WO2023020459A1 - 靶向SIRPα的单克隆抗体及其用途 - Google Patents

靶向SIRPα的单克隆抗体及其用途 Download PDF

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WO2023020459A1
WO2023020459A1 PCT/CN2022/112677 CN2022112677W WO2023020459A1 WO 2023020459 A1 WO2023020459 A1 WO 2023020459A1 CN 2022112677 W CN2022112677 W CN 2022112677W WO 2023020459 A1 WO2023020459 A1 WO 2023020459A1
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seq
antibody
sequence
variable region
sirpα
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PCT/CN2022/112677
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French (fr)
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何立珍
俞品
徐飞虎
周亮
郭宣诚
彭雍博
陈虹
王同映
孙汉栋
李晨
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杭州九源基因工程有限公司
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Publication of WO2023020459A1 publication Critical patent/WO2023020459A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to the field of biomedicine, in particular to a monoclonal antibody targeting SIRP ⁇ and its preparation method and application.
  • SIRP ⁇ Signal regulatory protein
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the CD47-SIRP ⁇ axis has been shown to play a key role in some homeostatic processes, such as clearing senescent red blood cells, protecting hematopoietic stem cells, and pruning neuronal synapses, etc. (Logtenberg et al., 2020). However, the vast majority of tumor cells upregulate CD47 expression, that is, through the so-called "don't eat me” signal to avoid being swallowed.
  • the CD47-SIRP ⁇ axis as an inhibitory signaling pathway, requires the presence of an activating signal as a prerequisite, otherwise its blockade is considered an invalid event (Logtenberg et al., 2020). Binding of CD47 to SIRP ⁇ has been shown to counteract or attenuate agonistic signals received by myeloid cells through various membrane receptors, including: 1) Fc gamma receptors (FcgRs), which bind the Fc fragment of mAbs; 2 ) lipoprotein-related protein (LRP), which binds to calreticulin (CRT); 3) SLAMF7 self-associates (Chao et al., 2010b; Oldenborg et al., 2001; Logtenberg et al., 2020).
  • FcgRs Fc gamma receptors
  • LRP lipoprotein-related protein
  • CRT calreticulin
  • blocking CD47-SIRP ⁇ "don't eat me” signaling alone is not sufficient to eliminate tumor cells, while the presence of "eat me” signaling activated by interactions such as Fc-Fc ⁇ Rs or CRT-LRP is required to initiate efficient phagocytosis.
  • CD47 or SIRP ⁇ blockade combined with opsonic antibodies targeting tumor cells [tumor-opsonized antibody, defined herein as having antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis ADCP, complement-mediated cytotoxicity (CDC) and other effector functions of IgG1 subtype monoclonal antibodies, such as anti-CD20 rituximab or anti-EGFR cetuximab, etc.], or combined with the apparent up-regulation of CRT expression
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-mediated cytotoxicity
  • IgG1 subtype monoclonal antibodies such as anti-CD20 rituximab or anti-EGFR cetuximab, etc.
  • Genetically modulated drugs such as azacitidine, are currently the main strategy for clinical development (Weiskopf et al., 2017; Advani et al., 2018, Feng et al., 2019, Sallman et al., 2020).
  • blocking CD47-SIRP ⁇ signaling can promote neutrophil "trogocytosis" (trogocytosis) of target cells that are brought closer by tumor opsonizing antibodies, causing them to undergo apoptosis (Matlung et al., 2018; Bouti et al., 2021; Mart ⁇ nez-Sanz P et al., 2021).
  • NK cells that do not normally express SIRP ⁇ can upregulate the expression of SIRP ⁇ to counteract inflammatory signals and downregulate NK cell activity when they are incubated with IL-2, and blocking CD47-SIRP ⁇ signaling can enhance the antitumor cytotoxicity of NK cells (Deuse et al., 2021).
  • blocking CD47-SIRP ⁇ signaling increased the phagocytosis of tumor cells by antigen-presenting cells (APCs) in the tumor microenvironment, including dendritic cells, and activated cGAS-STING- IRF3-type I IFNs, an important axis of innate immunity (Liu et al., 2015; Xu et al., 2017), on the one hand up-regulate the expression and release of various pro-inflammatory cytokines and chemokines, activate NK cells and Promote the polarization of macrophages to M1 and increase the infiltration of lymphocytes in tumors; on the other hand, promote the maturation of dendritic cells, enhance antigen presentation and activate T cells, especially CD8-T cell responses, to achieve innate immunity to adaptive immunity bridging between (Liu et al., 2015; Tseng et al., 2013; Xu et al., 2017; Gauttier et al., 2020).
  • APCs anti
  • CD47 blockers Drug development related to the SIRP ⁇ -CD47 pathway mainly focused on blocking CD47 using monoclonal antibodies or SIRP ⁇ -Fc fusion proteins in the early stage.
  • CD47 blockers have entered clinical development worldwide. Since CD47 is expressed in normal tissue cells throughout the body, the hemotoxicity caused by the attack of normal somatic cells, especially red blood cells and platelets, and the sinking of a large number of drugs in the antigen sink in the body have become two major pain points in the development of CD47 blockers. It has been more than ten years since the earliest clinical trial of CD47 antibody, and no CD47 blocker has been approved for marketing.
  • SIRP ⁇ is gaining more attention and investment in recent years, including domestic and foreign companies such as Celgene/BMS Bristol-Myers Squibb, Fortyseven/Gilead Science, Arch Oncology, ALX Oncology and Innovent Biological, which have been committed to the development of CD47 inhibitors. Recently, SIRP ⁇ monoclonal antibody research is also carried out. Compared with CD47, the expression of SIRP ⁇ in the human body is limited to myeloid cells, so blocking the CD47-SIRP ⁇ inhibitory signal by targeting SIRP ⁇ can avoid serious drug-related effects such as anemia, thrombocytopenia, and coagulation abnormalities caused by targeting CD47. Sexual Adverse Events (TRAEs).
  • TEEs Sexual Adverse Events
  • Anti-SIRP ⁇ antibodies are described in published international patent application publications: eg WO2018/057669, WO2018/026600, WO2017/178653, WO2017/068164, WO2016/063233, WO2016/ 205042, WO2015/138600, WO2013/0956352, WO2009/091547, WO2009/131453, WO2009/046541, etc., are hereby incorporated by reference in their entirety.
  • SIRP ⁇ has allelic sequence polymorphisms in the population and high sequence homology among SIRP family members. It is reported in the literature that the gene polymorphism sequences that are absolutely dominant in the five major populations of East Asia, South Asia, America, Europe and Africa are the three variants of V1, V2 and V8 (Volts et al., 2019), and more big data analysis will follow It was verified that there were only two variant sequences of V1 and V2 in these five populations, and no V8 variant was detected (Treffers et al., 2018; Sim et al., 2019). Therefore, an ideal SIRP ⁇ mAb must be able to bind SIRP ⁇ -V1 and V2.
  • SIRP ⁇ is expressed on myeloid cells, but SIRP ⁇ does not bind to CD47, and its ligand remains unknown.
  • SIRP ⁇ can transduce agonistic signals by forming a complex with the transmembrane protein DAP12 with an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • SIRP ⁇ cross-linking of SIRP ⁇ on macrophages to its antibody or to a SIRP ⁇ antibody that cross-conjugates SIRP ⁇ enhances phagocytosis.
  • SIRP ⁇ is expressed on most T cells and a subset of NK cells.
  • SIRP ⁇ has no intracellular tail and thus no signaling activity.
  • the binding strength of SIRP ⁇ to CD47 is about one-tenth of that of CD47-SIRP ⁇ (Brooke, et al., 2004).
  • CD47-SIRP ⁇ interaction can enhance T cell migration (binding to CD47 on endothelial cells) and activation response (binding to APC or CD47 on tumor cells) by enhancing adhesion (Piccio, et al., 2005; Dehmani et al., 2021), SIRP ⁇ antibody KWAR23 non-selectively binds SIRP ⁇ and SIRP ⁇ , and may inhibit T cell activation by blocking CD47-SIRP ⁇ interaction (Piccio et al., 2005; Gauttier et al., 2020). Hence, if SIRP ⁇ antibodies inhibit T cell activation, all anti-CD47 antibodies have this inhibitory effect, because there is no difference in the blocking ability of CD47 antibodies to CD47-SIRP ⁇ or CD47-SIRP ⁇ .
  • SIRP ⁇ monoclonal antibody drugs So far, no SIRP ⁇ monoclonal antibody drug has been approved for marketing in the world, and only two SIRP ⁇ monoclonal antibody drugs have reported preliminary clinical data. They are BI 765063 and BI 765063 jointly developed by OSE Immunotherapeutics and Boehringer Ingelheim CC-95251 developed by Celgene (Xinji)/BMS (Bristol-Myers Squibb).
  • BI 765063 also known as OSE-172 or HEFLB, is a humanized IgG4 subtype SIRP ⁇ monoclonal antibody. Its limited binding to SIRP ⁇ -V1 but not SIRP ⁇ -V2 is its major defect (Gauttier et al., 2020; Kuo et al., 2020, Patients with non-SIRP ⁇ -V1 genotype must be excluded in clinical trials (NCT03990233). It was well tolerated alone in patients with advanced solid tumors and did not occur at the highest tested dose of 36 mg/kg DLT reported, MTD not reached.
  • BI 765063 selected two doses (18mg/kg, 24mg/kg Q3W) in combination with PD-1 monoclonal antibody, and 18 solid tumor patients who received multiple lines of therapy
  • PRs partial responses
  • MSS microsatellite stable
  • CC-95251 is a fully human IgG1 subtype monoclonal antibody modified to remove complement activation function, and it exhibits high affinity with SIRP ⁇ protein V1-V6 variants. Its combination with rituximab has shown a manageable safety profile in CD20 + R/R NHL patients who have received multiple lines of therapy, and the MTD has not yet been reached (Strati et al., ASH 2021). In this clinical trial, patients received 3, 10 or 20 mg/kg CC-95251 and 375 mg/m 2 rituximab once a week (QW) for a 28-day treatment cycle until disease progression or unresponsiveness. Accepted Toxicity.
  • SIRP ⁇ antibodies in this field.
  • it is required to have high affinity to SIRP ⁇ -V1 and V2 and block their interaction with the ligand CD47, inhibit the "don't eat me” signal, and not bind to SIRP or Weak binding, so as to target myeloid cells without affecting T cell immune response; on the other hand, bridging innate immunity and adaptive immunity to maximize drug efficacy under the premise of ensuring safety.
  • SIRP ⁇ ⁇ Seiffert et al.Signal-regulatory protein ⁇ (SIRP ⁇ )but not SIRP ⁇ is involved in T-cell activation,binds to CD47 with high affinity,and is expressed on immature CD34 + CD38 - hematopoietic cells.BLOOD 2001;97:2741-2749 .
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ or an antigen-binding portion thereof, comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises the same amino acid sequence as shown in IIWGX 1 X 2 STDYX 3 X 4 ALKS, wherein X 1 is D, E or N, X 2 is G, A or S, and X3 is N, Q or S, X4 is S, T or A;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 23 or SEQ ID NO: 24;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 8 or SEQ ID NO: 25;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 6;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 14;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the present invention provides a SIRP ⁇ -targeting monoclonal antibody or an antigen-binding portion thereof, the CDR sequences of the heavy chain variable region and the light chain variable region of which can be modified by conservative sequences, including one or more amino acid substitutions, additions, and deletions.
  • the one or more amino acid additions, deletions and/or substitutions are no more than five, preferably no more than three.
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising heavy and light chain variable region sequences:
  • a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18;
  • a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ or an antigen-binding portion thereof, the heavy chain variable region of which is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 18 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; Its light chain variable region has at least 90%, 91%, 92%, 93% of the amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the antibody is a full length antibody comprising the Fc domain of a human or murine IgG antibody.
  • the human constant region Fc domain is selected from the group consisting of IgG1, IgG2, IgG3, IgG4.
  • the human constant region Fc domain is IgGl, IgG2 or IgG4.
  • the human constant region Fc domain is IgG1w or IgG1 mutant (LALA).
  • an antibody or antibody fragment of the invention is a human antibody or human antibody fragment.
  • the antibody fragment of the invention is a Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2 antibody fragment.
  • antibody fragments of the invention are diabodies.
  • the invention provides a monoclonal antibody targeting SIRP ⁇ comprising:
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ , comprising:
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ , which comprises:
  • the present invention also provides an isolated polynucleotide encoding the monoclonal antibody or antibody fragment of SIRP ⁇ .
  • the present invention provides an expression vector comprising the isolated polynucleotide, and a host cell comprising the expression vector.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the SIRP ⁇ -targeting monoclonal antibody or an antigen-binding portion thereof and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of a monoclonal antibody targeting SIRP ⁇ or an antigen-binding portion thereof to a tumor patient or a patient with a microbial infectious disease in need of treatment.
  • the present invention provides the use of the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion in combination with Rituximab in the treatment of tumor diseases.
  • the present invention provides the use of the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion in combination with daratumumab in the treatment of tumor diseases.
  • the present invention provides the use of the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion in combination with PD-(L)1 antibody in the treatment of tumor diseases.
  • Fig. 1.FACS detects the combination of AD4-12 and SIRP ⁇ -V1, V2 (see embodiment 4)
  • Fig. 3.FACS detects the combination of AD4-12 and people's PBMC (seeing embodiment 6)
  • SIRP ⁇ refers to wild-type Signal Regulatory Protein Alpha, or the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of wild-type Signal Regulatory Protein Alpha, or a native or naturally occurring allelic variant of Signal Regulatory Protein Alpha .
  • SIRP ⁇ preferably refers to wild-type mammalian SIRP ⁇ , and the most common protein variants are SIRP ⁇ v1 and v2 (reference accession numbers NP_001035111, NP_542970 (P78324) and CAA71403), and also include SIRP ⁇ v8 and the like.
  • SIRP ⁇ V1 The amino acid sequence of the mature form of the major wild-type human SIRP ⁇ (SIRP ⁇ V1) is shown in SEQ ID NO:2.
  • SIRP ⁇ is the SIRP ⁇ extracellular domain, that is, a SIRP ⁇ protein engineered to remove the transmembrane and cellular domains, and the sequence of the extracellular domain of wild-type SIRP ⁇ V1 is SEQ ID NO: 2 residues 1- 348.
  • a "variant" of SIRP ⁇ is defined as a SIRP ⁇ amino acid sequence having one or more amino acid modifications compared to wild-type SIRP ⁇ .
  • a variant may have "conservative" modifications, including amino acid substitutions, deletions, or insertions, or both, wherein the substituted amino acids have similar structural or chemical properties.
  • SIRP ⁇ variants include at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of wild-type SIRP ⁇ or wild-type SIRP ⁇ extracellular domain , 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • SIRP ⁇ refers to a SIRP ⁇ protein (also known as Signal Regulatory Protein ⁇ -1, SIRP- ⁇ -1, CD172 antigen-like family member B or CD172b) from a mammalian species, preferably human SIRP ⁇ (reference accession number O00241 related sequence).
  • SIRPy relates to SIRPy from a mammalian species, preferably human SIRPy.
  • the reference sequence of the human SIRP ⁇ protein corresponds to the sequence of accession numbers AAH64532, Q9P1W8 or NM 018556.
  • Antibody refers to any form of antibody that exhibits a desired biological activity, such as inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling. Accordingly, “antibody” is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, nanobodies, and multispecific antibodies (such as bispecific antibodies) .
  • a whole antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may in some cases comprise fewer chains, eg naturally occurring antibodies in camelids may comprise only heavy chains.
  • bind and “specifically bind” refer to the binding of an antibody or antigen-binding portion to an antigenic epitope in an in vitro assay, preferably in bioluminescent interferometry (ForteBio) using purified wild-type antigen.
  • an antibody or antigen-binding portion is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • the "Fc" region contains the two heavy chain fragments comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.
  • Fc can be selected from the Fc domain of human IgG1, IgG2, IgG3 or IgG4 or the Fc domain of which one or several sites have been mutated.
  • “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • the majority of humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable region residues of the recipient antibody are replaced by hypervariable region residues of a non-human species (donor antibody) having the desired specificity, affinity, and capacity. Residue substitution, non-human species such as mouse, rat, rabbit or non-human primate.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody will comprise at least one, and usually substantially all of, two variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, all or substantially all FR regions FR regions of human immunoglobulin sequences.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin (typically human immunoglobulin) constant region (Fc).
  • an “isolated” antibody is one that has been identified and separated from components of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody, which may include enzymes, hormones, and other protein solute or non-protein solute.
  • the antibody is purified to greater than 95% purity, more preferably greater than 99% purity, as determined by the Lowry method. Isolated antibody will usually be prepared by at least one purification step.
  • An "isolated" nucleic acid molecule is one that has been identified and separated from at least one contaminating nucleic acid molecule.
  • An isolated nucleic acid molecule is different from the form or environment in which it occurs in nature.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Immune cell includes cells of hematopoietic origin and which play a role in the immune response.
  • Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • a sequence "variant” as used herein refers to a sequence that differs from the shown sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.
  • the term "about” means that a value is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, depending in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” or “comprising essentially” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “comprising essentially” should be assumed to be within an acceptable error range for the specific value.
  • administering and “treating” are used to refer to an animal, human, test subject, cell, tissue, organ or biological fluid, it means the administration of an exogenous drug, therapeutic agent, diagnostic agent or composition to the animal, human, subject, Treatment, cells, tissues, organs or biological fluid contact.
  • administering can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting an agent with the cells and contacting the agent with a fluid, wherein the fluid contacts the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, eg, by reagents, diagnostic agents, binding compositions or by other cells.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject will vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier for the antibody-containing composition is suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • Patient means a human or non-human animal (eg, mammal).
  • Cancer or “tumor” refers to a collection of cells that proliferate in an abnormal manner.
  • PD-(L)1 antibody refers to an antibody against PD-1 or PD-L1 target, including PD-1 antibody, PD-L1
  • Antibodies or double antibodies against PD-1 or PD-L1 targets are provided.
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ or an antigen-binding portion thereof, comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises the same amino acid sequence as shown in IIWGX 1 X 2 STDYX 3 X 4 ALKS, wherein X 1 is D, E or N, X 2 is G, A or S, and X3 is N, Q or S, X4 is S, T or A;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 23 or SEQ ID NO: 24;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 8 or SEQ ID NO: 25;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 6;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising:
  • heavy chain variable region CDR1 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 5;
  • heavy chain variable region CDR2 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 14;
  • heavy chain variable region CDR3 which comprises an amino acid sequence consistent with being selected from SEQ ID NO: 7;
  • light chain variable region CDR2 it comprises and is selected from the consistent aminoacid sequence shown in SEQ ID NO: 9;
  • the present invention provides a SIRP ⁇ -targeting monoclonal antibody or an antigen-binding portion thereof, whose CDR sequence homology between the heavy chain variable region and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • antibodies with high (i.e., 90% or higher) homology to the CDR region or variable region of a monoclonal antibody targeting SIRP ⁇ are obtained through conservative sequence modifications, including one or more amino acids substitutions, additions, and deletions.
  • the one or more amino acid additions, deletions and/or substitutions are no more than five, preferably no more than three.
  • NS deamidation site
  • DG isomerization site
  • the general sequence formula of the heavy chain variable region CDR2 can be summarized as IIWGX 1 X 2 STDYX 3 X 4 ALKS (SEQ ID NO: 41), wherein X 1 is D, E or N, X 2 is G, A or S, X3 is N, Q or S, and X4 is S, T or A.
  • the general formula of the light chain variable region CDR1 can be summarized as RASESVDSYGX 5 X 6 FM (SEQ ID NO: 42), wherein X 5 is N, Q or S, and X 6 is S, T or A.
  • Example 18 For the exemplary description of the CDR sequence mutation of the heavy chain variable region and the light chain variable region, refer to Example 18.
  • two site mutations can be performed on the heavy chain CDR2, and the general formula can be summarized as IIWGDX 1 STDYNX 2 ALKS( SEQ ID NO:43), wherein X 1 is G or A, X 2 is S or A, it comprises and is selected from SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:23 or SEQ ID NO:24 Consensus amino acid sequences shown.
  • One site can be mutated to the light chain CDR1, and the general formula can be summarized as RASESVDSYGX 3 SFM (SEQ ID NO: 44), wherein X 3 is N or S, which comprises and is selected from SEQ ID NO: 8 or SEQ ID NO : The consensus amino acid sequence shown in 25.
  • the isomerization and deamidation sites found in the CDR sequence were mutated to eliminate the PTM risk (post-translational modification) and hopefully preserve the structure of the CDR loop.
