitol and sorbitol, or sodium chloride. Extending the absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0188] Sterile injection solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent, along with one or a combination of the components listed above as needed, and then performing sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components derived from the components listed herein. For sterile powders for the preparation of sterile injection solutions, some preparation methods involve vacuum drying and freeze-drying (lyophilization) to obtain the powder of the active component and any further desired components from its pre-sterile filtered solution.

[0189] The amount of active ingredient that can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient that can be combined with the carrier material is the amount that produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient.

[0190] The pharmaceutical compositions disclosed herein can be prepared using carriers that protect the active ingredient from rapid release, including controlled-release formulations, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, edited by JR Robinson, Marcel Dekker, New York, 1978.

[0191] Provided herein are also kits for the preparation of pharmaceutical compositions having the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the kit comprises the anti-CTLA4 / CD47 bispecific antibody disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may include the anti-CTLA4 / CD47 bispecific antibody disclosed herein for administration to a subject. In a specific embodiment, the kit includes instructions relating to the preparation and / or administration of the anti-CTLA4 / CD47 bispecific antibody.

[0192] (6.6 Method and Use) The antibodies, compositions, and methods described herein have many in vitro and in vivo applications, for example, by inhibiting (or antagonizing) CTLA4 and / or CD47 (e.g., signaling), resulting in enhancement of the immune response. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein can be administered to human subjects, for example, in vivo, to enhance immunity in various diseases. Provided herein are methods for modifying the immune response of a subject, comprising administering the anti-CTLA4 / CD47 bispecific antibodies described herein to the subject so as to modify the immune response of the subject. In some embodiments, the response is enhanced, stimulated, or upregulated.

[0193] In some embodiments, the foregoing provides a method for inducing or stimulating immune cell activation, comprising contacting immune cells with an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the foregoing provides a method for inducing or stimulating immune cell proliferation, comprising contacting immune cells with an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the foregoing provides a method for mitigating CTLA4 and / or CD47-mediated inhibition of immune cell proliferation, comprising contacting immune cells with an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the foregoing provides a method for inhibiting the interaction of CTLA4 and / or CD47 and its ligand on immune cells, comprising contacting immune cells with an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein.

[0194] Suitable subjects for this method include human patients for whom an enhancement of the immune response is desirable. This method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response (e.g., an immune response mediated by T cells, e.g., an antigen-specific T cell response). In some embodiments, this method is particularly suitable for the in vivo treatment of cancer. In some embodiments, provided herein is a method for enhancing the immune response of a subject in need thereof, comprising administering to the subject an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein. To achieve antigen-specific enhancement of immunity, the anti-CTLA4 / CD47 bispecific antibody described herein may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or a virus).

[0195] Anti-CTLA4 / CD47 bispecific antibodies or those described herein, for example, immunosuppressive T in TEM regConsidering the ability to stimulate or co-stimulate a T cell response, such as an antigen-specific T cell response, by depleting cells, provided herein are in vitro and in vivo methods for stimulating, enhancing, or upmodulating an antigen-specific T cell response, such as an antitumor T cell response, using the anti-CTLA4 / CD47 bispecific antibody described herein. The antigen-specific T cell response can be measured using any preferred indicator of the antigen-specific T cell response. Non-limiting examples of such preferred indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In some embodiments, interleukin-2 and / or interferon-γ production by antigen-specific T cells is stimulated.

[0196] Further encompassing are methods for stimulating an immune response to a target (e.g., an antigen-specific T cell response), comprising administering an anti-CTLA4 / CD47 bispecific antibody described herein to a target such that an immune response to the target (e.g., an antigen-specific T cell response) is stimulated. In some embodiments, the target is a subject having a tumor, and an immune response to the tumor is stimulated. The tumor may be a solid tumor or a humoral tumor, e.g., a hematological malignancy. In some embodiments, the tumor is an immunogenic tumor. In some embodiments, the tumor is non-immunogenic. In some embodiments, the tumor is PD-L1 positive. In some embodiments, the tumor is PD-L1 negative. In some embodiments, the tumor is non-immunogenic. In some embodiments, the tumor is CTLA4 positive. In some embodiments, the tumor is CD47 positive. In some embodiments, the tumor is both CTLA4 positive and CD47 positive. The target may also be a subject having a virus, and an immune response to the virus is stimulated.

[0197] In some embodiments, a method is provided for inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject an anti-CTLA4 / CD47 bispecific antibody described herein such that tumor growth is inhibited in the subject. Also provided is a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody disclosed herein. Also provided is the use of the bispecific antibody disclosed herein as a pharmaceutical agent. Also provided is the use of the bispecific antibody disclosed herein in the treatment of cancer. Also provided is the use of the bispecific antibody disclosed herein for the preparation of pharmaceuticals for the treatment of cancer.

[0198] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein are administered to subjects as adjuvant therapy. Treatment of subjects with cancer with the anti-CTLA4 / CD47 bispecific antibodies described herein can result in extended survival, e.g., a long-term, persistent response to current standard treatment; long-term survival of at least 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years or more, or recurrence-free survival of at least 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years or more. In some embodiments, treatment of subjects with cancer with the anti-CTLA4 / CD47 bispecific antibodies described herein prevents cancer recurrence or delays cancer recurrence for, for example, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years or more.

[0199] Treatment of patients with cancer using the anti-CTLA4 / CD47 bispecific antibodies described herein may result in, for example, disease stabilization, partial response, extended overall survival, extended disease-free survival, or improved progression-free survival.

[0200] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein do not exhibit significant toxicity. For example, the anti-CTLA4 / CD47 bispecific antibodies described herein do not exhibit significant toxicity to one or more human organs, such as the liver, kidneys, brain, lungs, and heart, as determined, for example, in clinical trials. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein have limited hematological toxicity. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein have limited irAEs. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies do not significantly induce undesirable immune responses, such as autoimmunity or inflammation. In some embodiments, treatment of a subject with the anti-CTLA4 / CD47 bispecific antibodies described herein does not result in excessive stimulation of the immune system to the extent that the subject's immune system subsequently attacks the subject itself (e.g., an autoimmune response) or, for example, leads to anaphylaxis. Therefore, in some embodiments, anti-CTLA4 / CD47 bispecific antibodies do not cause anaphylaxis.

[0201] In some embodiments, treatment of a subject with the anti-CTLA4 / CD47 bispecific antibodies described herein does not cause significant inflammatory responses, such as immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated pituitary inflammation, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, treatment of a subject with the anti-CTLA4 / CD47 bispecific antibody described herein does not cause significant cardiac disorders, e.g., ventricular arrhythmias; ocular disorders, e.g., iridocyclitis; infusion-related reactions; increased amylase, increased lipase; nervous system disorders, e.g., dizziness, peripheral neuropathy, and sensory neuropathy; skin and subcutaneous tissue disorders, e.g., rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo, or psoriasis; respiratory, chest, and mediastinal disorders, e.g., cough; fatigue; nausea; loss of appetite; constipation; arthralgia; or diarrhea.

[0202] In some embodiments, anti-CTLA4 / CD47 bispecific antibodies provide synergistic antitumor effects when combined with other cancer therapies, such as compounds that stimulate the immune system (e.g., immuno-oncological agents).

[0203] This disclosure also provides methods for using the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of cancer, polynucleotides encoding such antibodies, vectors comprising such polynucleotides, or pharmaceutical compositions having such antibodies or cells.

