CN115785268B

CN115785268B - Anti-CD47 antibodies and uses thereof

Info

Publication number: CN115785268B
Application number: CN202111067062.0A
Authority: CN
Inventors: 郎国竣; 胡宇豪; 谭永聪; 闫鑫甜
Original assignee: Sanyou Biopharmaceuticals Co Ltd
Current assignee: Sanyou Biopharmaceuticals Co Ltd
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2025-06-24
Anticipated expiration: 2041-09-13
Also published as: CN115785268A; WO2023036281A1

Abstract

The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-CD47 antibodies and uses thereof.

Description

Anti-CD 47 antibodies and uses thereof

Technical Field

The invention belongs to the field of biological medicine. In particular, the invention relates to anti-CD 47 antibodies and uses thereof.

Background

CD47 is a transmembrane glycoprotein widely expressed on the cell surface, belongs to the immunoglobulin superfamily, and can interact with signal regulatory protein α (sirpa), thrombospondin (TSP 1) and integrin (Integrin) to mediate a series of reactions such as apoptosis, proliferation, immunity, etc. In the innate immune system, CD47 functions by binding sirpa expressed by myeloid cells such as macrophages, neutrophils and dendritic cells to transmit inhibitory "do-it-yourself" signals, thereby inhibiting phagocytosis of target cells expressing CD47 by myeloid cells, especially macrophages. Thus, the role of CD47 in its broad expression under physiological conditions is to protect healthy cells from being eliminated by the innate immune system, whereas tumor cells effectively escape immune surveillance by over-expressing CD 47. On the other hand, macrophages infiltrating in tumor tissue, also known as tumor-associated macrophages (TAMs), often lose the immune effector function of phagocytizing and clearing tumor cells, even have immunosuppressive effects, promoting tumor proliferation and invasion. Blocking the CD 47-sirpa pathway by using anti-CD 47 antibodies has been demonstrated to be effective in mediating phagocytosis of tumor cells, thereby inhibiting the growth of various hematological and solid tumors in vivo. However, CD47 is highly expressed not only on tumor cells, but also on normal cells, e.g. erythrocytes, with a large amount of CD47 expression, and CD 47-targeting therapies may cause undesired side effects.

Some of the anti-CD 47 antibodies disclosed in the prior art (see e.g. US20160304609 A1) bind to erythrocytes, which not only causes severe anemic reactions, but also requires dosing up to 30mg/kg on administration, which properties present a great challenge for the clinical use of anti-CD 47 antibodies. There is a need in the art to develop new therapies and drugs targeting CD47 to expand the use of CD47 as a therapeutic target and macrophages as immunomodulating and effector cells.

Disclosure of Invention

In one aspect, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 4 or a variant thereof, the HCDR2 sequence of SEQ ID NO. 5 or a variant thereof, and the HCDR3 sequence of SEQ ID NO. 6 or a variant thereof;

The light chain variable region comprises the LCDR1 sequence of SEQ ID NO.7 or a variant thereof, the LCDR2 sequence of SEQ ID NO. 8 or a variant thereof, and the LCDR3 sequence of SEQ ID NO. 9 or a variant thereof;

wherein each of the variants independently comprises a substitution, addition or deletion of 1 amino acid relative to the sequence from which it is derived.

In some embodiments, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 4 or SEQ ID NO. 18, the HCDR2 sequence of SEQ ID NO. 5 or SEQ ID NO. 19, and the HCDR3 sequence of SEQ ID NO. 6 or SEQ ID NO. 23;

The light chain variable region comprises an LCDR1 sequence shown as SEQ ID NO. 7 or SEQ ID NO. 24, an LCDR2 sequence shown as SEQ ID NO. 8, SEQ ID NO. 16 or SEQ ID NO. 20 and an LCDR3 sequence shown as SEQ ID NO. 9 or SEQ ID NO. 25.

In some embodiments, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO. 4, the HCDR2 sequence shown in SEQ ID NO. 5 and the HCDR3 sequence shown in SEQ ID NO. 6, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO. 7, the LCDR2 sequence shown in SEQ ID NO. 8 and the LCDR3 sequence shown in SEQ ID NO. 9, or

The heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO. 4, the HCDR2 sequence shown in SEQ ID NO. 5 and the HCDR3 sequence shown in SEQ ID NO. 6, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO. 7, the LCDR2 sequence shown in SEQ ID NO. 16 and the LCDR3 sequence shown in SEQ ID NO. 9, or

The heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO. 18, the HCDR2 sequence shown in SEQ ID NO. 19 and the HCDR3 sequence shown in SEQ ID NO. 6, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO. 7, the LCDR2 sequence shown in SEQ ID NO. 20 and the LCDR3 sequence shown in SEQ ID NO. 9, or

The heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO. 4, the HCDR2 sequence shown in SEQ ID NO. 5 and the HCDR3 sequence shown in SEQ ID NO. 23, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO. 24, the LCDR2 sequence shown in SEQ ID NO. 8 and the LCDR3 sequence shown in SEQ ID NO. 25.

In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 11, or

The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 14 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 15, or

The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 14 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 17, or

The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 22, or

The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 26 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 27.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention further comprise a heavy chain constant region and/or a light chain constant region. In one embodiment, the heavy chain constant region is a human IgG4 heavy chain constant region and/or the light chain constant region is a human kappa light chain constant region.

The invention also provides a multispecific antibody comprising a first antigen-binding portion which binds CD47 and a second antigen-binding portion which binds a second antigen, wherein the first antigen-binding portion comprises an anti-CD 47 antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention also provides a chimeric antigen receptor comprising an anti-CD 47 antibody or antigen-binding fragment thereof of the invention. Accordingly, the present invention also provides an immune effector cell expressing the chimeric antigen receptor of the present invention on the surface.

In yet another aspect, the invention provides a polynucleotide encoding an anti-CD 47 antibody or antigen-binding fragment thereof of the invention. The invention also relates to expression vectors comprising the polynucleotides of the invention. The invention also relates to host cells comprising the polynucleotides or expression vectors of the invention.

The invention also provides an antibody conjugate comprising an anti-CD 47 antibody or antigen-binding fragment thereof or a multispecific antibody of the invention conjugated to at least one therapeutic agent.

In another aspect, the invention also provides a pharmaceutical composition comprising an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, immune effector cell, or antibody conjugate of the invention, and a pharmaceutically acceptable carrier.

The invention also relates to the use of an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, antibody conjugate, or pharmaceutical composition for the manufacture of a medicament for the treatment of cancer.

Drawings

FIGS. 1A-1B show the binding activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to CD47 on CCRF-CEM cells.

FIGS. 2A-2B show the binding activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to CD47 on erythrocytes.

FIG. 3 shows the activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to block human CD47 binding to SIRPalpha on CCRF-CEM cells.

FIGS. 4A-4B show hemagglutination of antibodies A7, A7H3L3, A7-44, A7-28 and A7-47 on erythrocytes.

FIG. 5 shows that antibodies A7-28 and A7-47 promote macrophage phagocytosis of CCRF-CEM cells.

Detailed Description

Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of" does not include any element not specified steps or components. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or components, plus any optional elements, steps or components that do not significantly affect the basic and novel properties of the claimed subject matter. It is to be understood that the expression "consisting essentially of the expression" comprising "and" consisting of the expression "comprising" is encompassed within the meaning of the expression "comprising".

As used herein, the term "and/or" in connection with a plurality of recited elements should be understood to include both individual and combined options.

Any numerical value or range of numerical values, such as concentration or range of concentration, should be construed as modified by the term "about" in any event, unless otherwise indicated. Thus, a numerical value typically includes ±10% of the value. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range explicitly includes all possible subranges, all individual values within the range including integers and fractions within the range unless the context clearly indicates otherwise.

As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope through at least one antigen binding site. Herein, the definition of antibody encompasses antigen binding fragments. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments. Antibodies may be synthetic (e.g., produced by chemical or biological coupling), enzymatically treated, or recombinantly produced. Antibodies provided herein include any immunoglobulin type (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b). The antibody may be a "monovalent", "bivalent", "trivalent" or "tetravalent" or more multivalent antibody, meaning that it comprises 1,2, 3, 4 or more antigen binding sites.

As used herein, an "antigen binding fragment" refers to a portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region of the full-length antibody (e.g., comprises one or more CDRs and/or one or more antigen binding sites), and thus retains at least a portion of the ability of the full-length antibody to specifically bind an antigen. Antigen binding fragments may, for example, include antibody derivatives produced by enzymatic treatment of full length antibodies, synthetically produced derivatives, recombinantly produced derivatives. Examples of antigen binding fragments include, but are not limited to, sdabs (e.g., variable domains of heavy chain antibodies), fv, scFv, dsFv, scdsFv, fab, scFab, fab ', F (ab ') ₂, diabodies, fd and Fd ' fragments, and other fragments (e.g., fragments comprising modifications).

As used herein, a "full length antibody" generally comprises four polypeptides, two Heavy Chains (HC) and two Light Chains (LC). Each light chain comprises a "light chain variable region (VL)" and a "light chain constant region (CL)" from the N-terminus (amino acid terminus) to the C-terminus (carboxy terminus). Each heavy chain comprises, from the N-terminus to the C-terminus, a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)". In general, the heavy chain constant region of a full length antibody may comprise a CH 1-hinge region (range) -CH2-CH3 from the N-terminus to the C-terminus. In certain immunoglobulin types (e.g., igM and IgE), the heavy chain constant region may comprise, from N-terminus to C-terminus, a CH 1-hinge region-CH 2-CH3-CH4.