  • the invention provides a monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ comprising heavy and light chain variable region sequences:
  • a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18;
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ or an antigen-binding portion thereof, the heavy chain variable region of which is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 18 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; Its light chain variable region has at least 90%, 91%, 92%, 93% of the amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • Antibodies with high (i.e., 90% or greater) homology between the heavy chain variable region (VH) and light chain variable region (VL) to the VH and VL regions of the above sequences were obtained by conservative sequence modifications, including amino acid substitutions , additions and deletions, etc.
  • the term "conservative sequence modification” is intended to mean that an amino acid modification does not significantly affect or alter the binding characteristics of an antibody comprising that amino acid sequence. Modifications can be made by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis of nucleic acid molecules encoding variable region sequences. Conservative amino acid substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains.
  • Families of amino acid residues having similar side chains have been specified in the art. These families include those with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine , proline, phenylalanine, methionine), ⁇ -branched side chains (e.g.
  • basic side chains e.g. lysine, arginine, histidine
  • acidic side chains e.g. aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, glycine, asparagine, glutamine, serine, thre
  • Amino acids can be grouped by common side chain properties:
  • hydrophobicity norleucine, Met, Ala, Val, Leu, Ile;
  • Non-conservative substitutions entail exchanging a member of one of these classes for another.
  • the heavy chain and light chain variable region sequences of the murine antibody AD4-12 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Compare the AD4-12 sequence with the human germline sequence library IMGT (Lefranc, 2003), and select the human germline sequence with the least amino acid difference from the corresponding position of the antibody AD4-12 framework as a humanized template. Refer to Example 16 for AD4-12 was humanized and backmutated, and the optimized humanized variant was basically consistent with the affinity of the murine antibody AD4-12 (refer to Example 17), among which the heavy chain and light chain of the humanized antibody were preferred.
  • the chain variable region sequence is:
  • the heavy chain variable region sequence is SEQ ID NO: 16, and the light chain variable region sequence is SEQ ID NO: 20;
  • the heavy chain variable region sequence is SEQ ID NO: 16, and the light chain variable region sequence is SEQ ID NO: 22;
  • the heavy chain variable region sequence is SEQ ID NO: 17, and the light chain variable region sequence is SEQ ID NO: 20;
  • the heavy chain variable region sequence is SEQ ID NO: 17, and the light chain variable region sequence is SEQ ID NO: 21;
  • the heavy chain variable region sequence is SEQ ID NO: 17, and the light chain variable region sequence is SEQ ID NO: 22;
  • the heavy chain variable region sequence is SEQ ID NO: 18, and the light chain variable region sequence is SEQ ID NO: 20;
  • the heavy chain variable region sequence is SEQ ID NO: 18, and the light chain variable region sequence is SEQ ID NO: 21;
  • the heavy chain variable region sequence is SEQ ID NO: 18, and the light chain variable region sequence is SEQ ID NO: 22;
  • the antibody is a full length antibody comprising a human IgG constant region.
  • the antibody is a full-length antibody comprising a mouse IgG constant region.
  • a full-length heavy chain gene can be formed by linking the gene sequence encoding the heavy chain variable region to the gene sequence encoding the human antibody heavy chain constant region (CH1, CH2, and CH3), and the gene sequence encoding the light chain variable region to Gene sequences encoding human antibody light chain constant regions are formed as full-length light chain genes.
  • the sequences of the human heavy chain constant region gene and the light chain constant region are known in the art (see e.g. Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • the heavy chain constant region can be human IgG1, IgG2, IgG3, IgG4 constant region, in another embodiment, the human constant region Fc domain is IgG1, IgG2 or IgG4. In another embodiment, the human constant region Fc domain is IgG1w or IgG1 mutant (LALA).
  • the light chain constant region can be a kappa or lambda constant region, but is most preferably a kappa constant region.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more commonly, both genes can be inserted into the same expression vector, and the expression vectors encoding the heavy and light chains can be transfected into the host by standard techniques Cells express full-length antibodies.
  • an antibody or antibody fragment of the invention is a human antibody or human antibody fragment.
  • the antibody fragment of the invention is a Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2 antibody fragment.
  • antibody fragments of the invention are diabodies.
  • the invention provides a monoclonal antibody targeting SIRP ⁇ comprising:
  • the invention provides an isolated monoclonal antibody whose heavy chain (HC) is selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
  • HC heavy chain
  • the amino acid sequence shown in NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology; its light chain (LC) has at least 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence selected from SEQ ID NO: 26 or SEQ ID NO: 27 %, 97%, 98%, 99% or 100% sequence identity.
  • Antibodies with high (i.e., 90% or more) homology to the heavy and light chains of the above sequences can be mutagenized (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) to nucleic acids encoding heavy and light chain amino acids molecules, the encoded altered antibodies are then tested for retained gain of function using the functional assays described herein.
  • Preferred sites for site-directed mutagenesis or PCR-mediated mutagenesis are located outside of the heavy chain variable region CDR1-CDR3 and the light chain variable region CDR1-CDR3.
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ , comprising:
  • the heavy chain and light chain sequences of the preferred humanized antibodies of the present invention are shown in the following table:
  • the present invention provides a monoclonal antibody targeting SIRP ⁇ , which comprises:
  • the monoclonal antibody or antigen-binding portion thereof targeting SIRP ⁇ used has the following characteristics:
  • the SIRP ⁇ antibody or its antigen-binding part has high affinity with SIRP ⁇ V1, the affinity is at least on the order of 10 -11 M or higher;
  • the SIRP ⁇ antibody or its antigen-binding part can efficiently block the binding of CD47-SIRP ⁇ V1, with an IC50 value of 4.7nM or lower;
  • SIRP ⁇ antibody or its antigen-binding part has high affinity with SIRP ⁇ V2 and SIRP ⁇ V8, and can simultaneously block the binding of CD47-SIRP ⁇ V2 and CD47-SIRP ⁇ V8;
  • the affinity between the SIRP ⁇ antibody or its antigen-binding portion and SIRP ⁇ is relatively weak, on the order of 10 -9 M, and the affinity data between the SIRP ⁇ antibody and SIRP ⁇ is comparable to the affinity data between the SIRP ⁇ antibody and SIRP ⁇ V1 2 orders of magnitude worse than approx.
  • the SIRP ⁇ antibody or antigen-binding portion thereof does not cause erythrocyte agglutination.
  • the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion used may also have at least one of the following characteristics (especially at least two of the following characteristics, especially all of the following characteristics characteristic):
  • the SIRP ⁇ antibody or antigen-binding portion thereof does not inhibit (especially in vivo) proliferation and/or activation of human T cells;
  • the SIRP ⁇ antibody or its antigen-binding part can promote the activation of macrophages, especially the phagocytosis of tumor cells by macrophages, by blocking the SIRP ⁇ -CD47 signaling pathway; and/or
  • the combination of the SIRP ⁇ antibody or its antigen-binding part with Rituximab can significantly promote the phagocytosis of Macrophage on tumor cells; it can also greatly improve the phagocytosis of Macrophage when used in combination with Daratumumab; and PD-(L)1 monoclonal antibody Combined use has a synergistic antitumor effect.
  • the present invention also provides an isolated polynucleotide encoding the SIRP ⁇ -targeting monoclonal antibody or an antigen-binding portion thereof.
  • a nucleic acid molecule of the invention may be DNA or RNA, and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • nucleic acid molecules of the invention can be obtained using standard molecular biology techniques.
  • 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.
  • nucleic acid encoding the antibody can be recovered from the library.
  • Preferred nucleic acid molecules of the present invention are those encoding the amino acid sequences of CDR regions, variable regions or full-length antibodies of SIRP ⁇ -targeting monoclonal antibodies shown in the present invention.
  • these DNA fragments are further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody gene, Fab fragment gene or scFv gene.
  • a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked” as used herein is intended to mean the joining of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain in the same reading frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3).
  • the sequence of the human heavy chain constant region gene is known in the art.
  • the heavy chain constant region may be an IgGl, IgG2, IgG3, IgG4 constant region, preferably an IgGl or IgG4 constant region.
  • the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequence of the human light chain constant region gene is known in the art, and the light chain constant region may be a kappa or lambda constant region, but is most preferably a kappa constant region.
  • DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as adjacent single Chain protein, wherein the VL and VH regions are connected by a flexible linker, and the general sequence formula can be (G 4 S)n, (SG 4 )n, G 4 (SG 4 )n or (G 4 S)nG, etc., wherein n It is an integer of 1-6, preferably 3-5.
  • the present invention provides an expression vector comprising the isolated polynucleotide, and a host cell comprising the expression vector.
  • vector when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • Vectors include plasmids, viruses, cosmids and artificial chromosomes.
  • engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker.
  • Vectors can also include other features besides transgene inserts and backbones: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags.
  • Vectors called expression vectors (expression constructs) are specifically used to express the transgene in target cells and usually have control sequences.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.
  • Antibody genes are inserted into expression vectors by standard methods.
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes for any antibody isotype by inserting them into regions already encoding the heavy and light chain constant regions of the desired isotype.
  • the VH segment is operably linked to the CH segment in the vector
  • the VK segment is operably linked to the CL segment in the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chains from the host cell.
  • the antibody chain genes can be cloned into a vector such that the signal peptide is linked in the same reading frame as the amino terminus of the antibody chain genes.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).
  • expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.
  • it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells it is most preferred to express the antibodies in eukaryotic cells, most preferably in mammalian host cells.
  • Preferred mammalian host cells for expressing the recombinant antibody of the present invention include Chinese hamster ovary cells (CHO cells), NSO myeloma cells, COS cells and SP2 cells, etc., preferably CHO cells.
  • the antibody When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for expression of the antibody in the host cell, or, more preferably, secreted into the medium in which the host cell is grown .
  • Antibodies can be recovered from the culture broth using standard protein purification methods.
  • the present invention provides a pharmaceutical composition, comprising the monoclonal antibody or antibody fragment targeting SIRP ⁇ and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition, which comprises one or a group of SIRP ⁇ -targeting monoclonal antibodies or antigen-binding portions thereof formulated together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the present invention provides a pharmaceutical composition, which includes the SIRP ⁇ -targeting monoclonal antibody or its antigen-binding portion of the present invention, Rituximab and a pharmaceutically acceptable carrier, which can be used to treat tumors related diseases, especially hematologic malignancies.
  • the present invention provides a pharmaceutical composition, which includes the SIRP ⁇ -targeting monoclonal antibody or its antigen-binding portion of the present invention, Daratumumab and a pharmaceutically acceptable carrier, which can be used to treat tumors related diseases, especially hematologic malignancies.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the SIRP ⁇ -targeting monoclonal antibody or its antigen-binding portion of the present invention, PD-(L)1 antibody, and a pharmaceutically acceptable carrier , which can be used to treat tumor-related diseases, especially tumor immunotherapy.
  • the PD-(L)1 antibody includes Nivolumab, Pembrolizumab, Cemiplimab-rwlc, Camrelizumab ( Camrelizumab), Sintilimab, Toripalimab, Tislelizumab, Zimberelimab, Penpulimab , Serplulimab, Pucotenlimab, Atezolizumab, Durvalumab, Avelumab, Enviro Envafolimab, Sugemalimab, and Cadonilimab.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated into dosage forms such as solution, microemulsion, liposome or freeze-dried powder injection.
  • Preferred routes of administration of the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal/spinal or other parenteral routes of administration, eg, by injection or infusion.
  • the dosage of the antibody of the present invention is in the range of about 0.01-100 mg/kg, more usually 0.1 mg/kg-100 mg/kg, or 0.5 mg/kg-50 mg/kg, or 1 mg/kg-25 mg/kg, Or 2mg/kg-10mg/kg, or 5mg/kg-10mg/kg dosage.
  • Exemplary treatment regimens call for administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a "therapeutically effective amount" of a SIRP ⁇ mAb of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of damage or disability resulting from disease.
  • a "therapeutically effective amount” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, relative to untreated subjects , and still more preferably up to at least about 80%.
  • the ability of compounds to inhibit tumor growth can be assessed in animal model systems that are predictive of efficacy in human tumors. Those of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.
  • the present invention provides a method for treating diseases, the method comprising administering a therapeutically effective amount of the SIRP ⁇ -targeting monoclonal antibody or antibody fragment of the present invention to a tumor patient or a microbial infectious disease patient in need of treatment.
  • the monoclonal antibody targeting SIRP ⁇ or its antigen-binding part of the present invention has high affinity with SIRP ⁇ V1, SIRP ⁇ V2, and SIRP ⁇ V8, and can promote the activation of macrophages by blocking the SIRP ⁇ -CD47 signaling pathway, especially the promotion of macrophages.
  • the phagocytosis of tumor cells by cells especially in combination with rituximab or daratumumab, can significantly promote the phagocytosis of tumor cells by Macrophage; in combination with PD-(L)1 monoclonal antibody, it has a synergistic anti-tumor effect.
  • the antibody of the present invention does not cause erythrocyte agglutination, and does not inhibit (especially in vivo) the proliferation and/or activation of human T cells, thus it is expected to reduce the unnecessary side effects of such drugs in tumor treatment .
  • the present invention administers the SIRP ⁇ antibody or formulation thereof to a subject in need of treatment.
  • subject includes humans and non-human animals.
  • Non-human animals include all vertebrates, eg mammals and non-mammals such as non-human primates, sheep, dogs, mice, rats, cats, cows, horses, chickens, amphibians and reptiles.
  • Preferred subjects or individuals to be administered are mammals, such as mice, monkeys, dogs, cows, horses or humans, more preferably humans.
  • Administration of a monoclonal antibody or antibody fragment targeting SIRP ⁇ to a subject can eliminate or inhibit or interfere with the expression, activity and/or signaling function of SIRP ⁇ mediated by ligand binding (eg, CD47 binding).
  • the disease or disorder associated with SIRP ⁇ expression is cancer.
  • a SIRP ⁇ antibody is administered to a patient with a SIRP ⁇ -expressing cancer, such as a blood proliferative disorder of myeloid cells.
  • an anti-SIRP ⁇ antibody is administered to a patient with a CD47-expressing cancer.
  • the present invention provides the use of a monoclonal antibody or antibody fragment targeting SIRP ⁇ for treating cancer or for inhibiting tumor growth.
  • the tumor or cancer is selected from squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer , liver cancer, colorectal cancer, endometrial cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma, cervical cancer, stomach cancer, bladder cancer, head and neck cancer, melanoma, bone cancer, skin Carcinoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML ), multiple myeloma, etc
  • the present invention provides the use of the SIRP ⁇ -targeting monoclonal antibody or its antigen-binding portion in combination with Rituximab in the treatment of tumor diseases.
  • said neoplastic disease is a hematological tumor.
  • the hematological neoplastic disease is diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, etc.
  • the present invention provides the use of the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion in combination with daratumumab in the treatment of tumor diseases.
  • said neoplastic disease is a hematological tumor.
  • the hematological neoplastic disease is diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, etc.
  • the present invention provides the use of the monoclonal antibody targeting SIRP ⁇ or its antigen-binding portion in combination with PD-(L)1 antibody in the treatment of tumor diseases.
  • the PD-(L)1 antibody includes PD-1 antibody, PD-L1 antibody or double antibody against PD-1/PD-L1; wherein, the PD-1 antibody includes Nivolumab , Pembrolizumab, Cemiplimab-rwlc, Camrelizumab, Sintilimab, Toripalimab , Tislelizumab, Zimberelimab, Penpulimab, Serplimab, Pucotenlimab, etc.
  • PD-L1 monoclonal antibodies include Atezolizumab, Durvalumab, Avelumab, Envafolimab, Sugemalimab ( Sugemalimab) and so on.
  • Dual antibodies against PD-1/PD-L1 include Cadonilimab (Cadonilimab), which targets PD-1 and CTLA-4.
  • the tumor disease is blood tumor, malignant melanoma, breast cancer, small cell lung cancer, non-small cell lung cancer, liver cancer, gastric cancer, kidney cancer, colorectal cancer, bladder cancer, head and neck tumor , cervical cancer, Merkel cell carcinoma and all solid tumors with microsatellite instability (MSI-H), etc.
  • the hematological neoplastic disease is diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, etc.
  • the present invention provides the use of monoclonal antibodies or antibody fragments targeting SIRP ⁇ for treating microbial infectious diseases.
  • the monoclonal antibody targeting SIRP ⁇ or the antigen-binding part thereof of the present invention can promote the activation of macrophages, thereby inducing or maintaining phagocytosis in an individual.
  • Phagocytosis involves professional phagocytes (eg, monocytes, macrophages, neutrophils, dendritic cells, or mast cells), non-professional phagocytes (eg, epithelial, endothelial, fibroblast, or mesenchymal plasmocytes) or both.
  • the invention provides methods for treating a viral infection or bacterial infectious disease in an individual comprising administering to an individual suffering from a viral infection or bacterial infectious disease a monoclonal antibody or an antigen-binding portion thereof described herein that targets SIRP ⁇ .
  • the viral infection or bacterial infection disorder or condition is chronic or acute.
  • the viral infectious diseases include adenovirus, herpes virus, papillomavirus, coronavirus, human immunodeficiency virus (HIV), human cytomegalovirus, Epstein-Barr virus, hepatitis C virus or hepatitis B virus, etc. disease.
  • the pathogenic infectious diseases include diseases caused by bacillus, Chlamydia pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas, Salmonella, Staphylococcus, Streptococcus, Treponema and the like.
  • Optimal dosages of SIRP ⁇ -targeting monoclonal antibodies or antibody fragments of the invention will depend on the disease being treated, the severity of the disease, and the presence or absence of side effects. Optimal dosages can be determined by routine experimentation. For parenteral administration, give 0.1 mg/kg-100 mg/kg, or 0.5 mg/kg-50 mg/kg, or 1 mg/kg-25 mg/kg, or 2 mg/kg-10 mg/kg, or 5 mg/kg-10 mg /kg dose. Exemplary treatment regimens may be administered weekly, every two weeks, every three weeks, every four weeks, monthly, every 3 months, or every 3-6 months.
  • Example 1 Obtaining murine antibody sequences by immunizing mice
  • SIRP ⁇ V1-Fc Fc fusion protein SIRP ⁇ V1-Fc (SEQ ID NO: 1) purchased from ACRO (Cat#SIA-H5251), and then use SIRP ⁇ V1 (SEQ ID NO:2) Transfected CHO cells were used as antigens to immunize BALB/C mice, and the immune response of mice was monitored by ELISA method.
  • the cDNAs of the VL and VH regions obtained from AD4-12 were connected to the human ⁇ light chain and the constant regions of IgG1, IgG2 and IgG4 heavy chains respectively, and the Fc segments of IgG1 and IgG2 heavy chain constant regions were designed to enhance or weaken the interaction with Fc receptors, respectively. body-binding mutant sequences.
  • the constructed AD4-12 human-mouse chimeric light chain and heavy chain were cloned into the expression vector pCDNA3.1(+).
  • the expression vector plasmid was transiently transfected into ExpiCHO-S cells for expression, the medium was (Gibco, Cat#A29100-01), and the transfection kit was (Gibco, Cat#A29129).
  • the specific method is as follows: subculture the ExpiCHO-S cells one day before transfection, mix the constructed plasmid and transfection reagent in a 25mL system, drop into the ExpiCHO-S cell culture, mix well, and incubate at 37°C After 18-22 hours, add the feed medium according to the instructions in the kit, place it in a 5% CO2 shaker incubator at 32°C, add the second feed on the 5th day, collect the supernatant after 10-12 days, and use
  • the conventional Protein A column method was used to purify the target antibodies of different IgG subtypes: AD4-12-G1 and AD4-12-G1lala.
  • the following examples focus on evaluating the IgG1 wild type (AD4-12-G1) and the mutant type (AD4-12-G1 lala ) containing amino acid substitutions of L234A/L235A (LALA) to eliminate binding to Fc receptors.
  • AD4-12-G1 and AD4-12-G1 lala the two are collectively marked as AD4-12, and only when comparing AD4-12-G1 and AD4-12-G1 lala are they marked separately Both.
  • Biofilm interferometry detects the affinity of AD4-12 to SIRP ⁇ -V1, SIRP ⁇ -V2, SIRP ⁇ , SIRP ⁇ and cynomolgus monkey SIRP ⁇
  • the binding affinity of AD4-12 to SIRP ⁇ , SIRP ⁇ , and SIRP ⁇ was determined by ForteBio Octet RED 96.
  • AD4-12 and reference product 18D5 were serially diluted by 2 times to a total of 7 concentration points.
