Antibodies targeting SIRPα and uses thereof

Abstract

An isolated monoclonal antibody that specifically binds to human SIRPα or an antigen-binding site thereof. Also provided are nucleic acid molecules encoding the antibody or antigen-binding portion thereof, expression vectors, host cells and methods for expressing the antibody or antigen-binding portion thereof. The disclosure further provides bispecific molecules, immunoconjugates, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody or antigen-binding portion thereof, as well as methods of treatment using the anti-SIRPα antibodies or antigen-binding portions thereof of the disclosure.

Description

[Technical field]

[0001] This application claims priority to International Patent Application No. PCT CN2022 / 088000, filed April 20, 2022.

[0002] The aforementioned application and all documents cited therein or cited during prosecution thereof ("Application Cited Documents"), and all documents cited or referenced herein, including but not limited to all literature documents, patents, published patent applications cited herein ("Documents Cited Herein"), and manufacturer's instructions, descriptions, product specifications, and product sheets for products mentioned herein or referenced in documents incorporated herein by reference, are hereby incorporated by reference and may be employed in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences referred to in this disclosure are incorporated by reference by making them the Genbank sequences as of the earliest effective filing date of this disclosure.

[0003] The present disclosure generally relates to isolated monoclonal antibodies, particularly chimeric or human monoclonal antibodies, or antigen-binding portions thereof, that bind to SIRPα with high affinity and functionality. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions thereof. The present disclosure further provides bispecific molecules, immunoconjugates, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions that can include the antibodies or antigen-binding portions thereof, as well as methods of treatment using the anti-SIRPα antibodies or antigen-binding portions thereof of the present disclosure. [Background technology]

[0004] Immune checkpoints are regulators of the immune system, and immune cells must pass stimulatory and / or inhibitory immune checkpoints to become activated or progress to anergy. Inhibitory immune checkpoints are particularly important for self-tolerance, preventing the immune system from attacking normal cells. PD-1 and CTLA-4 are often manipulated by tumor cells to evade immune surveillance. Over the past few decades, inhibitory immune checkpoint blockade has been widely studied in cancer immunotherapy, and PD-1 and / or CTLA-4 inhibitors have been clinically approved as treatments for various cancers.

[0005] Signal Regulatory Protein Alpha (SIRPα) is an inhibitory immune checkpoint for innate immune cells and is mainly expressed in myeloid cells such as macrophages, dendritic cells, and neutrophils. It has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. When SIRPα binds to CD47, a membrane protein expressed on almost all cell types, the ITIM is phosphorylated and recruits inhibitory molecules such as SHP-1 and SHP-2, exerting an inhibitory effect on cell activation (Veillette A, Chen J. (2018) Trends Immunol.39(3):173-184). Many tumor cells overexpress CD47 molecules and suppress phagocytosis by immune cells.

[0006] To date, 10 types of human SIRPα alleles have been discovered, and human SIRPα isoform 1 (or V1), isoform 2 (or V2), and isoform 8 (or V8) account for 80% of SIRPα expressed in humans.

[0007] SIRPα has two closely related SIRP family members, SIRPβ expressed in macrophages and neutrophils, and SIRPγ expressed in lymphocytes and natural killer cells. SIRPβ is a stimulatory receptor that does not bind to CD47, whereas SIRPγ is a non-receptor that binds to CD47 with low affinity (Barclay, AN and Brown, MH(2006) Nat. Rev. Immunol.6(6):457-464; Takahashi S. (2018) Biomedical Reports.9(1):3-7).

[0008] Preclinical studies have shown that disruption of SIRPα-CD47 interaction promotes phagocytic uptake of cancer cells by macrophages, including by aiding phagocytosis-promoting receptors on cancer cells such as SLAMF7 and Mac-1 (Chen, J. et al. (2017) 544:493-497). Antigenic peptides from cancer cells generated during phagocytosis can subsequently trigger adaptive immune responses. Most of the pharmaceuticals for SIRPα-CD47 blockade currently in clinical trials target the CD47 molecule and can induce FcR-mediated phagocytosis, such as recombinant SIRPα-Fc fusion protein and IgG1 anti-CD47 antibodies. Such agents may also harm normal CD47-expressing hematopoietic and nonhematopoietic cells, causing anemia, thrombocytopenia, and / or leukopenia, as observed in nonhuman models (Veillette A, Chen J. (2018) supra). Thus, there may be some advantage in using blocking antibodies against SIRPα, which have a more restricted histological distribution and limited toxicity. SIRPα is aberrantly expressed in solid tumors, such as renal cell carcinoma and melanoma, and anti-SIRPα antibodies may directly initiate FcR-mediated phagocytosis of these tumor cells (Yanagita, T. et al.(2017) JCI Insight 2(1): e89140).

[0009] KWAR23, an anti-SIRPα antibody that binds to an epitope at the CD47-SIRPα interface, has been reported to enhance macrophage-mediated phagocytosis of colorectal adenocarcinoma cells in a concentration-dependent manner in vitro, alone or in combination with cetuximab. BI 765063 (BOehringer Ingelheim / OSE) is an anti-SIRPα antibody that selectively inhibits SIRPα and restores the activity of tumor-associated macrophages and dendritic cells against tumor cells, and is currently in phase I clinical trials in patients with advanced solid tumors, both as a single agent and in combination with a PD-1 antagonist. CC-95251 (Celgene) is also in phase I clinical trials, alone or in combination with cetuximab or rituximab, for the treatment of advanced solid and hematological cancers (Uger R, Johnson L. (2020) Expert Opin Biol Ther.20(1):5-8; Zhang W et al. (2020) Front Immunol.11:18).

[0010] Blocking antibodies against SIRPα may affect the function of other members of the SIRP family, and studies have shown that polymorphisms in human SIRPα may reduce the efficacy of antibodies in some subjects (Yanagita, T. et al.(2017) supra; Barclay, AN and Van den Berg, TK(2014) Annu.Rev. Immunol.32:25-50). Therefore, there is a constant need for multiple monoclonal antibodies with desired properties.

[0011] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0012] The inventors of the present application have discovered antagonistic anti-SIRPα antibodies, or antigen-binding portions thereof, that (a) specifically bind to SIRPα (e.g., specifically binds to human and monkey SIRPα (including isoform 1 (V1), isoform 2 (V2) and isoform 8 (V8)) with similar, if not greater, binding affinity / activity compared to prior art anti-SIRPα antibodies, such as KWAR23 and BI-765063; (b) cross-reacts with SIRPβ with similar, if not greater, binding affinity / activity compared to prior art anti-SIRPα antibodies, such as KWAR23 and BI-765063; (c) does not bind to SIRPγ; (d) does not bind to mouse SIRPα; (e) blocks SIRPα binding to CD47 with blocking activity comparable, if not greater, than prior art antibodies; (f) can be internalized by SIRPα-expressing cells; (g) induces phagocytosis of tumor cells by macrophages with activity comparable, if not greater, than prior art anti-SIRPα antibodies such as KWAR23 and BI-765063; and / or (h) exhibits in vivo anti-tumor activity.

[0013] The antibodies or antigen-binding portions of the disclosure may be used in a variety of applications, such as detecting human or monkey SIRPα protein in vitro and for treating cancers, including solid tumors and hematological cancers.

[0014] Accordingly, in one aspect, the disclosure relates to an isolated monoclonal antibody (e.g., a chimeric or fully human antibody) or an antigen-binding portion thereof that binds to SIRPα, comprising: (i) a heavy chain variable region that may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following: (1) SEQ ID NOs: 1, 2 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G) and 3; (2) SEQ ID NOs: 9 (X=Y), 10 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G) and 11, respectively; or (3) SEQ ID NOs: 9 (X=F), 10 (X1=N, X2=G) and 11, respectively; (ii) a light chain variable region that may include a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, wherein the VL CDR1 region, the VL CDR2 region and the VL CDR3 region have a homology ratio of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to the following: or may comprise amino acid sequences with 100% identity: (1) SEQ ID NOs:4, 5 (X=N; X=A), and 6, or (2) SEQ ID NOs:12, 13, and 14, respectively.

[0015] An isolated monoclonal antibody, or antigen-binding portion thereof, of the disclosure can comprise a heavy chain variable region having a VH CDR 1 region, a VH CDR 2 region, and a VH CDR 3 region, and a light chain variable region having a VL CDR 1 region, a VL CDR 2 region, and a VL CDR 3 region, where VH CDR 1, VH CDR 2, VH CDR 3, VL CDR 1, VL CDR 2, and VL CDR 3 can comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (1) SEQ ID NOs: 1, 2 (X1=N, X2=G), 3, 4, 5 (X=N), and 6, respectively; (2) SEQ ID NOs: 1, 2 (X1=N, X2=A; or X1=A, X2=G), 3, 4, 5 (X=A) and 6, respectively; (3) SEQ ID NOs: 9 (X=Y), 10 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G), 11, 12, 13 and 14, respectively; or (4) SEQ ID NOs: 9 (X=F), 10 (X1=N, X2=G), 11, 12, 13 and 14, respectively.

[0016] An isolated monoclonal antibody of the disclosure, or an antigen-binding portion thereof, can comprise a heavy chain variable region that can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 38, 39 (X1=I, X2=I, X3=S, X4=F; X1=L, X2=M, X3=G, X4=L), 40, 41 (X1=T, X2=N; X1=S, X2=F), 42 (X1=R; X1=K) or 43.

[0017] The isolated monoclonal antibody, or antigen-binding portion thereof, of the present disclosure can comprise a light chain variable region that can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 44 (X1=S, X2=S, X3=F, X1=S, X2=S, X3=F; X1=T, X2=H, X3=I), 45, 46, 47, 48, or 59.

[0018] The isolated monoclonal antibody or antigen-binding portion thereof of the disclosure may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following: (1) SEQ ID NO:7 (X1=N, X2=G) and 8 (X=N), respectively; (2) SEQ ID NO:7 (X1=N, X2=A) and 8 (X=A), respectively; (3) SEQ ID NO:7 (X1=A, X2=G) and 8 (X=A), respectively; (4) SEQ ID NO:15 (X1=N, X2=G) and 16 (X=N), respectively; (5) SEQ ID NO:15 (X1=N, X2=A) and 16 (X=S), respectively; (6) SEQ ID NO:15 (X1=A, X2=G) and 16 (X=S), respectively; or (7) SEQ ID NO:17 and 18, respectively.

[0019] The isolated monoclonal antibody or antigen-binding portion thereof of the disclosure may comprise a heavy chain and a light chain linked by a disulfide bond, the heavy chain may comprise a heavy chain variable region and a heavy chain constant region, and the light chain may comprise a light chain variable region and a light chain constant region, the isolated monoclonal antibody or antigen-binding portion thereof of the disclosure may comprise a heavy chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region, the heavy chain variable region and the light chain variable region may comprise the amino acid sequences described above, and the antibody or antigen-binding portion thereof binds to SIRPα.

[0020] The heavy chain constant region may have FcR binding affinity, such as an engineered human IgG4 constant region or a human IgG1 constant region. The heavy chain constant region may be modified to have enhanced FcR binding affinity and / or enhanced antibody structural stability. In certain embodiments, the heavy chain constant region may be, for example, a human IgG4 constant region having the amino acid sequence set forth in SEQ ID NO:19, or a functional fragment thereof. In certain embodiments, the heavy chain constant region may be, for example, a human IgG1 constant region having the amino acid sequence set forth in SEQ ID NO:38 (X1=M, X2=S, X3=T; X1=Y, X2=T, X3=E). The heavy chain constant region may also have weak or no FcR binding affinity, such as an IgG4 constant region, or a modified IgG1 constant region. The light chain constant region may be, for example, a human kappa constant region having the amino acid sequence set forth in SEQ ID NO:20.

