CN112979782B - Polypeptide and application thereof - Google Patents

Polypeptide and application thereof Download PDF

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CN112979782B
CN112979782B CN202110250408.4A CN202110250408A CN112979782B CN 112979782 B CN112979782 B CN 112979782B CN 202110250408 A CN202110250408 A CN 202110250408A CN 112979782 B CN112979782 B CN 112979782B
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variant
polypeptide
ser
amino acid
domain
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CN112979782A (en
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毛新
苏彦景
李光磊
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Shanghai Letu Life Technology Co ltd
Shenzhen Letu Biomedical Co ltd
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Shanghai Letu Life Technology Co ltd
Shenzhen Letu Biomedical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C07K16/2827—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 against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

A polypeptide comprising a signal regulatory protein αd1 variant, or a fragment thereof, said signal regulatory protein αd1 variant having an amino acid mutation relative to a wild type signal regulatory protein αd1 domain, and uses thereof. The signal regulatory protein alpha D1 variant provided by the invention has excellent CD47 binding force, and can be used for treating diseases caused by CD47 over-expression.

Description

Polypeptide and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide and application thereof.
Background
Currently, cancer cells have developed several mechanisms to evade host immune surveillance, including: 1. immune surveillance of T lymphocytes is avoided by high expression of the membrane proteins PD-L1 and PD-L2, wherein both membrane proteins PD-L1 and PD-L2 bind to PD-1 on the surface of T cells, triggering T cell apoptosis; 2. immune monitoring that evades Natural Killer (NK) cells, NKG2D proteins on the surface of NK cells, when bound to MICA/MICB proteins on the surface of cancer cells, can activate NK cells to kill cancer cells, however, cancer cells have developed a mechanism that promotes the detachment of MICA/MICB from cancer cells, which detached MICA/MICB binds to NKG2D, blocking its activation of NK cells; 3. immune surveillance by macrophages was avoided, and almost all cancer cells expressed high levels of CD47 on their surface. However, only PD-L1 or PD-1 monoclonal antibodies are marketed for treating cancers, T cells and macrophages cannot be activated simultaneously, and the immune function of the organism is exerted to realize double killing.
Disclosure of Invention
According to a first aspect, in some embodiments, there is provided a polypeptide comprising a signal regulatory protein alpha (SIRP-alpha) D1 variant, or a fragment thereof, said signal regulatory protein alpha D1 variant having an amino acid mutation relative to a wild type signal regulatory protein alpha D1 domain.
According to a second aspect, in some embodiments there is provided a polypeptide comprising:
(a) A signal regulatory protein αd1 variant, the signal regulatory protein αd1 variant as defined in the first aspect;
(b) An Fc variant comprising an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer is independently a mutant comprising a human IgG1, igG2, or IgG4 Fc region.
According to a third aspect, in some embodiments there is provided a polypeptide comprising: an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer independently belongs to the IgG class and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
According to a fourth aspect, in some embodiments, there is provided an isolated polynucleotide encoding a polypeptide of the first aspect, or a polypeptide of the second aspect, or a polypeptide of the third aspect.
According to a fifth aspect, in some embodiments, there is provided a construct comprising a polynucleotide of the fourth aspect.
According to a sixth aspect, in some embodiments, there is provided an expression system comprising a construct according to the fifth aspect or a polynucleotide according to the fourth aspect integrated with an exogenous source in the genome.
According to a seventh aspect, in some embodiments, there is provided a composition comprising a polypeptide according to the first aspect, or a polypeptide according to the second aspect, or a polypeptide according to the third aspect, or a polynucleotide according to the fourth aspect, and at least one of a pharmaceutically acceptable carrier, diluent, excipient, adjuvant.
According to an eighth aspect, in some embodiments, there is provided a kit comprising a composition according to the seventh aspect, further comprising a container containing the composition according to the seventh aspect.
According to a ninth aspect, in some embodiments there is provided the use of a polypeptide according to the first aspect, or a polypeptide according to the second aspect, or a polypeptide according to the third aspect, or a polynucleotide according to the fourth aspect, or a construct according to the fifth aspect, or an expression system according to the sixth aspect, or a composition according to the seventh aspect, in the manufacture of a medicament for the treatment of a disease caused by overexpression of CD47 and/or PD-L1.
According to the polypeptide and the application thereof in the embodiment, the signal regulatory protein alpha D1 variant provided by the invention has excellent CD47 binding force and can be used for treating diseases caused by over-expression of CD 47.
Drawings
FIG. 1 is a schematic diagram of an antibody structure according to an embodiment;
FIG. 2 is a graph showing the results of protein detection according to one embodiment;
FIG. 3 is a graph of FACS analysis of CD47 binding capacity for one example;
FIG. 4 is a graph of FACS analysis of PD-L1 binding capacity for one example.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. Wherein like elements in different embodiments are numbered alike in association. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "coupled" and "connected," as used herein, are intended to encompass both direct and indirect coupling (coupling), unless otherwise indicated.
The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is conventional in the art. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly in the case of biological systems or methods, the term may refer to values within an order of magnitude, preferably within a factor of 5, and more preferably within a factor of 2. Where specific values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" is intended to be within the acceptable error range for the specific value.
The terminology used herein is for the purpose of describing particular situations only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," has, "" with, "or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.
As used herein, the term "antibody" refers to an intact antibody; antibody fragments, provided that they exhibit the desired biological activity (e.g., epitope binding); a monoclonal antibody; a polyclonal antibody; a monospecific antibody; multispecific antibodies (e.g., bispecific antibodies); an antibody-like protein.
As used herein, the term "antibody variable domain" refers to the portion of the light and heavy chains of an antibody that includes the amino acid sequences of complementarity determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and Framework Regions (FR).
As used herein, the term "linker" refers to a linkage between two elements (e.g., protein domains). In some embodiments, the linker may be a covalent bond or a spacer. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1-200 amino acids) that is present between two polypeptides or polypeptide domains to provide space or flexibility (or both) between the two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is part of the primary sequence of the polypeptide (e.g., linked to the spacer polypeptide or polypeptide domain via a polypeptide backbone).
SIRP and CD47 are defined as follows:
signal regulator proteins (SIRPs) are transmembrane glycoproteins that include three family members, SIRPalpha (CD 172 a), SIRPaleta (CD 172 b) and SIRPalpha (CD 172 g). All three proteins contain similar extracellular regions, but have different intracellular domains. The extracellular region comprises three immunoglobulin-like domains, one Ig-V and two Ig-C domains. The intracellular domain of SIR pα (CD 172 a) comprises two inhibitory signal transduction regions, which can inhibit signal transduction and corresponding cellular functions. The intracellular regions of sirpβ (CD 172 b) and sirpγ (CD 172 g) are very short and contain no signal transduction domains. However, sirpβ (CD 172 b) is able to function as signal transduction via an adapter protein such as DAP 12. SIRP is mainly expressed in macrophagesDendritic Cells (DCs) 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 integrin, thrombospondin-1 and SIRP. CD47 signals "do not eat me" by interacting with sirpa, and can inhibit phagocytosis of macrophages and thereby protect cells such as blood cells from attack by macrophages.
Previous studies have shown that many tumors or cancer cells that overexpress CD47 prevent phagocytosis of cancer cells by macrophages through binding to sirpa on the cell surface of the macrophages. Cancer cells that overexpress CD47 include Acute Myeloid Leukemia (AML), chronic Myeloid Leukemia (CML), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma (NHL), multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, and pancreatic cancer cells. It has been reported that tumor growth can be significantly inhibited by injecting a CD 47-specific antibody that blocks binding of CD47 to sirpa into tumor-bearing mice. When the same antibody is injected into mice bearing human leukemia cells, tumor cells or cancer cells are completely eliminated (theocharis APA, et al 2012).
Herein, PD-L1 refers to programmed death-ligand 1 (Programmed cell death ligand 1), also known as surface antigen cluster 274 (cluster of differentiation, CD 274) or B7 homolog (B7 homolog 1, B7-H1), a protein in humans, encoded by the CD274 gene. PD-L1 is a type I transmembrane protein of 40kDa and is believed to be involved in the suppression of the immune system in certain specific situations (e.g., pregnancy, tissue transplantation, autoimmune diseases, and certain diseases such as hepatitis). The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, triggering antigen-specific cytotoxic T cells (cd8+ Tcell proliferation). And the apoptosis receptor-1 (PD-1) is combined with the apoptosis-ligand 1 (PD-L1) to transmit an inhibitory signal and reduce proliferation of lymph node CD8+ T cells, and the PD-1 can control accumulation of antigen-specific T cells in lymph nodes by regulating Bcl-2 genes.