  • the initial antibody concentration and reaction dissociation time used to immobilize protein molecules are different: for SIRP ⁇ V1, SIRP ⁇ V2 and SIRP ⁇ immobilized sensors, the initial antibody concentration is 25nM, the binding time is 200 seconds, and the dissociation time is 1000 seconds; For the SIRP ⁇ -immobilized sensor, the initial antibody concentration is 200 nM, the binding time is 200 seconds, and the dissociation time is 800 seconds; for the cynomolgus monkey SIRP ⁇ -immobilized sensor, the binding time is 200 seconds, and the dissociation time is 600 seconds. Data fitting to calculate affinities.
  • AD4-12 of the present invention has high affinity with SIRP ⁇ -V1, which is an order of magnitude higher than that of the reference product 18D5; AD4-12 also has high affinity with SIRP ⁇ -V2; AD4-12
  • the binding affinity to SIRP ⁇ is slightly weaker than that to SIRP ⁇ ; AD4-12 can also bind to SIRP ⁇ , but the affinity is two orders of magnitude lower than that to SIRP ⁇ .
  • AD4-12 was able to cross-bind cynomolgus monkey SIRP ⁇ molecules with an affinity similar to that of human SIRP ⁇ molecules.
  • the antibody AD4-12 of the present invention has high affinity to SIRP ⁇ -V1, V2 and V8, and its EC50 value is significantly lower than that of the control antibody 18D5; and the binding of antibody AD4-12 to SIRP ⁇ is weaker than that of the control antibody 18D5 .
  • Example 4 Detection of AD4-12 binding to SIRP ⁇ -V1 and V2 by flow cytometry (FACS).
  • AD4-12 has a very good binding activity to the stable cell line expressing SIRP ⁇ -V1 protein, slightly better than 18D5; AD4-12 can well bind to the stable cell line expressing SIRP ⁇ -V2 protein strain, its binding activity was significantly better than that of 18D5.
  • Example 6 FACS detection of the binding of AD4-12 antibody to human PBMC
  • PBMC peripheral blood mononuclear cells
  • T cells CD3+CD56-
  • NK cells CD3-CD56+
  • NKT cells CD3+ CD56+
  • Example 7 Enzyme-linked immunosorbent assay (ELISA) detection of AD4-12 blocking SIRP ⁇ -V1, V2 binding to CD47
  • Streptavdin-HRP (Thermo, Cat#434323) was added to each well, the substrate TMB (Beyotime, Cat#P0209-100mL) was used for color development, and the microplate reader was read.
  • AD4-12 can block the binding of SIRP ⁇ -V1 and SIRP ⁇ -V2 to CD47, respectively, with IC50 values of 5.258nM and 9.322nM, and the blocking effects are better than 18D5.
  • AD4-12 has good blocking activity on the interaction of SIRP ⁇ -V1-CD47 and SIRP ⁇ -V2-CD47, with IC50 values of 1.742nM and 6.417nM, respectively.
  • the reference product 18D5 was weaker in blocking SIRP ⁇ -V1-CD47 than AD4-12, and had no blocking activity on SIRP ⁇ -V2-CD47.
  • Fresh PBMC (Miaoshun Biotechnology, Cat#PB003F), washed with the prepared 1 ⁇ BD IMag TM buffer (BD Pharmingen, Cat#552362), add 50 ⁇ L BD IMag TM Anti-Human CD14 Magnetic Particles per 1 ⁇ 10 7 cells –DM (BD Pharmingen, Cat#557769), incubate at room temperature for 30 minutes; after rinsing the cells, separate CD14 + monocytes with Cell Separation Magnet, resuspend in IMDM medium (containing 10% FBS+50uM ⁇ -mercaptoethanol+70ng/ ml M-CSF), 1 ⁇ 10 5 cells per well were spread on a 96-well plate, and placed in a 5% CO 2 incubator at 37°C.
  • IMDM medium containing 10% FBS+50uM ⁇ -mercaptoethanol+70ng/ ml M-CSF
  • Fc blocker was prepared in IgG1 with a final concentration of 1 mg/mL as a blocking solution, incubated at 4°C for 15 minutes, and then stained with fluorescein-labeled CD11b antibody, CD14 antibody, and SIRP ⁇ antibody (Santa Cruz, Cat#sc-23863PE). Samples were loaded on the flow cytometer and analyzed by FlowJo V10 and Graphpad Prism6 software.
  • Example 10 Phagocytosis-promoting effect of AD4-12 combined with rituximab and daratumumab
  • Collect Raji cells in the logarithmic growth phase (CD20, CD38, and CD47 are all positive), count and adjust the cell density to 8 ⁇ 10 6 cells/mL.
  • AD4-12 at a final concentration of 10 ⁇ g/mL to each well of the 96-well plate (see Example 9) in which differentiated macrophages were pre-induced, and continue to incubate at 37° C. in a 5% CO 2 incubator for 30 minutes.
  • CFSE-labeled Raji cells were incubated with or without rituximab (RTX) or daratoy (Dara) at a final concentration of 0.1 ⁇ g/mL in a 5% CO 2 incubator at 37°C for 30 minutes.
  • RTX rituximab
  • Dara daratoy
  • the Raji cells were transferred into the above-mentioned 96-well plate that had been plated with macrophages according to the effector-target cell ratio of 1:5, that is, 1 ⁇ 10 5 /well of macrophages and 5 ⁇ 10 5 /well of Raji cells, and placed at 37°C for 5 Incubate for 2 hours in a % CO 2 incubator.
  • AD4-12 has no phagocytic effect by itself, but it can significantly enhance the two antibody-dependent cellular phagocytosis (ADCP) when combined with rituximab (A) or daratoyol (B) .
  • Example 11 Induction of dendritic cell differentiation and identification
  • BD IMag TM Anti-Human CD14 Magnetic Particles–DM (BD Pharmingen, Cat#557769) per 1 ⁇ 10 7 cells, and incubate at room temperature for 30 minutes; after rinsing the cells, separate them with a Cell Separation Magnet to obtain CD14 + single Nucleated cells were resuspended in IMDM medium (containing 10% FBS + 50uM ⁇ -mercaptoethanol + 100ng/ml GM-CSF + 70ng/ml IL-4), inoculated with 5 ⁇ 106 cells in each bottle, and placed at 37°C for 5 %CO 2 incubator, after 3 days, the same medium was replenished, and the culture was continued to 7 days.
  • IMDM medium containing 10% FBS + 50uM ⁇ -mercaptoethanol + 100ng/ml GM-CSF + 70ng/ml IL-4
  • Fc blocker was prepared in IgG1 with a final concentration of 1 mg/mL as a blocking solution, incubated at 4°C for 15 minutes, then stained with fluorescein-labeled CD11b antibody and CD14 antibody, loaded on the flow cytometer, and analyzed by FlowJo V10 software.
  • Example 12 Mixed Lymphocyte Reaction (MLR) Evaluation of the Effect of AD4-12 on T Cell Activation
  • pHrodo Thermo, Cat#Z25611
  • the dye to be tested can be labeled with the dye to detect its endocytosis on a flow cytometer.
  • CD fusion medium to dilute the antibody to a final concentration of 160nM, and dilute the pHrodo stock solution 12.5 times.
  • Example 14 Antitumor activity of AD4-12 combined with rituximab
  • Raji cells were cultured in RPMI-1640 complete medium (89% RPMI1640+10% FBS+1% sodium pyruvate), and cells in logarithmic growth phase were taken to prepare cell suspension.
  • SIRP ⁇ humanized completely immunodeficient hSIRP ⁇ -B-NDG mice Biocytogen
  • 100 ⁇ L of 5 ⁇ 10 5 cell suspension was subcutaneously inoculated. Tumor volumes were measured twice a week with calipers.
  • mice divide mice into 3 groups, 7 in each group, start intraperitoneal injection after the tumor grows to a volume of ⁇ 100mm3 , 150 ⁇ g rituximab alone or combined with 200 ⁇ g AD4-12-G1 or AD4-12-G1 lala , 2 times a week for a total of 6 times.
  • Intraperitoneal injection was started on the day of inoculation, and 5 mice in each group were given normal saline, 20 ⁇ g rituximab alone or combined with 200 ⁇ g AD4-12-G1 or AD4-12-G1 lala , once a week for a total of 3 times.
  • RESULTS As shown in Figure 11B, in this mouse model of immune effector cells (including T cells, B cells, NK cells, and myeloid cells) provided by PBMCs, such a low dose of rituximab compared Saline can significantly delay tumor growth, and AD4-12-G1 lala or AD4-12-G1 combined with rituximab can significantly enhance the efficacy of rituximab (**P ⁇ 0.01, ****P ⁇ 0.0001) , and the efficacy of AD4-12-G1 combined with rituximab was stronger than that of AD4-12-G1 lala combined with rituximab (*P ⁇ 0.05).
  • Example 15 Anti-tumor activity of AD4-12 combined with daratumumab
  • mice Divide the mice into 3 groups, 7 in each group, and start intraperitoneal injection after the tumor grows to a volume of ⁇ 100mm3 , 150 ⁇ g daratuyil alone or combined with 200 ⁇ g AD4-12-G1 or AD4-12-G1 lala Medication, 2 times a week, a total of 6 times.
  • Intraperitoneal injection was started on the day of inoculation, and 5 mice in each group were given normal saline, 15 ⁇ g daratoyol alone or combined with 200 ⁇ g AD4-12-G1 or AD4-12-G1 lala , once a week 3 times in total.
  • the AD4-12 sequence was compared with the human immunoglobulin sequence library IMGT (Lefranc, 2003), and the human germline sequence with the least difference with the amino acid sequence of the VH and VL framework regions of AD4-12 was selected as the humanization template. See Table 4 for the V and J germlines with the closest AD4-12 sequence match.
  • the CDR region of AD4-12 is directly replaced with the CDR region of the humanized template to form the variant Z0 heavy chain variable region VH (SEQ ID NO: 15) and light chain variable region VL (SEQ ID NO: 19).
  • the second step reverse mutations were performed on the sites in the VH and VL framework sequences that may be necessary to maintain the conformation of the CDR, and the VH and VL carrying different mutations were combined in pairs to form humanized variants Z0-Z9 (Table 5-6).
  • these VL and VH were connected to the human ⁇ light chain and IgG1 heavy chain constant region, respectively, and the Fc segment of the heavy chain constant region contained the L234A/L235A (lala) mutation (SEQ ID NO: 13).
  • the activator was prepared by mixing 400mM EDC and 100mM NHS using the Biacore analysis system, and the CM5 sensor chip was activated with the activator at a flow rate of 10 ⁇ L/min for 420 seconds.
  • 30 ⁇ g/mL anti-Fc antibody dissolved in 10 mM NaAc (pH 4.5) was injected into the channel at a flow rate of 10 ⁇ L/min, and then blocked with 1M ethanolamine-HCl at a flow rate of 10 ⁇ L/min for 420 seconds.
  • AD4-12 Various humanized variants of AD4-12 were bound to the CM5 sensor chip at a flow rate of 10 ⁇ L/min, followed by injection of 50 nM SIRP ⁇ V1-his (ACRO, Cat#SIA-H5225) at a flow rate of 30 ⁇ L/min into channel 1- 8 for 120 s followed by 300 s dissociation, injecting 10 mM glycine at pH 1.5 as regeneration buffer after each dissociation stage. All experimental data were fitted using a 1:1 binding model.
  • the heavy chain CDR2 can be mutated at two sites, and the general formula can be summarized as IIWGDX 1 STDYNX 2 ALKS, wherein X 1 is G or A, X 2 is S or A, and the result is identical to SEQ ID NO: 6, SEQ ID NO : 14, the consensus amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.
  • the light chain CDR1 can be mutated at one site, the general formula can be summarized as RASESVDSYGX 3 SFM, wherein X 3 is N or S, and the amino acid sequence consistent with SEQ ID NO: 8 or SEQ ID NO: 25 can be obtained.
  • the mutated sequences were connected to the human ⁇ light chain and the IgG1 heavy chain constant region containing the L234A/L235A (lala) mutation, and their binding ability to SIRP family members was compared by flow cytometry, and finally the CDR2 of the heavy chain was selected.
  • H7L4 with both the NS site and the DG site eliminated was used as our antibody drug candidate sequence (Table 9-11).
  • VL and VH fragment cDNA of H7L4 were combined with human kappa light chain and wild-type IgG1 (SEQ ID NO:32), IgG2 (SEQ ID NO:33), IgG4 (SEQ ID NO:34), and L234A/L235A ( lala) mutated IgG1 heavy chain constant regions were connected to construct antibody subtypes with different effector function activities (Table 12).
  • the light and heavy chains of H7L4 were respectively cloned into the pCDNA3.1(+) expression vector, and the plasmid was transiently transferred into ExpiCHO-S cells, cultured and purified.
  • H7L4-G1 SEQ ID NO: 35, SEQ ID NO:27
  • H7L4-G2 SEQ ID NO:36, SEQ ID NO:27
  • H7L4-G4 SEQ ID NO:37, SEQ ID NO:27
  • H7L4-G1 lala SEQ ID NO: 31, SEQ ID NO: 27
  • the reference product BI 765063 was synthesized according to the sequence published in the patent CN201780023581.2 of OSE Immunotherapeutics, and the Fc fragment was IgG4.
  • Example 20 FACS comparison before and after humanization (AD4-12 and H7L4) binding ability of SIRP ⁇ -V1, V2, V8
  • Example 22 ELISA detection of the combination of H7L4 and SIRP ⁇ -V1, V2, V8
  • H7L4-G1, H7L4-G1 lala can bind well to SIRP ⁇ -V1, SIRP ⁇ -V2, and SIRP ⁇ -V8, while the reference product BI 765063 can only bind to SIRP ⁇ -V1, but cannot bind to SIRP ⁇ -V1. V2, V8.
  • Example 23 FACS detection of the binding of H7L4 to SIRP ⁇ -V1, V2, V8 and SIRP ⁇ , SIRP ⁇
  • H7L4-G1 and H7L4-G1 lala can bind well to SIRP ⁇ -V1, SIRP ⁇ -V2, and SIRP ⁇ -V8, and bind to SIRP ⁇ and SIRP ⁇ relatively weakly, while the reference product BI 765063 binds to SIRP ⁇
  • the binding of -V1 and SIRP ⁇ is close to that of H7L4, and it does not bind to SIRP ⁇ -V2 and SIRP ⁇ at all.
  • Example 24 FACS detection of H7L4 binding to human PBMC subpopulation cells
  • Example 25 ELISA detection of the blocking effect of H7L4 on the binding of SIRP ⁇ -V1 and V2 to CD47
  • H7L4-G1 and H7L4-G1 lala can block the binding of SIRP ⁇ -V1, V2 and CD47.
  • V2-CD47 has no blocking activity.
  • the cells 293T-SIRP ⁇ -V1, CHOZN-SIRP ⁇ -V2, and 293T-SIRP ⁇ -V8 in the logarithmic growth phase were taken, and 2 ⁇ 105 cells per well were added to a 96-well U-shaped culture plate.
  • FITC-labeled CD47 0.5 ⁇ g/well
  • samples H7L4-G1 and H7L4-G1 lala with concentration gradient dilution, reference product BI 765063 and isotype control incubated at 4°C for 30 minutes; cells were loaded on flow cytometer after washing, FlowJo V10 and Graphpad Prism6 software analyze.
  • both H7L4-G1 and H7L4-G1 lala can block the binding of SIRP ⁇ -V1, V2, V8 and CD47; while the reference product BI 765063 can only block the binding of SIRP ⁇ -V1 and CD47, and has no effect on SIRP ⁇ -V2, V8 can't block well.
  • Example 27 Phagocytosis-promoting effect of H7L4 combined with rituximab and daratumumab
  • the experimental method is the same as in Example 10, the antibodies used and their concentrations are: 10 ⁇ g/mL of H7L4-G1, H7L4-G1 lala or reference products BI 765063, Hu5F9-G4 (CD47 antibody Magrolimab, according to WHO Drug Information, Vol.33, No. .3, 2019 published sequence construction plasmid expression), and isotype control hIgG1; 0.1 ⁇ g/mL rituximab (RTX) or 0.2 ⁇ g/mL daratoy (Dara).
  • Example 28 Detection of the ADCP effect of H7L4 on SIRP ⁇ + cells by macrophage phagocytosis
  • the experimental method is the same as in Example 10, the target cells are CFSE-labeled U937 cells (both SIRP ⁇ and CD47 are highly expressed), and the antibodies used and their concentrations are: 10 ⁇ g/mL of H7L4-G1, H7L4-G1 lala or the reference product BI 765063 , Hu5F9-G4, and the isotype control hIgG1.
  • SIRP ⁇ antibodies including H7L4-G1, H7L4-G1 lala and the reference product BI 765063 basically did not mediate ADCP on U937 cells that were strongly positive for SIRP ⁇ , while the reference product Hu5F9-G4 showed a significant ADCP effect .
  • SIRP ⁇ and CD47 double humanized C57BL/6 mice (hSIRP ⁇ -hCD47-B6, purchased from Biocytogen) were intraperitoneally injected with starch broth to induce macrophage extravasation. Three days later, 200 ⁇ g of H7L4-G1, H7L4-G1 lala or normal saline were injected intraperitoneally, with 3 mice in each group. After another 3 days, peripheral blood, spleen and peritoneal exudate were collected for flow cytometry analysis.
  • H7L4-G1 slightly reduced CD11b + myeloid cells in peripheral blood (did not reach significance), but not F4/80 + macrophages in spleen.
  • CD11b + myeloid cells in peritoneal effusion did not decrease but increased (p ⁇ 0.05).
  • the red blood cell samples were washed and resuspended with DPBS to form a 6% (v/v) red blood cell suspension.
  • Chemi Doc XRS+Imaging System was used to image and record the results.
  • Hu5F9-G4 can significantly induce erythrocyte agglutination, while H7L4-G1 does not cause erythrocyte agglutination.
  • Fresh PBMCs (Miaoshun Biotech, Cat#PB003F) from 3 donors were placed in 3 duplicate wells in a U-bottom 96-well plate, with 0.5x106 cells per well, and H7L4-G1 was added at a final concentration of 10 ⁇ g/mL , H7L4-G1 lala or the reference product BI 765063 and the blank control without antibody were incubated for 48 hours, the supernatant was collected and frozen, and sent to Union Biotechnology (CRO Company) to detect 27 major components by the multifactor platform Luminex LXR-M500KCAF0Y Cytokines released by macrophages (monocytes), T cells.
  • CRO Company Union Biotechnology
  • Example 33 Anti-tumor effect of H7L4 in immune-competent mice (A20 lymphoma cell line)
  • SIRP ⁇ and CD47 double humanized BALB/c mice (hSIRP ⁇ -hCD47-Balb/c, purchased from Jiangsu Jicui Yaokang) were used as hosts, and 5x105 human CD47-transfected A20 lymphoma cells were subcutaneously inoculated, with 6 mice in each group. mouse.
  • the tumor volume reached 100-200 mm 3
  • 200 ⁇ g of H7L4-G1 or H7L4-G1 lala was injected intraperitoneally, twice a week for a total of 4 times. Tumor volume was measured twice a week.
  • hSIRP ⁇ -hCD47-B6 mice purchased from Biocytogen
  • 5 ⁇ 10 5 human CD47-transfected MC38 tumor cells were subcutaneously inoculated, with 6 mice in each group.
  • the tumor volume reached 100-200 mm 3
  • 200 ⁇ g of H7L4-G1, H7L4-G1 lala and reference product Hu5F9-G4 were injected intraperitoneally, twice a week for 3 weeks. Tumor volume was measured twice a week.
  • Example 35 Synergistic anti-tumor effect of H7L4 and PD-L1 monoclonal antibody in immunocompetent mice (MC38 colon cancer cell line)
  • hSIRP ⁇ -hCD47-B6 mice purchased from Biocytogen
  • 1x10 6 human CD47-transfected MC38 tumor cells were subcutaneously inoculated, with 6 mice in each group.
  • the tumor volume reached 100-200 mm 3
  • 100 ⁇ g PD-L1 monoclonal antibody avelumab or a combination of the two were injected intraperitoneally, once every 3 days for a total of 3 times. Tumor volume was measured twice a week.
  • Example 36 Detection of the binding of H7L4 to cynomolgus monkey SIRP ⁇ by ELISA and FACS
  • Resuscitate frozen cynomolgus monkey PBMC (gifted by Union Biotech), add 4 ⁇ 10 5 cells to each well, prepare Fc blocker in IgG1 with a final concentration of 1 mg/mL as a blocking solution, and incubate at 4°C for 15 minutes; Then add biotin-labeled samples H7L4-G1, H7L4-G1 lala and isotype control, and incubate at 4°C for 30 minutes; after washing, add secondary antibody 0.2 ⁇ g Streptavidin-PE and fluorescein-labeled CD11b antibody for staining, and incubate at 4°C for 30 minutes; After rinsing, add 0.5 ⁇ g of 7-ADD in DPBS/1% FBS and incubate for 5 minutes to distinguish dead and living cells. Samples were loaded on the flow cytometer and analyzed by FlowJo V10 software.