[0021] In certain embodiments, the antibodies of the disclosure may comprise or consist of two heavy chains and two light chains, each heavy chain may comprise a heavy chain constant region, heavy chain variable region or CDR sequence as described above, and each light chain may comprise a light chain constant region, light chain variable region or CDR sequence as described above, where the antibody binds to SIRPα2. In other embodiments, the antibodies or antigen-binding portions thereof of the disclosure may be single chain variable fragment (scFv) antibodies, or antibody fragments such as Fab or F(ab')2 fragments.

[0022] The present disclosure also provides bispecific molecules that may include an antibody of the present disclosure, or an antigen-binding portion thereof, linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than the antibody or antigen-binding portion thereof, e.g., a second functional moiety that targets a tumor-associated antigen, such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2. The present disclosure also provides immunoconjugates, such as antibody-drug conjugates, that may include an antibody of the present disclosure, or an antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin, e.g., a recombinant protein designated DT3C having the amino acid sequence of SEQ ID NO:36. In another aspect, the antibody or antigen-binding portion thereof of the present disclosure can be part of a chimeric antigen receptor (CAR). Also provided are immune cells that can form an antigen chimeric receptor, such as T cells and NK cells. The antibody or antigen-binding portion thereof of the present disclosure can also be encoded by or used in combination with an oncolytic virus.

[0023] Also included in the disclosure are nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the disclosure, as well as expression vectors that may contain such nucleic acid molecules, and host cells that may contain such expression vectors or have the nucleic acid molecules integrated into their genome. Methods of preparing an anti-SIRPα antibody or antigen-binding portion thereof of the disclosure using a host cell are also provided, which may include (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or a cell culture thereof.

[0024] Also provided is a composition, e.g., a pharmaceutical composition, comprising the antibody or antigen-binding portion thereof of the present disclosure, or an immunoconjugate, bispecific molecule, immune cell with CAR, oncolytic virus, nucleic acid molecule, expression vector or host cell, and optionally a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may further comprise a therapeutic agent for treating cancer, such as an anti-cancer agent. In certain embodiments, the composition further comprises an anti-Claudin18.2 antibody or an anti-CD20 antibody.

[0025] In yet another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, which may comprise administering to the subject a therapeutically effective amount of a composition of the present disclosure. The cancer may be a solid cancer, including but not limited to non-small cell lung cancer, breast cancer, ovarian cancer, renal cell cancer, colorectal cancer, pancreatic cancer, and the like, or a hematological cancer. The composition may comprise an antibody or antigen-binding portion thereof, a bispecific molecule, an immunoconjugate, an immune cell bearing a CAR, an oncolytic virus, a nucleic acid molecule, an expression vector, or a host cell of the present disclosure. When the composition comprises an antibody or antigen-binding portion thereof with strong FcR binding affinity, an immunoconjugate, an immune cell bearing a CAR, or an oncolytic virus, local delivery of the composition to the tumor site(s) is preferred. In some embodiments, at least one additional anti-cancer antibody can be further administered, such as an antibody targeting a tumor-associated antigen, such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, or an antibody targeting an inhibitory immune checkpoint, such as LAG-3, PD-1, VISTA, or CTLA-4. In yet another embodiment, the antibody or antigen-binding portion thereof of the present disclosure is administered with a cytokine (e.g., IL-2 and / or IL-21), or a costimulatory antibody (e.g., an anti-CD137 antibody and / or an anti-GITR antibody). In another embodiment, the antibody of the present disclosure, or an antigen-binding portion thereof, is administered with a chemotherapeutic agent, which may be a cytotoxic agent such as epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU). In certain embodiments, the antibody of the present disclosure, or an antigen-binding portion thereof, may be administered with an anti-Claudin18.2 antibody or an anti-CD20 antibody. The antibody or antigen-binding portion of the present disclosure may be, for example, murine, chimeric, or fully human. In certain embodiments, the subject is human.

[0026] In yet another aspect, the present disclosure provides a method for modulating or enhancing an immune response in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, such that the immune response in the subject is modulated / enhanced. The composition comprises an antibody with a weak FcR binding heavy chain constant region, or an antigen-binding portion thereof, a bispecific molecule, a nucleic acid molecule, or an expression vector of the present disclosure. The antibody or antigen-binding portion of the present disclosure may be, for example, murine, chimeric, or fully human. In certain embodiments, the subject is human.

[0027] Other features and advantages of the instant disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0028] Accordingly, it is the object of the present invention not to encompass any previously known products, methods of making products, or methods of using products, and the applicant hereby reserves the right to disclaim any previously known products, processes, or methods. Furthermore, it is noted that the present invention does not intend to encompass within its scope any products, processes, or methods of making products or methods of using products that do not meet the written description and enablement requirements of the USPTO (35 USC §112, first paragraph) or the EPO (Article 83 EPC), and the applicant hereby reserves the right to disclose the previously described disclaimers of products, methods of making products, or methods of using products. In the practice of the invention, it may be advantageous to comply with Art. 53(c). In the practice of the invention, it is advantageous to comply with Art. 53(c) EPC and Rules 28(b) and (c) EPC. All rights to expressly disclaim any embodiment that is the subject of the applicant's granted patents in this line, in other lines, or in prior applications of third parties are expressly reserved. Nothing herein should be construed as a commitment.

[0029] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "composed," "comprising," and the like can have the meaning ascribed to them in U.S. Patent Law, e.g., they mean "include," "contain," "comprises," and the like, and terms such as "consisting essentially of," "consisting essentially of," and the like have the meaning ascribed to them in U.S. Patent Law, e.g., they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0030] The following detailed description is given by way of example, and is not intended to limit the invention to only the particular embodiments described, and can be best understood in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0031] [Figure 1] 1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to human SIRPα in a capture ELISA.

[0032] [Diagram 2] 1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to monkey SIRPα in indirect ELISA.

[0033] [Diagram 3] 1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3, and E1H10B7C7 to human SIRPα-isoform 2 in indirect ELISA.

[0034] [Figure 4] 1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to HEK293 cells expressing human SIRPα in a cell-based binding FACS assay.

[0035] [Diagram 5]1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to HEK293 cells expressing human SIRPβ in a cell-based binding FACS assay.

[0036] [Figure 6] 1 shows the binding ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to Jurkat cells expressing human SIRPγ in a cell-based binding FACS assay.

[0037] [Figure 7] 1 shows the ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to inhibit binding of human CD47 to cell surface SIRPα in a cell-based blocking FACS assay.

[0038] [Figure 8] 1 shows the ability of antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 to inhibit the binding of human SIRPα to CD47 in a competitive ELISA.

[0039] [Figure 9] 1 shows the binding ability of E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG1 to monkey SIRPα in indirect ELISA.

[0040] [Figure 10] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to monkey SIRPα in indirect ELISA.

[0041] [Figure 11] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG1 to mouse SIRPα in indirect ELISA.

[0042] [Figure 12]1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to mouse SIRPα in indirect ELISA.

[0043] [Figure 13] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG1 to human SIRPα-V8 in indirect ELISA.

[0044] [Figure 14] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to human SIRPα-V8 in indirect ELISA.

[0045] [Figure 15] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1 (YTE) and E1H10B7-CDRV1-IgG1 to human SIRPα-isoform2 in indirect ELISA.

[0046] [Figure 16] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 to human SIRPα-isoform2 in indirect ELISA.

[0047] [Figure 17] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG to 293F cells expressing human SIRPα in a cell-based binding FACS assay.

[0048] [Figure 18] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to 293F cells expressing human SIRPα in a cell-based binding FACS assay.

[0049] [Figure 19] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG to 293F cells expressing human SIRPβ in a cell-based binding FACS assay.

[0050] [Figure 20] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to 293F cells expressing human SIRPβ in a cell-based binding FACS assay.

[0051] [Figure 21] 1 shows the binding ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG to Jurkat cells expressing human SIRPγ in a cell-based binding FACS assay.

[0052] [Figure 22] 1 shows the binding ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to Jurkat cells expressing human SIRPγ in a cell-based binding FACS assay.

[0053] [Diagram 23] 1 shows the ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG to inhibit binding of human CD47 to cell surface SIRPα in a cell-based blocking FACS assay.

[0054] [Figure 24] FIG. 1 shows the ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to inhibit binding of human CD47 to cell surface SIRPα in a cell-based blocking FACS assay.

[0055] [Diagram 25] 1 shows the ability of antibodies E1H10B7-CDRV1-IgG1(YTE) and E1H10B7-CDRV1-IgG to inhibit binding of human SIRPα to cell surface CD47 in a cell-based blocking FACS assay.

[0056] [Figure 26] FIG. 1 shows the ability of antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 to inhibit binding of human SIRPα to cell surface CD47 in a cell-based blocking FACS assay.

[0057] [Figure 27] FIG. 1 shows the ability of antibodies E1H10B7-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 (YTE) to induce macrophage-mediated phagocytosis of Raji cells.

[0058] [Figure 28] FIG. 1 shows the effect of antibodies E1H10B7-CDRV1-IgG1 and E1F1B1C3-CDRV1-IgG1 on SIRPα phosphorylation in THP1 cells.

[0059] [Figure 29] FIG. 1 shows the effect of antibodies E1H10B7-CDRV1-IgG1, E1H10B7-CDRV1-IgG1(YTE), E1F1B1C3-CDRV1-IgG1 and E1F1B1C3-CDRV1-IgG1(YTE) on CD47-mediated SIRPα phosphorylation in THP1 cells.

[0060] [Diagram 30] 1 shows internalization of antibodies E1H10B7-CDRV1-IgG1, E1H10B7-CDRV1-IgG1(YTE), E1F1B1C3-CDRV1-IgG1 and E1F1B1C3-CDRV1-IgG1(YTE) by SIRPα expressing cells.

[0061] [Diagram 31] Shown is the body weight of B-NDG hSIRPA mice injected with B-luc-GFP-Raji lymphoma cells and treated with antibodies of the disclosure.

[0062] [Diagram 32] 1 shows tumor signal intensity in B-NDG hSIRPA mice injected with B-luc-GFP-Raji lymphoma cells and treated with antibodies of the disclosure.

[0063] [Diagram 33] 1 shows tumor signal intensity in B-NDG hSIRP mice injected with B-luc-GFP-Raji lymphoma cells and treated with antibodies of the disclosure at different doses.

[0064] [Diagram 34] 1 shows tumor size in C57BL / 6-hSIRPA(2) / hCD47 mice injected with mouse colon cancer cells and treated with antibodies of the disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] In order that this disclosure may be more readily understood, certain terms are defined first. Additional definitions are set forth throughout the detailed description.

[0066] The term "SIRPα" refers to signal regulatory protein alpha, also known as CD172a or Src homology 2 domain-containing phosphatase substrate-1. The term "SIRPα" may include variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human SIRPα protein may in certain cases cross-react with SIRPα protein from species other than human, such as monkeys. In other embodiments, an antibody specific for human SIRPα protein may be completely specific for human SIRPα protein and not cross-react with other species or types, or may cross-react with SIRPα from certain other species but not all other species.

