WO2022262783A1 - Anticorps entièrement humain anti-cd22 ou fragment de liaison à l'antigène de celui-ci, son procédé de préparation et son utilisation - Google Patents

Anticorps entièrement humain anti-cd22 ou fragment de liaison à l'antigène de celui-ci, son procédé de préparation et son utilisation Download PDF

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WO2022262783A1
WO2022262783A1 PCT/CN2022/099016 CN2022099016W WO2022262783A1 WO 2022262783 A1 WO2022262783 A1 WO 2022262783A1 CN 2022099016 W CN2022099016 W CN 2022099016W WO 2022262783 A1 WO2022262783 A1 WO 2022262783A1
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cdr
antibody
seq
antigen
binding fragment
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PCT/CN2022/099016
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Chinese (zh)
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龙飞
S· 蒂米特鲁夫迪米特尔
尤拉马辛
彼得森埃里克
麦劳斯约翰
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西安宇繁生物科技有限责任公司
阿邦德生物有限公司
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Publication of WO2022262783A1 publication Critical patent/WO2022262783A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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  • the invention relates to the technical field of biomedicine, in particular to an anti-CD22 fully human single-chain antibody or an antigen-binding fragment thereof and a preparation method and application thereof.
  • CD22 (Siglec-2) is a sialic acid-binding Ig-like lectins (Sialic acid-binding Ig-like lectins or Siglecs), which is an important member of the sialic acid-binding immunoglobulin-like lectins family. Its molecular weight is 140kDa, it belongs to type I transmembrane protein, the extracellular domain is composed of 7 IgG-like domains, and the most distal V-set Ig domain plays a major role in binding ⁇ 2,6-sialic acid ( ⁇ 2,6sia) ligand The function of the linked C2-set Ig domain may be to allow the V-set Ig domain to fold correctly.
  • the intracellular domain of CD22 includes an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine activation motif (ITAM).
  • ITIM immunoreceptor tyrosine inhibitory motif
  • ITAM immunoreceptor tyrosine
  • CD22 molecule is one of the inhibitory co-receptors on the surface of B cells, which is closely related to the development, differentiation and function of B cells. CD22 is restricted to the surface of mature B cells and most B lymphoma cells. In most cases, CD22 molecules are still expressed during the transformation of normal B cells into tumor cells. Relevant data show that about 60-80% of B-cell lymphoma and leukemia cells express CD22 molecules, including acute B-lymphoblastic leukemia (B-ALL), indolent and high-grade non-Hodgkin's lymphoma, chronic lymphocytic leukemia and Hairy cell leukemia. Therefore, targeting CD22 for tumor immunotherapy has become one of the hotspots in immune research.
  • B-ALL acute B-lymphoblastic leukemia
  • indolent and high-grade non-Hodgkin's lymphoma chronic lymphocytic leukemia and Hairy cell leukemia. Therefore, targeting CD22 for tumor immunotherapy has become one of the hots
  • CD22 is highly expressed in both B-ALL and mature lymphocyte malignancies, for B-ALL cells with low expression of CD19 or loss of CD19 molecules, CD22 is expected to be an alternative target of CD19 in B-ALL.
  • Antibodies as targeted therapeutics have proven to be a reliable and effective option in the treatment of tumors and autoimmune diseases.
  • single chain variable fragments scFv
  • scFv single chain variable fragments
  • the single-chain antibody obtained by using the fully human phage display technology greatly avoids the severe body rejection caused by traditional antibodies during treatment, and at the same time can ensure the safety and therapeutic effect of clinical application.
  • CAR-T chimeric antigen receptor T cell
  • the object of the present invention is to provide an anti-CD22 fully human single-chain antibody or an antigen-binding fragment thereof, a preparation method and application thereof.
  • the anti-CD22 antibody or its antigen-binding fragment has strong specificity and low immunogenicity; it can specifically bind to CD22 antigen protein and has excellent affinity; it can effectively bind to CD22-positive target cells and can be used to treat diseases related to CD22 expression diseases, especially for the development of B-ALL treatment or early diagnosis reagents.