Herein, PD-L2, i.e. programmed death-ligand 2 or PDCDL2 or B7-DG, is a ligand of PD-1, a member of the B7 family of ligands, and can inhibit T cell proliferation and cytokine production, involved in the development of peripheral autoimmune tolerance.
Herein, PD-1 refers to programmed death receptor 1, also known as CD279, or cluster of differentiation 279.PD-1 is a cell surface receptor about 268 amino acids in length. When bound to PD-L1 or PD-L2, PD-1 can down regulate the immune system and increase self-tolerance by inhibiting T cell inflammatory responses. The inhibitory effect of PD-1 on the immune system prevents autoimmune diseases, but also prevents the immune system from killing cancer cells.
Fc and FcR
The crystallizable section (Fc region) is the tail region of an antibody, a domain that determines the effector function of an antibody (i.e., how an antibody is associated with a particular cellular receptor or other defensin).
Fc receptors (FcR) are proteins on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, and the like. 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, activating the immune system.
Herein, amino acid sequences having at least 80% identity include, but are not limited to, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, and the like.
Regarding therapeutic bispecific or multispecific fusion proteins/antibodies, antibodies targeting a single tumor-associated antigen have limited efficacy due to their limited efficacy. For example, the approved PD-L1 antibody, avelumab (BAVENCIO), had a total remission rate of only 33%. Therefore, there is a need to design and develop therapeutic bispecific or multispecific fusion proteins/antibodies to improve therapeutic efficacy.
Existing therapeutic bispecific or multispecific fusion proteins/antibodies suffer from a number of drawbacks including, e.g., high degree of aggregation, large molecular weight (> 200 kDa) and shorter half-life (< 12 hours, biTE, DART and TandAb), thereby reducing their efficacy.
According to a first aspect, in some embodiments, there is provided a polypeptide comprising a signal regulatory protein alpha (SIRP-alpha) D1 variant, or a fragment thereof, said signal regulatory protein alpha D1 variant having an amino acid mutation relative to a wild type signal regulatory protein alpha D1 domain.
In some embodiments, the signal regulatory protein αd1 variant has an amino acid mutation at residue 70 relative to the wild type signal regulatory protein αd1 domain, and further has at least one of the following amino acid mutations relative to the wild type signal regulatory protein αd1 domain: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, residue 92.
In some embodiments, the wild-type signal regulatory protein αd1 domain has the amino acid sequence of SEQ ID NO:14, and a nucleotide sequence shown in seq id no.
In some embodiments, at least one of the N-terminus, C-terminus of the signal regulatory protein αd1 variant is further linked to at least one of the following amino acid sequences:
1)ASCAWSGVAG；
2)PVVSGPAARATPQH；
alternatively, at least one of the N-terminal and C-terminal of the signal regulatory protein alpha D1 variant is linked with at least one of the amino acid sequences having at least 80% identity to the two amino acid sequences.
In some embodiments, the polypeptide further comprises the following amino acid sequence linked to the N-terminus of the signal regulatory protein αd1 variant: ASCAWSGVAG, or an amino acid sequence having at least 80% identity to the amino acid sequence.
In some embodiments, the polypeptide further comprises the following amino acid sequence linked to the C-terminus of the signal regulatory protein αd1 variant: PVVSGPAARATPQH, or an amino acid sequence having at least 80% identity to the amino acid sequence.
In some embodiments, the polypeptide comprises at least one of the following amino acid sequences:
EEELQX 1 IQPDKSVLVAAGETATLRCTX 2 TSLX 3 PVGPIQWFRGAGPGRX 4 LIYNQX 5 X 6 GX 7 FPRVTTVSDX 8 TKRX 9 NMDFSIRIGNITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPSA(SEQ ID NO：16)；
ASCAWSGVAGEEELQX 1 IQPDKSVLVAAGETATLRCTX 2 TSLX 3 PVGPIQWFRGAGPGRX 4 LIYNQX 5 X 6 GX 7 FPRVTTVSDX 8 TKRX 9 NMDFSIRIGNITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQH(SEQ ID NO：17)；
X 1 i, V or L, X 2 I, A or V, X 3 F, I, S or T, X 4 V, E or L, X 5 R or K, X 6 Is Q, or E, X 7 P, H or R, X 8 T, L, S or G, X 9 Is E, X 10 I or V.
Signal regulatory protein α ("SIRP- α" or "SIRP- α") is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed on the membranes of bone marrow cells. SIRP- α interacts with CD47, a protein widely expressed on many cell types in vivo. The interaction of SIRP- α with CD47 prevents phagocytosis of "autologous" cells that can otherwise be recognized by the immune system. It has been observed that high CD47 expression on tumor cells can play a role as a negative prognostic factor for survival in acute myelogenous leukemia and several solid tumor cancers.
Natural SIRP- α comprises 3 highly homologous immunoglobulin (Ig) -like extracellular domains-D1, D2, and D3. The SIRP-alpha D1 domain ("D1 domain") refers to the membrane distal extracellular domain of SIRP-alpha and mediates SIRP-alpha binding to CD 47. As used herein, the term "SIRP-a polypeptide" refers to any SIRP-a polypeptide or fragment thereof that is capable of binding to CD 47. There are at least ten variants of wild-type human SIRP-a. In some embodiments, the SIRP-a polypeptide comprises a SIRP-a D1 domain. In some embodiments, the SIRP-a polypeptide comprises a wild-type D1 domain, such as SEQ ID NO:14, and those wild-type D1 domains provided in seq id no. In some embodiments, the SIRP-a polypeptide includes a D2 or D3 domain (or both D2 and D3 domains) of wild-type human SIRP-a.
In some embodiments, further comprising a first Fc domain monomer bonded to the N-terminus or C-terminus of the signal regulatory protein αd1 variant, the first Fc domain monomer being a human IgG1, igG2, or IgG4 Fc region.
In some embodiments, the signal regulatory protein αd1 variant is linked to the C-terminus of the first Fc domain monomer.
In some embodiments, the N-terminus of the signal regulatory protein αd1 variant is linked to the C-terminus of the first Fc domain monomer.
Fc domain monomer refers to a polypeptide chain comprising the constant domains of the second and third antibodies (e.g., CH2 and CH 3). In some embodiments, the Fc domain monomer further comprises a hinge domain. In some embodiments, the Fc domain monomer belongs to any immunoglobulin antibody isotype, including IgG, igE, igM, igA and IgD. In addition, in some embodiments, the Fc domain monomers belong to any IgG subtype (e.g., igG1, igG2a, igG2b, igG2c, igG3, and IgG 4). In some embodiments, the Fc domain monomer comprises up to ten changes (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions, or combinations thereof) as compared to the wild-type Fc domain monomer sequence, which changes alter the interaction between the Fc domain and the Fc receptor.
In some embodiments, the first Fc domain monomer comprises at least one mutation relative to a wild-type human IgG1, igG2, or IgG4 Fc region.
In some embodiments, the first Fc domain monomer belongs to the IgG class and comprises at least one amino acid substitution at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, a327, a330, P331, or P329.
In some embodiments, the first Fc domain monomer independently belongs to the IgG class and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, the first Fc domain monomer belongs to the IgG class and comprises at least one amino acid substitution at L234, L235, P329.
In some embodiments, the first Fc domain monomer belongs to an IgG1 Fc region and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, the first Fc domain monomer belongs to a human IgG1 Fc region and contains a) a P329G mutation, and b) at least one of the following amino acid mutations: L234A, L235A, G237A, N297A.
In some embodiments, the first Fc domain monomer has at least one of the following amino acid substitutions relative to the wild-type human IgG1 Fc region: L234A, L235A, P G.
In some embodiments, the first Fc domain monomer has the following amino acid substitutions relative to a wild-type human IgG1 Fc region: L234A, L235A, P G.
In some embodiments, the Fc domain comprising the first Fc domain monomer does not have the ability to affect a biological response to a target cell, or has a reduced ability to affect a biological response to a target cell, which may be effective in reducing side effects of the product.
In some embodiments, the biological response includes, but is not limited to, at least one of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytolysis (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), release of inflammatory mediators, placental transfer, immunoglobulin production.
