WO2023040667A1 - 靶向cd47和pd-l1的重组融合蛋白及其制备和用途 - Google Patents

靶向cd47和pd-l1的重组融合蛋白及其制备和用途 Download PDF

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WO2023040667A1
WO2023040667A1 PCT/CN2022/116312 CN2022116312W WO2023040667A1 WO 2023040667 A1 WO2023040667 A1 WO 2023040667A1 CN 2022116312 W CN2022116312 W CN 2022116312W WO 2023040667 A1 WO2023040667 A1 WO 2023040667A1
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antibody
seq
fusion protein
recombinant fusion
heavy chain
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French (fr)
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田文志
李松
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宜明昂科生物医药技术（上海）股份有限公司
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Priority to KR1020247008548A priority Critical patent/KR20240055756A/ko
Publication of WO2023040667A1 publication Critical patent/WO2023040667A1/zh

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Definitions

  • This application relates to a recombinant fusion protein targeting CD47, PD-L1 and/or FcR, its preparation and use, especially its use in tumor treatment.
  • Cancer cells have developed some mechanisms to evade the host's immune surveillance, including: 1) evading the immune surveillance of T lymphocytes by highly expressing membrane proteins PD-L1 and PD-L2, both of which are associated with PD-1 binding on the surface of T cells triggers apoptosis of T cells; 2) escape the immune surveillance of natural killer (NK) cells by shedding MICA/MICB on the cancer cell membrane, and the shed MICA/MICB binds to NKG2D on the surface of NK cells , blocking the killing of MICA/MICB + cancer cells by NK cells; 3) Evading the immune surveillance of macrophages (M ⁇ ) through high expression of CD47, which binds to signal regulatory protein ⁇ (SIRP ⁇ ) on the surface of macrophages , thereby triggering the generation of an inhibitory signal that inhibits the phagocytosis of cancer cells by macrophages.
  • SIRP ⁇ signal regulatory protein ⁇
  • SIRP Signal regulatory protein
  • CD172a is a transmembrane glycoprotein that includes three family members, SIRP ⁇ (CD172a), SIRP ⁇ (CD172b) and SIRP ⁇ (CD172g). All three proteins contain similar extracellular domains but have distinct intracellular domains. The extracellular region contains three immunoglobulin-like domains, one Ig-V and two Ig-C domains.
  • the intracellular domain of SIRP ⁇ (CD172a) contains two inhibitory signal transduction domains, which can inhibit signal transduction and corresponding cellular functions.
  • the intracellular regions of SIRP ⁇ (CD172b) and SIRP ⁇ (CD172g) are very short and do not contain signaling domains. However, SIRP ⁇ (CD172b) is able to function in signal transduction via adapter proteins such as DAP12. SIRP is mainly expressed in macrophages (M ⁇ ), dendritic cells (DC) and neurons.
  • CD47 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily expressed on the surface of all cell types including erythrocytes.
  • Ligands for CD47 include integrins, thrombospondin-1 and SIRP.
  • Cancer cells overexpressing CD47 include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM) , bladder, ovarian, prostate, lung, colon, breast, and pancreatic cancer cells. It has been reported that injecting tumor-bearing mice with a CD47-specific antibody that blocks the binding of CD47 to SIRP ⁇ can significantly inhibit tumor growth. When the same antibody was injected into mice bearing human leukemia cells, tumor cells or cancer cells were completely eliminated (Theocharides APA, et al., 2012).
  • PD-L1 also known as programmed death-ligand 1 or CD274, is a transmembrane protein that plays an important role in suppressing the immune system in certain situations such as allogeneic tissue transplantation, autoimmune diseases and tumorigenesis .
  • loss of feedback restriction between transcription factors such as STAT3 and NF- ⁇ B will cause an increase in local PD-L1 expression, which will limit the effectiveness of systemic therapy with drugs targeting PD-L1 (Vlahopoulos SA, 2017 ).
  • An analysis of 196 tumor samples from patients with renal cell carcinoma found that high expression of tumor PD-L1 was associated with enhanced tumor aggressiveness and a 4.5-fold increased risk of death (Thompson RH et al., 2004).
  • PD-1 is a cell surface receptor of approximately 268 amino acids. When combined with PD-L1 or PD-L2, PD-1 can down-regulate the immune system and improve self-tolerance by inhibiting T-cell inflammatory responses. The suppressive effect of PD-1 on the immune system prevents autoimmune disease, but it also prevents the immune system from killing cancer cells.
  • the crystallizable region is the tail region of an antibody, the domain that determines the antibody's effector function (ie, how the antibody associates with specific cellular receptors or other defense proteins).
  • Fc receptors are found on certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells Proteins on the surface of other. These cells contribute to the protective function of the immune system.
  • the Fc region can interact with Fc receptors and some proteins of the complement system to activate the immune system.
  • Antibodies targeting a single antigen have limited efficacy.
  • the approved PD-L1 antibody, Avelumab The overall response rate was only 33%.
  • US 10,800,821 B2 discloses a recombinant bispecific fusion protein of about 90k Daltons targeting CD47 and FcR, and enhanced anti-tumor effect was observed in Balb/c nude mice carrying HL cells.
  • US 10,973,878 B2 discloses a fusion protein IMM2505 targeting CD47, PD-L1 and FcR simultaneously, which has low molecular weight and long half-life.
  • This application provides a new recombinant fusion protein, which is similar in structure to IMM2505 in 10,973,878 B2, but has a new PD-L1 antibody sequence, showing better anti-tumor effect than IMM2505.
  • the present application discloses a recombinant fusion protein comprising an antibody specifically binding to PD-L1 or an antibody fragment thereof, and a peptide specifically binding to CD47, wherein the CD47 binding peptide is linked to the PD-L1 antibody or an antibody fragment thereof,
  • the PD-L1 antibody or antibody fragment thereof comprises a heavy chain variable region, a heavy chain constant region, and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 19
  • the light chain variable region comprises The amino acid sequence of SEQ ID NO: 20
  • the heavy chain constant region has FcR binding ability and is connected with the C-terminus of the heavy chain variable region
  • the CD47 binding peptide comprises a mutated signal regulatory protein (SIRP) extracellular Ig-like domain
  • the mutated signal regulatory protein (SIRP) extracellular Ig-like domain comprises the amino acid sequence of SEQ ID NO: 2, wherein the recombinant fusion protein can simultaneously bind CD47 and PD
  • the CD47-binding peptide can bind to the N-terminus of the heavy chain variable region or the light chain variable region of the PD-L1 antibody or antibody fragment thereof.
  • the amino acid sequence of SEQ ID NO:2 can be encoded by the nucleic acid sequence of SEQ ID NO:1.
  • At least one paratope of the PD-L1 antibody or antibody fragment thereof is linked to the CD47-binding peptide at the N-terminus of the heavy chain variable region or the light chain variable region constituting the paratope.
  • each paratope of the PD-L1 antibody or antibody fragment thereof is linked to the CD47-binding peptide at the N-terminus of the heavy chain and variable region or light chain variable region constituting the paratope.
  • each paratope of the PD-L1 antibody or antibody fragment thereof is linked to the CD47-binding peptide at the N-terminus of the heavy chain variable region constituting the paratope.
  • each paratope of the PD-L1 antibody or antibody fragment thereof is linked to the CD47-binding peptide at the N-terminus of the light chain variable region constituting the paratope.
  • the FcR-binding heavy chain constant region may be a naturally occurring or engineered human IgGl, IgG2, IgG3 or IgG4 heavy chain constant region, or a functional fragment thereof.
  • the FcR-binding heavy chain constant region is a human IgG1 heavy chain constant region, or a functional fragment thereof.
  • the FcR-binding heavy chain constant region has the amino acid sequence shown in SEQ ID NO:21.
  • the PD-L1 antibody or antibody fragment thereof may comprise a light chain constant region, such as a human kappa light chain constant region, or a functional fragment thereof, linked to the C-terminus of the light chain variable region.
  • the PD-L1 antibody or antibody fragment thereof may comprise a heavy chain and a light chain, the heavy chain comprising at least 80%, 85%, 90%, 91%, 92%, 93% of SEQ ID NO:6 , 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence, the light chain comprising at least 80%, 85%, 90%, 91%, 92% with SEQ ID NO:20 , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences.
  • the PD-L1 antibody or antibody fragment thereof can comprise a heavy chain comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 and a light chain
  • the light chain comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8.
  • the PD-L1 antibody or antibody fragment thereof may comprise a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:6, and the light chain comprising the amino acid sequence shown in SEQ ID NO:20.
  • the PD-L1 antibody or antibody fragment thereof may comprise a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:6, and the light chain comprising the amino acid sequence shown in SEQ ID NO:8.
  • the PD-L1 antibody or antibody fragment thereof of the present application can be linked to the CD47-binding peptide via a linker.
  • the linker can be a peptide of 5-30, 10-30, 10-20, or 15 amino acids in length.
  • the linker can be, for example, -(Gly-Gly-Gly-Gly-Ser) 2 -(SEQ ID NO: 17), -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO: 4), or - (Gly-Gly-Gly-Gly-Ser) 4 - (SEQ ID NO: 18).
  • the linker is -(Gly-Gly-Gly-Gly-Ser) 3 - (SEQ ID NO: 4).
  • the amino acid sequence of SEQ ID NO:4 can be encoded by the nucleic acid sequence of SEQ ID NO:3.
  • the recombinant fusion protein of the present application comprises a CD47 binding peptide-joint-PD-L1 antibody heavy chain and a PD-L1 antibody light chain, wherein the CD47 binding peptide-joint-PD-L1 antibody heavy chain contains the same sequence as SEQ ID NO: 10 amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, PD- The L1 antibody light chain contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8 degree of amino acid sequence.
  • the recombinant fusion protein of the present application comprises a CD47 binding peptide-joint-PD-L1 antibody heavy chain and a PD-L1 antibody light chain, wherein the CD47 binding peptide-joint-PD-L1 antibody heavy chain contains the same sequence as SEQ ID NO: 10 amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, PD- The L1 antibody light chain contains an amino acid sequence with at least 95% sequence identity to SEQ ID NO:20.
  • the amino acid sequences of SEQ ID NOs: 8 and 10 can be encoded by the nucleic acid sequences of SEQ ID NOs: 7 and 9, respectively.
  • the recombinant fusion protein of the present application comprises a PD-L1 antibody heavy chain, and a CD47 binding peptide-linker-PD-L1 antibody light chain, wherein the PD-L1 antibody heavy chain contains at least Amino acid sequence with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, CD47 binding peptide-linker-PD-
  • the L1 antibody light chain contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16 degree of amino acid sequence.
  • the amino acid sequences of SEQ ID NOs: 6 and 16 can be encoded by the nucleic acid sequences of SEQ ID NOs: 5 and 15, respectively.
  • the present application also provides a nucleic acid molecule encoding the recombinant fusion protein of the present application, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector. Also provided is a method for preparing a recombinant fusion protein using the host cell of the present application, comprising (i) expressing the recombinant fusion protein in the host cell, and (ii) isolating the recombinant fusion protein from the host cell or its cell culture.
  • the present application also provides a pharmaceutical composition, which may comprise the recombinant fusion protein, nucleic acid molecule, expression vector or host cell of the present application, and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises at least one pharmaceutically acceptable adjuvant.
  • the recombinant fusion protein or pharmaceutical composition of the present application can be used to treat diseases related to overexpression of CD47 and/or PD-L1, or to prepare medicines for treating diseases related to overexpression of CD47 and/or PD-L1.
  • the application provides a method for treating or alleviating diseases associated with CD47 and/or PD-L1 overexpression in a subject in need, comprising administering a therapeutically effective amount of the drug of the application to the subject combination.