[0204] In some embodiments, anti-CTLA4 / CD47 bispecific antibodies can reduce the immunosuppressive effects mediated by the CTLA4 and / or CD47 signaling pathway, thereby promoting the activity of immune cells in eliminating, lysing, and / or killing cancer cells. In some embodiments, the method comprises administering a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein to a subject in need.

[0205] The anti-CTLA4 / CD47 bispecific antibodies described herein can block the interaction between CTLA4 and B7-1(CD80) / B7-2(CD86), and the interaction between CD47 and SIRPα. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies can specifically target cancer cells expressing CTLA4 / CD47 in vivo, thereby delivering their therapeutic effect of eliminating, lysing, and / or killing the cancer cells.

[0206] In some embodiments, provided herein are methods for treating a tumor or cancer of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, provided herein are the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of a tumor or cancer. In some embodiments, provided herein are the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein for the preparation of a medicament for the treatment of a tumor or cancer. In some embodiments, the tumor or cancer is CTLA4-positive. In some embodiments, the tumor or cancer is CD47-positive. In some embodiments, the tumor or cancer is both CTLA4-positive and CD47-positive.

[0207] In some embodiments, provided herein are methods for treating a tumor or cancer of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, provided herein are the use of the pharmaceutical composition disclosed herein in the treatment of a tumor or cancer. In some embodiments, provided herein are the use of the pharmaceutical composition disclosed herein for the preparation of a medicament for the treatment of a tumor or cancer.

[0208] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein (i.e., the anti-CTLA4 / CD47 bispecific antibody in the pharmaceutical compositions described herein) can be varied to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage is determined by various pharmacokinetic factors, including the activity of the particular composition described herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition being used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical field. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody described herein can be administered in a dosage that provides therapeutic benefit without causing high levels of immune-related adverse effects or hematological toxicity (anemia and / or thrombocytopenia).

[0209] The anti-CTLA4 / CD47 bispecific antibodies described herein can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the anti-CTLA4 / CD47 bispecific antibody in the patient. For therapeutic use, relatively high doses at relatively short intervals may be required until disease progression slows or stops, and until the patient shows partial or complete improvement of disease symptoms.

[0210] The anti-CTLA4 / CD47 bispecific antibodies or pharmaceutical compositions provided herein can be administered to a target by any method known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes, such as by injection or infusion, or by direct administration to the thymus. As used herein, the term "parenteral administration" usually means, and is not limited to, injection, intramuscular, intraarterial, intrathecal, intracapsular, orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, subcutaneous administration is employed. In some embodiments, intravenous administration is employed. In some embodiments, oral administration is employed. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein can be administered to a target by intratumor injection, peritumor injection, peritumor injection, intralesional injection, and / or injection into tumor inflow area lymph nodes, or by essentially any tumor-targeted injection where leakage into primary lymph nodes adjacent to the solid tumor targeted by the antitumor agent is expected. In some embodiments, the antibodies provided herein can be delivered locally to the tumor by known methods, including, but not limited to, hepatic or aortic pumps; limb, lung, or hepatic perfusion; in the portal vein; through venous shunts; or in cavities or veins near the tumor. In other embodiments, the antibodies provided herein can be administered systemically. In preferred embodiments, the antibodies are administered locally to the site of the tumor. The antibodies can also be administered intratumor, for example, by direct injection of cells into the site of the tumor and / or the tumor vascular system. For example, in cases of malignant pleural disease, mesothelioma, or lung cancer, administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6(261):261ra151(2014)). Those skilled in the art can select a suitable mode of administration based on the type of cancer to be treated and / or the location of the tumor.Antibodies can be introduced by injection or via catheter. In one embodiment, the antibody is administered pleurally to the target subject using, for example, an intrapleural catheter.

[0211] The cancers or tumors to be treated with the anti-CTLA4 / CD47 bispecific antibodies or pharmaceutical compositions provided herein include those that typically respond to immunotherapy and those that do not typically respond to immunotherapy. In some embodiments, the cancers have a high degree of microsatellite instability. In some embodiments, the cancers are metastatic, refractory, or recurrent cancers.

[0212] In some embodiments, the cancer or tumor that can be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is a hematological cancer. In some embodiments, the cancer or tumor that can be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is a solid tumor. In some embodiments, the solid tumor that will be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is melanoma. In some embodiments, the solid tumor that will be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is colon cancer. Accordingly, in some embodiments, what is provided herein is a method for treating melanoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, what is provided herein is the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of melanoma. In some embodiments, provided herein is the use of the anti-CTLA4 / CD47 bispecific antibody provided herein for the preparation of a pharmaceutical for the treatment of melanoma. In some embodiments, the melanoma is CTLA4-positive. In some embodiments, the melanoma is CD47-positive. In some embodiments, the melanoma is both CTLA4-positive and CD47-positive. In some embodiments, provided herein is a method for treating colon cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, provided herein is the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of colon cancer. In some embodiments, provided herein is the use of the anti-CTLA4 / CD47 bispecific antibody provided herein for the preparation of a pharmaceutical for the treatment of colon cancer. In some embodiments, the colon cancer is CTLA4-positive. In some embodiments, the colon cancer is CD47-positive.In some embodiments, colon cancer is both CTLA4-positive and CD47-positive.

[0213] In cancer treatment, while the elimination of target cancer cells or tumor cells may occur, any clinical improvement is also beneficial. Antitumor effects may be manifested by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an extension of life expectancy, or improvement of various physiological symptoms associated with the cancerous state. Antitumor effects may also be manifested by the ability of the antibodies or pharmaceutical compositions provided herein to prevent the development of tumors in the first place. In some embodiments, “antitumor effect” may be manifested by a reduction in cancer-induced immunosuppression. Clinical improvements include a reduction in the risk or rate of cancer or tumor progression or a reduction in pathological outcomes. It is also understood that a method of treating cancer may include any effect of improving signs or symptoms associated with cancer. Such signs or symptoms include, but are not limited to, reducing the tumor load, including inhibiting tumor growth, slowing the rate of tumor growth, reducing tumor size, reducing the number of tumors, and removing tumors, all of which can be measured using routine tumor imaging techniques well known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

[0214] In some embodiments, the methods or uses provided herein can reduce tumor burden. Therefore, administration of the anti-CTLA4 / CD47 bispecific antibodies or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, decrease tumor size, and / or eradicate tumors in a subject. Methods for monitoring patient responses to the administration of the pharmaceutical compositions disclosed herein are known in the art and can be utilized according to the methods disclosed herein.

[0215] In some embodiments, an antitumor effect is observed in patients with tumors or cancer who are administered the anti-CTLA4 / CD47 bispecific antibody described herein as a monotherapy, i.e., without combination with another therapeutic agent. In some embodiments, tumor burden is reduced in patients with tumors or cancer who are administered the anti-CTLA4 / CD47 bispecific antibody described herein as a monotherapy, i.e., without combination with another therapeutic agent.

[0216] In the methods disclosed herein, a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is administered to a subject in need of cancer treatment. The subject may be a mammal. In some embodiments, the subject is human. In some embodiments, these individuals do not have clinically measurable cancer. However, these individuals are suspected to be at risk of disease progression near the original tumor site or due to metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before and after initial treatment. These features are known in the clinical field and are suitably defined for various types of cancer. Features specific to the high-risk subgroup include tumor invasion into adjacent tissue or lymph node involvement.

