Antibodies targeting ctla4 and cd47 and uses thereof

Abstract

Disclosed herein are dual-specific antibodies that target both human CTLA4 and CD47. Also provided herein are polynucleotides encoding the dual-specific antibodies, pharmaceutical compositions comprising the dual-specific antibodies, and methods of producing the dual-specific antibodies. Also disclosed are medical uses of the dual-specific antibodies described herein in the treatment of cancer.

Description

[0001] This application claims priority to PCT International Patent Application No. PCT / CN2023 / 119498, filed on September 18, 2023, which is incorporated herein by reference in its entirety. 1. Reference to the electronically submitted sequence list

[0002] This application, by way of reference, incorporates a sequence list as an XML file, titled "720A001WO02_SL", created on September 18, 2024, and 66,646 bytes in size. Technical Field

[0003] This invention relates to molecular biology and cell biology. The present invention provides for bispecific antibodies that specifically bind to both human CTLA4 and human CD47, and their use in, for example, cancer treatment. Background Technology

[0004] Immune checkpoint inhibitors have emerged as a promising class of molecules for therapeutic development, such as those targeting PD-1, Tim-3, and CTLA4. Despite the challenges faced by checkpoint inhibitors like Keytruda... ® and Opdivo ® While some treatments have achieved success, current therapies targeting CTLA4 or CD47 have only seen limited success (primarily in melanoma) due to their narrow therapeutic window. Consequently, there remains a pressing unmet need for other cancer therapies, particularly those targeting CTLA4 and / or CD47. The compositions and methods provided in this disclosure address this need and offer relevant advantages. Summary of the Invention

[0005] This document provides a bispecific antibody (“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 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively, and wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NO: 4, 5, and 6, respectively; and (ii) a CD47-binding domain comprising an extracellular domain of SIRPα or a variant thereof. In some embodiments, the VL has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 7, and the VH has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the VL and VH have the amino acid sequences shown in SEQ ID NO: 7 and 8, respectively. In some embodiments, the CD47-binding domain comprises the extracellular domain of human SIRPα variant 2 or a variant thereof. In some embodiments, the CD47-binding domain has an amino acid sequence having at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the CD47-binding domain has the amino acid sequence shown in SEQ ID NO: 9.

[0006] In some embodiments, the bispecific antibody provided herein comprises (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 mortise-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47 binding domain and a mortise-Fc region from the N-terminus to the C-terminus. In some embodiments, the CD47 binding domain and the mortise-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 a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region has the amino acid sequence shown in SEQ ID NO: 46.

[0007] In some embodiments, the bispecific antibody provided herein comprises (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 mortise-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47 binding domain and a mortise-Fc region from the N-terminus to the C-terminus. In some embodiments, the CD47 binding domain and the mortise-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 a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region has the amino acid sequence shown in SEQ ID NO: 46.

[0008] In some embodiments of the bispecific antibody provided herein, the mortar-Fc region is a human IgG1 Fc region variant with up to 10 amino acid substitutions (including T366W substitution); and the mortar-Fc region is a human IgG1 Fc region variant with up to 10 amino acid substitutions (including T366S, L368A, Y407V substitution).

[0009] In some embodiments, the pestle-Fc region further includes an S354C substitution, and the mortar-Fc region further includes a Y349C substitution. In some embodiments, the pestle-Fc region further includes a Y349C substitution, and the mortar-Fc region further includes an S354C substitution.

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

[0011] In some embodiments of the bispecific antibody provided herein, (i) the CL region is κ CL (Cκ; SEQ ID NO: 21) or λ CL (Cλ; SEQ ID NO: 22), or a variant 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 mortar-Fc region and the gluten-Fc region have the following amino acid sequences, respectively: (1) SEQ ID NO: 31 and 35; (2) SEQ ID NO: 32 and 36; (3) SEQ ID NO: 33 and 37; or (4) SEQ ID NO: 34 and 38; or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CH1 domain, mortar-Fc region and stolon-Fc region have the following amino acid sequences: (1) SEQ ID NO: 21, 41, 31 and 35; (2) SEQ ID NO: 21, 41, 32 and 36; (3) SEQ ID NO: 21, 41, 33 and 37; or (4) SEQ ID NO: 21, 41, 34 and 38.

[0012] In some embodiments of the bispecific antibody provided herein, C1 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 51; C2 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 52; and C3 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 53; in some embodiments, C1, C2, and C3 have the amino acid sequences shown in SEQ ID NO: 51, 52, and 53, respectively.

[0013] In some embodiments, the bispecific antibody (1) has a high affinity for CTLA4 and CD47 double-positive 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)-(6). In some embodiments, the bispecific antibody (1) has a higher affinity for CTLA4 and CD47 double-positive cells than for CTLA4 or CD47 single-positive cells; (2) selectively eliminates CTLA4 and CD47 positive cells via antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematologic toxicity; or (4) has limited immune-related adverse events (irAEs); or any combination of (1)-(4). In some embodiments, the bispecific antibody has limited mismatch impurities.

[0014] This document also provides compositions comprising the bispecific antibodies disclosed herein, wherein the purity of the bispecific antibodies is at least 95%, and wherein the purity is measured by size exclusion chromatography (SEC) or non-reducing SDS-PAGE.

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

[0016] In some embodiments, this document provides polynucleotides encoding peptide chains of the bispecific antibodies disclosed herein. In some embodiments, the polynucleotides may encode all peptide chains of the bispecific antibody. In some embodiments, this document provides a variety of polynucleotides disclosed herein that collectively encode all peptide chains of the bispecific antibody.

[0017] In some embodiments, this document provides a vector comprising the polynucleotides disclosed herein.

[0018] In some embodiments, this document provides cells comprising the polynucleotides or multiple polynucleotides disclosed herein, or the vectors disclosed herein. In some embodiments, this document provides a method for preparing a bispecific antibody that specifically binds to human CTLA4 and human CD47, the method comprising culturing the cells disclosed herein under conditions that allow expression of the bispecific antibody.

[0019] In some embodiments, this document provides a method for treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody disclosed herein. In some embodiments, the subject is a human being.

[0020] In some embodiments, this document provides the use of the bispecific antibodies disclosed herein as pharmaceutical agents. In some embodiments, this document provides the use of the bispecific antibodies disclosed herein in cancer treatment. In some embodiments, this document provides the use of the bispecific antibodies disclosed herein in the preparation of cancer therapeutic agents. Attached Figure Description

[0021] Figure 1A-1D A schematic diagram of the bispecific antibody (bsAb) presented in this article is provided. Figure 1A The bsAb is shown in the Fab-Sirpα, mortar and pestle (KIH) configuration; Figure 1B The bsAbs in the Sirpα-IgG configuration were shown; Figure 1C The scFv-Sirpα and KIH configurations are shown. Figure 1D bsAb in the IgG-Sirpα configuration is shown.

[0022] Figure 2 Flow cytometry results showing the binding of HX044 to CD47-positive Jurkat cell lines are provided. The percentage of positive cells (top panel) and mean fluorescence intensity (MFI, bottom panel) are set on the y-axis. Prilimumab and SIRPα-Fc are used as reference antibodies / proteins.

[0023] Figure 3 Flow cytometry results showing the binding of HX044 to three CHO cell lines (CTLA4+CD47-) expressing hCTLA4 are provided. The binding EC was determined based on the 4-parameter equation fitting curve. 50 Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0024] Figure 4 Flow cytometry results showing the binding of HX044 to CD47+ / CTLA4- HEK293T cells or CTLA4-transfected HEK293T cells (CD47 and CTLA4 double positive) are provided. "+" indicates membrane CTLA4 expression levels verified by flow cytometry. Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

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

[0026] Figure 6 Flow cytometry results showing the binding of HX044 to isolated human erythrocytes, which was undetectable, are provided. Prilimumab, molololimumab, and SIRPα-Fc were used as reference antibodies / proteins.

[0027] Figure 7 Flow cytometry results showing the binding of HX044 to human FcRn receptor-positive HEK293T cells are provided. Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0028] Figure 8 ADCC assay results are provided, showing the ADCC activity of HX044 in CD47+ HEK293T cells with different CTLA4 expression levels. "+" indicates membrane CTLA4 expression levels verified by flow cytometry. Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0029] Figure 9 Results are provided in a humanized syngeneic mouse colon cancer model of the target gene (MC38-hCD47 model inoculated into hCTLA4×hCD47×hSIRPαHuGEMM mice), demonstrating the in vivo antitumor activity of HX044. Tumor growth was measured twice weekly and is shown as mean tumor size ± SEM for each group. Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0030] Figure 10 Results are provided in a humanized syngeneic mouse melanoma model of the target gene (a B16F10-hCD47 model inoculated into hCTLA4×hCD47×hSIRPαHuGEMM mice), demonstrating the in vivo antitumor activity of HX044. Tumor growth was measured twice weekly and is shown as mean tumor size ± SEM for each group. Prilimumab and SIRPα-Fc were used as reference antibodies / proteins.

[0031] Figure 11 Flow cytometry results are provided showing tumor-infiltrating lymphocytes (TILs) collected from mice at the end of treatment in an syngeneic mouse model (a B16F10-hCD47 model inoculated into hCTLA4×hCD47×hSIRPα mice). Prilimumab analogues and SIRPα-Fc were used as reference antibodies / proteins.

[0032] Figure 12 Flow cytometry results are provided showing the effect of HX044 treatment on lymphocyte composition in an syngeneic mouse model (hCTLA4×hCD47×hSIRPα HuGemm C57BL / 6J mice). Prilimumab analogues and SIRPα-Fc were used as reference antibodies / proteins.

[0033] Figures 13A-13B HX044 is provided ( Figure 13A ) and reference bispecific antibody ( Figure 13B Size exclusion chromatography (SEC) results at 214 nm and 280 nm.

[0034] Figure 14 The A-14B provides HX044 ( Figure 14 A) and reference bispecific antibody ( Figure 14 B) Results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 6. Detailed Explanation

[0035] This document provides bispecific antibodies that specifically bind to both human CTLA4 and human CD47. It also discloses pharmaceutical compositions comprising therapeutically effective amounts of these antibodies. Furthermore, it discloses the use of these antibodies and pharmaceutical compositions in the treatment of cancer.

[0036] Cytotoxic T-lymphocyte-associated protein-4 (CTLA4) is a classic immune checkpoint that inhibits T-cell function by blocking the co-stimulatory receptors B7-1 (CD80) / B7-2 (CD86) on antigen-presenting cells (APCs) that interact with the co-activator CD28 on T cells. CTLA4 plays a crucial role in tumor-infiltrating (TIL) T cells. reg The levels of CD8 activated in the blood are higher than in normal blood chambers and also higher than in the tumor microenvironment (TME). + Effector T cells (T cells) eff High-level constitutive expression of CTLA4. It should also be understood that anti-CTLA4 antibodies block the binding of CTLA4 to its CD80 / CD86 ligands on APCs, leading to the release of CD80 / CD86-mediated secondary signals and reactivation of effective T cells. On the other hand, tumor-infiltrating regulatory T cells (T cells) have also been confirmed to be involved. reg The ADCC effect and / or macrophage-mediated depletion, along with innate immune remodeling in the TME via Fcγ receptor (FcγR) binding, 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 window, primarily because of high systemic immune-related adverse events (irAEs).

[0037] CD47 is the "don't eat me" receptor, overexpressed in various human cancers. Together with its ligands on the surface of phagocytes, including macrophages and dendritic cells (DCs), CD47 constitutes an important innate and adaptive immune checkpoint protein and a promising target for immunotherapy. Targeting of CD47 via anti-CD47 antibodies or CD47-trap proteins (i.e., signal regulatory protein α or SIRPα) has been extensively tested, but has not yet been clinically successful due to its narrow therapeutic window. Firstly, due to the widespread expression of CD47 on megakaryocytes and erythrocytes, targeting CD47 has been found to be associated with dose-limited hematologic toxicities (DLTs), such as anemia and thrombocytopenia.

[0038] Compared to reagents targeting either hCTLA4 or hCD47, the anti-hCTLA4 / hCD47 bispecific antibody disclosed in this paper promotes TIL-T... reg It exhibits synergistic activity in terms of depletion and excellent antitumor activity, as well as very limited hematologic toxicity and immune-related adverse events (irAEs). With a larger therapeutic window than single-target agents, the dual-targeting bsAbs disclosed in this article can be used as a safer and more effective anticancer therapeutic agent.

[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 terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting. 6.1 Definition

[0040] Unless otherwise defined herein, the scientific and technical terms used in this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. Generally, the terminology used in conjunction with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, as well as the techniques used in these fields, are those well known and commonly used in the art.

[0041] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated as being incorporated by reference, and these publications and patents are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. References to any publication are for its disclosure prior to the filing date and should not be construed as an admission of non-existence of the invention prior to that publication. Furthermore, the publication dates provided may differ from the actual publication dates, which require independent verification.

[0042] The term “one” refers to one or more of the same entity; for example, “antibody” should be understood to mean one or more antibodies.

[0043] When used herein, the term “and / or” will be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, as used herein, the term “and / or” as in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A alone”; and “B alone”. Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover each of the following: 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 alone; B alone; and C alone.

[0044] As used herein, the term "about" is used to indicate the inherent error variation of a value, including the apparatus used to determine the value, the method used to determine the value, or the variation present in the study subjects. The term "about" encompasses the exact numerical values ​​listed. In some embodiments, "about" means within plus or minus 10% of a given value or range. In some embodiments, "about" means the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value represented by "about". In some embodiments, "about" means the variation is ±1%, ±0.5%, ±0.2%, or ±0.1% of the value represented by "about".

[0045] As used interchangeably herein, the terms “peptide chain,” “peptide,” “polypeptide,” “protein,” and their grammatical equivalents refer to a polymer of amino acids of any length, which may be linear or branched. It may include non-natural or modified amino acids or may be broken down into non-amino acid components. Polypeptides, peptides, polypeptide chains, or proteins may also be modified by, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification.

[0046] As used interchangeably herein, the terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents refer to polymers of nucleotides of any length and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be introduced into the polymer by DNA or RNA polymerase.