  • AML Acute Myeloid Leukemia
  • CML Chronic Myeloid Leukemia
  • ALL Acute Lymphoblastic Leukemia
  • NHL Non-Hodgkin Lymphoma
  • MM multiple myeloma
  • bladder cancer ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, or renal cell carcinoma.
  • FIGS. 1A and 1B are structural schematic diagrams of the recombinant fusion proteins IMM2520 and IMM2521 of the present application.
  • the circular structure at the top of the structure represents the mutated SIRP ⁇ first extracellular domain (SIRP ⁇ D1), which is linked to the N-terminus of the heavy chain (A) or light chain (B) of the PD-L1 antibody via a linker.
  • the mutated SIRP ⁇ D1 has the nucleic acid sequence and amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2 respectively.
  • the linker has an amino acid sequence shown in SEQ ID NO:4, which can be encoded by a nucleic acid sequence shown in SEQ ID NO:3.
  • the heavy chain of the PD-L1 antibody comprises the nucleic acid sequence and amino acid sequence shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.
  • the light chain of the PD-L1 antibody comprises the nucleic acid sequence and the amino acid sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • Figure 2 is a schematic diagram of the mechanism of action of the recombinant fusion protein of the present application.
  • Figure 3 shows the binding activity of IMM2520 and IMM2521 to PD-L1 on CHO cells expressing human PD-L1 (CHO-PDL1).
  • IMM2505 is a fusion protein mentioned in US 10,973,878 B2, which has a similar overall design to IMM2520 and IMM2521, including mutated SIRP ⁇ D1 (SEQ ID NO: 2) and different PD-L1 antibodies.
  • IMM2515 is a PD-L1 antibody constituting IMM2520 and IMM2521, comprising a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID NO:8.
  • Atezolizumab is a commercially available PD-L1 antibody, and hIgG1-Fc was used as a negative control.
  • Fig. 4 shows the binding activity of IMM2520 and IMM2521 to CD47 on Jurkat cells.
  • IMM2505 is a fusion protein mentioned in US10,973,878 B2, which has a similar overall design to IMM2520 and IMM2521, and contains mutated SIRP ⁇ D1 (SEQ ID NO: 2) and different PD-L1 antibodies.
  • IMM01 is described in US2021/0024598 A1, comprising two mutations SIRP ⁇ D1 (SEQ ID NO: 2), connected to the Fc dimer fragment, wherein the monomers contain respectively as shown in SEQ ID NO: 11 and SEQ ID NO: 12 Nucleic acid sequence and amino acid sequence.
  • hIgG1-Fc was used as a negative control.
  • Figures 5A and 5B show the ability of IMM2520 and IMM2521 to block the binding of PD-1-Fc to PD-L1 on CD47 ⁇ CHO-PD-L1 cells (A) or CD47 + Raji-PD-L1 cells (B).
  • IMM2515 is a PD-L1 antibody constituting IMM2520 and IMM2521, comprising a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID NO:8. hIgG1-Fc was used as a negative control.
  • Figures 6A and 6B show the ability of IMM2520 and IMM2521 to block the binding of SIRP ⁇ -Fc to CD47 on PD-L1 - Raji cells (A) or PD-L1 + Raji cells (B).
  • IMM01 is described in US 2021/0024598 A1, which contains two mutant SIRP ⁇ D1 (SEQ ID NO: 2), which is connected to the Fc dimer fragment, and the monomers thereof are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Nucleic acid and amino acid sequences. hIgG1-Fc was used as a negative control.
  • Figure 7 shows the ability of IMM2520 to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) on Raji-PD-L1 cells.
  • IMM2515 is a PD-L1 antibody constituting IMM2520 and IMM2521, comprising a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID NO:8.
  • FIG. 8 shows the ability of IMM2520 to trigger antibody-dependent cellular phagocytosis (ADCP) on Raji-PD-L1 cells.
  • IMM2515 is a PD-L1 antibody constituting IMM2520 and IMM2521, comprising a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID NO:8.
  • IMM01 is described in US 2021/0024598 A1, which contains two mutant SIRP ⁇ D1 (SEQ ID NO: 2), connected to the Fc dimer fragment, and the monomers in it are shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively Nucleic acid and amino acid sequences.
  • hIgG1-Fc was used as a negative control.
  • Figure 9 shows the in vivo anti-tumor effect of IMM2520 in syngeneic BALB/c-hPD1/hSIRP ⁇ mice bearing CT26-hPDL1 hCD47 tumors.
  • IMM01 is described in US 2021/0024598 A1, which contains two mutant SIRP ⁇ D1 (SEQ ID NO: 2), connected to the Fc dimer fragment, and the monomers in it are shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively Nucleic acid and amino acid sequences.
  • IMM2505 is a fusion protein mentioned in US 10,973,878 B2, which has a similar overall design to IMM2520 and IMM2521, including mutated SIRP ⁇ D1 (SEQ ID NO: 2) and different PD-L1 antibodies.
  • FIG. 10 shows that mice treated with IMM2520 had a higher survival rate than mice treated with IMM2505.
  • IMM2505 is a fusion protein mentioned in US 10,973,878 B2, which has a similar overall design to IMM2520 and IMM2521, including mutated SIRP ⁇ D1 (SEQ ID NO: 2) and different PD-L1 antibodies.
  • Figure 11 shows the binding force of PD-L1 binding saturated IMM2520 to CD47.
  • Figure 12 shows the binding force of CD47 binding saturated IMM2520 to PD-L1.
  • the second approach is to use fixed-dose combinations of multiple drugs in a single dosage form. This pathway reduces the drug quantity burden and improves patient compliance.
  • the disadvantage of fixed-dose combinations is mainly that the choice of possible dose ratios between the active ingredients is limited, which makes it more difficult to properly tailor the dose to the individual patient for maximum efficacy and minimum adverse effects.
  • the metabolic kinetics of the different drugs in the combination may introduce complex timing shifts in each target patient, thereby compromising overall efficacy.
  • a third approach is the use of multifunctional drugs that combine two or more pharmacological mechanisms in a single compound.
  • the design and characterization of these multifunctional molecules is more complex and requires extensive studies to confirm the optimal ratio of on-target activities in the molecules, whereas combined pharmacokinetics may result in matching pharmacokinetic activities at the molecular target level.
  • Multifunctional molecules can also be engineered into fixed-dose combinations with other drugs, thereby combining three, or even four, pharmacological mechanisms in a single tablet to produce further gains in efficacy.
  • the current inventors have invented a new recombinant multifunctional fusion protein, which can attack tumors through three mechanisms of action, one is to release the inhibitory signal mediated by PD-1 to check T cells or Inhibition, one is to release the SIRP-mediated inhibitory signal to check macrophages, and the other is to activate NK cells and/or macrophages to kill cancer cells.
  • the recombinant fusion protein of the present application comprises a PD-L1 antibody or an antibody fragment thereof, at least one paratope of the PD-L1 antibody or an antibody fragment thereof is connected to a signal regulation protein via a linker at the N-terminus of the heavy chain or light chain constituting the paratope (SIRP) extracellular Ig-like domain linkage.
  • SIRP paratope
  • the recombinant protein can simultaneously bind to CD47, PD-L1 and FcR, i) block the interaction between PD-L1 on cancer cells and PD-1 on T cells, thereby releasing the inhibitory signal mediated by PD-1 on T Cell inspection; ii) blocking the interaction between CD47 on cancer cells and SIRP on macrophages, and releasing SIRP-mediated inhibitory signals to macrophage inspection; and iii) antibody Fc region interaction with NK cells or macrophages FcR binding activates NK cells or macrophages to kill cancer cells.
  • a paratope of the PD-L1 antibody or antibody fragment thereof is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) via a linker at the N-terminus of the heavy chain or light chain constituting the paratope.
  • SIRP signal regulatory protein
  • each paratope of the PD-L1 antibody or antibody fragment thereof is connected to the extracellular Ig-like domain of the signal regulatory protein (SIRP) via a linker at the N-terminal of the heavy chain or light chain constituting the paratope .
  • each paratope of the PD-L1 antibody or antibody fragment thereof is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) at the N-terminal of the heavy chain constituting the paratope via a linker.
  • each paratope of the PD-L1 antibody or antibody fragment thereof is connected to the extracellular Ig-like domain of a signal regulatory protein (SIRP) at the N-terminal of the light chain constituting the paratope via a linker.
  • SIRP signal regulatory protein
  • the recombinant fusion protein of the present application is smaller in size (150-180kDa) and has a longer half-life of 5-10 days.
  • the three main components contained in the fusion protein of the present application are the extracellular Ig-like domain of the signal regulatory protein (SIRP), the linker, and the PD-L1 antibody or its antibody fragment.
  • SIRP signal regulatory protein
  • the linker the linker
  • the PD-L1 antibody or its antibody fragment there are many design options for the three components described above.
  • sequences of human origin are used in the treatment of human cancers, since the strong immunogenicity of non-human animal proteins or peptides may cause allergic and other adverse reactions.
  • other animal proteins or peptides can also be used in this application and can be humanized.
  • any extracellular Ig-like domain of any SIRP capable of binding to CD47 can be selected for the construction of the fusion protein.
  • the signal regulator protein in the recombinant fusion protein is SIRP ⁇
  • the extracellular Ig-like domain of the signal regulator protein is the first extracellular Ig-like domain of SIRP ⁇ (SIRP ⁇ D1).
  • SIRP ⁇ D1 is a mutant SIRP ⁇ D1. Compared with wild-type SIRP ⁇ D1, there is an N80A mutation at position 80 of SEQ ID NO: 2, and the mutation at this position can achieve the effect of deglycosylation.
  • the recombinant fusion protein comprises SIRP ⁇ D1 whose nucleic acid sequence and amino acid sequence are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • SIRP ⁇ D1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2, wherein SIRP ⁇ D1 is capable of interacting with cancer/tumor cells
  • Surface CD47 binds and blocks the interaction of CD47 with SIRP on the surface of macrophages.
  • the linker mainly functions as a spacer between the extracellular Ig-like domain of SIRP and the N-terminus of the heavy or light chain of the PD-L1 antibody.
  • the linker may consist of amino acids linked by peptide bonds, preferably 5-30, 10-30, 10-20, or 15 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids.
  • One or more of these amino acids may be glycosylated or deglycosylated, as known to those skilled in the art.
  • 5-30, 10-30, 10-20, or 15 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine .
  • the linker is composed of amino acids mostly hindered by vacant bonds, such as glycine and alanine.
  • exemplary linkers are polyglycine (particularly Gly, poly(Gly-Ala)), and polyalanine.
  • An exemplary suitable linker shown in the Examples below is (Gly-Ser), eg -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO: 4).
  • Linkers can also be non-peptide linkers.
  • These alkyl linkers may also be substituted with any non-sterically hindering groups such as lower alkyl (eg C 1-4 lower acyl), halogen (eg Cl, Br), CN, NH 2 , phenyl and the like.
  • the PD-L1 antibody is an isolated monoclonal antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:6, and each light chain having the amino acid sequence of SEQ ID NO:8
  • the amino acid sequence of these two amino acid sequences can be coded by SEQ ID NO:5 and SEQ ID NO:7 respectively.
  • the Fab part (or paratope) of the PD-L1 antibody can bind to PD-L1 on the surface of cancer/tumor cells to block the interaction between PD-L1 and PD-1 on the surface of T cells, thereby releasing PD-1-mediated Inhibitory signal checks on T cells, while the Fc part of the PD-L1 antibody can bind to FcR on the surface of NK cells and/or macrophages to stimulate the killing of cancer cells by NK cells or macrophages.