In some embodiments, a SIRP-a polypeptide or construct provided herein includes a SIRP-a D1 domain or variant thereof linked to a first Fc domain monomer (preferably C-terminal), a second Fc domain monomer (preferably C-terminal), and an antibody heavy chain constant domain, antibody heavy chain variable domain linked to a first Fc domain monomer (typically N-terminal), a second Fc domain monomer (typically N-terminal), wherein the first and second Fc domain monomers combine to form an Fc domain (e.g., a heterodimeric Fc domain). The Fc domain is the protein structure present at the C-terminus of an immunoglobulin. The Fc domain comprises two Fc domain monomers that dimerize by interaction between CH3 antibody constant domains. The wild-type Fc domain forms the smallest structure that binds to Fc receptors (e.g., fcγri, fcγriia, fcγriib, fcγriiia, fcγriiib, and fcγriv).
The Fc domain is not directly involved in binding an antibody to its target, but may be involved in various effector functions, such as antibody involvement in antibody-dependent cytotoxicity. In some embodiments, the Fc domain in a SIRP-a polypeptide or construct of the disclosure comprises an amino acid substitution, addition or insertion, deletion, or any combination thereof, that results in reduced effector function, such as reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cytolysis (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, a SIRP-a polypeptide or construct of the disclosure is characterized by reduced binding (e.g., little or no binding) to a human Fc receptor and reduced binding (e.g., little or no binding) to complement protein C1 q. In some embodiments, a SIRP-a construct of the disclosure is characterized by reduced (e.g., minimal) or no binding to human fcyri, fcyriia, fcyriib, fcyriiib, or any combination thereof, and C1 q. To alter or reduce antibody dependent effector functions, such as ADCC, CDC, ADCP or any combination thereof, in some embodiments the Fc domain in the SIRP-a constructs disclosed herein belongs to the IgG class and comprises one or more amino acid substitutions at EU index numbering of E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, a327, a330, P331, or P329 (according to Kabat (Sequences of Proteins of Immunological Interest, 5 th edition, publicHealth Service, national Institutes of Health, bethesda, MD. (1991)).
Antibodies targeting cell surface antigens have been known in the art to trigger immune stimulation and effector functions associated with Fc receptor (FcR) binding on immune cells. There are many Fc receptors specific for a particular class of antibodies, including IgG (gamma receptor), igE (eta receptor), igA (alpha receptor), igM (mu receptor), and the like. Binding of the Fc region to Fc receptors on the cell surface can trigger a number of biological responses including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, also known as ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, also known as ADCC), and release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In addition, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement can be important for the lysis of cellular pathogens. However, activation of complement can also stimulate inflammatory responses and may also be involved in autoimmune hypersensitivity reactions or other immune disorders. Variant Fc regions with reduced or eliminated ability to bind to certain Fc receptors may be useful in the development of therapeutic antibodies and Fc-fusion polypeptide constructs that function by targeting, activating, or neutralizing ligand functions without damaging or destroying local cells or tissues.
In some embodiments, the signal regulatory protein αd1 variant is linked to the first Fc domain monomer by a linker.
In some embodiments, the signal regulatory protein αd1 variant is linked to the C-terminus of the first Fc domain monomer by a linker.
In some embodiments, the linker is a short peptide containing only G (Gly, i.e., glycine), S (i.e., ser, serine).
In some embodiments, the linker is a short peptide containing 5-30 amino acid residues.
In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, a second polypeptide that can be linked to a first Fc domain monomer is also included, the second polypeptide comprising a second Fc domain monomer, the first Fc domain monomer being capable of being linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain dimer.
In some embodiments, the signal modulating protein αd1 variant is bonded to the N-terminus or C-terminus of at least one of the first Fc domain monomer, the second Fc domain monomer.
In some embodiments, the signal regulatory protein αd1 variant is bound to the C-terminus of at least one of the first Fc domain monomer, the second Fc domain monomer.
In some embodiments, the first Fc domain monomer, the second Fc domain monomer are each linked to the C-terminus of the signal regulatory protein αd1 variant.
In some embodiments, the first Fc domain monomer, the second Fc domain monomer are the same or different.
In some embodiments, the first Fc domain monomer, the second Fc domain monomer are the same.
In some embodiments, the first Fc domain monomer is linked to a third polypeptide.
In some embodiments, the second Fc domain monomer is linked to a fourth polypeptide.
In some embodiments, the first Fc domain monomer is N-terminal to a third polypeptide.
In some embodiments, the second Fc domain monomer is N-terminal to a fourth polypeptide.
In some embodiments, at least one of the third polypeptide, fourth polypeptide comprises an antibody heavy chain variable domain VH.
In some embodiments, at least one of the third polypeptide, the fourth polypeptide comprises an antibody heavy chain constant domain CH1.
In some embodiments, at least one of the third polypeptide, the fourth polypeptide comprises an antibody heavy chain variable domain VH and an antibody heavy chain constant domain CH1.
In some embodiments, the third polypeptide, fourth polypeptide are the same or different;
In some embodiments, the third polypeptide, fourth polypeptide are the same;
in some embodiments, at least one of the third polypeptide, the fourth polypeptide comprises SEQ ID NO:2, or an amino acid sequence having at least 80% identity thereto.
In some embodiments, at least one of the third polypeptide, the fourth polypeptide comprises SEQ ID NO:3, or an amino acid sequence having at least 80% identity thereto.
In some embodiments, at least one of the third polypeptide, the fourth polypeptide, and the fourth polypeptide, in sequence, comprises, from the N-terminus to the C-terminus, SEQ ID NO: 2. SEQ ID NO:3, or an amino acid sequence as set forth in SEQ ID NO: 2. SEQ ID NO:3, and an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no.
In some embodiments, the third polypeptide, the fourth polypeptide each independently comprises, in order from the N-terminus to the C-terminus, SEQ ID NO: 2. SEQ ID NO:3, or an amino acid sequence as set forth in SEQ ID NO: 2. SEQ ID NO:3, and an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no.
In some embodiments, SEQ ID NO:3 is linked to the N-terminus of the Fc domain monomer.
In some embodiments, at least one of the antibody variable domain contained in the third polypeptide linked on the first Fc domain monomer, the antibody variable domain contained in the fourth polypeptide linked on the second Fc domain monomer targets an antigen expressed on a cell.
In some embodiments, the cell is a cancer cell.
In some embodiments, the antibody variable domain targets a cell surface protein involved in immune cell regulation.
Antibody variable domains refer to the portions of the antibody light and heavy chains that comprise the amino acid sequences of complementarity determining regions (CDRs, e.g., CDR L1, CDRL2, CDR L3, CDR H1, CDR H2, and CDR H3) and Framework Regions (FR). The variable domains of an antibody may confer the ability of the antibody to bind to a particular antigen. Many different antibody variable domain molecules can be constructed. In some embodiments, antibody variable domain molecules used include, but are not limited to, single chain Fv.
In some embodiments, the third polypeptide, fourth polypeptide independently comprises a therapeutic protein.
In some embodiments, the therapeutic protein is a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulatory agent.
In some embodiments, at least one of the third polypeptide, the fourth polypeptide has a first signal peptide bonded to the N-terminus or the C-terminus.
In some embodiments, the first signal peptide is linked to the N-terminus of at least one of the third polypeptide and the fourth polypeptide.
In some embodiments, the first signal peptide comprises an IgG1 heavy chain of an animal.
In some embodiments, the first signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:1, or an amino acid sequence having at least 80% identity thereto.
In some embodiments, the signal regulatory protein αd1 variant can bind to CD47.
In some embodiments, the polypeptide of the invention has a longer half-life than BiTE.
In some embodiments, the polypeptides of the invention are of fully symmetrical molecular structure, with no risk of mismatch.
According to a second aspect, in some embodiments there is provided a polypeptide comprising:
(a) A signal regulatory protein αd1 variant, the signal regulatory protein αd1 variant being as defined in the first aspect;
(b) An Fc variant comprising an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer is independently a mutant comprising a human IgG1, igG2, or IgG4 Fc region.
In some embodiments, the signal regulatory protein αd1 variant has an amino acid mutation at residue 70 relative to the wild type signal regulatory protein αd1 domain, and further has at least one of the following amino acid mutations relative to the wild type signal regulatory protein αd1 domain: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, residue 92.
In some embodiments, the wild-type signal regulatory protein αd1 domain has the amino acid sequence of SEQ ID NO: 13.
In some embodiments, at least one of the N-terminus, C-terminus of the signal regulatory protein αd1 variant is further linked to at least one of the following amino acid sequences:
1)ASCAWSGVAG；
2)PVVSGPAARATPQH；
alternatively, at least one of the N-terminal and C-terminal of the signal regulatory protein alpha D1 variant is linked with at least one of the amino acid sequences having at least 80% identity to the two amino acid sequences.