[0047] As used herein with respect to a protein or polypeptide with a specific sequence characteristic (“reference protein” or “reference polypeptide”), the term “variant” refers to a different protein or polypeptide compared to the reference protein or reference 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. Changes in the amino acid sequence can be amino acid substitutions. Changes in the amino acid sequence can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0048] As used herein, the term "specific binding" means that a peptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in some combination of the foregoing substances, including related and unrelated proteins. For example, the binding moiety (e.g., antibody) of a specifically binding target molecule (e.g., antigen) can be identified by immunoassay, ELISA, biomembrane interference technique ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific reaction will be at least twice the background signal or noise and may be greater than 10 times the background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989. Fundamental Immunology Second Edition Raven Press, New York, pp. 332-336. In some embodiments, "specific binding" means, for example, that the binding site is at a Kc of about 0.1 mM or less. D Binding to molecular targets. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration of about 10 μM or less, or about 1 μM or less, of K+. D Binding to the target. In some embodiments, "specific binding" refers to the peptide or molecule binding at a concentration of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. D Target binding. Due to sequence identity between homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target.

[0049] As used herein, the term "binding affinity" generally refers to the sum of the strengths of non-covalent interactions between the binding moiety and the target molecule (e.g., antigen). Binding between the binding moiety and the target molecule is a reversible process, and binding affinity is typically reported as the equilibrium dissociation constant (K0). D K D It is the dissociation rate (koff or K d ) and binding rate (k on or k a The ratio of ) to. Combined with the pair K D The lower the value, the higher the affinity. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes disclosed herein. Specific illustrative embodiments include the following. In some embodiments, “K…” D "or "K D The "value" can be measured by methods known in the art, for example, by binding assays. K can be measured in radiolabeled antigen binding assays (RIA). D (Chen et al., (1999) J. Mol Biol 293:865-881). K can also be measured using biomembrane interferometry (BLI) by using, for example, the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K D The value can also be measured using surface plasmon resonance (SPR) measurements via Biacore, for example, using the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). D or K D Value. EC can also be used. 50 Quantitative binding affinity, EC50 is the concentration of ligands present in a bound state at half the target level in a binding assay.

[0050] As used herein, the cell-related term "CD47 positive" refers to a cell with detectable CD47 expression. In some embodiments, the cells have detectable CD47 expression on their surface. As used herein, the cancer- or tumor-related term "CD47 positive" refers to a cancer or tumor with cells exhibiting detectable CD47 expression. As used herein, the cell-related term "CTLA4 positive" refers to a cell with detectable CTLA4 expression. In some embodiments, the cells have detectable CTLA4 expression on their surface. As used herein, the cancer- or tumor-related term "CTLA4 positive" refers to a cancer or tumor with cells exhibiting detectable CTLA4 expression. Those skilled in the art can readily determine whether a cancer or tumor has CD47 and / or CTLA4 expression using any method known and available in the art, including, for example, immunohistochemistry (IHC), immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), etc.

[0051] As used herein in the context of two or more polynucleotides or polypeptides, the terms "identical," "identity percentage," and their syntactic equivalents refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence (with gaps introduced where necessary) and without considering any conserved amino acid substitutions as part of sequence identity. Percentage identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include (but are not limited to) the BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin software packages, and their variations. In some embodiments, the two polynucleotides or polypeptides provided herein are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection, they have at least 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% nucleotide or amino acid residue identity. In some embodiments, there is identity within amino acid sequence regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-860 residues, or any integer value between them. In some embodiments, there is identity within regions of more than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (such as the coding region of a target protein or antibody). In some embodiments, there is identity within nucleotide sequence regions 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 between them. In some embodiments, there is identity within regions of more than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (such as the nucleotide sequence encoding the target protein).

[0052] As used herein, the term "vector" and its grammatical equivalents refer to a medium that can be introduced into a host cell to carry genetic material (e.g., a polynucleotide sequence), which can be replicated and / or expressed in the host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes may provide, for example, antibiotic or toxin resistance, supplement nutrient deficiencies, or provide critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well known in the art. When two or more polynucleotides are co-expressed, the two polynucleotides may be inserted, for example, into a single expression vector or different expression vectors. For single-vector expression, the encoding polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the polynucleotide into the host cell can be confirmed using methods well known in the art. Those skilled in the art will understand that the polynucleotide is expressed in sufficient quantities to produce the desired product, and will also understand that methods well known in the art can be used to optimize the expression level to obtain sufficient expression.

[0053] As used herein, the term “encoding” and its syntactic equivalents refer to the inherent property of a specific nucleotide sequence in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties, in biological processes. Thus, if transcription and translation of the mRNA corresponding to a gene yields a protein, then the gene encodes that protein. Unless otherwise stated, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0054] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they are no longer in their naturally occurring form. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure.

[0055] As used herein, the term "treatment" in relation to a disease or condition, or a subject suffering from a disease or condition, and its grammatical equivalents, refers to actions, interventions, and / or measures that inhibit, eliminate, reduce, and / or improve symptoms, severity, and / or frequency of symptoms associated with the disease or condition being treated. Treatment for osteoporosis can inhibit, eliminate, reduce, or improve symptoms associated with osteoporosis. These symptoms primarily include bone loss, decreased bone density, and increased risk of fracture, and treatment aims to slow the rate of bone loss, strengthen bones, and reduce the likelihood of fractures.

[0056] As used herein, the term "administration" and its grammatical equivalents refer to the act of delivering or causing the delivery of a therapeutic agent or pharmaceutical composition to a subject by means described herein or otherwise known in the art. The therapeutic agent may be a compound, peptide, antibody, cell, or cell population. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered to 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) injectable dosage forms; transdermal dosage forms, including creams, gels, powders, or patches; oral dosage forms; and inhaled powders, sprays, suspensions, and rectal suppositories.

[0057] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to the amount of an agent administered to a subject, alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, in an amount capable of having any detectable, positive effect on any symptom, aspect, or characteristic of a disease, symptom, or condition. Therapeutic effective amounts can be determined by measuring relevant physiological effects. The exact amount required varies from subject to subject, based on factors such as the subject's age, weight and general condition, the severity of the condition to be treated, and the clinician's judgment. Those skilled in the art can determine the appropriate "effective amount" for any individual case using routine laboratory methods.

[0058] As used herein, the term “mismatch impurity” and its grammatical equivalents refer to non-target antibody material resulting from incorrect pairing of components or immunoglobulin chains during production, particularly in recombinant or engineered antibody systems. These impurities occur when heavy and light chains do not pair correctly, resulting in the formation of heavy chain homodimers, light chain homodimers, or heavy chains that do not pair with their corresponding light chains. For illustrative purposes, in the bispecific antibody consisting of three peptide chains described herein: (1) a first peptide chain (C1) comprising an IgG light chain containing anti-CTLA4 VL; (2) a second peptide chain (C2) comprising an IgG heavy chain containing anti-CTLA4 VL and a “pump” Fc region; and (3) a third peptide chain (C3) comprising a “hole” Fc region fused with a CD47 binding domain – a mismatch impurity, can be shown when C2 and C3 pair together without an accompanying light chain, resulting in a heterodimer with a short light chain, or when C1, C2, and C3 each form a homodimer. The presence of these mismatched impurities can adversely affect the structural integrity, specificity, potency, and safety of antibody products.

[0059] As used herein, the term "subject" refers to any animal (e.g., a mammal), including (but not limited to) humans, non-human primates, dogs, cats, rodents, etc., that will be a recipient of a specific treatment. Mammals include (but are not limited to) livestock, poultry, pets, primates, horses, dogs, cats, mice, and rats. A subject in need of treatment may be a person who has a disease, is at risk of having a disease, or is suspected of having a disease. A subject with a disease can be identified through routine medical examinations, such as physical examination, laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any such disease may exhibit one or more symptoms of the disease. A subject at risk of having a disease may be a subject who has one or more risk factors for the disease. A subject may be human. A subject may have a specific disease or condition.

[0060] Scope: Throughout this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be considered a rigid limitation on the scope of the invention. Therefore, a description of the scope should be considered to have all possible sub-scopes specifically disclosed, as well as the individual values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered to have specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual values ​​within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is applicable regardless of the width of the scope.

[0061] Exemplary genes and peptides are described herein with reference to GenBank numbers, GI numbers, and / or SEQ ID NOs. It should be understood that those skilled in the art can readily identify homologous sequences from the reference sequence sources, including (but not limited to) GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ). 6.2 Bispecific antibodies targeting human CTLA4 and human CD47

[0062] This document provides 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, an "antibody" is an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein) through at least one antigen-binding fragment typically located within a variable region of an immunoglobulin molecule. Antibodies can have a variety of 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. Based on the characteristics of their heavy chain constant domains, referred to as α, δ, ε, γ, and μ, respectively, 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). Unless otherwise explicitly stated, the term "antibody" as used herein includes the "antigen-binding fragment" of the complete antibody. As used herein, the term "antigen-binding fragment" refers to a portion or fragment of a whole antibody that is the antigen-determining variable region of a whole antibody. Examples of antigen-binding fragments include (but are not limited to) Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide-linked scFv (dsscFv), double-chain antibodies, triple-chain antibodies, tetra-chain antibodies, microantibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAb; e.g., camel antibodies, alpaca antibodies), and single variable domain (VHH) of heavy chain antibodies.

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

[0065] The structures of immunoglobulins have been well identified (see, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). Typically, immunoglobulins consist of two pairs of polypeptide chains, one pair of light chains (L; low molecular weight) and one pair of heavy chains (L; high molecular weight), with all four chains linked together by disulfide bonds.

[0066] Each light chain of an immunoglobulin typically comprises a variable region (“VL region”) and a constant region (“CL region”). Two distinct light chain types exist, based on the amino acid sequence of the CL region, and are referred to as κ (κ) or λ (λ). The amino acid sequence of the CL region is well known in the art.

[0067] Each heavy chain typically comprises a variable region (“VH region”) and a constant region (“CH region”). Based on the amino acid sequence, the VH region can be one of five different types, referred to as α, δ, ε, γ, and μ. When combined with a light chain, these different types of heavy chains produce five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM. IgG exists in four subclasses: IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the CH regions of the different antibody classes are well known in the art.

[0068] The CH region of an immunoglobulin contains more than one domain. 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 bonds in the hinge region are part of the interaction between the two heavy chains in an immunoglobulin. The "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing 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, and subtypes IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. The native Fc region can be modified. Modification of the Fc region is further described below. In some embodiments, the bispecific antibodies provided herein may comprise paired Fc domains containing paired different modifications that promote their binding to each other rather than forming homodimers.

[0069] Unless otherwise stated or contradicted by the context, the amino acid positions in constant regions are based on EU-numbers (Edelman et al., PNAS. 1969; 63:78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 1991 NIH Publication No. 91-3242). A list of exemplary amino acid sequences of constant domains / regions of human IgG antibodies is provided below. Several exemplary variants are also included, with further variants disclosed in the following sections.

[0070] Table 1. Constant regions / domains of natural human IgG.

[0071] The term "variable region" refers to a portion of the light or heavy chain of an immunoglobulin, typically located at the amino terminus of the light or heavy chain and used by each specific antibody for the binding and specificity of its particular antigen. The variable region of the light chain is called the "light chain variable region" or "VL region," which includes at least one, typically a "light chain variable domain" or "VL." The variable region of the heavy chain is called the "heavy chain variable region" or "VH region," which includes at least one, typically a "heavy chain variable domain" or "VH." The sequences of the variable domains vary widely between different antibodies. "VL and VH pairs" can bind to each other and form binding sites that specifically bind to target antigens or epitopes.

[0072] VH and VL regions can be further divided into hypervariable regions (or regions that are highly variable in the sequence and / or form of a structurally defined loop), also known as complementarity-determining regions (CDRs), and more conserved regions called frame regions (FRs). Sequence variations are concentrated in the CDRs, while the less variable portions of the variable domains are called frame regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. Each VH and VL typically consists of 3 CDRs and 4 FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J MolBiol 1987;196:901-17).

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

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

[0075] The bispecific antibodies presented herein comprise VL and VH pairs that specifically bind to the human CTLA4 and human CD47 binding domains containing the SIRPα extracellular domain (ECD). Exemplary VL / VH pairs that specifically bind to human CTLA4 are provided below, as well as exemplary SIRPα ECDs. In addition to the specific CTLA4 targeting the VL / VH pairs and CD47 binding domains illustrated below, variants of these VL / VH pairs and CD47 binding domains that retain their binding to CTLA4 and CD47, respectively, are explicitly considered herein.

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

[0077] Table 3B: SIRPα domains

[0078] In some embodiments, this document provides a bispecific antibody 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 VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively, according to the Kabat definition scheme, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NO: 4, 5, and 6, respectively, according to the Kabat definition scheme; and (ii) a CD47-binding domain comprising an extracellular domain of SIRPα or a variant thereof.

[0079] In some embodiments of the bispecific antibody disclosed herein, the VL has an amino acid sequence having 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% identity with SEQ ID NO: 7. The bispecific antibody may have a VL having at least 85% sequence identity with SEQ ID NO: 7. The bispecific antibody may have a VL having at least 90% sequence identity with SEQ ID NO: 7. The bispecific antibody may have a VL having at least 95% sequence identity with SEQ ID NO: 7. The bispecific antibody may have a VL having at least 98% sequence identity with SEQ ID NO: 7. The bispecific antibody may have a VL having at least 99% sequence identity with SEQ ID NO: 7. The bispecific antibody may have a VL having the amino acid sequence shown in SEQ ID NO: 7.

[0080] In some embodiments of the bispecific antibody disclosed herein, the VH has an amino acid sequence having 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% identity with SEQ ID NO: 8. The bispecific antibody may have a VH having at least 85% sequence identity with SEQ ID NO: 8. The bispecific antibody may have a VH having at least 90% sequence identity with SEQ ID NO: 8. The bispecific antibody may have a VH having at least 95% sequence identity with SEQ ID NO: 8. The bispecific antibody may have a VH having at least 98% sequence identity with SEQ ID NO: 8. The bispecific antibody may have a VH having at least 99% sequence identity with SEQ ID NO: 8. The bispecific antibody may have a VH having the amino acid sequence shown in SEQ ID NO: 8.