  • the heavy chain can comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 6, wherein the PD-L1 antibody is capable of interacting with PD-L1 binds and blocks the interaction between PD-L1 and PD-1 on the surface of T cells, and can bind to FcR on the surface of NK cells or macrophages to activate NK cells and/or macrophages against cancer cells of killing.
  • the light chain can have an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 8, wherein the PD-L1 antibody is capable of interacting with PD-L1 binds and blocks the interaction between PD-L1 and PD-1 on the surface of T cells.
  • antibody herein includes whole antibodies such as IgG, IgA, IgD, IgE and IgM, and any antigen-binding fragment (or antigen-binding portion) or single chain thereof.
  • Whole antibodies are glycoproteins comprising at least two heavy chains and two light chains linked by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region ( VH ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3 .
  • Each light chain comprises a light chain variable region (V L ) and a light chain constant region.
  • the light chain constant region comprises one domain, CL .
  • the VH and VL regions can be further divided into highly variable regions, that is, CDR regions, and relatively conserved framework regions (FRs) are distributed between the CDR regions.
  • Each VH and VL consists of three CDRs and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of the heavy and light chains contain binding domains that react with antigen.
  • the constant regions of the antibodies can be linked to the binding of the immune protein to host tissues or factors, including various immune system cells (eg, effector cells) and the first component of the complement system (Clq).
  • antibody fragment refers to a part or fragment of the antibody of the present application that retains the ability to specifically bind to an antigen (such as PD-L1), and optionally to FcR.
  • sequence identity refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in the reference sequence after sequence alignment, if necessary, the sequence introduced in the sequence alignment spaces to achieve the maximum percent sequence identity between the two sequences.
  • sequence identity refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in the reference sequence after sequence alignment, if necessary, the sequence introduced in the sequence alignment spaces to achieve the maximum percent sequence identity between the two sequences.
  • Those skilled in the art can use various methods, such as using computer software, to perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences.
  • Such computer software For example, ClustalOmega, T-coffee, Kalign, MAFFT and the like.
  • the present application provides polynucleotides encoding recombinant fusion proteins and expression vectors expressing recombinant fusion proteins.
  • vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformable artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and artificial additional chromosomes (HAECs).
  • the present application provides host cells comprising the above expression vectors.
  • Host cells can be transformed or transfected with expression vectors.
  • Suitable host cells include E. coli, yeast and other eukaryotes.
  • E. coli, yeast or mammalian cell lines eg COS or CHO are used.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the application formulated together with a pharmaceutically acceptable adjuvant.
  • the composition may optionally contain one or more other pharmaceutically active ingredients, such as another antibody or drug.
  • the pharmaceutical compositions of the present application may also be administered in combination therapy with, for example, another immunostimulant, anticancer drug, antiviral agent or vaccine.
  • compositions can contain any number of excipients.
  • Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersion or suspension aids, stabilizers, colorants, flavoring agents, coatings, disintegrants, lubricants , sweeteners, preservatives, isotonic agents, and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
  • the primary vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in injections.
  • the vehicle or carrier can be neutral buffered saline or saline mixed with serum albumin.
  • Other exemplary pharmaceutical compositions comprise Tris buffer, or acetate buffer, which may also contain sorbitol or a suitable substitute thereof.
  • the compositions can be prepared for storage by mixing selected components of the desired purity with any formulation (Remington's Pharmaceutical Sciences, supra) in lyophilized or aqueous form.
  • therapeutic compositions can be formulated as lyophilizates using suitable excipients such as sucrose.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or bolus injection).
  • the active molecule can be encapsulated in a material that protects it from acids and other natural conditions that could inactivate it.
  • parenteral administration refers to modes of administration other than enteral and topical administration usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, Intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, dural, and intrasternal injections and infusions.
  • the antibodies of the present application may be administered by non-injective routes, such as topical, epidermal, or mucosal modes of administration, eg, intranasal, oral, vaginal, rectal, sublingual, or topical administration.
  • compositions may be in the form of sterile aqueous solutions or suspensions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentrations.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject to be treated and the particular route of administration, and will generally be that amount of the composition which produces a therapeutic effect. Generally, as a percentage, this amount will be from about 0.01% to about 99% of the active ingredient, in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens can be adjusted to achieve the optimum desired response (eg, a therapeutic response). For example, divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited for unitary administration of the subjects to be treated; each unit containing a quantity of active compound calculated in advance to produce the desired therapeutic effect in association with the pharmaceutical carrier.
  • the fusion protein can be administered in a sustained release dosage form, in which case the administration is less frequent.
  • the dosage range is about 0.0001-100 mg/kg body weight of the recipient.
  • An exemplary treatment regimen is twice a week.
  • a "therapeutically effective amount" of the fusion protein of the present application preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevents damage or disability caused by the disease.
  • a "therapeutically effective amount” means, relative to untreated subjects, preferably inhibits tumor growth by at least about 40%, more preferably inhibits by at least about 60%, more preferably inhibits At least about 80%, more preferably at least about 99% inhibition.
  • a therapeutically effective amount of the fusion protein of the present application can reduce tumor volume or alleviate symptoms in a subject (usually a human, or another mammal).
  • the pharmaceutical composition can be a controlled sustained release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered by medical devices such as (1) needle-free hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. 4,487,603); (3) transdermal devices (US Patent 4,486,194); (4) infusion devices (US Patents 4,447,233 and 4,447,224); and (5) osmotic devices (US Patents 4,439,196 and 4,475,196), the above disclosures of which are incorporated by reference way incorporated into this article.
  • medical devices such as (1) needle-free hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. 4,487,603)
  • the fusion proteins of the present application can be formulated to ensure proper in vivo distribution.
  • the fusion protein in order to ensure that the therapeutic fusion protein of the present application crosses the blood-brain barrier, the fusion protein is formulated in liposomes, and may additionally contain targeting groups to enhance selective delivery to specific cells or organs. See, eg, US Patents 4,522,811, 5,374,548, 5,416,016, and 5,399,331.
  • the present application also relates to gene therapy in vivo, wherein a nucleic acid molecule encoding a fusion protein of the present application or a derivative thereof is directly introduced into a subject.
  • the nucleic acid sequence encoding the recombinant fusion protein of the present application is introduced into target cells via local injection of the nucleic acid construct with or without a suitable delivery vehicle such as an adeno-associated virus vector.
  • a suitable delivery vehicle such as an adeno-associated virus vector.
  • viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, and papillomavirus vectors.
  • In vivo physical transfer of viral vectors can be by local injection of the desired nucleic acid construct or other suitable delivery vehicle containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or particle bombardment (gene gun ) and realized.
  • compositions of the present disclosure may be used alone or in combination with other therapeutic agents to enhance their efficacy or reduce potential side effects.
  • Another object of the present application is to provide a method for preparing the above-mentioned recombinant fusion protein and a pharmaceutical composition comprising the recombinant fusion protein.
  • the preparation method comprises the following steps: (1) providing a nucleic acid molecule encoding a fusion protein; (2) constructing an expression vector comprising the nucleic acid molecule of (1); (3) using the expression vector in (2) to transform transfecting or transforming suitable host cells and culturing these host cells to express the protein; and (4) purifying the protein. Preparation can be carried out by techniques well known to those of ordinary skill.
  • Another object of the present application is to provide a method of using the pharmaceutical composition of the present application to treat cancer, comprising administering an effective amount of the above pharmaceutical composition to a patient or subject in need.
  • the pharmaceutical composition is used to treat tumors or cancers overexpressing CD47 and/or PD-L1, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoid Cellular leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, and kidney cancer.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • ALL acute lymphoid Cellular leukemia
  • NHL non-Hodgkin's lymphoma
  • MM multiple myeloma
  • bladder cancer ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer,
  • diseases associated with overexpression of CD47 and/or PD-L1 include, but are not limited to, Crohn's disease, allergic asthma, and rheumatoid arthritis.
  • IMM2515 is a monoclonal antibody targeting PD-L1.
  • the antibody has two heavy chains and two light chains, each heavy chain has the amino acid sequence of SEQ ID NO: 6, and each light chain has the amino acid sequence of SEQ ID NO: 8, and these two amino acid sequences can be respectively obtained from the nucleic acid sequence of SEQ ID NO: ID NO:5 and SEQ ID NO:7 encoding.
  • IMM01 is described in US 2021/0024598 A1, which contains two mutant SIRP ⁇ D1 (SEQ ID NO: 2), connected to the Fc dimer fragment, wherein the nucleic acid sequence and amino acid sequence of each monomer are as SEQ ID NO: 11 and SEQ ID NO:12 shown.
  • IMM2520 is a recombinant fusion protein comprising two mutant SIRP ⁇ D1s, each SIRP ⁇ D1 is connected to the N-terminus of each heavy chain of IMM2515 via a GS-linker, wherein the mutated SIRP ⁇ D1 contains two mutants as shown in SEQ ID NO:1 and SEQ ID NO:2 respectively Nucleic acid sequence and amino acid sequence, linker comprises the amino acid sequence shown in SEQ ID NO:4, and it can be encoded by the nucleic acid sequence of SEQ ID NO:3.
  • IMM2521 is a recombinant fusion protein, comprising two mutant SIRP ⁇ D1s, each SIRP ⁇ D1 is connected to the N-terminus of each light chain of IMM2515 via a GS-linker, wherein the mutated SIRP ⁇ D1 contains two mutants as shown in SEQ ID NO: 1 and SEQ ID NO: 2
  • Nucleic acid sequence and amino acid sequence, linker comprises the amino acid sequence shown in SEQ ID NO:4, and it can be encoded by the nucleic acid sequence of SEQ ID NO:3.
  • IMM2505 is a recombinant fusion protein described in US 10,973,878 B2. Its overall design is similar to IMM2520 and IMM2521, including mutated SIRP ⁇ D1 (SEQ ID NO: 2) and different PD-L1 antibodies.
  • Embodiment 1 Construction of IMM2520 and IMM2521 expression vectors
  • IMM2520 and IMM2521 are shown in Figures 1A and 1B.
  • the full-length coding sequences of recombinant fusion proteins IMM2520 and IMM2521 were artificially designed.
  • the coding sequence (SEQ ID NO: 1) of the mutant SIRP ⁇ D1 was combined with the PD-L1 in IMM2515 through the GS-linker coding sequence (SEQ ID NO: 3)
  • the 5' end of the coding sequence (SEQ ID NO:5) of the antibody heavy chain variable region was ligated; 57 nucleotides (SEQ ID NO:13) encoding the mouse IgG1 heavy chain signal peptide were added to the mutant SIRP ⁇ D1 coding sequence to the 5' end of the signal peptide sequence, and a Kozak sequence (SEQ ID NO: 14) was added to the 5' end of the signal peptide sequence.
  • HindIII and NheI restriction sites were added to the 5' and 3' ends of the resulting sequence, respectively.
  • the same signal peptide sequence and Kozak sequence were added to the 5' end of the PD-L1 light chain coding sequence (SEQ ID NO:7) and restricted by HindIII and XbaI sites were added to the 5' and 3' ends of the resulting sequence, respectively.
  • the coding sequence of mutant SIRP ⁇ D1 (SEQ ID NO:1) is encoded by the coding sequence of GS-linker (SEQ ID NO:3) and the light chain of PD-L1 antibody in IMM2515 The 5' end of the sequence (SEQ ID NO:7) was ligated.
  • the same signal peptide sequence and Kozak sequence were added to the 5' end of the PD-L1 heavy chain coding sequence (SEQ ID NO:5).
  • the resulting sequences were synthesized by GenScript and cloned into pMac-H and pMac-L vectors, respectively.
  • the expression vectors were electroporated into Chinese hamster ovary (CHO) cells (ATCC, Cat#CCL-61), after which these CHO cells were subjected to several rounds of neomycin pressure selection. Selected stable expressing cells were adapted in serum-free Balan CD CHO Growth A medium (Irvine Scientific, Cat#94120).