[0217] The anti-CTLA4 / CD47 bispecific antibodies or pharmaceutical compositions provided herein can be administered using medical devices known in the art. For example, in some embodiments, needleless subcutaneous injection devices, such as those disclosed in U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556, can be used. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233 disclosing a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing a variable-flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing a permeable drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196 disclosing a permeable drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0218] In some embodiments, anti-CTLA4 / CD47 bispecific antibodies are administered to patients with cancer who have shown an inadequate response to prior treatment, such as prior treatment with an immunotumor agent or immunotherapy agent, or whose cancer has progressed after such prior treatment, or who are refractory or resistant, essentially refractory or resistant, or who acquire a refractory or refractory state. For example, subjects who do not respond or do not respond adequately to initial therapy, or who experience disease progression after treatment, can be treated with anti-CTLA4 / CD47 bispecific antibodies, either alone or in combination with another therapy.

[0219] In some embodiments, anti-CTLA4 / CD47 bispecific antibodies are administered to patients who have not previously received (i.e., have not been treated with) immuno-oncological agents, such as PD-1 pathway antagonists or PD-L1 pathway antagonists. A method of treating a subject with cancer with anti-CTLA4 / CD47 bispecific antibodies may include administering a therapeutically effective dose of anti-CTLA4 / CD47 bispecific antibodies to a subject having cancer cells or TIL cells expressing CTLA4 and / or CD47.

[0220] Anti-CTLA4 / CD47 bispecific antibodies can be administered in conjunction with standard therapy. Anti-CTLA4 / CD47 bispecific antibodies can also be administered as maintenance therapy, for example, therapy intended to prevent tumor development or recurrence. Anti-CTLA4 / CD47 bispecific antibodies can also be administered in conjunction with other therapies, such as radiation, surgery, or chemotherapy. For example, adjuvant therapy with anti-CTLA4 / CD47 bispecific antibodies may be administered when there is a risk of micrometastasis and / or to reduce the risk of recurrence.

[0221] Anti-CTLA4 / CD47 bispecific antibodies can be administered as monotherapy or as sole immunostimulatory therapy. Anti-CTLA4 / CD47 bispecific antibodies can also be combined with immunogens, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides, such as gp100, MAGE antigen, Trp-2, MARTI, and / or tyrosinase peptides, or tumor cells transfected to express the cytokine GM-CSF.

[0222] Combination therapies using drugs with different mechanisms of action can produce additive or synergistic effects. Combination therapies allow for lower doses of each drug than those used in monotherapy, thereby reducing the toxic side effects of the drugs disclosed herein and / or increasing their therapeutic index. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. In some embodiments, the additional therapy results in an increase in the therapeutic index of the antibody or pharmaceutical composition described herein. In some embodiments, the additional therapy results in a reduction in the toxicity and / or side effects of the antibody or pharmaceutical composition described herein. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition described herein can be administered in combination with an additional therapy. In some embodiments, the additional therapy may be surgical resection, radiotherapy, or chemotherapy.

[0223] Add-on therapy may be administered before, concurrently with, or after the administration of the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition described herein. Combination administration may include co-administration in a single pharmaceutical formulation or using separate formulations, or in either order, but usually within a time frame that allows all activators to exert their biological activity simultaneously. Those skilled in the art can readily determine, based on the requirements of the subject being treated, an appropriate regimen for combining the pharmaceutical compositions and add-on therapies described herein, including the timing and administration of any additional agents to be used in combination therapy.

[0224] (6.7 Exemplary Embodiments) Embodiment 1. (i) A bispecific antibody comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) (wherein the VL / VH pair specifically binds to human CTLA4, and wherein the VL comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively); and (ii) a CD47-binding domain comprising the extracellular domain of SIRPα or a variant thereof.

[0225] Embodiment 2. The bispecific antibody according to Embodiment 1, wherein VL has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 7, and VH has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 8.

[0226] Embodiment 3. The bispecific antibody according to Embodiment 2, wherein VL and VH each have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0227] Embodiment 4. A bispecific antibody according to any one of Embodiments 1 to 3, wherein the CD47-binding domain comprises the extracellular domain of human SIRPα variant 2 or a variant thereof.

[0228] Embodiment 5. The bispecific antibody according to Embodiment 4, wherein the CD47-binding domain has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence-identical to SEQ ID NO: 9.

[0229] Embodiment 6. The bispecific antibody according to Embodiment 5, wherein the CD47-binding domain has the amino acid sequence of SEQ ID NO: 9.

[0230] Embodiment 7. A bispecific antibody according to any one of Embodiments 1 to 6, comprising: (1) a first peptide chain (C1) comprising the VL and light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising the VH, heavy chain constant domain 1 (CH1), and knob-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising the CD47 binding domain and hole-Fc region from the N-terminus to the C-terminus.

[0231] Embodiment 8. The bispecific antibody according to Embodiment 7, wherein the CD47-binding domain and the hole-Fc region are directly linked without a linker.

[0232] Embodiment 9. A bispecific antibody according to any one of Embodiments 1 to 6, comprising: (1) a first peptide chain (C1) comprising a VL and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising a VH, a heavy chain constant domain 1 (CH1), and a hole-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising the CD47 binding domain and the knob-Fc region from the N-terminus to the C-terminus.

[0233] Embodiment 10. The bispecific antibody according to Embodiment 9, wherein the CD47-binding domain and the knob-Fc region are directly linked without a linker.

[0234] Embodiment 11. A bispecific antibody according to any one of Embodiments 7 to 10, wherein the knob-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including a T366W substitution; and the hole-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including a T366S, L368A, Y407V substitution.

[0235] Embodiment 12. The bispecific antibody according to Embodiment 11, wherein the knob-Fc region further comprises an S354C substitution, and the hole-Fc region further comprises a Y349C substitution.

[0236] Embodiment 13. The bispecific antibody according to Embodiment 11, wherein the knob-Fc region further comprises a Y349C substitution, and the hole-Fc region further comprises an S354C substitution.

[0237] Embodiment 14. A bispecific antibody according to any one of Embodiments 11 to 13, wherein the knob-Fc region further comprises E357K and D399K substitutions, and the hole-region further comprises K370E and K409D substitutions.

[0238] Embodiment 15. A bispecific antibody according to any one of Embodiments 11 to 13, wherein the knob-Fc region further comprises K370E and K409D substitutions, and the hole-region further comprises E357K and D399K substitutions.

[0239] Embodiment 16. A bispecific antibody according to any one of Embodiments 7 to 10, wherein the CL region is kappa CL (Cκ; SEQ ID NO: 21) or lambda CL (Cλ; SEQ ID NO: 22), or a variant thereof having up to 10 amino acid substitutions; (ii) the CH1 domain is human IgG1 CH1 domain (SEQ ID NO: 41) or a variant thereof having up to 10 amino acid substitutions; and / or (iii) the knob-Fc region and the hole-Fc region are (1) the amino acid sequences of SEQ ID NOs: 31 and 35, respectively; (2) SEQ ID NOs: 32 and 36, respectively; (3) SEQ ID NOs: 33 and 37, respectively; or (4) the amino acid sequences of SEQ ID NOs: 34 and 38, respectively; or a variant thereof having up to 10 amino acid substitutions.

