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WO2024153193A1 - Molécules bi-spécifiques ciblant sirpa et claudine 18.2 - Google Patents

Molécules bi-spécifiques ciblant sirpa et claudine 18.2 Download PDF

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Publication number
WO2024153193A1
WO2024153193A1 PCT/CN2024/073050 CN2024073050W WO2024153193A1 WO 2024153193 A1 WO2024153193 A1 WO 2024153193A1 CN 2024073050 W CN2024073050 W CN 2024073050W WO 2024153193 A1 WO2024153193 A1 WO 2024153193A1
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amino acid
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set forth
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Inventor
Dawei Sun
Wenqiang Lu
Ziqiao DING
Yanan GENG
Haixia JIANG
Rui Gao
Hongtao Lu
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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Priority to EP24744363.3A priority Critical patent/EP4652202A1/fr
Priority to CN202480008424.4A priority patent/CN120569409A/zh
Publication of WO2024153193A1 publication Critical patent/WO2024153193A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to an anti-SIRP ⁇ antibody or antigen binding fragment thereof and the bi-specific molecules targeting SIRP ⁇ (signal regulatory protein alpha) and Claudin 18.2 (claudin 18 isotype 2) and uses thereof.
  • tumor cells can continually divide and grow by modifying their own surface antigens and changing the microenvironment around the tumor tissue to escape the surveillance, recognition and attack of the innate immune system, i.e., the so-called tumor immune escape.
  • tumor cells inhibit the immune function of macrophages and significantly inhibit the activity of immune cells by highly expressing CD47 which binds to the inhibitory receptor signal-regulatory protein ⁇ (SIRP ⁇ , or SIRPA) on the surface of macrophages.
  • SIRP ⁇ inhibitory receptor signal-regulatory protein ⁇
  • SIRPA inhibitory receptor signal-regulatory protein Alpha
  • SIRP ⁇ Signal regulatory protein ⁇ , SIRPA
  • SIRP ⁇ is a regulatory membrane glycoprotein from SIRP family. It is mainly expressed by myeloid cells and also by stem cells or neurons. SIRP ⁇ acts as inhibitory receptor and interacts with a broadly expressed transmembrane protein CD47. This interaction negatively controls effector function of innate immune cells such as host cell phagocytosis.
  • the inhibitory effect of CD47 on immune cells can be relieved by blocking the CD47-SIRP ⁇ signaling pathway, such as, SIRP ⁇ -Fc fusion protein, and exhibit certain anti-tumor activity.
  • the wild-type SIRP ⁇ -Fc fusion protein does not have potent efficacy due to its low affinity to CD47.
  • Claudin 18 belongs to the claudin family, which has at least 27 members in mammals (Furuse M. et al., J Cell Biol., 1998, 141, 1539) .
  • Claudin 18 has two different splice variants, Claudin 18.1 or CLDN 18.1 and Claudin 18.2 or CLDN 18.2. (Sanada Y. et al., J Pathol., 2006, 208, 633) .
  • CLDN 18.2 is a CD20-like differentiation protein overly expressed in various types of cancers, for example, gastric, esophageal, pancreatic, and non-small cell lung cancers. This molecule therefore is a valuable target for treatment of such cancers.
  • the present disclosure relates to anti-SIRP ⁇ antibodies as describes herein.
  • the present disclosure relates to anti-SIRP ⁇ single-chain fragment variable, bi-specific molecules and pharmaceutical compositions based on the anti-SIRP ⁇ single-chain fragment variable and uses thereof.
  • novel anti-SIRP ⁇ antibody or antigen binding fragment thereof which includes a heavy chain variable domain (VH) and a heavy chain variable domain (VL) .
  • the VH of anti-SIRP ⁇ antibody or antigen binding fragment thereof includes HCDR1, HCDR2, and HCDR3 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; (3) SEQ ID NOs: 20, 47, and 48, respectively.
  • the VL of anti-SIRP ⁇ antibody or antigen binding fragment thereof includes LCDR1, LCDR2, and LCDR3 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively.
  • the HCDR1 has the amino acid sequence set forth in SEQ ID NO: 20
  • the HCDR2 has the amino acid sequence shown as X 1 DPEDX 2 ETK (SEQ ID NO: 60)
  • the HCDR3 has the amino acid sequence shown as DRGLX 3 Y (SEQ ID NO: 61)
  • the LCDR1 has the amino acid sequence shown as X 4 ASX 5 SVSSSYLY (SEQ ID NO: 45)
  • the LCDR2 has the amino acid sequence shown as YSX 6 SNX 7 AS (SEQ ID NO: 46)
  • the LCDR3 has the amino acid sequence set forth in SEQ ID NO: 28, X 1 is V or I, X 2 is A or G, X 3 is V or A, X 4 is R or S, X 5 is Q or S, X 6 is A or T, X 7 is R or L.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 24, 26 and 28, respectively.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the VH of the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 29, 30, 32, and 33, respectively; (2) SEQ ID NOs: 29, 31, 32, and 33, respectively; (3) SEQ ID NOs: 49, 50, 51, and 33, respectively.
  • the VL of the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 34, 35, 36, and 37, respectively; (2) SEQ ID NOs: 34, 35, 36, and 38, respectively; (3) SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes the VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 14, 16, 18, and 54; and/or the VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 15, 17, 19, and 55.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 14; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 15.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 16; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 17
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 18; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 19.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 54; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 55.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof binds to human and/or mouse SIRP ⁇ .
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof is a Fab, a Fab', a F (ab') 2, an Fv fragment, or a single-chain fragment variable (scFv) .
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof is scFv, of which the VH and VL are linked via a first peptide linker, optionally the first peptide linker includes (Gly4Ser) 4.
  • N-terminus of the VH is linked to C-terminus of the VL.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof is a chimeric antibody or a humanized antibody.
  • a bi-specific molecule which includes
  • a SIRP ⁇ binding domain which contains a heavy chain variable domain (VH) and a heavy chain variable domain (VL) , wherein VH includes HCDR1, HCDR2, and HCDR3 having amino acid sequences about 80%to about 100%, e.g., about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more to about 100%, identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; (3) SEQ ID NOs: 20, 47, and 48, respectively.
  • VL includes LCDR1, LCDR2, and LCDR3 having amino acid sequences about 80%to about 100%, e.g., about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively; and
  • the HCDR1 has the amino acid sequence set forth in SEQ ID NO: 20
  • the HCDR2 has the amino acid sequence shown as X 1 DPEDX 2 ETK (SEQ ID NO: 60)
  • the HCDR3 has the amino acid sequence shown as DRGLX 3 Y (SEQ ID NO: 61)
  • the LCDR1 has the amino acid sequence shown as X 4 ASX 5 SVSSSYLY (SEQ ID NO: 45)
  • the LCDR2 has the amino acid sequence shown as YSX 6 SNX 7 AS (SEQ ID NO: 46)
  • the LCDR3 has the amino acid sequence set forth in SEQ ID NO: 28, X 1 is V or I, X 2 is A or G, X 3 is V or A, X 4 is R or S, X 5 is Q or S, X 6 is A or T, X 7 is R or L.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 24, 26 and 28, respectively; (2) the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively; or (3) the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the VH of SIRP ⁇ binding domain includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 29, 30, 32, and 33, respectively; (2) SEQ ID NOs: 29, 31, 32, and 33, respectively; (3) SEQ ID NOs: 49, 50, 51, and 33, respectively; and/or the VL of SIRP ⁇ binding domain includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 34, 35, 36, and 37, respectively; (2) SEQ ID NOs: 34, 35, 36, and 38
  • the VH of SIRP ⁇ binding domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 14, 16, 18, and 54, respectively; and/or the VL of SIRP ⁇ binding domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 15, 17, 19, and 55, respectively.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 14; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 15; (2) the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 16; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 17; (3) the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO
  • the SIRP ⁇ binding domain is scFv, of which the VH and VL are linked via a first polypeptide linker, optionally the first polypeptide linker includes (Gly4Ser) 4.