The light chain variable region and the heavy chain variable region may each comprise three highly variable "Complementarity Determining Regions (CDRs)" and four relatively conserved "Framework Regions (FR)", and are connected from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Herein, CDRs of the light chain variable region (CDRL or LCDR) may be referred to as LCDR1, LCDR2 and LCDR3, and CDRs of the heavy chain variable region (CDRH or HCDR) may be referred to as HCDR1, HCDR2 and HCDR3.

In the present invention, the amino acid sequences of the CDRs are all shown according to the AbM definition rules (the sequences shown according to the AbM definition rules are also in the claims of the present invention). It is well known in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (see, e.g., chothia, c.et Al., nature,342,877-883 (1989)), and Al-Lazikani, b.et Al., j.mol. Biol.,273,927-948 (1997)), kabat (see, e.g., Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)、AbM(Martin,A.C.R.and J.Allen(2007)"Bioinformatics tools for antibody engineering,"in S.Dübel(ed.),Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag,pp.95–118)、Contact(MacCallum,R.M.et al.,(1996)J.Mol.Biol.262:732-745)、IMGT(Lefranc,M.-P.,2011(6),IMGT,the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, m. -p.et Al., dev.comp. Immunol.,27,55-77 (2003)), based on antibody sequence variability, and North CDR definition based on neighbor-transmitted clusters (affinity propagation clustering) using a large number of crystal structures. It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the claimed invention is based on the sequences shown by the AbM definition rules, the amino acid sequences corresponding to the definition rules according to other CDRs should also fall within the scope of the claimed invention.

Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).

As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the term "framework region", "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR sequences as defined above.

Generally, an "Fv" fragment consisting of one VH and one VL by non-covalent interactions is considered to be the smallest antigen-binding fragment that contains an antigen-binding site. But single variable domains (single domain antibodies) also have antigen binding capacity. "Single chain Fv (scFv)" may be obtained by linking VH and VL via a peptide linker. "disulfide stabilized Fv (dsFv)" or "single chain disulfide stabilized Fv (scdsFv or dsscFv)", respectively, can be obtained by introducing disulfide bonds into Fv or scFv.

As used herein, "Fab" comprises one complete antibody light chain (VL-CL) and antibody heavy chain variable region and one heavy chain constant region (VH-CH 1, also known as Fd). A single chain "Fab (scFab)" can be obtained by ligating CL and CH1 in "Fab" with a peptide linker. "F (ab') ₂" essentially comprises two Fab fragments linked by a disulfide bridge of the hinge region. "Fab '" is half of F (ab ') ₂, which can be obtained by reducing the disulfide bond of the F (ab ') ₂ hinge region.

"Hinge region" refers to the portion of an antibody that links immunoglobulin Fab and Fc fragments. The hinge region may be a complete hinge region or a portion thereof. For IgG, the Fc region typically comprises a portion of the hinge region linked to CH2, as well as CH3. In this context, when used with respect to a chimeric antigen receptor, a hinge region may also refer to any functional equivalent, such as the portion of a T cell receptor that connects a constant region and a transmembrane domain. The person skilled in the art can determine VH, VL, CL, CH the positions of 1, CH2, CH3 and hinge regions in antibodies based on known algorithms and software, which can be used as described, for example, in William R.Strohl,Lila M.Strohl,(2012),Antibody structure–function relationships,In Woodhead Publishing Series in Biomedicine,Therapeutic Antibody Engineering,Woodhead Publishing,pp.37-56.

As used herein, "diabody" refers to an antibody comprising two scFv wherein the VH and VL in each scFv are linked by a short peptide linker (about 5-10 amino acid residues) such that the VH and VL pairs (i.e., the VH of the first scFv and the VL of the second scFv pair, the VL of the first scFv and the VH of the second scFv pair) form an antigen binding site. The diabody may be a bispecific antibody.

As used herein, "chimeric antibody" refers to an antibody in which a portion (e.g., CDR, FR, variable region, constant region, or a combination thereof) is identical or homologous to a corresponding sequence in an antibody derived from a particular species, and the remainder is identical or homologous to a corresponding sequence in an antibody derived from another species. In some embodiments of the invention, chimeric antibodies comprise variable regions derived from a non-human species (e.g., mouse) and constant regions derived from a different species (e.g., human). Chimeric antibodies may also refer to multispecific antibodies that are specific for at least two different antigens. Chimeric antibodies may be produced by antibody engineering. Methods of antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies may be generated by DNA recombination techniques (e.g., to participate in Sambrook,J.,et al.(1989).Molecular cloning:a laboratory manual,2nd ed.Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y).

As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen binding capacity of the non-human antibody from which they are derived and have low immunogenicity to humans. Humanized antibodies may be obtained by antibody engineering any non-human species antibody or antibodies (e.g., chimeric antibodies) comprising sequences derived from a non-human species therein. The non-human species may include, for example, mice, rats, rabbits, alpacas, sharks, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. For example, CDR sequences of a non-human antibody (e.g., a murine antibody) are grafted into human antibody framework regions. In some cases, to preserve the antigen binding capacity and/or stability of a humanized antibody, critical amino acid residues of a non-human antibody (e.g., murine antibody) framework sequence may be retained in the human antibody framework region, i.e., subjected to "back-mutations" (see, e.g., morrison et al (1984) Proc. Natl. Acad. Sci.81 (21): 6851-6855;Neuberger et al (1984) Nature 312:604-608).

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of human antibody encompasses whole or full length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.

As used herein, an "affinity matured" antibody comprises one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity of the affinity matured antibody for the antigen is improved as compared to a parent antibody that does not comprise such modifications. Methods of affinity maturation of antibodies are known in the art, see, e.g., Marks et al.,Bio/Technology 10:779-783(1992);Barbas et al.,Proc.Nat.Acad.Sci.USA 91:3809-3813(1994);Scier et al.,Gene 169:147-155(1995); and HAWKINS ET al, J.mol.biol.226:889-896 (1992).

As used herein, "percent (%) sequence identity" of amino acid sequences, sequence identity "has art-recognized definitions that refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by a known algorithm). The determination may be made using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, clustal Omega and FASTA software.

Herein, an amino acid sequence "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from a heavy chain constant region of a human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

Non-critical regions in the polypeptide (e.g., CDR regions of antibodies, non-critical amino acids of framework regions, amino acids of constant regions) may be modified, e.g., substitutions, additions and/or deletions of one or more amino acids may be made, without altering the function of the polypeptide. Those skilled in the art will appreciate that amino acids in non-critical regions of a polypeptide may be substituted with suitable conserved amino acids and generally do not alter their biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. In some cases, amino acid substitutions are non-conservative substitutions. It will be appreciated by those skilled in the art that amino acid mutations or modifications may be made to antibodies or antibody fragments to alter their properties, for example to alter the type of antibody glycosylation modification, to alter the ability to form interchain disulfide bonds, or to provide reactive groups for the preparation of antibody conjugates. Antibodies or antigen binding fragments thereof comprising such amino acid mutations or modifications are also encompassed within the scope of the antibodies or antigen binding fragments thereof of the invention.

"Affinity" or "binding affinity" is used to measure the strength of the interaction between an antibody and an antigen through non-covalent interactions. The magnitude of "affinity" can be reported as equilibrium dissociation constant K _D or EC ₅₀.K_D can be calculated by measuring the equilibrium association constant (ka) and equilibrium dissociation constant (kd): K _D =kd/ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interference techniques (e.g., octet Fortebio detection systems can be employed), radioimmunoassay, surface plasmon resonance, enzyme-linked immunoassay (ELISA), or flow cytometry (FACS), among others.

The anti-CD 47 antibodies or antigen-binding fragments, multispecific antibodies, or polynucleotides encoding the same of the invention may be isolated. As used herein, the expression "isolated" means that a substance (e.g., a polynucleotide or polypeptide) is isolated from the source or environment in which it is present, i.e., does not substantially comprise any other components.

In this context, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which may generally include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Herein, a "vector" is a vector used to introduce an exogenous polynucleotide into a host cell, which is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, poxviral vectors, and baculovirus vectors, among others.

As used herein, the term "expression" refers to the production of RNA and/or polypeptides.

As used herein, an "expression vector" refers to a vector capable of expressing a polynucleotide of interest (including DNA and RNA). For example, in an expression vector, polynucleotide sequences encoding a polypeptide of interest (including DNA and RNA) may be operably linked to regulatory sequences capable of affecting the expression of the polynucleotide sequences (e.g., promoters and ribosome binding sites). Regulatory sequences may comprise promoter and terminator sequences, and optionally may comprise origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. The expression vector may be a plasmid, phage vector, recombinant virus or other vector that, when introduced into an appropriate host cell, results in expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. The expression vector can be prepared by one skilled in the art as a vector that is replicable in a host cell, remains episomal in a host cell, or is integrated into the host cell genome, as desired.

As used herein, a "host cell" is a cell that is used to receive, hold, replicate, or amplify a vector. Host cells may also be used to express polypeptides encoded by polynucleotides or vectors. The host cell may be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells such as E.coli (E.coli) or B.subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, heLa cells, NSO cells or HEK293 cells.