[0240] Embodiment 17. The bispecific antibody according to Embodiment 16, wherein the CL region, CH1 domain, knob-Fc region, and hole-Fc region each have the amino acid sequences of (1) SEQ ID NOs. 21, 41, 31, and 35; (2) SEQ ID NOs. 21, 41, 32, and 36; (3) SEQ ID NOs. 21, 41, 33, and 37; or (4) SEQ ID NOs. 21, 41, 34, and 38.

[0241] Embodiment 18. A bispecific antibody according to Embodiment 7, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 51; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 52; and C3 has an amino acid sequence that is at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 53.

[0242] Embodiment 19. The bispecific antibody according to Embodiment 18, wherein C1, C2, and C3 each have the amino acid sequences of SEQ ID NOs. 51, 52, and 53, respectively.

[0243] Embodiment 20. The bispecific antibody according to any one of Embodiments 1 to 19, wherein the bispecific antibody (1) has high binding affinity to CTLA4 and CD47 bipositive cells; (2) depletes tumor-infiltrating lymphocytes (TILs) regulatory T cells (Treg cells) or Treg cells in the tumor microenvironment (TME); (3) increases cytokine levels in the TME; or (4) enhances T cell proliferation and / or activity against cancer; (5) enhances macrophage-mediated phagocytosis; (6) enhances dendritic cell-mediated antigen presentation; or any combination of (1) to (6).

[0244] Embodiment 21. The bispecific antibody according to any one of Embodiments 1 to 20, wherein the bispecific antibody (1) has a higher affinity for CTLA4 and CD47 bipositive cells than for CTLA4 or CD47 monopositive cells; (2) selectively removes CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematological toxicity; or (4) has limited immune-related adverse events (irAEs); or any combination of (1) to (4).

[0245] Embodiment 22. The bispecific compound according to any one of Embodiments 1 to 21, wherein the bispecific antibody has a limited number of mispairing impurities.

[0246] Embodiment 23. A composition comprising the bispecific antibody according to any one of Embodiments 1 to 22, wherein the purity of the bispecific antibody is at least 95%, and the purity is measured by size exclusion chromatography (SEC) or non-reducing SDS-PAGE.

[0247] Embodiment 24. A pharmaceutical composition comprising a therapeutically effective amount of a bispecific antibody according to any one of Embodiments 1 to 22 and a pharmaceutically acceptable carrier.

[0248] Embodiment 25. A polynucleotide encoding the peptide chain of a bispecific antibody according to any one of Embodiments 1 to 22.

[0249] Embodiment 26. The polynucleotide according to Embodiment 25, which encodes all of the peptide chains of the bispecific antibody.

[0250] Embodiment 27. A plurality of polynucleotides according to Embodiment 25, which collectively encode all the peptide chains of the bispecific antibody.

[0251] Embodiment 28. A vector comprising the polynucleotide described in Embodiment 25 or 26.

[0252] Embodiment 29. A cell comprising a polynucleotide or a plurality of polynucleotides as described in any one of Embodiments 25 to 27, or a vector as described in any one of Embodiment 28.

[0253] Embodiment 30. A method for producing a bispecific antibody that specifically binds to human CTLA4 and human CD47, comprising culturing the cells described in Embodiment 29 under conditions that enable the expression of the bispecific antibody.

[0254] Embodiment 31. A method for treating cancer in a subject that requires the treatment thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of Embodiments 1 to 22.

[0255] Embodiment 32. The method of Embodiment 31, wherein the subject is a human.

[0256] Embodiment 33. Use of the bispecific antibody according to any one of Embodiments 1 to 22, as a medicament.

[0257] Embodiment 34. Use of the bispecific antibody according to any one of Embodiments 1 to 22, in the treatment of cancer.

[0258] Embodiment 35. Use of the bispecific antibody according to any one of Embodiments 1 to 22, for the preparation of a medicament for treating cancer.

[0259] (6.8 Experiment) The examples provided below are for illustrative purposes only, and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0260] Briefly described, the data below show that the anti-CTLA4 / CD47 antibody HX044, which has a 1+1 asymmetric structure composed of an anti-CTLA4 VL / VH pair and a CD47-binding SIRPα domain, binds to respective targets with reduced affinity compared to the reference antibody ipilimumab and the reference protein SIRPα-Fc, and blocks ligand binding to the targets. Nevertheless, HX044 was found to bind to cells with high expression of CTLA4 and CD47 with particularly enhanced affinity and avidity. Consistent with this, HX044 has low binding to peripheral T cells that do not express CTLA4 and CD47 at high levels (e.g., CD4, CD8, and T reg ) and almost no binding to RBCs, but was found to have high binding to TIL-T that express CTLA4 at high levels reg .

[0261] HX044 also exhibited strong ADCC activity against cells with high CTLA4 expression levels. In a humanized syngeneic model, HX044 decreased TIL-T reg and also demonstrated potent anti-tumor activity against hCD47-MC38 tumors at very low doses (<0.2 mg / kg) and against huB16F10 melanoma tumors (traditionally cold tumors) at 5 mg / kg. Toxicity evaluation indicated limited hematological toxicity of HX044 when administered at high dose levels (~10 mg / kg), while no such toxicity was observed at low dose levels.

[0262] (6.8.1 Example 1: Construction of HX044) Bispecific antibodies (BsAbs) that bind to human CTLA4 and CD47 were prepared as follows. Recombinant expression of the three peptide chains (C1, C2, and C3; see Table 5B) of the bispecific antibody HX044 was achieved using a three-vector system encoding the three polypeptide chains. The coding genes of the three peptide chains were cloned separately into vectors. The BsAb expression vector was prepared using an endotoxin-free plasmid DNA purification method (EndoFree Plasmid Kit, TransGen Biotech). After preparing the DNA vectors, HEK293 suspension cells were used for transient expression. DNA and PEI solutions were combined, and the mixture was added to the cells. Six days after transfection, HEK293 cells were harvested by centrifugation. Subsequently, the target molecule was purified by Protein A affinity chromatography.

[0263] The BsAb targeting human CTLA4 and CD47 (HX044) adopts a knob-in-hole configuration (Figure 1A). Specifically, HX044 has an asymmetric "1+1" structure composed of a CTLA4-binding domain and a SIRPα (CD47-binding) domain. The first heavy chain constant region contains a knob-Fc region comprising the amino acid mutation T366W, and the second heavy chain constant region contains a hole-Fc region comprising the amino acid mutations T366S, L368A, and Y407V.

[0264] Different morphologies of BsAb, including those shown in Figures 1B (Sirpα-IgG), 1C (scFv-Sirpα, KIH), and 1D (IgG-Sirpα), were also prepared and tested. Surprisingly, only the morphology shown in Figure 1A (Fab-Sirpα, KIH) exhibited the desired properties, namely strong antitumor activity, at low dose levels that did not cause hematological toxicity or irAEs.