  • the N-terminus of the VH is linked to C-terminus of the VL.
  • the second domain binds to target antigen expressing on surface of cancer cells.
  • the target antigen may be immune checkpoint molecules, such as CD47, PD1, Claudin 18.2, or CTLA-4.
  • the second domain binds to Claudin 18.2 and/or CD47, preferably Claudin 18.2.
  • the second domain includes: a) a heavy chain variable region (VH) including CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 have amino acid sequences about 80%to about 100%identical to amino acid sequences set forth in SEQ ID NOs: 39, 40, and 41, respectively; and b) a light chain variable region (VL) including CDR1, CDR2, and CDR3, wherein the VL CDR1, CDR2, and CDR3 have amino acid sequences about 80%to about 100%identical to amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively.
  • VH heavy chain variable region
  • CDR1, CDR2, and CDR3 have amino acid sequences about 80%to about 100%identical to amino acid sequences set forth in SEQ ID NOs: 39, 40, and 41, respectively
  • VL light chain variable region
  • the second domain includes a VH having amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to amino acid sequence set forth in SEQ ID NO: 9, and a VL having amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to amino acid sequence set forth in SEQ ID NO: 10.
  • the second domain includes a light chain constant region (CL) , which is a kappa ( ⁇ ) or lamda ( ⁇ ) light chain.
  • CL includes an amino acid sequence set forth in SEQ ID NO: 12 or 13.
  • the second domain includes a heavy chain constant region (CH) having an amino acid sequence set forth in SEQ ID NO: 11.
  • CH heavy chain constant region
  • the bi-specific molecule is a symmetric bispecific molecule, of which the SIRP ⁇ binding domains are fused to C-terminus of the heavy chain constant region of the second domains, at the N-terminus of the heavy chain variable domain of SIRP ⁇ binding domains.
  • the SIRP ⁇ binding domains and the second domain are covalently linked via a second peptide linker having the formula (Gly4Ser) n, where n is an integer from 1 to 5, e.g., 1, 2, 3, 4, 5.
  • the first domain and the second domain are covalently linked via (Gly4Ser) 3.
  • the bi-specific molecule includes a heavy chain and a light chain
  • the heavy chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 56, 58 or 59
  • the light chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 57.
  • the heavy chain of the bi-specific molecule includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 7; and the light chain of the bi-specific molecule includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 8.
  • the heavy chain of the bi-specific molecule includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 5; and the light chain of the bi-specific molecule includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 6.
  • the bi-specific molecules block the interaction of CD47 and SIRP ⁇ , remove SHP-1/2 inhibition, and/or engage Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis.
  • immune effector cells e.g., macrophages
  • the bi-specific molecule enhances immune effector cells phagocytosis of cancer cells expressing target antigen to which the second domain binds, such as human Claudin 18.2 and human SIRP ⁇ .
  • the present disclosure provides an isolated polynucleotide encoding an anti-SIRP ⁇ antibody or antigen binding fragment thereof or a bi-specific molecule described above.
  • the present disclosure provides a construct including a polynucleotide described above.
  • the present disclosure provides an antibody expressing system including the construct described above or having a genome integrated with an exogenous polynucleotide described above, preferably, the expressing system is a cell expression system.
  • the present disclosure provides a method for producing the anti-SIRP ⁇ antibody or antigen binding fragment thereof or the bi-specific molecule described above, which includes: under conditions suitable for expressing the antibody, expressing the antibody or protein using the antibody expressing system described above.
  • the present disclosure provides a pharmaceutical composition including an anti-SIRP ⁇ antibody or antigen binding fragment thereof or the bi-specific molecule described above, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a kit including the anti-SIRP ⁇ antibody or antigen binding fragment thereof or the bi-specific molecule, the isolated polynucleotide, or a construct provided herein.
  • the present disclosure provides the use of an anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule or a pharmaceutical composition provided herein in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease, disorder, or condition.
  • the disease, disorder, or condition is cancer.
  • at least a cancer cell expresses Claudin 18.2, preferably human Claudin 18.2.
  • the cancer is a solid tumor.
  • the cancer includes esophageal cancer, hepatic cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classic Hodgkin lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis, and/or lymph node metastasis.
  • the present disclosure provides a method of treating a subject having cancer, including administrating to the subject a therapeutically effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or a pharmaceutical composition provided herein.
  • the subject is mammal animals comprising human, mouse, and cynomolgus monkey.
  • the present disclosure provides a method of decreasing the rate of tumor growth, including contacting a tumor cell with an effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or a pharmaceutical composition provided herein.
  • the present disclosure provides a method of killing a tumor cell, including contacting a tumor cell with an effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or a pharmaceutical composition provided herein.
  • Figure 1 shows antigen binding profile of wildtype control group and library2 in the 1 st round of yeast library sorting detected by FACS (Fluorescence activated Cell Sorting) .
  • Figure 2 shows antigen binding profile of wildtype control group and library2 in the 1 st round of yeast library sorting detected by FACS (Fluorescence activated Cell Sorting) .
  • Figure 3 shows comparation of binding potency between the wildtype control group and clone 201 detected by FACS (Fluorescence activated Cell Sorting) .
  • Figure 4A is a schematic drawing of an anti-Claudin18.2/SIRP ⁇ bispecific molecule, of which two anti-SIRP ⁇ scFv are fused to the C terminus of the anti-Claudin18.2 antibody heavy chain via a linker.
  • Figure 4B shows SEC (Size-Exclusion Chromatography) spectrum of AE016_201, the purity of each composition in the protein samples is listed in the table below the SEC spectrum.
  • Figure 5 shows binding curve of AE016_201 to SIRP ⁇ detected by the Octet.
  • Figure 6 shows CHO-K1 SIRP ⁇ binding curves of AE016_201 and anti-SIRP ⁇ antibody in the IgG4 isotype detected by FACS. EC50 values are listed in the table below the binding curves.
  • Figure 7 shows long term stability of the AE016_201 at 4°C.
  • Figure 8A shows the binding curves of representative bispecific antibodies to Raji/hClaudin18.2 cells by FACS detection.
  • Anti-Claudin18.2 antibody hu26. H1L2 is used as control.
  • Figure 8B shows the binding curves of representative bispecific antibodies to CHOK1/SIRP ⁇ cells by FACS detection.
  • Anti-SIRP ⁇ antibody hu025.060 is used as control.
  • Figure 9 shows that the anti-Claudin18.2/SIRP ⁇ bispecific antibodies stimulate BMDM phagocytosis against MC38/hCD47/hClausin18.2 cell better than single or combination treatment.
  • Figure 10 shows that in vivo anti-tumor efficacy of anti-Claudin18.2/SIRP ⁇ in MC38/hClaudin18.2/hSIRP ⁇ syngeneic model.
  • antibody (used interchangeably in the plural) is an immunoglobulin molecule capable of specifically binding to a target, such as carbohydrate, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • antibody includes not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F (ab') 2, Fv) , single-chain (scFv) , mutants thereof, fusion proteins comprising antibody moieties, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (eg, bispecific antibodies) , and any other modified constructs of immunoglobulin molecules comprising antigen recognition sites with the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • antigen-binding fragments thereof e.g., Fab, Fab', F (ab') 2, Fv
  • scFv single-chain mutants thereof, fusion proteins comprising antibody moieties, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific
  • Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof) , and the antibody need not be of any particular class.
  • immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) , such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • a typical antibody molecule includes a heavy chain variable region (VH) and a light chain variable region (VL) .
  • the variable region is a region with large changes in amino acid composition and arrangement at the N-terminal of the antibody molecule.
  • the site of specific binding that is, the antigen-binding site, is used to determine the specificity of antibody recognition.
  • the VH and VL regions can be further subdivided into hypervariable regions, also known as “complementarity determining regions” (CDRs) , interspersed with more conserved regions known as “framework regions” (FR) .
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is generally composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: : VL heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker or spacer, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.
  • bispecific molecule refers to an antibody that displays a double binding specificity and affinity for two particular epitopes or a composition of antibodies in which all antibodies display a double binding specificity and affinity for two particular epitopes.
  • an “Fc region” fragment crystallizable region or “Fc domain” or “Fc fragment” refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system.
  • an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL) .
  • Fc may also refer to this region in isolation or in the context of an Fc-comprising protein polypeptide.
  • fusion refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence.
  • a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
  • percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For example, the identity can be determined using the BLAST program of the NCBI database.
  • humanized antibody refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, where the rest of the immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin.
  • the antigen binding site may include a complete variable domain fused to a constant domain, or only include a complementarity determining region (CDR) grafted into an appropriate framework region in the variable domain.
  • CDR complementarity determining region
  • the antigen binding site can be wild-type, or modified by one or more amino acid substitutions. For example, modifications are made to allow the antibody to be more similar to human immunoglobulins.
  • Some forms of humanized antibodies retain all CDR sequences (e.g., a humanized single-domain antibody including all three CDRs from alpaca) . Other forms have one or more CDRs that have been altered relative to the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse or alpaca antibody and the constant regions are derived from a human antibody.
  • a “therapeutically effective amount” of an agent refers to an amount effective for achieving the desired therapeutic or prophylactic result at a dosage level and for a period of time.
  • a therapeutically effective amount of an agent can, for example, eliminate, reduce, delay, minimize or prevent the adverse effects of a disease.
  • pharmaceutically acceptable carrier refers to an ingredient other than the active ingredient in a pharmaceutical composition that is not toxic to the subject.
  • the pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • treating/preventing refers to an attempt to alter the natural progress of a disease in a treated individual and can be prevention or clinical intervention implemented during the course of clinical pathology.
  • the desired effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of diseases, alleviating the symptoms, relieving any direct or indirect pathological consequences of the disease, preventing the metastasis, slowing the rate of disease progression, improving or lessening the disease states, and relieving or improving the prognosis.
  • the antibody of the present disclosure is useful for delaying the development of a disease or delaying the progression of a disorder.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, identity, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, identity, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 20%, 15%, 10%, or 5%.
  • amino acid refers to an organic compound containing amine (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.
  • amine -NH 2
  • -COOH carboxyl
  • novel anti-SIRP ⁇ antibody or antigen binding fragment thereof which includes a heavy chain variable domain (VH) and a heavy chain variable domain (VL) .
  • VH includes complementarity determining regions (CDRs) 1, 2, and 3
  • VL includes CDR1, CDR2, and CDR3.
  • the CDR1 of VH (i.e., HCDR1) is different from an amino acid sequence of SEQ ID NO: 20 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the CDR2 of VH (i.e., HCDR2) is different from an amino acid sequence of SEQ ID NO: 21, 22 or 47 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the CDR3 of VH (i.e., HCDR3) is different from an amino acid sequence of SEQ ID NO: 23 or 48 by no more than 3, 2, 1, 0 amino acids.
  • the CDR1 of VL is different from an amino acid sequence of SEQ ID NO: 24 or 25 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the CDR2 of VL i.e., LCDR2 is different from an amino acid sequence of SEQ ID NO: 26 or 27 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the CDR3 of VL i.e., LCDR3 is different from an amino acid sequence of SEQ ID NO: 28 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the VH of anti-SIRP ⁇ antibody or antigen binding fragment thereof includes CDR1, CDR2, and CDR3 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; (3) SEQ ID NOs: 20, 47, and 48, respectively.
  • CDR1, CDR2, and CDR3 of VH of anti-SIRP ⁇ antibody or antigen binding fragment thereof have an amino acid sequence set forth in SEQ ID NOs: 20, 21, and 23, respectively; SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively.
  • the VL of anti-SIRP ⁇ antibody or antigen binding fragment thereof includes CDR1, CDR2, and CDR3 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively.
  • CDR1, CDR2, and CDR3 of VL of anti-SIRP ⁇ antibody or antigen binding fragment thereof have an amino acid sequence set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively.
  • the HCDR1 has the amino acid sequence set forth in SEQ ID NO: 20
  • the HCDR2 has the amino acid sequence shown as X 1 DPEDX 2 ETK (SEQ ID NO: 60)
  • the HCDR3 has the amino acid sequence shown as DRGLX 3 Y (SEQ ID NO: 61)
  • the LCDR1 has the amino acid sequence shown as X 4 ASX 5 SVSSSYLY (SEQ ID NO: 45)
  • the LCDR2 has the amino acid sequence shown as YSX 6 SNX 7 AS (SEQ ID NO: 46)
  • the LCDR3 has the amino acid sequence set forth in SEQ ID NO: 28, X 1 is V or I, X 2 is A or G, X 3 is V or A, X 4 is R or S, X 5 is Q or S, X 6 is A or T, X 7 is R or L.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 24, 26 and 28, respectively.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the VH of the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes FR1, FR2, FR3, and FR4.
  • the FR1 of VH is different from an amino acid sequence of SEQ ID NO: 29 or 49 by no more than 3, 2, 1, 0 amino acids.
  • the FR2 of VH is different from an amino acid sequence of SEQ ID NO: 30, 31 or 50 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • the FR3 of VH is different from an amino acid sequence set forth in SEQ ID NO: 32 or 51 by no more than 3, 2, 1, 0 amino acids.
  • the FR4 of VH is different from an amino acid sequence of SEQ ID NO: 33 by no more than 3, 2, 1, 0 amino acids.
  • FR1, FR2, FR3, and FR4 of the VH of the anti-SIRP ⁇ antibody or antigen binding fragment thereof have amino acid sequences about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.
  • FR1, FR2, FR3, and FR4 of the VH of the anti-SIRP ⁇ antibody or antigen binding fragment thereof have an amino acid sequence set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.
  • the VL of the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes FR1, FR2, FR3, and FR4.
  • the FR1 of VL is different from an amino acid sequence of SEQ ID NO: 34 by no more than 3, 2, 1, 0 amino acids.
  • the FR2 of VL is different from an amino acid sequence of SEQ ID NO: 35 or 52 by no more than 3, 2, 1, 0 amino acids.
  • the FR3 of VL is different from an amino acid sequence set forth in SEQ ID NO: 36 or 53 by no more than 3, 2, 1, 0 amino acids.
  • the FR4 of VL is different from an amino acid sequence of SEQ ID NO: 37 or 38 by no more than 5, 4, 3, 2, 1, 0 amino acids.
  • FR1, FR2, FR3, and FR4 of the VL of the anti-SIRP ⁇ antibody or antigen binding fragment thereof have an amino acid sequence about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • FR1, FR2, FR3, and FR4 of the VL of the anti-SIRP ⁇ antibody or antigen binding fragment thereof have an amino acid sequence set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the VH comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively.
  • the VH comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 29, 31, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36 and 38, respectively.