As used herein, the term "treatment" refers to an improvement in a disease/symptom, e.g., alleviation or disappearance of a disease/symptom, prevention or slowing of the occurrence, progression, and/or worsening of a disease/symptom. Thus, treatment includes prophylaxis, treatment and/or cure.

An "effective amount" refers to that amount required to prevent, cure, ameliorate, block or partially block a disease or condition. For example, for the treatment of a tumor, an "effective amount" of an anti-CD 47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, relative to an untreated subject. The efficacy of inhibiting tumor growth can be assessed using tumor animal models conventional in the art, e.g., spontaneous tumors, evoked tumors, transplantable tumor animal models. Alternatively, the ability to inhibit cell growth can be examined using in vitro assays known in the art. An effective amount of an antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the invention is capable of reducing the tumor size, or otherwise alleviating a symptom (e.g., preventing and/or treating metastasis or recurrence) in a subject. The effective amount can be determined by one skilled in the art based on factors such as the age, physical condition, sex, severity of symptoms, particular composition or route of administration of the subject, and the like. An effective amount may be administered in one or more administrations.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: for example, pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80, ionic strength enhancers include, but are not limited to, sodium chloride, preservatives include, but are not limited to, various antibacterial and antifungal agents, the agents that maintain osmotic pressure include, but are not limited to, sugars, sodium chloride, and the like, agents that retard absorption include, but are not limited to, mono-stearates and gelatin, diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), and the like, preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like Glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

Anti-CD 47 antibodies or antigen binding fragments thereof

In a general aspect, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof that specifically recognizes and binds CD47.

As used herein, the term "CD47" refers to the leukocyte surface antigen CD47 (Leukocyte surface ANTIGEN CD 47), also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, rh-associated antigen, or protein MER6. In some embodiments, the anti-CD 47 antibody or antigen-binding fragment thereof specifically binds human CD47. The term "human CD47" refers to CD47 derived from a human. An exemplary amino acid sequence for human CD47 is shown in GenBank accession No. np_001768.1.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention are chimeric antibodies, humanized antibodies, human antibodies, scFv, fab, fab ', F (ab') ₂, fv fragments, disulfide stabilized Fv (dsFv), or diabodies.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention

1) Specifically bind to CD47 positive cancer cells but do not bind or do not substantially bind to erythrocytes;

2) Does not cause or substantially does not cause hemagglutination;

3) Blocking the binding of CD47 to SIRPalpha, and/or

4) Promoting phagocytosis of CD47 positive tumor cells by macrophages.

Variable region

In some embodiments, an anti-CD 47 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3.

In some embodiments, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO.4 (GFNIKDIYIY), the HCDR2 sequence shown in SEQ ID NO. 5 (KIDPANGNTK) and the HCDR3 sequence shown in SEQ ID NO. 6 (GYGSGFAY). In some embodiments, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 7 (RASQDISNHLN), the LCDR2 sequence of SEQ ID NO. 8 (YTSRIHS) and the LCDR3 sequence of SEQ ID NO. 9 (QQGYTLPFT).

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention are obtained by affinity maturation engineering. Such anti-CD 47 antibodies, or antigen binding fragments thereof, may comprise one or more of the variants of the HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 6), LCDR1 (SEQ ID NO: 7), LCDR2 (SEQ ID NO: 8) and LCDR3 (SEQ ID NO: 9) sequences described above. In one embodiment, the variants each independently comprise a substitution, addition or deletion of 1 amino acid relative to the sequence from which they are derived. Preferably, the variants each independently comprise 1 amino acid substitution relative to the sequence from which they are derived.

In some embodiments, an anti-CD 47 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein

In some embodiments, the HCDR1 sequence comprises the amino acid sequence depicted in SEQ ID NO. 4 wherein amino acid 7 is substituted. In one embodiment, the amino acid at position 7 of SEQ ID NO. 4 is substituted with V (SEQ ID NO. 18; GFNIKDV YIY).

In some embodiments, the HCDR2 sequence comprises the amino acid sequence depicted in SEQ ID NO. 5 wherein amino acid 10 is substituted. In one embodiment, the amino acid at position 10 of SEQ ID NO. 5 is substituted with H (SEQ ID NO. 19; KIDPANGNTH).

In some embodiments, the HCDR3 sequence comprises the amino acid sequence depicted in SEQ ID NO. 6 wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO. 6 is substituted with V (SEQ ID NO. 23; GYGSVFAY).

In some embodiments, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 7 wherein amino acid 10 is substituted. In one embodiment, amino acid 10 of SEQ ID NO. 7 is substituted with I (SEQ ID NO. 24; RASQDISNHIN).

In some embodiments, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 8 wherein amino acid 7 is substituted. In one embodiment, amino acid 7 in SEQ ID NO. 8 is substituted with L (SEQ ID NO. 16; YTS RIHI). In one embodiment, the amino acid at position 7 of SEQ ID NO. 8 is substituted with K (SEQ ID NO. 20; YTS RIHK).

In some embodiments, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 9, wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO. 9 is substituted with H (SEQ ID NO. 25; QQGYHLPFT).

The heavy chain variable region comprises an HCDR1 sequence shown as SEQ ID NO. 4 or SEQ ID NO. 18, an HCDR2 sequence shown as SEQ ID NO. 5 or SEQ ID NO. 19 and an HCDR3 sequence shown as SEQ ID NO. 6 or SEQ ID NO. 23;

In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 4, the HCDR2 sequence of SEQ ID NO. 5 and the HCDR3 sequence of SEQ ID NO. 6, and the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 7, the LCDR2 sequence of SEQ ID NO. 8 and the LCDR3 sequence of SEQ ID NO. 9.

In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 4, the HCDR2 sequence of SEQ ID NO. 5 and the HCDR3 sequence of SEQ ID NO.6, and the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 7, the LCDR2 sequence of SEQ ID NO. 16 and the LCDR3 sequence of SEQ ID NO. 9.

In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 18, the HCDR2 sequence of SEQ ID NO. 19 and the HCDR3 sequence of SEQ ID NO. 6, and the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 7, the LCDR2 sequence of SEQ ID NO. 20 and the LCDR3 sequence of SEQ ID NO. 9.

In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 4, the HCDR2 sequence of SEQ ID NO. 5 and the HCDR3 sequence of SEQ ID NO. 23, and the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 24, the LCDR2 sequence of SEQ ID NO. 8 and the LCDR3 sequence of SEQ ID NO. 25.

In some embodiments, the VH as described above further comprises a heavy chain framework region. In some embodiments, the VL as described above further comprises a light chain framework region. In some embodiments, the VH as described above further comprises a heavy chain framework region, and the VL as described above further comprises a light chain framework region. The heavy chain framework region and/or the light chain framework region may each be independently derived from the heavy chain framework region and the light chain framework region of an immunoglobulin of any species.

In some embodiments, the VH comprises a heavy chain framework region derived from a murine immunoglobulin, and/or the VL comprises a light chain framework region derived from a murine immunoglobulin.

In certain preferred embodiments, the VH comprises a heavy chain framework region derived from a human immunoglobulin, and/or the VL comprises a light chain framework region derived from a human immunoglobulin. Thus, in certain preferred embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention are humanized. The heavy and/or light chain framework regions of the humanized anti-CD 47 antibody or antigen-binding fragment thereof may comprise one or more non-human (e.g., murine) amino acid residues, e.g., the heavy and/or light chain framework regions may comprise one or more amino acid back mutations comprising the corresponding murine amino acid residues therein.

In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 14, SEQ ID NO. 21 or SEQ ID NO. 26. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 22 or SEQ ID NO. 27.

In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 14, SEQ ID NO. 21 or SEQ ID NO. 26. In some embodiments, the heavy chain variable region comprises an amino acid sequence having one or more (e.g., 1, 2,3, 4, 5, 6,7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21, or SEQ ID NO: 26. Preferably, the amino acid substitutions, additions and/or deletions do not occur in the CDR regions.

In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 22, or SEQ ID NO. 27. In some embodiments, the light chain variable region comprises an amino acid sequence having one or more (e.g., 1,2, 3, 4, 5, 6,7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to SEQ ID NO. 11, 15, 17, 22, or 27. Preferably, the amino acid substitutions, additions and/or deletions do not occur in the CDR regions.

In some embodiments, the heavy chain variable region comprises 1) the amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21, or SEQ ID NO 26, 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21, or SEQ ID NO 26, 2) an amino acid sequence having substitution, addition, and/or deletion of one or more (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, or 10) amino acids as compared to the amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21, or SEQ ID NO 26, and

The light chain variable region comprises 1) an amino acid sequence of SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 22 or SEQ ID NO. 27, 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 22 or SEQ ID NO. 27, or 3) an amino acid sequence having one or more (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions compared to SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 22 or SEQ ID NO. 27. Preferably, the amino acid substitutions, additions and/or deletions do not occur in the CDR regions.

In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 11. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 14 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 15. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 14 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 17. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 22. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 26 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 27.

Constant region

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention further comprise a heavy chain constant region and/or a light chain constant region.

The heavy chain constant region and the light chain constant region may each be independently derived from the heavy chain constant region and the light chain constant region of an immunoglobulin of any species. The heavy chain constant region may be derived from the heavy chain constant region of immunoglobulins of any subclass (e.g., igA, igD, igE, igG and IgM), class (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2) or subclass (e.g., igG2a and IgG2 b), or a combination thereof. The light chain constant region may be derived from a Lambda (Lambda) light chain or a Kappa (Kappa) light chain constant region.