[0265] (6.8.2 Example 2: Ligand binding of HX044) 1. CD47 antigen binding by ELISA: A 96-well polyvinyl microtiter plate was coated overnight at 2–8°C with 100 μL of 0.5 μg / mL CD47-his in carbonate-bicarbonate buffer (200 mM, pH 9.4). The plate was washed three times with PBS-T buffer, and the plate coated with the antigen (CD47-his) was blocked in 1% BSA-PBS at 37°C for 60 minutes. The plate was washed four times with PBS-T buffer and incubated at 37°C for 1 hour with 100 μL / well of a 5-fold serial dilution of BsAb(HX044) provided herein (approximately 20 nM–0.000256 nM). The plates were then washed five times with PBS-T and incubated at 37°C for 1 hour with 100 μL of HRP-goat anti-human IgG (H+L) (minimal cross-reactivity) (10000X) in sample dilution buffer (1‰BSA in PBS). The washed plates were then incubated at 37°C for 10 minutes with 100 μL / well of substrate solution. The reaction was stopped by adding 50 μL / well of 2M sulfuric acid. Absorbance at 450 nm was recorded using a microplate reader.

[0266] When measured by ELISA assay, HX044 bound to recombinant human CD47 protein at an EC50 of 8.12 nM, while SIRPα-Fc bound to the same protein at an EC50 of approximately 0.05 nM. [Table 7]

[0267] 2. CTLA4 antigen-binding ELISA: 96-well polyvinyl microtiter plates were coated overnight at 2–8°C with 100 μL of 0.5 μg / mL CTLA4-his in carbonate-bicarbonate buffer (200 mM, pH 9.4). The plates were washed three times with PBS-T buffer, and the plates coated with antigen (CTLA4-his) were blocked in 1% BSA-PBS at 37°C for 60 minutes. The plates were washed four times with PBS-T buffer and incubated at 37°C for 1 hour with 100 μL / well of the reference antibody ipilimumab and a 5-fold serial dilution of BsAb(HX044) provided herein (approximately 20 nM–0.000256 nM). The plates were then washed five times with PBS-T and incubated at 37°C for 1 hour with 100 μL of HRP-goat anti-human IgG (H+L) (minimal cross-reactivity) (10000X) in sample dilution buffer (1‰BSA in PBS). The washed plates were then incubated at 37°C for 10 minutes with 100 μL / well of substrate solution. The reaction was stopped by adding 50 μL / well of 2M sulfuric acid. Absorbance at 450 nm was recorded using a microplate reader.

[0268] When measured by ELISA assay, HX044 bound to recombinant human CTLA4 receptor protein with an EC50 of 0.77 nM, showing significantly lower affinity for CTLA4 compared to ipilimumab (EC50: 0.01 nM). [Table 8]

[0269] 3. CD47 Ligand Blockade ELISA: A 96-well polyvinyl microtiter plate was coated overnight at 2–8°C with 100 μL of 0.5 μg / mL CD47-his in carbonate-bicarbonate buffer (200 mM, pH 9.4). The plate was washed three times with PBS-T buffer, and the plate coated with the antigen (CD47-his) was blocked with 1% BSA-PBS at 37°C for 60 minutes. The plate was washed four times with PBS-T buffer and incubated at 37°C for 1 hour with 50 μL / well of BsAb(HX044) provided herein and 50 μL / well of a 5-fold serial dilution of 0.2 μg / mL SIRPα-mFc (approximately 40 nM–0.000512 nM). The plates were then washed five times with PBS-T and incubated with 100 μL of HRP-goat anti-mouse (5000X) in sample dilution buffer (1‰BSA in PBS) at 37°C for 1 hour. The washed plates were then incubated with 100 μL / well of substrate solution at 37°C for 10 minutes. The reaction was stopped by adding 50 μL / well of 2M sulfuric acid. Absorbance at 450 nm was recorded using a microplate reader.

[0270] HX044 competitively blocked CD47 binding to recombinant SIRPα with an IC50 of ~0.4 nM. [Table 9]

[0271] 4. CTLA4 Ligand Blockade ELISA: 96-well polyvinyl microtiter plates were coated overnight at 2–8°C with 100 μL of 0.5 μg / mL CTLA4-his in carbonate-bicarbonate buffer (200 mM, pH 9.4). The plates were washed three times with PBS-T buffer, and the plates coated with the antigen (CTLA4-his) were blocked with 1% BSA-PBS at 37°C for 60 minutes. The plates were washed four times with PBS-T buffer and incubated at 37°C for 1 hour with 50 μL / well of a 5-fold serial dilution of the reference antibody (ipilimumab) (approximately 40 nM to 0.000512 nM), a 5-fold serial dilution of the BsAb (HX044) provided herein (approximately 1000 nM to 0.0128 nM), and 50 μL / well of 0.2 μg / mL B7-1 / CD80-mFc. The plates were then washed five times with PBS-T buffer and incubated at 37°C for 1 hour with 100 μL of HRP-goat anti-mouse (5000X) in sample dilution buffer (1‰ BSA in PBS). The washed plates were then incubated at 37°C for 10 minutes with 100 μL / well of substrate solution. The reaction was stopped by adding 50 μL / well of 2 M sulfuric acid. The absorbance at 450 nm was recorded using a microplate reader.

[0272] HX044 competitively blocked CTLA4 binding to its ligand CD80 with an IC50 of 8.8 nM. [Table 10]

[0273] 5. HX044 binding to CD47-positive Jurkat cells: Jurkat cells endogenously express human CD47. Jurkat cells were incubated with titrated doses of reference antibody proteins (ipilimumab and SIRPα-Fc) and serial dilutions (from about 300 nM to 0.00012 nM) of the bsAb (HX044) provided herein at 4°C for 1 hour. Bound antibodies were detected with Alexa Fluor® 647 (AF647)-conjugated AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. EC50 values were calculated from the best-fit binding curve using GraphPad Prism software.

[0274] As shown in Figure 2, HX044 bound to Jurkat cells with an EC50 of 1024 nM, and the affinity for CD47 was markedly lower compared to the affinity of SIRPα-Fc (EC50: 311.2 nM). Table 11

[0275] 6. HX044 binding to CHO-K1-hCTLA4 cells: CHO-K1-CTLA4 cells modified to express human CTLA4. These cells were confirmed to specifically express human CTLA4, rather than human CD47. Three monoclones with similar hCTLA4 expression (CTLA4-4C4, CTLA4-8F8, and CTLA4-7C11) were incubated at 4°C for 1 hour with titration-dose reference antibodies / proteins (ipilimumab and SIRPα-Fc) and serial dilutions (approximately 300 nM to 0.00012 nM) of bsAb(HX044) provided herein. The bound antibody was detected using Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. The EC50 value was calculated from the best-fit binding curve using GraphPad prism software.

[0276] As shown in Figure 3, all three clones exhibited similar binding characteristics. While SIRPα-Fc did not bind to any of the clones, HX044 bound to all three clones with an EC50 of 11.28–14.90 nM and showed lower affinity for hCTLA4 compared to ipilimumab, which has an EC50 of 1.168–3.035 nM. [Table 12]

[0277] 7. HX044 conjugation to 293T-hCTLA4 cells: 293T cells and four 293T-hCTLA4 cell clones modified to stably express increased levels of human CTLA4 (marked from - to ++++, see table below). All five cell lines were CD47-positive and expressed membrane human CD47 at similar levels. These cells were incubated at 4°C for 1 hour with titration doses of reference antibody / protein (ipilimumab and SIRPα-Fc) and serial dilutions (approximately 300 nM to 0.00012 nM) of BsAb(HX044) provided herein. The conjugated antibodies were detected with Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG(H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. The EC50 value was calculated from the best-fit coupling curve using GraphPad prism software. [Table 13]

[0278] As shown in Figure 4, when CTLA4 expression was negative or low, HX044 bound to cells weaker than SIRPα-Fc. With increasing CTLA4 expression, HX044 bound to CTLA4+ / CD47+ bipositive cells with progressively enhanced affinity (EC50 of 5–35 nM), and with significantly stronger binding affinity than ipilimumab and SIRPα-Fc when CTLA4 was expressed at high levels (Figure 4). These results indicate that HX044 can bind strongly to cells highly expressing both targets and relatively weakly to cells that do not express CTLA4 or express low levels of CTLA4.