  • the first aspect of the present invention provides an anti-CD22 antibody or an antigen-binding fragment thereof.
  • the anti-CD22 antibody or antigen-binding fragment thereof specifically binds to the human CD22 antigen and inhibits or competes for other antibodies (preferably the same or overlapping epitopes as the anti-CD22 antibody of the present invention) to bind to human CD22 Antigen binding.
  • the anti-CD22 antibody or antigen-binding fragment thereof is a single chain.
  • amino acid sequences of the heavy chain variable region and/or light chain variable region are derived from humans.
  • the anti-CD22 antibody or antigen-binding fragment thereof is fully human.
  • the anti-CD22 antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region and/or CDR-H1, CDR-H2, and CDR of the heavy chain variable region. -H3.
  • amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequentially comprise SEQ ID NO: 7-9 or 10-12, or have the same sequence as SEQ ID NO: 7-9 or 10-12 Amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.
  • amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 comprise SEQ ID NO: 1-3 or 4-6 in sequence, or have the same sequence as SEQ ID NO: 1-3 or 4-6 Amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.
  • the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequentially comprise SEQ ID NO: 1-3, or have at least 80% of SEQ ID NO: 1-3 , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and, the CDR-L1, CDR- The amino acid sequences of L2 and CDR-L3 comprise SEQ ID NO: 7-9 sequentially, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequences of 95%, 96%, 97%, 98%, 99% identity.
  • the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO: 1-3, respectively. shown; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are respectively shown in SEQ ID NO: 7-9.
  • the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequentially comprise SEQ ID NO: 4-6, or have at least 80% of SEQ ID NO: 4-6 , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and, the CDR-L1, CDR- The amino acid sequences of L2 and CDR-L3 comprise SEQ ID NO: 10-12 sequentially, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequences of 95%, 96%, 97%, 98%, 99% identity.
  • the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO: 4-6, respectively. shown; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 10-12, respectively.
  • amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminal to C-terminal.
  • amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are arranged in order from N-terminal to C-terminal.
  • the amino acid sequence of the light chain variable region comprises SEQ ID NO: 15 or 16, or at least 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NO: 15 or 16 Amino acid sequences with %, 94%, 95%, 96%, 97%, 98%, 99% identity. Further preferably as shown in SEQ ID NO: 15 or 16.
  • the amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 13 or 14, or has at least 80%, 85%, 90%, 91%, 92%, 93% of SEQ ID NO: 13 or 14 Amino acid sequences with %, 94%, 95%, 96%, 97%, 98%, 99% identity. Further preferably as shown in SEQ ID NO: 13 or 14.
  • the amino acid sequence of the light chain variable region comprises SEQ ID NO: 15, or at least 80%, 85%, 90%, 91%, 92% with SEQ ID NO: 15 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence
  • the amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 13, or with SEQ ID NO: 13 is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the amino acid sequence of the light chain variable region comprises SEQ ID NO: 16, or at least 80%, 85%, 90%, 91%, 92% of SEQ ID NO: 16 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence
  • the amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 14, or with SEQ ID NO: 14 is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region As shown in SEQ ID NO:15.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region As shown in SEQ ID NO:16.
  • the light chain variable region is directly connected to the heavy chain variable region or connected through a linker, preferably, the linker is a connecting peptide.
  • the connecting peptide is a flexible connecting peptide.
  • the connecting peptide can be GGGGS (SEQ ID NO: 37), GGGGSGGGGSGGGGS (SEQ ID NO: 38) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 39).
  • the anti-CD22 antibody or antigen-binding fragment thereof includes a heavy chain variable region, a connecting peptide and a light chain variable region in sequence from N-terminus to C-terminus.
  • the anti-CD22 antibody or antigen-binding fragment thereof comprises a light chain variable region, a connecting peptide and a heavy chain variable region in sequence from N-terminus to C-terminus.
  • the anti-CD22 antibody or antigen-binding fragment thereof is a single-chain antibody (scFv).
  • the anti-CD22 antibody or its antigen-binding fragment can also be Fab, Fab', Fab'-SH, Fv, F(ab') 2 , single-domain antibody, diabody (dAb) or linear antibody, etc. form.