[0081] In some embodiments of the bispecific antibody disclosed herein, the VL and VH that specifically bind to human CTLA4 have the amino acid sequences shown in 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 to transmit a "don't eat me" signal, helping to prevent phagocytosis by macrophages. SIRPα contains an extracellular region with three immunoglobulin superfamily (IgSF) domains, including an NH2-terminal ligand-binding V-domain. The intracellular region of SIRPα contains both the ITIM and ITSM motifs, essential for the inhibitory activity of the receptor. For illustrative purposes, full-length human SIRPα can 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). Despite their similarities, the two variants of human SIRPα differ by 13 amino acids, all within their extracellular domains. To date, there are no approved therapeutic products containing either variant 1 or variant 2 of human SIRPα, although several investigational therapies targeting the CD47-SIRPα pathway are in clinical trials. For treatment design, the choice between variant 1 and variant 2 is highly unpredictable due to the complex balance and complexity of cancer immunology. 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 skew the therapeutic balance towards undesirable immunosuppression or overactivation. As discussed further below, bispecific antibodies containing ECD variant 2 of human SIRPα achieve a good balance and realize synergistic anti-tumor 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 having 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% identity with the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the CD47-binding domain has the amino acid sequence shown in 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 that of SEQ ID NO: 12. In some embodiments, the CD47-binding domain has the amino acid sequence shown in SEQ ID NO: 12.

[0084] In some embodiments, the CD47-binding domain comprises an extracellular domain of human SIRPα or a variant thereof that retains its binding to CD47. In some embodiments, the CD47-binding domain comprises an 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: 12. 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. In some embodiments, the CD47-binding domain has the amino acid sequence of 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 having 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% identity with SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9. The CD47-binding domain may have an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 9. In some embodiments, the CD47 binding domain has the amino acid sequence shown in SEQ ID NO: 9.

[0086] In some embodiments, the CD47-binding domain comprises 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 having 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% identity with SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. The CD47-binding domain may have an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11. The CD47 binding domain may have an amino acid sequence that is at least 98% sequence identical to that of SEQ ID NO: 11. In some embodiments, the CD47 binding domain has the amino acid sequence shown in SEQ ID NO: 11.

[0087] Those skilled in the art can select variants with known SIRPa amino acid sequences by referring to references such as LEE et al., The Journal of Immunology, 179, 7741-7750, 2007 and HATHERLEY et al., The Journal of Biological Chemistry, 282:19, pp. 14567-14575, 2007, each of which is incorporated herein by reference in its entirety. More information about 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. For the human SIRPa gene (Uniprot NO:P78324-1, P78324-2 and P78324-4), three (3) alternative splicing transcript variants encoding different isotypes are described.

[0088] In some embodiments, the bispecific antibodies provided herein have a "mortar and pestle" or "KIH" structure (e.g., Figure 1A and 1C The “KIH” model promotes the formation of heterodimers of engineered bispecific antibodies, rather than homodimers of heavy chains.

[0089] Modifications that promote binding of Fc domain pairs in bispecific antibodies include so-called "mortar and pestle" modifications, which consist of a "pestle" modification in one Fc domain and a "mortar" modification in the other. Mortar and pestle techniques are described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot. Eng. 9, 617-621 (1996) and Carter, JImmunol. Meth. 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("pestle") at the interface of the first Fc ("pestle-Fc") and a corresponding cavity ("mortar") at the interface of the second Fc ("mortar-Fc"), thereby allowing the protrusion to be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), complementary cavities with the same or similar size as the protrusions are created at the interface of the second polypeptide.

[0090] Therefore, "groove-Fc regions" and "mortar-Fc regions" are designed to form heterodimer pairs. A groove-Fc region refers to a region where an amino acid in the CH3 domain is replaced by an amino acid residue with a larger side chain volume, thereby creating a protrusion within the CH3 domain that can be positioned within a cavity in the CH3 domain of the mortar-Fc region. In the mortar-Fc region, an amino acid residue in the CH3 domain is replaced by an amino acid residue with a smaller side chain volume, thereby creating a cavity within the CH3 domain, within which the protrusion of the first subunit in the CH3 domain is positionable. Preferably, the amino acid residue with the larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue with the smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutations or by peptide synthesis.

[0091] In some embodiments, the threonine residue at position 366 of the mordone-Fc region is replaced by a tryptophan residue (T366W), and the tyrosine residue at position 407 of the mordone-Fc region is replaced by a valine residue (Y407V). Optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A). In some embodiments, the mordone-Fc region further has a serine residue at position 354 replaced by a cysteine ​​residue (S354C) or a glutamate residue at position 356 replaced by a cysteine ​​residue (E356C), and the mordone-Fc region further has a tyrosine residue at position 349 replaced by a cysteine ​​residue (Y349C). In some embodiments, the mortar-Fc region further comprises a serine residue at position 354 (S354C) replaced by a cysteine ​​residue, or a glutamic acid residue at position 356 (E356C) replaced by a cysteine ​​residue, and the mortar-Fc region further comprises a tyrosine residue at position 349 (Y349C) replaced by a cysteine ​​residue. In some embodiments, the mortar-Fc region contains amino acid substitutions for S354C and T366W, and the mortar-Fc region contains amino acid substitutions for Y349C, T366S, L368A, and Y407V. In some embodiments, the mortar-Fc region contains amino acid substitutions for Y349C and T366W, and the mortar-Fc region contains amino acid substitutions for 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 pair with three positively charged residues in the CH3 domain of another chain. These specific charged residue pairs are: E356-K439, E357-K370, D399-K409, and vice versa. In some embodiments, two of the following three mutations are introduced into the mortar-Fc region: E356K, E357K, and D399K, and two of the following three mutations are introduced into the mortar-Fc region: K370E, K409D, and K439E. In some embodiments, two of the following three mutations are introduced into the mortar-Fc region: E356K, E357K, and D399K, and two of the following three mutations are introduced into the mortar-Fc region: K370E, K409D, and K439E.

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

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

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

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

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

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

[0099] In some embodiments, this document provides a bispecific antibody 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 (ii) a CD47-binding domain comprising an extracellular domain of SIRPα or a variant thereof. Figure 1A As shown, 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 mortise-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47 binding domain and a mortise-Fc region from the N-terminus to the C-terminus.

[0100] As those skilled in the art will understand, in the bispecific antibodies with the KIH configuration described above, the mortar-Fc and cladodes-Fc regions can be interchanged. In other words, in the C2 and C3 pair of a bispecific antibody designed with KIH, C2 may include a mortar-Fc region and C3 may include a cladodes-Fc region; alternatively, in some embodiments, C2 may include a cladodes-Fc region and C3 may include a mortar-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 cladodes-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a CD47 binding domain and a mortar-Fc region from the N-terminus to the C-terminus.

[0101] In some embodiments of the bispecific antibodies disclosed herein, the Fc region (grooved Fc region or mortared Fc region) may include 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 a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region is an IgG1 hinge (SEQ ID NO: 45) or a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region has the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the hinge region is an IgG2 hinge (SEQ ID NO: 47) or a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region is an IgG3 hinge (SEQ ID NO: 48) or a variant thereof having a mutation of up to 5 amino acids. In some embodiments, the hinge region is an IgG4 hinge (SEQ ID NO: 49) or a variant thereof having a mutation of up to 5 amino acids.

[0102] In some embodiments, the CD47 bonding domain and the Fc region are connected via a flexible joint, such as a GS joint. In other embodiments, the CD47 bonding domain and the Fc region are directly connected, i.e., the C-end of the CD47 bonding domain is directly connected to the N-end of the Fc region (typically the hinge of the Fc region), without any other joints.

[0103] Flexible linkers are a common choice in therapeutic molecule design, enhancing flexibility by introducing a spatial spacer between the SIRPα domain and the Fc region, allowing each domain to function independently. Increased flexibility can also improve the binding efficiency of each domain to its respective target (CD47 or immune cells) and facilitate simultaneous binding with CD47 and CTLA4, especially when these two targets are located at different distances on different cells (e.g., tumor cells and T-cells). Flexible linkers can also minimize steric conflict, particularly in crowded TMEs, improving the accessibility of both the SIRPα domain and the CTLA4-targeting domain, thus enhancing the overall potency of dual-specific molecules. Furthermore, GS linkers are frequently used in protein engineering to facilitate better protein folding and stability during expression, enabling higher production yields and reduced aggregation of dual-specific molecules in recombinant systems, thereby improving their manufacturability.

[0104] In this paper, the inventors unexpectedly discovered that bispecific antibodies lacking a flexible linker between the CD47 binding domain and the Fc region, while exhibiting reduced affinity for CD47+ / CTLA4- cells compared to their counterparts with flexible linkers, maintain comparable binding affinity for CD47+ / CTLA4+ cells. Unbound from theoretical constraints, the increased rigidity allows for optimal positioning of the two binders to simultaneously bind to both antigens. This spatial confinement enhances the selectivity of the molecule, ensuring efficient binding and activation only in tumor cells or immune cells co-expressing both CD47 and CTLA4, such as Tregs in the tumor microenvironment (TME). Furthermore, it was unexpectedly found that bispecific antibodies lacking a flexible linker between the CD47 binding domain and the Fc region actually demonstrate excellent production capabilities, resulting in higher expression levels and lower mismatch impurities. Therefore, in some preferred embodiments of the bispecific antibodies disclosed herein, the CD47 binding domain (e.g., the ECD of human Sirpα variant 2) and the Fc region are directly linked without a linker.

[0105] The bispecific antibody in the KIH configuration described herein comprises a CL region, a CH1 domain, and two Fc regions (a mortar-and-palm Fc, each comprising a hinge, a CH2 domain, and a CH3 domain). The amino acid sequences of the CH1, CL, and Fc regions of the bispecific antibody disclosed herein can be derived from any suitable source, such as the constant regions of antibodies like IgG1, IgG2, IgG3, or IgG4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art, for example, those available in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0106] In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL, and Fc regions (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein may contain one or more amino acid substitutions different from those of wild-type immunoglobulins. These substitutions are known in the art (see, for example, US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727).

[0107] In some embodiments, the CH1 domain of the bispecific antibody provided herein may be selected from the following: human IgG1 CH1 domain (SEQ ID NO: 41), human IgG2 CH1 domain (SEQ ID NO: 42), human IgG3 CH1 domain (SEQ ID NO: 43), or human IgG4 CH1 domain (SEQ ID NO: 44); or variants thereof having up to 10 amino acid substitutions. The CH1 domain may be human IgG1 CH1 domain (SEQ ID NO: 41) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be human IgG2 CH1 domain (SEQ ID NO: 42) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be human IgG3 CH1 domain (SEQ ID NO: 43) or a variant thereof having up to 10 amino acid substitutions. The CH1 domain may be 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 κ CL (Cκ; SEQ ID NO: 21). In some embodiments, the CL region of the bispecific antibody provided herein may be λ CL (Cλ; SEQ ID NO: 22).

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

[0110] In some embodiments, the pestle-Fc region and the mortar-Fc region may each have the following amino acid sequences: (1) SEQ ID NO: 31 and 35; (2) SEQ ID NO: 32 and 36; (3) SEQ ID NO: 33 and 37; or (4) SEQ ID NO: 34 and 38. The pestle-Fc region and the mortar-Fc region may each have the amino acid sequences shown in SEQ ID NO: 31 and 35. The pestle-Fc region and the mortar-Fc region may each have the amino acid sequences shown in SEQ ID NO: 32 and 36. The pestle-Fc region and the mortar-Fc region may each have the amino acid sequences shown in SEQ ID NO: 33 and 37. The pestle-Fc region and the mortar-Fc region may each have the amino acid sequences shown in SEQ ID NO: 34 and 38.

[0111] Table 4: Examples of Fc sequences in the KIH model (excluding hinges).

[0112] In some embodiments of the bispecific antibody provided herein, (i) the CL region is κ CL (Cκ; SEQ ID NO: 21) or λ CL (Cλ; SEQ ID NO: 22), or a variant 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 mortar-Fc and clathrate-Fc regions have the following amino acid sequences, respectively: (1) SEQ ID NO: 31 and 35; (2) SEQ ID NO: 32 and 36; (3) SEQ ID NO: 33 and 37; or (4) SEQ ID NO: 34 and 38; or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CH1 domain, mortar-Fc region, and clathrate-Fc region have the amino acid sequences shown in SEQ ID NO: 21, 41, 31, and 35, respectively. In some embodiments, the CL region, CH1 domain, pestle-Fc region, and mortar-Fc region have the amino acid sequences shown in SEQ ID NO: 21, 41, 32, and 36, respectively. In some embodiments, the CL region, CH1 domain, pestle-Fc region, and mortar-Fc region have the amino acid sequences shown in SEQ ID NO: 21, 41, 33, and 37, respectively. In some embodiments, the CL region, CH1 domain, pestle-Fc region, and mortar-Fc region have the amino acid sequences shown in SEQ ID NO: 21, 41, 34, and 38, respectively.

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

[0114] Table 5A: Peptide chains of exemplary bispecific antibodies (KIH) Note: VL1 / VH1 specifically binds to CTLA4 (e.g., SEQ ID NO: 7 and 8); SIRPα specifically binds to CD47 (e.g., SEQ ID NO: 9 or SEQ ID NO: 11); Cκ refers to κ CL. (H): Hinge region Table 5B: Sequences of Exemplary Bispecific Antibodies (KIH)

[0115] In some embodiments, this document provides a bispecific antibody that specifically binds to human CTLA4 and human CD47, wherein the bispecific antibody has a first peptide chain (C1), a second peptide chain (C2), and a third peptide chain (C3), wherein C1 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 51; C2 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 52; and C3 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 53; in some embodiments, C1, C2, and C3 have the amino acid sequences shown in SEQ ID NO: 51, 52, and 53, respectively.

[0116] This invention also considers other variants and equivalent forms 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, it is desirable to modulate the biological properties of the antibody, including (but not limited to) specificity, thermostability, 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, such as changing the number or location of glycosylation sites or altering membrane anchoring characteristics.

[0117] This document also provides antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of the antibodies described in the examples. Functional variants of the heavy chain, light chain, VL, VH, or CDR used in the antibody context still allow the antibody to retain at least a significant proportion (at least about 90%, 95%, or more) of the functional characteristics of the “reference” and / or “parental” antibody, including affinity and / or specificity / selectivity, Fc inertness, and PK parameters such as half-life, Tmax, and Cmax. These functional variants typically retain significant sequence identity to the parental antibody and / or have substantially similar heavy and light chain lengths. Exemplary variants include those primarily through conserved substitutions of the heavy chain and / or light chain, VH and / or VL and / or CDR regions that differ from the parental antibody sequence; for example, 10 substitutions, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the variant may be conserved amino acid residue substitutions.

[0118] In some embodiments, variants of the bispecific antibodies disclosed herein may retain their ability to bind CTLA4 and CD47 to a similar, identical, or greater degree than that of the parental bispecific antibody. In some embodiments, the variants may have 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 higher identity with the parental antibody in terms of amino acid sequence. In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parental bispecific antibody disclosed herein, having one or more conserved amino acid substitutions. Conserved amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical properties.