[0279] 8. CD4+ / CD8+ T or regulatory T(T regHX044 binding to cells: PBMCs were isolated from healthy donors using Ficol density gradient separation (GE) and incubated with 200 nM reference antibodies / proteins (ipilimumab, maglorimab, and SIRPα-Fc) and the BsAb (HX044) provided herein at 4°C for 1 hour. The bound antibodies were detected with Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003). Subsequently, PBMCs were stained with anti-human CD3-BV-786 (BD), anti-human CD4-BV510 (BD), anti-human CD25-BV421 (BD), and anti-human CD8 (BD) at 4°C for 30 minutes. For intracellular staining, PBMCs were fixed, permeabilized with Fix / Perm buffer at 4°C for 30 minutes, and then stained with anti-human FoxP3 (1:10) at 4°C for 45 minutes. The explanation followed cell detection by flow cytometry.

[0280] As shown in Figure 5 (top panel and bottom left panel), both HX044 and SIRPα-Fc express isolated human peripheral CD4+ lymphocytes, CD8+ lymphocytes, and T1A4, which express CTLA4 and CD47 at low levels. reg It bound to cells. At the same concentration, HX044 showed reduced binding to such cells compared to maglorimab (Phase-III anti-CD47 antibody). HX044 also showed stronger binding to T lymphocytes than ipilimumab.

[0281] 9. RBC cell binding assay: Red blood cells (RBCs) isolated from healthy donors were incubated at 4°C for 1 hour with titration doses of reference antibody / protein (ipilimumab, maglorimab, and SIRPα-Fc) and serial dilutions (approximately 1500 nM to 1.2 nM) of bsAb (HX044) provided herein. The bound antibodies were detected with Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. The data were calculated from the best-fit binding curve using GraphPad Prism software.

[0282] HX044 showed little binding to RBCs compared to the reference antibody maglorimab (Figure 6).

[0283] 10. Platelet binding assay: Platelets were isolated from healthy donors using Ficol density gradient separation (GE) and incubated with 200 nM reference antibodies / proteins (ipilimumab, maglorimab, and SIRPα-Fc) and BsAb (HX044) provided herein at 4°C for 1 hour. The bound antibodies were detected with Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003). Subsequently, platelets were stained with anti-human CD45-BV605 and anti-human CD41-BV421 at 4°C for 30 minutes. Platelets were gated with CD45-CD41+.

[0284] As shown in Figure 5 (bottom right panel), both HX044 and SIRPα-Fc bound to isolated human platelets expressing human CD47. At the same concentration, HX044 showed reduced binding to such cells compared to SIRPα-Fc or maglorimab.

[0285] In summary, these data showed that HX044 could bind to membrane CD47 and CTLA4, but with lower affinities compared to SIRPα-Fc and ipilimumab, respectively. Nevertheless, HX044 bound with high affinity and with higher binding affinity than ipilimumab to CD47+ / CTLA4+ cells with high CTLA4 expression. Furthermore, HX044 showed significantly weaker binding affinity to human RBCs, platelets, and T lymphocytes compared to SIRPα and maglorimab, supporting its reduced hematological toxicity. HX044 also showed stronger binding to T lymphocytes compared to ipilimumab, further supporting its enhanced potency in activated T cells.

[0286] (6.8.3 Example 3: Fc activity of HX044) 1. HX044 binding to 293T-FcRn cells: 293T-FcRn cells modified to express human FCGRT-β2M were incubated with titration doses of reference antibody / protein (ipilimumab and SIRPα-Fc) and 5-fold serial dilutions (approximately 300 nM to 0.00012 nM) of bsAb(HX044) provided herein at pH 6.0 and 4°C for 1 hour. The bound antibody was detected with Alexa Fluor® 647 (AF647) conjugated AffiniPure goat anti-human IgG(H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. EC50 values ​​were calculated from the best-fit binding curve using a GraphPad prism.

[0287] As shown in Figure 7, HX044 exhibits FcRn binding comparable to ipilimumab at pH 6.0, demonstrating similar self-recyclability in serum and similar pharmacokinetic properties.

[0288] 2. ADCC assay: 7.5 × 10⁶ cells modified to express human FCγRIII and NFAT factor-driven luciferase. 4Three types of Jurkat-NFAT-CD16A cells were modified to express different levels of human CTLA4, resulting in three distinct sets of 1.25 × 10¹⁶ cells. 4 293T / 293T-CTLA4 cells (25 μL) were plated with 293T cells in a 96-well solid white flat-bottom polystyrene TC-treated microplate. The cells were then incubated at 37°C in 5% CO2 with titration doses of reference antibody / protein (ipilimumab and SIRPα-Fc) and serial dilutions (approximately 60 nM to 0.00012 nM) of bsAb (HX044) provided herein. After 18 hours, 100 μL / well of Stable-Lite® luciferase assay reagent (Vazyme-DD1202-02) was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. EC50 values ​​were calculated from the best-fit coupling curve using GraphPad prism software.

[0289] As shown in Figure 8, in CD47+ / CTLA4- 293T cells, HX044 showed slightly stronger ADCC compared to SIRPα-Fc. In CD47+ / CTLA4+ cells, HX044 showed significantly greater ADCC than ipilimumab and SIRPα-Fc, and the ADCC activity of HX044 appeared to be dependent on the expression level of CTLA4 (Figure 8).

[0290] (6.8.4 Example 4: Antitumor activity of HX044) 1. In vivo MC-38-hCD47 humanized syngeneic model (HuGeMM): The hCD47 gene was knocked into the mouse colon cancer cell line MC-38 (KI), and the tumor cell line was inoculated into hCTLA4×hCD47×hSIRPα HuGeMM mice to construct the humanized syngeneic mouse model MC38-hCD47 HuGeMM. Mice were randomly divided into groups, and tumor size was ~70mm. 3Treatment was administered when the tumor volume reached a certain level. Different groups of mice were separately administered low, medium, or high doses of HX044 or reference antibodies / proteins (ipilimumab and SIRPα-Fc). Treatment groups included HX044 (0.132, 1.98, and 6.6 mg / kg IP twice weekly, respectively), SIRPα-Fc (0.092, 1.38, and 4.6 mg / kg IP twice weekly, respectively), ipilimumab analogs (0.172, 2.58, and 8.6 mg / kg IP twice weekly, respectively), and vehicle (PBS). All treatments were molecularly equivalent. Tumor volume was 2000 mm², with an average tumor volume of 2000 mm². 3 Evaluations were performed twice a week until the target was reached. Tumor growth inhibition (TGI) was calculated as TGI% = (1 - V treatment / V control) × 100.

[0291] As shown in Figure 9, strong antitumor activity of HX044 was observed in hCTLA4×hCD47×hSIRPα C57 / B6-based HuGEMM mice (MC38-hCD47 model), which was significantly higher than the antitumor activity of either SIRPα-Fc or ipilimumab, particularly at low dose levels (<0.2 mg / kg).