  • CHO Chinese hamster ovary
  • Selected stable expressing cells were adapted in serum-free Balan CD CHO Growth A medium (Irvine Scientific, Cat#94120).
  • cells were seeded into 3-liter bioreactors and cultured in fed-batch culture. When the cell viability dropped to -80%, the cell culture supernatant in the bioreactor was collected and subjected to protein purification by affinity chromatography. The purity of the recombinant protein is higher than 95%, and the amount of endotoxin is lower than 0.5U/g.
  • CHO-PD-L1 cells overexpressing PD-L1 (manufactured by the company) or Jurkat cells naturally expressing CD47 were incubated in serially diluted IMM2520, IMM2521 and control solutions at 4°C for 1 hour, respectively.
  • Cells were washed twice with cold PBS and then incubated with FITC-conjugated secondary antibody against human IgG-Fc (Cat#F9512, Sigma) for 45 minutes. Cells were washed twice and resuspended in 200ml PBS. Afterwards, the cells were analyzed by flow cytometry (Merck Millipore, easyCyte 5HT) for FACS analysis.
  • IMM2520 binds to PD-L1 on CHO cells with an EC 50 value of 0.09nM ( Figure 3), and binds to CD47 on Jurkat cells with an EC 50 value of 0.80nM ( Figure 4), which is more effective than traditional single-antigen targeting proteins slightly worse.
  • IMM2521 binds to PD-L1 on CHO cells with an EC 50 value of 0.11nM ( Figure 3), and binds to CD47 on Jurkat cells with an EC 50 value of 0.70nM ( Figure 4), which is more effective than traditional single-antigen targeting proteins slightly worse.
  • Example 4 IMM2520 and IMM2521 block the interaction between PD-L1 and PD-1
  • Biotin-hPD1-mFc (SEQ ID NO:22) was mixed with serially diluted IMM2520, IMM2521, IMM2515, and hIgG1-Fc, and the mixture was added to a 96-well plate containing CD47 + or CD47 - CHO-PD-L1 cells middle. Cells were incubated at 4°C for 45 minutes, washed with PBS, and incubated with PE-conjugated mouse anti-human CD279 (Cat#557946, BD BioScience) for 45 minutes at 4°C. Cells were washed and resuspended in 200ml PBS, and PD1-Fc-PD-L1 binding/interaction was analyzed by FACS.
  • IMM2520, IMM2521 and IMM2515 were all able to block the binding of PD1-mFc to CD47 - PD-L1 + cells with IC50 values below 1nM.
  • the PD-L1 and CD47 bispecific molecules namely, IMM2520 and IMM2521, showed higher inhibitory activity than the monospecific PD-L1 antibody IMM2515.
  • SIRP ⁇ -Fc wild-type human SIRP ⁇ +human IgG1Fc, SEQ ID NO: 23
  • serially diluted IMM2520, IMM2521, IMM01, and IgG1-Fc respectively.
  • the mixture was added to 96-well plates containing CD47-expressing PD- L1- or PD-L1 + Jurkat cells, and the plates were incubated at 4°C for 45 minutes. Cells were washed with PBS, and SIRP ⁇ -Fc-CD47 interaction was analyzed by FACS.
  • IMM2520 inhibited the binding of SIRP ⁇ -Fc to PD-L1 - CD47 + cells with an IC50 value of 127.7nM
  • IMM2521 inhibited the binding of SIRP ⁇ -Fc to CD47 + cells with an IC50 value of 139.8nM.
  • the PD-L1 and CD47 bispecific molecules namely, IMM2520 and IMM2521, showed higher inhibitory activity than the monospecific PD-L1 antibody IMM2515.
  • Example 6.IMM2520 triggers high levels of antibody-dependent cell-mediated cytotoxicity against PD-L1 positive cells (ADCC)
  • CFSE-labeled Raji-PD-L1 cells (used as target cells) were mixed with NK92MI cells stably expressing Fc ⁇ RIIIa (effector cells) at a ratio of 1:2, and the mixed cells were mixed with serially diluted IMM2520 or IMM2515 under 5% CO 2 Incubate at 37°C for 4 hours. Then, propidium iodide (PI) (Cat#P4170, Sigma) was added to the cell culture medium at a concentration of 5 ⁇ g/ml, and the cell culture medium was analyzed for PI signal by FACS. The percentage of cell lysis by ADCC was calculated based on the following formula:
  • % Lysis (% IMM2515 or IMM2520 treated PI positive cells-% negative control protein treated PI positive cells)/(100-% negative control protein treated PI positive cells)*100
  • IMM2520 elicited higher levels of ADCC compared to the monospecific PD-L1 antibody IMM2515.
  • Example 7 IMM2520 triggers higher levels of antibody-dependent cellular phagocytosis (ADCP) against PD-L1 positive cells
  • the mouse macrophage cell line Ana-1 (as effector cells) was seeded into a 96-well cell culture plate with 1x105 cells per well, and cultured at 37°C and 5% CO2 for 16-18 hours.
  • Raji-PD-L1 cells (as targeted cells) were labeled with CFSE and incubated with serial dilutions of IMM2520, IMM2515, IMM01, a combination of IMM01 and IMM2515, and hIgG1-Fc for 45 minutes, respectively.
  • FIG. 8 shows that IMM2520 induces high levels of antibody-dependent cellular phagocytosis (ADCP) on PD-L1 + tumor cells.
  • ADCP antibody-dependent cellular phagocytosis
  • Example 8.IMM2520 shows strong anti-tumor activity
  • mice Twenty-four SCID mice aged 5-7 weeks were subcutaneously injected with CT26-hPDL1/hCD47 colorectal cancer cells in the right abdomen, 2x106 cells per mouse.
  • the tumor volume reached 100-150 mm
  • the mice were randomly divided into 4 groups with 6 mice in each group, and the day of grouping was defined as D0. From this day, mice in each group were injected with PBS, IMM2505 (6.0 mg/kg), IMM2520 (6.0 mg/kg), and IMM01 (3.0 mg/kg) intraperitoneally for 4 weeks, twice a week. After 4 weeks, the administration was terminated, and the mice were continuously observed until the end of the experiment.
  • the experiment was terminated when the average tumor volume of the PBS group reached 3000 mm 3 , and the experiment was terminated on D60 in the drug treatment group. Tumor volume and mouse body weight were measured every 3-4 days.
  • Tumor volume (V) was calculated as (length x width 2 )/2.