[0119] In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of a parental antibody having one or more non-conserved amino acid substitutions. In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of a parental binding antibody having one or more non-conserved amino acid substitutions, wherein the one or more non-conserved amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In some embodiments, the one or more conserved amino acid substitutions and / or the one or more non-conserved amino acid substitutions can enhance the biological activity of the variant, thereby increasing the biological activity of the functional variant compared to the parental antibody.

[0120] In some embodiments, the variants may have 1, 2, 3, 4 or 5 amino acid substitutions in the CDRs of the binding portion (e.g., VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3).

[0121] In some embodiments, the bispecific antibodies provided herein include modifications located in their Fc regions. In some embodiments, the modified antibodies (e.g., modified Fc regions) provide altered effector function, which in turn affects the antibody's biological profile. For example, in some embodiments, the deletion or inactivation of a constant region (through point mutation or other means) reduces the Fc receptor binding of the modified antibody when it cycles. In some embodiments, the constant region modification reduces the immunogenicity of the antibody. In some embodiments, the constant region modification increases the serum half-life of the antibody. In some embodiments, the constant region modification reduces the serum half-life of the antibody. In some embodiments, the constant region modification reduces or removes the antibody's ADCC and / or complement-dependent cytotoxicity (CDC). In some embodiments, specific amino acid substitutions in the human IgG1 Fc region having corresponding IgG1 or IgG4 residues reduce the effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the constant region modification increases or enhances the antibody's ADCC and / or CDC. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some implementations, the constant region is modified to add / replace one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate linkage sites.

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

[0123] The variant antibodies described herein can be generated 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 naturally or through interventional chemical modification. In some embodiments, the bispecific antibodies have been chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or binding to cellular ligands or other proteins. Any variety of chemical modifications can be performed using known techniques. The bispecific antibodies provided herein may comprise one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0125] The physical, chemical, and / or biological properties of the bispecific antibodies disclosed herein can be analyzed using a variety of methods known in the art. In some embodiments, the ability of the bispecific antibodies provided herein to bind to human CTLA4 and CD47 was tested. Binding assays include (but are not limited to) BLI, SPR (e.g., Biacore), ELISA, and FACS. Additionally, the antibody's solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency can be evaluated.

[0126] In some embodiments, the bispecific antibodies disclosed herein can be conjugated to a detectable substance or molecule, allowing the reagent to be used for detection. Detectable substances may 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, luciferin, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazineamine luciferin, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials such as luciferase; radioactive substances; positron-emitting metals; and magnetic metal ions.

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

[0128] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein offer several technical advantages compared to reagents that target CTLA4 only (e.g., prilimumab) or CD47 only (e.g., Sirpα). Compared to corresponding molecules with different CD47-binding domains (e.g., different Sirpα variants) or more flexible structures, the anti-CTLA4 / CD47 bispecific antibodies provided herein can achieve several technical advantages by, for example, including additional flexible linkers. For illustrative purposes, in some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) exhibit greater selectivity for both CTLA4 and CD47 double-positive cells. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) exhibit greater stability. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies provided herein (e.g., HX044) have fewer mismatch impurities (e.g., homodimers and / or incorrect 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 antibody (e.g., HX044) provided herein has limited mismatch impurities.

[0130] In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) selectively eliminate CTLA4 and CD47 positive cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Hematologic toxicity refers to side 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 in the TME and avoid nonspecific binding to peripheral cells, in some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) exhibit low hematologic toxicity. In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have improved safety (e.g., lower hematologic toxicity) compared to antibodies that target CD47 alone (e.g., Sirpα). Immune-related adverse events (irAEs) are inflammatory toxicities that occur when immune checkpoint inhibitors—such as anti-CTLA-4, anti-PD-1, and anti-PD-L1 therapies—disrupt immune autotolerance 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 multiple organ systems, including the skin, gastrointestinal tract, liver, endocrine glands, lungs, and nervous system. Clinically, irAEs can range from mild to severe and can manifest as dermatitis, colitis, hepatitis, endocrine disorders, pneumonia, or neuropathy. In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a low risk of triggering irAEs by reducing affinity for CTLA4+ / CD47- cells. In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a low risk of triggering cytokine release syndrome (CRS). In some embodiments, the bispecific antibodies disclosed herein (e.g., HX044) have a lower risk of triggering irAEs than immune checkpoint inhibitors with a single target (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 triggering CRS than immune checkpoint inhibitors with a single target (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4).

[0131] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein (1) has a high affinity for CTLA4 and CD47 double-positive 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; (5) has a higher affinity for CTLA4 and CD47 double-positive cells than for CTLA4 or CD47 single-positive cells; (6) selectively eliminates CTLA4 and CD47 positive cells via antibody-dependent cell-mediated cytotoxicity (ADCC); (7) has limited hematologic toxicity; (8) has limited immune-related adverse events (irAEs); (9) enhances macrophage-mediated phagocytosis; (10) enhances dendritic cell-mediated antigen presentation; or (11) has limited mismatch impurities; or any combination of (1)-(11).

[0132] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein (1) has a high affinity for CTLA4 and CD47 double-positive 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; (5) enhances macrophage-mediated phagocytosis; (6) enhances dendritic cell-mediated antigen presentation; or any combination of (1)-(6).

[0133] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein (1) has a higher affinity for CTLA4 and CD47 dual-positive cells than for CTLA4 or CD47 single-positive cells; (2) selectively eliminates CTLA4 and CD47 positive cells via antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematologic toxicity; (4) has limited immune-related adverse events (irAEs); or any combination of (1)-(4).

[0134] The technical efficacy of the anti-CTLA4 / CD47 bispecific antibody (e.g., HX044) provided herein can be measured in 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 Experimental Section below) for cells expressing only CD47, only CTLA4, or both CD47 and CTLA4 can be measured by, for example, ELISA or FACS. We expect to confirm that HX044 has a lower affinity for cells expressing CD47 but not CTLA4 than the reference bsAb, but the same or higher affinity for cells expressing both CD47 and CTLA4 than the reference bsAb.

[0135] The improved safety of HX044 could be measured by FACS analysis, which showed its effects on RBC, HGB, and platelet counts in an syngeneic mouse model (hCTLA4×hCD47×hSIRPα HuGemm C57BL / 6J mice) (see exemplary studies described in the Experimental section below). We expect to confirm that HX044 has a milder effect on RBC / PLT depletion and a reduction in CTLA-4-related irAEs compared to the reference bsAb. 6.3 Polynucleotides, Carriers, and Cells

[0136] This document provides polynucleotides encoding at least one light chain or one heavy chain of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the polynucleotides provided herein encode a polypeptide, such as a light chain or a heavy chain, of the bispecific antibody. In some embodiments, the polynucleotides provided herein encode more than one polypeptide. In some embodiments, the polynucleotides provided herein may encode, for example, the light chain and heavy chain of the bispecific antibody provided herein. Cistrons can be separated by, for example, internal ribosome entry sites (IRES) or 2A elements. As understood in the art, an IRES refers to a nucleotide sequence in the expression cassette that, when transcribed into mRNA, can directly recruit ribosomes without prior scanning of the untranslated region of the mRNA by ribosomes. As understood in the art, 2A elements encoding self-cleaving short 2A peptides (approximately 20 amino acids) provide a mechanism for the subsequent separation of target polypeptides produced in equimolar amounts. The self-cleaving 2A peptide family 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 adapters recognized in the art may be suitable for the constructs of this disclosure (e.g., constructs encoded by the nucleic acids of this disclosure). Those skilled in the art will also understand that other polycistronic constructs may be suitable for the uses provided herein.

[0137] In some embodiments, this document provides polynucleotides encoding peptide chains C1, C2, C3, or any combination thereof of the disclosed anti-CTLA4 / CD47 bispecific antibody having a KIH structure. In some embodiments, this document provides polynucleotides encoding C1, C2, C3, or any combination thereof of a bispecific antibody represented as HX044. In some embodiments, this document provides a plurality of polynucleotides collectively encoding C1, C2, and C3 of a bispecific antibody represented as HX044.

[0138] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of a polypeptide, as well as polynucleotides that include other coding and / or non-coding sequences. The polynucleotides disclosed in this invention can be in the form of RNA or DNA. The DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be a coding strand or a non-coding (antisense) strand. The polynucleotides disclosed herein can be mRNA.

[0139] This disclosure also provides variants of the polynucleotide described herein, wherein said variants have a polynucleotide sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with a 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 having at least about 95% identity with a polynucleotide sequence" means that the nucleotide sequence of said polynucleotide is identical to the reference sequence, except that said polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence having at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in said reference sequence may be deleted or substituted with another nucleotide, or some nucleotides of up to 5% of the total nucleotides in said reference sequence may be inserted into said reference sequence. These mutations in the reference sequence can occur at the 5' or 3' end of the reference nucleotide sequence or anywhere between those end positions, either alone in the nucleotides of the reference sequence or in one or more adjacent groups within the reference sequence.

[0140] The polynucleotide variant may contain alterations in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variant contains alterations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, the polynucleotide variant contains silent substitutions that result in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a specific host (e.g., changing codons in human mRNA for bacterial hosts, such as those preferred in Escherichia coli). In some embodiments, the polynucleotide variant contains at least one silent mutation in a non-coding or coding region of the sequence.

[0141] In some embodiments, polynucleotide variants are generated to regulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has decreased expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0142] In some embodiments, the polynucleotide includes the coding sequence of a polypeptide (e.g., an antibody) fused within the same reading frame to a polynucleotide (e.g., a leader sequence that functions as a secretory sequence for controlling polypeptide transport) expressed and secreted from the host cell. 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 contains the coding sequence of a polypeptide (e.g., an antibody) fused to a marker or tag sequence within the same reading frame. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS-tag), which allows for efficient purification of the polypeptide fused to the marker. In some embodiments, when using a mammalian host (e.g., COS-7 cells), 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 may 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, vectors comprising the polynucleotides disclosed herein are also provided. As used herein, the term "vector" and its grammatical equivalents refer to a medium that can be introduced into a host cell to carry genetic material (e.g., a polynucleotide sequence) that can be replicated and / or expressed in the host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes may provide, for example, antibiotic or toxin resistance, supplement nutrient deficiencies, or provide critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well known in the art. When two or more polynucleotides are co-expressed, the two polynucleotides may be inserted, for example, into a single expression vector or different expression vectors. For single-vector expression, the encoding polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the polynucleotide into the host cell can be confirmed using methods well known in the art. Those skilled in the art will understand that the polynucleotide is expressed in sufficient quantities to produce the desired product, and will also understand that expression levels 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 comprise polynucleotides encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, recombinant expression vectors are provided herein that can be used to amplify and express polynucleotides encoding at least one polypeptide chain of the anti-CTLA4 / CD47 bispecific antibody described herein. For example, the recombinant expression vector may be a reproducible 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, operably linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. In some embodiments, viral vectors are used. The DNA regions are “operably linked” when they are functionally related to each other. For example, if a promoter controls transcription of a sequence, the promoter is operably linked to a coding sequence; or if the location of a ribosome-binding site allows translation, it is operably linked to a coding sequence. In some embodiments, structural elements designed for use in certain expression systems include leader sequences that enable the host cell to secrete the translated protein extracellularly. In some embodiments, where the recombinant protein is expressed in the absence of a leader sequence or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0147] Examples of vectors are plasmids, autonomously replicating sequences, and translocation factors. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from *E. coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with broader host ranges, such as M13 and other filamentous single-stranded DNA phages. Other exemplary vectors include, without limitation, plasmids, phages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), phages such as λ phage or M13 phage, and animal viruses. Examples of animal virus classes useful as vectors include, without limitation, retroviruses (including lentiviruses), adenoviruses, adenovirus-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (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 lentivirus-mediated gene transfer and expression in mammalian cells. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Exemplary transposon systems, such as Sleeping Beauty and PiggyBac, can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4): 501–510 (1997); Cadiñanos et al., (2007) Nucleic AcidsResearch. 35 (12): e87).

[0148] In some embodiments, the vector is a cell-free gene vector or an extrachromosomally maintained vector. As used herein, the term "cell-free gene" refers to a vector capable of replication but not integrated into the host chromosomal DNA and not gradually lost from dividing host cells, and also indicates extrachromosomal or cell-free gene replication of the vector. The vector is engineered to have a sequence encoding a DNA replication origin or "ori" from a lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically corresponding to the oriP of EBV as the replication origin of a lymphotropic herpesvirus or gamma herpesvirus. In some embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), squirrel monkey herpesvirus (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, the host cell contains a viral replication transactivator protein that activates replication.

[0149] The “expression control sequences,” “control elements,” or “regulatory sequences” present in expression vectors are those vector untranslated regions—origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (ribosome binding site sequences or Kozak sequences), introns, polyadenylated sequences, and 5' and 3' untranslated regions—that interact with host cell proteins to carry out transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and host used, any number of suitable transcriptional and translational elements can be used, including ubiquitous promoters and inducible promoters.

[0150] The illustrative and ubiquitous expression control sequences that can be used in this disclosure include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) promoter (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Raúl's sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-α (EF1α) promoter, the early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa β, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin (β-KIN), and 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] Illustrative examples of inducible promoters / systems include (but are not limited to) steroid-inducible promoters, such as promoters for genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the “GeneSwitch” mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, etc. The bispecific antibodies described herein can be generated by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise stated, the practice of this invention utilizes conventional techniques in molecular biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of this art. These techniques are described in the references cited herein and are fully explained therein.See, 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 ed., 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 annual update); Current Protocols in Immunology, John Wiley & Sons (1987 and annual update); 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 ed., Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc.; Each of the above references is incorporated herein by reference in its entirety.

[0152] This invention also discloses cells comprising the 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 C1, C2, and C3 encoding the anti-CTLA4 / CD47 bispecific antibody disclosed herein that has a KIH structure. In some embodiments, the cells provided herein comprise multiple polynucleotides C1, C2, and C3 that collectively encode the anti-CTLA4 / CD47 bispecific antibody disclosed herein that has a KIH structure.

[0153] Cells comprising the vectors disclosed herein are also considered. In some embodiments, a host cell is provided herein that comprises a vector containing the polynucleotides disclosed herein. In some embodiments, the host cell provided herein comprises a vector or multiple vectors that collectively contain a polypeptide chain encoding the anti-CTLA4 / CD47 bispecific antibody described herein. In some embodiments, the host cell provided herein produces the anti-CTLA4 / CD47 bispecific antibody described herein.