Suitable immunoglobulin constant regions (e.g., CH1 and light chain constant regions, hinge region-CH 2-CH3, CH 1-hinge region-CH 2-CH3 and light chain constant regions, or Fc regions), and types (e.g., igG, e.g., igG1, igG2, igG3, and IgG 4) can be selected and optionally modified to obtain antibodies having the desired properties.

In preferred embodiments, the heavy chain constant region comprises at least an Fc region, e.g., the heavy chain constant region of IgG may include 1) all or part of the hinge region-CH 2-CH3, or 2) the CH 1-hinge region-CH 2-CH3. In some embodiments, the heavy chain constant region is a heavy chain constant region (e.g., an Fc region or a CH 1-hinge region-CH 2-CH 3) of a human IgG (e.g., igG1, igG2a, igG2b, igG3, or IgG 4). In one embodiment, the heavy chain constant region is a heavy chain constant region of human IgG 1. In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO. 1. In a preferred embodiment, the heavy chain constant region is a heavy chain constant region of human IgG 4. In one embodiment, the heavy chain constant region comprises 1) the amino acid sequence of SEQ ID NO. 12, or 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 12.

In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In one embodiment, the light chain constant region comprises 1) the amino acid sequence of SEQ ID NO. 13, or 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 13.

Antibodies or antigen binding fragments thereof may be prepared and produced using methods known in the art. Such methods may include, for example, preparation and isolation of antibodies or antigen binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridoma cells or EBV immortalized B cells). It is also possible to immunize an animal, for example, an animal (e.g., a mouse) with an antigen or a DNA encoding an antigen, and then isolate B cells expressing the antibody from the immunized animal. Preferably, the B cells expressing the antibody are immortalized, e.g. prepared as hybridoma or EBV immortalized B cells. Polynucleotides encoding antibodies or antigen binding fragments thereof may also be isolated from immunized animals or prepared by chemical synthesis and then used to construct expression vectors.

Multispecific antibodies

In one aspect, the invention provides a multispecific antibody comprising a first antigen-binding portion which binds CD47 and a second antigen-binding portion which binds a second antigen, wherein the first antigen-binding portion comprises an anti-CD 47 antibody or antigen-binding fragment thereof of the invention.

As used herein, the term "multispecific antibody" refers to an antibody that is capable of specifically binding to two or more (e.g., 2, 3, 4, 5, or 6) different epitopes. The multispecific antibody may be, for example, a bispecific, trispecific or tetraspecific antibody, which is capable of specifically binding 2, 3 or 4 epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to an antigen binding site of an antibody. An epitope is typically composed of chemically active surface groups (e.g., amino acids or sugar side chains) of an antigen and typically has specific three-dimensional structural properties as well as specific charge properties. The second antigen may be an antigen other than CD 47. The second antigen may also be CD47, which binds to a different epitope on CD47 than the anti-CD 47 antibodies or antigen binding fragments thereof of the invention. The determination of whether the epitope bound by the two antibodies is identical can be made using methods conventional in the art, for example, by ELISA, flow cytometry, or surface plasmon resonance to determine the competitive binding of the two antibodies to the same epitope.

The multispecific antibody may be a multivalent (e.g., 2,3, 4 valent) antibody, i.e., it has multiple antigen binding sites. The multispecific antibody may be, for example, a chimeric antibody, a humanized antibody, a scFab, a F (ab') ₂, or a diabody.

Methods for constructing multispecific antibodies using an antibody or antigen-binding fragment of interest are well known to those skilled in the art (see, e.g., WO 93/08829;Suresh et al, (1986) Methods in Enzymology,121:210; and Traunecker et al, (1991) EMBO, 10:3655-3659). Multispecific antibodies can be generated and isolated using a variety of techniques known in the art. For example, polynucleotides encoding the multispecific antibodies may be obtained by recombinant DNA techniques, optionally cloning the polynucleotides into an expression vector, then transforming a host cell with the polynucleotides or expression vector, culturing the transformed host cell under suitable conditions to allow expression of the polynucleotides or expression vector, and finally isolating and purifying the multispecific antibodies from the host cell or culture medium. It is also possible to obtain each part of the multispecific antibody separately, e.g. to obtain a first antigen-binding portion and a second antigen-binding portion as described herein separately, and then to couple each part, optionally via a linker, by enzymatic or chemical coupling techniques to obtain a multispecific antibody that specifically binds CD47 and other antigens.

As used herein, "first antigen binding portion" and "second antigen binding portion" refer to an amino acid sequence comprising an antigen binding site that is capable of binding to an epitope of an antigen, which definition falls within the meaning of an antibody or antigen binding fragment.

The first antigen binding portion may be any form of antibody or antigen binding fragment, including but not limited to Fv, scFv, dsFv, scdsFv, fab, scFab, fab 'and F (ab') ₂. In some embodiments, the first antigen binding portion comprises an anti-CD 47 antibody or antigen binding fragment thereof of the invention. In one embodiment, the first antigen-binding portion comprises VH and VL comprising HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, respectively, of an anti-CD 47 antibody of the invention or antigen-binding fragment thereof as described above. In one embodiment, the first antigen-binding portion comprises VH and VL comprising VH and VL, respectively, of an anti-CD 47 antibody or antigen-binding fragment thereof of the invention as described above.

The second antigen binding portion may be an antibody or antigen binding fragment that binds any epitope of interest. In one embodiment, the second antigen is an antigen other than CD 47. Antigens to which the second antigen binding portion may specifically bind may include tumor antigens (e.g., tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors, and immune checkpoint molecules. As used herein, "tumor-associated antigen" refers to an antigen that is highly expressed in tumor cells, but is also present in healthy cells but expressed at a lower level. As used herein, "tumor-specific antigen" refers to an antigen that is specifically expressed in tumor cells and hardly expressed in healthy cells. Non-limiting examples of tumor antigens may include CD19, CD20, EGFR, GPC3, HER-2, and FOLR1. Non-limiting examples of immune checkpoint molecules may include CTLA-4, LAG-3, PD-1, PD-L1 and TIM-3. Immunomodulatory receptors may include, for example, immune activating receptors (e.g., CD27, CD137, CD40, GITR, and OX 40) and immune inhibitory receptors (e.g., BTLA, CTLA4, LAG-3, and PD-1). For example, the second antigen binding portion may be an agonist antibody of an immune activating receptor or an antagonist antibody of an immune inhibitory receptor.

In one embodiment, the second antigen binding portion binds a tumor antigen, an immunomodulatory receptor, and an immune checkpoint molecule. In one embodiment, the second antigen binding portion specifically binds SIRPα、PD-1、PD-L1、LAG3、TIM-3、CTLA-4、VISTA、GPC3、EGFR、HER-2、CD19、CD20、CD33、CD40、CD73、OX40、CD3、DLL-3、TIP-1、 folate receptor alpha (FOLR 1) and/or other tumor antigens.

The second antigen binding portion may be any form of antibody or antigen binding fragment, including but not limited to a single variable domain of an immunoglobulin (e.g., a VHH comprising alpaca or IgNAR variable domain of shark), fv, scFv, dsFv, scdsFv, fab, scFab, fab 'and F (ab') ₂. In one embodiment, the second antigen binding portion comprises a single variable domain of an immunoglobulin.

The first antigen binding portion and the second antigen binding portion may optionally be connected by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not connected by a linker. In other embodiments, the first antigen binding portion and the second antigen binding portion are linked by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding portion and the second antigen binding portion are linked by a peptide linker. Exemplary peptide linkers can include, but are not limited to, poly glycine (G), poly alanine (a), poly serine (S), or combinations thereof, such as GGAS, GGGS, GGGSG or (G ₄S)_n), where n is an integer from 1 to 20.

In some embodiments, the second antigen is a different antigen than CD47, and the binding affinity of the first antigen-binding portion to CD47 is weaker than the binding affinity of the second antigen-binding portion to the other antigen. In such embodiments, the multispecific antibodies of the present invention may preferentially target other antigens, such that the multispecific antibodies specifically recognize target cells (e.g., cancer cells) that express other antigens, and the target cells may or may not express CD47. In a specific embodiment, when the target cell expresses both the CD47 antigen and a second antigen different from CD47, the multispecific antibody of the present invention binds CD47 more strongly than CD47 alone, and blocks the binding of CD47 and SIRPa more strongly.

CAR and immune effector cells expressing the same

In another aspect, the invention also provides a Chimeric Antigen Receptor (CAR) comprising an anti-CD 47 antibody or antigen-binding fragment thereof of the invention. CARs are recombinant receptors that simultaneously provide antigen binding and activate T cell function. CAR structures and engineering are described, for example, in Dotti G.et al, (2014) Immunol Rev.257 (1): 107-126, which is incorporated herein by reference.

In an exemplary embodiment, the CAR of the invention comprises, from N-terminus to C-terminus:

(a) An extracellular region comprising an anti-CD 47 antibody or antigen-binding fragment thereof of the invention;

(b) A transmembrane region which connects the extracellular region and the intracellular signal region and anchors the CAR to the cell membrane, and

(C) An intracellular signal region.

The extracellular region comprises an anti-CD 47 antibody or antigen-binding fragment thereof of the invention, such that a CAR of the invention binds to a CD 47-expressing cell (e.g., a cancer cell). In a preferred embodiment, the extracellular region in the CAR is in the form of an scFv.

The transmembrane region connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane.