  • the tumor inhibition rate of Group 4 was 97.89%, much higher than other groups, including the group treated with IMM2505, whose overall structure is very similar to IMM2520 and contains mutations SIRP ⁇ D1 (SEQ ID NO:2) and different PD-L1 antibodies.
  • IMM2505 showed a better anti-tumor effect than a monospecific drug combination (ie, PD-L1 antibody plus IMM01) in a mouse model.
  • the survival rates of groups 2 and 3 were comparable, dropping to about 80% at about day 40 after the start of dosing; the survival rate of group 4 remained at 100% up to day 60.
  • Example 9 IMM2520 simultaneously binds to PD-L1 and CD47
  • a molecular interaction instrument (Gator, Probe Life) was used to detect the simultaneous binding of IMM2520 to CD47 and PD-L1.
  • An anti-human IgG probe was used to capture 10 ⁇ g/ml IMM2520 until a responsivity of ⁇ 1.0 nm was reached.
  • the probes were then rinsed in buffer for 30 seconds before being transferred to 10 ⁇ g/ml PD-L1-His ( Figure 11) or 10 ⁇ g/ml CD47-His ( Figure 12) until the binding strength reached saturation levels. Afterwards, transfer the probe to 10 ⁇ g/ml CD47-His ( Figure 11) or 10 ⁇ g/ml PD-L1-His solution ( Figure 12) and let it stand for 120 seconds.
  • IMM2520 can still bind CD47 even after PD-L1 binding is saturated. As shown in Figure 12, IMM2520 can still bind PD-L1 after CD47 binding is saturated.
  • Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly. J 10 Cell 3 Biol. 1179–1189

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Abstract

提供了一种重组融合蛋白，包含PD-L1抗体或其抗体片段，该PD-L1抗体或其抗体片段的至少一个互补位在构成该互补位的重链或轻链的N端通过接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接，其中该重组融合蛋白可以同时与CD47、PD-L1和FcR结合。还提供了编码该重组融合蛋白的核酸分子、包含该核酸分子的表达载体、制备该重组融合蛋白的方法、以及使用重组融合蛋白来治疗与CD47和/或PD-L1过表达相关的疾病。

Description

靶向CD47和PD-L1的重组融合蛋白及其制备和用途 发明领域
本申请涉及一种靶向CD47、PD-L1和/或FcR的重组融合蛋白、以其制备和用途，特别是其在肿瘤治疗中的用途。
背景技术
癌细胞已经发展出一些逃避宿主免疫监视的机制，包括：1)通过高表达膜蛋白PD-L1和PD-L2来逃避T淋巴细胞的免疫监视，其中膜蛋白PD-L1和PD-L2均与T细胞表面的PD-1结合，引发T细胞凋亡；2)通过癌细胞膜上MICA/MICB的脱落来逃避自然杀伤(NK)细胞的免疫监视，脱落的MICA/MICB与NK细胞表面的NKG2D结合，阻断NK细胞对MICA/MICB +癌细胞的杀灭；3)通过高表达CD47来逃避巨噬细胞(Mφ)的免疫监视，CD47与巨噬细胞表面上的信号调节蛋白α(SIRPα)结合，从而引发抑制性信号的生成，该抑制信性号抑制巨噬细胞对癌细胞的吞噬作用。可以看出，癌细胞相当□聪明□，可以基于它们发展出的逃避机制而迅速增殖。因此，有效杀灭所有癌细胞的抗癌药物的开发可以针对这些机制。
SIRP和CD47
信号调节蛋白(SIRP)是跨膜糖蛋白，包括三个家族成员，SIRPα(CD172a)、SIRPβ(CD172b)和SIRPγ(CD172g)。这三种蛋白均包含相似的胞外区域，但是具有不同的胞内结构域。胞外区域包含三个免疫球蛋白样结构域，一个Ig-V和两个Ig-C结构域。SIRPα(CD172a)的胞内结构域包含两个抑制性信号转导区域，其可以抑制信号转导以及相应的细胞功能。SIRPβ(CD172b)和SIRPγ(CD172g)的胞内区域非常短，不含信号转导结构域。但是，SIRPβ(CD172b)能够经由衔接蛋白例如DAP12而起到信号转导的功能。SIRP主要表达于巨噬细胞(Mφ)、树突状细胞(DC)和神经元。
CD47是属于免疫球蛋白超家族的跨膜糖蛋白，在包括红细胞在内的所有细胞类型的表面上表达。CD47的配体包括整联蛋白、血小板反应蛋白-1和SIRP。CD47，通过与SIRPα相互作用发出“不要吃我”的信号，可以抑制巨噬细胞的吞噬作用，并从而保护例如血细胞等免收巨噬细胞的攻击。
已经有研究表明，很多过表达CD47的肿瘤或癌细胞可以抑制巨噬细胞对癌细胞的吞噬作用。过表达CD47的癌细胞包括急性髓细胞样白血病(AML)、慢性髓细胞样白血病(CML)、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、膀胱癌、卵巢癌、前列腺癌、肺癌、结肠癌、乳腺癌和胰腺癌细胞。有报道称，向荷肿瘤的小鼠注射阻碍CD47与SIRPα结合的CD47特异性抗体，可以显著地抑制肿瘤生长。当将同一种抗体注射到携带人白血病细胞的小鼠中时，肿瘤细胞或癌细胞完全消除(Theocharides APA,et al.,2012)。
PD-L1和PD-1
PD-L1，又称为程序性死亡-配体1或CD274，是一种跨膜蛋白，在一些特定情况例如异体组织移植、自身免疫疾病和肿瘤发生的过程中起到抑制免疫系统的重要作用。在癌症中，丧失转录因子例如STAT3与NF-κB之间的反馈限制将引起局部PD-L1表达的增加，这会限制靶向PD-L1的药物的全身性治疗的有效性(Vlahopoulos SA,2017)。对来自肾细胞癌患者的196个肿瘤样本分析发现，肿瘤PD-L1的高表达与增强的肿瘤侵袭性以及增加4.5倍的死亡风险相关(Thompson RH et al.,2004)。
PD-1是长约268个氨基酸的细胞表面受体。当与PD-L1或PD-L2结合时，PD-1可以通过抑制T细胞炎性反应，下调免疫系统，并提高自身耐受性。PD-1对于免疫系统的抑制性作用可防止自身免疫疾病，但也同时阻止免疫系统杀灭癌细胞。一种PD-1抗体，BMS-936558，在大约1/5-1/4的小细胞肺癌、黑色素瘤或肾细胞癌病患中产生客观性缓解(Suzanne L.Topalian et al.,2012)。
Fc和FcR
可结晶区段(Fc区)是抗体的尾部区，是决定抗体的效应子功能(即，抗体如何与特定细胞受体或其他防御蛋白建立关系)的结构域。
Fc受体(FcR)是在某些细胞，包括B淋巴细胞、滤泡树突状细胞、自然杀伤细胞、巨噬细胞、中性粒细胞、嗜酸粒细胞、嗜碱粒细胞、和肥大细胞等的表面上的蛋白。这些细胞有助于免疫系统的保护功能。
Fc区可以与Fc受体以及补体系统的一些蛋白相互作用，激活免疫系统。
治疗性双特异性或多特异性融合蛋白/抗体
靶向单一抗原的抗体具有有限的疗效。例如，获批的PD-L1抗体，Avelumab
Figure PCTCN2022116312-appb-000001
总的缓解率仅为33％。
近年来已经开发出双特异性或三特异性融合蛋白，且这些融合蛋白已经在临床前和临床试验中显示出相当不错的效果。
尽管从概念上讲，在传统抗体上附加额外的结合基团不是很复杂的事，然而，这种改造会显著地改变抗体的结构，抗体和额外结合基团间可能会相互影响结合力和/或药效(Wang S et al.,2021)。为优化体内疗效和药物性质，需要在主次基团(序列)的选择、目标物结合力的平衡、结合位点(重链或轻链的N-或C-端)的选择、结构稳定性、接头长度和序列方面做出精心的设计和改造(Shim H.2020)。
US 10,800,821 B2公开了一种约90k道尔顿的重组双特异性融合蛋白，靶向CD47和FcR，在治疗携带HL细胞的Balb/c裸鼠中观察到增强的抗肿瘤效果。US 10,973,878 B2公开了一种同时靶向CD47、PD-L1和FcR的融合蛋白IMM2505，其具有低分子量和较长的半衰期。
对于本申请中任何文件的引用，并不等同于承认这些文件是本申请的现有技术。
发明内容
本申请提供一种新的重组融合蛋白，与10,973,878 B2中的IMM2505结构相似，但具有新的PD-L1抗体序列，显示出比IMM2505更优的抗肿瘤效果。
具体地，本申请公开一种重组融合蛋白，其包含特异结合PD-L1的抗体或其抗体片段、以及特异结合CD47的肽，其中该CD47结合肽与该PD-L1抗体或其抗体片段连接，其中该PD-L1抗体或其抗体片段包含重链可变区、重链恒定区、和轻链可变区，重链可变区包含SEQ ID NO:19的氨基酸序列，轻链可变区包含SEQ ID NO:20的氨基酸序列，重链恒定区具有FcR结合力且与重链可变区的C端连接，其中该CD47结合肽包含突变的信号调节蛋白(SIRP)胞外Ig样结构域，该突变的信号调节蛋白(SIRP)胞外Ig样结构域包含SEQ ID NO:2的氨基酸序列，其中该重组融合蛋白能够同时结合CD47和PD-L1。该CD47结合肽可以与该PD-L1抗体或其抗体片段的重链可变区或轻链可变区的N端结合。SEQ ID NO:2的氨基酸序列可以由SEQ ID NO:1的核酸序列编码。
在一些实施方式中，PD-L1抗体或其抗体片段的至少一个互补位在构成该互补位的重链可变区或轻链可变区的N端与CD47结合肽连接。在一些实施方式中，PD-L1抗体或其抗体片段的各个互补位在构成该互补位的重链及可变区或轻链可变区的N端与CD47结合肽连接。在一些实施方式中，PD-L1抗体或其抗体片段的各个互补位在构成该互补位的重链可变区的N端与CD47结合肽连接。在一些实施方式中，PD-L1抗体或其抗体片段的各个互补位在构成该互补位的轻链可变区的N端与CD47结合肽连接。
具有FcR结合力的重链恒定区可以是天然存在的或经改造的人IgG1、IgG2、IgG3或IgG4重链恒定区，或其功能片段。在一些实施方式中，具有FcR结合力的重链恒定区是人IgG1重链恒定区，或其功能片段。在一些实施方式中，具有FcR结合力的重链恒定区具有SEQ ID NO:21所示的氨基酸序列。
PD-L1抗体或其抗体片段可以包含轻链恒定区，例如人κ轻链恒定区，或其功能片段，与轻链可变区的C端连接。
在一些实施方式中，PD-L1抗体或其抗体片段可以包含重链和轻链，重链包含与SEQ ID NO:6具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，轻链包含与SEQ ID NO:20具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。在一些实施方式中，PD-L1抗体或其抗体片段可以包含重链和轻链，重链包含与SEQ ID NO:6具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，轻链包含与SEQ ID NO:8具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。在一些实施方式中，PD-L1抗体或其抗体片段可以包含重链和轻链，重链包含SEQ ID NO:6所示的氨基酸序列，轻链包含SEQ ID NO:20所示的氨基酸序列。在一些实施方式中，PD-L1抗体或其抗体片段可以包含重链和轻链，重链包含SEQ ID NO:6所示的氨基酸序列，轻链包含SEQ ID NO:8所示的氨基酸序列。
本申请的PD-L1抗体或其抗体片段可以与CD47结合肽经接头而连接。接头可以是5-30、10-30、10-20、或15个氨基酸长度的肽。接头可以是例如-(Gly-Gly-Gly-Gly-Ser) 2-(SEQ ID NO:17)、-(Gly-Gly-Gly-Gly-Ser) 3-(SEQ ID NO:4)、或-(Gly-Gly-Gly-Gly-Ser) 4-(SEQ ID NO:18)。在一些实施方式中，接头是-(Gly-Gly-Gly-Gly-Ser) 3-(SEQ ID NO:4)。SEQ ID NO:4的氨基酸序列可以由SEQ ID NO:3的核酸序列编码。