[0067] The term "human SIRPα" refers to a SIRPα protein having an amino acid sequence derived from a human, such as the amino acid sequence of human SIRPα having NCBI Reference Number: NP_001035111.1 (Burgess TL et al., (2020) , PLoS ONE 15 (4) :e0226661). The term "monkey SIRPα" or "cynomolgus monkey SIRPα" refers to a SIRPα protein having an amino acid sequence derived from a monkey, such as the amino acid sequence of NCBI Reference Number: NP_001271679.1 (Wang HY et al., (2007) , PLoS Biol.5 (2) :e13). The term "mouse SIRPα" refers to a SIRPα protein having an amino acid sequence derived from mouse, such as the amino acid sequence of mouse SIRPα having GenBank Reference Number: AAH62197.1 (Strausberg, RLet al., (2002) Proc. Natl. Acad. Sci. USA 99 (26):16899-16903).

[0068] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as SIRPα, through at least one antigen-binding site, where the antigen-binding site is typically located within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monovalent antibodies, fusion proteins containing the antigen-binding site of an antibody, and any other modified immunoglobulin molecule that contains an antigen-binding site, and further, an antigen-binding site (e.g., a dual variable domain immunoglobulin molecule), so long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) based on the identity of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, such as toxins or radioisotopes. Unless expressly indicated otherwise, the term "antibody" as used herein includes the "antigen-binding portion" of an intact antibody. IgG is a glycoprotein consisting of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain may be composed of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region may be composed of three domains: CH1, CH2, and CH3. Each light chain may be composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may be composed of one domain CL. The VH and VL regions may be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and regions of high conservation called framework regions (FRs).Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0069] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a SIRPα protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a monovalent Fab fragment consisting of the VL, VH, CL and CH1 domains. (ii) F(ab')2 fragment, a bivalent fragment that may include two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); (viii) nanobodies, a heavy chain variable region comprising one variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be made into a single protein chain, using recombinant methods, in which the VL and VH domains pair to form a monovalent molecule. Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0070] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a SIRPα protein is substantially free of antibodies that specifically bind to antigens other than the SIRPα protein). However, an isolated antibody that specifically binds to a human SIRPα protein may have cross-reactivity to other antigens, e.g., SIRPα proteins from other species. Additionally, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0071] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or minor post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific antibodies directed against a single antigenic site. Whereas polyclonal antibody preparations usually contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, for example, the hybridoma method.

[0072] The term "human antibody" or "fully human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences.

[0073] The term "chimeric antibody" refers to an antibody made by combining genetic material of non-human origin with genetic material of human origin, or, more commonly, a chimeric antibody is an antibody that has genetic material from one species with genetic material from another species.

[0074] The term "isotype" refers to the antibody class (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

[0075] As used herein, the terms "an antibody that recognizes an antigen" and "an antibody specific to an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0076] As used herein, an antibody that "specifically binds to SIRPα5" is intended to refer to an antibody that binds to SIRPα protein (and optionally SIRPα proteins from one or more non-human species), but does not substantially bind to non-SIRPα proteins. Preferably, the antibody is "high affinity," i.e., binds to SIRPα5 with a specific affinity greater than 1.0 x 10 -7 M or less, preferably 5.0 x 10 -8 M or less, more preferably 1.0 x 10 -8 It binds to human SIRPα protein with a KD of less than M.

[0077] As used herein, the term "does not substantially bind" to a protein or cell means that it does not bind to a protein or cell or does not bind with high affinity, i.e., less than 1.0×10 -6 M or more, preferably 1.0×10 -5 M or more, and more preferably 1.0×10 -4 M or more, and more preferably 1.0×10 -3 M or more, and even more preferably 1.0×10 -2 This means that there is binding to a protein or cell with a KD of M or greater.

[0078] The term "high affinity" for an IgG antibody is defined as having an affinity of 1.0 × 10 for the target antigen. -7 M or less, preferably 1.0×10 -8 K below M D By "high affinity" is meant an antibody having a KD of 10-6M or less, more preferably 10-7M or less, and even more preferably 10-8M or less. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10-6M or less, more preferably 10-7M or less, and even more preferably 10-8M or less.

[0079] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K dis " or "K d " is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "dissociation constant" refers to the ratio of Kd to Ka (i.e., Kd / Ka) and is intended to be expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art. D A preferred method for determining is by using surface plasmon resonance, preferably Biacore. TM The method uses a biosensor system such as the system.

[0080] The term "EC50", also called the half maximal effective concentration, refers to the concentration of antibody that induces a response halfway between the baseline and maximum after a particular exposure time.

[0081] The term "IC50", also known as the half maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody.

[0082] The term "antagonistic" means that an antibody, or antigen-binding portion thereof, of the disclosure is capable of binding to SIRPα and inhibiting the binding of CD47 to CD47.

[0083] The term "subject" includes a human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0084] The term "therapeutically effective amount" refers to an amount of an antibody or antigen-binding portion of the disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as chronic inflammation) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in the context of the condition being treated, where the actual effective amount is readily discernible by one of skill in the art.

[0085] The antibodies, or antigen-binding portions thereof, of the present disclosure (a) specifically bind to SIRPα (e.g., human and monkey SIRPα) with binding affinity / activity that is comparable, if not greater, than prior art anti-SIRPα antibodies, such as KWAR23 and BI-765063; (b) cross-react with SIRPβ with binding affinity / activity that is comparable, if not greater, than prior art anti-SIRPα antibodies, such as KWAR23 and BI-765063; (c) do not bind SIRPγ; (d) do not bind mouse SIRPα; (e) inhibit binding of SIRPα to CD47 with binding inhibitory activity that is comparable, if not greater, than prior art antibodies; (f) can be internalized by SIRPα-expressing cells; and / or (g) in The antibodies of the present disclosure are chimeric and human monoclonal antibodies.

[0086] The antibodies or antigen-binding portions thereof of the present disclosure are structurally and chemically characterized below and in the Examples below. The amino acid sequence ID numbers of the heavy / light chain variable regions and CDRs of the present disclosure are summarized in Table 1 below, although some antibodies have the same V H Or V L The heavy chain constant region of the antibody may be, for example, a human IgG4 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 19, or a human IgG1 heavy chain constant region having the amino acid sequence of SEQ ID NO: 38 (X1=M, X2=S, X3=T; X1=Y, X2=T, X3=E) modified to enhance FcR binding affinity and / or antibody stability. The heavy chain constant region may also have weak FcR binding affinity. The light chain constant region of the antibody may be, for example, a human κ constant region having the amino acid sequence set forth in SEQ ID NO: 20. The antibody of the present disclosure may also include a human λ light chain constant region. [Table 1]

[0087] The heavy and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system, however, as is known in the art, CDR regions can also be determined based on the heavy / light chain variable region sequences by other systems such as Chothia, and the IMGT, AbM, or Contact numbering systems / methods.

[0088] V of other anti-PD-1 antibodies that bind human SIRPα H and V L The sequence (or CDR sequence) of the anti-SIRPα antibody of the disclosure H and V L Preferably, when VH and VL chains (or CDRs within such chains) are mixed and matched, a VH sequence from a particular VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, preferably, a particular VH sequence is replaced with a structurally similar VH sequence. H / V L V from pairing L The sequence is structurally similar to V L It is replaced with an array.

[0089] Thus, in one embodiment, an antibody or antigen-binding portion thereof of the disclosure may comprise: (a) a heavy chain variable region, which can comprise an amino acid sequence listed in Table 1; and (b) a light chain variable region that may comprise an amino acid sequence listed in Table 1 above, or the V of another anti-SIRPα antibody. L wherein the antibody specifically binds to human SIRPα. In another embodiment, an antibody or antigen-binding portion thereof of the present disclosure may comprise: (a) the CDR1, CDR2, and CDR3 regions of a heavy chain variable region listed in Table 1; and (b) the CDR1, CDR2, and CDR3 regions of the light chain variable region listed in Table 1 above, or the CDRs of another anti-SIRPα antibody, wherein the antibody specifically binds to human SIRPα.

[0090] In yet another embodiment, the antibody, or antigen-binding portion thereof, comprises the heavy chain variable CDR2 region of an anti-C5 antibody combined with CDRs from another antibody that binds SIRPα, e.g., CDR1 and / or CDR3 from the heavy chain variable region, and / or CDR1, CDR2, and / or CDR3 from the light chain variable region of a different anti-SIRPα antibody.

[0091] Furthermore, it is well known in the art that the CDR3 domain can solely determine the binding specificity of an antibody to its cognate antigen, independent of the CDR1 and / or CDR2 domains, and that multiple antibodies with identical binding specificity can be predictably generated based on a common CDR3 sequence. For example, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol.Biol.296:833-849 (2000); Rader et al., Proc.Natl.Acad.Sci.USA95:8910-8915 (1998); Barbas et al.,, J. Am. Chem.Soc.116:2161-2162 (1994); Barbas et al., Proc.Natl.Acad.Sci.USA92:2529-2533 (1995); Ditzel et al., J. Immunol.157:739-749 (1996); Berezov et al., BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0092] Thus, in another embodiment, the antibody of the present disclosure may comprise the CDR2 of the heavy chain variable region of an anti-SIRPα antibody and at least the CDR3 of the heavy and / or light chain variable region of an anti-SIRPα antibody, or the CDR3 of the heavy and / or light chain variable region of another anti-SIRPα antibody, where the antibody can specifically bind to SIRPαC5. These antibodies preferably (a) compete for binding with SIRPα; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity as the anti-SIRPα antibody of the present disclosure. In yet another embodiment, the antibody may further comprise the CDR2 of the light chain variable region of an anti-SIRPα antibody, or the CDR2 of the light chain variable region of another anti-SIRPα antibody, where the antibody can specifically bind to human SIRPα. In another embodiment, an antibody of the present disclosure may comprise CDR1 of the heavy and / or light chain variable region of an anti-SIRPα antibody, or CDR1 of the heavy and / or light chain variable region of another anti-SIRPα antibody, wherein the antibody is capable of specifically binding to human C5.

[0093] In another embodiment, an antibody of the disclosure may comprise heavy and / or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences that differ from those of an anti-SIRPα antibody of the disclosure by one or more conservative modifications. It will be understood by those of skill in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0094] Thus, in one embodiment, an antibody may comprise a heavy chain variable region, which may comprise CDR1, CDR2, and CDR3 sequences, and / or a light chain variable region, which may comprise CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR1 sequence may comprise a sequence set forth in Table 1 above, and / or a conservative modification thereof; and / or (b) the heavy chain variable region CDR2 sequence may comprise a sequence set forth in Table 1 above, and / or a conservative modification thereof; and / or (c) the heavy chain variable region CDR3 sequence may comprise a sequence set forth in Table 1 above, and / or a conservative modification thereof; and / or (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 sequences may comprise a sequence set forth in Table 1 above, and / or a conservative modification thereof; and / or (e) The antibody specifically binds to human SIRPα.

[0095] The antibodies of the present disclosure have one or more of the above functional properties, such as high affinity binding to human SIRPα and blocking activity against SIRPα-PD-L1 binding.

[0096] In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0097] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those that replace an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the disclosure can be substituted with another amino acid residue from the same side chain family, and the altered antibodies can be tested for retained function (i.e., as defined above) using the functional assays described herein.

[0098] The V of the anti-SIRPα antibody of the present disclosure H / V L The antibodies of the present disclosure can be prepared and modified antibodies engineered using as a starting material an antibody having one or more of the sequences. H and / or V L ), for example, one or more residues in one or more CDR regions and / or one or more framework regions. Additionally, or alternatively, antibodies can be designed by altering residues in the constant region, for example, to alter the effector functions of the antibody.