In some embodiments, the polypeptide further comprises the following amino acid sequence linked to the N-terminus of the signal regulatory protein αd1 variant: ASCAWSGVAG, or an amino acid sequence having at least 80% identity to the amino acid sequence.
In some embodiments, the polypeptide further comprises the following amino acid sequence linked to the C-terminus of the signal regulatory protein αd1 variant: PVVSGPAARATPQH, or an amino acid sequence having at least 80% identity to the amino acid sequence.
In some embodiments, the polypeptide comprises at least one of the following amino acid sequences:
EEELQX 1 IQPDKSVLVAAGETATLRCTX 2 TSLX 3 PVGPIQWFRGAGPGRX 4 LIYNQX 5 X 6 GX 7 FPRVTTVSDX 8 TKRX 9 NMDFSIRIGNITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPSA(SEQ ID NO：16)；
ASCAWSGVAGEEELQX 1 IQPDKSVLVAAGETATLRCTX 2 TSLX 3 PVGPIQWFRGAGPGRX 4 LIYNQX 5 X 6 GX 7 FPRVTTVSDX 8 TKRX 9 NMDFSIRIGNITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQH(SEQ ID NO：17)；
X 1 i, V or L, X 2 I, A or V, X 3 F, I, S or T, X 4 V, E or L, X 5 R or K, X 6 Is Q, or E, X 7 P, H or R, X 8 T, L, S or G, X 9 Is E, X 10 I or V.
In some embodiments, the signal regulatory protein αd1 variant is linked to the C-terminus of the first Fc domain monomer.
In some embodiments, the N-terminus of the signal regulatory protein αd1 variant is linked to the C-terminus of the Fc domain monomer.
In some embodiments, each Fc domain monomer independently belongs to the IgG class and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, each Fc domain monomer independently belongs to an IgG1 Fc region and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, each Fc domain monomer independently belongs to the IgG class and comprises at least one amino acid substitution at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, a327, a330, P331, or P329.
In some embodiments, each Fc domain monomer independently belongs to the IgG class and comprises at least one amino acid substitution at L234, L235, P329.
In some embodiments, each Fc domain monomer independently belongs to a human IgG1 Fc region and comprises at least one amino acid substitution at L234, L235, P329.
In some embodiments, each Fc domain monomer independently belongs to a human IgG1 Fc region and contains a) a P329G mutation, and b) at least one of the following amino acid mutations: L234A, L235A, G237A, N297A.
In some embodiments, at least one of the Fc domain monomers is a human IgG1 Fc region consisting of the mutation L234A, L235A, G237A, N297A, P329G.
In some embodiments, at least one of the Fc domain monomers is a human IgG1 Fc region consisting of the mutation L234A, L235A, P329G.
In some embodiments, each Fc domain monomer independently has the following amino acid substitutions relative to the wild-type human IgG1 Fc region: L234A, L235A, P G.
In some embodiments, each Fc domain monomer independently comprises SEQ ID NO:5-6, or at least one of the amino acid sequences set forth in SEQ ID NO:5-6, wherein at least one of the amino acid sequences shown in figures 1-6 has an amino acid sequence with at least 80% identity.
In some embodiments, each Fc domain monomer independently comprises SEQ ID NO:4-6, or at least one of the amino acid sequences set forth in SEQ ID NO:4-6, wherein at least one of the amino acid sequences shown in figures 4-6 has an amino acid sequence having at least 80% identity.
In some embodiments, each Fc domain monomer independently comprises SEQ ID NO: 4-6.
In some embodiments, the two Fc domain monomers are identical.
In some embodiments, the Fc variant exhibits an elimination or reduced binding to an fcγ receptor compared to a wild-type version of a human IgG Fc region.
In some embodiments, the IgG1 Fc variant exhibits an elimination or a reduction in binding to CD16a, CD32b, CD32c, and CD64 fcγ receptor as compared to the wild-type version of the human IgG1 Fc region.
In some embodiments, the IgG1 Fc variant is present at greater than about 5x10 -6 The KD of M binds to fcγ receptor.
In some embodiments, the IgG1 Fc variant exhibits an elimination or reduced binding to C1q compared to the wild-type version of the human IgG1 Fc fusion.
In some embodiments, the signal regulatory protein αd1 variant can bind to CD 47.
In some embodiments, the Fc variant is linked to an antigen binding portion.
In some embodiments, the N-terminal of the Fc variant is linked to an antigen binding moiety.
In some embodiments, the Fc variant is linked to a fifth polypeptide that binds to PD-L1.
In some embodiments, the N-terminal of the Fc variant is linked to a fifth polypeptide that can bind to PD-L1.
In some embodiments, the fifth polypeptide comprises at least one of an antibody heavy chain first constant domain (CH 1), an antibody heavy chain variable domain (VH), an antibody light chain constant domain (CL), an antibody light chain variable domain (VL).
In some embodiments, the fifth polypeptide comprises SEQ ID NO: 2. 3, 10 or 11, or an amino acid sequence having at least 80% identity to at least one of the four amino acid sequences set forth above.
In some embodiments, the pentapeptide further comprises a second signal peptide that can be linked to an antibody light chain variable domain (VL).
In some embodiments, the second signal peptide is linked to the N-terminus of an antibody light chain variable domain (VL).
In some embodiments, the second signal peptide bond comprises SEQ ID NO:9, or an amino acid sequence having at least 80% identity thereto.
According to a third aspect, in some embodiments there is provided a polypeptide comprising: an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer independently belongs to the IgG class and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, each Fc domain monomer independently belongs to an IgG1 Fc region and comprises a) a mutation at P329, and b) at least one of the following position mutations: e233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331.
In some embodiments, each Fc domain monomer independently belongs to a human IgG1 Fc region and contains a) a P329G mutation, and b) at least one of the following amino acid mutations: L234A, L235A, G237A, N297A.
In some embodiments, wherein each Fc domain monomer is independently selected from the human IgG1 Fc region consisting of the mutations L234A, L235A, P329G.
In some embodiments, the two Fc domain monomers are identical.
In some embodiments, the Fc variant exhibits an elimination or a reduction in binding to fcγ receptor compared to the wild-type version of the human IgG Fc region.
In some embodiments, the Fc variant exhibits an elimination or a reduction in binding to CD16a, CD32b, CD32c, and CD64 fcγ receptor as compared to the wild-type version of the human IgG Fc region.
In some embodiments, wherein the Fc variant exhibits an elimination or reduced binding to C1q compared to the wild-type version of the human IgG Fc fusion.
In some embodiments, the Fc variant exhibits an abrogated or reduced binding to CD16a, CD32b, CD32c, and CD64 fcγ receptor as compared to the wild-type version of the human IgG1 Fc region.
In some embodiments, the Fc variant exhibits an elimination or reduced binding to C1q compared to the wild-type version of the human IgG1 Fc fusion.
In some embodiments, the Fc variant binds to an Fcγ receptor with a KD of greater than about 5x 10-6M.
In some embodiments, a polypeptide that binds CD47 is also included.
In some embodiments, wherein the Fc variant exhibits an elimination or a reduction in binding to fcγ receptor compared to a wild-type version of the human IgG Fc region.
In some embodiments, the CD 47-binding polypeptide does not cause acute anemia in rodents and non-human primates.
In some embodiments, the CD 47-binding polypeptide does not cause acute anemia in humans.
In some embodiments, the CD 47-binding polypeptide is a signal regulatory protein alpha (SIRP-alpha) polypeptide or fragment thereof.
In some embodiments, wherein the signal regulatory protein a polypeptide comprises a SIRP-a D1 variant, the SIRP-a D1 variant being as defined in the first aspect.
According to a fourth aspect, in some embodiments, there is provided an isolated polynucleotide encoding a polypeptide of the first aspect, or a polypeptide of the second aspect, or a polypeptide of the third aspect.
In some embodiments, the nucleotide encoding the polypeptide comprises one cleavage site at each end.
In some embodiments, the nucleotide encoding the antibody heavy chain of the polypeptide comprises a Kozak sequence located between the 5' cleavage site and the nucleotide encoding the first signal peptide.
In some embodiments, the Kozak sequence is a sequence present in eukaryotic mRNA that plays an important role in the initiation of translation. Specifically, a Kozak sequence is a nucleic acid sequence located behind the 5 'cap structure of eukaryotic mRNA that can bind to a translation initiation factor to mediate translation initiation of mRNA containing the 5' cap structure. Corresponding to the SD sequence of prokaryotes.