在一些实施方式中，本申请的重组融合蛋白包含CD47结合肽-接头-PD-L1抗体重链、和PD-L1抗体轻链，其中CD47结合肽-接头-PD-L1抗体重链含有与SEQ ID NO:10具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致度的氨基酸序列，PD-L1抗体轻链含有与SEQ ID NO:8具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致度的氨基酸序列。在一些实施方式中，本申请的重组融合蛋白包含CD47结合肽-接头-PD-L1抗体重链、和PD-L1抗体轻链，其中CD47结合肽-接头-PD-L1抗体重链含有与SEQ ID NO:10具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致度的氨基酸序列，PD-L1抗体轻链含有与SEQ ID NO:20具有至少95％序列一致度的氨基酸序列。SEQ ID NOs:8和10的氨基酸序列可以分别由SEQ ID NOs:7和9的核酸序列编码。
在一些实施方式中，本申请的重组融合蛋白包含PD-L1抗体重链、和CD47结合肽-接头-PD-L1抗体轻链，其中PD-L1抗体重链含有与SEQ ID NO:6具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致度的氨基酸序列，CD47结合肽-接头-PD-L1抗体轻链含有与SEQ ID NO:16具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致度的氨基酸序列。SEQ ID NOs:6和16的氨基酸序列可以分别由SEQ ID NOs:5和15的核酸序列编码。
本申请还提供编码本申请重组融合蛋白的核酸分子，以及包含该核酸分子的表达载体、和包含该表达载体的宿主细胞。还提供一种使用本申请的宿主细胞来制备重组融合蛋白的方法，包括(i)在宿主细胞中表达重组融合蛋白，以及(ii)从宿主细胞或其细胞培养物中分离重组融合蛋白。
本申请还提供一种药物组合物，其可以包含本申请的重组融合蛋白、核酸分子、表达载体或宿主细胞，以及至少一种药学上可接受的赋形剂。在一些实施方式中，药物组合物包含至少一种药学上可接受的佐剂。
本申请的重组融合蛋白或药物组合物可以用于治疗与CD47和/或PD-L1过表达相关的疾病，或用于制备治疗与CD47和/或PD-L1过表达相关的疾病的药物。
一方面，本申请提供一种用于在有需求受试者中治疗或减轻与CD47和/或PD-L1过表达相关的疾病的方法，包括向该受试者施用治疗有效量的本申请药物组合物。
与CD47和/或PD-L1过表达相关的疾病可以是急性髓细胞样白血病(AML)、慢性髓细胞样白血病(CML)、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、膀胱癌、卵巢癌、前列腺癌、肺癌、结肠癌、乳腺癌、胰腺癌或肾细胞癌。
基于以下的详细描述和实施例，当前公开的其他特征和有利之处将是非常明晰可见的，详细描述和实施例不应当解读为是限制性的。所有在说明书中引用的文献、Genbank登记号、专利和公开专利申请均通过引用的方式并入本文。
附图说明
详细描述在以下以示例方式给出但不意在将本申请限制于所述具体实施方式，可以结 合附图更好地进行理解。
图1A和1B是本申请重组融合蛋白IMM2520和IMM2521的结构示意图。结构最上方的圆形结构表示突变的SIRPα第一胞外结构域(SIRPαD1)，其经接头与PD-L1抗体的重链(A)或轻链(B)的N端连接。突变的SIRPαD1分别具有SEQ ID NO:1和SEQ ID NO:2所示的核酸序列和氨基酸序列。接头具有SEQ ID NO:4所示氨基酸序列，其可以由SEQ ID NO:3所示的核酸序列编码。PD-L1抗体的重链包含分别如SEQ ID NO:5和SEQ ID NO:6所示的核酸序列和氨基酸序列。PD-L1抗体的轻链包含分别如SEQ ID NO:7和SEQ ID NO:8所示的核酸序列和氨基酸序列。
图2是本申请重组融合蛋白的作用机制示意图。
图3示出IMM2520和IMM2521对表达人PD-L1的CHO细胞(CHO-PDL1)上PD-L1的结合活性。IMM2505是US 10,973,878 B2中提及的融合蛋白，具有与IMM2520以及IMM2521相似的整体设计，包含突变的SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。IMM2515是构成IMM2520和IMM2521的PD-L1抗体，包含SEQ ID NO:6的重链和SEQ ID NO:8的轻链。阿替利珠单抗(atezolizumab)是市售可得的PD-L1抗体，hIgG1-Fc用作阴性对照。
图4示出IMM2520和IMM2521对Jurkat细胞上CD47的结合活性。IMM2505是US10,973,878 B2中提及的融合蛋白，具有与IMM2520以及IMM2521相似的整体设计，并包含突变的SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。IMM01记载于US2021/0024598 A1，包含两个突变SIRPαD1(SEQ ID NO:2)，与Fc二聚体片段连接，其中的单体含有分别如SEQ ID NO:11和SEQ ID NO:12所示的核酸序列和氨基酸序列。hIgG1-Fc用作阴性对照。
图5A和5B示出IMM2520和IMM2521阻断PD-1-Fc与CD47 -CHO-PD-L1细胞(A)或CD47 +Raji-PD-L1细胞(B)上PD-L1结合的能力。IMM2515是构成IMM2520和IMM2521的PD-L1抗体，包含SEQ ID NO:6的重链和SEQ ID NO:8的轻链。hIgG1-Fc用作阴性对照。
图6A和6B示出IMM2520和IMM2521阻断SIRPα-Fc与PD-L1 -Raji细胞(A)或PD-L1 +Raji细胞(B)上CD47结合的能力。IMM01记载于US 2021/0024598 A1，包含两个突变SIRPαD1(SEQ ID NO:2)，与Fc二聚体片段连接，其中的单体含有分别如SEQ ID NO:11和SEQ ID NO:12所示的核酸序列和氨基酸序列。hIgG1-Fc用作阴性对照。
图7示出IMM2520对Raji-PD-L1细胞引发抗体依赖性细胞介导的细胞毒性(ADCC)的能力。IMM2515是构成IMM2520和IMM2521的PD-L1抗体，包含SEQ ID NO:6的重链和SEQ ID NO:8的轻链。
图8示出IMM2520对Raji-PD-L1细胞引发抗体依赖性细胞吞噬(ADCP)的能力。IMM2515是构成IMM2520和IMM2521的PD-L1抗体，包含SEQ ID NO:6的重链和SEQ ID NO:8的轻链。IMM01记载于US 2021/0024598 A1，包含两个突变SIRPαD1(SEQ ID NO:2)，与Fc二聚体片段连接，其中的单体含有分别如SEQ ID NO:11和SEQ ID NO:12所示的核酸序列和氨基酸序列。hIgG1-Fc用作阴性对照。
图9示出IMM2520在携带CT26-hPDL1hCD47肿瘤的同系移植BALB/c-hPD1/hSIRPα小鼠中的体内抗肿瘤效果。IMM01记载于US 2021/0024598 A1，包含两个突变SIRPαD1(SEQ ID NO:2)，与Fc二聚体片段连接，其中的单体含有分别如SEQ ID NO:11和SEQ ID NO:12所示的核酸序列和氨基酸序列。IMM2505是US 10,973,878 B2中提及的融合蛋白，具有与IMM2520以及IMM2521相似的整体设计，包含突变的SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。
图10示出用IMM2520处理的小鼠具有比IMM2505处理小鼠更高的存活率。IMM2505是US 10,973,878 B2中提及的融合蛋白，具有与IMM2520以及IMM2521相似的整体设计，包含突变的SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。
图11示出PD-L1结合饱和的IMM2520与CD47的结合力。
图12示出CD47结合饱和的IMM2520与PD-L1的结合力。
具体实施方式
从原理上讲，主要有三种不同的途径来靶向两种或更多种肿瘤生长的药理机制。最常见的，可以给予患者两种或更多种不同药物的组合。尽管这种选择使得对于可能的药物组合和不同剂量有着最大化的灵活度，其面临的问题是：a)由于药物变多且各个药物有不同的剂量安排，患者依从性变差；b)由于药物-药物相互作用，存在可能的不相容性；以及c)药物副作用风险增加。这些问题会降低治疗的有效性并妨碍治疗目标的达成，尤其在慢性疾病例如癌症的管理中。
第二个途径是在单剂型中使用多种药物的固定剂量组合。该途径减少药物数量负担，患者依从性改善。固定剂量组合的不利之处主要在于，对于活性成分之间可能的剂量比的选择是有限的，这使得更加难以恰当地针对个体患者将剂量调至最大功效和最小不利作用。此外，组合中不同药物的代谢动力学特性可能在各目标患者中引起复杂的药效时间错位，从而使总的功效折中。
第三个途径是使用在单个化合物中结合两种或更多种药理机制的多功能药物。这些多功能分子的设计和鉴定更加复杂，并需要大量的研究来确认分子中靶向活性的最佳比，而联合的药物代谢动力学可能会在分子靶标级别产生匹配的药物代谢动力学活性。多功能分子也可以改造成与其他药物的固定剂量组合，从而在单一的药片中结合三种、甚至四种药理机制，以产生功效的进一步增长。
经过大量的实验，当前的发明人发明出了一种新的重组多功能融合蛋白，其能够通过三种作用机制来攻击肿瘤，一个是解除PD-1介导的抑制信号对T细胞的检查或抑制，一个是解除SIRP介导的抑制信号对巨噬细胞的检查，另一个是激活NK细胞和/或巨噬细胞等对癌细胞的杀灭。
本申请的重组融合蛋白包含PD-L1抗体或其抗体片段，该PD-L1抗体或其抗体片段的至少一个互补位在构成该互补位的重链或轻链的N端经接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接。该重组蛋白可以同时与CD47、PD-L1以及FcR结合，i)阻断癌细胞上PD-L1与T细胞上PD-1之间的相互作用，从而解除PD-1介导的抑制信号对T 细胞的检查；ii)阻断癌细胞上CD47与巨噬细胞上SIRP的相互作用，解除SIRP介导的抑制信号对巨噬细胞的检查；以及iii)抗体Fc区与NK细胞或巨噬细胞上FcR结合，激活NK细胞或巨噬细胞对癌细胞的灭杀。在一个实施方式中，PD-L1抗体或其抗体片段的一个互补位在构成该互补位的重链或轻链的N端经接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接。在另一实施方式中，PD-L1抗体或其抗体片段的各互补位在构成该互补位的重链或轻链的N端经接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接。在一个实施方式中，PD-L1抗体或其抗体片段的各互补位在构成该互补位的重链的N端经接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接。在一个实施方式中，PD-L1抗体或其抗体片段的各互补位在构成该互补位的轻链的N端经接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接。本申请的重组融合蛋白尺寸较小(150-180kDa)，具有5-10天的较长半衰期。
包含在本申请融合蛋白中的三个主要组成部分为信号调节蛋白(SIRP)的胞外Ig样结构域、接头、和PD-L1抗体或其抗体片段。本领域技术人员将意识到，对于上述三个组成部分，存在很多种设计选择。优选地，在人癌症治疗中使用人源序列，因为非人动物蛋白或肽的强烈免疫原性可能会引起过敏反应和其他不良反应。然而，基于不同的应用目的，也可以在本申请中使用其他动物蛋白或肽，可以进行人源化。
能够与CD47结合的任何SIRP(SIRPα、SIRPβ和SIRPγ)的任何胞外Ig样结构域均可以选择用于融合蛋白的构建。在一个实施方式中，重组融合蛋白中的信号调节蛋白是SIRPα，且信号调节蛋白的胞外Ig样结构域为SIRPα的第一胞外Ig样结构域(SIRPαD1)。在一个实施方式中，SIRPαD1为突变SIRPαD1，相比于野生型SIRPαD1，在SEQ ID NO:2的第80位处存在N80A突变，该位点的突变可实现去糖基化的效果。
在一个实施方式中，重组融合蛋白包含核酸序列和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO:2所示的SIRPαD1。在另一实施方式中，SIRPαD1可以包含与SEQ ID NO:2具有至少80％、85％、90％、95％、98％或99％序列同一性的氨基酸序列，其中SIRPαD1能够与癌/肿瘤细胞表面的CD47结合并阻断CD47与巨噬细胞表面SIRP的相互作用。
接头主要起到SIRP的胞外Ig样结构域与PD-L1抗体重链或轻链N端之间的间隔的作用。接头可以由肽键连接的氨基酸构成，优选肽键连接的5-30个、10-30个、10-20个、或15个氨基酸，其中氨基酸选自20种天然存在的氨基酸。这些氨基酸中的一个或多个可以糖基化或去糖基化，如本领域技术人员所了解的。在一个实施方式中，5-30个10-30个、10-20个、或15个氨基酸可以选自甘氨酸、丙氨酸、脯氨酸、天冬酰胺、谷氨酰胺、丝氨酸和赖氨酸。在一个实施方式中，接头由大部分有空键位阻的氨基酸构成，例如甘氨酸和丙氨酸。示例性的接头为多聚甘氨酸(特别是Gly、多聚(Gly-Ala))、以及多聚丙氨酸。以下实施例中示出的示例性合适接头为(Gly-Ser)，例如-(Gly-Gly-Gly-Gly-Ser) 3-(SEQ ID NO:4)。