  • the VH comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 49, 50, 51 and 33, respectively; and the VL comprises FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes the VH, which has an amino acid sequence 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to one of amino acid sequences set forth in SEQ ID NOs: 14, 16, 18, and 54.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes the VL, which has an amino acid sequence 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to one of amino acid sequences set forth in SEQ ID NOs: 15, 17, 19, and 55.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 14; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical the amino acid sequence set forth in SEQ ID NO: 15.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 16; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical the amino acid sequence set forth in SEQ ID NO: 17.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 18; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical the amino acid sequence set forth in SEQ ID NO: 19.
  • the VH includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 54; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical the amino acid sequence set forth in SEQ ID NO: 55.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof includes a VH having an amino acid sequence set forth in SEQ ID NO: 14, and a VL having an amino acid sequence set forth in SEQ ID NO: 15; or VH having an amino acid sequence set forth in SEQ ID NO: 16, and a VL having an amino acid sequence set forth in SEQ ID NO: 17; or a VH having an amino acid sequence set forth in SEQ ID NO: 18, and a VL having an amino acid sequence set forth in SEQ ID NO: 19; or a VH having an amino acid sequence set forth in SEQ ID NO: 54, and a VL having an amino acid sequence set forth in SEQ ID NO: 55.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof binds to human and/or mouse SIRP ⁇ , preferably binds to human SIRP ⁇ .
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof is a Fab, a Fab', a F (ab') 2, an Fv fragment, or a single-chain fragment variable (scFv) , preferably, scFv, and the VH and VL are linked via a first peptide linker.
  • scFv single-chain fragment variable
  • N-terminus of the VH is fused to C-terminus of the VL.
  • the first peptide linker includes (Gly4Ser) 4. In some embodiments, the first peptide linker is (Gly4Ser) 4.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof is a chimeric antibody or a humanized antibody.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof provided herein specifically binds to human SIRP ⁇ , and thus blocks the interaction between CD47 and SIRP ⁇ , upregulating the immune response and enhancing the unwanted host cell (such as tumor cells) phagocytosis.
  • humanized antibodies designed according to techniques in the art are all embraced in the present disclosure.
  • the humanized antibody further improves the drug safety, and effectively reduces the immunogenicity of the antibody.
  • the humanized antibody with modified framework region obtained in the present disclosure still maintains a high affinity, making it feasible for use in practical clinical application.
  • the present disclosure relates to a bi-specific molecule based on the anti-SIRP ⁇ antibody or antigen binding fragment thereof described above.
  • the bi-specific molecule includes a SIRP ⁇ binding domain and a second domain binding to a target antigen, wherein the second domain is fused to the SIRP ⁇ binding domain.
  • the SIRP ⁇ binding domain contains a heavy chain variable domain (VH) and a heavy chain variable domain (VL) .
  • the VH of SIRP ⁇ binding domain includes HCDR1, HCDR2, and HCDR3 having amino acid sequences at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; or SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively.
  • the HCDR1, HCDR2, and HCDR3 of SIRP ⁇ binding domain have amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; or SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively.
  • the VL of SIRP ⁇ binding domain includes LCDR1, LCDR2, and LCDR3 having amino acid sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively.
  • the LCDR1, LCDR2, and LCDR3 of SIRP ⁇ binding domain have amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively.
  • the HCDR1 has the amino acid sequence set forth in SEQ ID NO: 20
  • the HCDR2 has the amino acid sequence shown as X 1 DPEDX 2 ETK (SEQ ID NO: 60)
  • the HCDR3 has the amino acid sequence shown as DRGLX 3 Y (SEQ ID NO: 61)
  • the LCDR1 has the amino acid sequence shown as X 4 ASX 5 SVSSSYLY (SEQ ID NO: 45)
  • the LCDR2 has the amino acid sequence shown as YSX 6 SNX 7 AS (SEQ ID NO: 46)
  • the LCDR3 has the amino acid sequence set forth in SEQ ID NO: 28, X 1 is V or I, X 2 is A or G, X 3 is V or A, X 4 is R or S, X 5 is Q or S, X 6 is A or T, X 7 is R or L.
  • the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 24, 26 and 28, respectively; (2) the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively; or (3) the HCDR1, HCDR2 and HCDR3 have amino acid sequences set forth in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have amino acid sequences set forth in SEQ ID NOs: 25, 27 and 28, respectively.
  • the VH of SIRP ⁇ binding domain includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: SEQ ID NOs: 29, 30, 32, and 33, respectively; or SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.
  • the VH FR1, FR2, FR3, and FR4 of SIRP ⁇ binding domain have amino acid sequences set forth in: SEQ ID NOs: 29, 30, 32, and 33, respectively; or SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.
  • the VL of SIRP ⁇ binding domain includes FR1, FR2, FR3, and FR4 having amino acid sequences at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequences set forth in: SEQ ID NOs: 34, 35, 36, and 37, respectively; or SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the VL FR1, FR2, FR3, and FR4 of SIRP ⁇ binding domain have amino acid sequences set forth in: SEQ ID NOs: 34, 35, 36, and 37, respectively; or SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the VH in the SIRP ⁇ binding domain, includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; and the VL includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively.
  • the VH in the SIRP ⁇ binding domain, includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 29, 31, 32, and 33, respectively; and the VL includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36 and 38, respectively.
  • the VH in the SIRP ⁇ binding domain, includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 49, 50, 51 and 33, respectively; and the VL includes FR1, FR2, FR3 and FR4 having amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in SEQ ID NOs: 34, 52, 53, and 37, respectively.
  • the VH of SIRP ⁇ binding domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to one of amino acid sequences set forth in SEQ ID NOs: 14, 16, 18, and 54, respectively. In some embodiments, the VH of SIRP ⁇ binding domain have an amino acid sequence set forth in SEQ ID NOs: 14, 16, 18, and 54, respectively.
  • the VL of SIRP ⁇ binding domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 15, 17, 19, and 55, respectively. In some embodiments, the VL of SIRP ⁇ binding domain have an amino acid sequence set forth in SEQ ID NOs: 15, 17, and 19, respectively.
  • the VH in the SIRP ⁇ binding domain, includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 14; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 15.
  • the VH in the SIRP ⁇ binding domain, includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 16; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 17.
  • the VH in the SIRP ⁇ binding domain, includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 18; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 19.
  • the VH in the SIRP ⁇ binding domain, includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 54; and the VL includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical the amino acid sequence set forth in SEQ ID NO: 55.
  • the VH of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 14, and the VL of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 15; or the VH of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 16, and the VL of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 17; or the VH of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 18, and the VL of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 19; or the VH of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 54, and the VL of SIRP ⁇ binding domain includes or has an amino acid sequence set forth in SEQ ID NO: 55.
  • the SIRP ⁇ binding domain is a Fab, a Fab', a F (ab') 2, an Fv fragment, or a single-chain fragment variable (scFv) , preferably, the SIRP ⁇ binding domain is scFv, of which the VH and VL are linked via a first polypeptide linker.
  • the first polypeptide linker includes or is (Gly4Ser) 4.
  • the N-terminus of the VH is linked to C-terminus of the VL.
  • the second domain binds to target antigen expressing on surface of cancer cells.
  • the target antigen may be immune checkpoint molecules, such as CD47, PD1, Claudin 18.2, or CTLA-4.
  • the second domain binds to Claudin 18.2 and/or CD47, preferably Claudin 18.2.