In some embodiments, the Kozak sequence contains the following nucleotide sequence: GCCACC.
In some embodiments, the 5 'end of the nucleotide encoding the heavy chain of the antibody of the polypeptide contains a HindIII cleavage site and the 3' end contains an EcoRI cleavage site.
In some embodiments, the 5 'end of the nucleotide encoding the antibody light chain of the polypeptide contains an XbalI cleavage site and the 3' end contains a NotI cleavage site.
In some embodiments, the nucleotide encoding the antibody light chain of the polypeptide comprises a Kozac sequence located between the 5' cleavage site and the nucleotide encoding the second signal peptide.
In some embodiments, the Kozac sequence contains the following nucleotide sequence: GCCACC.
According to a fifth aspect, in some embodiments, there is provided a construct comprising a polynucleotide of the fourth aspect. The construct may generally be obtained by inserting the isolated polynucleotide into a suitable vector, which may be a phage, plasmid, viral vector or artificial chromosome, such as a bacterial or yeast artificial chromosome, which may be selected by a person skilled in the art. In other words, the vectors of embodiments of the invention comprise a polynucleotide of interest capable of being expressed in a host cell or an isolated fraction thereof. Vectors are also generally suitable as cloning vectors, i.e.replicable in microbial systems; cloning vectors may be designed for replication in one host, while constructs are designed for expression in a different host. Vectors comprising the polypeptides and proteins of embodiments of the invention may also comprise a selectable marker for propagation or selection in a host cell. The vector may be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques.
According to a sixth aspect, in some embodiments, there is provided an expression system comprising a construct according to the fifth aspect or a polynucleotide according to the fourth aspect integrated with an exogenous source in the genome. The expression system may be a host cell which may express a polypeptide according to the first aspect, or a polypeptide according to the second aspect, or a polypeptide according to the third aspect. In another embodiment of the invention, the host cell may be a eukaryotic cell and/or a prokaryotic cell, more particularly a mouse cell, a human cell, etc.
According to a seventh aspect, in some embodiments, there is provided a composition comprising a polypeptide according to the first aspect, or a polypeptide according to the second aspect, or a polypeptide according to the third aspect, or a polynucleotide according to the fourth aspect, and at least one of a pharmaceutically acceptable carrier, diluent, excipient, adjuvant.
The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used in this specification includes liquid or solid fillers and may also include, but is not limited to, at least one of solvents and encapsulating materials; and refers to a pharmaceutically or physiologically acceptable material, composition, substance, or medium.
According to an eighth aspect, in some embodiments, there is provided a kit comprising a composition according to the seventh aspect.
In some embodiments, the composition of the seventh aspect is further included in a container.
According to a ninth aspect, in some embodiments there is provided the use of a polypeptide according to the first aspect, or a polypeptide according to the second aspect, or a polypeptide according to the third aspect, or a polynucleotide according to the fourth aspect, or a construct according to the fifth aspect, or an expression system according to the sixth aspect, or a composition according to the seventh aspect, in the manufacture of a medicament for the treatment of a disease caused by overexpression of CD47 and/or PD-L1.
In some embodiments, the disease includes, but is not limited to, at least one of Acute Myeloid Leukemia (AML), chronic Myeloid Leukemia (CML), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma (NHL), multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, renal cell carcinoma.
In some embodiments, the disease includes, but is not limited to, at least one of crohn's disease, allergic asthma, rheumatoid arthritis.
In some embodiments, the recombinant fusion proteins provided herein can bind to CD47, PD-L1 simultaneously, but do not bind to Fc receptors, or have reduced Fc receptor binding.
In some embodiments, the polypeptide containing the signal regulatory protein alpha D1 variant and the Fc variant can target the PD-L1 antigen and the CD47 molecule at the same time, break through the limitation of single antibody independent treatment, have stronger double antibody functions, remove ADCC function, reduce possible side effects of the product, and can be cloned to an expression vector at the same time, thereby improving the expression efficiency.
In some embodiments, the binding of a signal regulatory protein alpha (SIRP-alpha) D1 variant to CD47 on a cancer cell or tumor cell can block the interaction of CD47 with SIRP on a macrophage, thus relieving SIRP-mediated inhibition signal examination of macrophages.
In some embodiments, the polypeptides provided herein are PD-L1 antibodies, the antigen binding (Fab) portion (or paratope) of which can bind to PD-L1 on the surface of a cancer or tumor cell, blocking the interaction of PD-L1 with PD-1 on the surface of a T cell, thereby relieving examination of T cells for PD-1 mediated inhibition signals.
The term "antigen binding portion" of an antibody (or simply "antibody portion") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-L1). It has been demonstrated that the antigen binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include: (i) Fab fragments, i.e. monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab') 2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (v) dAb fragments consisting of VH domains (Ward et al (1989) Nature 341:544-546); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made into one protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al (1988) Science 242:423-426; and Huston et al (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883). Such single chain antibodies are also included within the term "antigen binding portion" of an antibody. These antibody fragments are obtained by conventional techniques well known to those skilled in the art and screened for utility in the same manner as the whole antibody.
In some embodiments, the invention also provides polynucleotides encoding the recombinant fusion proteins, expression vectors comprising the polynucleotides, methods of making the recombinant proteins, and methods of using the recombinant proteins to treat diseases caused by overexpression of CD47 and/or PD-L1.
In some embodiments, the PD-L1/SIRP alpha double antibody provided by the invention can target PDL1 antigen and CD47 molecules at the same time, breaks through the limitation of single antibody independent treatment, and has stronger double antibody function.
In some embodiments, the PD-L1/SIRPalpha diabodies provided by the invention adopt the FC segment of IgG1, so that ADCC functions (antibody-dependent, cell-mediated cytotoxicity, antibody dependent cell-mediated cytotoxicity, abbreviated as ADCC) are removed, and possible side effects of the product are reduced.
ADCC, antibody-dependent cell-mediated cytotoxicity (ADCC), refers to the binding of the Fab fragment of an antibody to an epitope of a virus-infected or tumor cell, and the binding of the Fc fragment to an FcR on the surface of a killer cell (NK cell, macrophage, etc.), which mediates direct killing of the killer cell to the target cell.
In some embodiments, PDL 1/SIRPalpha diabody sequences are cloned into an expression vector at the same time, thereby improving expression efficiency.
In some embodiments, the invention includes the following stages:
the first stage: bispecific antibody structural design was performed. PDL1 and SIRP alpha mutants are respectively constructed, and then double-antibody vector construction and identification are carried out. Transformation experiments were performed to extract plasmids.
And a second stage: and (5) stable transgenic plant construction. Plasmid transfection of CHO-K1 cells constructs a transient expression system, followed by the construction of a stable expression system.
And a third stage: and (5) purifying the antibody. Purifying by using ProteinA, purifying the transient system, and performing primary analysis and identification; and then carrying out purification process development on the stable transgenic plant, and finally purifying to obtain qualified stock solution.
Fourth stage: and (5) establishing an analysis method. Development of an analysis method is carried out aiming at the double-antibody molecules, and a related detection method is established.
Fifth stage: antibody function verification. In vitro functional assays include binding capacity assays for diabodies to target cells and detection of TDCC killing activity. In vivo functional assays include anti-tumor activity assays and the like.
In some embodiments, the invention comprises the steps of:
(1) The SIRPalpha fusion protein is connected at the C end of the PDL1 antibody, and the heavy chain and the light chain are respectively cloned into the same expression vector containing two promoters. So that the constructed expression vector successfully expresses the target protein.
(2) An effective stable transgenic (stable transfected) cell strain screening method is established. After screening the monoclonal cell strain, detecting whether the molecular weight of the target clone is about 180KD by adopting an immunoblotting experimental technology, and judging whether stable rotation is successful.
(3) The Y-trap has a completely symmetrical molecular structure, and the purification process is simple without the problem of heterodimer. Purifying by using ProteinA to prepare the standard product meeting the requirements.
(4) A reliable analysis method is established.
In some embodiments, PSK001 (QuaCell, cat.No.A13201) expression vectors are constructed, transiently transfected into ExpiCHO-S TM Cells (Thermo, cat. No. A29127) CellsIn the strain, the cultivation period was 7 days and the cultivation temperature was 32 ℃. When the cell viability was reduced below 80%, the reaction in the reactor was terminated, the cell culture supernatant was collected, and affinity purification was performed using Hitrap Mabselect SuRe LX mL (GE, cat.No.17547401) pre-packed column, with the purity of recombinant protein being higher than 95%.