接头也可以是非肽类接头。例如，可以使用烷基接头，例如-NH-、-(CH 2)s-C(O)-，其中s＝2-20。这些烷基接头还可以经任何非空间位阻基团例如低级烷基(例如C 1-4低级酰基)、卤素(例如Cl、Br)、CN、NH 2、苯基等进行取代。
在一些实施方式中，PD-L1抗体是分离的单克隆抗体，包含两条重链和两条轻链，各重链具有SEQ ID NO:6的氨基酸序列，各轻链具有SEQ ID NO:8的氨基酸序列，这两个氨基酸序列可以分别由SEQ ID NO:5和SEQ ID NO:7编码。PD-L1抗体的Fab部分(或互补位)可以与癌/肿瘤细胞表面的PD-L1结合，以阻断PD-L1与T细胞表面PD-1的相互作用，从而解除PD-1介导的抑制性信号对T细胞的检查，而PD-L1抗体的Fc部分可以与NK细胞和/或巨噬细胞表面的FcR结合，以刺激NK细胞或巨噬细胞对癌细胞的杀灭。在一些实施方式中，重链可包含与SEQ ID NO:6具有至少80％、85％、90％、95％、98％或99％序列同一性的氨基酸序列，其中该PD-L1抗体能够与PD-L1结合并阻断PD-L1与T细胞表面PD-1之间的相互作用，并且能够与NK细胞或巨噬细胞表面的FcR结合，以激活NK细胞和/或巨噬细胞对癌细胞的杀灭。在一些实施方式中，轻链可以具有与SEQ ID NO:8具有至少80％、85％、90％、95％、98％或99％序列同一性的氨基酸序列，其中该PD-L1抗体能够与PD-L1结合，并阻断PD-L1与T细胞表面PD-1之间的相互作用。
本文中的术语“抗体”包括例如IgG、IgA、IgD、IgE和IgM的全抗体、及其任意的抗原结合片段(或抗原结合部分)或单链。全抗体是包含至少两条重链和两条轻链的糖蛋白，重链和轻链间经二硫键连接。每一重链包含重链可变区(V H)和重链恒定区。重链恒定区包含三个结构域，C H1、C H2和C H3。每一轻链包含轻链可变区(V L)和轻链恒定区。轻链恒定区包含一个结构域C L。V H和V L区域还可以再分成高度变化区，即CDR区，CDR区之间分布有较为保守的框架区(FR)。每一V H和V L由三个CDR和四个FR区构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排列。重链和轻链的可变区包含与抗原反应的结合域。抗体的恒定区可以接到免疫蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如效应细胞)和补体系统第一成分(C1q)。
本文的术语“抗体片段”是指本申请抗体中保持有特异结合抗原(例如PD-L1)、以及任选地结合FcR的能力的部分或片段。
本文中提到的“序列一致度”是指在进行序列比对后，一条序列中与参照序列中核苷酸/氨基酸残基相同的核苷酸/氨基酸百分比，如果需要的话，在序列对比中引入空格来达到两条序列间最大的序列一致性百分比。本领域技术人员可以通过多种方法，例如使用计算机软件，来进行两两序列对比或多序列比对，以确定两条或多条核酸或氨基酸序列之间的序列一致性百分比，此类计算机软件为例如ClustalOmega、T-coffee、Kalign和MAFFT等。
同时，本申请提供编码重组融合蛋白的多核苷酸和表达重组融合蛋白的表达载体。载体的例子包括但不限于质粒、病毒载体、酵母人工染色体(YAC)、细菌人工染色体(BAC)、可转化人工染色体(TAC)、哺乳动物人工染色体(MAC)和人工附加染色体(HAEC)。
本申请提供包含上述表达载体的宿主细胞。宿主细胞可以用表达载体进行转化或转染。合适的宿主细胞包括大肠杆菌(E.coli)、酵母和其他真核生物。优选地，使用大肠杆菌、酵母或哺乳动物细胞系(例如COS或CHO)。
另一方面，本申请提供一种药物组合物，其包含本申请的与药学上可接受佐剂配制 在一起的融合蛋白。组合物可以任选地包含一种或多种其他药学活性成分，例如另一种抗体或药物。本申请的药物组合物也可以与例如另一种免疫刺激剂、抗癌药物、抗病毒剂或疫苗一起在联合疗法中进行施用。
药物组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬助剂、稳定剂、着色剂、矫味剂、包衣、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂、及其组合。合适赋形剂的选择和使用在Gennaro,ed.,Remington:The Science and Practice of Pharmacy,20th Ed.(Lippincott Williams&Wilkins 2003)中有过教导，其公开内容通过引用的方式并入本文。
药物组合物中的主要媒介物或载体可以本质上是水性或非水性的。例如，合适的媒介物或载体可以是注射用水、生理盐水或人工脑脊液，可以补充有注射中常见的其他材料。例如，媒介物或载体可以是中性缓冲盐溶液或混有血清白蛋白的盐溶液。其他示例性的药物组合物包含Tris缓冲液、或醋酸盐缓冲液，其还可以包含山梨醇或其合适的替代物。在本申请的一个实施方式中，组合物可以通过混合具有所需纯度的所选组分与任意的配制剂(Remington’s Pharmaceutical Sciences，如上)以冻干或水溶液形式制备而用于储存。此外，治疗组合物可以使用合适的赋形剂例如蔗糖配制为冻干剂。
优选地，药物组合物适用于静脉、肌内、皮下、非肠道、脊柱、或表皮给药(例如，通过注射或推注)。取决于给药途径的不同，活性分子可以包裹在材料中以保护其免受酸和可能使其失活的其他自然条件的作用。本文所用的术语“非肠道给药”是指除通常通过注射进行的肠道和局部给药外的给药模式，包括而不限于，静脉、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜、和胸骨内注射和输注。或者，本申请的抗体可以通过非注射途径给药，例如局部的、表皮的或粘膜的给药模式，例如，鼻内、经口、阴道、直肠、舌下、或局部给药。
药物组合物可以是无菌水溶液或混悬液的形式。它们也可以配制成微乳剂、脂质体、或其他适用于高浓度药物的有序结构。
可以与载体材料结合来制备单剂型的活性成分的量，取决于待治疗的受试者和特定的给药途径而各异，且通常是产生疗效的组合物的量。通常而言，以百分比计，该量为约0.01％-约99％的活性成分，与药学上可接受的载体组合在一起。
给药方案可以调整以达成最优的所需反应(例如，治疗反应)。例如，随着时间的推进施用多个分剂量，或者根据治疗情况的危急程度而成比例地减少或增加剂量。特别有利的是，以剂量单位配制非肠道组合物，以方便给药且有利于剂量的均一性。本文所用的剂量单位型是指适用于待治疗受试者的单次给药的物理上分离的单元；各单元包含事先计算出的与药物载体一起产生所需的疗效的活性化合物的量。或者，融合蛋白可以以持续释放剂型进行给药，在这种情况下，给药的频率降低。
对于融合蛋白的给药，剂量范围为约0.0001-100mg/kg受体体重。示例性的治疗方案为每周两次。
“治疗有效量”的本申请融合蛋白优选地引起疾病症状严重程度的降低、无疾病症 状时期的频率和持续时间的上升、或防止由疾病引起的损伤或失能。例如，对于荷肿瘤受试者的治疗，“治疗有效量”是指，相对于未治疗的受试者，优选地，肿瘤生长抑制至少约40％，更优选抑制至少约60％，更优选抑制至少约80％，更优选抑制至少约99％。治疗有效量的本申请融合蛋白可以在受试者(通常为人，或者可以为另一种哺乳动物)中减小肿瘤体积、或者减轻症状。
药物组合物可以是受控的缓释制剂，包括植入体、透皮贴剂、和微囊化递送系统。可以使用可生物降解的生物相容性多聚物，例如乙烯-醋酸乙烯共聚物、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。参见，例如Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978。
药物组合物可以通过以下医疗装置进行施用，例如(1)无针皮下注射装置(例如，美国专利5,399,163、5,383,851、5,312,335、5,064,413、4,941,880、4,790,824、和4,596,556)；(2)微输注泵(美国专利4,487,603)；(3)透皮装置(美国专利4,486,194)；(4)输注装置(美国专利4,447,233和4,447,224)；和(5)渗透装置(美国专利4,439,196和4,475,196)，以上公开内容通过引用的方式并入本文。
在某些实施方式中，本申请的融合蛋白可以配制成保证合适的体内分布。例如，为保证本申请的治疗融合蛋白跨过血脑屏障，将融合蛋白配制在脂质体中，还可以额外地包含靶向基团以加强对特定细胞或器官的选择性传输。参见，例如，美国专利4,522,811、5,374,548、5,416,016、和5,399,331。
本申请还涉及体内基因疗法，其中将编码本申请融合蛋白或其衍生物的核酸分子直接引入受试者中。例如，将编码本申请重组融合蛋白的核酸序列经由带有或不带有合适递送载体例如腺相关病毒载体的核酸构建体经局部注射而引入目标细胞。其他可供选择的病毒载体包括但不限于逆转录病毒、腺病毒、单纯疱疹病毒、和乳头状瘤病毒载体。病毒载体的体内物理转移可以通过所需核酸构建体或包含所需核酸序列的其他合适递送载体的局部注射、脂质体介导的转移、直接注射(裸露的DNA)、或微粒轰击(基因枪)而实现。
本公开的组合物可以单独使用，或者与其他用于增强其疗效或降低潜在副作用的治疗剂联合使用。
本申请的另一目的是提供制备上述重组融合蛋白以及包含该重组融合蛋白的药物组合物的方法。在一个实施方式中，制备方法包括以下步骤：(1)提供编码融合蛋白的核酸分子；(2)构建包含(1)的核酸分子的表达载体；(3)用(2)中的表达载体转染或转化合适的宿主细胞并培养这些宿主细胞以表达蛋白；以及(4)纯化蛋白。制备可以由普通技术人员以熟知的技术进行。
本申请的另一目标是提供使用本申请药物组合物来治疗癌症的方法，包括向有该需求的患者或受试者施用有效量的上述药物组合物。在一个实施方式中，药物组合物用于治疗过表达CD47和/或PD-L1的肿瘤或癌症，包括但不限于急性髓细胞样白血病(AML)、慢性髓细胞样白血病(CML)、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、膀胱癌、卵巢癌、前列腺癌、肺癌、结肠癌、乳腺癌、胰腺癌和肾癌。
在一个实施方式中，与过表达CD47和/或PD-L1相关的疾病包括但不限于克罗恩病、过敏性哮喘和类风湿性关节炎。
本申请将参照以下非限制性实施例进行进一步说明。
实施例
IMM2515是靶向PD-L1的单克隆抗体。该抗体具有两条重链和两条轻链，各重链具有SEQ ID NO:6的氨基酸序列，各轻链具有SEQ ID NO:8的氨基酸序列，这两个氨基酸序列可以分别由核酸序列SEQ ID NO:5和SEQ ID NO:7编码。
IMM01记载于US 2021/0024598 A1，包含两个突变SIRPαD1(SEQ ID NO:2)，与Fc二聚体片段连接，其中各单体的核酸序列以及氨基酸序列分别如SEQ ID NO:11和SEQ ID NO:12所示。
IMM2520是重组融合蛋白，包含两个突变SIRPαD1，各SIRPαD1经GS-接头与IMM2515在各重链的N端连接，其中突变的SIRPαD1包含分别如SEQ ID NO:1和SEQ ID NO:2所示的核酸序列和氨基酸序列，接头包含SEQ ID NO:4所示的氨基酸序列，其可以由SEQ ID NO:3的核酸序列编码。
IMM2521是重组融合蛋白，包含两个突变SIRPαD1，各SIRPαD1经GS-接头与IMM2515在各轻链的N端连接，其中突变的SIRPαD1包含分别如SEQ ID NO:1和SEQ ID NO:2所示的核酸序列和氨基酸序列，接头包含SEQ ID NO:4所示的氨基酸序列，其可以由SEQ ID NO:3的核酸序列编码。
IMM2505是US 10,973,878 B2中记载的重组融合蛋白，其整体设计与IMM2520以及IMM2521较为相似，包含突变的SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。
实施例1.IMM2520和IMM2521表达载体的构建
IMM2520和IMM2521的结构如图1A和1B所示。人工设计出重组融合蛋白IMM2520和IMM2521的全长编码序列。
具体地，对于IMM2520中的SIRPαD1-接头-PD-L1抗体重链，将突变SIRPαD1的编码序列(SEQ ID NO:1)经GS-接头编码序列(SEQ ID NO:3)与IMM2515中PD-L1抗体重链可变区的编码序列(SEQ ID NO:5)的5’端连接；将编码小鼠IgG1重链信号肽的57个核苷酸(SEQ ID NO:13)加至突变SIRPαD1编码序列的5’端，并将Kozak序列(SEQ ID NO:14)加至信号肽序列的5’端。最后，将HindIII和NheI限制性酶切位点分别加至所得序列的5’和3’端。对于IMM2520中的PD-L1抗体轻链，将相同的信号肽序列以及Kozak序列加至PD-L1轻链编码序列(SEQ ID NO:7)的5’端，并将HindIII和XbaI限制性酶切位点分别加至所得序列的5’和3’端。