  • the second domain includes:
  • VH heavy chain variable region
  • CDR1, CDR2, and CDR3 wherein the VH CDR1 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 39;
  • VH CDR2 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 40;
  • VH CDR3 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 41;
  • VL light chain variable region
  • CDR1, CDR2, and CDR3 a light chain variable region including CDR1, CDR2, and CDR3, wherein the VL CDR1 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 42; the VL CDR2 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 43; the VL CDR3 have amino acid sequences at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identical to amino acid sequence of SEQ ID NO: 44.
  • the second domain includes VH CDR1, CDR2, and CDR3 having amino acid sequences set forth in SEQ ID NOs: 39, 40, and 41, respectively; and VL CDR1, CDR2, and CDR3 having amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively.
  • the second domain includes a VH having amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to amino acid sequence set forth in SEQ ID NO: 9, and a VL having amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to amino acid sequence set forth in SEQ ID NO: 10.
  • the second domain includes a VH having amino acid sequence set forth in SEQ ID NO: 9, and a VL having amino acid sequence set forth in SEQ ID NO: 10.
  • the second domain includes a light chain constant region (CL) , which is a kappa ( ⁇ ) or lamda ( ⁇ ) light chain.
  • CL light chain constant region
  • the light chain constant region (kappa) has an amino acid sequence of SEQ ID NO: 12
  • the light chain constant region (Lamda) has an amino acid sequence of SEQ ID NO: 13:
  • the second domain includes a heavy chain constant region (CH) , preferably a human IgG1 CH.
  • the human IgG1 CH has an amino acid sequence of SEQ ID NO: 11.
  • the bi-specific molecule is a symmetric bispecific molecule, of which the N-terminus of the heavy chain variable domain of the SIRP ⁇ binding domain is fused to C-terminus of the heavy chain constant region of the second domains.
  • the SIRP ⁇ binding domains and the second domain are covalently linked via a second peptide linker having the formula (Gly4Ser) n, where n is an integer from 1 to 5, e.g., 1, 2, 3, 4, 5.
  • the SIRP ⁇ binding domain and the second domain are covalently linked via (Gly4Ser) 3.
  • the bi-specific molecule includes a heavy chain and a light chain.
  • the heavy chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 56, 58 or 59; and the light chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 57.
  • the heavy chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 7; and the light chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 8.
  • the heavy chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 5; and the light chain includes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence set forth in SEQ ID NO: 6.
  • the heavy chain has an amino acid sequence set forth in SEQ ID NO: 56, 58 or 59; and the light chain has an amino acid sequence set forth in SEQ ID NO: 57.
  • the heavy chain has an amino acid sequence set forth in SEQ ID NO: 7; and the light chain has an amino acid sequence set forth in SEQ ID NO: 8.
  • the heavy chain has an amino acid sequence set forth in SEQ ID NO: 5; and the light chain has an amino acid sequence set forth in SEQ ID NO: 6.
  • the bi-specific molecules block the interaction of CD47 and SIRP ⁇ , remove SHP-1/2 inhibition, and/or engage Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis.
  • immune effector cells e.g., macrophages
  • SHP refers to tyrosine phosphatase inhibitor.
  • effector cells are immune cells that can perform immune effector functions, such as macrophages, natural killing cells, monocytes, cytotoxic T cells.
  • the bi-specific molecule enhances immune effector cells phagocytosis of cancer cells expressing target antigen to which the second domain binds, such as human Claudin 18.2 and human SIRP ⁇ .
  • the bi-specific molecule of the present application has the advantages of high dual target binding affinity and specificity, and thereby further enhancing the anti-tumor immune function.
  • bi-specific molecule shows higher affinity to SIRP ⁇ (e.g., human SIRP ⁇ ) ,
  • bi-specific molecule binds to human SIRP ⁇ with KD no more than 9.9E-7 M, or no more than 9.9E-8 M detected by Octet rule.
  • the KD value, or affinity constant is the equilibrium dissociation constant, or a ratio of Kd/Ka, between the antibody and its target antigen.
  • Kd refers to dissociation constant
  • Ka refers to association constant.
  • the bi-specific molecule binds to SIRP ⁇ (e.g., human SIRP ⁇ , CHOK1 cells overexpressing human SIRP ⁇ ) with EC50 no more than 3 nM, 2 nM, 1.5 nM, 1 nM detected by FACS (Fluorescence activated Cell Sorting) .
  • SIRP ⁇ e.g., human SIRP ⁇ , CHOK1 cells overexpressing human SIRP ⁇
  • FACS Fluorescence activated Cell Sorting
  • Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environment and is an intrinsic property of antibodies that can influence product stability, such as aggregation, during manufacturing and storage.
  • the melting temperature (Tm) value may predict their thermal stability in general. Tm values of the bi-specific molecule provided herein range from 40-70°C, preferably 55-60°C, such as 48°C, 50°C, 55°C, or 57°C, indicating that the antibodies have good thermal stability.
  • the bi-specific molecule provided herein has good freeze-thaw stability. Freeze-thaw stability is often explored to determine the susceptibility of antibodies to temperature cycling which products are frequently exposed. For example, drug substance is often frozen to enable long-term storage. Drug product may be exposed to frozen temperature as part of the lyophilization process.
  • the major degradation pathway of freeze-thaw is aggregates, including precipitates, particles, and soluble particles.
  • the bi-specific molecule provided herein has good stability under a 4°C condition for at least 7, 14, 28, 28 days, and under a 40°C condition for at least 14 days, the purity of the bi-specific molecule changes little.
  • the present disclosure provides an isolated polynucleotide encoding an anti-SIRP ⁇ antibody or antigen binding fragment thereof or a bi-specific molecule described above.
  • the polynucleotide is polymers of DNA, RNA, DNA/RNA hybrids, or modifications thereof.
  • the polynucleotide is polymers of DNA.
  • the polynucleotide is polymers of RNA.
  • DNA or RNA encoding an anti-SIRP ⁇ antibody or antigen binding fragment thereof or a bi-specific molecule described above is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA or RNA may also be obtained by synthetic methods.
  • the isolated polynucleotide encoding an anti-SIRP ⁇ antibody or antigen binding fragment thereof or a bi-specific molecule described above can be inserted into a construct for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • construct components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • a construct includes the isolated polynucleotide provided above.
  • a method of constructing the construct is known to those skilled in the art.
  • the construct can be obtained by in-vitro recombinant DNA technology, DNA synthesis technology, or in-vivo recombinant technology. More specifically, it can be constructed by inserting the isolated polynucleotide into a polyclonal site of an expression vector.
  • the expression vector in the present disclosure generally refers to various commercially available expression vectors well known in the art, for example, bacterial plasm ids, bacteriophages, yeast plasmids, plant cell-infected viruses, mammalian cell-infected viruses such as adenovirus, retrovirus or other vectors.
  • the vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, where the regulatory sequence may include a suitable promoter sequence.
  • the promoter sequence is usually operably linked to a sequence coding the amino acid sequence to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutated, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell.
  • the regulatory sequence may further include a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate the transcription. The terminator sequence is linked to the 3′end or terminus of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used in the present disclosure.
  • a suitable vector may contain an origin of replication capable in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.
  • these promoters may include, but not limited to, the lac or trp promoter of Escherichia coli (E. coli) ; the lambda phage PL promoter; and eukaryotic promoters (including CMV immediate-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, methanol oxidase promoter of Pichia pastoris) , and some other known promoters that are capable of controlling gene expression in prokaryotic cells or eukaryotic cells or viruses.
  • Marker genes can be used to provide phenotypic characters for selection of transformed host cells.
  • marker genes may include, but not limited to, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for E. coli.