Example 1
Construction of vector expressing recombinant protein CLA014
The full-length coding sequence of recombinant fusion protein CLA014 was designed artificially.
As shown in fig. 1, the recombinant fusion protein CLA014 in this example has a schematic structure, which includes two recombinant heavy chain monomers on the inner side and two recombinant light chain monomers on the outer side, wherein the two recombinant heavy chain monomers on the inner side are linked by disulfide bonds to form a dimer.
For each recombinant heavy chain monomer, from N-terminus to C-terminus, there is in order an antibody heavy chain variable domain (VH), an antibody heavy chain first constant domain (CH 1), a HINGE (HINGE), an antibody heavy chain second constant domain (CH 2), an antibody heavy chain third constant domain (CH 3), a linker, a sirpa D1 variant.
For each recombinant light chain monomer, from N-terminus to C-terminus, there is an antibody light chain variable domain, an antibody light chain constant domain in sequence.
The recombinant light chain constant domain (CL) is linked to the recombinant heavy chain constant domain (CH 1-CH2-CH 3) by disulfide bonds.
In fig. 1, sirpa D1 variants can bind to CD47, with the antigen-binding portion formed by the variable domain linkages of the heavy and light chains of the antibody (i.e., the antigen-binding portion formed by the VH and VL regions linkages of fig. 1) being the PD-L1 binding region.
Specifically, for the heavy chain, the coding sequence of the first extracellular domain of sirpa (sirpa D1) variant was linked to the 3' end of the heavy chain Fc-segment coding sequence of CLA014 by a GS linker. 57 nucleotides of a signal peptide encoding a mouse IgG1 heavy chain (i.e., a first signal peptide) were added to the 5 'end of the antibody heavy chain constant domain (VH) coding sequence, and a Kozak sequence was added to the 5' end of the signal peptide coding sequence. Finally, hindIII and EcoRI restriction sites were added to the 5 'and 3' ends of the resulting sequence, respectively.
For the light chain, another signal peptide coding sequence (i.e., a second signal peptide coding sequence) and a Kozac sequence were used, but Xbal and NotI restriction sites were added at the 5 'and 3' ends of the resulting sequence, respectively. The two resulting sequences were synthesized by Huada gene (ID #: C5119FJ290-CL 004-04) and cloned into pSK001 vector, respectively. The pSK001 vector was purchased from the manufacturer: zhongshan Kang Tian is available from biotechnology limited; vector original name QuaCell pKS001, cat: A13201.
from N-terminal to C-terminal, heavy chain fragment amino acid sequence (insertion site HindIII, ecoRI):
the amino acid sequence of the first signal peptide (also called the chain signal peptide) is as follows:
MELGLSWIFLLAILKGVQC(SEQ ID NO：1)。
the amino acid sequence of the VH segment is as follows:
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS(SEQ ID NO：2)。
the amino acid sequence of the CH1 segment is as follows:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQ ID NO：3)。
in this embodiment, the monomer segment of the Fc domain is hinge+ch2+ch3, and the amino acid sequence of each partial region is specifically as follows:
the amino acid sequence of the HINGE (HINGE) is as follows:
EPKSCDKTHTCPPCP(SEQ ID NO：4)。
the amino acid sequence of the CH2 segment is as follows:
the amino acid sequence of the CH3 segment is as follows:
for the Fc domain monomer segment, the two AA amino groups indicated by the single straight line drawn below in the CH2 and CH3 segments are mutant amino groups, specifically the mutation L234A, L a (i.e., with an amino acid mutation at residues 234, 235 relative to the wild-type IgG1 Fc region, from LL amino group to AA amino group); the G amino group marked by a single wavy line is mutant P329G (i.e., relative to the wild-type heavy chain, there is an amino acid mutation at residue 329, in this example, P amino group is mutated to G amino group), and the Fc segment is mutated in the three foregoing places, thereby achieving the function of removing ADCC; the terminal amino acid A marked by double straight lines is mutated from K to A, so that oxidation and shedding are prevented. The Fc region amino acid residue positions 234, 235, 329 mentioned herein may be specified according to the EU numbering system of Kabat et al, 1991.
The amino acid sequence of the monomer segment of the Fc domain consisting of the hinge +CH2+CH3 is as follows:
EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO：18)。
the amino acid sequence of the GS Linker (LINK) is as follows:
GGGGSGGGGSGGGGSGGGGSG(SEQ ID NO：7)。
the amino acid sequence of the first extracellular domain (sirpa D1) variant is as follows:
ASCAWSGVAGEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRENMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQH(SEQ ID NO：8)。
from the N-terminus to the C-terminus, the antibody light chain amino acid sequence comprises, in order, a second signal peptide (also known as a light chain signal peptide), a light chain variable region (VL), and a light chain constant region (CL).
The method comprises the following steps:
a second signal peptide:
MDMRVPAQLLGLLLLWLSGARC(SEQ ID NO：9)。
light chain variable region (VL):
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPTFGQGTKVEIK(SEQ ID NO：10)。
light chain constant region (CL):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：11)。
the nucleotide sequence of the heavy chain of the antibody is specifically as follows:
SEQ ID NO:11, comprising, in order from the 5 'end to the 3' end, a HindIII cleavage site, a Kozak sequence, a first signal peptide coding sequence, a VH coding sequence, a CH1 coding sequence, a hinge coding sequence, a CH2 coding sequence, a CH3 coding sequence, a GS linker coding sequence, a sirpa variant coding sequence, a translation termination codon, an EcoRI cleavage site, each of which is set forth in SEQ ID NO:11, for example, the first underlined wavy line indicates a HindIII cleavage site, the first underlined wavy line indicates a Kozac sequence, the second underlined wavy line indicates a first signal peptide coding sequence, the second underlined wavy line indicates a VH coding sequence, and so on.
From the 5 'end to the 3' end, the nucleotide sequence of the antibody light chain sequentially comprises an XbalI enzyme cutting site, a Kozac sequence, a second signal peptide coding sequence, a light chain variable region (VL) coding sequence, a light chain constant region (CL) coding sequence, a stop codon and a NotI enzyme cutting site, which are marked by a lower single wavy line and a lower single straight line interval, and the nucleotide sequence is specifically as follows:
SEQ ID NO:13, from the 5 'end to the 3' end, the first underlined nucleotide sequence is an XbalI cleavage site, the first underlined nucleotide sequence is a Kozac sequence, the second underlined nucleotide sequence is a second signal peptide coding sequence, the second underlined nucleotide sequence is a light chain variable region (VL) coding sequence, the third underlined nucleotide sequence is a light chain constant region (CL) coding sequence, the third underlined nucleotide sequence is a stop codon, and the fourth underlined nucleotide sequence is a NotI cleavage site.
PDL 1-sirpa-Native refers to wild-type sirpa D1, and according to the result of PDL 1-sirpa-Native flow analysis, the amino acid sequence of wild-type sirpa D1 from N-terminus to C-terminus is as follows:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSA(SEQ ID NO：14)。
The nucleotide sequence of wild type sirpa D1 is as follows:
5’-GAAGAAGAACTGCAAGTGATTCAGCCCGACAAGTCCGTGCTGGTGGCCGCTGGAGAAACAGCTACATTGAGATGTACAGCTACATCCCTGATCCCCGTGGGCCCTATTCAATGGTTCAGGGGAGCTGGACCTGGAAGGGAACTGATTTACAACCAGAAGGAGGGCCACTTCCCCAGGGTGACAACAGTGTCTGATCTGACAAAAAGGAACAACATGGACTTCTCCATCAGGATCGGCAACATCACCCCCGCCGATGCTGGAACATATTATTGTGTGAAGTTCCGGAAGGGCTCCCCCGACGATGTGGAGTTTAAATCCGGAGCTGGCACAGAGCTGTCCGTGAGGGCTAAACCCTCCGCT-3’(SEQ ID NO：15)。
example 2
Protein expression and purification
To prepare recombinant protein CLA014, 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 MSX pressure selection. Selected stable expressing cells were adapted in serum-free CD04 CHO growth a medium (IrvineScientific, cat#94120). For protein expression, cells were inoculated into a 3 liter bioreactor and cultured in a fed-batch culture. When the cell viability was reduced to about 80%, the reaction in the reactor was terminated, and the cell culture supernatant was collected and protein purified by affinity chromatography. The purity of the recombinant protein is higher than 90%. FIG. 2 is a graph showing the results of protein detection, and in FIG. 2, the right-hand value indicates the molecular weight of the protein, including non-reduced groups and reduced groups.