[0099] In certain embodiments, CDR grafting can be used to engineer the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse in individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that contain the CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from another antibody with different properties (e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen, (1989) Proc. Natl. Acad. USA 86:10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0100] Thus, another embodiment of the present disclosure relates to isolated monoclonal antibodies, or antigen-binding portions thereof, that may comprise a heavy chain variable region that may comprise CDR1, CDR2, and CDR3 sequences that may comprise sequences of the present disclosure as described above, and / or a light chain variable region that may comprise CDR1, CDR2, and CDR3 sequences that may comprise sequences of the present disclosure as described above. These antibodies comprise the VH and VL CDR sequences of the monoclonal antibodies of the present disclosure, but they may comprise different framework sequences.

[0101] Such framework sequences can be obtained from public DNA databases or published literature that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al., (1991), cited supra; Tomlinson et al., (1992) J. Mol.Biol.227:776-798; and Cox et al., (1994) Eur.J. Immunol.24:827-836, supra; the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available at attached Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 & BC070333), 3-33 (NG--0010109 & NT--024637) and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available under the attached Genbank Accession Nos: 1-69 (NG--0010109, NT--024637 & BC070333), 5-51 (NG--0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).1-69 (NG--0010109, NT--024637 & BC070333), 5-51 (NG--0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0102] The antibody protein sequence is compared against compiled protein sequence databases using one of the sequence similarity search methods known as Gapped BLAST (Altschul et al., (1997), supra), which is well known to those skilled in the art.

[0103] Preferred framework sequences for use in the antibodies of the present disclosure are those that are structurally similar to the framework sequences used by the antibodies of the present disclosure. The VH CDR1, CDR2, and CDR3 sequences can be grafted into framework regions that have identical sequences to those found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequences. For example, in some cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0104] Another type of variable region modification is the V H and / or V LThe aim is to mutate amino acid residues in the CDR1, CDR2 and / or CDR3 regions, thereby improving one or more binding properties (e.g. affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays, as known in the art. Preferably, conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. Furthermore, typically no more than 1, 2, 3, 4 or 5 residues in the CDR regions are altered.

[0105] Thus, in another embodiment, the present disclosure provides an isolated anti-SIRPα-1 monoclonal antibody, or antigen-binding portion thereof, which can include a heavy chain variable region comprising: (a) a V H (b) a CDR1 region or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H (c) a CDR2 region, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H (d) a CDR3 region, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; L (e) a CDR1 region or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; L (f) a CDR2 region, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; L A CDR3 region or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.

[0106] Engineered antibodies of the present disclosure include those in which modifications have been made to framework residues within VH and / or VL, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "mutate" one or more framework residues to their corresponding germline residues. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequence of the antibody to the germline sequence from which the antibody is derived.

[0107] Another type of framework modification involves mutating one or more residues within the framework regions, or within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0108] Additionally, or as an alternative to modifications made within the framework or CDR regions, the antibodies of the disclosure can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Further, the antibodies of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody.

[0109] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0110] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment so that the antibody has impaired staphylococcal protein A (SpA) binding compared to native Fc hinge domain SpA binding. This method is described in more detail in U.S. Patent No. 6,165,745.

[0111] In yet another embodiment, the glycosylation of the antibody is modified. For example, a glycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such glycosylation can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0112] Additionally, antibodies can be made with altered glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such glycosylation modifications have been shown to increase or decrease the ADCC activity of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present disclosure, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and antibodies expressed in Ms704, Ms705, and Ms709 cell lines will lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Onuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes cell lines in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α-1,6 linkage-related enzymes. EP 1,176,195 also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrate, which also results in hypofucosylation of antibodies expressed in the host cells (Shields et al., (2002) J. Biol. Chem. 277:26733-26740).Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as Lemna. Methods for producing antibodies in plant systems are disclosed in U.S. Patent Application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. Fucose residues of antibodies can be cleaved using fucosidase enzymes; for example, fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (see also Tarentino et al., (1975) Biochem. 14:5516-23). ​​See also 14:5516-23).

[0113] Another modification of the antibodies herein contemplated by the present disclosure is pegylation. Antibodies may be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C 10 ) is intended to include any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycols or polyethylene glycol-maleimides. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See EP 0 154 316 and EP 0 401 384.

[0114] The antibodies of the present disclosure can be characterized by their various physical properties in order to detect and / or distinguish their different classes.

[0115] For example, an antibody can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites can result in increased immunogenicity of the antibody or alteration of the antibody's pK by altering antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some instances, it may be preferable to have an anti-SIRPα antibody that does not contain variable region glycosylation, which can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues in the glycosylated region.

[0116] In a preferred embodiment, the antibody does not contain an asparagine isomerization site: deamidation of asparagine occurs on NG or DG sequences, leading to the generation of isoaspartic acid residues that introduce links into the polypeptide chain and reduce its stability (isoaspartic acid effect).

[0117] Each antibody has a unique isoelectric point (pI), which generally ranges from pH 6 to 9.5. The pI of IgG1 antibodies generally falls within the pH range of 7-9.5, and the pI of IgG4 antibodies generally falls within the pH range of 6-8. It has been speculated that antibodies with pIs outside the normal range may have some unfolding or instability under in vivo conditions. Therefore, it is preferable to have an anti-SIRPα antibody with a pI value that falls within the normal range. This can be achieved by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0118] In another aspect, the present disclosure provides a nucleic acid molecule encoding the heavy and / or light chain variable region, or CDR, of the antibody of the present disclosure. The nucleic acid can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" if it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0119] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as further described below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display deep Ls), nucleic acids encoding such antibodies can be retrieved from the gene library.

[0120] Preferred nucleic acid molecules of the present disclosure include the V H and V LOnce DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L - or V H The DNA fragment encoding - is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operatively linked" means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by them remain in frame.

[0121] The isolated DNA encoding the VH region may be prepared by cloning the VH-encoding DNA to a heavy chain constant region (C H1 , C H2 , C H3 A full-length heavy chain gene can be converted to a full-length heavy chain gene by operatively linking to another DNA molecule encoding a human heavy chain constant region gene. Sequences of human heavy chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. In the case of a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain C H1 It can be operatively linked to another DNA molecule encoding only the constant region.

[0122] V L The isolated DNA encoding the V L The DNA encoding the light chain constant region C LA light chain constant region can be converted to a full-length light chain gene (similar to a Fab light chain gene) by operatively linking it to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0123] To generate the scFv gene, H and V L The DNA fragment encoding V H and V L The sequence is V L and V H For example, it is operably linked to another fragment encoding the amino acid sequence (Gly4-Ser)3 so that the regions can be expressed as a contiguous single-chain protein joined by a flexible linker (see e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0124] The monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. 495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display deep Ls. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are expressly incorporated herein by reference in their entirety.

[0125] Antibodies of the disclosure can also be produced in host cell transfectants, for example, using a combination of recombinant DNA technology and gene transfection methods as are well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

[0126] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, e.g., adenovirus major late promoter (AdMLP) and polymath virus enhancer. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter and β-globin promoter, can be used. Additionally, regulatory elements comprised of sequences from different sources include, for example, the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472; see also Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0127] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same expression vector or into separate expression vectors. In a preferred embodiment, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding the heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH segment(s) in the vector and the VL segment is operably linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0128] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). The selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0129] For expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to include a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, particularly mammalian host cells, is most preferred, since such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0130] Preferred mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese Hamster Ovary (CHO) cells (Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036 and EP338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0131] The antibodies or antigen-binding portions thereof of the present disclosure can be conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Publication Nos. 20060024317; 20060004081; and 20060247295, the disclosures of which are incorporated herein by reference.

[0132] In another aspect, the disclosure features bispecific molecules that can include one or more antibodies of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or a ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities.

[0133] In one embodiment, the bispecific molecule has a third specificity in addition to the Fc binding specificity and the anti-SIRPα binding specificity, the third specificity being directed against a tumor associated antigen such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, HER-2, etc.

[0134] Bispecific molecules can be in many different formats and sizes. At one end of the size spectrum, bispecific molecules retain the format of a traditional antibody, except that instead of having two binding arms of the same specificity, they have two binding arms, each with a different specificity. At the other extreme are bispecific molecules in which two single chain antibody fragments (scFv) are linked by a peptide chain, the so-called Bs(scFv) construct. Bispecific molecules of intermediate size include two F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Kufer et al., Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), cited above, and references cited therein.

[0135] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present disclosure can be used in combination with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present invention can be introduced into the human body.

[0136] Also provided herein is a chimeric antigen receptor (CAR) comprising an anti-SIRPα scFv, where the anti-SIRPα scFv can be composed of the CDRs and heavy / light chain variable regions described herein.

[0137] The anti-SIRPα CAR may be composed of (a) an extracellular antigen-binding domain, which may comprise an anti-SIRPα scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0138] CAR may contain a signal peptide at the N-terminus of the extracellular antigen binding domain that guides the nascent receptor to the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen binding domain that allows the receptor to bind more easily. CAR is preferably composed of a primary intracellular signaling domain and one or more costimulatory signaling domains in the intracellular signaling domain. The predominantly used and most effective intracellular primary signaling domain is the CD3-zeta cytoplasmic domain containing ITAM, the phosphorylation of which leads to the activation of T cells. The costimulatory signaling domain may be derived from costimulatory proteins such as CD28, CD137 and OX40.

[0139] CARs may further include additional factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines and costimulatory ligands.

[0140] Also provided are engineered immune effector cells that may comprise the CARs provided herein. In certain embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In certain embodiments, the immune effector cells are T cells.

[0141] In another aspect, the present disclosure provides pharmaceutical compositions that may include one or more antibodies (or antigen-binding portions thereof, or bispecifics, immune cells with CAR, immunoconjugates, oncolytic viruses, or nucleic acid molecules or expression vectors expressing same) of the present disclosure formulated with a pharma- ceutically acceptable carrier. The antibodies (or antigen-binding portions thereof, bispecifics, immune cells with CAR, oncolytic viruses, immunoconjugates, nucleic acid molecules or expression vectors expressing same) can be administered separately when the composition includes one or more antibodies (or antigen-binding portions thereof, bispecifics, immune cells with CAR, oncolytic viruses, immunoconjugates, or nucleic acid molecules or expression vectors expressing same). The compositions may optionally include one or more additional pharma- ceutical active ingredients, such as another antibody or an agent, such as an antitumor drug.

[0142] Pharmaceutical compositions may be comprised of any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (L iPPiNCoTT WiL L iAMS & WiL kiNS 2003), the disclosure of which is incorporated herein by reference.

[0143] Preferably, the pharmaceutical composition 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 may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" refers to a mode of administration other than enteral administration and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intraarticular, putamen, subarachnoid, intraspinal, epidural and intraperiosteal injection and infusion. Alternatively, the antibody of the present disclosure may be administered via a parenteral route, such as a topical, epidermal or mucosal route of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0144] The pharmaceutical compositions can be in the form of a sterile aqueous solution or dispersion, or can be formulated as a microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0145] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration, and will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99%, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient combined with a pharma- ceutically acceptable carrier.

[0146] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dosage to the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

[0147] For administration of the composition, the dosage may range from about 0.0001 to 100 mg / kg. An exemplary treatment regime is once weekly administration.

[0148] A "therapeutically effective amount" of an anti-SIRPα antibody or antigen-binding portion thereof, or a bispecific, CAR-bearing immune cell, immunoconjugate, oncolytic virus, or nucleic acid molecule or expression vector expressing the same of the present disclosure preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of injury or disability due to disease. For example, for the treatment of a cancer-bearing subject, a "therapeutically effective amount" preferably eliminates inflammation by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to an untreated subject.