  • GFP green fluorescent protein
  • the expression vector may further include an enhancer sequence. If an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancer is a cis- acting factor of DNA, typically containing about 10 to 300 base pairs. Enhancer acts on a promoter to enhance gene transcription.
  • the present disclosure provides an antibody expression system, which includes a construct provided above or incorporates an exogenous polynucleotide provided above in the genome.
  • Any cell suitable for the expression of an expression vector can be used as a host cell.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including, but not limited to, Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; or fungal cells such as yeast, and filamentous fungi; plant cells; insect cells derived from Drosophila S2 or Sf9; animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or a combination thereof.
  • Methods for constructing the expression system should be known to those skilled in the art, for example, including, but not limited to, microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, precipitation with calcium phosphate, or a combination thereof.
  • the present disclosure relates to a pharmaceutical composition including an anti-SIRP ⁇ antibody or antigen binding fragment thereof described above or a bi-specific molecule described above, and a pharmaceutically acceptable carrier.
  • the composition is a pharmaceutical composition, which contains the anti-SIRP ⁇ antibody or antigen binding fragment thereof described above or a bi-specific molecule described above and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH can be changed according to properties of the formulated substances and disease conditions to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to) intratumoral administration, intraperitoneal administration, intravenous administration, or topical administration.
  • the pharmaceutical composition of the present disclosure contains a safe and effective amount (such as 0.001-99 wt %, preferably 0.01-95 wt %, more preferably 0.1-90 wt %) of the single domain antibody or the fusion protein provided and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient includes (but is not limited to) saline, buffer, glucose, water, glycerol, ethanol and combinations thereof.
  • a pharmaceutical preparation should be matched with the administration mode.
  • the pharmaceutical composition of the present application can be prepared into an injection form, for example, the pharmaceutical composition is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition such as an injection and a solution should be manufactured under sterile conditions.
  • the dosage of active ingredients is a therapeutically effective amount, such as about 10 ⁇ g/kg body weight to about 100 mg/kg body weight per day.
  • the anti-SIRP ⁇ antibody or antigen binding fragment thereof described above or a bi-specific molecule described above can also be used with other therapeutic agents.
  • kits containing the anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, the isolated polynucleotide, the construct, and/or the pharmaceutical composition provided herein.
  • kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • Instructions, either as inserts or a labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the present disclosure provides a use of the anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or the pharmaceutical composition described above in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease, disorder, or condition.
  • the disease, disorder, or condition is a tumor, and at least a tumor cell expresses SIRP ⁇ , preferably human SIRP ⁇ . In some embodiments, at least a tumor cell expresses Claudin 18.2, preferably human Claudin 18.2. In certain embodiments, the tumor is a solid tumor. Generally, the tumor includes benign tumors and malignant tumors (also called as cancer) .
  • diseases associated with Claudin 18.2 expressing cells may include all Claudin 18.2 expressing cancers and tumor entities.
  • the diseases include, but not limited to, esophageal cancer, hepatic cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classic Hodgkin lymphoma, hematological malignancies, head and neck cancer nasopharyngeal cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis, and lymph node metastasis, which may be early, intermediate, or advanced, such as, metastatic cancers.
  • provided herein is a method of treating a subject having cancer, including administrating to the subject a therapeutically effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or the pharmaceutical composition provided herein.
  • the “therapeutically effective amount” of the anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule described above, or a pharmaceutical composition provided in the present disclosure preferably causes a reduction in the severity of disease symptoms and increased frequency and duration of asymptomatic period of a disease, disorder or condition, or prevents injury or disability due to illness or suffering.
  • the “therapeutically effective amount” preferably inhibits the cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy against human tumors, or evaluated by detecting the ability to inhibit cell growth. Such inhibition can be determined in vitro by assays well known to those skilled in the art.
  • the therapeutically effective amount of the anti-SIRP ⁇ antibody or antigen binding fragment thereof described above, the bi-specific molecule, and the pharmaceutical compositions is often able to reduce the tumor size, or otherwise relieve the symptoms of a subject.
  • Those skilled in the art can select an appropriate therapeutically effective dose according to the actual situation, for example, the tumor size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.
  • a prescription for treatment (e.g., decision on dosage, etc. ) may be determined by a physician commonly considering factors including, but not limited to, the disease being treated, status of the patient, delivery site, route of administration and other factors.
  • a prophylactically effective amount refers to an amount effective for achieving the desired prophylactic effect at a dose and for a period of time required. Usually, but not necessarily, since a prophylactic dose is administered to a subject before the onset of a disease or at an early stage of the disease, the “prophylactically effective amount” is usually lower than the “therapeutically effective amount” .
  • the subject is mammal animals, such as human, mouse, and cynomolgus monkey.
  • the present disclosure provides a method of decreasing the rate of tumor growth, including contacting a tumor cell with an effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or the pharmaceutical composition described above.
  • the present disclosure provides a method of killing a tumor cell, including contacting a tumor cell with an effective amount of anti-SIRP ⁇ antibody or antigen binding fragment thereof, the bi-specific molecule, or the pharmaceutical composition described above.
  • Human SIRP ⁇ stably expressing cell line CHOK1/SIRP ⁇ has been generated for chimeric antibody in vitro assays.
  • CHOK1 cell was transfected with human-SIRP ⁇ (Uniprot Access# P78324, AA Met1-Lys504) expression lentivirus and selectively cultured in medium containing 10 g/mL puromycin for 2 weeks. Single cell clones were then isolated by limited dilution and screened by FACS to obtain the monoclonal cell lines stably expressing SIRP ⁇ .
  • the mutations were randomly introduced into template of anti-SIRP ⁇ scFv 2534m1V3. Firstly, random mutation PCR were carried out with the GeneMorph II Random Mutagenesis Kit. Then the PCR products were separated on a 1%agarose gel. A DNA marker DL2000 was used to indicate the length of the DNA band. The DNA band around 750bp were excised and purified by the NucleoSpin Gel and PCR Clean-up Kit according to its protocol. In the next step, the mutated DNA was amplified by the same primers with the PrimeSTAR Max DNA Polymerase kit. The PCR products were separated on a 1%agarose gel and purified by the NucleoSpin Gel and PCR Clean-up kit according to its protocol.
  • Yeast library was induced in SGRCAA medium at 30°C for overnight and then was used for the 1 st round MACS (Magnetic-Activated Cell Sorting) .
  • 2.8E9 induced yeasts from the initial library were washed twice with 0.1%PBSA (Phosphate NaCl Buffer) and incubated with biotinylated SIRP ⁇ (bio-SIRP ⁇ ) in 50mL 0.1%PBSA at R. T. (room temperature) for 2h. The mixture was then washed with 0.1%PBSA and was incubated with streptavidin labeled magnetic beads in 50mL 0.1%PBSA at R. T. for 1h.
  • the yeasts that displayed SIRP ⁇ -binder were captured by the magnetic beads and the yeast-magnetic beads complexes were separated by a magnet set from the library.
  • the yeasts captured by beads were expanded in SDCAA medium and induced in the SG medium, forming a new library, labeled as library1.
  • yeasts from the library 2 were washed and resuspended in 3M guanidine and incubate at 4°C for overnight on a rotator with a proper-speed rotation.
  • yeasts were washed and incubated with 5nM bio-SIRP ⁇ in 0.5M guanidine at R. T. for 2h.
  • the mixture was then washed and resuspended in 0.1%PBSA containing PE-streptavidin (PE-conjugated streptavidin) and FITC-anti-HA antibody (fluorescein isothiocyanate conjugated hemagglutinin Tag antibody) and incubated at R. T. for 0.5h.
  • the wildtype of 2534m1V3 was displayed on a yeast as a control (wildtype control in the following text) and do the same treatment.