The following is described for each group of fig. 2:
culture (natural): when the SIRP alpha sequence is a natural sequence, transiently transfecting the SIRP alpha sequence into CHO cells, and sampling and detecting when the viability of the cultured living cells is reduced to about 80%;
flow through (natural): when SIRP alpha sequence is natural sequence, transiently transfecting into CHO cells, and when the viability of cultured living cells is reduced to about 80%, carrying out protein A loading and purification, collecting a flow-through sample, sampling and detecting;
Culture (mutation): when the SIRP alpha sequence is a SIRR alpha variant sequence, transiently transfecting the SIRP alpha variant sequence into CHO cells, and sampling and detecting when the viability of the cultured living cells is reduced to about 80%;
flow through (mutation): when the SIRPalpha sequence is a SIRRalpha variant sequence, transiently transfecting the SIRPalpha to CHO cells, and when the viability of the cultured living cell is reduced to about 80%, carrying out protein A loading and purification, collecting a flow-through sample, and sampling and detecting;
eluent (natural): when the activity of cultured living cells is reduced to about 80%, protein A is loaded and purified, and samples are collected by eluting and are sampled and detected;
eluent (mutation): when the SIRPalpha sequence is the SIRRapha variant sequence, the SIRPalpha is transiently transfected into CHO cells, and when the viability of the cultured living cell is reduced to about 80%, protein A loading and purification are carried out, and samples are collected by eluting and sampling detection.
As can be seen from FIG. 2, the molecular weight of the target clone was around 180KD, indicating successful transformation.
Example 3
CLA014 binds to PD-L1 or CD47
Jurkat cells highly expressing CD47 were incubated with CLA014 at 4 ℃ for 1 hour, positive control Jurkat cells were incubated with sirp α -his (11612-H08H, san-a) at 4 ℃ for 1 hour; after the incubation was completed, the cells were washed twice with pre-chilled PBS, after which the experimental group was incubated with APC (Allophycocyanin) labeled secondary antibody against human IgG Fc (Biolegend, cat# 409306) for 45 minutes, and the control group was incubated with APC labeled secondary antibody against his (Biolegend, cat# 362605) for 45 minutes; after incubation was completed, washed twice with PBS pre-chilled to 4deg.C, resuspended in 400 μL of PBS and FACS analyzed (flow cytometric fluorescence sorting, fluorescenceactivated Cell Sorting) with a flow cytometer (Sony, MA 900) over 1 hour.
This experiment was designed to compare with the natural sequence of SIRP, wherein the coding sequences of PD-L1 binding regions, as well as the Fc region, linker and hinge region, are identical for PDL1-SIRP alpha-Native (meaning that the SIRP alpha sequence is a natural sequence) and PDL1-SIRP alpha-variant (meaning that the SIRP alpha sequence is a variant sequence, the specific structure and sequence are as described in example 1).
In FIG. 3, blank control (CHO-S culture supernatant, secondary antibody was anti-human FC fluorescent secondary antibody) of Blank-CHO-S hFc-APC experimental group.
Blank-CHO-S his-APC represents a Blank control of the experimental group (CHO-S culture supernatant, secondary antibody is anti-his fluorescent secondary antibody).
Positive-sirpαhis-APC represents a Jurkat cell positive control, demonstrating binding to SIRPalpha.
negative-PDL1-APC by negative-control of Jurkat cells demonstrated that the cells were unable to bind PDL 1.
PDL 1-SIRPa-Native hFc-APC represents the experimental group (Native).
PDL 1-SIRPalpha-Mutant hFc-APC represents the experimental group (mutation).
As a result, it was found that the sirpa D1 variant in CLA014 has a greater CD47 binding capacity than the CD47 binding capacity of the native sequence.
U251 cells highly expressing PD-L1 were incubated with CLA014 or control for 1 hr at 4deg.C, cells were washed twice with pre-chilled PBS, then incubated with APC-labeled secondary antibody to human IgG Fc (Biolegend, cat # 409306) for 45 min, washed twice with pre-chilled PBS, resuspended in 400 μL of PBS, and FACS analyzed with a flow cytometer (Sony, MA 900) for 1 hr.
FIG. 4 shows a flow cytometric fluorescence sorting graph of PD-L1 binding assays, which revealed that CLA014 had a stronger PD-L1 binding capacity than that of the native sequence.
Example 4
CLA014 blocks the interaction of PD-L1 with PD-1
biotin-PD 1-Fc was added to a 96-well plate containing CHO-PD-L1 cells with serial dilutions of CLA014-Mutant, CLA014-Nature, TTI-622. Cells were incubated at 4℃for 45 min, washed with PBS, and further incubated with PE-conjugated mouse anti-human CD279 (BD BioScience, cat # 557946) at 4℃for 45 min. Cells were resuspended in 200mL of PBS after washing and analyzed by FACS for binding affinity of PD1-Fc to PD-L1 on the membrane.
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
SEQUENCE LISTING
<110> Shenzhen Dimethom biological medicine Co., ltd
<120> a polypeptide and use thereof
<130> 20I31163
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> PRT
<213> artificial sequence
<400> 1
Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 2
<211> 123
<212> PRT
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 3
<211> 98
<212> PRT
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val
<210> 4
<211> 15
<212> PRT
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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
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Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
<210> 6
<211> 107
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<400> 6
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
1 5 10 15
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
50 55 60
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
65 70 75 80
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala
100 105
<210> 7
<211> 21
<212> PRT
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<400> 7
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly
20
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<212> PRT
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<400> 8
Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu Glu Leu Gln Ile
1 5 10 15
Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr
20 25 30
Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro Val Gly Pro Ile Gln Trp
35 40 45
Phe Arg Gly Ala Gly Pro Gly Arg Val Leu Ile Tyr Asn Gln Arg Gln
50 55 60
Gly Pro Phe Pro Arg Val Thr Thr Val Ser Asp Thr Thr Lys Arg Glu
65 70 75 80
Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala
85 90 95
Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys Gly Ser Pro Asp Asp Val
100 105 110
Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro
115 120 125
Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His
130 135 140
<210> 9
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<212> PRT
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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
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Leu Ser Gly Ala Arg Cys
20
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<212> PRT
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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 11
<211> 107
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<400> 11
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 12
<211> 1935
<212> DNA
<213> artificial sequence
<400> 12
aagcttgcca ccatggagct gggcctgtcc tggatcttcc tgctggccat tctgaagggc 60
gtgcagtgcc aagtgcagct ggttcaatcc ggagctgagg tgaaaaagcc cggatcctcc 120
gtgaaagtgt cctgcaagac ctccggcgac accttctcca catacgccat ttcctgggtg 180
aggcaggccc ctggacaagg actggaatgg atgggaggaa tcatccctat cttcggcaaa 240
gcccactacg cccagaagtt ccagggcagg gtgacaatca ccgccgacga atccacatcc 300
accgcctata tggaactgtc ctccctgagg tccgaggaca ccgctgtgta tttctgcgcc 360
aggaaattcc acttcgtgtc cggctccccc tttggcatgg atgtgtgggg acagggaacc 420
acagtgaccg tgtcctccgc ttctacaaag ggcccttccg tgtttcctct ggctccctcc 480
tctaaatcca cctccggagg aacagctgct ctgggctgtt tagtgaaaga ttattttccc 540
gagcccgtga ccgtgtcctg gaattccgga gctctgacct ccggcgtgca tacctttccc 600
gctgttctgc aatcctccgg cctgtattcc ctgtcctccg tggtgacagt gccttcctcc 660
tccctgggaa cacaaacata catttgcaac gtgaaccaca agccctccaa caccaaggtg 720
gacaagaagg tggagcccaa gtcctgcgac aagacccaca cctgtcctcc ctgtcctgct 780