[0292] 2. In vivo B16F10-hCD47 HuGeMM syngeneic model: The hCD47 gene was knocked into the mouse melanoma cell line B16F10 (KI), and the tumor cell line was seeded into hCTLA4×hCD47×hSIRPα HuGeMM mice to construct the humanized syngeneic mouse model B16F10-hCD47 HuGEMM. Mice were randomly divided into groups, and tumor size was ~70mm. 3 Treatment was administered when the tumor volume reached a certain level. The treatment groups included HX044 (two doses of 3.3 mg / kg IP followed by four doses of 6.6 mg / kg), SIRPα-Fc (two doses of 2.3 mg / kg IP followed by four doses of 4.6 mg / kg), ipilimumab analog (two doses of 4.3 mg / kg IP followed by four doses of 8.6 mg / kg), and vehicle (PBS). All treatments were molecularly equivalent. The mean tumor volume was 2500 mm³. 3The mice were evaluated twice a week until the target was reached. Tumor growth inhibition (TGI) was calculated as TGI% = (1 - V treatment / V control) × 100. At the end of this experiment, tumors and spleens were removed from three mice per group, and immune cells were analyzed using flow cytometry. reg Cells were sorted as CD3+CD4+Foxp3+ cells, while helper T cells were sorted as CD3+CD4+Foxp3-, and cytotoxic T cells were sorted as CD3+CD8+Foxp3-.

[0293] As shown in Figure 10, HX044 also demonstrated significantly higher antitumor activity (5 mg / kg, then 10 mg / kg) than either ipilimumab or SIRPα-Fc in the B16F10-hCD47 melanoma model, which is traditionally a "cold" tumor.

[0294] Furthermore, as shown in Figure 11, the increase in TIL-T cells and TIL-T reg A decrease in TIL-T was also observed in tumors treated with HX044, and this increase was significantly greater than the increases in the ipilimumab-treated or SIRPα-Fc-treated groups. This observation is related to TIL-T reg This reduction also demonstrated that it contributes to the antitumor activity of HX044.

[0295] (6.8.5 Example 5: Hematological toxicity of HX044) To evaluate the hematological toxicity of HX044, hCTLA4×hCD47×hSIRPα HuGeMM C57BL / 6J mice were randomly divided into groups according to body weight and treated with either HX044 (9.2 mg / kg IP twice weekly), SIRPα-Fc (6.4 mg / kg IP twice weekly), ipilimumab analog (12 mg / kg IP twice weekly), or vehicle (PBS) for 2 weeks (5 doses). Group assignment was set as day 0. Blood cell analysis was performed on days 4 and 11. All blood samples were collected at the end of administration (day 15) and analyzed for lymphocyte composition. regCells were sorted as CD3+CD4+Foxp3+ cells; helper T cells were sorted as CD45+CD4+Foxp3-; and cytotoxic T cells were sorted as CD45+CD8+Foxp3-.

[0296] As shown, in non-tumor-bearing HuGEMM mice treated with high doses, minimal toxicity to peripheral T cells was observed (Figure 12), minimal adverse effects on lymphocyte composition, and therefore minimal hematological toxicity.

[0297] All in vivo mouse experiments were conducted under sterile conditions at the Crown Bioscience SPF facility, strictly adhering to the National Institutes of Health's guidelines for the management and use of laboratory animals. The protocols were approved by the Crown Bioscience IACUC Committee. All study designs followed the ARRIVE guidelines.

[0298] (6.8.6 Example 6: Production efficiency and stability of HX044) HX044 was prepared and analyzed with reference bispecific antibodies ("reference bsAb") targeting CTLA4 and CD47, each possessing an ECD of hSirpα variant 1 as the CD47-binding domain and a flexible GS linker between the CD47-binding domain and the Fc domain. The reference bsAb consists of three peptides, each having the amino acid sequences of SEQ ID NOs. 54, 55, and 56.

[0299] Specifically, HX044 and the reference bsAb were prepared as follows: Recombinant expression of the three peptide chains of the bispecific antibody HX044 and the reference bsAb was achieved using three vector systems encoding the three polypeptide chains. The encoding genes for the three peptide chains were cloned separately into the vectors. The BsAb expression vector was prepared using an endotoxin-free plasmid DNA purification method (EndoFree Plasmid kit, TransGen Biotech). After preparing the DNA vector, CHO suspension cells were used for transient expression. The DNA and PEI solution were combined and the mixture was added to the cells. Six days after transfection, the CHO cells were harvested by centrifugation. Subsequently, the target molecule was purified by protein A affinity chromatography.

[0300] Both HX044 and the reference bsAb were subjected to stability analysis. Specifically, their purity was measured using SEC chromatography and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For SEC chromatography, an LC-20AT chromatography column was used under the following experimental conditions: [Table 14]

[0301] Deionized water was used as the liquid phase, and the flow rate was slowly increased to 1.0 ml / min to a stable baseline. The sample immersion time was set to 15 minutes. The data was analyzed and saved. The liquid phase was replaced with deionized water and washed for 1 hour.

[0302] SDS-PAGE was performed as follows: (1) the samples were denatured under reducing and non-reducing conditions; (2) electrophoresis was performed at 180V for 40 minutes; (3) staining and unbound dyes were washed for 30 minutes; and (4) photographs were taken.

[0303] As shown in Figures 13A-13B (SEC) and 14A-14B (SDS-PAGE), HX044 exhibited superior heterodimerization compared to the reference bsAb. Specifically, HX044 had higher purity (96.031% at 214 nM and 96.501% at 280 nM, measured by SEC) than the reference bsAb (93.985% at 214 nM, 96.501% at 280 nM, and 94.883% at 280 nm, measured by SEC) (Figures 13A-13B). When measured by SDS-PAGE, mispairing byproducts were observed for the reference bsAb, but not for HX044 (Figures 14A-14B). As shown in Figure 14A, HX044 appeared as a single clean band on the non-reducing gel and as three clean bands (C1, C2, and C3) on the reducing gel. In contrast, as shown in Figure 14B, for the reference bsAb, bands with higher or lower molecular weights were observed on the non-reducing gel, which corresponded to homodimer byproducts such as CTLA4-CTLA4 mAb (estimated MW 145 kD) and Sirpα-Fc homodimer (estimated MW 78.5 kD), as well as inaccurate heterodimers without light chains (estimated 90-100 kD).

[0304] The expression levels of both HX044 and the reference bsAb were also measured, and HX044 showed higher expression (423.15 mg) than the reference bsAb (381.76 mg).

[0305] The results are summarized in the table below: [Table 15]

[0306] Due to their asymmetric structure, the expression of the two bispecific antibodies (HX044 and reference bsAb) was susceptible to impurities. These fragments or aggregates could significantly impact both the efficacy and safety of the molecules. As shown, HX044 unexpectedly exhibited superior stability compared to reference bsAb, along with reduced mispairing impurities and greater productivity. This enhanced stability and efficiency highlight the strong therapeutic potential of HX044. ***

[0307] While the aforementioned invention has been described in some detail by illustration and examples for the purpose of clarifying understanding, it will be readily apparent to those skilled in the art that certain modifications and alterations can be made thereto without departing from the spirit or scope of the appended claims, in light of the teachings of the present invention.