  • H7L4-G1 and H7L4-G1 lala can well bind to cynomolgus monkey SIRP ⁇ at the protein molecular level and on the cell membrane surface.

Abstract

本发明涉及生物医药领域，具体涉及一种靶向SIRPα的单克隆抗体及其制备方法与应用。本发明制备的SIRPα抗体或其抗原结合部分与SIRPα具有高亲和力，与SIRPγ具有低亲和力，两者存在两个数量级的差异，能够高效阻断CD47与SIRPα-V1、SIRPα-V2和SIRPα-V8的结合，从而促进巨噬细胞的活化，特别是增强靶向肿瘤细胞的调理性抗体依赖性细胞吞噬作用[Antibody-dependent cellular phagocytosis(ADCP)]，桥接先天性免疫与适应性免疫反应，以及与免疫检查点抑制剂PD-(L)1单抗的协同抗癌效应。该抗体可用于治疗多种癌症和微生物感染性疾病。

Description

靶向SIRPα的单克隆抗体及其用途 技术领域
本发明涉及生物医药领域，具体涉及一种靶向SIRPα的单克隆抗体及其制备方法与应用。
背景技术
信号调节蛋白(SIRPα)，也称为CD172a、SHPS-1、PTPNS1、BIT或P84，是一种跨膜蛋白，局限性地表达在所有类型的髓系细胞，包括单核细胞、巨噬细胞、中性粒细胞、树突状细胞亚群(Adams et al.,1998；Seiffert et al.,1999；Veillette et al.,1998)。它的胞外区域包含三个Ig样结构域(V、C1和C2域)和胞内区域包含免疫受体基于酪氨酸的抑制基序(ITIM)(Fujioka et al.,1996；Kharitonenkov et al,1997；Liu et al.,2015)，与配体CD47(Genbank Accession：NP_942088)结合后，SIRPα胞内ITIM上的酪氨酸残基被磷酸化，募集并激活含有SH2-结构域的蛋白酪氨酸磷酸酶SHP-1和SHP-2，传导抑制性信号，致使髓系细胞的效应功能降低，尤其是吞噬作用减弱(Fujioka et al.,1996；Kharitonenkov et al.,1997；Tsai and Discher,2008)。CD47-SIRPα轴已被证明在一些内稳态过程中发挥关键作用，比如清除衰老红细胞、保护造血干细胞和修剪神经元突触等(Logtenberg et al.,2020)。然而，绝大多数肿瘤细胞上调CD47表达，即通过所谓的“别吃我”信号来逃避被吞噬。因此，开发针对CD47或SIRPα的单克隆抗体或融合蛋白来阻断“别吃我”信号，从而增强巨噬细胞的吞噬作用，已经是一个被广泛接受的抗肿瘤治疗概念(Chao et al.,2010；Zhao et al.,2011；Feng,et al.,2019)。
CD47-SIRPα轴，作为一条抑制性信号通路，需要有激动信号存在作为前提，否则对它的阻断被认为是无效事件(Logtenberg et al.,2020)。CD47与SIRPα的结合已被证明可以抵消或削弱髓系细胞通过各种膜受体接收到的激动信号，包括：1) Fc伽马受体(FcgRs)，它与单抗的Fc片段结合；2)脂蛋白相关蛋白(LRP)，它与钙网蛋白(CRT)结合；3)SLAMF7自我结合(Chao et al.,2010b；Oldenborg et al.,2001；Logtenberg et al.,2020)。换句话说，单单阻断CD47-SIRPα"别吃我"信号不足以消灭肿瘤细胞，而由Fc-FcγRs或CRT-LRP等相互作用激活"吃我"信号的存在是启动有效吞噬所必需的。因此，CD47或SIRPα阻断剂联合靶向肿瘤细胞的调理性抗体[tumor-opsonized antibody，本文定义为具有抗体依赖性细胞介导的细胞毒作用(ADCC)、抗体依赖性细胞介导的吞噬作用ADCP、补体介导的细胞毒作用(CDC)等效应功能的IgG1亚型单抗，比如抗CD20的利妥昔或抗EGFR的西妥昔，等等]，或联合可以上调CRT表达的表观遗传学调控药物如阿扎胞苷，是目前临床开发的主要策略(Weiskopf et al.,2017；Advani et al.,2018,Feng et al.,2019,Sallman et al.,2020)。
越来越多的证据表明，阻断CD47-SIRPα这一信号通路的作用机制(MOA)不单纯停留在增强巨噬细胞的吞噬作用。首先，阻断CD47-SIRPα信号可促进中性粒细胞“胞啃”(trogocytosis)被肿瘤调理抗体拉近的靶细胞，使其凋亡(Matlung et al.,2018；Bouti et al.,2021；Martínez-Sanz P et al.,2021)。其次，正常情况下不表达SIRPα的NK细胞，当它们与IL-2孵育后可上调表达SIRPα来对抗激动信号、下调NK细胞活性，而阻断CD47-SIRPα信号可增强NK细胞的抗肿瘤细胞毒性(Deuse et al.，2021)。更重要的是，阻断CD47-SIRPα信号使得包括树突状细胞在内的肿瘤微环境中的抗原呈递细胞(APC)增加对肿瘤细胞的吞噬，由肿瘤细胞来源的DNA驱动活化cGAS-STING-IRF3-type I IFNs这一重要固有免疫轴(Liu et al.,2015；Xu et al.,2017)，一方面上调多种促炎性细胞因子和趋化因子的表达和释放，激活NK细胞和促进巨噬细胞向M1极化，增加肿瘤中淋巴细胞浸润；另一方面促进树突状细胞的成熟，增强抗原呈递以及激活T细胞，特别是CD8-T细胞反应，实现固有免疫到适应性免疫之间的桥接(Liu et al.,2015；Tseng et al.,2013；Xu et al.,2017；Gauttier et al.,2020)。同时阻断CD47-SIRPα和PD-L1-PD-1两条通路在动物实验和初步临床数据已展示出抗肿瘤协同效应(Sockolosky et al.,2016；Gauttier et al.,2020；Kuo et al.,2020)，该联合用药在治疗PD-(L)1难治性癌症或所谓的 “冷肿瘤”方面前景可期。
有关SIRPα-CD47通路的药物开发，早期主要集中在使用单克隆抗体或SIRPα-Fc融合蛋白靶向CD47的阻断，全球已经有近20款CD47阻断剂进入临床开发。由于CD47在全身的正常组织细胞中均有表达，正常体细胞尤其是红细胞和血小板受到攻击导致的血液毒性以及体内抗原池(antigen sink)沉没大量药物成为CD47阻断剂开发的两大痛点。从最早的CD47抗体开始临床试验至今走过了坑坑洼洼的十多个年头，目前还没有任何一款CD47阻断剂获批上市。
近年来靶向SIRPα正在获得更多的关注和投入，包括Celgene新基/BMS百时美施贵宝、Fortyseven/Gilead Science、Arch Oncology、ALX Oncology和信达生物等国内外公司，原先一直致力于开发CD47阻断剂，最近也在开展SIRPα单抗研究。相较于CD47，SIRPα在人体内的表达仅局限于髓系细胞，因此通过靶向SIRPα阻断CD47-SIRPα抑制信号可以避免靶向CD47所带来的贫血、血小板减少和凝血异常等严重药物相关性不良事件(TRAE)。抗SIRPα抗体在已公开的国际专利申请公开中有所描述：例如第WO2018/057669号、第WO2018/026600号、第WO2017/178653号、第WO2017/068164号、第WO2016/063233号、第WO2016/205042号、第WO2015/138600号、第WO2013/0956352号、第WO2009/091547号、第WO2009/131453号及第WO2009/046541号等，在此全部引入作为参考。
已有研究发现，SIRPα在人群中具有等位基因的序列多态性以及SIRP家族成员之间的序列高度同源性。文献报道，在东亚、南亚、美洲、欧洲和非洲五大人群中占绝对优势的基因多态性序列为V1、V2和V8三个变体(Volts et al.,2019)，后续更多大数据分析验证在这五大人群中只有V1和V2两个变体序列，没有检测到V8变体(Treffers et al.,2018；Sim et al.,2019)。因此，理想的SIRPα单抗必须能够结合SIRPα-V1和V2。
关于人体SIRP家族，已经识别有SIRPα、SIRPβ和SIRPγ三个成员，它们的胞外区氨基酸序列，尤其是与配体结合的V结构域氨基酸序列高度同源(Barclay et al.,2006)。SIRPβ与SIRPα一样在髓系细胞上表达，但是SIRPβ并不与CD47结合，其配体仍然未知。在功能上，SIRPβ可以通过与具有基于免疫受体酪氨酸的激活 基序(ITAM)的跨膜蛋白DAP12形成复合物来转导激动信号。有研究报道，巨噬细胞上的SIRPβ与其抗体或与交叉结合SIRPβ的SIRPα抗体交联会增强吞噬作用(Hayashi et al.,2004；Sakamoto et al.,2022)。相反，SIRPγ表达于大多数T细胞和一部分NK细胞上。SIRPγ没有胞内尾段，因此没有信号转导活性。SIRPγ与CD47的结合强度约为CD47-SIRPα结合强度的十分之一(Brooke,et al.,2004)。有报道CD47-SIRPγ相互作用可通过增强粘附性从而增强T细胞迁移(与内皮细胞上的CD47结合)和激活反应(与APC或肿瘤细胞上的CD47结合)(Piccio,et al.,2005；Dehmani et al.,2021)，SIRPα抗体KWAR23非选择性地结合SIRPα和SIRPγ，可能通过阻断CD47-SIRPγ相互作用而抑制T细胞活化(Piccio et al.,2005；Gauttier et al.,2020)。显然，如果SIRPα抗体抑制T细胞活化作用存在的话，所有抗CD47抗体都有这个抑制作用，因为CD47抗体对CD47-SIRPα或CD47-SIRPγ的阻断能力无差异。
到目前为止，全球没有SIRPα单抗药物获批上市，仅有两款SIRPα单抗药物报告了初步临床数据，它们就是OSE Immunotherapeutics和Boehringer Ingelheim(勃林格殷格翰生物药业)联合开发的BI 765063和Celgene(新基)/BMS(百时美施贵宝)开发的CC-95251。
BI 765063，又名OSE-172或HEFLB，是一款人源化IgG4亚型SIRPα单抗，局限性结合SIRPα-V1而不能结合SIRPα-V2是它的一大缺陷(Gauttier et al.,2020；Kuo et al.,2020，在临床试验中必须排除非SIRPα-V1基因型的患者(NCT03990233)。它单独给药在晚期实体瘤患者中耐受性良好，并且在36mg/kg最高测试剂量下没有报告DLT，未达MTD。50名患者接受了至少一次BI 765063给药，在可根据RECIST 1.1进行评估的47名患者中观察到初步临床获益，一位肝细胞癌患者达到PR并持续9个月以上(Champiat et al.,ASCO 2021)。随后，BI 765063选择两个剂量(18mg/kg、24mg/kg Q3W)与PD-1单抗联用，18位接受过多线治疗的实体瘤患者临床数据令人鼓舞，有三例携带微卫星稳定(MSS)的晚期子宫内膜癌或结直肠癌患者取得部分缓解(PR)(Kotecki et al.,ESMO 2021)。当前，一个扩展队列正在招募具有上述诊断和MSS特征的患者。
CC-95251是做过祛除补体活化功能修饰的全人源IgG1亚型单抗，与SIRPα蛋白V1-V6变体均展现出高亲和力。它与利妥昔联用在已接受多线治疗的CD20 +R/R NHL患者中表现出可控的安全性，尚未达到MTD(Strati et al.,ASH 2021)。在这项临床试验中，患者每周一次(QW)接受3、10或20mg/kg CC-95251和375mg/m 2利妥昔单抗治疗，28天为一个治疗周期，直至疾病进展或出现不可接受的毒性。最常见任何级别或级别≥3的治疗期间不良事件(TEAE)都是中性粒细胞减少[12/17(70.6％)，10/17(58.8％)]和感染[10/17(58.8％)，5/17(29.4％)]。在可根据RECIST1.1进行评估的16名患者中观察到BOR 68.8％，ORR 56.3％，其中达到完全缓解CR的患者为25％(4/16)。该联合用药研究在继续入组病人，另外一臂评估CC-95251与西妥昔单抗联合治疗晚期实体瘤也在进行中(NCT03783403)。
因此，本领域仍然需要开发新型的SIRPα抗体，一方面要求其对SIRPα-V1、V2具有高亲和力并且阻断它们与配体CD47的相互作用，抑制“别吃我”信号，对SIRP不结合或弱结合，从而靶向髓系细胞但不影响T细胞免疫应答；另一方面，桥接先天性免疫与适应性免疫，在保障安全性的前提下实现药效最大化。
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发明概述
本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与IIWGX 1X 2STDYX 3X 4ALKS所示一致的氨基酸序列，其中X 1为D、E或N，X 2为G、A或S，X3为N、Q或S，X 4为S、T或A；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与RASESVDSYGX 5X 6FM所示一致的氨基酸序列，其中 X 5为N、Q或S，X 6为S、T或者A；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：6、SEQ ID NO：14、SEQ ID NO：23或SEQ ID NO：24所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8或SEQ ID NO：25所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：6所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：14所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其重链可变区和轻链可变区的CDR序列可通过保守序列修饰，包括一处或多处氨基酸的取代、添加和缺失等。所述一处或多处氨基酸的添加、缺失和/或取代(例如，保守性取代)不超过五处，优选地不超过三处。
在又一个实施方案中，本发明提供靶向SIRPα的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
(a)重链可变区，其包含与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18所示一致的氨基酸序列；
(b)轻链可变区，其包含与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其重链可变区与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
在另一实施方案中，所述抗体为全长抗体,包括人或鼠IgG抗体的Fc结构域。又一方面，所述人恒定区Fc结构域选自由IgGl、IgG2、IgG3、IgG4组成的组。在另一实施方案中，所述人恒定区Fc结构域是IgG1、IgG2或IgG4。在另一实施方案中，所述人恒定区Fc结构域是IgG1w或IgG1突变体(LALA)。
在另一实施方案中，本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中，本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
在另一实施方案中，本发明抗体片段为双抗体。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30或SEQ ID NO：31所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：26或SEQ ID NO：27所示的氨基酸序列一致。
在另一优选实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
在本发明一个特别优选的实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：31所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致；或
(b)重链，其序列与SEQ ID NO：35所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
在另一实施方案中，本发明还提供了编码所述SIRPα的单克隆抗体抗体或抗体片段的分离的多核苷酸。本发明提供了包含所述分离的多核苷酸的表达载体，以及包含所述表达载体的宿主细胞。
在另一实施方案中，本发明提供了药物组合物，包含所述靶向SIRPα的单克隆抗体或其抗原结合部分以及可药用载体。
在另一实施方案中，本发明提供了治疗疾病的方法，所述方法包括将治疗有效量的靶向SIRPα的单克隆抗体或其抗原结合部分给予需要治疗的肿瘤患者或微生物感染性疾病患者。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合Rituximab在治疗肿瘤疾病中的用途。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合Daratumumab在治疗肿瘤疾病中的用途。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合PD-(L)1抗体在治疗肿瘤疾病中的用途。
附图说明
图1.FACS检测AD4-12与SIRPα-V1、V2的结合(参见实施例4)
图2.FACS检测AD4-12对比其它抗体与SIRPγ的结合(参见实施例5)
图3.FACS检测AD4-12与人PBMC的结合(参见实施例6)
图4.ELISA检测AD4-12对SIRPα-V1、V2结合CD47的阻断作用(参见实施例7)
图5.FACS检测AD4-12对SIRPα-V1、V2结合CD47的阻断作用(参见实施例8)
图6.FACS鉴定从单核细胞诱导分化衍生的巨噬细胞(参见实施例9)
图7.AD4-12与利妥昔单抗、达雷妥尤单抗联用的促吞噬作用(参见实施例10)
图8.FACS鉴定从单核细胞诱导分化衍生的树突状细胞(参见实施例11)
图9.MLR评价AD4-12对T细胞活化的影响(参见实施例12)
图10.FACS检测AD4-12的内吞(参见实施例13)
图11.AD4-12与利妥昔单抗联合应用的体内抗淋巴瘤活性(参见实施例14)
图12.AD4-12与达雷妥尤单抗联合应用的体内抗淋巴瘤活性(参见实施例15)
图13.FACS比较AD4-12人源化前后与SIRPα-V1、V2、V8的结合(参见实施例20)
图14.Octet检测H7L4与SIRPα-V1、V2亲和力(参见实施例21)
图15.ELISA检测H7L4与SIRPα-V1、V2、V8的结合(参见实施例22)
图16.FACS检测H7L4与SIRPα-V1、V2、V8以及SIRPβ、SIRPγ的结合(参见实施例23)
图17.FACS检测H7L4与人PBMC细胞亚群的结合(参见实施例24)
图18.ELISA检测H7L4对SIRPα-V1、V2结合CD47的阻断作用(参见实施例25)
图19.FACS检测H7L4对SIRPα-V1、V2、V8结合CD47的阻断作用(参见实施例26)
图20.H7L4与利妥昔单抗、达雷妥尤单抗联用的促吞噬作用(参见实施例27)
图21.巨噬细胞吞噬法检测H7L4对SIRPα +细胞的ADCP效应(参见实施例28)
图22.FACS检测H7L4在小鼠体内对SIRPα +细胞的削减作用(参见实施例29)
图23.MLR评价H7L4对T细胞活化的影响(参见实施例30)
图24.红细胞凝集实验(参见实施例31)
图25.H7L4孵育PBMC的上清液中细胞因子检测(参见实施例32)
图26.H7L4在免疫健全小鼠的抗癌效应(A20淋巴瘤细胞株)(参见实施例33)
图27.H7L4在免疫健全小鼠的抗癌效应(MC38结肠癌细胞株)(参见实施例34)
图28.H7L4与PD-L1单抗联用在免疫健全小鼠的抗癌效应(MC38结肠癌细胞株)(参见实施例35)
图29.ELISA和FACS检测H7L4与食蟹猴SIRPα的结合(参见实施例36)
发明详述
定义
为使本发明更易于理解，首先定义某些术语。别的定义将在整个详述中阐明。
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内，这些技术在本领域的技术文献和通用教科书中有充分解释，诸如Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)等。
术语“SIRPα”是指野生型信号调节蛋白α、或具有野生型信号调节蛋白α的氨基酸序列的重组或非重组多肽的氨基酸序列、或天然或天然存在的信号调节蛋白α的等位基因变体。SIRPα优选指野生型哺乳动物SIRPα，最常见的蛋白质变体是SIRPαv1和v2(参考登录号NP_001035111、NP_542970(P78324)和CAA71403)，另外还包括SIRPαv8等。人SIRP的多态性导致表面暴露的氨基酸发生变化，但这不影响与CD47的结合。主要野生型人SIRPα(SIRPα V1)的成熟形式的氨基酸序列如SEQ ID NO：2所示。根据一个实施方案，SIRPα是SIRPα胞外结构域，即经改造以去除跨膜和细胞结构域的SIRPα蛋白，野生型SIRPα V1的胞外结构域的序列是SEQ ID NO：2的残基1-348。SIRPα的“变体”定义为与野生型SIRPα相比具有一个或多个氨基酸修饰的SIRPα氨基酸序列。变体可以具有“保守”修饰，包括氨基酸的取代、缺失或插入，或同时包括这数者，其中取代的氨基酸具有相似的结构或化学特性。SIRPα变体包括与野生型SIRPα或野生型SIRPα胞外结构域具有至少70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列一致性的多肽。
术语“SIRPβ”是指来自哺乳动物物种的SIRPβ蛋白(也称为信号调节蛋白β-1，SIRP-β-1，CD172抗原样家族成员B或CD172b)，优选人SIRPβ(参考登录号O00241相关的序列)。
术语“SIRPγ”涉及来自哺乳动物物种的SIRPγ，优选为人SIRPγ。人SIRPγ蛋白的参考序列对应于登录号AAH64532、Q9P1W8或NM 018556的序列。
“抗体”是指表现出所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此，“抗体”以其最广泛的意义来使用，并明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、纳米抗体和多特异性抗体(例如双特异性抗体)。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼科动物中天然存在的抗体可仅包含重链。
如本文所用，术语“结合”和“特异性结合”指抗体或抗原结合部分在体外测定法中，优选地在采用纯化的野生型抗原的生物光干涉测量(ForteBio)中与抗原表位结合。在某些实施方案中，在抗体或抗原结合部分优选地识别蛋白质和/或大分子的复杂混合物中其靶抗原时，将抗体或抗原结合部分称作特异性结合抗原。