[0154] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (mouse fibroblast-derived), C127 (mouse mammary tumor-derived), 3T3 (mouse fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and their variants. Mammalian expression vectors may contain non-transcriptional elements such as origin of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanked non-transcriptional sequences, and 5' or 3' non-translational sequences such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust approach for producing correctly folded and biologically functional proteins. Baculovirus systems used to produce heterologous proteins in insect cells are well known to those skilled in the art. 6.4 Production Method

[0155] This document also provides methods for generating the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, the bispecific antibody disclosed herein consists of more than one polypeptide chain, which may be generated separately or together. In some embodiments, the methods provided herein generate at least one polypeptide chain of the bispecific antibody disclosed herein. In some embodiments, the methods provided herein generate all polypeptide chains of the bispecific antibody disclosed herein.

[0156] Bispecific antibodies or peptides described herein can be generated and isolated using methods known in the art. Peptides can be synthesized completely 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 performed using a variety of solid-phase techniques (see, for example, Roberge, Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)) and, for example, automated synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Combinatorial methods can also be used to synthesize peptides. Synthetic residues and peptides can be synthesized using a variety of procedures and methods known in the art (see, for example, Organic Syntheses Collective Volumes, Gilman et al. (eds.), 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 alterations, derivatizations, 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 peptide sequences, variants, fusions and chimeras of the present invention, as well as alterations, derivatives, substitutions and modifications thereof.

[0157] A variety of host-expression vector systems can be used to recombinantly express the bispecific antibodies described herein or one or more of their polypeptide chains. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells controlled by appropriate promoters. Cloning and expression vectors suitable 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. These host-expression systems represent, through which the coding sequences of the bispecific antibodies described herein can be produced and subsequently purified, and also represent cells that can express the bispecific antibodies described herein in situ when transformed or transfected with suitable polynucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing sequences encoding compounds described herein (e.g., *Escherichia coli* and *Bacillus subtilis*); and yeast transformed with recombinant yeast expression vectors containing sequences encoding compounds described herein (e.g., *Pichia pastoris*). (pichia); insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing a sequence encoding a compound described herein; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) containing a sequence encoding a molecular compound described herein, or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing a sequence encoding a molecular compound described herein; or mammalian cell systems having 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) (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytes (see U.S. Patent No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell).

[0158] In bacterial systems, a variety of expression vectors can be advantageously selected based on their intended use for the protein to be expressed. For example, when producing large quantities of such a protein, a vector that guides high-level expression of an easily purified protein product may be desirable for the production of the pharmaceutical composition of the bispecific antibody described herein. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983), EMBO J. 2: 1791-1794); the pIN vector (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), J. Biol. Chem. 24:5503-5509); etc. The pGEX vector can also be used to express peptides as fusion proteins with glutathione S-transferase (GST). Typically, these proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites, thereby allowing the release of cloned target gene products from the GST moiety.

[0159] Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. In mammalian host cells, several virus-based expression systems can be used. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (mouse fibroblast-derived), C127 (mouse mammary tumor-derived), 3T3 (mouse fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and their variants. Mammalian expression vectors may contain non-transcriptional elements such as origin of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanked non-transcriptional sequences, and 5' or 3' non-translational sequences such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. The alfalfa silver-striped armyworm (Autographa californica) nucleopolyhedrovirus (AcNPV) was used as a vector to express exogenous genes.

[0160] Additionally, the expression of the inserted sequence can be selectively modulated, or the host cell line of the gene product can be altered and processed in a desired specific manner. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for protein function. For example, in some embodiments, the antibody described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a multi-protein precursor) that requires proteolytic cleavage via natural or recombinant cellular mechanisms to form a single polypeptide of the bispecific antibody described herein. Therefore, this invention discloses multi-protein precursor molecules engineered with nucleic acid sequences to encode polypeptides comprising the bispecific antibodies described herein, including coding sequences capable of directing post-translational cleavage of said multi-protein precursor. Post-translational cleavage of the multi-protein precursor results in the production of the polypeptide of the bispecific antibody described herein. Post-translational cleavage of precursor molecules of peptides comprising the compounds described herein can occur in vivo (i.e., within host cells via natural or recombinant cellular systems / mechanisms, e.g., cleavage by furin protease at appropriate sites) or in vitro (e.g., incubation of the polypeptide chain in a composition comprising a protease or peptidase of known activity and / or in a composition comprising conditions or reagents known to promote the desired proteolytic activity). Purification and modification of recombinant proteins are well known in the art, and thus the design of multi-protein precursors can include several implementations readily understood by those skilled in the art. Any known protease or peptidase in the art can be used for the described modifications of the precursor molecule.

[0161] Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the proper modification and processing of expressed exogenous proteins. For this purpose, eukaryotic host cells with appropriate cellular mechanisms for the processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. These 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, and 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 engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. Following the introduction of exogenous DNA, engineered cells can be allowed to grow in enrichment media for 1–2 days and then transferred to selection media. Selectable markers in the recombinant plasmid confer selection tolerance and allow cells to stably integrate the plasmid into their chromosomes and grow to form focal points (foci), which in turn can be cloned and amplified into cell lines. This approach can be advantageously used to engineer cell lines expressing the compounds described herein. These engineered cell lines can be particularly useful in the screening and evaluation of compounds that interact directly or indirectly with the compounds described herein.

[0163] Several selection systems can be used, including (but not limited to) herpes simplex virus thymidine kinase (Wigler et al., (1977), Cell 11: 223-232), hypoxanthine-guanine transphosphoribosylase (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. Additionally, antimetabolite tolerance can be used as a selection basis for the following genes: dhfr, which confers tolerance to methotrexate (Wigler et al. (1980) PNAS 77:3567-3570; O'Hare et al. (1981) PNAS, 78: 1527-1531); gpt, which confers tolerance to mycophenolic acid (Mulligan et al. (1981) PNAS, 78: 2072-2076); neo, which confers tolerance to the aminoglycoside G-418 (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 hygro, which confers resistance to hygromycin (Santerre et al. (1984) Gene 30: 147-156). Methods commonly known in the field of recombinant DNA technology that can be used are described in Chapters 12 and 13 of Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.

[0164] The expression levels of the bispecific antibodies or their polypeptide chains described herein can be increased by vector amplification (for the review, see Bebbington and Hentschel, The use of vectors based on geneamplification for the expression of cloned genes in mammalian cells in DNAcloning, Vol. 3 (Academic Press, New York, 1987)). When the markers in the vector systems described herein are amplifiable, increased levels of inhibitors present in host cell culture will increase the number of marker gene copies. Since the amplified region is associated with the nucleotide sequence of the target protein, this will also increase the production of the target protein (Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0165] Host cells can be co-transfected with more than one expression vector, each encoding a polypeptide chain of the bispecific antibody described herein. The vectors may contain the same optional markers, enabling all polypeptides to be expressed equally. Alternatively, a single vector encoding two or more polypeptides may be used. The coding sequence of the polypeptides of the compounds described herein may comprise cDNA or genomic DNA.

[0166] Once the bispecific antibody or peptide described herein has been recombinantly expressed, it can be purified by any method known in the art for the purification of peptides, multiproteins, or antibodies (e.g., similar to antigen-selective antibody purification protocols), such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, specifically by affinity for a specific antigen (optionally, after protein A selection when said compound contains an Fc domain (or a portion thereof), and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for peptide or antibody purification.

[0167] This document provides a method for generating the anti-CTLA4 / CD47 bispecific antibody or the polypeptide chain of the bispecific antibody described herein, the method comprising obtaining the cells described herein and expressing the polynucleotides described herein in the cells. In some embodiments, the method provided herein includes culturing the cells under conditions that allow expression of the bispecific antibody. In some embodiments, the method further includes isolating and purifying the bispecific antibody or polypeptide chain described herein.

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

[0169] The reactivity of the anti-CTLA4 / CD47 bispecific antibodies described herein with the target antigen can be further tested by Western blotting. Other methods known in the art for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-CTLA4 / CD47 bispecific antibodies described herein include, for example, biomembrane 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] Unless otherwise stated, the practice of this invention utilizes conventional techniques from 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 this art. These techniques are described in the references cited herein and are fully explained therein.See, 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 ed., 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 annual update); Current Protocols in Immunology, John Wiley & Sons (1987 and annual update); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRLPress; Birren et al. (ed.) (1999) Genome Analysis: A Laboratory Manual, ColdSpring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, 2nd ed., Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc.; The entire contents of each of the above references are incorporated herein by reference. 6.5 Composition

[0171] This document provides compositions comprising the anti-CTLA4 / CD47 bispecific antibody disclosed herein, wherein the bispecific antibody has a purity of 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-reducing 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 mismatch 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 less than 10% mismatch impurities. In some embodiments, the compositions provided herein have less than 8% mismatch impurities. In some embodiments, the compositions provided herein have less than 5% mismatch impurities. In some embodiments, the compositions provided herein have less than 3% mismatch impurities. In some embodiments, the compositions provided herein have less than 2% mismatch impurities. In some embodiments, the compositions provided herein have less than 1% mismatch impurities. In some embodiments, the compositions provided herein have no detectable mismatch impurities when measured by non-reducing SDS-PAGE. The mismatch impurities include, for example, homodimers and / or incorrect heterodimers. In some embodiments, the compositions provided herein have no detectable homodimers when measured by non-reducing SDS-PAGE. In some embodiments, the compositions provided herein have no detectable incorrect heterodimers when measured by non-reducing SDS-PAGE.

[0172] This document also provides 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 available carrier. In some embodiments, the pharmaceutical composition is useful in treating inflammatory diseases or autoimmune diseases.

[0173] The terms "drug-compatible carrier" or "drug-compatible excipient" refer to materials suitable for administration to an individual medicament together with an active agent without causing unwanted biological effects or interactions in a harmful manner with any other component of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions disclosed herein may also comprise one or more buffer systems, preservatives, isotropic agents, chelating agents, stabilizers, and / or surfactants, and various combinations thereof. The use of preservatives, isotropic 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 comprise the anti-CTLA4 / CD47 bispecific antibody provided herein. The anti-CTLA4 / CD47 bispecific antibody may be present in various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise 1-1000 mg / mL of the anti-CTLA4 / CD47 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions comprise 10-500 mg / mL, 10-400 mg / mL, 10-300 mg / mL, 10-200 mg / mL, 10-100 mg / mL, 20-100 mg / mL, or 50-100 mg / mL of the anti-CTLA4 / CD47 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions provided herein comprise about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 150 mg / mL, about 180 mg / mL, about 200 mg / mL, about 300 mg / mL, about 500 mg / mL, about 800 mg / mL, or about 1000 mg / mL of the anti-CTLA4 / CD47 bispecific antibody provided herein. Those skilled in the art can readily adjust the dosage; for example, a decrease in purity may necessitate a dose increase.

[0175] Available carriers for pharmaceuticals that can be used in the compositions provided herein include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc. 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., the anti-CTLA4 / CD47 bispecific antibody) may be coated into the material to protect the active ingredient from the effects of acids and other natural conditions that could inactivate it.

[0176] This document also provides pharmaceutical compositions or formulations for improving 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 swelling agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0177] Buffers useful in the pharmaceutical compositions or formulations disclosed herein may be weak acids or bases used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheological, viscosity, and other properties may also be based 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) and an isotonic agent, and potentially pH-adjusted by 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 between about 2 and about 10, or between about 4 and about 8.

[0178] Stabilizers are added to pharmaceutical products to stabilize them. These agents can stabilize proteins in different 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, raffinose, or maltose; polyols such as glycerol, mannitol, sorbitol, cyclodextrin, or glucans of any type and molecular weight; or PEG. In some embodiments, stabilizers are selected to maximize the stability of the antibody in the lyophilized formulation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0179] Expanding agents can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby aiding in accurate measurement and handling. Common expanding agents include (but are not limited to) lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0180] Surfactants are amphiphilic substances having both 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 oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocoamide MEA, cocoamide DEA, polysorbate, or dodecyl dimethylamine 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, dispersions, 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 composition disclosed herein is freeze-dried, and a physician or patient adds a solvent and / or diluent thereto before use.

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

[0184] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate flowability can be maintained, for example, by using coating materials such as lecithin, in relation to the dispersion system, by maintaining the desired particle size, and by using surfactants.

[0185] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial presence can be ensured by sterilization procedures as described above and by including multiple antibacterial and antifungal agents, such as p-hydroxybenzoic acid, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Additionally, prolonged absorption in injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0186] Suitable carriers for pharmaceuticals include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Such carriers and reagents for the use of pharmaceutically active substances are known in the art. In some embodiments, pharmaceutical compositions comprising the anti-CTLA4 / CD47 bispecific antibody or cells provided herein are provided, wherein said compositions are suitable for topical application.

[0187] Pharmaceutical compositions or formulations must generally be sterile and stable under manufacturing and storage conditions. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating, such as lecithin, to maintain the desired particle size and by using a surfactant. In most cases, the composition may include an isotonic agent, such as sugars, polyols, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate and gelatin.

[0188] As needed, a sterile injectable solution can be prepared by incorporating the required amount of the active compound into a suitable solvent having one or a combination of the components listed above, followed by microfiltration sterilization. Typically, a dispersion system is prepared by incorporating the active compound into a sterile medium comprising a base dispersion medium and other required components from those listed herein. In the case of sterile powders used to prepare sterile injectable solutions, some preparation methods include vacuum drying and freeze-drying (lyophilization), which yield a powder containing the active ingredient plus any desired components from a previously 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 may vary. In some embodiments, the amount of active ingredient that can be combined with a carrier material is the amount that produces a therapeutic effect. Typically, this amount will be in the range of about 0.01% to about 99% of the active ingredient, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient combined with the carrier in which the drug is available.

[0190] The pharmaceutical compositions disclosed herein can be prepared together with a carrier that protects the active ingredient from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Various methods for preparing these 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, Inc., New York, 1978.

[0191] This document also provides kits for preparing 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 available carrier in one or more containers. In another embodiment, the kit may comprise the anti-CTLA4 / CD47 bispecific antibody disclosed herein for administration to a subject. In specific embodiments, the kit comprises instructions for the preparation and / or administration of the anti-CTLA4 / CD47 bispecific antibody. 6.6 Methods and Applications

[0192] The antibodies, compositions, and methods described herein have a variety of in vitro and in vivo applications, including, for example, enhancement of immune responses, such as by inhibiting (or antagonizing) CTLA4 and / or CD47 (e.g., signal transduction). 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 a variety of diseases. Therefore, methods for altering immune responses in subjects are provided herein, the methods comprising administering the anti-CTLA4 / CD47 bispecific antibodies described herein to the subject, thereby altering the immune response in the subject. In some embodiments, the response is enhanced, stimulated, or upregulated.