  • the amino acid sequence of the anti-CD22 antibody or antigen-binding fragment thereof comprises SEQ ID NO: 17 or 18, or has at least 80%, 85%, or Amino acid sequences of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • amino acid sequence of the anti-CD22 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 17 or 18.
  • the second aspect of the present invention provides a nucleic acid encoding the anti-CD22 antibody or antigen-binding fragment thereof of the present invention.
  • a nucleic acid which comprises a nucleotide sequence encoding a light chain variable region, a nucleotide sequence encoding a heavy chain variable region, and/or encoding CDR-H1, CDR - Nucleotide sequences of H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3.
  • the nucleotide sequences encoding CDR-H1, CDR-H2, and CDR-H3 sequentially comprise SEQ ID NO: 19-21, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences; or,
  • the nucleotide sequence encoding CDR-H1, CDR-H2, CDR-H3 comprises SEQ ID NO: 22-24 sequentially, or has at least 80%, 85%, 90%, 91%, Nucleotide sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • nucleotide sequences encoding CDR-H1, CDR-H2, and CDR-H3 are sequentially shown in SEQ ID NO: 19-21 or shown in SEQ ID NO: 22-24.
  • the nucleotide sequence encoding CDR-L1, CDR-L2, CDR-L3 comprises SEQ ID NO: 25-27, or has at least 80%, 85%, 90%, 91% of SEQ ID NO: 25-27 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences; or,
  • the nucleotide sequence encoding CDR-L1, CDR-L2, CDR-L3 comprises SEQ ID NO: 28-30, or has at least 80%, 85%, 90%, 91%, 92% of SEQ ID NO: 28-30 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences.
  • nucleotide sequences encoding CDR-L1, CDR-L2, and CDR-L3 are sequentially shown in SEQ ID NO: 25-27 or shown in SEQ ID NO: 28-30.
  • the nucleotide sequence encoding the heavy chain variable region comprises SEQ ID NO: 31 or 32, or has at least 80%, 85%, 90%, 91%, 92%, 93% of SEQ ID NO: 31 or 32 %, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences. Further preferably, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 31 or 32.
  • the nucleotide sequence encoding the light chain variable region comprises SEQ ID NO: 33 or 34, or at least 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NO: 33 or 34 %, 94%, 95%, 96%, 97%, 98%, 99% identity and encode the same amino acid sequence. Further preferably, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 33 or 34.
  • the nucleic acid encoding the anti-CD22 antibody or its antigen-binding fragment comprises the nucleotide sequence shown in SEQ ID NO: 31, and, the nucleotide sequence shown in SEQ ID NO: 33 acid sequence.
  • the nucleic acid encoding an anti-CD22 antibody or an antigen-binding fragment thereof comprises the nucleotide sequence shown in SEQ ID NO: 32, and the nucleotide sequence shown in SEQ ID NO: 34 acid sequence.
  • the nucleotide sequence of the nucleic acid encoding an anti-CD22 antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 35, or at least 80%, 85%, 90% with SEQ ID NO: 35 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleotide sequences that are identical and encode the same amino acid sequence.
  • the nucleotide sequence of the nucleic acid encoding an anti-CD22 antibody or an antigen-binding fragment thereof comprises SEQ ID NO: 36, or has at least 80%, 85%, 90% of SEQ ID NO: 36 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleotide sequences that are identical and encode the same amino acid sequence.
  • nucleotide sequence of the nucleic acid encoding the anti-CD22 antibody or an antigen-binding fragment thereof is shown in SEQ ID NO: 35 or 36.
  • the nucleotide sequences encoding CDR-H1, CDR-H2, and CDR-H3 are arranged sequentially in the direction from 5' to 3'.
  • the nucleotide sequences encoding CDR-L1, CDR-L2, and CDR-L3 are arranged sequentially in the direction from 5' to 3'.
  • the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are aligned in the direction of 5' to 3' or 3' to 5'.
  • the nucleic acid further comprises a connecting nucleic acid fragment, and the connecting nucleic acid fragment connects the nucleotide sequence encoding the variable region of the heavy chain and the nucleotide sequence encoding the variable region of the light chain.