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。Fc可选自人IgGl、IgG2、IgG3或IgG4的Fc结构域或经过突变其中1个或几个位点的Fc结构域。
非人类(例如鼠)抗体的“人源化”形式为含有最小限度的来源于非人类免疫球蛋白序列的嵌合抗体。人源化抗体的大部分为人免疫球蛋白(受体抗体)，其中受体抗体的高变区残基被具有所需特异性、亲和力和能力的非人类物种(供体抗体)高变区的残基置换，非人类物种例如有小鼠、大鼠、兔或非人类灵长类。在某些情况下，人免疫球蛋白的Fv构架区(FR)残基被相应的非人类残基取代。此外，人源化抗体可包含不在受体抗体或供体抗体中存在的残基。进行这些修饰以进一步改进抗体性能。一般而言，人源化抗体包含至少一个且通常为两个可变结构域的几乎全部，其中全部或几乎全部超变环对应于非人类免疫球蛋白的超变环，全部或几乎全部FR区为人免疫球蛋白序列的FR区。人源化抗体还任选包含至少部分免疫球蛋白(通常为人免疫球蛋白) 恒定区(Fc)。
“分离的”抗体为业已被鉴定并与其天然环境组分相分离的抗体，其天然环境的污染组分是会干扰所述抗体的诊断性或治疗性应用的物质，可包括酶、激素和其它蛋白质溶质或非蛋白质溶质。在一些实施方案中，将所述抗体纯化到超过95％纯度，更优选超过99％纯度，其由Lowry法测定。分离的抗体通常由至少一个纯化步骤制备。
“分离的”核酸分子为被鉴定并与至少一种污染性核酸分子分离的核酸分子。分离的核酸分子不同于其天然存在的形式或环境。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
本文所用术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
本文所用序列“变体”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
“有效量”包括足以改善或防止医学疾病的症状或病症的量。有效量还意指足以使得可以诊断或促进诊断的量。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。
“药学上可接受载体”包括生理上相容的任何和全部溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等。优选，用于含有抗体的组合物的载体适用于静脉内(IV)、肌内、皮下(SC)、胃肠外、脊髓或表皮施用(例如，通过注射或输注)。
“患者”表示人或非人动物(例如哺乳动物)。
“癌症”或“肿瘤”是指以异常方式增殖的细胞的集合。
“PD-(L)1抗体”是指针对PD-1或PD-L1靶点的抗体，包括PD-1抗体、PD-L1
抗体或包含针对PD-1或PD-L1靶点的双抗。
本发明的各个方面将在下述分部中进一步详细描述。
SIRPα抗体
本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与IIWGX 1X 2STDYX 3X 4ALKS所示一致的氨基酸序列，其中X 1为D、E或N，X 2为G、A或S，X3为N、Q或S，X 4为S、T或A；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与RASESVDSYGX 5X 6FM所示一致的氨基酸序列，其中X 5为N、Q或S，X 6为S、T或者A；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：6、SEQ ID NO：14、SEQ ID NO：23或SEQ ID NO：24所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8或SEQ ID NO：25所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：6所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：14所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其重链可变区和轻链可变区的CDR序列同源性至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。在一些优选的实施方案中，与靶向SIRPα的单克隆抗体CDR区或可变区具有高(即90％或更高)同源性的抗体通过保守序列修饰获得，包括一处或多处氨基酸的取代、添加和缺失等。所述一处或多处氨基酸的添加、缺失和/或取代(例如，保守性取代)不超过五处，优选地不超过三处。术语“保守序列修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的诱变编码可变区序列的核酸分子。示例性的，本发明制备的抗体一个脱酰胺位点(NS)和一个异构化位点(DG)位于重链CDR2上(SEQ ID NO:6)，一个脱酰胺位点(NS)位于轻链CDR1上(SEQ ID NO:7)。因此重链可变区CDR2的序列通式可概括为IIWGX 1X 2STDYX 3X 4ALKS(SEQ ID NO:41)，其中X 1为D、E或N，X 2为G、A或S，X 3为N、Q或S，X 4为S、T或A。轻链可变区CDR1的通式可概括为RASESVDSYGX 5X 6FM(SEQ ID NO:42)，其中X 5为N、Q或S，X 6为S、T或者A。
对于重链可变区和轻链可变区的CDR序列突变的示范性说明参考实施例18，例如对重链CDR2可进行两个位点的突变，通式可概括为IIWGDX 1STDYNX 2ALKS(SEQ ID NO:43)，其中X 1为G或A，X 2为S或A，其包含与选自SEQ ID NO：6、SEQ ID NO：14、SEQ ID NO：23或SEQ ID NO：24所示一致的氨基酸序列。对轻链CDR1可进行一个位点的突变，通式可概括为RASESVDSYGX 3SFM(SEQ ID NO:44)，其中X 3为N或S，其包含与选自SEQ ID NO：8或SEQ ID NO：25所示一致的氨基酸序列。对CDR序列中发现的异构化位点和脱酰胺位点进行突变以消除PTM风险(post-translational modification)，并有望保留CDR环的结构。
在另一实施方案中，本发明提供靶向SIRPα的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
(a)重链可变区，其包含与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18所示一致的氨基酸序列；
(b)轻链可变区，其包含与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21 或SEQ ID NO：22所示一致的氨基酸序列。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体或其抗原结合部分，其重链可变区与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
重链可变区(VH)和轻链可变区(VL)与上述序列的VH和VL区具有高(即90％或更高)同源性的抗体通过保守序列修饰获得，包括氨基酸的取代、添加和缺失等。术语“保守序列修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的诱变编码可变区序列的核酸分子。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区域外的一个或多个氨基酸残基，并使用本文所述的功能测定法对改变后的抗体测试保留的功能。优选的定点诱变或PCR介导的诱变位点位于可变区CDR1-CDR3之外的位点。
示例性的取代
原残基 示例性的取代 保守取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp，Lys；Arg Gln
Asp(D) Glu；Asn Glu
原残基 示例性的取代 保守取代
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe； Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala； Leu
氨基酸可以按照常见侧链的性质进行分组：
(1)疏水性:正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性亲水性:Cys，Ser，Thr，Asn，Gln；
(3)酸性:Asp，Glu；
(4)碱性:His，Lys，Arg；
(5)影响链取向的残基:Gly，Pro；
(6)芳族的:Trp，Tyr，Phe。
非保守性取代需要将这些类别中之一的成员交换为另一类。
在本发明中，鼠源抗体AD4-12包含的重链和轻链可变区序列分别如SEQ ID NO： 3和SEQ ID NO：4所示。将AD4-12序列与人类生殖系序列库IMGT(Lefranc，2003)进行比对，选择与抗体AD4-12框架相对应位置氨基酸差异最少的人类种系序列作为人源化模板，参考实施例16对AD4-12进行人源化改造及回复突变，优化后的人源化变体基本与鼠源抗体AD4-12的亲和力保持一致(参考实施例17)，其中优选人源化抗体的重链和轻链可变区序列为：
(1)重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：20；
(2)重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：22；
(3)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：20；
(4)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：21；
(5)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：22；
(6)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20；
(7)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：21；
(8)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：22；
在另一实施方案中，所述抗体为含有人IgG恒定区的全长抗体。在另一些实施例中，所述抗体为含有小鼠IgG恒定区的全长抗体。可以通过将编码重链可变区的基因序列连接至编码人抗体重链恒定区(CH1、CH2和CH3)基因序列形成为全长重链基因，将编码轻链可变区的基因序列连接至编码人抗体轻链恒定区基因序列形成为全长轻链基因。人重链恒定区基因和轻链恒定区的序列是本领域已知的(参见例如Kabat，E.A.等人(1991)，Sequences of Proteins of Immunological Interest，Fifth Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242)。重链恒定区可以是人IgG1、IgG2、IgG3、IgG4恒定区，在另一实施方案中，所述人恒定区Fc结构域是IgG1、IgG2或IgG4。在另一实施方案中，所述人恒定区Fc结构域是IgG1w或IgG1突变体(LALA)。轻链恒定区可以是κ或λ恒定区，但最优选为κ恒定区。可将抗体轻链基因和抗体重链基因插入到不同的载体中，或者更通常的将两个基因插入到同一表达载体中，通过标准技术将编码重链和轻链的表达载体转染到宿主细胞中表达出全长抗体。
在另一实施方案中，本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中，本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
在另一实施方案中，本发明抗体片段为双抗体。
在另一实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：26或SEQ ID NO：27所示的氨基酸序列一致。
在优选的实施方案中，本发明提供了分离的单克隆抗体，其重链(HC)与选自SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30SEQ ID NO：31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链(LC)与选自SEQ ID NO：26或SEQ ID NO：27的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。与上述序列的重链和轻链具有高(即90％或更高)同源性的抗体可以通过诱变(例如定点诱变或PCR介导的诱变)编码重链和轻链氨基酸的核酸分子，然后使用本文所述的功能测定法对所编码的改变后的抗体测试所保留的功能获得。优选的定点诱变或PCR介导的诱变位点位于重链可变区CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
在另一优选实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO:31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
在另一优选实施方案中，本发明优选的人源化抗体的重链和轻链序列如下表所示：
ID 重链序列 轻链序列
H4L3 SEQ ID NO:28 SEQ ID NO:26
H5L3 SEQ ID NO:29 SEQ ID NO:26
H6L3 SEQ ID NO:30 SEQ ID NO:26
H7L3 SEQ ID NO:31 SEQ ID NO:26
H4L4 SEQ ID NO:28 SEQ ID NO:27
H5L4 SEQ ID NO:29 SEQ ID NO:27
H6L4 SEQ ID NO:30 SEQ ID NO:27
H7L4 SEQ ID NO:31 SEQ ID NO:27
H7L4-G1w SEQ ID NO:35 SEQ ID NO:27
H7L4-G2 SEQ ID NO:36 SEQ ID NO:27
H7L4-G4 SEQ ID NO:37 SEQ ID NO:27
在本发明一个特别优选的实施方案中，本发明提供了靶向SIRPα的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：31所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致；或
(b)重链，其序列与SEQ ID NO：35所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
在本发明的一个具体实施方式中，使用的靶向SIRPα的单克隆抗体或其抗原结合部分具有以下特性：
--所述的SIRPα抗体或其抗原结合部分与SIRPα V1具有高亲和力，亲和力至少为10 -11M量级或更高；
--所述的SIRPα抗体或其抗原结合部分能够高效阻断CD47-SIRPα V1的结合， IC50值达到4.7nM或更低；
--所述的SIRPα抗体或其抗原结合部分与SIRPα V2、SIRPα V8具有高亲和力，能够同时阻断CD47-SIRPα V2和CD47-SIRPα V8的结合；
--所述的SIRPα抗体或其抗原结合部分与SIRPγ的亲和力相对较弱，亲和力约为10 -9M量级，且所述SIRPα抗体与SIRPγ的亲和力数据与SIRPα抗体与SIRPα V1的亲和力数据相比约差2个数量级。
--所述的SIRPα抗体或其抗原结合部分不会引起红细胞凝集。
在本发明的另一具体实施方式中，使用的靶向SIRPα的单克隆抗体或其抗原结合部分还可具有以下特性中的至少一种(特别是以下特性中的至少两种、特别是以下所有特性)：
--所述的SIRPα抗体或其抗原结合部分不抑制(特别是体内)人T细胞的增殖和/或活化；和/或
--所述的SIRPα抗体或其抗原结合部分通过阻断SIRPα-CD47信号通路，能促进巨噬细胞的活化，特别是促进巨噬细胞对肿瘤细胞的吞噬；和/或
--所述的SIRPα抗体或其抗原结合部分与Rituximab联用，有明显促进Macrophage对肿瘤细胞的吞噬作用；与Daratumumab联用，也有大幅度提高Macrophage吞噬作用；与PD-(L)1单抗联用具有协同抗肿瘤效应。
编码本发明抗体的核酸分子
本发明还提供了编码所述靶向SIRPα的单克隆抗体或其抗原结合部分的分离的多核苷酸。本发明的核酸分子可以是DNA或RNA，而且可以含有或不含内含子序列。在一优选的实施方式中，核酸是cDNA分子。
可以使用标准分子生物学技术来获得本发明的核酸分子。对于由杂交瘤表达的抗体，可以通过标准PCR扩增或cDNA克隆技术获得编码杂交瘤所制备的抗体轻链和重链的cDNAs。对于从免疫球蛋白基因库(例如使用噬菌体展示技术)中获得的抗体，可以从文库中回收编码抗体的核酸。
本发明优选的核酸分子是那些编码本发明中所示靶向SIRPα的单克隆抗体CDR 区、可变区或者全长抗体的氨基酸序列的核酸分子。在获得编码本发明所述的靶向SIRPα的单克隆抗体的VH和VL区段的DNA片段后，进一步通过标准重组DNA技术操作这些DNA片段，例如将可变区基因转变为全长抗体基因、Fab片段基因或scFv基因。在这些操作中，将编码VL或VH的DNA片段可操作的连接至编码另一蛋白质，诸如抗体恒定区或柔性接头的另一DNA片段。术语“可操作的连接”在用于本文时意图表示连接两DNA片段从而使得这两个DNA片段所编码的氨基酸序列保持在同一读码框中。
通过将编码VH的DNA可操作的连接至编码重链恒定区(CH1、CH2和CH3)的另一DNA分子可以将编码VH区的分离的DNA转变为全长重链基因。人重链恒定区基因的序列是本领域已知的。重链恒定区可以是IgG1、IgG2、IgG3、IgG4恒定区，优选为IgG1或IgG4恒定区。为获得Fab片段重链基因，可以将编码VH的DNA可操作的连接至仅编码重链CH1恒定区的另一DNA分子。
通过将编码VL的DNA可操作的连接至编码轻链恒定区CL另一DNA分子可以将编码VL区的分离的DNA转变为全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的，轻链恒定区可以是κ或λ恒定区，但最优选为κ恒定区。
为创造scFv基因，将编码VH和VL的DNA片段可操作的连接至编码柔性接头，例如编码氨基酸序列(Gly4-Ser)3的另一片段，从而使得VH和VL序列可以表达成相邻的单链蛋白质，其中VL和VH区通过柔性接头连接，序列通式可以为为(G 4S)n、(SG 4)n、G 4(SG 4)n或(G 4S)nG等，其中n为1-6的整数，优选为3-5。
表达载体及宿主细胞
本发明提供了包含所述分离的多核苷酸的表达载体，以及包含所述表达载体的宿主细胞。
如本文所用，术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。载体包括质粒、病毒、粘粒和人工染色体。通常，工程化载体包含复制起点、多克隆位点和选择标记。载体还可以包括除转基因插入物和骨架之外的其他特征：启动子、遗传标记、抗生素抗性、报告基因、靶向序列、蛋白质纯化标签。称 为表达载体(表达构建体)的载体特异性地用于在靶细胞中表达转基因，并且通常具有控制序列。
可将抗体轻链基因和抗体重链基因插入到不同的载体中，或者更通常的，将两个基因插入到同一表达载体中。通过标准方法将抗体基因插入到表达载体中。可以使用本文所述抗体的轻链和重链可变区来创造任何抗体同种型的全长抗体基因，其通过将它们插入已编码期望同种型的重链恒定区和轻链恒定区的表达载体中，从而使得VH区段与载体中的CH区段可操作的连接，VK区段与载体中的CL区段可操作的连接。或者，重组表达载体可以编码信号肽，其利于宿主细胞中抗体链的分泌。可将抗体链基因克隆到载体中以使信号肽与抗体链基因的氨基末端连接于同一读码框中。信号肽可以是免疫球蛋白信号肽或异源信号肽(即来自非免疫球蛋白的信号肽)。
为表达轻链和重链，通过标准技术将编码重链和轻链的表达载体转染到宿主细胞中。各种形式的术语“转染”意图涵盖多种通常用于将外源DNA导入原核或真核宿主细胞的技术，例如电穿孔、磷酸钙沉淀、DEAE-右旋糖苷转染等。虽然理论上在原核或真核宿主细胞中都有可能表达本发明的抗体，但是最优选在真核细胞中(最优选在哺乳动物宿主细胞中)表达抗体。用于表达本发明重组抗体的优选哺乳动物宿主细胞包括中国仓鼠卵巢细胞(CHO细胞)、NSO骨髓瘤细胞、COS细胞和SP2细胞等，优选CHO细胞。
当将编码抗体基因的重组表达载体导入哺乳动物宿主细胞时，通过培养宿主细胞一段足以使宿主细胞中抗体表达的时间生产抗体，或者更优选的是，将抗体分泌到培养宿主细胞的培养基中。可以使用标准蛋白质纯化方法从培养物的培养液中回收抗体。
药物组合物
本发明提供了药物组合物，包含所述靶向SIRPα的单克隆抗体或抗体片段以及药学上可接受载体。
一方面，本发明提供了一种药用组合物，其包含一种或一组本发明的靶向SIRPα的单克隆抗体或其抗原结合部分，与药学可接受载体配制在一起。药剂学可接受载体 包括任何和所有溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等生理学相容的载体。优选的是，载体适于静脉内、肌肉内、皮下、胃肠外、脊髓或表皮施用(例如通过注射或输注)。
在另一实施方案中，本发明提供了一种药物组合物，其包括本发明所述的靶向SIRPα的单克隆抗体或其抗原结合部分，Rituximab以及药学上可接受载体，其可用于治疗肿瘤相关疾病，尤其是血液肿瘤疾病。
在另一实施方案中，本发明提供了一种药物组合物，其包括本发明所述的靶向SIRPα的单克隆抗体或其抗原结合部分，Daratumumab以及药学上可接受载体，其可用于治疗肿瘤相关疾病，尤其是血液肿瘤疾病。
在另一实施方案中，本发明提供了一种药物组合物，其包括本发明所述的靶向SIRPα的单克隆抗体或其抗原结合部分，PD-(L)1抗体以及药学上可接受载体，其可用于治疗肿瘤相关疾病，尤其是肿瘤免疫治疗。其中，所述的PD-(L)1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab-rwlc)、卡瑞利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、特瑞普利单抗(Toripalimab)、替雷利珠单抗(Tislelizumab)、赛帕利单抗(Zimberelimab)、派安普利单抗(Penpulimab)、斯鲁利单抗(Serplulimab)、普特利单抗(Pucotenlimab)、阿替利珠单抗(Atezolizumab)、度伐利尤单抗(durvalumab)、阿维鲁单抗(Avelumab)、恩沃利单抗(Envafolimab)、舒格利单抗(Sugemalimab)和卡度尼利单抗(Cadonilimab)。
药物组合物在生产和贮存条件下通常必须是无菌的和稳定的。可以将组合物配制成溶液、微乳液、脂质体或冻干粉针等剂型。本发明药物组合物的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊髓/脊柱或其它胃肠外施用路径，例如通过注射或输注。