[0193] In some embodiments, this document provides methods for inducing or stimulating immune cell activation, the methods comprising contacting immune cells with an effective amount of an anti-CTLA4 / CD47 bispecific antibody as described herein. In some embodiments, this document provides methods for inducing or stimulating immune cell proliferation, the methods comprising contacting immune cells with an effective amount of an anti-CTLA4 / CD47 bispecific antibody as described herein. In some embodiments, this document provides methods for reducing CTLA4 and / or CD47-mediated inhibition of immune cell proliferation, the methods comprising contacting immune cells with an effective amount of an anti-CTLA4 / CD47 bispecific antibody as described herein. In some embodiments, this document provides methods for inhibiting the interaction between CTLA4 and / or CD47 and their ligands on immune cells, the methods comprising contacting the immune cells with an effective amount of an anti-CTLA4 / CD47 bispecific antibody as described herein.

[0194] Subjects suitable for the methods of the present invention include patients in whom an enhanced immune response is desired. The methods are particularly suitable for treating patients with conditions that can be treated by enhancing an immune response, such as a T-cell-mediated immune response, e.g., an antigen-specific T-cell response. In some embodiments, the methods are particularly suitable for in vivo treatment of cancer. In some embodiments, methods for enhancing an immune response in subjects in need are provided herein, the methods comprising administering to the subject an effective amount of the anti-CTLA4 / CD47 bispecific antibody described herein. To achieve immune antigen-specific enhancement, the anti-CTLA4 / CD47 bispecific antibody described herein may be administered together with a target antigen, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or a virus).

[0195] Given the anti-CTLA4 / CD47 bispecific antibody described herein that stimulates or co-stimulates T cell responses, such as the ability of antigen-specific T cell responses, as well as the ability to deplete immunosuppressive T cells in the TEM,reg The ability to stimulate or co-stimulate T cell responses is described herein. This document provides in vitro and in vivo methods for stimulating, enhancing, or upregulating antigen-specific T cell responses, such as anti-tumor T cell responses, using the anti-CTLA4 / CD47 bispecific antibodies described herein. Any suitable indicator of antigen-specific T cell response can be used to measure it. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of said antibody and / or increased cytokine production in the presence of said antibody. In some embodiments, stimulation is achieved via interleukin-2 and / or interferon-γ production by antigen-specific T cells.

[0196] It also covers methods for stimulating an immune response (e.g., antigen-specific T-cell response) in a subject, the methods comprising administering the subject an anti-CTLA4 / CD47 bispecific antibody as described herein, thereby stimulating an immune response (e.g., antigen-specific T-cell response) in the subject. In some embodiments, the subject is a subject with a tumor and an immune response against the tumor is stimulated. The tumor may be a solid tumor or a liquid tumor, such as a hematologic 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 and CD47 positive. The subject may also be a subject with a virus and an immune response against the virus is stimulated.

[0197] In some embodiments, a method for inhibiting tumor cell growth in a subject is provided, the method comprising administering to the subject an anti-CTLA4 / CD47 bispecific antibody as described herein, thereby inhibiting tumor growth in the subject. A method for treating cancer in a subject in need is also provided herein, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody disclosed herein. The use of the bispecific antibody disclosed herein as a pharmaceutical agent is also provided herein. The use of the bispecific antibody disclosed herein in the treatment of cancer is further provided herein. The use of the bispecific antibody disclosed herein in the preparation of a pharmaceutical agent for treating cancer is also provided herein.

[0198] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody described herein is provided to subjects as adjuvant therapy. Treatment of subjects with cancer using the anti-CTLA4 / CD47 bispecific antibody described herein can result in prolonged survival, such as a long-lasting response relative to current standard of care; long-term survival for at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 years or more; or recurrence-free survival for at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5 or 10 years or more. In some embodiments, treatment of subjects with cancer using the anti-CTLA4 / CD47 bispecific antibody described herein prevents cancer recurrence or delays cancer recurrence (e.g.) for 3 months, 6 months, 9 months, 1, 2, 3, 4, 5 or 10 years or more.

[0199] Treatment of subjects with cancer with the anti-CTLA4 / CD47 bispecific antibody described in this article can result in, for example, stable disease, partial response, improved overall survival, improved disease-free survival, or enhanced progression-free survival.

[0200] In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein are not significantly toxic. 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 hematologic toxicity. In some embodiments, the anti-CTLA4 / CD47 bispecific antibodies described herein have limited intraepithelial adverse events (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 the immune system reaching the subject's immune system and then attacking the subject itself (e.g., an autoimmune response) or causing an overstimulation to the extent of, for example, anaphylaxis. Therefore, in some embodiments, the anti-CTLA4 / CD47 bispecific antibodies do not induce anaphylactic reactions.

[0201] In some embodiments, treatment of subjects with the anti-CTLA4 / CD47 bispecific antibody described herein does not induce significant inflammatory responses, such as immune-mediated pneumonia, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, treatment of subjects with the anti-CTLA4 / CD47 bispecific antibody described herein does not induce significant cardiac symptoms, such as ventricular arrhythmias; eye conditions, such as iridocyclitis; infusion-related reactions; elevated amylase or lipase levels; neurological disorders, such as vertigo, peripheral neuropathy, and sensory neuropathy; skin and subcutaneous tissue disorders, such as rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo, or psoriasis; respiratory, thoracic, and mediastinal disorders, such as cough; fatigue; nausea; decreased appetite; constipation; arthralgia; or diarrhea.

[0202] In some implementations, the combination of anti-CTLA4 / CD47 bispecific antibodies with another cancer therapy, such as compounds that stimulate the immune system (e.g., immunotumor agents), provides a synergistic antitumor effect.

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

[0204] In some embodiments, the anti-CTLA4 / CD47 bispecific antibody can reduce immunosuppression mediated by the CTLA4 and / or CD47 signaling pathways, thereby promoting the activity of immune cells in eliminating, lysing, and / or killing cancer cells. In some embodiments, the method includes 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 antibody described herein can block the interaction between CTLA4 and B7-1 (CD80) / B7-2 (CD86) as well as the interaction between CD47 and SIRPα. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody can specifically target cancer cells expressing CTLA4 / CD47 in vivo, thereby delivering therapeutic effects that eliminate, lyse, and / or kill cancer cells.

[0206] In some embodiments, this document provides a method of treating a tumor or cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of tumors or cancer. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody provided herein in the preparation of a tumor or cancer therapeutic agent. 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, this document provides a method of treating a tumor or cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, this document provides the use of the pharmaceutical compositions disclosed herein in treating a tumor or cancer. In some embodiments, this document provides the use of the pharmaceutical compositions provided herein in preparing an agent for treating a tumor or cancer.

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

[0209] The anti-CTLA4 / CD47 bispecific antibody described herein can be administered as a sustained-release formulation, in which case frequent dosing is not required. Dosage and frequency can be varied based on the half-life of the anti-CTLA4 / CD47 bispecific antibody in the patient. In therapeutic applications, sometimes relatively high doses at relatively short intervals are required until disease progression is reduced or terminated and until the patient shows partial or complete improvement in disease symptoms.

[0210] The anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition provided herein can be administered to a subject 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 administration routes, such as by injection or infusion, or direct administration to the thymus. As used herein, the phrase "parenteral administration" refers to a form of administration other than enteral and local administration, which is generally by injection and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intrasacral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. In some embodiments, subcutaneous administration is used. In some embodiments, intravenous administration is used. In some embodiments, oral administration is used. In some embodiments, the anti-CTLA4 / CD47 bispecific antibody provided herein can be administered to a subject via intratumoral injection, peritumoral injection, juxtatumoralinjection, intralesional injection, and / or injection into the tumor-draining lymph nodes, or any injection of the antitumor agent intended to penetrate into the primary lymph nodes adjacent to the targeted solid tumor, or virtually any targeting tumor. In some embodiments, the antibody provided herein can be locally delivered to the tumor using well-known methods, including (but not limited to) hepatic or aortic pumps; perfusion of the extremities, lungs, or liver; in the portal vein; via venous shunt; in a cavity; or in a vein near the tumor. In another embodiment, the antibody provided herein can be administered systemically. In a preferred embodiment, the antibody is administered locally at the tumor site. Intratumoral administration of the antibody is also possible, for example, by direct injection of cells at the tumor site and / or direct injection of cells into the tumor vascular system. For example, in the case of malignant pleural diseases, mesothelioma, or lung cancer, administration is preferably performed via intrapleural delivery (see Adusumilli et al., Science Translational Medicine 6(261):261ra151 (2014)). Those skilled in the art can select a suitable delivery method based on the type of cancer to be treated and / or the location of the tumor. The antibody can be introduced by injection or catheter. In one embodiment, the antibody is administered pleurally to the recipient, for example, using an intrapleural catheter.

[0211] Cancers or tumors treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition provided herein include those that typically respond to immunotherapy and those that typically do not. In some embodiments, the cancer has high microsatellite instability. In some embodiments, the cancer is metastatic, refractory, or recurrent.

[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 hematologic 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 to be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is melanoma. In some embodiments, the solid tumor to be treated with the anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition disclosed herein is colon cancer. Accordingly, in some embodiments, this document provides a method of treating melanoma in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of melanoma. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody provided herein in the preparation of a melanoma therapeutic agent. In some embodiments, the melanoma is CTLA4 positive. In some embodiments, the melanoma is CD47 positive. In some embodiments, the melanoma is both CTLA4 and CD47 positive. In some embodiments, this document provides a method of treating colon cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody disclosed herein. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody disclosed herein in the treatment of colon cancer. In some embodiments, this document provides the use of the anti-CTLA4 / CD47 bispecific antibody provided herein in the preparation of a colon cancer therapeutic agent. In some embodiments, the colon cancer is CTLA4 positive. In some embodiments, the colon cancer is CD47 positive. In some embodiments, the colon cancer is both CTLA4 and CD47 positive.

[0213] In cancer treatment, the elimination of cancer or tumor cells in a subject can occur, but any clinical improvement constitutes a benefit. Antitumor effects can be demonstrated by a reduction in tumor volume, a decrease in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancerous condition. Antitumor effects can also be demonstrated primarily by the ability of the antibodies or pharmaceutical compositions described herein to prevent tumorigenesis. In some embodiments, an "antitumor effect" can be demonstrated by a reduction in cancer-induced immunosuppression. Clinical improvement includes a reduction in the risk or rate of cancer or tumor development or a reduction in the pathological consequences of cancer or tumor. It should also be understood that methods of treating cancer can include any effect of improving cancer-related signs or symptoms. These signs or symptoms include (but are not limited to) a reduction in tumor burden, including inhibition of tumor growth, slowing of tumor growth rate, reduction of tumor size, reduction of tumor number, and elimination of tumor, all of which can be measured using conventional tumor imaging techniques well known in the art. Other cancer-related signs or symptoms 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 antibody or pharmaceutical composition disclosed herein can reduce the number of tumor cells in a subject, reduce tumor size, and / or eradicate the tumor. Methods for monitoring a patient's response to administration of the pharmaceutical composition disclosed herein are known in the art and can be used according to the methods disclosed herein.

[0215] In some embodiments, antitumor effects have been observed in subjects with tumors or cancer who have received the anti-CTLA4 / CD47 bispecific antibody described herein as a monotherapy (i.e., not in combination with another therapeutic agent). In some embodiments, tumor burden has been reduced in subjects with tumors or cancer who have received the anti-CTLA4 / CD47 bispecific antibody described herein as a monotherapy (i.e., not in combination with another therapeutic agent).

[0216] In the methods disclosed herein, a therapeutically effective amount of the disclosed anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition is administered to a subject requiring cancer treatment. The subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, these individuals do not have clinically measurable tumors. However, they are suspected of being at risk of disease progression near the primary tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk individuals. This subdivision is based on characteristics observed before or after initial treatment. These characteristics are known in the clinical field and are appropriately defined for different types of cancer. Typical characteristics of the high-risk subgroup include those in which tumors have invaded adjacent tissues or show involvement of lymph nodes.

[0217] The anti-CTLA4 / CD47 bispecific antibody or pharmaceutical composition provided herein can be administered using medical devices known in the art. For example, in some embodiments, a needle-free subcutaneous injection device, such as those disclosed in the following: U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules used for the purposes described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system with multiple compartments; and U.S. Patent No. 4,475,196, which discloses 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 implementations, anti-CTLA4 / CD47 bispecific antibodies are administered to patients with cancer that shows an inadequate response to prior treatment or continues to progress after prior treatment, such prior treatment being, for example, prior treatment with immuno-oncology or immunotherapy drugs. Alternatively, anti-CTLA4 / CD47 bispecific antibodies may be administered to patients with cancer that is refractory or tolerable, either inherently refractory or tolerable, or in which a tolerable or refractory state has been acquired. For example, anti-CTLA4 / CD47 bispecific antibodies may be administered alone or in combination with another therapy to treat subjects who do not respond or respond inadequately to a first therapy or to subjects whose disease progression has been observed.

[0219] In some implementations, the anti-CTLA4 / CD47 bispecific antibody is administered to patients who have not previously received immuno-oncology agents, such as PD-1 pathway antagonists or PD-L1 pathway antagonists (i.e., those not treated with immuno-oncology agents). Methods of treating subjects with cancer with the anti-CTLA4 / CD47 bispecific antibody may include administering a therapeutically effective amount of the anti-CTLA4 / CD47 bispecific antibody to a subject with cancer cells or TIL cells expressing CTLA4 and / or CD47.

[0220] Anti-CTLA4 / CD47 bispecific antibodies can be administered in conjunction with standard care. They can also be used as maintenance therapy, for example, in treatments aimed at preventing tumor development or recurrence. Furthermore, they can be administered in conjunction with another treatment, such as radiation therapy, surgery, or chemotherapy. For instance, anti-CTLA4 / CD47 bispecific antibodies can be used as adjuvant therapy 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 a monotherapy or as the sole immunostimulatory therapy. Anti-CTLA4 / CD47 bispecific antibodies can also be combined with immunogenic agents 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 peptides of melanoma antigens such as gp100, MAGE antigen, Trp-2, MARTI, and / or tyrosinase, or peptides transfected with tumor cells expressing the cytokine GM-CSF.