[0149] The pharmaceutical composition may be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0150] Therapeutic compositions can be administered via medical devices, such as: (1) needleless hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. Patent No. 4,487,603); (3) transdermal devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0151] In certain embodiments, the monoclonal antibodies or antigen-binding portions thereof of the present disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present disclosure cross the blood-brain barrier, they can be formulated in liposomes that may additionally contain a targeting moiety to enhance selective delivery to specific cells or organs. See, e.g., U.S. Patents U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331. VV Ranade(1989) J. Clin.Pharmacol.29:685; Umezawa et al., (1988) Biochem.Biophys.Res.Commun.153:1038; Bloeman et al., (1995) FEBS Lett.357:140; M. Owais et al., (1995) Antimicrob.Agents Chemother.39:180; Briscoe et al., (1995) Am. J. Physiol.1233:134; Schreier et al., (1994) J. Biol.Chem.269:9090; Keinanen and Laukkanen (1994) FEBS Lett.346:123; and Killion and Fidler (1994) See Immunomethods 4:273.

[0152] Pharmaceutical compositions that may include the antibodies or antigen-binding portions thereof of the present disclosure, or bispecifics, CAR-bearing immune cells, immunoconjugates, oncolytic viruses, or nucleic acid molecules or expression vectors expressing the same, have many in vitro and in vivo utilities, including, for example, those involving the treatment of cancer.

[0153] The present disclosure provides methods of treating cancers associated with SIRPα signaling, which may include administering to a subject a therapeutically effective amount of a composition of the present disclosure.

[0154] The cancer may be a solid or hematological cancer, including, but not limited to, non-small cell lung cancer, breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, and pancreatic cancer. The composition comprises an antibody or antigen-binding portion thereof with a weak or strong FcR binding heavy chain constant region, a bispecific molecule, an immunoconjugate, an immune cell with a CAR, an oncolytic virus, a nucleic acid molecule, or an expression vector of the present disclosure. When the composition comprises an antibody or antigen-binding portion thereof with a strong FcR binding affinity, an immunoconjugate, an immune cell with a CAR, or an oncolytic virus, local delivery of the composition to the tumor site(s) is preferred. The antibody of the present disclosure may be, for example, a chimeric antibody, or a human antibody. In certain embodiments, the subject is a human. In some embodiments, at least one additional anti-cancer antibody can be further administered, such as an antibody targeting a tumor-associated antigen, such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, or an antibody targeting an inhibitory immune checkpoint, such as LAG-3, PD-1, VISTA, or CTLA-4. In yet another embodiment, the antibody or antigen-binding portion thereof of the present disclosure is administered with a cytokine (e.g., IL-2 and / or IL-21), or a costimulatory antibody (e.g., an anti-CD137 antibody and / or an anti-GITR antibody). In another embodiment, an antibody of the disclosure, or an antigen-binding portion thereof, is administered with a chemotherapeutic agent, which may be a cytotoxic agent such as epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU).

[0155] In yet another aspect, the present disclosure provides a method for modulating or enhancing an immune response in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, such that the immune response in the subject is modulated / enhanced. The composition comprises an antibody with a weak FcR binding heavy chain constant region, or an antigen-binding portion thereof, a bispecific molecule, a nucleic acid molecule, or an expression vector of the present disclosure. The antibody of the present disclosure may be, for example, a chimeric antibody or a human antibody. In certain embodiments, the subject is a human.

[0156] In another aspect, the present disclosure provides a method of combination therapy, in which the pharmaceutical composition of the present disclosure is co-administered with one or more additional antibodies effective in inhibiting tumor growth in a subject. In some embodiments, at least one additional anti-cancer antibody can be further administered, such as an antibody targeting a tumor-associated antigen, such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, or an antibody targeting an inhibitory immune checkpoint, such as LAG-3, PD-1, VISTA, or CTLA-4. SIRPα pathway blockade can also be further combined with standard cancer treatment. For example, SIRPα pathway blockade can be combined with a chemotherapy regime. For example, a chemotherapeutic agent can be administered together with the anti-SIRPα antibody, and the chemotherapeutic agent can be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-FU are administered to patients undergoing anti-SIRPα therapy. Optionally, the combination of anti-SIRPα and one or more additional antibodies can be further combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immune-stimulating cytokines (He et al., (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be used include tumor cells transfected to express peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or the cytokine GM-CSF. Other treatments that can be used in combination with anti-SIRPα antibodies include, but are not limited to, administration of interleukin-2 (IL-2), radiation, surgery, and hormone blockade.

[0157] The combinations of therapeutic agents discussed herein can be co-administered as a single composition in a pharma- ceutically acceptable carrier, or as separate compositions with each agent in a pharma- ceutically acceptable carrier, hi another embodiment, the combination of therapeutic agents can be administered sequentially.

[0158] Furthermore, when multiple doses of the combination therapy are administered sequentially, the order of sequential administration can be reinstated at each time point of administration or can be kept in the same order, sequential administration can be combined with simultaneous administration, or any combination thereof.

[0159] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0160] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures, and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Working Example Example 1: Production of anti-SIRPα monoclonal antibodies using hybridoma technology vaccination

[0161] AceMouse genetically engineered to produce antibodies with human heavy and light chain variable regions and mouse constant regions TMMice (AceMab Ltd., Hunan Province, China) were vaccinated according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human SIRPα-Fc protein (SEQ ID NO: 21, produced in-house) was used as the immunogen and was also used to measure antiserum titers and screen hybridomas secreting antigen-specific antibodies.

[0162] The inoculation dose included 22.5 μg of recombinant human SIRPα-Fc protein per mouse per injection for both primary and booster inoculations. To enhance the immune response, complete Freud's adjuvant was used for the primary immunization and incomplete Freud's adjuvant (Sigma, St. Louis, Mo.) was used for the boost immunization. Briefly, antigen was prepared in PBS or saline at a concentration of 0.23–0.3 mg / ml, a calculated amount of antigen was added to the desired amount of adjuvant, and the resulting mixture was mixed by gentle vortexing for 2 min to generate a water-in-oil emulsion. The adjuvant-antigen emulsion was then drawn into a syringe suitable for animal injection. A total of 22.5 μg of antigen was injected in a volume of 150–200 μl. Each animal was immunized and then boosted three to four times depending on the antiserum titer. Animals that showed good titers by ELISA were given a final boost by intraperitoneal injection prior to fusion. Hybridoma fusion and screening

[0163] Cells of a mouse myeloma cell line (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured to reach log phase just prior to fusion. Spleen cells from immunized mice were prepared aseptically and fused with mouse myeloma cells according to the method described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256:495-497 (1975). The fused "hybrid cells" were then dispensed into 96-well plates in DMEM / 20% FCS / HAT medium. Viable hybridoma colonies were observed under a microscope 7 to 10 days after fusion. After 2 weeks, the supernatant from each well was subjected to capture ELISA using biotin-labeled human SIRPα-Fc protein made in-house. Positive hybridomas secreting antibodies that bind to human SIRPα-Fc protein were selected and transferred to 24-well plates. These hybridomas were further tested for their ability to inhibit the binding of human SIRPα to cell surface human CD47. Hybridoma cell clones that produced antibodies that showed high specificity human SIRPα-Fc binding and human SIRPα-cell surface human CD47 inhibitory activity were subcloned by limiting dilution to ensure the clonality of the cell line, and monoclonal antibodies were purified. Briefly, a Protein A Sepharose column (bestchrom (Shanghai) Biosciences, catalog number AA0273) was washed using 5-10 column volumes of PBS buffer. After the cell supernatant of the monoclonal hybridoma was passed through the column, the column was washed using PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) and immediately collected in a tube with neutralization buffer (1 M Tris-HCl, pH 9.0). The fractions containing immunoglobulins were pooled and dialyzed in PBS at 4°C overnight. Example 2: Binding activity of anti-SIRPα monoclonal antibodies

[0164] Purified anti-SIRPα monoclonal antibodies, including E1D5E1F1, E1F1B1C3, and E1H10B7C7, produced in Example 1, were characterized for binding activity to human SIRPα, cynomolgus monkey SIRPα, and human SIRPβ by capture ELISA, indirect ELISA, and flow cytometry (FACS). KWAR23 (described in Ring NG, et al. Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity. Proc Natl Acad Sci USA. 2017 Dec 5;114(49): E10578-E10585. BI-765063 (Boehringer Ingelheim International GmbH, referred to herein as BM, heavy and light chain amino acid sequences as set forth in SEQ ID NOs: 32 and 33, respectively, produced in-house) and BI-765063 (Boehringer Ingelheim International GmbH, referred to herein as BM5, heavy and light chain amino acid sequences as set forth in SEQ ID NOs: 34 and 35, respectively, produced in-house) were used as positive controls. 2.1 Capture ELISA

[0165] Briefly, 96-well plates were coated with 100 μl of 2 μg / ml AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch, Cat#115-005-072) in PBS for 2 hours at 37°C. Plates were washed once with wash buffer (PBS+0.05% Tween-20, PBST) and then blocked with 200 μl of blocking buffer (5% w / v nonfat milk in PBST) overnight at 4°C. Plates were washed again and 100 μl of serially diluted anti-SIRPα antibodies of the present disclosure, positive control, and negative control (hIgG, human immunoglobulin for intravenous injection (pH4), Hualan Biological Engineering Inc.) (diluted 5-fold in 2.5% nonfat milk in PBST, starting at 66.7 nM) were added for 40 minutes at 37°C, followed by washing again 4 times. The resulting plates containing anti-SIRPα antibodies were incubated with 100 μl of biotin-labeled human SIRPα-Fc protein (SEQ ID NO:21, in-house production, 3.8 ng / mL dissolved in 2.5% non-fat milk in PBST) for 40 min at 37°C, washed four times, and incubated with streptavidin-conjugated HRP (1:10000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 μl / well) for 40 min at 37°C. After the final wash, the plates were incubated with 100 μl TMB (Innoreagents, Cat#TMB-S-002) at room temperature. The reaction was stopped with 50 μl / well 1 M H2SO4 for 3 min, and the absorbance of each well was read on a microplate reader using a dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. OD (450-630) values ​​were plotted against antibody concentration. Data were analyzed using Graphpad Prism and EC 50 The results are shown in Figure 1. 2.2 Indirect immunoassay (Indirect ELISA)

[0166] Briefly, 96-well microplates were coated with 100 μl of 2 μg / ml cynomolgus monkey SIRPα-is protein (SEQ ID NO:25, in-house) or 2 μg / ml human SIRPα-isoform 2-is protein (SEQ ID NO:28, in-house) in carbonate / bicarbonate buffer (pH 9.6) for 2 hours at 37° C. Plates were washed once with washing buffer (PBS+0.05% Tween-20, PBST) and then blocked with 200 μl / well of blocking buffer (5% w / v nonfat milk in PBST) overnight at 4° C. Plates were washed again and incubated with 100 μl of serially diluted disclosure or control anti-SIRPα antibodies (starting at 66.7 nM, 5-fold serial dilutions in 2.5% nonfat milk in PBST) for 40 minutes at 37° C. Plates were washed four times and incubated with Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific (1:5000 dilution in PBST buffer, Jackson Immunoresearch, Cat#115-035-071, 100 μl / well) for 40 min at 37°C. After the final wash, plates were incubated with 100 μl TMB (Innoreagents) at room temperature. The reaction was stopped after 3-10 min with 50 μl / well 1M H2SO4 and the absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as reference wavelength. OD (450-630) values ​​were plotted against antibody concentration. Data was analyzed using Graphpad Prism and EC 50 The results are shown in Figures 2 and 3. 2.3 Cell-based coupled FACS

[0167] The binding activity of the disclosed anti-SIRPα antibodies to cell surface human SIRPα, human SIRPβ, or human SIRPγ was tested by flow cytometry (FACS) using HEK293-SIRPα cells (uniprot#P78324 expressing full-length human SIRPα on the cell membrane), HEK293-SIRPβ cells (uniprot#O00241 expressing full-length human SIRPβ on the cell membrane) prepared in-house by Biosion, and Jurkat cells (endogenously expressing full-length human SIRPγ on the cell membrane) purchased from ATCC. HEK293-SIRPα and HEK293-SIRPβ cells were prepared by transfecting HEK293 cells with pCMV-TP plasmids containing human SIRPα and human SIRPβ coding sequences, respectively, according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher). Cells were harvested from cell culture flasks, washed twice, and suspended in PBS containing 2% v / v Fetal Bovine Serum (FACS buffer), then plated at 1 × 10 cells per well in a 96-well plate. 5 Cells were incubated with 100 μl of serially diluted anti-SIRPα antibodies of disclosure or control (starting at 10 μg / mL, 5-fold serial dilutions) in FACS buffer for 50 min on ice. Cells were washed twice with FACS buffer and 100 μl of R-Phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG(H+L) (1:1000 dilution in FACS buffer, Jackson Immuno Research, Cat#115-116-146) was added. After 50 min incubation at 4°C in the dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument and MFI (mean fluorescence intensity) was plotted against antibody concentration. Data was analyzed using Graphpad Prism and EC 50 The results are shown in Figures 4 to 6.

[0168] From Figure 1 and Figure 3, it can be seen that the antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 specifically bind to human SIRPα and cross-react with human SIRPα isoform 2 with a higher Bmax (maximum binding) and lower EC50 than the positive control. Unlike BM and BM5, the antibodies E1D5E1F1 and E1H10B7C7 showed high binding activity to cynomolgus monkey SIRPα (Figure 2). In particular, the antibody E1F1B1C3 showed high binding activity to both cells expressing human SIRPα (Figure 4) and cells expressing human SIRPβ (Figure 5) in a cell-based binding FACS assay. According to Figure 6, the antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 did not bind to human SIRPγ. Example 3: Blocking activity of anti-SIRPα antibodies against SIRPα-CD47 interaction 3.1 Cell-based ligand blocking FACS

[0169] The ability of the disclosed anti-SIRPα antibodies to block binding of CD47 protein to cell surface SIRPα was assessed by FACS using in-house produced 293F-SIRPα cells. Briefly, 293F cells were transfected with a pCMV-TP plasmid construct carrying the nucleotide sequence encoding human SIRPα (NP_542970.1) between EcoRI and XbaI according to the instructions of Lipofectamine 3000 transfection reagent (Thermo Fisher).

[0170] 293F-SIRPα cells were harvested from cell culture flasks, washed twice, and resuspended in PBS containing 2% v / v Fetal Bovine Serum (FACS buffer), then plated at 1 × 10 per well in a 96-well plate. 5Cells were incubated with 100 μl of serially diluted anti-SIRPα antibodies of disclosure or control (starting at 10 μg / mL, 5-fold serial dilutions) in FACS buffer for 50 min at 4°C. After washing twice in FACS buffer, 100 μl / well of biotin-labeled human CD47-Fc protein (SEQ ID NO: 31, made in-house at 148 ng / mL) in FACS buffer was added to the plate and the plate was incubated for 60 min at 4°C. Plates were washed twice with FACS buffer, then 100 μl of R-Phycoerythrin Streptavidin (diluted 1:500 in FACS buffer, Jackson Immunoresearch, Cat#016-110-084) was added and incubated for 40 min at 4°C in the dark. Cells were washed twice and then resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument and MFI (mean fluorescence intensity) was plotted against antibody concentration. Data was analyzed using Graphpad Prism and IC 50 The results are shown in Figure 7. 3.2 Ligand blocking ELISA

[0171] The ability of anti-SIRPα antibodies to block human SIRPα-CD47 binding was also measured by competitive ELISA assay. Briefly, 100 μl of human SIRPα-his protein (SEQ ID NO: 22, in-house produced) at 2 μg / mL in PBS was coated onto a 96-well microplate for 2 hours at 37° C. The plate was washed once with washing buffer (PBS+0.05% Tween-20, PBST) and then blocked with 200 μl of blocking buffer (5% w / v non-fat milk in PBST) for 2 hours at 37° C. While blocking, the disclosed or control anti-SIRPα antibodies were diluted in 2.5% non-fat milk in PBST, 5-fold serial dilutions starting from 10 μg / ml, added to the plate at 100 μl per well and incubated at 37° C. for 40 minutes. After washing the plate three times, 0.8 mg / ml biotin-labeled human CD47-his protein (SEQ ID NO: 30) was diluted 1:5000 in 2.5% milk and the resulting solution was added to the plate at 100 μl per well. After incubation at 37°C for 40 min, the plate was washed with washing buffer and 100 μl of streptavidin-conjugated HRP was added and incubated at 37°C for 40 min to detect biotin-labeled human CD47-his bound to SIRPα. The plate was washed again with washing buffer, TMB was added, and the reaction was stopped with 1 M H2SO4. The absorbance of each well was read on a microplate reader using dual wavelength mode with TMB at 450 nm and 630 nm as the reference wavelength, and the OD(450-630) values ​​were plotted against the antibody concentration. The data was analyzed using Graphpad Prism and IC 50 The values ​​were reported and are shown in Figure 8.

[0172] FIG. 7 shows that the anti-SIRPα antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 can block the binding of human CD47 to cell surface human SIRPα with blocking activity comparable to that of BM5.

[0173] FIG. 8 showed that the anti-SIRPα antibodies E1D5E1F1, E1F1B1C3 and E1H10B7C7 were able to block human SIRPα-CD47 binding with blocking activity comparable to that of BM and BM5. Example 4: Engineering of anti-SIRPα antibodies

[0174] The anti-SIRPα antibodies E1F1B1C3 and E1H10B7C7 were designed to have an asparagine (Asn, N) residue immediately followed by a glycine (Gly, G) residue in the CDR and framework regions, making them susceptible to deamination. In particular, the antibody E1H10B7C7 originally had a combination of NG residues in the heavy chain CDR2 region and also in the light chain framework 3 region, which is also a glycosylation site, so it was genetically engineered at these two sites to eliminate the risk of deamination and glycosylation, as shown in Table 2-1 below. The antibody E1F1B1C3 has two high-risk deamination sites, a combination of NG residues in the heavy chain CDR2 region and a combination of NG residues in the light chain CDR2-FR3 region, and was genetically engineered at these two sites, as shown in Table 2-2 below. The amino acid sequences of the heavy and light chain variable regions after genetic engineering are shown in Table 1.

[0175] The engineered antibody was expressed with a human IgG4 (S228P+YTE+K447 Del) constant region (SEQ ID NO: 19) and a human kappa constant region (SEQ ID NO: 20), which may reduce the occurrence of arm exchange in IgG4 antibodies and extend the half-life of the antibody compared to the wild-type one. [Table 2-1] [Table 2-2]

[0176] Engineered antibodies were also expressed with a human IgG1 (AQQ+K447 Del or AQQ+YTE+K447 Del) constant region (SEQ ID NO:38 (X1=M, X2=S, X3=T; or X1=Y, X2=T, X3=E)) and a human kappa constant region (SEQ ID NO:20), as shown in Tables 3-1 and 3-2, where the IgG1 constant region with said modifications has enhanced Fcγ receptor binding affinity (and thus extended half-life) and enhanced antibody stability (i.e., reduced antibody heterogeneity). [Table 3-1] [Table 3-2] Example 5: Characterization of the generated antibodies

[0177] The antibodies prepared in Example 4, namely E1H10B7-CDRV1-IgG1 (YTE), E1H10B7-CDRV1-IgG1, E1F1B1C3-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1, were purified as described above and tested in BIAcore, indirect ELISA, cell-based binding FACS and cell-based ligand blocking FACS according to the protocols described in the previous examples or below. 5.1 Determining Binding Affinity Using BIACORE Surface Plasmon Resonance

[0178] The binding affinity and binding kinetics of the anti-SIRPα antibodies produced were evaluated using a Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0179] Briefly, a Protein A chip (GE healthcare, Cat#Cytiva 29127555) was used. 2 μg / ml of engineered or control antibodies of the present disclosure were flowed over the chip at a flow rate of 10 μL / min. Then, serially diluted human SIRPα-his protein (made in-house, amino acid sequence shown in SEQ ID NO: 22), human SIRPβ-his protein (made in-house, amino acid sequence shown in SEQ ID NO: 23), human SIRPγ-his protein (made in-house, amino acid sequence shown in SEQ ID NO: 24), or cynomolgus monkey SIRPα-his (made in-house, amino acid sequence shown in SEQ ID NO: 25) were diluted 2-fold in HBS-EP+ buffer (provided by Biacore) starting from 200 nM and flowed over the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were followed for 2 min and dissociation kinetics for 10 min, and the binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore evaluation software. K D , K a and K d The values ​​were determined and are summarized in Tables 4-1 and 4-2 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0180] To test the binding affinity of the engineered anti-SIRPα monoclonal antibodies of the present disclosure to human FcRn protein (Cat#FCM-H5286, Acro biosystems Inc.), a CM5 chip (carboxymethyl dextran coated chip, GE healthcare, Cat#BR100530) covalently bound to human FcRn protein was used, and the engineered anti-SIRPα monoclonal antibodies, diluted 2-fold in HBS-EP+ buffer (provided by Biacore) starting at 62.5 μg / ml, were run over the chip at a flow rate of 30 μL / min. D , K A and KD The values ​​were determined and are summarized in Table 4-3 below.

[0181] BIAcore data showed that the antibodies E1H10B7-CDRV1-IgG1 (YTE) and E1H10B7-CDRV1-IgG1 bound specifically to human SIRPα, but not to human SIRPβ or human SIRPγ (data not shown). On the other hand, the antibodies E1F1B1C3-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 bound to both human SIRPα and human SIRPβ, but not to human SIRPγ. Furthermore, E1H10B7-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 (YTE) showed higher binding affinity to FcRn at pH 6.0 than the respective antibodies without YTE modification, suggesting that they may have a longer half-life in the human body. 5.2 Measurement of anti-SIRPα antibody binding activity using indirect ELISA, cell-based binding FACS, and cell-based ligand blocking FACS

[0182] Indirect ELISA to test the binding activity of antibodies against mouse SIRPα-his (in-house produced with SEQ ID NO: 37, mouse SIRPα (BAA20376.1, Met 1-Phe 363)) was performed according to the protocol in Example 2, where Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson Immunoresearch, Cat#109-036-098) was used instead of Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific 100 μl / well.

[0183] The anti-SIRPα antibodies of the present disclosure were also tested for cross-reactivity with human SIRPα-V8 by indirect ELISA according to the protocol of Example 2, where 96-well microplates were coated with 100 μl 2 μg / ml human SIRPα-V8-his protein (SEQ ID NO:29, made in-house) in carbonate / bicarbonate buffer (pH 9.6).

[0184] The results of the indirect ELISA are shown in Figures 9 to 16.

[0185] For cell-based binding FACS, R-Phycoerythrin AffiniPure goat anti-human IgG Fcγ fragment specific (Jackson Immunoresearch, Cat#109-115-098) was used instead of R-Phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG (H+L), diluted 1:1000 in FACS buffer, 100μl / well. The binding activity of anti-SIRPα antibodies to human SIRPα, human SIRPβ and human SIRPγ was also tested by flow cytometry (FACS) following a similar protocol. The results of cell-based binding FACS are shown in Figures 17-22.

[0186] For cell-based ligand blocking FACS, the ability of anti-SIRPα antibodies to block the binding of CD47 protein to human SIRPα on the cell surface and to block the binding of SIRPα protein to human CD47 on the cell surface was assessed by FACS using in-house made 293F-SIRPα cells and 293F-CD47 cells, respectively. Briefly, 293F cells were transfected with pCMV-TP plasmid constructs carrying nucleotide sequences encoding human SIRPα (NP_542970.1) or human CD47 (Uniprot#Q08722). The results of cell-based ligand blocking FACS studies are shown in Figures 23-26.

[0187] In indirect ELISA, E1H10B7-CDRV1-IgG1(YTE), E1H10B7-CDRV1-IgG1, E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 specifically bound to monkey SIRPα (Figures 9 and 10), human SIRPα-V8 (Figures 13 and 14) and human SIRPα-isotype 2 (Figures 15 and 16), but did not bind to mouse SIRPα (Figures 11 and 12).

[0188] In cell-based binding FACS studies, the data showed that E1H10B7-CDRV1-IgG1 (YTE) and E1H10B7-CDRV1-IgG1 showed comparable binding activity to cell surface human SIRPα (Figure 17) and weak binding activity to cell surface human SIRPβ (Figure 19) when compared to BM and BM5, while E1F1B1C3-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 showed higher binding activity to cell surface human SIRPα and human SIRPβ than BM and BM5 (see Figures 18 and 20). Similar to BM5, E1H10B7-CDRV1-IgG1(YTE), E1H10B7-CDRV1-IgG1, E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 did not bind to human SIRPγ expressed on Jurkat cells, whereas BM did (Figure 21, Figure 22).

[0189] Antibodies E1H10B7-CDRV1-IgG1 (YTE) and E1H10B7-CDRV1-IgG1 were able to block the binding of human CD47 to cell surface human SIRPα (Figure 23) and human SIRPα to cell surface human CD47 (Figure 25) with activity comparable to BM5. Antibodies E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 were able to block the binding of human CD47 to cell surface human SIRPα (FIG. 23) and human SIRPα to cell surface human CD47 (FIG. 25) with activity comparable to that of BM5. These results demonstrated that the disclosed anti-SIRPα monoclonal antibodies have high SIRPα-CD47 blocking activity. 5.3 Melting temperature of anti-SIRPα antibody

[0190] The antibodies E1H10B7-CDRV1-IgG1(YTE), E1H10B7-CDRV1-IgG1, E1F1B1C3-CDRV1-IgG1(YTE) and E1F1B1C3-CDRV1-IgG1 were also tested for their thermal stability.TM The protein melting temperature (Tm) was measured by protein thermal shift assay using the Thermal Shift Protein Stability Kit (Biotium, Cat#33022-T). TM The dye was thawed and allowed to warm to room temperature. The vial containing the dye was vortexed and centrifuged. Next, 5 μL of the 200x diluted dye was added to 95 μL PBS to prepare a 10x diluted dye. Next, 2 μL of the 10x diluted dye and 10 μg antibody were added, and PBS was added to bring the total reaction volume to 20 μL. The tube containing the dye and antibody was briefly swirled and placed in a real-time PCR thermocycler (Roche, LightCycler 480 II) with a melt curve program set with the parameters in Table 5. [Table 5]

[0191] The results, as shown in Table 6, suggest that the antibodies of the present disclosure are likely stable in the human body. [Table 6] Example 6: Cell-based functional activity of anti-SIRPα antibodies 6.1 Enhancement of phagocytosis of Raji cells by macrophages using anti-SIRPα antibodies

[0192] CD14+ monocytes were isolated from normal human PBMCs by negative selection and differentiated into macrophages in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 75ng / mL M-CSF. Macrophages were cultured on day 5 using Accutase TM The cells were harvested at 4°C and seeded overnight in a 96-well flat-bottom plate (104 cells / well, 100 μl). The next day, Raji cells (ATCC#CCL-86, 2 × 10 6 cells / mL) were labeled with 1.25 μM CFSE for 20 min at 37 °C and grown in RPMI-1640 medium supplemented with 10% FBS at a final cell density of 8 × 10 5The concentration was adjusted to 1000 cells / mL. Labeled Raji cells (40 μl) and rituximab (Roche Inc, 4 μg / mL, 40 μl) were added to a 96-well U-bottom plate and incubated at room temperature for 30 minutes. The supernatant of the plate with macrophages was discarded, and 20 μg / mL of the anti-SIRPα antibody of the present disclosure was added to the plate at 50 μl / well and incubated at room temperature for 30 minutes. A mixture of CFSE-labeled Raji cells and rituximab (50 μl / well) was added to the plate together with the macrophages and anti-SIRPα antibody and incubated at 37°C for 4 hours. All cells were then collected with Accutase and transferred to a 96-well V-bottom plate. The cells were incubated with Human FcX Blocker (Biolegend, Cat#422302) at 4°C for 20 minutes to block Fc receptors, and then incubated with APC-CD11b (Biolegend, Cat#B325845) at 4°C for 30 minutes. After centrifugation, cells were resuspended in 50 μl staining buffer and 10 μL PI (Propidium Iodide P3566, Thermo, Cat#2229176) was added. Phagocytosis was measured as the ratio of PI-CD11b+CFSE+ macrophages to total PI-CD11b+ macrophages. The results are shown in Figure 2 and Figure 3. The results are shown in Figure 27, where the antibody of the present disclosure enhanced the phagocytosis of Raji cells by macrophages, showing higher activity than BM5. 6.2 Anti-SIRPα antibody does not induce SIRPα phosphorylation in THP1 cells

[0193] The disclosed anti-SIRPα antibodies were tested for their agonist activity in a cell-based assay. Briefly, the disclosed anti-SIRPα antibodies or 100 μl of serially diluted human CD47-Fc (SEQ ID NO: 31, produced in-house) were diluted 3-fold in RPMI-1640 medium supplemented with 10% FBS to a final concentration of 3 nM to 0.004 nM and plated in a 96-well PCR plate. Then, 106 THP1 cells (ATCC#TIB-202) were added in 100 μl RPMI-1640 medium supplemented with 10% FBS and incubated at 4°C for 15 minutes. The plate was centrifuged to remove the supernatant and washed once with PBS. 220 μl / well of NP-40 lysis buffer with protease / phosphatase inhibitors (1:100) was added to the plate and incubated on ice for 15 minutes. The supernatant was collected and stored at -80°C.

[0194] To examine the phosphorylation of SIRPα protein in cell lysate samples, anti-SIRPα polyclonal antibody (INVITROGEN, Cat#PA5-81024, 1:1000 in PBS) was plated in 96-well ELISA plates at 100μl / well and incubated overnight at room temperature. The next day, the plates were washed four times with washing buffer (0.05% Tween-20 in PBS, pH7.2-7.4) and blocked with 5% milk (250μl / well) for 2 hours at room temperature. The plates were washed four times with washing buffer, and the cell lysates obtained above (100μl / well) were added and incubated for 2 hours at room temperature. After washing four times, anti-pY-HRP (RD systems Inc., Cat#HAM1676, 1:5000, 100μl / well) was added to the plates and incubated in the dark at room temperature for 2 hours. After washing, TMB (100 μl / well) was added to the plate and incubated at room temperature for about 20 minutes. OD450 values ​​were measured after adding 1 M H2SO4 (50 μl / well). The results are shown in Figure 28, which indicates that the antibodies of the present disclosure do not induce phosphorylation of SIRPα in THP1 cells and have no agonist activity. 6.3 Anti-SIRPα antibodies antagonize CD47-mediated phosphorylation of SIRPα in THP1 cells

[0195] The disclosed anti-SIRPα antibody serially diluted in RPMI-1640 medium supplemented with 10% FBS (four-fold dilution from final concentration 66.7 nM to 0.065 nM) was plated in a 96-well plate (50 μl / well). 10 THP1 cells were cultured in 100 μl of RPMI-1640 supplemented with 10% FBS. 6 After incubation at 4°C for 60 min, human CD47-Fc (final concentration 0.625 nM, 50 μl / well) was added in RPMI-1640 supplemented with 10% FBS and incubated at 4°C for 15 min. The plates were centrifuged to remove the supernatant, and the cells in each well were washed once with PBS. NP-40 lysis buffer supplemented with protease / phosphatase inhibitors (1:100) was then added (220 μl / well) and the plates were incubated on ice for 15 min. The supernatant was collected and stored at -80°C. SIRPα polyclonal antibody (INVITROGEN, Cat#PA5-81024, 1:1000 in PBS), 100 μl / well, was plated on a 96-well ELISA plate overnight at room temperature. The next day, the plate was washed four times with washing buffer (0.05% Tween-20 in PBS, pH 7.2-7.4) and blocked with 5% milk (250 μl / well) at room temperature for 2 hours. Next, 100 μl / well of the cell lysate obtained above was added to the plate and incubated at room temperature for 2 hours. After washing four times, anti-pY-HRP (RD systems Inc., Cat#HAM1676, 1:5000, 100 μl / well) was added and incubated in the dark at room temperature for 2 hours. After washing, TMB (100 μl / well) was added to the plate and incubated at room temperature for about 20 minutes. OD450 values ​​were measured after the addition of 1 M H2SO4 (50 μl / well).

[0196] As shown in FIG. 29, the antibodies of the present disclosure exhibited antagonistic activity. 6.4 Internalization assay

[0197] Briefly, 293F-SIRPα cells in 100 μl of FreeStyle 293 medium supplemented with 10% FBS were plated at 1500 cells / well in a 96-well white / clear bottom plate and incubated overnight at 37° C. under 5% CO2. The antibody of the present disclosure and recombinant toxic protein (DT3C, SEQ ID NO:36, in-house production) were each diluted in FreeStyle 293 medium, mixed (volume ratio 1:1, molar ratio 1:2.2), and incubated at room temperature for 30 minutes. 100 μl of the serially diluted mixture was added per well to the plate containing 293F-SIRPα cells and incubated at 37° C. under 5% CO2 for 3 days. Luminescent Cell Viability Assay reagent (Vendor#Vazyme Inc., Cat#DD1101-02) was added and RLU was measured (the bottom of the plate was covered with foil). The results are shown in FIG. 30. The antibodies of the present disclosure were shown to be internalized into cells at levels and rates similar to BM and BM5. Example 7: In vivo antitumor effect of anti-SIRPα antibody in mouse tumor model 7.1 Antitumor effect of combined use of anti-SIRPα antibody and anti-CD20 monoclonal antibody

[0198] The in vivo antitumor activity of antibodies E1H10B7-CDRV1-IgG1 (YTE) and E1F1B1C3-CDRV1-IgG1 (YTE) was tested in B-NDG hSIRPA mice (Beijing Biocytogen Co., Ltd.).

[0199] Raji cells were purchased from ATCC and genetically engineered to express luciferase (referred to as B-luc-GFP-Raji cells) by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. B-luc-GFP-Raji cells were cultured in RPMI1640 medium containing 10% inactivated fetal bovine serum at 37.0°C and 5% CO2, and then injected into 42 female B-NDG hSIRPA mice via the tail vein at 1 × 10 5 The mean tumor signal intensity was 1.5 × 106 When p / sec was reached, mice were randomly assigned into 4 groups based on tumor signal intensity and body weight, with 6 mice per group, and this day was designated as day 1. From day 1, the control group (G2) was injected with Dulbecco's phosphate-buffered saline (DPBS), and group 1 (G1) was injected intraperitoneally with E1H10B7-CDRV1-IgG1(YTE) at a dose of 10 mg / kg twice a week, and intravenously with the anti-CD20 antibody rituximab at a dose of 0.1 mg / kg every 2 weeks. Group 3 (G3) animals were injected intraperitoneally with DPBS twice a week, and intravenously with rituximab at 0.1 mg / kg every 2 weeks. Group 4 (G4) animals were injected intraperitoneally with E1F1B1C3-CDRV1-IgG1(YTE) at 10 mg / kg twice a week, and intravenously with rituximab at 0.1 mg / kg every 2 weeks.

[0200] Tumor signal intensity and mouse body weight were measured twice a week during the study. The study was terminated on day 27. Tumor inhibition rates (TGI) were calculated according to tumor signal intensity, and survival curves were generated at the end of the study.

[0201] On day 21, the body weight of the control group was significantly decreased (-2.2 g, 10.8%) compared to day 1. Furthermore, the changes in body weight of G1 and G3 mice were not significantly different from those of the control group, but the average body weight of G4 mice was significantly increased (Table 7-1). [Table 7-1] [Table 7-2]

[0202] The mean tumor signal intensity on the 21st day of administration was 3.58 × 10 9 p / sec, G1 group 8.39×10 8 p / sec, G4 group 5.94×10 8 p / sec. Furthermore, the TGITVs for G1 and G4 were 76.6% and 83.4%, respectively (Table 7-2).

[0203] As can be seen from Tables 7-1, 7-2 and Figures 31 and 32, the anti-SIRPα antibody of the present disclosure at a dose of 10 mg / kg was highly effective in combination with rituximab. As can be seen from Tables 7-1, 7-2 and Figures 31 and 32, the anti-SIRPα antibody of the present disclosure at a dose of 10 mg / kg was highly effective in combination with rituximab. 7.2 Dose-dependent antitumor activity of anti-SIRPα antibodies in animal models

[0204] The in vivo antitumor activity of E1F1B1C3-CDRV1-IgG1 (YTE) was further tested according to the protocol described in 7.1. Specifically, mice were randomly assigned into 5 groups (6 mice per group) based on tumor signal intensity and body weight. Starting from day 1, animals in the control group (G2) were injected with DPBS, animals in the third group (G3) were injected intraperitoneally with E1F1B1C3-CDRV1-IgG1 (YTE) at a dose of 10 mg / kg twice a week, and intravenously with rituximab at a dose of 0.1 mg / kg every 2 weeks. Animals in the fifth group (G5) were injected intraperitoneally with E1F1B1C3-CDRV1-IgG1 (YTE) at a dose of 3 mg / kg twice a week, and intravenously with rituximab at a dose of 0.1 mg / kg every 2 weeks. Group 1 (G1) animals were injected intraperitoneally with DPBS twice a week and intravenously with rituximab at 0.1 mg / kg every 2 weeks. Group 4 (G4) animals were injected intraperitoneally with BM5 at 10 mg / kg twice a week and intravenously with rituximab at 0.1 mg / kg every 2 weeks. The results are shown in Table 7-3. [Table 7-3]

[0205] The mean tumor signal intensity on the 17th day after administration was 3.32 × 10 9 p / sec, G3 group 9.12×10 8 p / sec, G5 group 6.23×10 8 p / sec. Furthermore, the TGI TV were 97.25% and 81.25%, respectively (Table 7-3).

[0206] As can be seen from Table 7-3 and Figure 33, the anti-SIRPα antibody of the present disclosure exhibited dose-dependent anti-tumor activity. Furthermore, the anti-tumor effect of the anti-SIRPα antibody of the present disclosure was higher than that of BM5 at a dose of 10 mg / kg when combined with rituximab. 7.3 Antitumor activity of anti-SIRPα antibodies in solid tumor models

[0207] The in vivo antitumor activity of E1F1B1C3-CDRV1-IgG1 (YTE) was tested in C57BL / 6-hSIRPA(2) / hCD47 mice (Shanghai Model Organisms Center, Inc.) bearing colon tumors.

[0208] MC38-hCLDN18.2 / hCD47 cells (NM-S13-TM19, Shanghai Model Organisms Center, Inc.), a mouse colon cancer cell line, were cultured in EMEM / High+10% fetal bovine serum medium at 37°C and 5% CO2 and injected subcutaneously into the right flank of 48 female C57BL / 6-hSIRPA(2) / hCD47 mice (3 × 10 per mouse). 6 cells / 0.1 mL). Mean tumor volume was 75-95 mm 3 At the time of reaching tumor volume 1000 mg / kg, the mice were randomly assigned into 4 groups based on tumor volume and body weight, with 8 mice per group, and this day was designated as day 1. From day 1, the animals in the control group (G2) were intraperitoneally injected with DPBS, and the animals in the first group (G1) were intraperitoneally injected with anti-CLDN18.2 antibody (heavy and light chains of SEQ ID NO: 39 and 40, produced in-house) at a dose of 10 mg / kg twice a week. The animals in the third group (G3) were intraperitoneally injected with E1F1B1C3-CDRV1-IgG1(YTE) at a dose of 10 mg / kg twice a week, and the animals in the fourth group (G4) were intraperitoneally injected with E1F1B1C3-CDRV1-IgG1(YTE) at a dose of 10 mg / kg twice a week and anti-CLDN18.2 antibody at a dose of 10 mg / kg twice a week.

[0209] During the study, tumor volume and body weight were measured twice a week. Tumor inhibition rate (TGI) was calculated from tumor volume, and the results are shown in Table 7-4. [Table 7-4]

[0210] The mean tumor volume on day 17 was 924.72 mm in the control group. 3 , G3 group 693.94 mm 3 , G5 group 355.15 mm 3 It was. TGI TV was 23.83% in the G3 group and 66.05% in the G4 group.

[0211] As can be seen from Table 7-4 and FIG. 34, the anti-SIRPα antibody of the present disclosure, when combined with an anti-CLDN18.2 antibody, exhibited high anti-tumor activity suppressing the growth of solid tumors.

[0212] While the present disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and that the description is intended to cover all alternatives, modifications, and equivalents that may be included within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety.

[0213] The sequences in this application are summarized below. JPEG2025514652000015.jpg61148JPEG2025514652000016.jpg219150JPEG2025514652000017.jpg218149JPEG2025514652000018.jpg215149 JPEG2025514652000019.jpg218147JPEG2025514652000020.jpg218148JPEG2025514652000021.jpg215149JPEG2025514652000022.jpg92147

[0214] Thus, while preferred embodiments of the invention have been described in detail, it will be understood that the invention defined by the above paragraphs is not limited to the specific details set forth in the above description, since many obvious variations thereof are possible without departing from the spirit or scope of the invention.

Claims

1. 1. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to signal regulatory protein alpha (SIRPα), comprising: (i) a heavy chain variable region that may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (1) SEQ ID NOs: 1, 2 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G) and 3, respectively; (2) SEQ ID NOs: 9 (X=Y), 10 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G) and 11, respectively; or (3) SEQ ID NOs: 9 (X=F), 10 (X1=N, X2=G) and 11, respectively; and / or (ii) a light chain variable region consisting of a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, wherein the VVL CDR1 region, the VL CDR2 region and the VL CDR3 region are The CDR3 region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (1) an isolated monoclonal antibody, or antigen-binding portion thereof, comprising SEQ ID NOs:4, 5 (X=N; X=A), and 6, respectively; or (2) SEQ ID NOs:12, 13, and 14, respectively.

2. 2. The antigen-binding portion of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region and the VL CDR3 region comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following:

2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising: (1) SEQ ID NO:1, 2 (X1=N, X2=G), 3, 4, 5 (X=N) and 6, respectively; (2) SEQ ID NO:1, 2 (X1=N, X2=A; or X1=A, X2=G), 3, 4, 5 (X=A) and 6, respectively; (3) SEQ ID NO:9 (X=Y), 10 (X1=N, X2=G; X1=N, X2=A; X1=A, X2=G), 11, 12, 13 and 14, respectively; or (4) SEQ ID NO:9 (X=F), 10 (X1=N, X2=G), 11, 12, 13 and 14, respectively.

3. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 38, 39 (X1=I, X2=I, X3=S, X4=F; X1=L, X2=M, X3=G, X4=L), 40, 41 (X1=T, X2=N; X1=S, X2=F), 42 (X1=R; X1=K) or 43.

4. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 44 (X1=S, X2=S, X3=F, X1=S, X2=S, X3=F; X1=T, X2=H, X3=I), 45, 46, 47, 48, or 59.

5. 1. An isolated monoclonal antibody, or an antigen-binding portion thereof, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (1) SEQ ID NO: 7 (X1=N, X2=G) and 8 (X=N), respectively; (2) SEQ ID NO: 7 (X1=N, X2=A) and 8 (X=A), respectively; (3) SEQ ID NO: 7 (X1=A, X2=G) and 8 (X=A), respectively; (4) SEQ ID NO: 15 (X1=N, X2=G) and 16 (X=N), respectively; (5) SEQ ID NO: 15 (X1=N, X2=A) and 16 (X=S), respectively; (6).

3. The isolated monoclonal antibody of claim 2, or an antigen-binding portion thereof, comprising: (1) SEQ ID NO: 15 (X1=A, X2=G) and 16 (X=S); or (7) SEQ ID NO: 17 and 18, respectively.

6. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which is of the IgG1, IgG2 or IgG4 isotype.

7. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 19 or 38 (X1=M, X2=S, X3=T; X1=Y, X2=T, X3=E) linked to the heavy chain variable region, and a light chain constant region having the amino acid sequence of SEQ ID NO: 20 linked to the light chain variable region.

8. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which (a) binds to human SIRPα; (b) binds to monkey SIRPα; (c) does not bind to mouse SIRPα; (d) binds to human SIRPβ; (e) does not bind to human SIRPγ; (f) inhibits the binding of SIRPα to CD47; (g) can be taken up by cells expressing SIRPα; (h) induces phagocytosis of tumor cells by macrophages; and / or (i) has in vivo anti-tumor activity.

9. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which is a chimeric or humanized antibody.

10. A nucleic acid molecule encoding the isolated monoclonal antibody or antigen-binding portion thereof of claim 1.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11 or having the nucleic acid molecule integrated into its genome.

13. A pharmaceutical composition comprising the isolated monoclonal antibody, or an antigen-binding portion thereof, of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12, and a pharma- ceutically acceptable carrier.

14. A method for treating a SIRPα-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 13.

15. 15. The method of claim 14, wherein the cancer is a solid cancer or a hematological cancer.

16. 16. The method of claim 15, wherein the cancer is non-small cell lung cancer, breast cancer, ovarian cancer, renal cell carcinoma, colorectal cancer, or pancreatic cancer.

17. The method of claim 14, wherein the subject is further administered an antibody that targets a tumor-associated antigen.

18. 18. The method of claim 17, wherein the tumor-associated antigen is selected from the group consisting of CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP and HER-2.

19. 15. The method of claim 14, wherein the subject is further administered an antibody that targets an inhibitory immune checkpoint.

20. 14. A method for enhancing an immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 13.

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