本发明的抗体的给药剂量的范围为大约0.01-100mg/kg，更加通常的为0.1mg/kg-100mg/kg，或0.5mg/kg-50mg/kg，或1mg/kg-25mg/kg，或2mg/kg-10mg/kg，或5mg/kg-10mg/kg的剂量。例示性的治疗方案要求每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。本发明药用组 合物中的活性成分的实际剂量水平可以变化，从而获得对于特定患者、组合物和施用模式有效实现期望治疗性应答而对患者是无毒的活性成分量。“治疗有效量”的本发明SIRPα单抗优选导致疾病症状严重性降低、无疾病症状期的频率和持续时间提高、或对患病所造成的损害或残疾的预防。例如，对于肿瘤的治疗而言，“治疗有效量”优选相对于未治疗受试者抑制细胞生长或肿瘤生长达至少大约20％、更优选达至少大约40％、甚至更优选达至少大约60％、且仍更优选达至少大约80％。可以在动物模型系统中评估化合物抑制肿瘤生长的能力，所述动物模型系统可以预测在人肿瘤中的功效。本领域普通技术人员将会能够基于诸如受试者体型大小、受试者症状的严重性、及所选择的特定组合物或施用路径的因素来确定此类量。
用途
本发明提供了治疗疾病的方法，所述方法包括将治疗有效量的本发明的靶向SIRPα的单克隆抗体或抗体片段给予需要治疗的肿瘤患者或微生物感染性疾病患者。
本发明的靶向SIRPα的单克隆抗体或其抗原结合部分与SIRPα V1、SIRPα V2、SIRPα V8具有高亲和力，通过阻断SIRPα-CD47信号通路，能促进巨噬细胞的活化，特别是促进巨噬细胞对肿瘤细胞的吞噬，特别是与rituximab或Daratumumab联用，有明显促进Macrophage对肿瘤细胞的吞噬作用；与PD-(L)1单抗联用具有协同抗肿瘤效应。研究还发现，本发明所述的抗体不会引起红细胞凝集，而且不抑制(特别是体内)人T细胞的增殖和/或活化，因而可望降低该类药物在肿瘤治疗中的不必要的副作用。
一方面，本发明将所述的SIRPα抗体或其制剂施用至给予需要治疗的受试者。术语“受试者”包括人和非人动物。非人动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、羊、犬、小鼠、大鼠、猫、牛、马、鸡、两栖类和爬行类。优选的施用对象或个体是哺乳动物，例如小鼠、猴子、狗、牛、马或人，更优选是人。给予受试者施用靶向SIRPα的单克隆抗体或抗体片段可消除或抑制或干扰由配体结合(例如CD47结合)介导的SIRPα的表达、活性和/或信号传导功能。在一 个实施方案中，与SIRPα表达相关联的疾病或病症为癌症。在一些实施方案中，将SIRPα抗体施用至具有表达SIRPα的癌症(诸如髓样细胞的血液增殖性病症)的患者。在典型的实施方案中，将抗SIRPα抗体施用至具有表达CD47的癌症的患者。
在另一实施方案中，本发明提供了将靶向SIRPα的单克隆抗体或抗体片段用于治疗癌症或用于抑制肿瘤生长的用途。所述的肿瘤或癌症选自鳞状细胞癌，小细胞肺癌，非小细胞肺癌，鳞状非小细胞肺癌(NSCLC)，非鳞状NSCLC，胶质瘤，胃肠癌，肾癌，卵巢癌，肝癌，结直肠癌，子宫内膜癌，前列腺癌，甲状腺癌，神经母细胞瘤，胰腺癌，成胶质细胞瘤，宫颈癌，胃癌，膀胱癌，头颈癌，黑色素瘤，骨癌，皮肤癌，弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等，以及所述癌症的任何组合。本发明的抗SIRPα抗体还可用于治疗转移性癌症。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合Rituximab在治疗肿瘤疾病中的用途。在另一实施方案中，所述的肿瘤疾病为血液肿瘤。在另一实施方案中，所述的血液肿瘤疾病为弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合Daratumumab在治疗肿瘤疾病中的用途。在另一实施方案中，所述的肿瘤疾病为血液肿瘤。在另一实施方案中，所述的血液肿瘤疾病为弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等。
在另一实施方案中，本发明提供了所述的靶向SIRPα的单克隆抗体或其抗原结合部分联合PD-(L)1抗体在治疗肿瘤疾病中的用途。其中，所述的PD-(L)1抗体包括PD-1抗体、PD-L1抗体或针对PD-1/PD-L1的双抗；其中，PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗 (Cemiplimab-rwlc)、卡瑞利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、特瑞普利单抗(Toripalimab)、替雷利珠单抗(Tislelizumab)、赛帕利单抗(Zimberelimab)、派安普利单抗(Penpulimab)、斯鲁利单抗(Serplulimab)、普特利单抗(Pucotenlimab)等。PD-L1单抗包括阿替利珠单抗(Atezolizumab)、度伐利尤单抗(durvalumab)、阿维鲁单抗(Avelumab)、恩沃利单抗(Envafolimab)、舒格利单抗(Sugemalimab)等。针对PD-1/PD-L1的双抗包括靶向PD-1和CTLA-4的双抗卡度尼利单抗(Cadonilimab)。在另一实施方案中，所述的肿瘤疾病为血液肿瘤、恶性黑色素瘤、乳腺癌、小细胞肺癌、非小细胞肺癌、肝癌、胃癌、肾癌、结直肠癌、膀胱癌、头颈部肿瘤、宫颈癌、Merkel细胞癌以及所有微卫星高度不稳定(MSI-H)的实体瘤治疗等。在另一实施方案中，所述的血液肿瘤疾病为弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等。在另一实施方案中，本发明提供了将靶向SIRPα的单克隆抗体或抗体片段用于治疗微生物感染性疾病的用途。本发明的靶向SIRPα的单克隆抗体或其抗原结合部分，能促进巨噬细胞的活化，从而能诱导或者维持个体中的吞噬作用。吞噬作用包括专业的吞噬细胞(例如单核细胞、巨噬细胞、嗜中性粒细胞、树突细胞或肥大细胞)、非专业的吞噬细胞(例如上皮细胞、内皮细胞、成纤维细胞或间充质细胞)或二者引起的吞噬作用。本发明提供用于治疗个体的病毒感染或细菌性感染疾病的方法，其包括向患有病毒感染或细菌性感染疾病的个体施用本文所述的靶向SIRPα的单克隆抗体或其抗原结合部分。该病毒感染或细菌性感染病症或病况是慢性的或者是急性的。所述的病毒性感染疾病包括腺病毒、疱疹病毒、乳头瘤病毒、冠状病毒、人免疫缺陷病毒(HIV)、人巨细胞病毒、EB病毒、丙型肝炎病毒或乙型肝炎病毒等感染引起的疾病。所述的病菌性感染疾病包括芽孢杆菌、肺炎衣原体、流感嗜血杆菌、结核分枝杆菌、假单胞菌、沙门氏菌、葡萄球菌、链球菌、密螺旋体等感染引起的疾病。
本发明的靶向SIRPα的单克隆抗体或抗体片段的最优剂量将取决于治疗中的疾 病、疾病的严重程度，和副作用的存在与否。最优剂量可通过常规实验确定。对于胃肠外给予，给予0.1mg/kg-100mg/kg，或0.5mg/kg-50mg/kg，或1mg/kg-25mg/kg，或2mg/kg-10mg/kg，或5mg/kg-10mg/kg的剂量。例示性的治疗方案可以每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。
具体实施方式
提供以下实施例旨在完整地公开和描述如何制备、筛选、鉴定和使用本发明，这些实施例不意在以任何方式限制本发明的范围，也不代表下述实验是所进行的所有或者仅有的实验。发明人保证实验数据的客观、准确，但应允许一定的实验误差和偏差。
实施例1：免疫小鼠获得鼠源抗体序列
制备针对人SIRPα的鼠源单克隆抗体，首先用购自ACRO(Cat#SIA-H5251)的重组SIRPα胞外区域Fc融合蛋白SIRPα V1-Fc(SEQ ID NO:1)、接着用SIRPα V1(SEQ ID NO:2)转染的CHO细胞作为抗原，免疫BALB/C小鼠，用ELISA方法监测小鼠免疫应答。在抗人SIRPα免疫球蛋白滴度达到要求时取小鼠脾脏提取RNA，RT-PCR扩增VH和Vκ基因并克隆入噬菌体进行展示、淘选,得到高SIRPα亲和力、且有阻断CD47-SIRPα功能的抗体片段。经测序，获得AD4-12的重链可变区(SEQ ID NO:3)、轻链可变区(SEQ ID NO:4)。使用软件ANARCI(Dunbar和Deane 2015)得到小鼠抗体序列的Kabat编号。CDR(表2，SEQ ID NO:5-10)是根据抗体网站http://www.bioinf.org.uk/(Martin，2018)上公布的定义来确定的。
表1
Figure PCTCN2022112677-appb-000001
表2.AD4-12可变区的CDR序列
Figure PCTCN2022112677-appb-000002
实施例2：AD4-12抗体表达纯化
将筛选获得AD4-12的VL和VH区cDNA分别与人κ轻链和IgG1、IgG2、IgG4重链恒定区连接，还对IgG1和IgG2重链恒定区的Fc段分别设计增强或减弱与Fc受体结合的突变序列。将构建好的AD4-12人鼠嵌合轻链和重链克隆到表达载体pCDNA3.1(+)。将表达载体质粒瞬转进入ExpiCHO-S细胞进行表达，培养基为(Gibco,Cat#A29100-01)，转染试剂盒为(Gibco，Cat#A29129)。具体方法如下：转染前一天将ExpiCHO-S细胞进行传代，在25mL体系内，将构建好的质粒与转染试剂混合之后滴加入ExpiCHO-S细胞培养物中，充分混匀，于37℃孵育18-22小时，根据试剂盒内说明添加补料培养基，置于32℃5％CO 2摇床培养箱中，第5天添加第二次补料，10-12天之后收集上清，采用常规Protein A过柱方法纯化得到不同IgG亚型的目的抗体：AD4-12-G1和AD4-12-G1lala。
下面实施例中重点评价IgG1野生型(AD4-12-G1)和包含有L234A/L235A(LALA)氨基酸替代以消除对Fc受体结合的突变型(AD4-12-G1 lala)。在AD4-12-G1和AD4-12-G1 lala无差异的实施例中两者统一标示为AD4-12，只有在对AD4-12-G1和AD4-12-G1 lala进行比较时才分别标示出这两者。
实施例3：生物膜干涉技术(BLI)检测AD4-12与SIRPα-V1、SIRPα-V2、SIRPβ、SIRPγ以及食蟹猴SIRPα的亲和力
通过ForteBio Octet RED 96测定AD4-12与SIRPα、SIRPβ、SIRPγ结合的亲和力。使用Ni-NTA sensor固化重组蛋白SIRPα V1-his(ACRO，Cat#SIA-H5225)、SIRPα V2-his(根据SEQ ID NO:38序列构建质粒表达，)、SIRPβ-his(Novoprotein，Cat#CS93)、SIRPγ-his(Sino Biological，Cat#11828-H08H)或食蟹猴SIRPα-his(ACRO，Cat#SIA-C52H7)，浓度均为5μg/mL，固化时间600秒。将AD4-12和参照品18D5(OSE Immunotherapeutics公司的SIRPα抗体BI 765063之母本鼠源抗体，合成序列参考专利CN201780023581.2，Fc片段为IgG4)进行2倍梯度稀释共7个浓度点，针对不同固化蛋白分子所使用的抗体起始浓度和反应解离时间有所不同：对于SIRPα V1、SIRPα V2和SIRPβ固化的sensor，抗体起始浓度为25nM，结合时间200秒，解离时间1000秒；对于SIRPγ固化的sensor，抗体起始浓度为200nM，结合时间200秒，解离时间800秒；对于食蟹猴SIRPα固化的sensor，结合时间200秒，解离时间600秒。数据拟合计算亲和力。
结果：如表3-1所示，本发明的AD4-12与SIRPα-V1具有高亲和力，与参照品18D5对比，提高了一个数量级；AD4-12与SIRPα-V2同样具有高亲和力；AD4-12与SIRPβ结合的亲和力稍弱于与SIRPα的结合；AD4-12与SIRPγ也能结合，但亲和力比与SIRPα的结合低两个数量级。此外，AD4-12能够交叉结合食蟹猴SIRPα分子，亲和力与结合人SIRPα分子相近。
表3-1：Octet检测AD4-12结合SIRP家族成员的亲和力
Figure PCTCN2022112677-appb-000003
如表3-2所示，本发明的抗体AD4-12与SIRPα-V1、V2和V8具有高亲和力，其EC50值显著低于对照抗体18D5；且抗体AD4-12与SIRPβ结合弱于对照抗体18D5。
表3-2 AD4-12结合SIRP家族成员的亲和力EC50值比较
Figure PCTCN2022112677-appb-000004
实施例4：流式细胞仪(FACS)检测AD4-12结合SIRPα-V1、V2。
采用稳转构建的高表达SIRPα-V1(SEQ ID NO:2)、V2(SEQ ID NO:38)或V8(SEQ ID NO:39)的细胞株，以流式细胞仪方法分析AD4-12结合胞膜上SIRPα分子的能力。取对数期生长的293T-SIRPα-V1细胞、CHOZN-SIRPα-V2细胞，在96孔U型培养板每孔加入2×10 5细胞，经Fc blocker(BD Pharmingen，Cat#564219)封闭后，每孔分别加入梯度稀释的AD4-12或参照品18D5，4℃孵育30分钟；漂洗细胞后，每孔加入0.2μg AF488标记的羊抗人IgG抗体(Jackson ImmunoResearch，Cat#109-546-170)，4℃孵育30分钟；漂洗后加入50μL的DPBS/1％FBS重悬细胞，流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图1所示，AD4-12与表达SIRPα-V1蛋白的稳转细胞株有非常好的结合活性，略优于18D5；AD4-12能很好结合表达SIRPα-V2蛋白的稳转细胞株，其结合活性明显优于18D5。
实施例5：FACS检测AD4-12与SIRPγ的结合
取对数生长期的Jurkat细胞，在96孔U型培养板每孔加入2×10 5细胞，经Fc blocker封闭后，每孔加入2μg/mL相应抗体，其中LSB2.20(SantaCruz，Cat#sc-53604)为SIRPγ特异性抗体，SE7C2(SantaCruz，Cat#sc-23863)为SIRPα特异性抗体，4℃孵育30分钟；漂洗细胞后，每孔加入0.2μg AF488标记的羊抗鼠IgG抗体(Jackson ImmunoResearch，Cat#115-545-003)或羊抗人IgG抗体，4℃孵育30分钟；漂洗后加入50μL的DPBS/1％FBS重悬细胞，流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图2所示，LSB2.20和SE7C2验证了Jurkat细胞为SIRPγ阳性SIRPα阴性。抗体AD4-12与18D5类似，基本不与Jurkat细胞结合。
实施例6：FACS检测AD4-12抗体与人PBMC的结合
复苏取自3位供者的冻存人外周血单个核细胞(PBMC)(妙顺生物，Cat#PB003F)，在96孔U型培养板每孔加入5×10 5细胞，经配制于1mg/mL mIgG(Invitrogen，Cat#31903)的Fc blocker封闭后，每孔分别加入3μg biotin标记的AD4-12、同型对照和0.2μg针对细胞表面标记分子的抗体，包括APC标记的CD56抗体(BD Pharmingen，Cat#555518)、eFlour 506标记的CD11b抗体(eBioscience TM，Cat#69-0118-42)、FITC标记的CD3抗体(eBioscience TM，Cat#17-0038-42)、APC-eFlour 780标记的CD14抗体(eBioscience TM，Cat#47-0149-42)和eFlour 450标记的CD19抗体(eBioscience TM，Cat#48-0198-42)，4℃孵育30分钟；漂洗细胞后，每孔加入二抗0.2μg PE Streptavidin(BD Pharmingen，Cat#554061)，4℃孵育30分钟；漂洗后加入含0.5μg的7-ADD(BD Pharmingen，Cat#559925)的DPBS/1％FBS孵育5分钟用以分辨死活细胞。流式细胞仪上样，FlowJo V10软件分析，并以荧光强度中位数(MFI)计算各细胞亚群的染色指数[staining index＝MFI(AD4-12)/MFI(isotype)]。
结果：如图3所示，AD4-12与PBMC中的单核细胞(CD11b+CD14+)高强度结合，与T细胞(CD3+CD56-)，NK细胞(CD3-CD56+)和NKT细胞(CD3+CD56+)有微弱结合，与B细胞(CD19+CD3-)不结合。(A)代表性流式细胞仪分析；(B)3位供者的染色指数mean±SD。
实施例7：酶联免疫吸附测定(ELISA)检测AD4-12阻断SIRPα-V1、V2结合CD47
取Maxisorp ELISA 96孔板，分别加入100μL的2.5μg/mL的重组SIRPα-V1-his和SIRPα-V2-his，4℃包被过夜。将梯度稀释的AD4-12和参照品18D5分别与0.25μg的biotin-CD47(CD47根据Genbank Accession NP_942088序列构建质粒表达，序列为QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP(SEQ ID NO：40：))混合，加入到包被封闭后的微孔板中，37℃孵育1小时。洗板后，各孔加入Streptavdin-HRP(Thermo，Cat#434323)，底物TMB(Beyotime，Cat#P0209-100mL)显色，酶标仪读数。
结果：如图4所示，AD4-12能够分别阻断SIRPα-V1、SIRPα-V2结合CD47，IC50值为5.258nM和9.322nM，阻断效果均优于18D5。
实施例8：FACS检测AD4-12阻断SIRPα-V1、V2与CD47结合
取对数期293T-SIRPα-V1细胞和CHOZN-SIRPα-V2细胞，以每孔2×10 5细胞加入 96孔U型培养板，封闭后，每孔加入0.5μg FITC标记的CD47(自制)和浓度梯度稀释的AD4-12或18D5，4℃孵育30分钟；细胞漂洗后流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图5所示，AD4-12对SIRPα-V1-CD47和SIRPα-V2-CD47的相互作用具有很好的阻断活性，IC50值分别为1.742nM和6.417nM。而参照品18D5对SIRPα-V1-CD47的阻断弱于AD4-12，对SIRPα-V2-CD47没有阻断活性。
实施例9：巨噬细胞的诱导分化和鉴定
新鲜PBMC(妙顺生物，Cat#PB003F)，用配制好的1×BD IMag TM缓冲液(BD Pharmingen，Cat#552362)漂洗，每1×10 7cells加入50μL BD IMag TM Anti-Human CD14 Magnetic Particles–DM(BD Pharmingen，Cat#557769)，室温孵育30分钟；漂洗细胞后用Cell Separation Magnet分离得到CD14 +单核细胞，重悬于IMDM培养基(含10％FBS+50uMβ-巯基乙醇+70ng/ml M-CSF)，以每孔1×10 5细胞铺于96孔板，置于37℃5％CO 2培养箱，3天后同样培养基补液，继续培养至7天。以0.25％Trpsin-EDTA消化收集1-2孔细胞，流式细胞仪鉴定诱导分化的巨噬细胞表型。将Fc blocker配制于终浓度为1mg/mL的IgG1中作为封闭液，4℃孵育15分钟，然后分别加荧光素标记的CD11b抗体、CD14抗体和SIRPα抗体(SantaCruz，Cat#sc-23863PE)染色，流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图6所示，诱导分化的巨噬细胞为CD11b +CD14 +SIRPα +，表明诱导分化成功。
实施例10：AD4-12与利妥昔单抗、达雷妥尤单抗联用的促吞噬作用
(一)Raji细胞CFSE染色
取对数生长期Raji细胞(CD20、CD38、CD47均为阳性)，计数并调节细胞密度到8×10 6cells/mL。取0.15mM CFSE工作液2μL与1mL的DPBS混匀，再加入到1mL上述细胞悬液中混匀，置于37℃培养箱15分钟；加入等体积即2mL的血清，混匀终止反应；细胞漂洗后加入RPMI1640培养基重悬，调整细胞密度到2×10 6/mL。
(二)巨噬细胞与Raji细胞共孵育
在预先诱导分化巨噬细胞的96孔板(见实施例9)，每孔加入终浓度10μg/mL的AD4-12,继续于37℃5％CO 2培养箱孵育30分钟。CFSE标记的Raji细胞,加或不加终浓度0.1μg/mL的利妥昔(RTX)或达雷妥尤(Dara)，于37℃5％CO 2培养箱孵育 30分钟。然后按照效靶细胞比例1：5将Raji细胞移入上述已经铺了巨噬细胞的96孔板，即巨噬细胞1×10 5/孔，Raji细胞5×10 5/孔，置于37℃5％CO 2培养箱孵育2小时。
(三)流式细胞仪检测吞噬活性
上述96孔细胞培养板弃上清，加入250μL的0.25％Trpsin-EDTA消化使细胞从96孔板脱离，漂洗，每孔加入1.5μg Fc blocker配制于终浓度为1mg/mL的IgG1溶液中封闭，然后加入每孔0.2μg APC标记的CD11b抗体4℃染色30分钟；漂洗，加入含0.5μg的7-ADD的DPBS/1％FBS孵育5分钟以分辨死活细胞。重悬细胞，流式细胞仪上样，FlowJo V10软件分析，比较去除死细胞和粘连细胞后的CD11b阳性群体中CFSE着色比例。
结果：如图7所示，AD4-12自身没有促吞噬作用，与利妥昔(A)或者达雷妥尤(B)联用，有明显增强这两个抗体依赖性细胞吞噬作用(ADCP)。
实施例11：树突状细胞诱导分化和鉴定
新鲜PBMC漂洗后，每1×10 7cells加入50μL BD IMag TM Anti-Human CD14 Magnetic Particles–DM(BD Pharmingen，Cat#557769)，室温孵育30分钟；漂洗细胞后用Cell Separation Magnet分离得到CD14 +单核细胞，重悬于IMDM培养基(含10％FBS+50uMβ-巯基乙醇+100ng/ml GM-CSF+70ng/ml IL-4)，每瓶接种5×10 6个细胞，置于37℃5％CO 2培养箱，3天后同样培养基补液，继续培养至7天。收集细胞，取2×10 5个细胞进行染色验证表型。将Fc blocker配制于终浓度为1mg/mL的IgG1中作为封闭液，4℃孵育15分钟，然后分别加荧光素标记的CD11b抗体、CD14抗体染色，流式细胞仪上样，FlowJo V10软件分析。
结果：如图8所示，诱导分化的DC细胞为CD11b +CD14 -，表明诱导分化成功。
实施例12：混合淋巴细胞反应(MLR)评价AD4-12对T细胞活化的影响