[0222] Combination therapies using agents with different mechanisms of action can result in additive or synergistic effects. Combination therapies can allow for lower doses of each agent than those used in single therapies, thereby reducing the toxicity and / or increasing the therapeutic index of the agents disclosed herein. Combination therapies can reduce the likelihood of the development of resistant cancer cells. In some embodiments, other therapies result in an increased therapeutic index of the antibody or pharmaceutical composition described herein. In some embodiments, other therapies result in reduced 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 other therapies. In some embodiments, the other therapies may be surgical resection, radiotherapy, or chemotherapy.

[0223] Other therapies 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 administration of a single pharmaceutical formulation or co-administration of different formulations, or in any order but generally within a time period that allows all active agents to exert their biological activity simultaneously. Those skilled in the art can readily determine appropriate regimens for combining the pharmaceutical compositions described herein with other therapies, including the timing and dosage of other agents used in the combination therapy, based on the needs of the subject to be treated. 6.7 Exemplary Implementation

[0224] Implementation 1. A bispecific antibody 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 comprises VL CDR1, VL CDR2 and VL CDR3 having the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and wherein the VH comprises VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively; and (ii) a CD47 binding domain comprising an extracellular domain of SIRPα or a variant thereof.

[0225] Implementation Method 2. The bispecific antibody according to Implementation Method 1, wherein the VL has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 7, and the VH has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 8.

[0226] Implementation Method 3. The bispecific antibody according to Implementation Method 2, wherein the VL and VH have the amino acid sequences shown in SEQ ID NO: 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] Implementation Method 5. The bispecific antibody according to Implementation Method 4, wherein the CD47 binding domain has an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9.

[0229] Implementation Method 6. The bispecific antibody according to Implementation Method 5, wherein the CD47 binding domain has the amino acid sequence shown in SEQ ID NO: 9.

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

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

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

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

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

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

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

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

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

[0239] Implementation 16. A bispecific antibody according to any one of Implementations 7 to 10, wherein (i) the CL region is κ CL (Cκ; SEQ ID NO: 21) or λ 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 mortar-Fc region and the gluten-Fc region each have the following amino acid sequences: (1) SEQ ID NO: 31 and 35; (2) SEQ ID NO: 32 and 36; (3) SEQ ID NO: 33 and 37; or (4) SEQ ID NO: 34 and 38; or a variant thereof having up to 10 amino acid substitutions.

[0240] Implementation Method 17. The bispecific antibody according to Implementation Method 16, wherein the CL region, CH1 domain, mortar-Fc region and stolon-Fc region have the following amino acid sequences: (1) SEQ ID NO: 21, 41, 31 and 35; (2) SEQ ID NO: 21, 41, 32 and 36; (3) SEQ ID NO: 21, 41, 33 and 37; or (4) SEQ ID NO: 21, 41, 34 and 38.

[0241] Implementation Method 18. The bispecific antibody according to Implementation Method 7, wherein C1 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 51; C2 has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 52; and C3 has an amino acid sequence having at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO: 53.

[0242] Implementation Method 19. The bispecific antibody according to Implementation Method 18, wherein C1, C2 and C3 have the amino acid sequences shown in SEQ ID NO: 51, 52 and 53, respectively.

[0243] Implementation 20. A bispecific antibody according to any one of Implementation 1 to 19, wherein the bispecific antibody (1) has a high affinity for CTLA4 and CD47 double positive cells; (2) consumes 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)-(6).

[0244] Implementation Method 21. A bispecific antibody according to any one of Implementation Methods 1 to 20, wherein the bispecific antibody (1) has a higher affinity for CTLA4 and CD47 dual-positive cells than for CTLA4 or CD47 single-positive cells; (2) selectively eliminates CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematologic toxicity; or (4) has limited immune-related adverse events (irAEs); or any combination of (1)-(4).

[0245] Embodiment 22. A bispecific antibody according to any one of Embodiments 1 to 21, wherein the bispecific antibody has limited mismatch impurities.

[0246] Example 23. A composition comprising a bispecific antibody according to any one of Examples 1 to 22, wherein the purity of the bispecific antibody is at least 95%, wherein 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 available carrier.

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

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

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

[0251] Embodiment 28. A carrier comprising the polynucleotides described in Embodiment 25 or 26.

[0252] Embodiment 29. A cell comprising one or more polynucleotides according to any one of Embodiments 25 to 27, or a carrier according to Embodiment 28.

[0253] Embodiment 30. A method for preparing a bispecific antibody that specifically binds to human CTLA4 and human CD47, the method comprising culturing cells according to Embodiment 29 while allowing expression of the bispecific antibody.

[0254] Implementation Method 31. A method for treating cancer in a subject in need of it, comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of Implementation Methods 1 to 22.

[0255] Implementation Method 32. The method according to Implementation Method 31, wherein the subject is a human being.

[0256] Embodiment 33. Use of a bispecific antibody according to any one of Embodiments 1 to 22 as a pharmaceutical agent.

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

[0258] Embodiment 35. Use of a bispecific antibody according to any one of Embodiments 1 to 22 for the preparation of a medicament for treating cancer. 6.8 Experiment

[0259] Unless otherwise stated, the following embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be considered as limited to the following embodiments, but should be considered as covering any and all changes that become apparent from the teachings provided herein.

[0260] In summary, the following data show that, compared to the reference antibody prilimumab and the reference protein SIRPα-Fc, the anti-CTLA4 / CD47 antibody HX044, with its 1+1 asymmetric structure consisting of an anti-CTLA4 VL / VH pair and a CD47-binding SIRPα domain, binds with reduced affinity and blocks ligand binding to the corresponding target. However, HX044 was found to bind with cells highly expressing CTLA4 and CD47 with particularly enhanced affinity and binding strength. Consistently, HX044 was found to bind with peripheral T cells (e.g., CD4, CD8, and T6 cells). reg It exhibits low binding, with CTLA4 and CD47 not expressed at high levels and hardly binding to RBCs, but binding to TIL-T. reg It has high binding and expresses CTLA4 at high levels.

[0261] HX044 also exhibited strong ADCC activity in cells with high CTLA4 expression levels. In humanized syngeneic models, HX044 showed strong antitumor activity against hCD47-MC38 tumors at very low doses (<0.2 mg / kg) and against huB16F10 melanoma tumors (conventional cryotherapy tumors) (5 mg / kg), while also exhibiting TIL-T activity. reg The reduction in [something]. Toxicological evaluation showed that HX044 had limited hematological toxicity at high dose levels (~10 mg / kg), but none at low dose levels. 6.8.1 Example 1: Construction of HX044

[0262] The following describes the generation of a bispecific antibody (BsAb) binding to human CTLA4 and CD47. Recombinant expression of the three peptide chains (C1, C2, and C3; Table 5B) of the bispecific antibody HX044 was achieved using three vector systems encoding three peptide chains. The genes encoding the three peptide chains were cloned into vectors respectively. BsAb expression vectors were prepared using an endotoxin-free plasmid DNA purification method (EndoFree plasmid kit, TransGen Biotech). After preparing the DNA vectors, HEK293 suspended cells were used for transient expression. DNA and PEI solution were mixed and added to the cells. HEK293 cells were harvested by centrifugation 6 days after transfection. The target molecule was then purified using protein A affinity chromatography.

[0263] BsAb targeting human CTLA4 and CD47 (HX044) adopts a club-and-mortar configuration. Figure 1A Specifically, HX044 has a “1+1” asymmetric structure consisting of a CTLA4-binding domain and a SIRPα (CD47-binding) domain. The first heavy chain constant region contains a mortar-Fc region containing amino acid mutations including T366W, and the second heavy chain constant region contains a mortar-Fc region containing amino acid mutations T366S, L368A, and Y407V.

[0264] BsAbs with different configurations were also generated and tested, including Figure 1B Those described in (Sirpα-IgG), 1C (scFv-Sirpα, KIH), and 1D (IgG-Sirpα). Surprisingly, only... Figure 1A The configuration shown (Fab-Sirpα, KIH) exhibits the desired properties, namely, no hematologic toxicity and strong antitumor activity of irAE at low dose levels. 6.8.2 Example 2: Ligand binding of HX044

[0265] 1. CD47 antigen binding by ELISA: Coat 96-well polyethylene microtiter plates overnight with 100 μL of 0.5 μg / mL CD47-his carbonate-bicarbonate buffer (200 mM, pH 9.4) at 2–8 °C. Wash the plates three times with PBS-T buffer and block the antigen-coated plates with 1% BSA-PBS at 37 °C for 60 min. Wash the plates four times with PBS-T buffer and incubate at 37 °C for 1 h with 100 μL / well of BsAb (HX044) provided herein at a 5-fold serial dilution (approximately 20 nM–0.000256 nM). Then wash the plates five times with PBS-T and incubate at 37 °C for 1 h with 100 μL of HRP-goat anti-human IgG (H+L) (minimum cross-reactivity) (10000×) in sample dilution buffer (1‰ BSA in PBS). Then, the washed wells were incubated with 100 μL / well of substrate solution at 37°C for 10 minutes. The reaction was terminated by adding 50 μL / well of 2M sulfuric acid. The absorbance at 450 nm was recorded using a microplate reader.

[0266] As measured by ELISA, HX044 binds to recombinant human CD47 protein at an EC50 of 8.12 nM, while SIRPα-Fc binds to it at an EC50 of approximately 0.05 nM.

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

[0268] As measured by ELISA, HX044 binds to the recombinant human CTLA4 receptor protein at an EC50 of 0.77 nM, indicating a significantly lower affinity for CTLA4 compared to prilimumab (EC50: 0.01 nM).

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

[0270] HX044 competitively blocks the binding of CD47 to recombinant SIRPα with an IC50 of ~0.4 nM.

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

[0272] HX044 competitively blocks the binding of CTLA4 to its ligand CD80 with an IC50 of 8.8 nM.

[0273] 5. Binding of HX044 to CD47-positive Jurkat cells: Jurkat cells endogenously express human CD47. Jurkat cells were incubated at 4°C for 1 hour with titrated doses of reference antibody proteins (Prilimumab and SIRPα-Fc) and the bsAb (HX044) provided herein, at serial dilutions (approximately 300 nM – 0.00012 nM). Antibody binding 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. EC50 values ​​were calculated from the best-fit binding curve using GraphPad Prism software.

[0274] like Figure 2 As shown, HX044 binds to Jurkat cells with an EC50 of 1024 nM, which has a significantly lower affinity for CD47 compared to SIRPα-Fc (EC50: 311.2 nM).

[0275] 6. Binding of HX044 to CHO-K1-hCTLA4 cells: CHO-K1-CTLA4 cells engineered to express human CTLA4 were confirmed to specifically express human CTLA4 but not human CD47. Three monoclonal antibodies (CTLA4-4C4, CTLA4-8F8, and CTLA4-7C11) with similar hCTLA4 expression were incubated at 4°C for 1 h with titrated doses of reference antibodies / proteins (prilimumab and SIRPα-Fc) and the bsAb (HX044) provided herein at serial dilutions (approximately 300 nM – 0.00012 nM). The binding antibodies were 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] like Figure 3 As shown, all three clones exhibited similar binding properties. SIRPα-Fc did not bind to any clone, while HX044 bound to all three clones with EC50 values ​​of 11.28–14.90 nM, indicating a lower affinity for hCTLA4 than for prilimumab (EC50 values ​​of 1.168–3.035 nM).

[0277] 7. Binding of HX044 to 293T-hCTLA4 cells: 293T cells were combined with four 293T-hCTLA4 cell clones engineered to stably express elevated levels of human CTLA4 (labeled from - to ++++, see table below). All five cell lines were CD47 positive and expressed membrane-bound human CD47 at similar levels. Cells were incubated at 4°C for 1 h with titrated doses of reference antibodies / proteins (prilimumab and SIRPα-Fc) and the BsAb (HX044) provided herein, at serial dilutions (approximately 300 nM – 0.00012 nM). Antibody binding 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. EC50 values ​​were calculated from the best-fit binding curves using GraphPad Prism software.

[0278] like Figure 4As shown, when CTLA4 expression was negative or low, HX044 bound to cells with a lower affinity than SIRPα-Fc. With increasing CTLA4 expression, HX044 bound to CTLA4+ / CD47+ double-positive cells with gradually increasing affinity (EC50 5–35 nM), and when CTLA4 expression was high, the affinity was significantly higher than that of prilimumab and SIRPα-Fc. Figure 4 This result indicates that HX044 can bind strongly to cells that highly express both targets, while binding relatively weakly to cells that do not express or express low levels of CTLA4.

[0279] 8. HX044 and CD4+ / CD8+ T or regulating T (T reg Cell binding: PBMCs were isolated from healthy donors using polysucrose density gradient separation (GE) and incubated at 4°C for 1 h with 200 nM of reference antibodies / proteins (prilimumab, morolimab, and SIRPα-Fc) and the BsAb (HX044) provided herein. Antibody binding was detected using Alexa Fluor® 647 (AF647)-conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003). PBMCs were then stained at 4°C for 30 min with anti-human CD3-BV-786 (BD), anti-human CD4-BV510 (BD), anti-human CD25-BV421 (BD), and anti-human CD8 (BD). For intracellular staining, PBMCs were fixed and permeabilized with fixation / permeabilization buffer at 4°C for 30 min, followed by staining with anti-human FoxP3 (1:10) at 4°C for 45 min. Cells were then analyzed by flow cytometry and described.

[0280] like Figure 5 As shown in the top and bottom left figures, both HX044 and SIRPα-Fc are associated with isolated human peripheral CD4+ lymphocytes, CD8+ lymphocytes, and T cells. reg HX044 binds to cells expressing low levels of CTLA4 and CD47. At the same concentration, HX044 showed reduced binding to these cells compared to morolimab (a phase III anti-CD47 antibody). HX044 also showed stronger binding to T lymphocytes compared to prilimumab.

[0281] 9. RBC Cell Binding Assay: Red blood cells (RBCs) isolated from healthy donors were incubated at 4°C for 1 hour with serially diluted reference antibodies / proteins (prilimumab, morolimab, and SIRPα-Fc) and bsAb (HX044) provided herein (approximately 1500 nM–1.2 nM). Antibody binding 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. Data were calculated from the best-fit binding curve using GraphPad Prism software.

[0282] Compared to the reference antibody molotovicillin, HX044 showed very little binding to RBCs. Figure 6 ).