  • the connecting nucleic acid fragment encodes the connecting peptide.
  • nucleotide sequence encoding the variable region of the heavy chain, the connecting nucleic acid fragment and the nucleotide sequence encoding the variable region of the light chain are arranged in a 5' to 3' direction.
  • nucleotide sequence encoding the variable region of the light chain, the connecting nucleic acid fragment and the nucleotide sequence encoding the variable region of the heavy chain are arranged in a 5' to 3' direction.
  • the fourth aspect of the present invention provides a chimeric antigen receptor, the extracellular domain of the chimeric antigen receptor comprises the anti-CD22 antibody or antigen-binding fragment thereof of the present invention.
  • the chimeric antigen receptor further comprises any conventional transmembrane region and/or intracellular signal transduction region in the prior art.
  • the fifth aspect of the present invention provides a nucleic acid encoding the chimeric antigen receptor of the present invention.
  • the nucleic acid encoding the chimeric antigen receptor comprises the nucleic acid of the anti-CD22 antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid encoding the extracellular domain of the chimeric antigen receptor is as shown in SEQ ID NO: 35, or has at least 80%, 85%, or 90% of SEQ ID NO: 35 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences that encode identical amino acid sequences.
  • the nucleic acid encoding the extracellular domain of the chimeric antigen receptor is as shown in SEQ ID NO: 36, or has at least 80%, 85%, or 90% of SEQ ID NO: 36 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences that encode identical amino acid sequences.
  • the sixth aspect of the present invention provides a vector comprising the nucleic acid of the present invention.
  • the vector can be expressed in vivo or in vitro or in vitro.
  • the expression vector is a prokaryotic expression vector or a lentiviral expression vector.
  • the prokaryotic expression vector is Escherichia coli series.
  • the vector is pComb3X.
  • the seventh aspect of the present invention provides a host cell comprising the nucleic acid or vector of the present invention.
  • said host cell can be eukaryotic or prokaryotic. More preferably, the host cells are yeast cells, 293 cells, CHO cells, Escherichia coli and the like.
  • described host cell is Top 10F 'escherichia coli.
  • the eighth aspect of the present invention provides an immune cell expressing the chimeric antigen receptor of the present invention.
  • the immune cells include, but are not limited to, lymphocytes (such as T cells, B cells, NK cells), dendritic cells, monocytes/macrophages, granulocytes, and mast cells.
  • the immune cells are CAR-immune cells.
  • the ninth aspect of the present invention provides a method for preparing immune cells, the preparation method comprising transfecting the nucleic acid sequence encoding the chimeric antigen receptor of the present invention into immune cells for expression.
  • said cell expresses said chimeric antigen receptor.
  • the immune cells include, but are not limited to, lymphocytes (such as T cells, B cells, NK cells), dendritic cells, monocytes/macrophages, granulocytes, and mast cells.
  • the immune cells are CAR-immune cells.
  • the tenth aspect of the present invention provides a T cell, the T cell comprising the anti-CD22 antibody or antigen-binding fragment thereof of the present invention.
  • the T cells are CAR-T cells.
  • the eleventh aspect of the present invention provides an antibody drug conjugate (antibody-drug conjugate, ADC), which comprises the anti-CD22 antibody or antigen-binding fragment thereof of the present invention covalently bound to a drug.
  • ADC antibody-drug conjugate
  • the drugs in the ADC can be chemically synthesized drugs, antibiotics or various biological drugs.
  • the twelfth aspect of the present invention provides an anti-CD22 antibody or its antigen-binding fragment, nucleic acid, chimeric antigen receptor, vector, host cell or immune cell described in the present invention in the preparation of an antibody drug conjugate (ADC) ) or applications in multifunctional antibodies.
  • ADC antibody drug conjugate
  • the thirteenth aspect of the present invention provides a pharmaceutical composition comprising any one or a combination of two or more of the following:
  • the pharmaceutical composition specifically targets tumor cells expressing CD22.
  • the pharmaceutical composition further includes pharmaceutically acceptable auxiliary materials.