取预先分化好的DC细胞(见实施例11)，漂洗后加入RPMI1640培养基(含10％FBS)调整密度到1.4×10 5/mL；利用CD4T细胞阴选试剂盒(Miltenyi，Cat#130-096-533)磁珠分选得到CD4 +T细胞，漂洗后将T细胞密度调整到1.4×10 6/mL，然后与DC细胞按体积1：1混合均匀，每孔加入150μL的DC:T(1:10)细胞混合液；再加入终浓度为10μg/mL的相应抗体和同型对照如图标所示，每个样品设置三个复 孔，置于37℃5％CO 2培养箱孵育2天后，取上清100μL，使用IL-2检测试剂盒(R&D Systems，Cat#D2050)进行定量检测；补液(含同浓度样品)后将孔板放回37℃5％CO 2培养箱继续孵育3天，收集上清检测IFNγ(试剂盒R&D Systems，Cat#DIF50C)。FlowJo V10软件分析。
结果：如图9所示，在PD-L1抗体avelumab(Ave)存在条件下，T细胞被激活释放IL-2和IFNγ水平增加，添加AD4-12-G1或AD4-12-G1 lala与同型对照比较未见上清液中IL-2和IFNγ水平减低，说明它们不影响T细胞活化释放细胞因子。
实施例13：FACS检测AD4-12内吞
(一)抗体标记：
利用pHrodo(Thermo，Cat#Z25611)染料在进入细胞内pH值低的溶酶体内才显示荧光的特点，将待检抗体标记该染料可在流式细胞仪上检测其内吞情况。使用CD fusion培养基稀释抗体至终浓度160nM、稀释pHrodo母液12.5倍。分别取稀释后抗体AD4-12或同型对照与稀释后pHrodo按照1:1混匀，37℃培养箱孵育10分钟。
(二)抗体内吞检测：
取对数期293T-SIRPα V1细胞以CD fusion悬浮并调整细胞密度，以每孔1×10 5铺于96孔板，取50μL抗体-pHrodo标记复合物加入细胞，冰上孵育20分钟，此时收集细胞为0小时样品，漂洗后存放于冰上待测。其余细胞孔板移入37℃5％CO 2培养箱孵育，分别于1、2、4、6、8小时收集细胞，漂洗后存放于冰上待测。集中全部样品流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图10所示，AD4-12能随着细胞在37℃孵育时间的延长，荧光信号值增加，表明内吞作用明显。
实施例14：AD4-12与利妥昔单抗联合应用的抗肿瘤活性
A.Raji细胞在RPMI-1640完全培养基中培养(89％RPMI1640+10％FBS+1％丙酮酸钠)，取对数生长期细胞配制细胞悬液。SIRPα人源化完全免疫缺陷的hSIRPα-B-NDG小鼠(百奥赛图)为宿主，皮下接种100μL含5×10 5细胞悬液。每周两次游标卡尺测量肿瘤体积。
将小鼠分成3组，每组7只，待肿瘤生长至体积～100mm 3开始腹腔内注射给药， 150μg利妥昔单药或与200μg AD4-12-G1或AD4-12-G1 lala联合用药，每周2次共6次。
结果：如图11A所示，在这个缺乏T细胞、B细胞和NK细胞，仅存髓系细胞为免疫效应细胞的小鼠模型中，肿瘤生长在AD4-12-G1联合利妥昔给药组与利妥昔单药组无差别，而添加AD4-12-G1lala能增强利妥昔的抑制肿瘤生长效果(*P＜0.05)。
B.取对数生长期Raji细胞配制2×10 7/mL细胞悬液，与1×10 7/mL PBMC(妙顺生物，Cat#PB003F-W)按体积1：1混匀，再与Matrigel按1：1混匀。完全免疫缺陷的B-NDG小鼠(百奥赛图)为宿主，皮下接种100μL细胞-Matrigel混悬液，即每只小鼠接种Raji细胞5×10 5混合PBMC 2.5×10 5。每周两次游标卡尺测量肿瘤体积。
接种当天开始腹腔内注射给药，每组5只小鼠，分别给予生理盐水、20μg利妥昔单药或与200μg AD4-12-G1或AD4-12-G1 lala联合用药，每周1次共3次。
结果：如图11B所示，在这个由PBMC提供免疫效应细胞(包括T细胞、B细胞、NK细胞和髓系细胞)的小鼠模型中，如此低剂量的利妥昔单用相较于生理盐水即有明显的延缓肿瘤生长作用，AD4-12-G1 lala或AD4-12-G1联合利妥昔都能够显著增强利妥昔药效(**P＜0.01，****P＜0.0001)，而AD4-12-G1联合利妥昔的药效比AD4-12-G1 lala联合利妥昔的药效更强(*P＜0.05)。
实施例15：AD4-12与达雷妥尤单抗联合应用的抗肿瘤活性
A.取对数生长期细胞配制细胞悬液，SIRPα人源化完全免疫缺陷的hSIRPα-B-NDG小鼠为宿主，皮下接种100μL含5×10 5细胞悬液。每周两次游标卡尺测量肿瘤体积。
将小鼠分成3组，每组7只，待肿瘤生长至体积～100mm 3开始腹腔内注射给药，150μg达雷妥尤单药或与200μg AD4-12-G1或AD4-12-G1 lala联合用药，每周2次共6次。
结果：如图12A所示，在这个缺乏T细胞、B细胞和NK细胞，仅存髓系细胞为免疫效应细胞的小鼠模型中，肿瘤生长在AD4-12-G1联合达雷妥尤给药组与达雷妥尤单药组无差别，而添加AD4-12-G1 lala能增强达雷妥尤的抑制肿瘤生长效果(*P＜0.05)。
B.取对数生长期Raji细胞配制2×10 7/mL细胞悬液，与2×10 7/mL PBMC按体积1：1混匀，再与Matrigel按1：1混匀。完全免疫缺陷的B-NDG小鼠为宿主，皮下接种100μL细胞-Matrigel混悬液，即每只小鼠接种Raji细胞5×10 5混合PBMC 5×10 5。每周两次游标卡尺测量肿瘤体积。
接种当天开始腹腔内注射给药，每组5只小鼠，分别给予生理盐水、15μg达雷妥尤单药或与200μg AD4-12-G1或AD4-12-G1 lala联合用药，每周1次共3次。
结果：如图12B所示，在这个由PBMC提供免疫效应细胞(包括T细胞、B细胞、NK细胞和髓系细胞)的小鼠模型中，如此低剂量的达雷妥尤单用未能延缓肿瘤生长，但添加AD4-12-G1 lala或AD4-12-G1都能够显著增强达雷妥尤药效(*P＜0.05，**P＜0.01)。
实施例16：AD4-12抗体人源化
AD4-12序列与人类免疫球蛋白序列库IMGT(Lefranc，2003)进行比对，选择与AD4-12之VH和VL框架区氨基酸序列差异最少的人类种系序列作为人源化模板。AD4-12序列匹配最相近的V和J种系见表4。
表4.AD4-12序列匹配最相近的V和J种系
序列 V种系 J种系
AD4-12重链 V4-30-4*01 J6*01
AD4-12轻链 V7-3*01 J4
第一步，将AD4-12之CDR区直接替换人源化模板的CDR区，形成变体Z0重链可变区VH(SEQ ID NO:15)和轻链可变区VL(SEQ ID NO:19)。第二步，分别针对VH和VL框架序列中可能对维持CDR构象所必需的位点进行回复突变，并将携带不同突变的VH和VL两两组合，形成人源化变体Z0-Z9(表5-6)。第三步，将这些VL和VH分别与人κ轻链和IgG1重链恒定区连接，重链恒定区的Fc段含有L234A/L235A(lala)突变(SEQ ID NO：13)。
表5.AD4-12人源化序列及回复突变
Figure PCTCN2022112677-appb-000005
表6 AD4-12人源化序列
Figure PCTCN2022112677-appb-000006
Figure PCTCN2022112677-appb-000007
实施例17：表面等离子共振技术(SPR)检测AD4-12人源化变体的亲和力
利用Biacore分析系统，混合400mM EDC和100mM NHS制备活化剂，CM5传感器芯片以10μL/min的流速与活化剂一起激活420秒。将溶解在10mM NaAc(pH 4.5)中的30μg/mL的抗Fc抗体以10μL/min的流速注入通道，然后以10μL/min的流速用1M ethanolamine-HCl封闭420秒。AD4-12各种人源化变体以10μL/min的流速结合到CM5传感器芯片上，接着以30μL/min的流速将50nM SIRPα V1-his(ACRO，Cat#SIA-H5225)注射到通道1-8中，持续120秒，然后是300秒分离，在每个解离阶段后注入pH值为1.5的10mM甘氨酸作为再生缓冲液。所有实验数据均采用1:1结合模型进行拟合。
结果：如表7所示，人源化变体Z1,Z3-Z9的亲和力与AD4-12的亲和力基本保持一致，选择Z4作为抗体AD4-12人源化序列。
表7.SPR检测AD4-12人源化变体结合SIRPα-V1亲和力。
Figure PCTCN2022112677-appb-000008
实施例18：祛除CDR序列中翻译后修饰(PTM)高风险位点的尝试
在Z4抗体的CDR序列中发现可能发生异构化或脱酰胺的高风险位点，其中一个脱酰胺位点(NS)和一个异构化位点(DG)位于重链CDR2上(SEQ ID NO:6)，一个脱酰胺位点(NS)位于轻链CDR1上(SEQ ID NO:7)。为了减少PTM风险，我们对Z4抗体序列中这3个潜在PTM位点进行氨基酸替换(表8)。对重链CDR2可进行两个位 点的突变，通式可概括为IIWGDX 1STDYNX 2ALKS，其中X 1为G或A，X 2为S或A，得到与SEQ ID NO：6、SEQ ID NO：14、SEQ ID NO：23或SEQ ID NO：24所示一致的氨基酸序列。对轻链CDR1可进行一个位点的突变，通式可概括为RASESVDSYGX 3SFM，其中X 3为N或S，得到与SEQ ID NO：8或SEQ ID NO：25所示一致的氨基酸序列。
表8 CDR上PTM位点进行氨基酸替换
Figure PCTCN2022112677-appb-000009
将突变后序列分别与人κ轻链和含有L234A/L235A(lala)突变的IgG1重链恒定区连接，并用流式细胞仪方法比较它们对SIRP家族成员的结合能力，最终选定重链CDR2的NS位点和DG位点都消除掉的H7L4作为我们的抗体药候选序列(表9-11)。
表9.对CDR序列中PTM高发位点进行氨基酸替换形成Z4变体
Figure PCTCN2022112677-appb-000010
表10.H7L4可变区的CDR序列
Figure PCTCN2022112677-appb-000011
表11.Z4变体可变区序列
Figure PCTCN2022112677-appb-000012
Figure PCTCN2022112677-appb-000013
Figure PCTCN2022112677-appb-000014
实施例19：构建不同Fc亚型的H7L4
将H7L4的VL和VH片段cDNA分别与人κ轻链和野生型IgG1(SEQ ID NO:32)、IgG2(SEQ ID NO:33)、IgG4(SEQ ID NO:34)，以及含有L234A/L235A(lala)突变的IgG1重链恒定区连接，构建具有不同效应功能活性的抗体亚型(表12)。将H7L4之轻、重链分别克隆到pCDNA3.1(+)表达载体，质粒瞬转进入ExpiCHO-S细胞、培养、纯化具体方法同上实例2，得到目的抗体H7L4-G1(SEQ ID NO:35，SEQ ID NO:27)、H7L4-G2(SEQ ID NO:36，SEQ ID NO:27)、H7L4-G4(SEQ ID NO:37，SEQ ID NO:27)和H7L4-G1 lala(SEQ ID NO:31，SEQ ID NO:27)。
参照品BI 765063根据OSE Immunotherapeutics公司的专利CN201780023581.2所公布序列合成，Fc片段为IgG4。
表12 不同Fc亚型的H7L4
Figure PCTCN2022112677-appb-000015
Figure PCTCN2022112677-appb-000016
Figure PCTCN2022112677-appb-000017
实施例20：FACS比较人源化前后(AD4-12与H7L4)结合SIRPα-V1、V2、V8的能力
取对数生长期的稳转构建细胞株293T-SIRPα-V1(SEQ ID NO:2)、CHOZN-SIRPα-V2(SEQ ID NO:38)，和CHOZN-SIRPα-V8(SEQ ID NO:39)，在96孔U型培养板每孔加入2×10 5细胞，经Fc blocker封闭后，每孔加入2μg/mL相应抗体，4℃孵育30分钟；漂洗细胞后，每孔加入0.2μg AF488标记的羊抗人IgG抗体，4℃孵育30分钟；漂洗后加入50μL的DPBS/1％FBS重悬细胞，流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图13所示，鼠源母本抗体AD4-12与人源化后的抗体H7L4对结合SIRPα-V1、SIRPα-V2、SIRPα-V8无差异。
表13 SIRPα-V2和SIRPα-V8序列
Figure PCTCN2022112677-appb-000018
实施例21：BLI检测H7L4与SIRPα-V1、V2亲和力
利用ForteBio Octet RED 96测定H7L4抗体与SIRPα-V1、SIRPα-V2结合的亲和力。使用Ni-NTA sensor固化重组蛋白SIRPα V1-his、SIRPα V2-his，浓度均为5μg/mL，固化时间600秒。将H7L4-G1、H7L4-G1 lala和BI 765063进行2倍梯度稀释共7个浓度点。设置程序为结合时间200秒，解离时间600秒。数据拟合计算亲和力。
结果：如图14所示，H7L4-G1和H7L4-G1 lala与SIRPα-V1、V2都具有高亲和力，而参照品BI 765063只结合SIRPα-V1，不能结合SIRPα-V2。
实施例22：ELISA检测H7L4与SIRPα-V1、V2、V8的结合
取Maxisorp ELISA 96孔板，每孔加入100μL的1μg/mL SIRPα-V1-his、 SIRPα-V2-his或SIRPα-V8-his(Sino Biological，Cat#30015-H08H)，4℃包被过夜。封闭后，将梯度稀释的H7L4-G1、H7L4-G1 lala和参照品BI 765063加入到微孔板中，37℃孵育2小时。洗板后，各孔加入100uL 1:5000稀释的羊抗人IgG Fc HRP(Invitrogen，Cat#A18823)，孵育1小时后，TMB底物显色，酶标仪读数。
结果：如图15所示，H7L4-G1、H7L4-G1 lala与SIRPα-V1、SIRPα-V2、SIRPα-V8都能够很好地结合，而参照品BI 765063只结合SIRPα-V1，不能结合SIRPα-V2、V8。
实施例23：FACS检测H7L4与SIRPα-V1、V2、V8以及SIRPβ、SIRPγ的结合
取对数生长期的稳转构建细胞株293T-SIRPα-V1(SEQ ID NO:2)细胞、CHOZN-SIRPα-V2(SEQ ID NO:38)细胞、293T-SIRPα-V8(SEQ ID NO:39)细胞、293T-SIRPβ-V1(Genbank Accession：O00241序列(30-371))细胞，以每孔2×10 5细胞加入96孔U型培养板，封闭后，加入浓度梯度稀释的样品H7L4-G1、H7L4-G1 lala和参照品BI 765063，4℃孵育30分钟；漂洗细胞后，每孔加入0.2μg AF488标记的羊抗人IgG抗体，4℃孵育30分钟；漂洗后加入50μL的DPBS/1％FBS重悬细胞，流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图16所示，H7L4-G1、H7L4-G1 lala与SIRPα-V1、SIRPα-V2、SIRPα-V8能够很好结合，与SIRPβ、SIRPγ的结合相对较弱，而参照品BI 765063与SIRPα-V1、SIRPβ的结合接近于H7L4，与SIRPα-V2和SIRPγ完全不结合。
实施例24：FACS检测H7L4与人PBMC亚群细胞的结合
复苏冻存的人PBMC(妙顺生物，Cat#PB003F)，在96孔U型培养板每孔加入5×10 5细胞，经配制于1mg/mL mIgG的Fc blocker封闭后，每孔分别加入3μg biotin标记的H7L4-G1、BI 765063、同型对照以及0.2μg细胞表面标记分子的抗体，包括APC标记的CD56抗体、eFlour 506标记的CD11b抗体、FITC标记的CD3抗体、APC-eFlour 780标记的CD14抗体和eFlour 450标记的CD19抗体，4℃孵育30分钟；漂洗细胞后，每孔加入二抗0.2μg PE Streptavidin，4℃孵育30分钟；漂洗后加入含0.5μg的7-ADD的DPBS/1％FBS孵育5分钟后流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析，并以荧光强度中位数(MFI)计算各细胞亚群的染色指数[staining index＝MFI(antibody)/MFI(isotype)]。
结果：如图17所示，H7L4或BI 765063对各亚群细胞的结合由强到弱排序依次为单核细胞(CD11b +CD14 +)、DC细胞(CD11b +CD14 -)和T细胞(CD3 +CD56 -)，不结 合B细胞(CD19 +CD3 -)和NK细胞(CD3 -CD56 +)。看起来H7L4的结合活性均高于BI 765063，不能排除biotin标记效率不同所致，而且供者的SIRPα基因型未知。
实施例25：ELISA检测H7L4对SIRPα-V1、V2结合CD47的阻断作用
取Maxisorp ELISA 96孔板，分别加入100μL的1μg/mL的重组SIRPα-V1-his和SIRPα-V2-his，4℃包被过夜。封闭后，将梯度稀释的H7L4-G1、H7L4-G1 lala和参照品BI 765063分别与0.5μg的biotin-CD47混合，加入到包被封闭后的微孔板中，37℃孵育1小时。洗板后，每孔加入0.2μg Streptavdin-HRP，TMB底物显色，酶标仪读数。
结果：如图18所示，H7L4-G1和H7L4-G1 lala能够阻断SIRPα-V1、V2与CD47的结合，参照品BI 765063对SIRPα-V1-CD47的阻断活性与H7L4相当，对SIRPα-V2-CD47则没有阻断活性。
实施例26：FACS检测H7L4对SIRPα-V1、V2、V8结合CD47的阻断作用
取对数生长期的细胞293T-SIRPα-V1、CHOZN-SIRPα-V2、293T-SIRPα-V8，以每孔2×10 5细胞加入96孔U型培养板，封闭后，加入FITC标记的CD47(0.5μg/孔)和浓度梯度稀释的样品H7L4-G1和H7L4-G1 lala、参照品BI 765063以及同型对照，4℃孵育30分钟；细胞漂洗后流式细胞仪上样，FlowJo V10和Graphpad Prism6软件分析。
结果：如图19所示，H7L4-G1、H7L4-G1 lala均能阻断SIRPα-V1、V2、V8与CD47的结合；而参照品BI 765063仅能阻断SIRPα-V1与CD47结合，对SIRPα-V2、V8不能很好阻断。
实施例27：H7L4与利妥昔单抗、达雷妥尤单抗联用的促吞噬作用
实验方法同实施例10，所用抗体及其浓度为：10μg/mL的H7L4-G1、H7L4-G1 lala或参照品BI 765063、Hu5F9-G4(CD47抗体Magrolimab，根据WHO Drug Information,Vol.33,No.3,2019公布序列构建质粒表达)、以及同型对照hIgG1；0.1μg/mL的利妥昔(RTX)或0.2μg/mL的达雷妥尤(Dara)。
结果：如图20所示，SIRPα抗体单用未见促吞噬作用，只有CD47抗体Hu5F9-G4显示明显的促吞噬效应；CD47或SIRPα抗体与RTX或Dara联用都能够显著提高后者的抗体依赖性细胞吞噬作用(ADCP)，H7L4-G1 lala的增强ADCP作用略优于H7L4-G1、BI 765063或Hu5F9-G4。
实施例28：巨噬细胞吞噬法检测H7L4对于SIRPα +细胞的ADCP效应
实验方法同实施例10，靶细胞为CFSE标记的U937细胞(SIRPα和CD47均为阳性高表达)，所用抗体及其浓度为：10μg/mL的H7L4-G1、H7L4-G1 lala或参照品BI 765063、Hu5F9-G4，以及同型对照hIgG1。
结果：如图21所示，SIRPα抗体包括H7L4-G1、H7L4-G1 lala和参照品BI 765063对SIRPα强阳性的U937细胞基本没有介导ADCP，而参照品Hu5F9-G4则表现出明显的ADCP效应。
实施例29：FACS检测H7L4对小鼠体内SIRPα +细胞的削减作用
SIRPα和CD47双人源化C57BL/6小鼠(hSIRPα-hCD47-B6,购自百奥赛图)腹腔注射淀粉肉汤诱导巨噬细胞渗出。3天后腹腔注射200μg的H7L4-G1、H7L4-G1 lala或生理盐水，每组各3只小鼠。再过3天采集外周血、脾脏和腹腔渗出液做流式细胞仪分析。取100μL每只小鼠外周血，低渗溶解红细胞，移置于96孔板；机械研磨方法制备脾脏单细胞悬液，每份样品取2x10 5细胞移置于96孔板；腹腔渗出液离心、重悬，每份样品取2x10 5细胞移置于96孔板。对96孔板中细胞加入Fc blocker封闭，加入荧光素标记的抗鼠CD11b抗体或F4/80抗体，4℃孵育30分钟，漂洗、重悬、流式细胞仪上样、分析。
结果：如图22所示，H7L4-G1稍微降低外周血中CD11b +髓系细胞(未达到显著性差异)，但不减少脾脏內F4/80 +巨噬细胞。腹腔渗出液中CD11b +髓系细胞不降反升(p<0.05)。
实施例30：MLR评价H7L4对T细胞活化的影响
取预先分化鉴定好的DC细胞(见实施例11)，细胞漂洗后加入RPMI1640培养基(含10％FBS)调整密度到1.4×10 5/mL；利用CD4T细胞阴选试剂盒(Miltenyi，Cat#130-096-533)磁珠分选得到CD4 +T细胞，漂洗后加入CFSE工作液(终浓度1μM)，置于37℃培养箱15分钟，随后加入等体积的血清终止反应，漂洗后将T细胞密度调整到1.4×10 6/mL，然后与DC细胞按体积1：1混合均匀，每孔加入150μL的DC与T细胞混合液；再加入终浓度为10μg/mL的样品H7L4-G1、BI 765063和同型对照，每个样品设置三个复孔，置于37℃5％CO 2培养箱孵育2天后，取上清100μL，使用IL-2检 测试剂盒进行定量；补液(含同浓度样品)后将孔板放回37℃5％CO 2培养箱继续孵育3天，漂洗后，流式细胞仪上样根据CFSE荧光随着细胞分裂被稀释的原理检测T细胞增殖情况，FlowJo V10软件分析。
结果：如图23所示，与未加抗体的空白对照或IgG1同型对照比较，H7L4-G1和参照品BI 765063均没有改变T细胞增殖(A)和活化(IL-2的释放)(B)。
实施例31：红细胞凝集实验
取红细胞样本漂洗后用DPBS重悬成6％(v/v)的红细胞悬液。用DPBS配制H7L4-G1、Hu5F9-G4和hIgG1溶液，最高浓度100μg/ml，3倍稀释，10个梯度。取配好的药液50μL/孔加入到96孔U底孔板中，再加入上述红细胞悬液50μL/孔，37℃孵育3小时，移置4℃孵育过夜。采用Chemi Doc XRS+Imaging System成像记录结果。
结果：如图24所示，Hu5F9-G4能显著引起红细胞凝集，而H7L4-G1不会引起红细凝集。
实施例32：H7L4孵育PBMC诱导细胞因子释放实验