When the extracellular region of the CAR binds to the antigen it recognizes, the intracellular signaling region mediates a T Cell Receptor (TCR) like signal into the cell and activates the immune effector cells expressing the CAR to exert its effector function.

In some embodiments, the intracellular signaling region further comprises at least one costimulatory domain. The co-stimulatory domain can promote activation of the CAR-expressing immune effector cell upon binding to the antigen targeted by the extracellular region.

In some embodiments, the CARs of the invention further comprise a spacer region connecting the extracellular region and the transmembrane region. In one embodiment, the spacer is derived from the heavy chain constant region of an immunoglobulin.

In a further aspect, the invention also relates to polynucleotides encoding the CARs of the invention and expression vectors comprising the polynucleotides.

In another aspect, the invention also provides an immune effector cell that expresses the CAR of the invention on the cell surface. Preferably, the immune effector cells are selected from T lymphocytes (e.g., cytotoxic T Cells (CTLs)), natural killer cells (NK), and natural killer T cells (NKT). Immune effector cells can target CD47 positive diseased cells (e.g., CD47 positive cancer cells) and be activated to initiate effector functions, e.g., cause death of CD47 positive cancer cells.

Polynucleotides, vectors and host cells

In another aspect, the invention provides a polynucleotide comprising a polynucleotide sequence encoding an anti-CD 47 antibody or antigen-binding fragment of the invention or a multispecific antibody of the invention.

The polynucleotides of the invention may be obtained using methods known in the art. For example, polynucleotides of the invention may be isolated from phage display libraries, yeast display libraries, immune animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The polynucleotides of the invention may be codon optimized for the host cell used for expression.

In yet another aspect, the invention also provides an expression vector comprising a polynucleotide of the invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. Polynucleotides of the invention may be present in one or more expression vectors. In one embodiment, the polynucleotides of the invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase Chain Reaction (PCR) techniques), and the like.

The invention also provides a host cell comprising a polynucleotide or expression vector of the invention. The polynucleotides or expression vectors of the invention may be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection, among others.

In a preferred embodiment, the host cell is used to express an anti-CD 47 antibody or antigen-binding fragment thereof of the invention or a multispecific antibody of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, heLa cells, HEK cells (e.g., HEK 293 cells), chinese Hamster Ovary (CHO) cells, and other mammalian cells suitable for expression of antibodies.

The invention also provides a method of producing an anti-CD 47 antibody or antigen-binding fragment thereof of the invention or a multispecific antibody of the invention comprising the steps of:

(I) Culturing a host cell of the invention under suitable conditions to express an anti-CD 47 antibody or antigen-binding fragment thereof of the invention or a multispecific antibody of the invention, and

(II) isolating the antibody or antigen-binding fragment thereof or the multispecific antibody of the invention from the host cell or culture thereof.

Antibody conjugates

The invention also provides an antibody conjugate comprising an anti-CD 47 antibody of the invention or an antigen-binding fragment thereof or a multispecific antibody of the invention conjugated to at least one therapeutic agent. An antibody-drug conjugate (ADC) is a typical antibody conjugate, wherein the therapeutic agent may be, for example, a cytotoxic agent.

As used herein, "conjugated" refers to the attachment of two or more moieties to each other by covalent or non-covalent interactions. In a preferred embodiment, the conjugation is covalent conjugation.

The therapeutic agent may be selected from the group consisting of cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen binding fragments thereof that specifically bind to additional antigens), radioisotopes, oligonucleotides and analogs thereof (e.g., interfering RNAs), bioactive peptides, protein toxins (e.g., diphtheria toxin, ricin), and enzymes (e.g., urease).

Cytotoxic agents refer to substances that inhibit or reduce the activity, function, and/or kill cells. Examples of cytotoxic agents may include, but are not limited to: maytansinoids (e.g., maytansinoids (maytansine), oridases (e.g., MMAF, MMAE, MMAD), spinosads (duostatin), candidiasis (cryptophycins), vinca alkaloids (e.g., vinblastine, vincristine), colchicines, dolastaxins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin (daunorubicin), dihydroxyanthracene dione (dihydroxyanthracindione), doxorubicin (doxorubicin)), cytotoxic antibiotics (e.g., mitomycin (mitomycin), actinomycin (actinomycin), betamycin (duocarmycins) (e.g., CC-1065), and combinations thereof aureomycin (auromycin), doxycycline (duomycin), calicheamicin (calicheamicin), endosporin (endomycin), phenomycin (phenomycin)), doxorubicin, daunorubicin, calicheamicin, cisplatin (cisplatin), ethidium bromide (ethidiumbromide), bleomycin, mitomycin, mithramycin, pladienolide, podophyllotoxin, etoposide (etoposide), mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pravastatin, fludarabine, cladribine, nelarabine, carmustine, lomustine, methotrexate, phenylalanine nitrogen mustard, teniposide (tenoposide), glucocorticoids and the like.

The radioisotope may be selected from, for example, ²¹²Bi、²¹³Bi、¹³¹I、¹²⁵I、¹¹¹In、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、⁹⁰Y. antibodies labeled with a radioisotope, which may also be referred to as radioimmunoconjugates.

In some embodiments, the therapeutic agent is selected from the group consisting of cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor agents. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In yet another preferred embodiment, the therapeutic agent is a radioisotope.

The therapeutic agent may be conjugated to an antibody or antigen-binding fragment of the invention or a multispecific antibody of the invention via a linker using any technique known in the art. The linker may contain reactive groups for covalent conjugation, such as amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, sulfhydryl or hydroxyl groups.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an anti-CD 47 antibody or antigen-binding fragment, an antibody conjugate, an immune effector cell expressing a CAR, or a multispecific antibody of the invention, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like), solubilizing agents, gelling agents, softeners, solvents (such as water, alcohols, acetic acid, and syrups), buffers (such as phosphate buffers, histidine buffers, and acetate buffers), surfactants (such as nonionic surfactants, such as polysorbate 80, polysorbate 20, poloxamers or polyethylene glycols), antibacterial agents, antifungal agents, isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents, and emulsifying agents. For compositions comprising an antibody or antibody conjugate, a suitable carrier may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamers), or combinations thereof.

The pharmaceutical compositions provided herein may be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). For compositions comprising antibodies or antibody conjugates, preferred dosage forms may generally be, for example, injectable solutions and lyophilized powders.

The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). It will be appreciated by those skilled in the art that the exact dosage administered will depend on various factors such as the pharmacokinetic properties of the pharmaceutical composition, the duration of the treatment, the rate of excretion of the particular compound, the purpose of the treatment, the route of administration and the condition of the subject, such as the age, health, weight, sex, diet, medical history of the patient, and other factors well known in the medical arts. The method of administration may be, for example, injection or infusion.

As a general guidance, the anti-CD 47 antibodies, or antigen-binding fragments thereof, antibody conjugates, or multispecific antibodies of the invention may be administered in a dosage range of from about 0.0001 to 100mg/kg, more typically from 0.01 to 20mg/kg of subject body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly dosing, biweekly dosing, tricyclically dosing, weekly dosing, monthly dosing, 3 months dosing, 3-6 months dosing, or slightly shorter initial dosing intervals followed by longer post dosing intervals. The mode of administration may be intravenous drip.

Treatment of

In a further aspect, the invention relates to the use of an anti-CD 47 antibody or antigen binding fragment thereof, a multispecific antibody, a somatic conjugate, an immune effector cell, or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a disease in a subject.

The invention also relates to an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate, an immune effector cell, or a pharmaceutical composition of the invention for use in treating a disease.

The invention also provides a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the invention.

In some embodiments, the disease is associated with aberrant expression of CD47. The term "abnormal expression" refers to an excessive or insufficient level of protein expression in a sample as compared to a sample in a normal state (or a standard sample, e.g., a sample of a subject not suffering from a disease associated with abnormal expression of CD 47). Preferably, the disease is characterized by high expression of CD47. For example, CD47 is highly expressed in tissues (e.g., gastric and paracancestral tissues) of a subject having or suspected of having a disease (e.g., gastric cancer), while CD47 is underexpressed in corresponding tissues of a subject not having the disease.

In one embodiment, the disease as described above is cancer. As used herein, "cancer" includes, but is not limited to, hematological tumors and solid tumors. Herein, "hematological cancer" means cancers of the blood, including leukemia, lymphoma, myeloma, and the like. As non-limiting examples, leukemias may include Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), and myeloproliferative disorders (e.g., myelodysplastic syndrome). Lymphomas may include, for example, hodgkin's lymphoma, non-hodgkin's lymphoma, burkitt's (Burkitt) lymphoma, follicular lymphoma, and the like. Myeloma can include, for example, multiple Myeloma (MM) and giant cell myeloma, among others. Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, smooth muscle tumor, glioma, glioblastoma, and the like. The cancer may also be metastatic cancer. "metastasis" refers to the spread of cancer cells from their original site to other parts of the body.

The invention also relates to an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate, an immune effector cell, or a pharmaceutical composition of the invention for use in treating cancer. In some embodiments, the cancer is selected from Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), myelodysplastic syndrome, hodgkin's lymphoma, non-hodgkin's lymphoma, burkitt's lymphoma, follicular lymphoma, multiple Myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, smooth muscle tumor, glioma, and glioblastoma.