[0308] Accordingly, the foregoing merely illustrates the principle of the present invention. Those skilled in the art will understand that various arrangements embodying the principle of the present invention and falling within its spirit and scope can be devised, even if not expressly described or indicated herein. Furthermore, all embodiments and conditional statements enumerated herein are intended primarily to help the reader understand the concepts to which the inventors have contributed to advance the principle and art of the present invention, and should be interpreted as not being limited to such specifically enumerated embodiments and conditions. Furthermore, all descriptions herein enumerating the principle, aspects, and embodiments of the present invention, and specific examples thereof, are intended to encompass both structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any elements to be developed that perform the same function regardless of structure. Furthermore, nothing disclosed herein, whether such disclosure is expressly enumerated in the claims or not, is intended to be dedicated to the public.

Claims

1. (i) light chain variable domain (VL) and heavy chain variable domain (VH) (wherein the VL / VH pair is specifically bound to human CTLA4, and wherein the VL comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively); and (ii) CD47 binding domain containing the extracellular domain of SIRPα or a variant thereof A bispecific antibody containing this antibody.

2. The bispecific antibody according to claim 1, wherein VL has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 7, and VH has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO:

8.

3. The bispecific antibody according to claim 2, wherein VL and VH each have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the CD47-binding domain comprises the extracellular domain of human SIRPα variant 2 or a variant thereof.

5. The bispecific antibody according to claim 4, wherein the CD47-binding domain has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence-identical to SEQ ID NO:

9.

6. The bispecific antibody according to claim 5, wherein the CD47-binding domain has the amino acid sequence of SEQ ID NO:

9.

7. (1) A first peptide chain (C1) extending from the N-terminus to the C-terminus, containing the VL and the light chain constant region (CL); (2) A second peptide chain (C2) extending from the N-terminus to the C-terminus, comprising the VH, heavy chain constant domain 1 (CH1), and the knob-Fc region; and (3) A third peptide chain (C3) extending from the N-terminus to the C-terminus, containing the CD47 binding domain and the hole-Fc region. A bispecific antibody according to any one of claims 1 to 6, comprising:

8. The bispecific antibody according to claim 7, wherein the CD47-binding domain and the hole-Fc region are directly linked without a linker.

9. (1) The first peptide chain (C1) from the N-terminus to the C-terminus, containing the VL and the light chain constant region (CL); (2) A second peptide chain (C2) comprising VH, heavy chain constant domain 1 (CH1), and a hole-Fc region, extending from the N-terminus to the C-terminus; and (3) A third peptide chain (C3) extending from the N-terminus to the C-terminus, containing the CD47 binding domain and the knob-Fc region. A bispecific antibody according to any one of claims 1 to 6, comprising:

10. The bispecific antibody according to claim 9, wherein the CD47-binding domain and the knob-Fc region are directly linked without a linker.

11. The bispecific antibody according to any one of claims 7 to 10, wherein the knob-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including a T366W substitution; and the hole-Fc region is a human IgG1 Fc region variant having up to 10 amino acid substitutions, including a T366S, L368A, Y407V substitution.

12. The bispecific antibody according to claim 11, wherein the knob-Fc region further comprises an S354C substitution, and the hole-Fc region further comprises a Y349C substitution.

13. The bispecific antibody according to claim 11, wherein the knob-Fc region further comprises a Y349C substitution, and the hole-Fc region further comprises an S354C substitution.

14. The bispecific antibody according to any one of claims 11 to 13, wherein the knob-Fc region further comprises E357K and D399K substitutions, and the hole-region further comprises K370E and K409D substitutions.

15. The bispecific antibody according to any one of claims 11 to 13, wherein the knob-Fc region further comprises K370E and K409D substitutions, and the hole-region further comprises E357K and D399K substitutions.

16. (i) The CL region is kappa CL (Cκ; SEQ ID NO: 21) or lambda CL (Cλ; SEQ ID NO: 22), or a variant thereof having up to 10 amino acid substitutions; (ii) The CH1 domain is a human IgG1 CH1 domain (SEQ ID NO: 41) or a variant thereof having up to 10 amino acid substitutions; and / or (iii) The knob-Fc region and the hole-Fc region have the amino acid sequences of (1) SEQ ID NOs. 31 and 35, respectively; (2) SEQ ID NOs. 32 and 36, respectively; (3) SEQ ID NOs. 33 and 37, respectively; or (4) SEQ ID NOs. 34 and 38, respectively; or variants thereof having up to 10 amino acid substitutions. A bispecific antibody according to any one of claims 7 to 10.

17. The bispecific antibody according to claim 16, wherein the CL region, CH1 domain, knob-Fc region, and hole-Fc region each have the amino acid sequences of (1) SEQ ID NOs. 21, 41, 31, and 35; (2) SEQ ID NOs. 21, 41, 32, and 36; (3) SEQ ID NOs. 21, 41, 33, and 37; or (4) SEQ ID NOs. 21, 41, 34, and 38.

18. The bispecific antibody according to claim 7, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 51; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO: 52; and C3 has an amino acid sequence that is at least 95%, at least 98%, or 100% sequence identical to SEQ ID NO:

53.

19. The bispecific antibody according to claim 18, wherein C1, C2, and C3 each have the amino acid sequences of SEQ ID NOs. 51, 52, and 53, respectively.

20. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody (1) has high binding affinity to CTLA4 and CD47 bipositive cells; (2) depletes tumor-infiltrating lymphocytes (TILs) regulatory T cells (Treg cells) or Treg cells in the tumor microenvironment (TME); (3) increases cytokine levels in the TME; or (4) enhances T cell proliferation and / or activity against cancer; (5) enhances macrophage-mediated phagocytosis; (6) enhances dendritic cell-mediated antigen presentation; or any combination of (1) to (6).

21. The bispecific antibody according to any one of claims 1 to 20, wherein the bispecific antibody (1) has a higher affinity for CTLA4 and CD47 bipositive cells than for CTLA4 or CD47 monopositive cells; (2) selectively removes CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematological toxicity; or (4) has limited immune-related adverse events (irAEs); or any combination of (1) to (4).

22. The bispecific antibody has a limited number of mispairing impurities, according to any one of claims 1 to 21.

23. A composition comprising the bispecific antibody according to any one of claims 1 to 22, wherein the purity of the bispecific antibody is at least 95%, and the purity is measured by size exclusion chromatography (SEC) or non-reducing SDS-PAGE.

24. A pharmaceutical composition comprising a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

25. A polynucleotide encoding the peptide chain of a bispecific antibody according to any one of claims 1 to 22.

26. The polynucleotide according to claim 25, which encodes all of the peptide chains of the bispecific antibody.

27. A plurality of polynucleotides according to claim 25, which collectively encode all the peptide chains of the bispecific antibody.

28. A vector comprising the polynucleotide according to claim 25 or 26.

29. A cell comprising a polynucleotide or a plurality of polynucleotides according to any one of claims 25 to 27, or a vector according to any one of claims 28.

30. A method for producing a bispecific antibody that specifically binds to human CTLA4 and human CD47, comprising culturing the cells described in claim 29 under conditions that enable the expression of the bispecific antibody.

31. A method for treating a cancer in which such treatment is needed, comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 22.

32. The method according to claim 31, wherein the subject is a human.

33. Use of a bispecific antibody as a pharmaceutical product, according to any one of claims 1 to 22.

34. Use of a bispecific antibody according to any one of claims 1 to 22 in the treatment of cancer.

35. Use of a bispecific antibody according to any one of claims 1 to 22 for the preparation of a pharmaceutical for the treatment of cancer.

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