对于IMM2521中SIRPαD1-接头-PD-L1抗体轻链，将突变SIRPαD1的编码序列(SEQ ID NO:1)经GS-接头编码序列(SEQ ID NO:3)与IMM2515中PD-L1抗体轻链编码序列(SEQ ID NO:7)的5’端连接。对于IMM2521中的PD-L1抗体重链，将相同的信号肽序列以及Kozak序列加至PD-L1重链编码序列(SEQ ID NO:5)的5’端。
所得的序列经金斯瑞合成，并分别克隆到pMac-H和pMac-L载体中。
实施例2.蛋白表达和纯化
为制备重组蛋白IMM2520和IMM2521，将表达载体经电穿孔导入中国仓鼠卵巢(CHO)细胞(ATCC，Cat#CCL-61)中，之后，使这些CHO细胞接受几轮新霉素压力选择。选出的稳定表达细胞在无血清Balan CD CHO生长A培养基(Irvine Scientific，Cat#94120)中适应。对于蛋白表达，将细胞接种到3升生物反应器中并以流加培养法培养。当细胞活力降到～80％时，收集生物反应器中的细胞培养上清液并通过亲和色谱法进行蛋白纯化。重组蛋白的纯度高于95％，内毒素的量低于0.5U/g。
实施例3.IMM2520和IMM2521与PD-L1以及CD47结合
过表达PD-L1的CHO-PD-L1细胞(公司自制)或自然表达CD47的Jurkat细胞分别在梯度稀释的IMM2520、IMM2521和对照液中4℃孵育1小时。细胞用冷的PBS清洗两遍，之后用结合FITC的针对人IgG-Fc的二抗(Cat#F9512，Sigma)孵育45分钟。细胞清洗两遍，并重悬浮于200ml PBS。之后，细胞用流式细胞仪(Merck Millipore,
Figure PCTCN2022116312-appb-000002
easyCyte 5HT)进行FACS分析。
IMM2520以0.09nM的EC 50值与CHO细胞上的PD-L1结合(图3)，并以0.80nM的EC 50值与Jurkat细胞上的CD47结合(图4)，比传统的单抗原靶向蛋白略差。IMM2521以0.11nM的EC 50值与CHO细胞上的PD-L1结合(图3)，并以0.70nM的EC 50值与Jurkat细胞上的CD47结合(图4)，比传统的单抗原靶向蛋白略差。
实施例4.IMM2520和IMM2521阻断PD-L1与PD-1的相互作用
生物素-hPD1-mFc(SEQ ID NO:22)分别与梯度稀释的IMM2520、IMM2521、IMM2515、以及hIgG1-Fc混合，将混合物添加到含有CD47 +或CD47 -CHO-PD-L1细胞的96孔板中。细胞于4℃孵育45分钟，用PBS洗，并再与结合PE的小鼠抗人CD279(Cat#557946，BD BioScience)于4℃孵育45分钟。细胞清洗后重悬于200ml PBS中，并经FACS分析PD1-Fc-PD-L1结合/相互作用。
如图5A所示，IMM2520、IMM2521和IMM2515均能阻断PD1-mFc与CD47 -PD-L1 +细胞的结合，IC 50值低于1nM。
在PD-L1和CD47双阳性细胞上，如图5B所示，PD-L1和CD47双特异性分子，即IMM2520和IMM2521，显示出比单特异PD-L1抗体IMM2515更高的抑制活性。
实施例5.IMM2520和IMM2521抑制CD47与SIRPα的相互作用
FITC结合的SIRPα-Fc(野生型人SIRPα+人IgG1Fc，SEQ ID NO:23)分别与梯度稀释的IMM2520、IMM2521、IMM01、以及IgG1-Fc混合。混合物添加到含表达CD47的PD-L1 -或PD-L1 +Jurkat细胞的96孔板中，板于4℃孵育45分钟。细胞用PBS洗，并经FACS分析SIRPα-Fc-CD47的相互作用。
如图6A所示，IMM2520抑制SIRPα-Fc与PD-L1 -CD47 +细胞的结合，IC 50值为127.7nM，而IMM2521抑制SIRPα-Fc与CD47 +细胞结合的IC 50值为139.8nM。
对于PD-L1和CD47双阳性的细胞，如图6B所示，PD-L1和CD47双特异性分子，即IMM2520和IMM2521，显示出比单特异PD-L1抗体IMM2515更高的抑制活性。
实施例6.IMM2520针对PD-L1阳性细胞引发高水平抗体依赖性细胞介导的细胞毒性 (ADCC)
CFSE标记的Raji-PD-L1细胞(用作靶向细胞)与稳定表达FcγRIIIa的NK92MI细胞(效应细胞)以1：2混合，且混合的细胞在5％CO 2下与梯度稀释的IMM2520或IMM2515于37℃培养4小时。之后向细胞培养液中加入碘化丙啶(PI)(Cat#P4170，Sigma)，浓度5μg/ml，细胞培养液经FACS分析PI信号。由ADCC造成的细胞裂解百分比基于以下公式进行计算：
％裂解＝(％IMM2515或IMM2520处理的PI阳性细胞-％阴性对照蛋白处理的PI阳性细胞)/(100-％阴性对照蛋白处理的PI阳性细胞)*100
如图7所示，相比于单特异性PD-L1抗体IMM2515，IMM2520引发更高水平的ADCC。
实施例7.IMM2520针对PD-L1阳性细胞引发更高水平的抗体依赖性细胞吞噬(ADCP)
将小鼠巨噬细胞系Ana-1(作为效应细胞)接种至96孔细胞培养板，每孔1x10 5个细胞，并于37℃和5％CO 2培养16-18个小时。Raji-PD-L1细胞(作为靶向细胞)用CFSE标记，并分别与梯度稀释的IMM2520、IMM2515、IMM01、IMM01和IMM2515的组合、以及hIgG1-Fc孵育45分钟。将靶向细胞培养液转移到含Ana-1细胞的板中，Ana-1细胞与Raji-PD-L1细胞的比为1:1。混合物在细胞培养孵育器中培养2小时，之后经FACS分析Ana-1细胞中CFSE的密度。
图8示出，IMM2520对PD-L1 +肿瘤细胞引发高水平的抗体依赖性细胞吞噬(ADCP)。
实施例8.IMM2520显示出强劲的抗肿瘤活性
24只5-7周龄的SCID小鼠在右侧腹皮下注射CT26-hPDL1/hCD47结直肠癌细胞，每只小鼠2x10 6个细胞。当肿瘤体积达到100-150mm 3时，小鼠随机分成4组，每组6只小鼠，分组当天定义为D0。从这一天开始，各组小鼠分别腹膜注射PBS、IMM2505(6.0mg/kg)、IMM2520(6.0mg/kg)、和IMM01(3.0mg/kg)，持续4周，每周2次。在4周后结束给药，持续观察小鼠直至实验结束。PBS组在平均肿瘤体积达到3000mm 3时终止实验，给药组在D60终止实验。每3-4天测量一次肿瘤体积和小鼠体重。
肿瘤体积(V)计算为(长×宽 2)/2。肿瘤生长抑制率(TGI)用以下公式计算：肿瘤生长抑制率＝(1-药物处理组肿瘤体积变化/对照组肿瘤体积变化)×100％。
测试的方案和结果总结在如下表1中。
表1.IMM2520和其他治疗剂的抗肿瘤效果
Figure PCTCN2022116312-appb-000003
如表1以及图9和图10所示，第4组的肿瘤抑制率为97.89％，比其他组高得多，包括用IMM2505处理的那组，其中IMM2505的整体结构和IMM2520很像，包含突变的 SIRPαD1(SEQ ID NO:2)和不同的PD-L1抗体。在US 10,973,878 B2中，IMM2505在小鼠模型中显示出了比单特异性药物组合(即PD-L1抗体加上IMM01)更优的抗肿瘤效果。
具体地，如图9所示，在施用PBS的第1组中，小鼠肿瘤体积不断增长；在第2和第3组中，在分别施用IMM01(突变SIRPαD1-Fc)和IMM2505后，小鼠的肿瘤体积仍呈上升态势，在开始给药后约第40天时开始下降，逐渐降低；而在第4组中，在施用IMM2520后，小鼠肿瘤体积不断下降。以上数据表明，IMM2520在抑制肿瘤方面，能够更快地发挥作用，且总体的抑瘤效果比其他两个给药组好。
如图10所示，第2和第3组的存活率相当，在给药开始后约第40天时降至约80％；第4组的存活率在截止第60天时一直保持在100％。
实施例9.IMM2520同时结合PD-L1和CD47
使用分子作用仪(Gator，Probe Life)来检测IMM2520与CD47以及PD-L1的同时结合。使用抗人IgG探针来捕捉10μg/ml IMM2520，直至响应度达到～1.0nm。随后将探针在缓冲液中漂洗30秒，之后转移至10μg/ml PD-L1-His(图11)或10μg/ml CD47-His液(图12)，直至结合强度达到饱和水平。之后，将探针转移至10μg/ml CD47-His(图11)或10μg/ml PD-L1-His液(图12)，放置120秒。
如图11所示，IMM2520即使在PD-L1结合饱和后，仍可以结合CD47。如图12所示，IMM2520在CD47结合饱和后，仍能够结合PD-L1。
以上结果表明，在与其中之一抗原结合饱和后，IMM2520能够结合另一抗原，表明其能够同时结合PD-L1和CD47。
本申请的序列信息总结如下。
Figure PCTCN2022116312-appb-000004
Figure PCTCN2022116312-appb-000005
Figure PCTCN2022116312-appb-000006
Figure PCTCN2022116312-appb-000007
Figure PCTCN2022116312-appb-000008
Figure PCTCN2022116312-appb-000009
尽管本申请已经结合一个或多个实施方式进行了描述，应当理解的是，本申请并不受限于这些实施方式。本申请中的描述意在涵盖所有变体形式以及等同物，均包含在所附权利要求的主旨和范围内。所有在本文中引用的文献通过引用的方式全部并入本文。
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Claims (15)

  1. 一种重组融合蛋白，包含PD-L1抗体或其抗体片段、以及CD47结合肽，
    其中所述PD-L1抗体或其抗体片段包含重链可变区、重链恒定区、和轻链可变区，重链可变区包含SEQ ID NO:19所示的氨基酸序列，轻链可变区包含SEQ ID NO:20所示的氨基酸序列，重链恒定区具有FcR结合力且与重链可变区的C端连接，
    其中所述CD47结合肽包含信号调节蛋白(SIRP)的胞外Ig样结构域，该信号调节蛋白(SIRP)的胞外Ig样结构域的氨基酸序列如SEQ ID NO:2所示，
    其中所述PD-L1抗体或其抗体片段的各互补位与所述CD47结合肽在构成该互补位的重链可变区或轻链可变区的N端连接，
    所述重组融合蛋白能够同时与CD47、PD-L1以及FcR结合。
  2. 根据权利要求1所述的重组融合蛋白，其中所述PD-L1抗体或其抗体片段的各互补位与所述CD47结合肽在构成该互补位的重链可变区的N端连接。
  3. 根据权利要求1所述的重组融合蛋白，其中所述PD-L1抗体或其抗体片段的各互补位与所述CD47结合肽在构成该互补位的轻链可变区的N端连接。
  4. 根据权利要求1所述的重组融合蛋白，其中所述PD-L1抗体或其抗体片段经接头与所述CD47结合肽连接。
  5. 根据权利要求4所述的重组融合蛋白，其中接头为–(Gly-Gly-Gly-Gly-Ser) 3-(SEQ ID NO:4)。
  6. 根据权利要求1所述的重组融合蛋白，其中重链恒定区包含SEQ ID NO:21所示的氨基酸序列。
  7. 根据权利要求2所述的重组融合蛋白，还包含轻链恒定区，
    其中所述重组融合蛋白包含具有SEQ ID NO:10所示的氨基酸序列的CD47结合肽-接头-PD-L1抗体重链可变区-重链恒定区、以及具有SEQ ID NO:8所示的氨基酸序列的PD-L1抗体轻链可变区-轻链恒定区。
  8. 根据权利要求3所述的重组融合蛋白，还包含轻链恒定区，
    其中所述重组融合蛋白包含具有SEQ ID NO:6所示的氨基酸序列的PD-L1抗体重链可变区-重链恒定区、以及具有SEQ ID NO:16所示的氨基酸序列的CD47结合肽-接头-PD-L1抗体轻链可变区-轻链恒定区。
  9. 一种核酸分子，其编码权利要求1-8中任一项所述的重组融合蛋白。
  10. 一种表达载体，其包含权利要求9所述的核酸分子。
  11. 一种宿主细胞，其包含权利要求10所述的表达载体。
  12. 一种药物组合物，其包含权利要求1-8中任一项所述的重组融合蛋白、权利要求9所述的核酸分子、权利要求10所述的表达载体、或权利要求11所述的宿主细胞，以及至少一种药学上可接受的赋形剂。
  13. 权利要求12所述的药物组合物，还包含至少一种药学上可接受的佐剂。
  14. 权利要求12或13所述的药物组合物在制备用于治疗与CD47和/或PD-L1过表达相关的疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其中所述疾病选自急性髓细胞样白血病(AML)、慢性髓细胞样白血病(CML)、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、膀胱癌、卵巢癌、前列腺癌、肺癌、结肠癌、乳腺癌、胰腺癌和肾细胞癌。
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CN113773401B (zh) 2023-06-20
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