The anti-CD 47 antibodies or antigen-binding fragments thereof of the invention may be used to treat CD47 positive hematological and solid tumors. In some embodiments, the cancer is selected from Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), myelodysplastic syndrome, hodgkin's lymphoma, non-hodgkin's lymphoma, burkitt's lymphoma, follicular lymphoma, multiple Myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, smooth muscle tumor, glioma, glioblastoma, and the like.

Depending on the second antigen binding portion, the multispecific antibodies of the invention may be used to treat a cancer selected from, for example, acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), myelodysplastic syndrome, hodgkin's lymphoma, non-Hodgkin's lymphoma, burkitt's lymphoma, follicular lymphoma, multiple Myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, smooth muscle tumor, glioma, glioblastoma, and the like.

The anti-CD 47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates, immune effector cells, or pharmaceutical compositions of the invention may be administered in combination with at least one or more therapeutic agents described herein. The mode of administration of the combination is not limited. For example, the therapeutic agents described above may be administered all at once or separately.

For cancer treatment, the anti-CD 47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates, or pharmaceutical compositions of the present invention may be used in combination with other therapeutic methods, including, but not limited to, surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibition, and palliative therapy.

In certain embodiments, the anti-CD 47 antibodies, or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates, or pharmaceutical compositions of the invention are further used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs. Chemotherapeutic agents may include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, microtubule inhibitors. Tumor antigen targeting drugs include, but are not limited to, drugs that target tumor-associated antigens and tumor-specific antigens. As used herein, "tumor-associated antigen" refers to an antigen that is highly expressed in tumor cells, but is also present in healthy cells but expressed at a lower level. As used herein, "tumor-specific antigen" refers to an antigen that is specifically expressed in tumor cells and hardly expressed in healthy cells. Other non-limiting examples of therapeutic agents may include, for example, angiogenesis inhibitors, deacetylase (HDAC) inhibitors, hedgehog signaling pathway blockers, mTOR inhibitors, p53/mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, marizomib, oprozomib), and tyrosine kinase inhibitors (e.g., BTK inhibitors).

In some embodiments, the anti-CD 47 antibodies, or antigen-binding fragments thereof, or multispecific antibodies of the invention are used in combination with one or more therapeutic agents selected from the group consisting of an anti-SIRPalpha antibody, an anti-CD 20 mono-gram antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-EGFR antibody, an anti-HER-2 antibody, an anti-CD 19 antibody, an anti-CD 33 antibody, an anti-CD 47 antibody, an anti-CD 73 antibody, an anti-DLL-3 antibody, an anti-TIP-1 antibody, an anti-FOLR 1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and an anti-PD-1 antibody.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof or multispecific antibodies of the invention are used in combination with a chemotherapeutic agent. In other embodiments, the antibody conjugates of the invention are used in combination with an immune checkpoint inhibitor. In still other embodiments, the antibodies or antigen-binding fragments, antibody conjugates, or pharmaceutical compositions of the invention are used in combination with a radioisotope.

Kit for detecting a substance in a sample

The invention also provides a kit comprising an anti-CD 47 antibody or antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate, an immune effector cell, or a pharmaceutical composition of the invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administering the drug. Typically, the kit further comprises a label for indicating the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with or otherwise with the kit.

Advantageous effects

The anti-CD 47 antibodies or antigen binding fragments thereof of the invention may achieve the beneficial effects of 1) specifically binding to CD47 positive tumor cells but not or substantially not binding to erythrocytes, 2) causing or substantially not causing hemagglutination, 3) blocking the binding of CD47 to SIRPalpha, and/or 4) promoting phagocytosis of CD47 positive tumor cells by macrophages.

Examples

The following examples are intended to be illustrative of the invention only and should not be construed as limiting the invention in any way.

EXAMPLE 1 animal immunization, construction of immune repertoire and antibody screening

1.1 Immunization of animals

Human IgG1 Fc or 6 XHis tag was fused at the carboxy terminus (C terminus) of the CD47-ECD in the extracellular region of CD47 to construct the CD47 antigen protein CD47-ECD-Fc (SEQ ID NO: 3) or CD47-ECD-His (SEQ ID NO: 2), respectively. CD47-ECD-Fc was used for immunization of C57BL/6 mice (Shanghai Ling Biotechnology Co., ltd.) by subcutaneous multipoint immunization, 50. Mu.g each time, once every two weeks, four times. Titers of immunization were determined four times after immunization by ELISA using CD47-ECD-His as ELISA antigen plates. The assay showed that the immunopotentiator reached 1:600000, at which time 100. Mu. gCD47-ECD-Fc was used for booster immunization, and spleen was taken 2-3 days later.

1.2 Construction of immune repertoire

B lymphocytes from the spleen of immunized mice described in example 1.1 were isolated, their RNA extracted and reverse transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A). Primers were designed to amplify the coding sequences of the light and heavy chain variable regions and the first constant region (CL and CH 1), respectively, and the coding sequence of the M13 phage GIII protein was ligated to the 3' end of the coding sequence of CH1, and then cloned into phage display vectors. The vector was then transformed into competent E.coli SS320 cells (Lucigen, MC 1061F) by electrotransfer (Bio-Rad, micropulser) and, after 1 hour of resuscitation, plated onto ampicillin-resistant 2-YT solid plates. The pool capacity of this immune pool was determined to be 1X 10 ⁹ cfu by gradient dilution plating. Packaging was performed with helper phage M13KO7 (NEB), and the final constructed immune repertoire was displayed as Fab on the coat protein of M13 phage.

1.3 Antibody screening

In the first round of screening, 4mL of 50 μg/mL of CD47-ECD-His was added to the immune tube, the coating was carried out at 4 ℃ overnight, the coating solution was discarded the next day, PBS containing 5% milk powder was added for 2 hours, the prepared phage was added for incubation for 2 hours after rinsing with PBS, rinsing to remove non-specifically bound phage, then 0.8mL of pancreatin digest containing 0.05% EDTA was added to the immune tube for eluting phage specifically bound to the target antigen, then the eluted phage was used to infect log-phase E.coli SS320 (Lucigen, 60512-1), standing at 37 ℃ for 30 minutes, then culturing at 220rpm for 1 hour, then VSCM helper phage was added for 30 minutes, culturing was continued at 220rpm for 1 hour, centrifugation and displacement to C ⁺/K⁺ -YT medium, and culturing was continued overnight at 30 ℃ and 220 rpm. The phage preparation on the next day was followed by the second round of screening, and the procedure was repeated, with 10 clones randomly selected for sequence analysis for each round. And selecting bacteria with obvious enrichment and high sequence polymorphism for monoclonal coating preparation.

Positive clones were selected for sequencing analysis by ELISA of the induced monoclonal Fab supernatant. As a result, it was found that this immune pool selection gave more than 60 positive clones with different sequences. After further performing a binding experiment on CCRF-CEM cells (a human acute lymphoblastic leukemia T lymphocyte, endogenously expressing CD47, purchased from China academy of sciences, cat# TCHu 147) and a binding blocking experiment on SIRPalpha by Fab-induced supernatants prepared by these clones, partial clones were screened to have functions of binding to both CCRF-CEM cells and blocking SIRPalpha binding on cells. Clone A7 is one of them, murine. The amino acid sequence of the heavy chain variable region of clone A7 is shown in SEQ ID NO. 10 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 11. The amino acid sequences of HCDR1, HCDR2 and HCDR3 of clone A7 defined by AbM are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 7, SEQ ID NO.8 and SEQ ID NO. 9, respectively.

Example 2 antibody engineering

The variable region of the humanized antibody A7H3L3 was obtained by humanization of the cloned A7 variable region. The variable regions of antibodies A7-44, A7-28 and A7-47 were obtained by affinity maturation engineering the variable regions of humanized antibody A7H3L 3.

2.1 Humanization of antibodies

The murine VH and VL sequences of clone A7 screened in example 1 were aligned with the database of known human antibodies to find the human germline (germline) gene VH and VL sequences that have the highest homology to the murine VH and VL sequences, respectively. The framework regions of the VH and VL sequences of the human germline gene (the CDR and framework regions are defined using AbM), then the murine amino acids in the framework region of clone A7 that have an important effect on antigen binding are retained by means of back-mutation by means of computer predictive modeling, and finally the Complementarity Determining Region (CDR) sequences of this germline gene are replaced with the corresponding CDR sequences in clone A7. Cloning of the A7 variable region the humanized antibody A7H3L3 variable region was obtained by humanization of the A7H3L3 heavy chain variable region (VH) amino acid sequence shown in SEQ ID NO. 14 and the light chain variable region (VL) amino acid sequence shown in SEQ ID NO. 15 (Table 1).

2.2 Affinity maturation engineering

Affinity maturation was engineered for antibody A7H3L3 for improved affinity and biological activity. The affinity maturation transformation is based on an M13 phage display technology, a codon-based primer (in the primer synthesis process, a single codon consists of NNK) is adopted to introduce CDR region mutation, 4 phage display libraries are constructed, wherein library 1 and library 2 are single-point combination mutation, library 1 is CDRL1+CDRL3+CDRH3 combination mutation, library 2 is CDRL2+CDRH1+CDRH2 combination mutation, library 3 and library 4 are double-point saturation mutation, library 3 is double-point saturation mutation of CDRL3, and library 4 is double-point saturation mutation of CDRH 3. The specific library construction method comprises the steps of firstly synthesizing a primer containing point mutation (Jin Weizhi Biotechnology Co., ltd.), secondly taking an antibody A7H3L3 to be modified as a PCR amplification template, amplifying a CDR region to contain sequences of designed mutation, combining fragments containing different CDR mutations through a bridging PCR method, connecting the point mutation antibody to a phage display carrier through double digestion (HindIII and NotI) and double sticky end connection, and finally transferring an antibody sequence with mutation sites into escherichia coli SS320 through electrotransformation. Library capacity calculation, phage library preparation and library screening procedures were detailed in example 1, and 3 anti-CD 47 affinity matured antibodies A7-44, A7-28 and A7-47 were selected (Table 1).