  • the two groups of yeast were washed and injected into the S3e cell sorter.
  • the desired yeast population, gate R2 in the Library2 was collected into a FACS tube.
  • the yeasts were then expanded in the SDCAA medium and induced in the SG medium, forming a new library, labeled as library 3.
  • 3E7 yeasts from the library 3 were harvested and washed twice with 0.1%PBSA.
  • the yeasts were resuspended in 2ml PBS (phosphate buffer saline) containing 1M guanidine and 1nM bio-SIRP ⁇ and incubated at R. T. for 2h on a rotator with a proper-speed rotation.
  • the mixture was then washed and resuspended in 0.1%PBSA containing PE-streptavidin and FITC-anti-HA antibody and incubated at R. T. for 0.5h.
  • the wildtype control was treated in the same procedure.
  • the two groups of yeast were injected into the S3e cell sorter.
  • the desired yeast population gate 2 in the library 3 was collected into a FACS tube. The yeasts were then expanded in the SDCAA medium and plated on to SD (Sabouraud Dextrose) agar plates.
  • Colonies on the plates were picked and induced in SG medium in a 96-deep-well plate at 30°Cfor overnight. Wildtype clone was also induced as a control. Induced yeasts were aliquoted into a new 96-well-low-absorption-plate and washed twice with 0.1%PBSA. The yeasts were resuspended with PBS containing 1M guanidine and 1nM bio-SIRP ⁇ and incubated at R. T. for 2h. The mixture was then washed with 0.1%PBSA and resuspended in 0.1%PBSA containing PE-streptavidin and FITC-anti-HA antibody and incubated at R. T. for 0.5h. The yeast was washed and injected into the BD Celesta for signal collection. 380 colonies were picked and screened.
  • the bispecific antibody is a symmetrical structure with an anti-SIRPa scFv (Seq. 2534m1v3) fused to the C terminal of the Fc of the Claudin 18.2 antibody in Example 1.1.
  • the wildtype bispecific antibody has been generated with the IgG1 isotype and used as a control.
  • the sequence of clone 201 were synthesized and cloned into the C terminal of the Fc fragment of a claudin 18.2 antibody with a (G4S) 3 linker, and co-electroporated with the light chain into CHO cell line and expressed in a 30mL medium, yield BsAb antibodies AE016_201.
  • the wt (short for wildtype) BsAb, 005-08 was also expressed in 30ml medium.
  • the sequences of AE016_201 are list in Table 2.
  • the expressed protein was harvested on day 5 and purified by one-step protein-A affinity chromatography. The protein was measured, and the productivity was shown in Table 3.
  • AE016_201 had a purity higher than 90%in the SEC characterization as Table 3 and Figure 4B shows.
  • the binding affinity of AE016_201 and wt BsAb to human SIRP ⁇ ECD recombinant protein was determined using Bio-Layer Interferometry (Octet) .
  • the association and dissociation curves were fit with 1: 1 binding model, and the Kon/Koff/KD value for AE016_201 was calculated and summarized in Table 4.
  • the binding curve was showed in Figure 5.
  • AE016_201 showed comparable affinity with wt BsAb.
  • the SIRP ⁇ overexpressed cells CHOK1/SIRP ⁇ was harvested by TrypLE digesting and centrifugating. Cells were resuspended in FACS buffer containing DPBS (Dulbecco's Phosphate-Buffered Saline) and 2%FBS (Fetal Bovine Serum) for 30 min. 1E5 cells were aliquoted into each well in the 96-well plate with diluted antibodies. The concentration of the antibodies used were 100 nM for first well and then 5 times diluted for the remaining wells. After incubating at 4 °C for 60 min, cells were washed twice with FACS buffer and resuspended with the secondary antibody AF647 anti human Fc antibody.
  • the cells were then incubated at 4 °C for 30 min in dark. After that the cells were washed twice with FACS buffer and resuspended in FACS buffer and analyzed on the flow cytometry. An anti-SIRP ⁇ antibody 2534m1V3 in the IgG4 isotype was used as control. The binding curve and EC 50 were calculated by the software GraphPad Prism 9 as shown in Figure 6.
  • Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environment and is an intrinsic property of antibodies that can influence product stability, such as aggregation, during manufacturing and storage.
  • the melting temperature (Tm) values of AE016_201 in 10 mM Histidine and 10 mM Glycine buffer (HG buffer) , PBS, or 20 mM Histidine buffer was measured to predict their thermal stability. Briefly, hit antibodies AE016_201 were resolved in HGS or PBS or 20 mM Histidine buffer. The Tm values were then detected by DSF (Differential Scanning Fluorimetry) using QuantStudio 7 Flex Real time PCR system. The Tm1 value of the AE016_201 in different buffer was listed in Table 5, indicating their good thermal stability in different buffers.
  • ⁇ monomer was the purity variation of samples after and before 3xFT treatment.
  • ⁇ monomer was the purity variation of samples after and before low pH treatment.
  • Thermal stress at temperatures exceeding normal storage conditions can accelerate degradation, thereby increasing the detectability of potential degradation pathways to provide information about long-term degradation at intended storage conditions.
  • the thermal stress tests of AE016_201 in HGS was performed at 40 °C for 14 days. SEC-purity were detected to monitor insoluble or soluble aggregates. As shown in Table 8, the purity of AE016_201 in HG buffer changed little after 14 days of incubation at 40 °C.
  • AE016_201.003 was R486S, Q489S, A514T, R517L, V641I, and A646G in the CDR and/or FR region of anti-SPRP ⁇ antibody.
  • the sequence of AE016_201.003 was shown in Table 9.
  • the mutant AE016_201.003 was expressed in CHO cell line in a 30 ml medium. The production was 91 mg/L with a purity of 97.3%after one-step purification. The affinity, Tm value, and freezing and thaw stability (FT) was assessed as described above. The results were summarized in the Table 10.
  • Anti-Claudin18.2 antibodies were generated from hybridoma and humanization by in-house development.
  • the DNA encoding the light chain and the heavy chain in either the same expression vector or separate expression vectors were used to transfect CHO cell for transfection.
  • the culture media were harvested and the fusion protein was purified by Protein A Sepharose column.
  • Table 11 shows the VH and VL of hu26. H1L2.
  • Anti-SIRP ⁇ antibodies were generated from hybridoma and humanization by in-house development.
  • the DNA encoding the light chain and the heavy chain in either the same expression vector or separate expression vectors were used to transfect CHO cell for transfection.
  • the culture media were harvested and the fusion protein was purified by Protein A Sepharose column.
  • Table 12 shows the VH and VL of hu025.060 (ES0040025) , hu025.201, hu025.003, and hu025.003. SS.
  • Anti-Claudin18.2/SIRP ⁇ bispecific antibodies were constructed as an anti-Claudin18.2 antibody (hu26. H1L2) with anti-SIRP ⁇ scFv (hu025.201, hu025.003, hu025.003. SS) at the C-terminus of heavy chain.
  • a flexible (Gly4Ser) 3 linker was genetically linked to the N-terminus of the anti-SIRP ⁇ scFv.
  • Figure 4A The schematic drawing of the anti-Claudin18.2/SIRP ⁇ bispecific antibodies was shown in Figure 4A. The amino acid sequence of the resultant bispecific antibodies were shown below:
  • the DNA encoding the light chain and the heavy chain in either the same expression vector or separate expression vectors were used to transfect CHO-K1 cells for transfection.
  • the culture media were harvested, and the fusion protein was purified by Protein A Sepharose column.
  • the Anti-Claudin18.2/SIRP ⁇ bispecific protein were characterized for binding affinity against human Claudin18.2 or SIRP ⁇ using Octet assay (ForeBio) according to manufacturer’s manual, separately. Briefly, the antibodies were coupled on sensors and then the sensors were dipped into Claudin18.2 or SIRP ⁇ protein gradients (start at 200 nM, with 2-fold dilution and totally 8 doses) . Their binding responses were measured in real-time, and results were fit globally. The affinity data of the tested antibody are summarized in Table 13 and Table 14.
  • Raji lymphoma cells overexpressing human Claudin18.2 (Raji/hClaudin18.2) , generated by lentivirus stable transfection, were washed with wash buffer and incubated with 100 ⁇ l serial dilution of Claudin18.2/SIRP ⁇ bispecific protein for 30 minutes on ice. Cells were then washed twice with wash buffer and incubated with 100 ⁇ l of AF647 anti-human Fc antibody for 30 minutes on ice. Cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences) .
  • BD Biosciences FACS Canto II analyzer
  • the anti-Claudin18.2/SIRP ⁇ bispecific antibodies bound to Raji/hClaudin18.2 cell in a dose-dependent manner.
  • SS bound Raji/hClaudin18.2 similar to anti-Claudin18.2 monoclonal antibody hu26. H1L2.
  • CHOK1 cells overexpressing human SIRP ⁇ (CHOK1/SIRP ⁇ ) were washed with wash buffer and incubated with 100 ⁇ l serial dilution of Claudin18.2/SIRP ⁇ bispecific protein for 30 minutes on ice. Cells were then washed twice with wash buffer and incubated with 100 ⁇ l of AF647 anti-human Fc antibody for 30 minutes on ice. Cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences) . As shown in Figure 8B, the anti-Claudin18.2/SIRP ⁇ bispecific antibodies ES028.26.201, ES028.26.003, ES028.26.003. SS bound to CHOK1/SIRP ⁇ cell in a dose-dependent manner, similar to anti-SIRP ⁇ monoclonal antibody hu025.060.
  • Mouse MC38 colon tumor cells expressing human CD47 and human Claudin18.2 were labeled with the fluorescent dye CFSE and incubated with mouse bone marrow derived macrophages (BMDMs) prepared from C57BL6/hCD47/hSIRP ⁇ knock-in mice either in the presence of anti-Claudin18.2 antibody hu26. H1L2, anti-SIRP ⁇ antibody hu025.060, the combination of anti-Claudin18.2 and anti-SIRP ⁇ antibody, anti-Claudin18.2/SIRP ⁇ bispecific antibody. After 2 hours, the macrophages were harvested, stained with a fluorescently labeled anti-mouse macrophage antibody, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cell. Phagocytic index was shown for three separate samples.
  • anti-Claudin18.2 antibody hu28H1L2 induced ⁇ 25%phagocytosis by antibody dependent cellular phagocytosis (ADCP) and anti-SIRP ⁇ antibody barely induce phagocytosis.
  • the combination of anti-Claudin18.2 and anti-SIRP ⁇ antibody significantly improve the phagocytosis.
  • SS induced stronger phagocytosis in a dose dependent manner compared to combination and mono-treatment.
  • Anti-Claudin18.2/SIRP ⁇ bispecific antibody enhances in vivo anti-tumor efficacy
  • mice Human SIRP ⁇ /CD47 double knock-in mice (Shanghai Model Organisms Center, Inc) were inoculated with hCD47/hCLDN18.2 overexpression MC38 cells (stably transfected with lentivirus in Shanghai Model Organisms Center) . Mice were grouped according to tumor volume into 5 groups when the mean tumor volume reached ⁇ 60-100 mm 3 . Mice were i.p. dosed with the same molar concentration of hu26. H1L2, ES028.26.201, ES028.26.003, ES028.26.003. SS, or vehicle. Dosing schedule was BIW for 5 doses. Tumor volumes were measured twice per week. 3 days post the 5th dosing, mice were sacrificed, and tumors were weighted. Statistics were carried out by 2-way anova comparing the mean tumor volume of different treatment groups to that of control group.
  • TGI Relative tumor inhibition rate
  • TGI% (1-T/C) ⁇ 100%.
  • T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively) .
  • T/C % TRTV /CRTV ⁇ 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the vehicle control group;
  • RTV Vt/V0, V0 is the tumor volume of the animal at the time of grouping, Vt is the tumor volume of animal after treatment) ;
  • T/C can also be calculated based tumor weight as below:
  • T/C % TTW /CTW ⁇ 100% (TTW: mean tumor weight of the treatment group at the end; CTW: mean tumor weight of the vehicle control group at the end) .
  • anti-Claudin18.2 antibody hu26. H1L2 monotherapy did not inhibit tumor growth similar to vehicle treatment.
  • SS induces strong tumor growth inhibition compared to monotherapy or vehicle treatment.

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Abstract

L'invention concerne des anticorps anti-SIRPα ou des fragments de liaison à l'antigène de ceux-ci, des molécules bispécifiques ciblant SIRPα et claudine 18.2, des compositions pharmaceutiques, et les utilisations de telles molécules dans la prévention, le diagnostic ou le traitement de maladies cancéreuses.
PCT/CN2024/073050 2023-01-18 2024-01-18 Molécules bi-spécifiques ciblant sirpa et claudine 18.2 Ceased WO2024153193A1 (fr)

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CN202480008424.4A CN120569409A (zh) 2023-01-18 2024-01-18 靶向sirpa和claudin 18.2的双特异性分子

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106456749A (zh) * 2014-03-11 2017-02-22 小利兰·斯坦福大学托管委员会 抗SIRPα抗体和双特异性巨噬细胞增强型抗体
CN111518214A (zh) * 2019-02-03 2020-08-11 上海健信生物医药科技有限公司 靶向cldn18.2的双特异性抗体及其制备方法和应用
CN111635458A (zh) * 2020-03-20 2020-09-08 上海健信生物医药科技有限公司 靶向Sirpα的抗体或其抗原结合片段及其制备和应用
WO2022063272A1 (fr) * 2020-09-28 2022-03-31 Elpiscience (Suzhou) Biopharma, Ltd. Nouveaux anticorps anti-claudin18
WO2023010100A1 (fr) * 2021-07-28 2023-02-02 Elpiscience (Suzhou) Biopharma, Ltd. NOUVEAUX ANTICORPS ANTI-SIRPα
WO2023010098A2 (fr) * 2021-07-28 2023-02-02 Elpiscience (Suzhou) Biopharma, Ltd. Nouvelles molécules multi-spécifiques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106456749A (zh) * 2014-03-11 2017-02-22 小利兰·斯坦福大学托管委员会 抗SIRPα抗体和双特异性巨噬细胞增强型抗体
CN111518214A (zh) * 2019-02-03 2020-08-11 上海健信生物医药科技有限公司 靶向cldn18.2的双特异性抗体及其制备方法和应用
CN111635458A (zh) * 2020-03-20 2020-09-08 上海健信生物医药科技有限公司 靶向Sirpα的抗体或其抗原结合片段及其制备和应用
WO2022063272A1 (fr) * 2020-09-28 2022-03-31 Elpiscience (Suzhou) Biopharma, Ltd. Nouveaux anticorps anti-claudin18
WO2023010100A1 (fr) * 2021-07-28 2023-02-02 Elpiscience (Suzhou) Biopharma, Ltd. NOUVEAUX ANTICORPS ANTI-SIRPα
WO2023010098A2 (fr) * 2021-07-28 2023-02-02 Elpiscience (Suzhou) Biopharma, Ltd. Nouvelles molécules multi-spécifiques

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