[0283] 10. Platelet Binding Assay: Platelets were isolated from healthy donors using a polysucrose density gradient separation (GE) apparatus and incubated at 4°C for 1 hour with 200 nM of reference antibodies / proteins (prilimumab, morolimab, and SIRPα-Fc) and the BsAb (HX044) provided herein. Binding antibodies were detected using Alexa Fluor® 647 (AF647)-conjugated AffiniPure goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003). Platelets were then stained with anti-human CD45-BV605 and anti-human CD41-BV421 at 4°C for 30 min. Platelets were gated by CD45-CD41+.

[0284] like Figure 5 As shown in the lower right panel, both HX044 and SIRPα-Fc bind to isolated human platelets expressing human CD47. At the same concentration, HX044 showed reduced binding to these cells compared to SIRPα-Fc or molotovicillin.

[0285] In summary, these data indicate that HX044 can bind to membrane CD47 and CTLA4, but with lower affinity compared to SIRPα-Fc and prilimumab, respectively. However, HX044 binds with high affinity in CD47+ / CTLA4+ cells with high CTLA4 expression, and this affinity is higher than that of prilimumab. Furthermore, HX044 exhibits significantly weaker affinity for human RBCs, platelets, and T lymphocytes compared to SIRPα and morolimab, thus supporting its reduced hematologic toxicity. HX044 also shows stronger binding to T lymphocytes compared to prilimumab, further supporting its enhanced potency in active T cells. 6.8.3 Example 3: Fc activity of HX044

[0286] 1. Binding of HX044 to 293T-FcRn cells: 293T-FcRn cells engineered to express human FCGRT-β2M were incubated for 1 hour at 4°C and pH 6.0 with titrated doses of reference antibodies / proteins (prilimumab and SIRPα-Fc) and the bsAb (HX044) provided herein, serially diluted 5-fold (approximately 300 nM - 0.00012 nM). Antibody binding 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. EC50 values ​​were calculated from the best-fit binding curve using GraphPad Prism.

[0287] like Figure 7 As shown, HX044 exhibits FcRn binding at pH 6.0 comparable to that of prilimumab, indicating that it possesses comparable self-circulating capacity and similar pharmacokinetic characteristics in serum.

[0288] 2. ADCC determination: 7.5 × 10 4 Jurkat-NFAT-CD16A cells engineered to express human FCγRIII and NFAT factor-driven luciferase were compared with three different cell types at 1.25 × 10⁻⁶ cells per cell line. 4 25 μL of engineered 293T / 293T-CTLA4 cells expressing different levels of human CTLA4 were plated together in 96-well solid white flat-bottomed TC-treated polystyrene microplates. Cells were then incubated at 37°C, 5% CO2 with titrated doses of reference antibodies / proteins (prilimumab and SIRPα-Fc) and bsAb (HX044) provided herein, serially diluted (approximately 60 nM – 0.00012 nM). After 18 hours, 100 μL / well of Stable-Lite™ luciferase assay system reagent (Vazyme-DD1202-02) was added, and the plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal. Luminescence was observed on a 2104 EnVision microplate reader. EC50 values ​​were calculated from the best-fit binding curve using GraphPad Prism software.

[0289] like Figure 8As shown, in CD47+ / CTLA4-293T cells, HX044 exhibited slightly stronger ADCC compared to SIRPα-Fc. In CD47+ / CTLA4+ cells, HX044 showed significantly higher ADCC than prilimumab and SIRPα-Fc, and the ADCC activity of HX044 appeared to depend on the expression level of CTLA4. Figure 8 ). 6.8.4 Example 4: Antitumor activity of HX044

[0290] 1. In vivo MC-38-hCD47 humanized syngeneic mouse model (HuGeMM): The MC38-hCD47 HuGeMM model was constructed by knocking in the hCD47 gene into the KI mouse colon cancer cell line MC-38 and then inoculating this tumor cell line into hCTLA4×hCD47×hSIRPα HuGeMM mice. When the tumor reached ~70mm... 3 Mice were randomly assigned to groups and treated accordingly. Different groups were administered low, medium, or high doses of HX044 or reference antibodies / proteins (prilimumab and SIRPα-Fc), respectively. Treatment groups included HX044 (0.132, 1.98, and 6.6 mg / kg, IP twice weekly), SIRPα-Fc (0.092, 1.38, and 4.6 mg / kg, IP twice weekly), prilimumab analogs (0.172, 2.58, and 8.6 mg / kg, IP twice weekly), and a mediator (PBS). All treatments were molecularly equivalent. Tumor volume was assessed twice weekly until the mean tumor volume reached 2000 mmHg. 3 Tumor growth inhibition (TGI) is calculated as TGI% = (1 - V treatment / V control) × 100.

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

[0292] 2. In vivo B16F10-hCD47 HuGeMM syngeneic model: A humanized syngeneic mouse model of B16F10-hCD47 HuGeMM was constructed by knocking in the hCD47 gene into the (KI) mouse melanoma cell line B16F10 and then inoculating this tumor cell line into hCTLA4×hCD47×hSIRPα HuGeMM mice. When the tumor reached ~70mm... 3Mice were randomly assigned to groups and treated accordingly. Treatment groups included HX044 (3.3 mg / kg IP dose twice, followed by 6.6 mg / kg IP dose four times), SIRPα-Fc (2.3 mg / kg IP dose twice, followed by 4.6 mg / kg IP dose four times), prilimumab analogue (4.3 mg / kg IP dose twice, followed by 8.6 mg / kg IP dose four times), and a mediator (PBS). All treatments were molecularly equivalent. Tumor volume was assessed twice weekly until the mean tumor volume reached 2500 mm. 3 Tumor growth inhibition (TGI) was calculated as TGI% = (1 - V treatment / V control) × 100. At the end of the experiment, tumors and spleens were harvested from three mice in each group for flow cytometry analysis of immune cells. reg Cells were sorted as CD3+CD4+Foxp3+ cells, helper T cells as CD3+CD4+Foxp3- cells, and cytotoxic T cells as CD3+CD8+Foxp3- cells.

[0293] like Figure 10 As shown, HX044 also exhibited strong antitumor activity in the B16F10-hCD47 melanoma model (a conventional “cold” tumor, 5 mg / kg followed by 10 mg / kg), which was significantly higher than that of prilimumab or SIRPα-Fc.

[0294] In addition, such as Figure 11 As shown, an increase in TIL-T cells and TIL-T... were also observed in the tumors of the HX044 treatment group. reg The reduction was observed, and this increase was significantly greater than in the prilimumab treatment group or the SIRPα-Fc treatment group. This observation indicates that TIL-T... reg Reduction also contributes to the antitumor activity of HX044. 6.8.5 Example 5: Blood Toxicology of HX044

[0295] To evaluate the hematologic toxicology of HX044, hCTLA4×hCD47×hSIRPα HuGeMMC57BL / 6J mice were randomly assigned to groups based on body weight and treated for 2 weeks (5 doses) with HX044 (9.2 mg / kg IP dose twice weekly), SIRPα-Fc (6.4 mg / kg IP dose twice weekly), prilimumab analogue (12 mg / kg IP dose twice weekly), or a mediator (PBS). Grouping was designated as day 0. Hematologic tests were performed on days 4 and 11. Whole blood samples were collected at the end of treatment (day 15), and lymphocyte composition was analyzed. regCells were sorted as CD3+CD4+Foxp3+ cells; helper T cells were sorted as CD45+CD4+Foxp3- cells; and cytotoxic T cells were sorted as CD45+CD8+Foxp3- cells.

[0296] As shown, low toxicity to peripheral T cells was observed in non-tumor-bearing HuGEMM mice treated with high doses. Figure 12 This indicates that the adverse effects on lymphocyte composition are minimal, and therefore the hematologic toxicity is minimal.

[0297] All in vivo experiments on mice were conducted under sterile conditions at the Crown Bioscience SPF facility, strictly adhering to the National Institutes of Health's guidelines for laboratory animal management and use. These procedures were approved by the Crown Bioscience IACUC Committee. Study designs followed the ARRIVE guidelines. 6.8.6 Example 6: Production efficiency and stability of HX044

[0298] HX044 and a reference bispecific antibody targeting CTLA4 and CD47 were generated and analyzed. This antibody possesses 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 (“reference bsAb”). The reference bsAb has three peptides with amino acid sequences of SEQ ID NO: 54, 55, and 56, respectively.

[0299] Specifically, HX044 and the reference bsAb were generated 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 three polypeptide chains. The genes encoding the three peptide chains were cloned into vectors respectively. The BsAb expression vector was prepared using an endotoxin-free plasmid DNA purification method (EndoFree plasmid kit, TransGen Biotech). After preparing the DNA vectors, CHO suspended cells were used for transient expression. DNA and PEI solution were mixed and the mixture was added to the cells. Six days after transfection, CHO cells were harvested by centrifugation. The target molecule was then purified using protein A affinity chromatography.

[0300] Stability analysis was performed on both HX044 and the reference bsAb. Specifically, SEC chromatography and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine their respective purities. For SEC chromatography, an LC-20AT column was used under the following experimental conditions:

[0301] Use deionized water as the liquid phase and slowly increase the flow rate to 1.0 ml / min until the baseline stabilizes. Set the sample ingress time to 15 min. Analyze and store the data. Replace the liquid phase with deionized water and wash for 1 h.

[0302] SDS-PAGE was performed as follows: (1) The sample was denatured under reducing and non-reducing conditions; (2) Electrophoresis was performed at 180V for 40 min; (3) The sample was stained and the unbound dye was washed for 30 min; (4) The sample was photographed.

[0303] like Figures 13A-13B (SEC) and Figure 14 As shown in A-14B (SDS-PAGE), HX044 exhibits better heterodimerization than the reference bsAb. Specifically, HX044 has a higher purity (96.031% at 214 nm and 96.501% at 280 nm) than the reference bsAb (93.985% at 214 nm, 96.501% at 280 nm, and 94.883% at 280 nm, as measured by SEC). Figures 13A-13B As measured by SDS-PAGE, mismatched byproducts were observed for the reference bsAb, but not for HX044. Figure 14 A-14B). (e.g.) Figure 14 As shown in Figure A, HX044 appears as a single clean band on a non-reducing gel and as three clean bands (C1, C2, and C3) on a reducing gel. Conversely, as... Figure 14 As shown in B, bands with higher or lower molecular weights were observed on the non-reducing gel of reference bsAb, corresponding to homodimer byproducts such as CTLA4-CTLA4 mAb (estimated MW 145kD) and Sirpα-Fc homodimer (estimated MW 78.5kD), as well as incorrect heterodimers without light chains (estimated MW 90~100kD).

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

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

[0306] Due to their asymmetric structures, the expression of both bispecific antibodies (HX044 and the reference bsAb) is sensitive to impurities. These fragmented or aggregated impurities can significantly affect both the efficacy and safety of the molecules. As shown, HX044 unexpectedly exhibits superior stability, reduced mismatch impurities, and higher production capacity compared to the reference bsAb. This enhanced stability and efficiency underscores the strong therapeutic potential of HX044.

[0307] Although the invention has been described in some detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes or modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0308] Therefore, the foregoing has only illustrated the principles of the invention. It will be understood that although not explicitly described or shown herein, those skilled in the art will be able to design various arrangements embodying the principles of the invention and included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to assist the reader in understanding the principles of the invention and the concepts contributed by the inventors to the field, and should be considered as not being limited to these specifically listed examples and conditions. Moreover, all statements herein listing the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Additionally, these equivalents are intended to include currently known equivalents and future development equivalents, i.e., any developed elements that perform the same function regardless of structure. Furthermore, nothing disclosed herein is intended for public use, whether or not such disclosure is expressly set forth in the claims.

Claims

1. A bispecific antibody, 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 comprises VL CDR1, VLCDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively, and wherein the VH comprises VHCDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NO: 4, 5, and 6, respectively; and (ii) CD47 binding domains containing an extracellular domain of SIRPα or a variant thereof.

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

8.

3. The bispecific antibody according to claim 2, wherein the VL and VH have the amino acid sequences shown in SEQ ID NO: 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 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

9.

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

9.

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

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

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

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

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

12. The bispecific antibody of claim 11, wherein the mortar-Fc region further comprises an S354C substitution, and the mortar-Fc region further comprises a Y349C substitution.

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

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

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

16. The bispecific antibody according to any one of claims 7 to 10, wherein (i) The CL region is κ CL (Cκ; SEQ ID NO: 21) or λ 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 pestle-Fc region and the mortar-Fc region respectively have the following amino acid sequences: (1) SEQ ID NO: 31 and 35; (2) SEQ ID NO: 32 and 36; (3) SEQ ID NO: 33 and 37; or (4) SEQ ID NO: 34 and 38; or variants thereof having up to 10 amino acid substitutions.

17. The bispecific antibody according to claim 16, wherein the CL region, CH1 domain, mortar-Fc region and stolon-Fc region have the following amino acid sequences respectively: (1) SEQ ID NO: 21, 41, 31 and 35; (2) SEQ ID NO: 21, 41, 32 and 36; (3) SEQ ID NO: 21, 41, 33 and 37; or (4) SEQ ID NO: 21, 41, 34 and 38.

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

53.

19. The bispecific antibody according to claim 18, wherein C1, C2 and C3 have the amino acid sequences shown in SEQ ID NO: 51, 52 and 53, respectively.

20. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody (1) has a high affinity for CTLA4 and CD47 double positive 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)-(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 dual-positive cells than for CTLA4 or CD47 single-positive cells; (2) selectively eliminates CTLA4 and CD47 positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC); (3) has limited hematologic toxicity; or (4) has limited immune-related adverse events (irAEs); or any combination of (1)-(4).

22. The bispecific antibody according to any one of claims 1 to 21, wherein the bispecific antibody has limited mismatch impurities.

23. A composition comprising a bispecific antibody according to any one of claims 1 to 22, wherein the purity of the bispecific antibody is at least 95%, wherein 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 available carrier.

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

26. The polynucleotide of claim 25, wherein all peptide chains of the bispecific antibody are encoded.

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

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

29. A cell comprising one or more polynucleotides according to any one of claims 25 to 27, or a carrier according to claim 28.

30. A method for preparing a bispecific antibody that specifically binds to human CTLA4 and human CD47, the method comprising culturing cells according to claim 29 while allowing expression of the bispecific antibody.

31. A method of treating cancer in a subject in need of treatment, 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 of claim 31, wherein the subject is a human being.

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

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 in the preparation of a medicament for treating cancer.

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