  • the fourteenth aspect of the present invention provides a method for preparing the anti-CD22 antibody or antigen-binding fragment thereof of the present invention, which includes the step of culturing host cells comprising the above-mentioned nucleic acid or vector of the present invention.
  • the preparation method comprises the steps of:
  • screening vectors such as recombinant plasmids
  • nucleic acids of the anti-CD22 antibody or its antigen-binding fragment containing nucleic acids of the anti-CD22 antibody or its antigen-binding fragment
  • step (b) transforming the vector (such as a recombinant plasmid) obtained by screening in step (a) into a host cell, and then inducing its expression and purification;
  • the screening in the step (a) includes screening the vector containing the nucleic acid of the anti-CD22 antibody or its antigen-binding fragment from a fully human natural phage antibody library, and then obtaining the vector encoding the variable region of the heavy chain and the light chain
  • the nucleotide sequence of the variable region determines the amino acid sequence of the variable region of the heavy chain and the variable region of the light chain.
  • using CD22 as the solid phase through 3 to 5 rounds of "adsorption-elution-amplification" process, the anti-CD22 antibody or its antigen-binding fragment is screened from the whole human natural phage antibody library. Nucleic acid carrier.
  • the vector is a prokaryotic expression vector.
  • the prokaryotic expression vector is Escherichia coli series.
  • the vector is pComb 3X.
  • said host cell can be eukaryotic or prokaryotic. More preferably, the host cells are yeast cells, 293 cells, CHO cells, Escherichia coli and the like.
  • the host cell is Top 10F'.
  • the preparation method further includes steps such as purification and/or concentration.
  • the fifteenth aspect of the present invention provides the anti-CD22 antibody of the present invention or its antigen-binding fragment, the chimeric antigen receptor of the present invention, the nucleic acid of the present invention, the carrier of the present invention, the present invention Application of the host cell of the invention, the immune cell of the invention or the pharmaceutical composition of the invention in the preparation of products for diagnosing, preventing and/or treating diseases related to CD22 expression.
  • the product is a targeted drug.
  • the product may be a kit, a chip, an antibody drug conjugate or a multifunctional antibody.
  • the disease associated with CD22 expression is selected from tumors.
  • the tumor is selected from acute B-lymphoblastic leukemia (B-ALL), indolent and high-grade non-Hodgkin's lymphoma, chronic lymphocytic leukemia and hairy cell leukemia.
  • B-ALL acute B-lymphoblastic leukemia
  • indolent and high-grade non-Hodgkin's lymphoma chronic lymphocytic leukemia and hairy cell leukemia.
  • the sixteenth aspect of the present invention provides a method for detecting CD22, the method comprising contacting the sample to be detected with the anti-CD22 antibody or antigen-binding fragment thereof of the present invention, and then detecting CD22 and the anti-CD22 antibody or Complexes formed by its antigen-binding fragments.
  • the detection of CD22 is the detection of the presence or content of CD22.
  • the presence means presence or absence, and the content may be expression level or protein concentration, etc.
  • said anti-CD22 antibody or antigen-binding fragment thereof includes a detectable label.
  • the marker may be Flag.
  • the method for detecting CD22 of the present invention can be used for disease diagnosis and treatment purposes, and can also be used for non-disease diagnosis and treatment purposes. It should be noted here that even if the CD22 detection method of the present invention detects the presence of CD22 in the isolated sample of the organism or contains a certain concentration or expression level of CD22, it cannot directly determine the diagnosis of the disease. The diagnosis of the disease needs to be comprehensively evaluated by clinicians in combination with other test results.
  • the seventeenth aspect of the present invention provides a method for diagnosing tumors, the method comprising taking a sample, contacting the sample with the anti-CD22 antibody or antigen-binding fragment thereof of the present invention, and then detecting the relationship between CD22 and the anti-CD22 antibody or Complexes formed by its antigen-binding fragments.
  • said antibody or antigen-binding fragment thereof includes a detectable label.
  • the tumor is acute B-lymphoblastic leukemia (B-ALL), indolent and high-grade non-Hodgkin's lymphoma, chronic lymphocytic leukemia or hairy cell leukemia and the like.
  • B-ALL acute B-lymphoblastic leukemia
  • indolent and high-grade non-Hodgkin's lymphoma chronic lymphocytic leukemia or hairy cell leukemia and the like.
  • the eighteenth aspect of the present invention provides a method for treating or preventing tumors, the method comprising administering to the subject an effective amount of the anti-CD22 antibody or antigen-binding fragment thereof, chimeric An antigen receptor, an immune cell (preferably a T cell) expressing a chimeric antigen receptor, or a pharmaceutical composition.
  • the tumor is acute B-lymphoblastic leukemia (B-ALL), indolent and high-grade non-Hodgkin's lymphoma, chronic lymphocytic leukemia or hairy cell leukemia.
  • B-ALL acute B-lymphoblastic leukemia
  • indolent and high-grade non-Hodgkin's lymphoma chronic lymphocytic leukemia or hairy cell leukemia.
  • the subject may be a mammal, for example, a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey or a human, especially a human.
  • the effective amount depends on many factors, including the patient's age, weight, gender, natural health status, nutritional status, taking time, metabolic rate, severity of the disease, and subjective judgment of the treating physician, etc.
  • the present invention uses a fully human antibody library to screen the specific antibody sequence of the CD22 protein, and conducts functional verification to determine the specific antibody that can treat cancer (such as B-ALL) and achieve the purpose of curing the disease.
  • cancer such as B-ALL
  • the "antigen-binding fragment" of the present invention is a part of the antibody that retains the specific binding activity of the whole antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the whole antibody. It includes eg Fab, Fab', F(ab')2 and variants of these fragments.
  • the protein or nucleic acid may consist of the sequence, or may have additional Amino acids or nucleotides, but still have the activity described in the present invention.
  • Diagnosis in the present invention refers to finding out whether a patient has a disease or disorder in the past, at the time of diagnosis or in the future, or to find out the progress of a disease or possible future progress, or to evaluate a patient's response to treatment.
  • Treatment means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, disorder, or disease, but not necessarily all disease-related signs, Complete elimination of a symptom, condition, or disorder, and refers to a therapeutic intervention that ameliorate the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.
  • prevention in the present invention refers to all actions of suppressing symptoms or delaying the tension of specific symptoms by administering the products described in the present invention.
  • the "effective amount” of the present invention refers to the amount or dose of the product of the present invention that provides the desired treatment or prevention after administration to a patient or an organ in single or multiple doses.
  • the "identity" mentioned in the present invention means that in terms of using amino acid sequence or nucleotide sequence, those skilled in the art can adjust the sequence according to actual work needs without changing the structure or activity of the original sequence, so that Compared with the specific sequence described in the present invention, the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27% , 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44 %, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%,
  • amino acid sequence having at least 80% identity with SEQ ID NO: 1 refers to SEQ ID NO: 1 making adjustments, such as substitution, deletion and/or insertion of one or more amino acids, etc.
  • said at least 80% includes but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100 %.
  • the present invention provides a fully human single-chain anti-CD22 antibody or an antigen-binding fragment thereof that has the following beneficial effects:
  • Figure 1 is a schematic diagram showing the affinity of ELISA detection antibody to CD22
  • Figure 2 is a schematic diagram of the competition ELISA detection between the detection antibody and the m971 antibody
  • Figure 3A is a schematic diagram showing the binding ability of antibodies to CD22 positive target cells and negative target cells by flow cytometry; specifically, the difference in the binding of target cell antibodies under different antibody concentrations;
  • Figure 3B is a schematic diagram showing the binding ability of the antibody to CD22-positive target cells and negative target cells by flow cytometry, specifically, the flow diagram of the target cells contacted with the antibody and the target cells not contacted with the antibody;
  • Figure 4 is a schematic diagram of DLS detection of antibody stability.
  • Figure 5A shows the verification of the tumor killing effect of CAR-T, where the abscissa is the effect-to-target ratio, the target cell is K562, and the ordinate is the cell lysis rate, abd141 represents the CAR-T cells prepared from As scFv, and abd142 represents the B CAR-T cells prepared by scFv, T represents negative target cells.
  • Figure 5B shows the verification of the tumor killing effect of CAR-T, where the abscissa is the effect-to-target ratio, the target cell is Raji, and the ordinate is the cell lysis rate, abd141 represents the CAR-T cells prepared from As scFv, and abd142 represents the CAR-T cells prepared from AscFv.
  • B CAR-T cells prepared by scFv, T represents negative target cells.
  • FIG. 6 shows the CAR-T cell structure, specifically
  • the fully human single-chain antibody against CD22 described herein is panned from a fully human phage antibody library (antibody library type is scFv). More than 1E+10cfu, the correct rate of target fragment insertion is high, and the library diversity is good, which can be used for screening of high-affinity antibodies.
  • PBMC peripheral blood mononuclear cells
  • the phage display library used in the present invention is a fully human scFv antibody library constructed by the inventors, with a library capacity of more than 1E+10 cfu and good diversity, which is sufficient for screening of various antibodies.
  • the fully human scFv antibody library was used for four rounds of screening and identification, and candidate scFv antibodies were obtained, which were named A scFv (represented by abd141 in the schematic diagram) and B scFv (represented by abd142 in the schematic diagram).
  • the monoclonal antibodies obtained from the screening were sequenced and analyzed. Using the IMGT database, the CDR regions of the heavy chain variable region and the light chain variable region of the screened sequences were divided and compared, and the A scFv and B scFv antibody sequences were identified. , and prepare corresponding antibodies or antigen-binding fragments.
  • the full-length nucleotide sequence of the prepared A scFv is shown in SEQ ID NO: 35, and its amino acid sequence is shown in SEQ ID NO: 17.
  • the full-length nucleotide sequence of the prepared B scFv is shown in SEQ ID NO: 36, and its amino acid sequence is shown in SEQ ID NO: 18.
  • the specific sequences are shown in Table 1 and Table 2.
  • Collect bacterial cells centrifuge at 12,000 rpm for 10 minutes to collect bacterial cells, discard the supernatant;
  • CD22-Fc fusion proteins Three different forms of CD22-Fc fusion proteins, CD22-Fc, CD22-d6d7-Fc, CD22-d7-Fc, were diluted to a final concentration of 2 ⁇ g/ ml, coat the ELISA plate at 100 ⁇ l/well overnight at 4°C, the control protein is TEM8-Fc, coat at the same concentration overnight at 4°C;
  • Blocking Discard the coating solution, wash 3 times with PBST, add 200 ⁇ l of blocking solution PBSM (PBS+3% Milk) to each well and block at 37°C for 1 hour;
  • PBSM blocking solution
  • Color development Discard the secondary antibody incubation solution, wash 3 times with PBST, add 100 ⁇ l color development solution ABTS to each well, and react at room temperature for 20 minutes in the dark;
  • Stop reading Add 50 ⁇ l of stop solution to each well to stop color development, and read the absorbance value of OD 405 with a Thermo MULTISKAN FC microplate reader.
  • the anti-CD22 antibody A scFv of the present invention has significant binding to three different forms of CD22-Fc fusion proteins, but has no specific binding to the control protein TEM8-Fc.
  • the EC 50 of the anti-CD22 antibody A scFv and the antigen CD22-Fc is 1 nM, indicating that the anti-CD22 antibody of the present invention can specifically bind to the CD22 protein with strong binding force.
  • Antibody m971 (a published monoclonal antibody targeting CD22 whose antigen binds to a membrane-proximal epitope) (prepared by the applicant) was diluted to a final pH 7.4 PBS buffer. Concentration is 2 ⁇ g/ml, according to 100 ⁇ l/well coated ELISA plate, 4 °C overnight;
  • Blocking Discard the coating solution, wash 3 times with PBST, add 200 ⁇ l of blocking solution PBSM (PBS+3% Milk) to each well and block at 37°C for 1 hour;
  • PBSM blocking solution
  • Competing antibody incubation Discard the blocking solution and wash 3 times with PBST. Add 100 ⁇ l of a mixture containing 10nM CD22-Fc protein and different concentrations of m971 antibody to each well as the control group, add 100 ⁇ l of a mixture containing 10nM CD22-Fc protein and different concentrations of AscFv antibody to each well as the experimental group, and incubate at 37°C for 1h , D2-scFv is the negative control antibody.
  • Color development Discard the secondary antibody incubation solution, wash 3 times with PBST, add 100 ⁇ l color development solution ABTS to each well, and react at room temperature for 20 minutes in the dark;
  • Stop reading add 50 ⁇ l of stop solution to each well to stop color development, and read the absorbance value of OD405 with a microplate reader.
  • the anti-CD22 antibody A scFv of the present invention can compete with the m971 antibody for binding to the CD22 protein, and it has an overlapping region with the epitope of the m971 antibody targeting CD22.
  • the anti-CD22 antibody A scFv can specifically recognize CD22-positive target cells, and has no obvious binding to negative cells, and the specific binding effect is significant with the increase of antibody concentration. It shows that the anti-CD22 antibody has good specificity.
  • the antibody A scFv prepared in Example 2 was diluted to 1 mg/ml in PBS, filtered aseptically, and then incubated at 37°C for the specified time (0 days, 7 days, 14 days), and then passed through a dynamic light scattering instrument (from Malvern Company) to analyze the stability of antibody A scFv.
  • the CAR-T cells (cell structure shown in Figure 6) prepared the corresponding antibody were tested for cytotoxicity by lactate dehydrogenase (LDH) method, and the cell killing results are shown in Figure 5A and Figure 5B.
  • LDH lactate dehydrogenase

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Abstract

L'invention concerne un anticorps entièrement humain anti-CD22 ou un fragment de liaison à l'antigène de celui-ci, son procédé de préparation et son utilisation. L'anticorps anti-CD22 ou le fragment de liaison à l'antigène de celui-ci est criblé à partir d'une bibliothèque de phages entièrement humains, et est formé au moyen d'un peptide lieur flexible liant une région variable de chaîne lourde et une région variable de chaîne légère. Des séquences de région de structure et des séquences de région de détermination de complémentarité (CDR) de la région variable de chaîne lourde et de la région variable de chaîne légère sont dérivées d'êtres humains.
PCT/CN2022/099016 2021-06-16 2022-06-15 Anticorps entièrement humain anti-cd22 ou fragment de liaison à l'antigène de celui-ci, son procédé de préparation et son utilisation WO2022262783A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027638A1 (fr) * 1993-05-21 1994-12-08 Dana-Farber Cancer Institute, Inc. Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
CA2484420A1 (fr) * 2002-05-02 2003-11-13 Celltech R&D Limited Anticorps specifiques au cd22 humain et leurs utilisations therapeutiques et diagnostiques
CN103588882A (zh) * 2012-08-13 2014-02-19 中国抗体制药有限公司 针对人cd22抗体的抗独特型抗体及其应用
CN109678965A (zh) * 2018-10-12 2019-04-26 中国人民解放军总医院 嵌合抗原受体及其基因和重组表达载体、cd22-cd19双靶向性的t细胞及其应用
CN109836495A (zh) * 2017-11-25 2019-06-04 深圳宾德生物技术有限公司 一种靶向cd22的单链抗体、嵌合抗原受体t细胞及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027638A1 (fr) * 1993-05-21 1994-12-08 Dana-Farber Cancer Institute, Inc. Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
CA2484420A1 (fr) * 2002-05-02 2003-11-13 Celltech R&D Limited Anticorps specifiques au cd22 humain et leurs utilisations therapeutiques et diagnostiques
CN1662558A (zh) * 2002-05-02 2005-08-31 细胞技术研究及开发有限公司 人cd22特异性抗体及其治疗和诊断应用
CN103588882A (zh) * 2012-08-13 2014-02-19 中国抗体制药有限公司 针对人cd22抗体的抗独特型抗体及其应用
CN109836495A (zh) * 2017-11-25 2019-06-04 深圳宾德生物技术有限公司 一种靶向cd22的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
CN109678965A (zh) * 2018-10-12 2019-04-26 中国人民解放军总医院 嵌合抗原受体及其基因和重组表达载体、cd22-cd19双靶向性的t细胞及其应用

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