TABLE 1 amino acid sequence of anti-CD 47 antibodies (SEQ ID NO:)

Antibody name	HCDR1	HCDR2	HCDR3	LCDR1	LCDR2	LCDR3	VH	VL
									A7H3L3	4	5	6	7	8	9	14	15
A7-44	4	5	6	7	16	9	14	17
									A7-28	18	19	6	7	20	9	21	22
A7-47	4	5	23	24	8	25	26	27

EXAMPLE 3 construction, expression and purification of anti-CD 47 antibodies

The coding sequence of VH (SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21 or SEQ ID NO: 26) of the anti-human CD47 antibody described in examples 1 and 2 was ligated to the coding sequence of the heavy chain constant region (SEQ ID NO: 12) of human IgG4 to construct a heavy chain coding sequence of the anti-CD 47 antibody, and the coding sequence of VL (SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO: 27) of the anti-human CD47 antibody was ligated to the coding sequence of the human Kappa constant region (SEQ ID NO: 13) to construct a light chain coding sequence of the anti-CD 47 antibody. Heavy and light chain coding sequences were inserted into expression plasmid pcdna3.4 (Invitrogen) to construct expression vectors for heavy and light chains of anti-CD 47 antibodies, respectively. The heavy chain and light chain expression vectors are respectively transformed into escherichia coli DH5 alpha, cultured at 37 ℃ overnight, and subjected to plasmid extraction by using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody light chain and heavy chain plasmids for eukaryotic expression.

Cell culture supernatants expressing the protein of interest (i.e., anti-CD 47 antibody) expressed by ExpiCHO transient expression system (Thermo Fisher, a 29133) were centrifuged at 15000g for 10min at high speed after 7-15 days of transfection, and the resulting supernatants were affinity purified by MabSelect SuRe LX (GE, 17547403), then eluted with 100mM sodium acetate (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting proteins were replaced into PBS buffer by ultrafiltration concentrate tube (Millipore, UFC 901096). The anti-CD 47 antibodies A7H3L3, A7-44, A7-28 and A7-47 with higher purity are obtained through SDS-PAGE identification and SEC purity detection.

Example 4 binding Activity of anti-CD 47 antibodies to CD47 on CCRF-CEM cells

Since CD47 at the cellular level is more similar to the native conformation relative to the recombinant protein, the binding capacity of the anti-CD 47 antibodies of the invention and the positive control antibody 1F8 (WO 2018075857A1, heavy chain variable region (VH) amino acid sequence shown in SEQ ID NO:28, light chain variable region (VL) amino acid sequence shown in SEQ ID NO:29, preparation as described in example 3) on CCRF-CEM cells was determined by flow cytometry in this example.

The specific method is that 1X 10 ⁵ CCRF-CEM cells are taken, the supernatant is removed by low-speed centrifugation (300 g), the cells at the bottom of the centrifuge tube are rinsed once by a formulated FACS buffer (1 XPBS buffer containing 2% FBS by volume), then the antibody to be tested is added to the rinsed cells for gradient dilution, incubated for 1h at 4 ℃, then rinsed three times with the above FACS buffer, PE-labeled goat anti-human IgG Fc antibody (Abcam, ab 98596) is added for 0.5 μg, incubated for 1h at 4 ℃, then the cells are rinsed three times with FACS buffer and resuspended with 200 μL of FACS buffer, and finally the amount of anti-CD 47 antibody bound on the CCRF-CEM cells (expressed as Mean Fluorescence Intensity (MFI)) is detected by a flow cytometer (Beckman, cytoFLEX AOO-1-1102).

The results are shown in FIGS. 1A and 1B, antibodies A7H3L3, A7-44, A7-28, and A7-47 bind to CD47 on CCRF-CEM cells with high binding activity, and the binding activity is superior to 1F8.

Example 5 binding Activity of anti-CD 47 antibodies to CD47 on erythrocytes

To determine whether the anti-CD 47 antibodies of the application bind to CD47 on erythrocytes, the binding activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to erythrocytes was determined by flow cytometry (FACS). As a comparison, the binding activity of positive control antibodies 1F8 and F4AM4 (another anti-CD 47 antibody, which the inventors have self-developed, heavy chain having the amino acid sequence of SEQ ID NO:30 and light chain having the amino acid sequence of SEQ ID NO: 31) to erythrocytes was also determined. Human IgG4 was used as isotype negative control.

The method comprises separating red blood cells from 1mL anticoagulated human blood, centrifuging, removing supernatant, rinsing with PBS twice, adding 1mL PBS, and re-suspending. Red blood cells are diluted to 1X 10 ⁷/mL by using PBS, 50 mu L of the red blood cells are sucked up from each hole and added into a 96-hole round-bottomed cell culture plate, then gradient diluted antibody to be detected is added into the culture plate in an equal volume, and the mixture is fully and uniformly mixed and placed at 4 ℃ for incubation for 1h. Then rinsed three times with FACS buffer, 0.5. Mu.g of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab 98596) was added and incubated at 4℃for 1h. Thereafter, the cells were resuspended by washing three times with FACS buffer and 200. Mu.L of FACS buffer was added to the cells, and finally the amount of bound anti-CD 47 antibody (MFI) on the erythrocytes was detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).

As a result, as shown in FIGS. 2A-2B, antibody A7H3L3 hardly bound to CD47 on erythrocytes and the binding activity was comparable to or weaker than that of positive control antibody 1F8, antibodies A7-44 and A7-47 weakly bound to CD47 on erythrocytes, and antibodies A7-28 bound to CD47 on erythrocytes (but also much weaker than positive control antibody F4AM 4). These results indicate that antibodies A7H3L3, A7-44, A7-47 do not substantially bind to erythrocytes while exhibiting a higher specificity for targeting CD47 positive tumor cells.

Example 6 blocking Activity of anti-CD 47 antibodies to block binding of CD47 to SIRPalpha

The blocking activity of the anti-CD 47 antibodies of the invention to block binding of CD47 to the receptor sirpa on tumor cells was determined by flow cytometry (FACS). Human IgG4 was used as isotype negative control.

The method is carried out by taking 1X 10 ⁵ CCRF-CEM cells, centrifuging at low speed (300 g) to remove the supernatant. Cells at the bottom of the centrifuge tube were rinsed once with formulated FACS buffer (1×pbs buffer containing 2% fbs), and then gradient diluted test antibody was added to the rinsed cells and incubated for 1h. After rinsing the cells twice with FACS buffer, 100. Mu.L of 1. Mu.g/mL SIRPalpha-mFc (ACRO, SIA-H52A 8) was added and incubated for 1H at 4 ℃. After three washes with FACS buffer, 100. Mu.L of PE-labeled goat anti-mouse Fc secondary antibody (1:200, abcam, ab98742) was added, the supernatant was centrifuged off after incubation at 4℃for 1h, 200. Mu.L of FACS buffer was added to the cells to resuspend the cells, and finally the amount of SIRPalpha-mFc bound on CCRF-CEM cells (MFI) was detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).

The results are shown in FIG. 3, where antibodies A7-44, A7-28 and A7-47 effectively block binding of CD47 to SIRP alpha on tumor cells and the blocking ability is superior to positive control antibody 1F8.

EXAMPLE 7 hemagglutination of anti-CD 47 antibodies on erythrocytes

The antibody drug reaches the tumor focus through blood circulation to exert drug effect, and the red blood cells in the blood express CD47 in a large amount. For antibodies that bind to CD47 on the surface of erythrocytes with high binding activity, aggregation of erythrocytes may be caused by crosslinking when the concentration of the antibody reaches a certain level, so that the aggregated erythrocytes are cleared by phagocytes, which is one of the important causes of toxic reactions such as anemia to some extent. This example compares the hemagglutination response of the anti-CD 47 antibodies of the present invention to red blood cells with positive control antibodies 1F8, hu5F9 (also known as magrolimab, see U.S. patent application US20160304609 A1). Human IgG4 was used as isotype negative control.

The method comprises separating red blood cells from 1mL anticoagulated human blood, centrifuging, removing supernatant, rinsing with PBS twice, adding 1mL PBS, and re-suspending. Red blood cells are diluted 20 times by PBS, 50 mu L of the red blood cells are sucked up by each hole and added into a 96-hole round-bottomed cell culture plate, then gradient diluted (0.05-100 mu g/mL) antibody to be detected is added into the culture plate in an equal volume, fully and uniformly mixed, and the mixture is placed into a 37 ℃ incubator for incubation for 3 hours. And then taking out the sample to observe the degree of the hemagglutination reaction, wherein the size of the area of erythrocyte deposition represents the strength of the hemagglutination, and the larger the area is, the stronger the hemagglutination is, and the weaker the area is, on the contrary, the weaker the hemagglutination is.