cctgaagctg ctggaggccc ttccgttttt ctgtttcccc ctaaacccaa agacaccctg 840
atgatctcca ggacccccga ggtgacatgt gtggtggttg atgtgtccca cgaggacccc 900
gaagtgaaat tcaattggta cgtggacggc gtggaggtgc acaacgctaa gacaaagccc 960
agggaagagc agtacaactc cacctacagg gtggtgtccg tgctgaccgt gttgcatcaa 1020
gattggctga acggcaaaga gtacaagtgc aaggtgtcca acaaggccct gggcgcccct 1080
atcgaaaaaa caatctccaa ggccaagggc cagcccaggg aacctcaagt gtataccctg 1140
cctccctcca gggatgagct gacaaaaaac caagtgtccc tgacctgcct ggtgaagggc 1200
ttttatccct ccgatatcgc cgtggagtgg gagtccaatg gccagcctga gaataattat 1260
aagaccaccc cccccgtgct ggactccgat ggatcctttt ttctgtattc caagctgacc 1320
gtggacaagt ccaggtggca gcagggcaat gtgttctcct gctccgtgat gcacgaggcc 1380
ttgcataatc attataccca gaagtccctg tccctgtccc ccggcgctgg aggaggagga 1440
tctggaggag gaggatccgg aggaggagga tctggaggag gaggatccgg agcctcctgt 1500
gcatggtctg gagtggctgg agaagaggaa ctgcagatta ttcagcccga caagtccgtg 1560
ctggtggccg ctggcgaaac agctacactg aggtgtacaa tcacctccct gttccccgtg 1620
ggccctatcc aatggtttag gggcgctgga cctggaaggg tgctgattta taatcagagg 1680
cagggcccct tccccagggt gacaacagtg tccgatacaa ccaagaggga gaacatggac 1740
ttctccatca ggatcggcaa catcaccccc gccgatgccg gaacatatta ttgcatcaag 1800
ttcaggaagg gctcccccga cgacgtggaa tttaagtccg gagctggaac cgaactgtcc 1860
gtgagggcta aaccctccgc ccctgtggtt tctggacctg ctgctagagc tacacctcaa 1920
cattaataag aattc 1935
<210> 13
<211> 728
<212> DNA
<213> artificial sequence
<400> 13
tctagagcca ccatggatat gagggtgccc gcccaactgc tgggactgct gctgctgtgg 60
ttgtccggag ctagatgcga aattgtgctg acacagtccc ctgccaccct gtccctgtcc 120
cctggagaaa gggctacact gtcctgtagg gcttcccagt ccgtgtcctc ctatctggcc 180
tggtatcagc agaagcccgg ccaggcacct aggctgctga tttatgatgc ctccaatagg 240
gccaccggca tccccgccag attttccgga tccggatccg gaacagattt taccctgaca 300
atctcctccc tggagcccga ggattttgcc gtgtattact gccagcagag gtccaactgg 360
cccacctttg gccaaggaac caaagtggaa atcaagagga ccgtggccgc cccctccgtg 420
tttatttttc ctccttccga tgagcagctg aagtccggca cagcttccgt ggtgtgtctg 480
ctgaacaatt tctaccccag ggaggccaag gtgcagtgga aagtggataa cgccctgcag 540
tccggaaact cccaggagtc tgtgacagaa caagattcca aggactccac ctactccctg 600
tcctccaccc tgactctgtc caaagctgat tatgagaagc acaaggtgta cgcctgcgag 660
gtgacccacc aaggcctgtc ctcccctgtt acaaaatcct ttaatagggg cgagtgctga 720
gcggccgc 728
<210> 14
<211> 120
<212> PRT
<213> artificial sequence
<400> 14
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala
115 120
<210> 15
<211> 360
<212> DNA
<213> artificial sequence
<400> 15
gaagaagaac tgcaagtgat tcagcccgac aagtccgtgc tggtggccgc tggagaaaca 60
gctacattga gatgtacagc tacatccctg atccccgtgg gccctattca atggttcagg 120
ggagctggac ctggaaggga actgatttac aaccagaagg agggccactt ccccagggtg 180
acaacagtgt ctgatctgac aaaaaggaac aacatggact tctccatcag gatcggcaac 240
atcacccccg ccgatgctgg aacatattat tgtgtgaagt tccggaaggg ctcccccgac 300
gatgtggagt ttaaatccgg agctggcaca gagctgtccg tgagggctaa accctccgct 360
<210> 16
<211> 120
<212> PRT
<213> artificial sequence
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (27)..(27)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (47)..(47)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (53)..(54)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (56)..(56)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (66)..(66)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (70)..(70)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (92)..(92)
<223> Xaa can be any naturally occurring amino acid
<400> 16
Glu Glu Glu Leu Gln Xaa Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Lys Arg Xaa Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala
115 120
<210> 17
<211> 144
<212> PRT
<213> artificial sequence
<220>
<221> misc_feature
<222> (16)..(16)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (37)..(37)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (41)..(41)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (57)..(57)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (63)..(64)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (66)..(66)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (76)..(76)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (80)..(80)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (102)..(102)
<223> Xaa can be any naturally occurring amino acid
<400> 17
Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu Glu Leu Gln Xaa
1 5 10 15
Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr
20 25 30
Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro Val Gly Pro Ile Gln Trp
35 40 45
Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu Ile Tyr Asn Gln Xaa Xaa
50 55 60
Gly Xaa Phe Pro Arg Val Thr Thr Val Ser Asp Xaa Thr Lys Arg Xaa
65 70 75 80
Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala
85 90 95
Gly Thr Tyr Tyr Cys Xaa Lys Phe Arg Lys Gly Ser Pro Asp Asp Val
100 105 110
Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro
115 120 125
Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His
130 135 140
<210> 18
<211> 232
<212> PRT
<213> artificial sequence
<400> 18
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Ala
225 230

Claims (11)

1. A signal regulatory protein alpha D1 variant, characterized in that the amino acid sequence of the signal regulatory protein alpha D1 variant is as set forth in SEQ ID NO: shown at 8.
2. A polypeptide comprising:
(a) The signal regulatory protein αd1 variant of claim 1;
(b) An Fc variant comprising an Fc domain dimer having two Fc domain monomers, each Fc domain monomer independently comprising the amino acid sequence of SEQ ID NO: 5-6.
3. The polypeptide of claim 2, wherein the signal regulatory protein αd1 variant is linked to the C-terminus of the Fc domain monomer.
4. An isolated polynucleotide encoding a variant of the signal conditioning protein αd1 according to claim 1 or the polypeptide according to any one of claims 2-3.
5. A construct comprising the polynucleotide of claim 4.
6. An expression system comprising the construct of claim 5, or the polynucleotide of claim 4 integrated exogenously into the genome.
7. A composition comprising a signal regulatory protein αd1 variant according to claim 1, or a polypeptide according to any one of claims 2-3, or a polynucleotide according to claim 4, and at least one of a pharmaceutically acceptable carrier, diluent, excipient, adjuvant.
8. A kit comprising the composition of claim 7 and a container containing the composition.
9. Use of a signal regulatory protein αd1 variant according to claim 1, or a polypeptide according to any one of claims 2-3, or a polynucleotide according to claim 4, or a construct according to claim 5, or an expression system according to claim 6, or a composition according to claim 7, for the preparation of a medicament for the treatment of tumors and autoimmune diseases caused by overexpression of CD 47.
10. The use of claim 9, wherein the tumor is at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, multiple myeloma, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, renal cell carcinoma.
11. The use according to claim 9, wherein the autoimmune disease is at least one of crohn's disease, allergic asthma, rheumatoid arthritis.
CN202110250408.4A 2021-03-08 2021-03-08 Polypeptide and application thereof Active CN112979782B (en)

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EP2906599B1 (en) * 2012-10-12 2019-07-17 Agency For Science, Technology And Research Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics
PT3331902T (en) * 2015-08-07 2021-07-26 Alx Oncology Inc Constructs having a sirp-alpha domain or variant thereof
AU2017214692B2 (en) * 2016-02-06 2021-11-04 Epimab Biotherapeutics, Inc. Fabs-in-tandem immunoglobulin and uses thereof
JP7035299B2 (en) * 2017-10-26 2022-03-15 イミューンオンコ バイオファーマシューティカルズ （シャンハイ） カンパニー リミテッド New recombinant fusion proteins, as well as their preparation and use
GB201805963D0 (en) * 2018-04-11 2018-05-23 Avacta Life Sciences Ltd PD-L1 Binding Affirmers and Uses Related Thereto
TW202035448A (en) * 2018-07-09 2020-10-01 中國大陸商上海岸邁生物科技有限公司 Efficiently expressed egfr and pd-l1 bispecific binding proteins
US20220089667A1 (en) * 2019-01-07 2022-03-24 Inhibrx, Inc. Polypeptides Comprising Modified IL-2 Polypeptides and Uses Thereof
KR20210143192A (en) * 2019-02-22 2021-11-26 우한 이지 바이오파마 씨오., 엘티디. Modified Fc fragments, antibodies comprising same, and applications thereof
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CN111808183B (en) * 2020-07-25 2022-07-08 北京吉尔麦迪生物医药科技有限公司 High-affinity SIRP alpha mutant targeting CD47 and fusion protein thereof

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