取自3个供者的新鲜PBMC(妙顺生物，Cat#PB003F)，分别在U型底96孔板设置3复孔，每孔0.5x10 6细胞，加入终浓度为10μg/mL的H7L4-G1、H7L4-G1 lala或参照品BI 765063以及未加抗体空白对照，孵育48小时，收集上清液冻存，送优宁维生物(CRO公司)用多因子平台Luminex LXR-M500KCAF0Y检测27个主要由巨噬细胞(单核细胞)、T细胞释放的细胞因子。
结果：如图25所示，与空白对照比较，添加H7L4-G1孵育的PBMC上清液中可见MCP-1、MIP-1α、MIP-1β、IL-1Ra、TNFα、IFNγ、IL-6因子升高，IP-10水平降低；而添加H7L4-G1 lala孵育的PBMC上清液中MIP-1α、IP-10水平减低。其它所检因子低于检测下限或与空白对照无差异。
实施例33：H7L4在免疫健全小鼠的抗肿瘤效应(A20淋巴瘤细胞株)
以SIRPα和CD47双人源化BALB/c小鼠(hSIRPα-hCD47-Balb/c,购自江苏集萃药康)为宿主，皮下接种5x10 5人CD47转染的A20淋巴瘤细胞，每组6只小鼠。待 肿瘤体积达到100～200mm 3,腹腔注射200μg H7L4-G1或H7L4-G1 lala，每周2次共4次给药。每周2次测量肿瘤体积。
结果：如图26所示，与生理盐水对照组比较，H7L4-G1或H7L4-G1 lala治疗组都可见抑制肿瘤生长的药效。
实施例34：H7L4在免疫健全小鼠的抗肿瘤效应(MC38结肠癌细胞株)
以hSIRPα-hCD47-B6小鼠(购自百奥赛图)为宿主，皮下接种5x10 5人CD47转染的MC38肿瘤细胞，每组6只小鼠。待肿瘤体积达到100～200mm 3,腹腔注射200μg H7L4-G1、H7L4-G1 lala、参照品Hu5F9-G4，每周2次共3周给药。每周2次测量肿瘤体积。
结果：如图27所示，与生理盐水对照组比较，H7L4-G1 lala治疗组4/6小鼠肿瘤消失，其它组都可见4/6小鼠肿瘤生长至安乐死终点。
实施例35：H7L4与PD-L1单抗联用在免疫健全小鼠的协同抗肿瘤效应(MC38结肠癌细胞株)
以hSIRPα-hCD47-B6小鼠(购自百奥赛图)为宿主，皮下接种1x10 6人CD47转染的MC38肿瘤细胞，每组6只小鼠。待肿瘤体积达到100～200mm 3,腹腔注射200μg H7L4-G1、100μg PD-L1单抗avelumab或两者联用，每隔3天给药1次共3次。每周2次测量肿瘤体积。
结果：如图28所示，H7L4-G1治疗组4/6小鼠肿瘤消失，虽然Avelumab单药使得全部小鼠肿瘤完全消退，H7L4-G1联合Avelumab治疗组肿瘤消退更快，提示协同效应。
实施例36：ELISA和FACS检测H7L4与食蟹猴SIRPα的结合
A.ELISA检测H7L4与食蟹猴SIRPα的结合
取Maxisorp ELISA 96孔板，加入100μL的1μg/mL食蟹猴SIRPα-his，4℃包被过夜。封闭后，将梯度稀释的H7L4-G1和H7L4-G1 lala加至孔板中，37℃孵育1小时。洗板后，各孔加入100μL 1:5000稀释的羊抗人IgG Fc HRP，37℃孵育1小时后，底物TMB 显色，酶标仪读数。
B.FACS检测H7L4与食蟹猴SIRPα的结合
复苏冻存的食蟹猴PBMC(由优宁维生物赠送)，每孔加入4×10 5细胞，将Fc blocker配制于终浓度为1mg/mL的IgG1中作为封闭液，4℃孵育15分钟；然后加入biotin标记的样品H7L4-G1、H7L4-G1 lala和同型对照，4℃孵育30分钟；漂洗后，加入二抗0.2μg Streptavidin-PE以及荧光素标记的CD11b抗体染色，4℃孵育30分钟；漂洗后加入含0.5μg的7-ADD的DPBS/1％FBS孵育5分钟用以分辨死活细胞。流式细胞仪上样，FlowJo V10软件分析。
结果：如图29所示，H7L4-G1、H7L4-G1 lala能够很好地结合蛋白分子水平和细胞膜表面的食蟹猴SIRPα。

Claims (56)

  1. 靶向SIRPα的单克隆抗体或其抗原结合部分，其包含：
    (a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
    (b)重链可变区CDR2，其包含与IIWGX 1X 2STDYX 3X 4ALKS所示一致的氨基酸序列，其中X 1为D、E或N，X 2为G、A或S，X3为N、Q或S，X 4为S、T或A；
    (c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
    (d)轻链可变区CDR1，其包含与RASESVDSYGX 5X 6FM所示一致的氨基酸序列，其中X 5为N、Q或S，X 6为S、T或者A；
    (e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
    (f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其特征在于，其包含：
    (a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
    (b)重链可变区CDR2，其包含与选自SEQ ID NO：6、SEQ ID NO：14、SEQ ID NO：23或SEQ ID NO：24所示一致的氨基酸序列；
    (c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
    (d)轻链可变区CDR1，其包含与选自SEQ ID NO：8或SEQ ID NO：25所示一致的氨基酸序列；
    (e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
    (f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
  3. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其特征在于，其包含：
    (a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
    (b)重链可变区CDR2，其包含与选自SEQ ID NO：6所示一致的氨基酸序列；
    (c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
    (d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
    (e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
    (f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
  4. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其特征在于，其包含：
    (a)重链可变区CDR1，其包含与选自SEQ ID NO：5所示一致的氨基酸序列；
    (b)重链可变区CDR2，其包含与选自SEQ ID NO：14所示一致的氨基酸序列；
    (c)重链可变区CDR3，其包含与选自SEQ ID NO：7所示一致的氨基酸序列；
    (d)轻链可变区CDR1，其包含与选自SEQ ID NO：8所示一致的氨基酸序列；
    (e)轻链可变区CDR2，其包含与选自SEQ ID NO：9所示一致的氨基酸序列；和
    (f)轻链可变区CDR3，其包含与选自SEQ ID NO：10所示一致的氨基酸序列。
  5. 根据权利要求1-4任一项所述的单克隆抗体或其抗原结合部分，其特征在于，与靶向SIRPα的单克隆抗体CDR区或可变区具有高同源性的抗体通过保守序列修饰获得，包括一处或多处氨基酸的取代、添加和缺失等。
  6. 根据权利要求5所述的单克隆抗体或其抗原结合部分，其特征在于，所述一处或多处氨基酸的添加、缺失和/或取代不超过五处。
  7. 根据权利要求6所述的单克隆抗体或其抗原结合部分，其特征在于，所述一处或多处氨基酸的添加、缺失和/或取代不超过三处。
  8. 靶向SIRPα的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
    (a)重链可变区，其包含与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18所示一致的氨基酸序列；
    (b)轻链可变区，其包含与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22所示一致的氨基酸序列。
  9. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，其重链可变区与选自SEQ ID NO：3、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：4、SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22的氨基酸序列具有至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
  10. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体包含的重链和轻链可变区序列分别如SEQ ID NO：3和SEQ ID NO：4所示。
  11. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体为人 源化抗体，其重链和轻链可变区序列选自如下的组：
    (1)重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：20；
    (2)重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：22；
    (3)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：20；
    (4)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：21；
    (5)重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：22；
    (6)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20；
    (7)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：21；
    (8)重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：22。
  12. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体为含有人IgG恒定区的全长抗体。
  13. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体为含有小鼠IgG恒定区的全长抗体。
  14. 根据权利要求12所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体重链恒定区选自人IgG1、IgG2、IgG3或IgG4恒定区。
  15. 根据权利要求14所述的单克隆抗体或其抗原结合部分，其特征在于，所述人恒定区Fc结构域是IgG1、IgG2或IgG4 Fc结构域。
  16. 根据权利要求15所述的单克隆抗体或其抗原结合部分，其特征在于，人恒定区Fc结构域是IgG1突变体(LALA)或IgG1野生型。
  17. 根据权利要求12所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体轻链恒定区可以是κ或λ恒定区，但最优选为κ恒定区。
  18. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体或抗体片段为人抗体或人抗体片段。
  19. 根据权利要求18所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
  20. 根据权利要求18所述的单克隆抗体或其抗原结合部分，其特征在于，所述抗体片段为双抗体。
  21. 靶向SIRPα的单克隆抗体，其包含：
    (a)重链，其序列与SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列一致；以及
    (b)轻链，其序列与SEQ ID NO：26或SEQ ID NO：27所示的氨基酸序列一致。
  22. 根据权利要求21所述的单克隆抗体，其特征在于，其重链(HC)与选自SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链(LC)与选自SEQ ID NO：26或SEQ ID NO：27的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
  23. 根据权利要求22所述的单克隆抗体，其特征在于，与所述序列的重链和轻链具有高90％或更高同源性的抗体可以通过诱变得到，优选的定点诱变或PCR介导的诱变位点位于重链可变区CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
  24. 靶向SIRPα的单克隆抗体，其包含：
    (a)重链，其序列与SEQ ID NO:31、SEQ ID NO：35、SEQ ID NO：36或SEQ ID NO：37所示的氨基酸序列一致；以及
    (b)轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
  25. 根据权利要求21所述的单克隆抗体，其特征在于：所述抗体的重链和轻链序选自如下的组：
    (1)重链序列与SEQ ID NO:28所示一致，轻链序列与SEQ ID NO:26所示一致；
    (2)重链序列与SEQ ID NO:29所示一致，轻链序列与SEQ ID NO:26所示一致；
    (3)重链序列与SEQ ID NO:30所示一致，轻链序列与SEQ ID NO:26所示一致；
    (4)重链序列与SEQ ID NO:31所示一致，轻链序列与SEQ ID NO:26所示一致；
    (5)重链序列与SEQ ID NO:28所示一致，轻链序列与SEQ ID NO:27所示一致；
    (6)重链序列与SEQ ID NO:29所示一致，轻链序列与SEQ ID NO:27所示一致；
    (7)重链序列与SEQ ID NO:30所示一致，轻链序列与SEQ ID NO:27所示一致；
    (8)重链序列与SEQ ID NO:31所示一致，轻链序列与SEQ ID NO:27所示一致；
    (9)重链序列与SEQ ID NO:35所示一致，轻链序列与SEQ ID NO:27所示一致；
    (10)重链序列与SEQ ID NO:36所示一致，轻链序列与SEQ ID NO:27所示一致；
    (11)重链序列与SEQ ID NO:37所示一致，轻链序列与SEQ ID NO:27所示一致。
  26. 靶向SIRPα的单克隆抗体，其包含：
    (a)重链，其序列与SEQ ID NO：31所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致；或
    (b)重链，其序列与SEQ ID NO：35所示的氨基酸序列一致；轻链，其序列与SEQ ID NO：27所示的氨基酸序列一致。
  27. 编码权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或其抗原结合部分的核酸分子。
  28. 根据权利要求27所述的核酸分子，其为DNA或RNA，而且可以含有或不含内含子序列。
  29. 根据权利要求28所述的核酸分子，其为cDNA分子。
  30. 一种表达载体，其含有如权利要求27-29任一项所述的核酸分子。
  31. 一种宿主细胞，其由权利要求30所述的载体经转化或转染原核生物或真核生物宿主细胞得到。
  32. 根据权利要求31所述的宿主细胞，其为细菌、酵母或哺乳动物细胞。
  33. 根据权利要求32所述的宿主细胞，其为哺乳动物细胞，选自中国仓鼠卵巢细胞(CHO细胞)、NSO骨髓瘤细胞、COS细胞和SP2细胞等。
  34. 根据权利要求33所述的宿主细胞，其为CHO细胞。
  35. 一种药物组合物，其包含权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段以及药学上可接受载体。
  36. 一种药物组合物，其包含权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段，Rituximab以及药学上可接受载体。
  37. 一种药物组合物，其包含权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段，Daratumumab以及药学上可接受载体。
  38. 一种药物组合物，其包含权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段，PD-(L)1抗体以及药学上可接受载体；其中，所述的PD-(L)1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab-rwlc)、卡瑞利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、特瑞普利单抗(Toripalimab)、替雷利珠单抗(Tislelizumab)、赛帕利单抗(Zimberelimab)、派安普利单抗(Penpulimab)、斯鲁利单抗(Serplulimab)、普特利单抗(Pucotenlimab)、阿替利珠单抗(Atezolizumab)、度伐利尤单抗(durvalumab)、阿维鲁单抗(Avelumab)、恩沃利单抗(Envafolimab)、舒格利单抗(Sugemalimab)和卡度尼利单抗(Cadonilimab)。
  39. 权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段在治疗肿瘤疾病或微生物感染性疾病中的用途。
  40. 根据权利要求39所述的用途，其特征在于，所述用途为用于治疗癌症或用于抑制肿瘤生长。
  41. 根据权利要求40所述的用途，其特征在于，所述的肿瘤或癌症选自鳞状细胞癌，小细胞肺癌，非小细胞肺癌，鳞状非小细胞肺癌(NSCLC)，非鳞状NSCLC，胶质瘤，胃肠癌，肾癌，卵巢癌，肝癌，结直肠癌，子宫内膜癌，前列腺癌，甲状腺癌，神经母细胞瘤，胰腺癌，成胶质细胞瘤，宫颈癌，胃癌，膀胱癌，头颈癌，黑色素瘤，骨癌，皮肤癌，弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等，以及所述癌症的任何组合。
  42. 权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段联合Rituximab或Daratumumab在治疗肿瘤疾病中的用途。
  43. 根据权利要求42所述的用途，其特征在于,所述的肿瘤疾病为血液肿瘤。
  44. 根据权利要求43所述的用途，其特征在于,所述的血液肿瘤疾病为弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等。
  45. 权利要求1-26任一项所述的靶向SIRPα的单克隆抗体或抗体片段联合 PD-(L)1抗体在治疗肿瘤疾病中的用途。
  46. 根据权利要求45所述的用途，其特征在于,所述的PD-(L)1抗体包括PD-1抗体、PD-L1抗体或针对PD-1/PD-L1的双抗。
  47. 根据权利要求46所述的用途，其特征在于,所述的PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab-rwlc)、卡瑞利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、特瑞普利单抗(Toripalimab)、替雷利珠单抗(Tislelizumab)、赛帕利单抗(Zimberelimab)、派安普利单抗(Penpulimab)、斯鲁利单抗(Serplulimab)、普特利单抗(Pucotenlimab)等；所述的PD-L1单抗包括阿替利珠单抗(Atezolizumab)、度伐利尤单抗(durvalumab)、阿维鲁单抗(Avelumab)、恩沃利单抗(Envafolimab)、舒格利单抗(Sugemalimab)等；所述的针对PD-1/PD-L1的双抗包括靶向PD-1和CTLA-4的双抗卡度尼利单抗(Cadonilimab)。
  48. 根据权利要求45所述的用途，其特征在于,所述的肿瘤疾病为血液肿瘤、恶性黑色素瘤、乳腺癌、小细胞肺癌、非小细胞肺癌、肝癌、胃癌、肾癌、结直肠癌、膀胱癌、头颈部肿瘤、宫颈癌、Merkel细胞癌以及所有微卫星高度不稳定(MSI-H)的实体瘤治疗等。
  49. 根据权利要求48所述的用途，其特征在于,所述的血液肿瘤疾病为弥漫性大B细胞淋巴瘤，非霍奇金氏淋巴瘤，急性成淋巴细胞白血病(ALL)，急性髓样白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性髓样白血病(CML)，多发性骨髓瘤等。50、根据权利要求39所述的用途，其特征在于，所述治疗微生物感染性疾病用途为用于治疗病毒感染或细菌性感染疾病。
  50. 根据权利要求50所述的用途，其特征在于，所述用途为用于治疗病毒感染或细菌性感染疾病，其中所述的病毒性感染疾病包括腺病毒、疱疹病毒、乳头瘤病毒、冠状病毒、人免疫缺陷病毒(HIV)、人巨细胞病毒、EB病毒、丙型肝炎病毒或乙型肝炎病毒等感染引起的疾病；所述的病菌性感染疾病包括芽孢杆菌、肺炎衣原体、流感嗜血杆菌、结核分枝杆菌、假单胞菌、沙门氏菌、葡萄球菌、链球菌、密螺旋体等感染引起的疾病。
  51. 根据权利要求39所述的用途，其特征在于，将治疗有效量的权利要求1-26任一项 所述的靶向SIRPα的单克隆抗体或抗体片段给予需要治疗的受试者。
  52. 根据权利要求52所述的用途，其特征在于，所述的受试者为人或非人动物。
  53. 根据权利要求53所述的用途，其特征在于，所述的非人动物包括非人灵长类、羊、犬、小鼠、大鼠、猫、牛、马、鸡、两栖类和爬行类。
  54. 根据权利要求52所述的用途，其特征在于，所述抗体的给药剂量的范围为0.01-100mg/kg。
  55. 根据权利要求55所述的用途，其特征在于：所述抗体的给药剂量的范围为0.1mg/kg-100mg/kg，或0.5mg/kg-50mg/kg，或1mg/kg-25mg/kg，或2mg/kg-10mg/kg，或5mg/kg-10mg/kg。
  56. 根据权利要求52所述的用途，其特征在于：所述抗体的治疗方案要求每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。
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