The results are shown in fig. 4A, where the antibody A7 treated erythrocytes aggregate into a small pellet which settles at the bottom of the round hole, consistent with the negative control results, indicating that they do not have hemagglutination, whereas the positive control antibody Hu5F9 treated erythrocytes have a large area of dispersion, indicating that the hemagglutination is very severe, consistent with the report results in patent US20160304609 A1. This suggests that chimeric antibody A7 is superior in hemagglutination response to positive control antibody Hu5F9, and is expected to show clinically lower anemia toxicity or side effects.

As shown in FIG. 4B, the results of the antibodies A7H3L3, A7-44 and A7-47 were shown to aggregate into a small pellet that settled at the bottom of the round hole, consistent with the negative control (IgG 4) results, indicating that it did not initiate a hemagglutination reaction, whereas the A7-28 treated erythrocytes had a degree of hemagglutination, essentially comparable to that of antibody 1F 8. The above results indicate that antibodies A7H3L3, A7-44 and A7-47 are superior to antibody 1F8 in terms of hemagglutination, and are expected to show clinically lower anemia toxicity or side effects.

Example 8 ability of anti-CD 47 antibodies to promote phagocytosis of tumor cells by macrophages

Antibodies that block binding of CD47 and sirpa can effectively promote phagocytosis of CD47 positive tumor cells by macrophages. This example compares the ability of the anti-CD 47 antibodies of the invention to the positive control antibody 1F8 to promote phagocytosis of tumor cells by macrophages. Human IgG4 was used as isotype negative control.

The specific method is that firstly, human peripheral blood mononuclear cells PBMC are isolated, 50ng/mL rhM-CSF (purchased from Peprotech, 300-25-10) is added for inducing the differentiation of cells into macrophages, after about 8 days of culture in a 37 ℃ cell culture incubator, the supernatant and the non-adherent cells are removed, then Ackutase ^TM cell digest (purchased from Sigma, A6964) is added, the cells are incubated for 45min at 37 ℃, the adherent cells are digested and prepared into a uniform cell suspension with complete medium (RPMI 1640+10% FBS), and 5×10 ⁴ cells are dispensed into 96-well plates per well. Gradient dilutions of the test antibody (1. Mu.g/mL-0.002. Mu.g/mL) were added, incubated at 37℃for 30min, followed by 2.5X10 ⁴ CFSE (from Abcam, ab 113853) labeled CCRF-CEM cells, and 0.25. Mu.g of APC labeled anti-CD 14 antibody (from eBioscience, 17-0149-42) after incubation at 37℃for 1 h. The cell suspension was then incubated at 4℃for 20min, then centrifuged at 500g for 5min, the cells were washed twice with FACS buffer and finally examined by flow cytometry (Beckman, cytoFLEX AOO-1-1102), wherein the ratio of CFSE-labeled CCRF-CEM cells phagocytosed in CD14 ⁺ macrophages was the phagocytosis rate.

The results are shown in FIG. 5, where antibodies A7-28 and A7-47 dose-dependently promote phagocytosis of tumor cells by macrophages, and both have better effects on promoting phagocytosis of tumor cells by macrophages than antibody 1F8.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

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<223> CD47-ECD-His

<400> 2

Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn

1 5 10 15

Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn

20 25 30

Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr

35 40 45

Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser

50 55 60

Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu

65 70 75 80

Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys

85 90 95

Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys

100 105 110

Tyr Arg Val Val Ser Trp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

115 120 125

Ser Ala His His His His His His

130 135

<210> 3

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> CD47-ECD-Fc

<400> 3

Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn

1 5 10 15

Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn

20 25 30

Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr

35 40 45

Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser

50 55 60

Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu

65 70 75 80

Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys

85 90 95

Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys

100 105 110

Tyr Arg Val Val Ser Trp Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-HCDR1

<400> 4

Gly Phe Asn Ile Lys Asp Ile Tyr Ile Tyr

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-HCDR2

<400> 5

Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-HCDR3

<400> 6

Gly Tyr Gly Ser Gly Phe Ala Tyr

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-LCDR1

<400> 7

Arg Ala Ser Gln Asp Ile Ser Asn His Leu Asn

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-LCDR2

<400> 8

Tyr Thr Ser Arg Ile His Ser

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-LCDR3

<400> 9

Gln Gln Gly Tyr Thr Leu Pro Phe Thr

1 5

<210> 10

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-VH

<400> 10

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Ile Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Thr

115

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-VL

<400> 11

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Phe Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Arg Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 327

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG4 heavy chain constant region

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser 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 Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 13

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Human light chain constant region

<400> 13

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> 14

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-VH/A7-44

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 15

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-VL

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 16

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-44 LCDR2

<400> 16

Tyr Thr Ser Arg Ile His Leu

1 5

<210> 17

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-44 VL

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Leu Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-28 HCDR1

<400> 18

Gly Phe Asn Ile Lys Asp Val Tyr Ile Tyr

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-28 HCDR2

<400> 19

Lys Ile Asp Pro Ala Asn Gly Asn Thr His

1 5 10

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-28 LCDR2

<400> 20

Tyr Thr Ser Arg Ile His Lys

1 5

<210> 21

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-28 VH

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Val

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr His Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-28 VL

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Lys Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-47 HCDR3

<400> 23

Gly Tyr Gly Ser Val Phe Ala Tyr

1 5

<210> 24

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-47 LCDR1

<400> 24

Arg Ala Ser Gln Asp Ile Ser Asn His Ile Asn

1 5 10

<210> 25

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-47 LCDR3

<400> 25

Gln Gln Gly Tyr His Leu Pro Phe Thr

1 5

<210> 26

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-47 VH

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Val Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 27

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7-47 VL

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Ile Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr His Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 1F8 VH

<400> 28

Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Arg Lys Thr Asp Gly Glu Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Gly Ser Asn Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ala

115

<210> 29

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> 1F8 VL

<400> 29

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Asn Gln Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile

65 70 75 80

Ile Ser Ser Leu His Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Thr Pro Pro Leu Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 30

<211> 452

<212> PRT

<213> Artificial Sequence

<220>

<223> F4AM4 heavy chain

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Thr Asp Gly Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Pro Tyr Tyr Gly Thr Arg Tyr Gly Ser Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 31

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> F4AM4 light chain

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Lys Asn Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. An anti-CD 47 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO.4, the HCDR2 sequence shown in SEQ ID NO. 5 and the HCDR3 sequence shown in SEQ ID NO.6, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO. 7, the LCDR2 sequence shown in SEQ ID NO. 8 and the LCDR3 sequence shown in SEQ ID NO. 9, or

2. The antibody or antigen-binding fragment thereof of claim 1, wherein

The heavy chain variable region comprises 1) an amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21 or SEQ ID NO 26, 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21 or SEQ ID NO 26, or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence of SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 21 or SEQ ID NO 26.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein

The light chain variable region comprises 1) an amino acid sequence of SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 22 or SEQ ID NO. 27, 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 22 or SEQ ID NO. 27, or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 22 or SEQ ID NO. 27.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein

The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 11, or

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a chimeric antibody, a humanized antibody, a human antibody, scFv, fab, fab ', F (ab') ₂, an Fv fragment, a disulfide stabilized Fv (dsFv), or a diabody.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a heavy chain constant region and/or a light chain constant region.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein

The heavy chain constant region is a heavy chain constant region of human IgG4 and/or the light chain constant region is a human kappa light chain constant region.

8. The antibody or antigen-binding fragment thereof of claim 6, wherein

The heavy chain constant region comprises 1) the amino acid sequence of SEQ ID NO. 12, or 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO. 12, and/or

The light chain constant region comprises 1) the amino acid sequence of SEQ ID NO. 13, or 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO. 13.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which

1) Specifically bind CD47 positive cancer cells but not erythrocytes;

2) Does not cause erythrocyte aggregation;

3) Blocking the binding of CD47 to SIRPalpha, and/or

4) Promoting phagocytosis of CD47 positive tumor cells by macrophages.

10. A multispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds a second antigen, wherein the first antigen-binding portion comprises the antibody or antigen-binding fragment thereof of any one of claims 1-5.

11. The multispecific antibody of claim 10, wherein the second antigen-binding portion specifically binds a tumor antigen, an immunomodulatory receptor, or an immune checkpoint molecule.

12. The multispecific antibody of claim 11, wherein

The second antigen binding portion specifically binds SIRPα、PD-1、PD-L1、LAG3、TIM-3、CTLA-4、VISTA、GPC3、EGFR、HER-2、CD19、CD20、CD33、CD40、CD73、OX40、CD3、DLL-3、TIP-1、 folate receptor alpha (FOLR 1) or other tumor antigens.

13. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5.

14. An immune effector cell expressing the chimeric antigen receptor of claim 13 on a surface.

15. The immune effector cell of claim 14, selected from the group consisting of cytotoxic T cells and natural killer cells.

16. The immune effector cell of claim 14, which is a natural killer T cell.

17. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.

18. An expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17 or the expression vector of claim 18.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9, the multispecific antibody of any one of claims 10-12, or the immune effector cell of any one of claims 14-16, and a pharmaceutically acceptable carrier.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-9, the multispecific antibody of any one of claims 10-12, or the pharmaceutical composition of claim 20 in the manufacture of a medicament for treating a cancer, wherein the cancer is selected from Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), myelodysplastic syndrome, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple Myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, stomach cancer, kidney cancer, smooth muscle tumor, and glioma.

22. The use of claim 21, wherein the cancer is selected from burkitt's lymphoma, follicular lymphoma, and glioblastoma.

23. The use of claim 21 or 22, wherein the medicament is used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs.