CN115197323A - SN38 antibody or antigen binding fragment thereof and application thereof - Google Patents

SN38 antibody or antigen binding fragment thereof and application thereof Download PDF

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CN115197323A
CN115197323A CN202110375203.9A CN202110375203A CN115197323A CN 115197323 A CN115197323 A CN 115197323A CN 202110375203 A CN202110375203 A CN 202110375203A CN 115197323 A CN115197323 A CN 115197323A
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amino acid
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acid sequence
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高贝
沈毅珺
吴芳
吴劲松
杨彤
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses an SN38 antibody or an antigen binding fragment thereof and application thereof. The SN38 antibody or antigen-binding fragment thereof comprises a sequence as set forth herein. The SN38 antibody can be used for recognizing the small molecule SN38 with high specificity, has high affinity to the SN38, and can be used for effectively detecting the SN38 content in a sample.

Description

SN38 antibody or antigen binding fragment thereof and application thereof
Technical Field
The application relates to the field of biomedicine, in particular to an SN38 antibody or an antigen-binding fragment thereof and application thereof.
Background
Antibody-small molecule drug conjugates (ADCs) are a new generation of Antibody-targeted therapeutic drugs, mainly applied to the treatment of cancer tumors. The ADC medicine consists of three parts, namely a small molecule cytotoxic medicine (Drug), an Antibody (Antibody) and a Linker (Linker) for connecting the Antibody and the cytotoxic medicine, wherein the small molecule cytotoxic medicine is combined on an Antibody protein by a chemical coupling method. Clinical research proves that ADC medicine has high drug effect and is relatively stable in blood, can effectively reduce the toxicity of small molecular cytotoxic drugs (chemical drugs) to the circulation system and healthy tissues, and is a hot research and development point of international anticancer drugs at present.
Camptothecin (CPT) is a drug with a pentacyclic backbone consisting of 1 (3, 4- β) quinoline group (ring a, B), 1 benzazole (ring C, D) and 1 α -hydroxy- δ -endocyclic ring (ring E), with 1 chiral carbon atom C-20.
SN38 (7-ethyl-10-hydroxycamptothecin) is a semi-synthetic natural anticancer alkaloid camptothecin analog, having the following molecular formula:
Figure BDA0003010900600000011
SN38 utilizes supercoiled DNA unwinding and promotes DNA replication to regulate the topology of DNA, inhibiting synthesis of TOP. By forming a stable ternary complex with TOP1, DNA, the cell cycle is arrested in S phase, thereby preventing DNA replication and causing cell death. SN38 may therefore serve as a small molecule cytotoxic drug preferred for ADCs (Antibody-small molecule drug conjugates).
In clinical trials, the concentration of the ADC in the body of the SN38 constructed ADC after administration needs to be monitored so as to determine the administration dosage in a targeted manner, and no detection reagent capable of well detecting the content of the ADC in a sample exists, so that the market urgently needs an antibody capable of effectively binding SN38 so as to quickly and accurately analyze the SN38 ADC.
Disclosure of Invention
Aiming at the technical problem that an SN38 antibody in the prior art does not exist and good detection of SN38 is difficult to realize, the invention provides the SN38 antibody or the antigen binding fragment thereof and application thereof. The SN38 antibody can be used for identifying the small molecule SN38 with high specificity, has higher affinity to the SN38, and can realize effective detection on the SN38 content in a sample.
In order to solve the technical problems, one of the technical schemes provided by the invention is as follows: an SN38 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and/or a light chain variable region;
the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 1 or variant 1 thereof; the HCDR2 comprises an amino acid sequence selected from the group consisting of the sequences shown as SEQ ID NO 2, SEQ ID NO 5 and SEQ ID NO 8; the HCDR3 comprises an amino acid sequence selected from the group consisting of the sequences shown as SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 9;
the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has an amino acid sequence shown in SEQ ID NO. 14 or a variant 2 thereof; the LCDR2 comprises an amino acid sequence as shown in GTN or GTY; the amino acid sequence of the LCDR3 comprises the amino acid sequence shown as SEQ ID NO. 15 or variant 3 thereof;
the variant has at least the function of the SN38 antibody or antigen-binding fragment thereof.
Preferably, the variant 1 is obtained after mutation at the 6 th position, the 7 th position and/or the 8 th position of the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation at the 6 th position is preferably T6G, the mutation at the 7 th position is preferably Y7A, and the mutation at the 8 th position is preferably W8G;
the variant 2 is obtained after mutation at 7 th position and/or 9 th position of the amino acid sequence shown in SEQ ID NO. 14, wherein the mutation at the 7 th position is preferably S7R, and the mutation at the 9 th position is preferably F8Y;
the variant 3 is obtained by mutating the 4 th position, the 5 th position and/or the 6 th position of the amino acid sequence shown in SEQ ID NO. 15, wherein the mutation of the 4 th position is preferably Y4H, the mutation of the 5 th position is preferably R5S or R5T, and the mutation of the 6 th position is preferably N6T.
In a preferred embodiment:
the amino acid sequence of the variant 1 is shown as SEQ ID NO. 4 or SEQ ID NO. 7;
the amino acid sequence of the variant 2 is shown as SEQ ID NO. 10 or SEQ ID NO. 12;
the amino acid sequence of the variant 3 is shown as SEQ ID NO. 11 or SEQ ID NO. 13.
Preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NOs: 1-3, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 4-6, respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 7-9, respectively;
and/or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO 10, GTN and SEQ ID NO 11 respectively; or LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 12, GTY and SEQ ID NO 13, respectively; or LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 14, GTN, SEQ ID NO. 15, respectively.
More preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO 1-3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO 10, GTN, SEQ ID NO 11, respectively; or
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 4-6 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 12, GTY and SEQ ID NO. 13 respectively;
the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO 7-9 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO 14, GTN and SEQ ID NO 15 respectively.
In the technical scheme, the heavy chain variable region can also comprise HFR1, HFR2, HFR3 and HFR4, and the amino acid sequences of the HFR1, the HFR2, the HFR3 and the HFR4 are respectively shown in SEQ ID NO. 16-18 and SEQ ID NO. 19; or the amino acid sequences of the HFR1, the HFR2, the HFR3 and the HFR4 are respectively shown in SEQ ID NO. 20-22 and SEQ ID NO. 19; or the amino acid sequences of the HFR1, the HFR2, the HFR3 and the HFR4 are respectively shown in SEQ ID NO. 23-25 and SEQ ID NO. 19.
And/or, the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4, wherein the LFR1 comprises the amino acid sequence shown in SEQ ID NO:26 or variant 4 thereof; the LFR2 comprises an amino acid sequence as shown in SEQ ID NO. 27 or variant 5 thereof; the LFR3 comprises an amino acid sequence as shown in SEQ ID NO. 28 or variant 6 thereof; the LFR4 comprises the amino acid sequence shown as SEQ ID NO. 29 or variant 7 thereof.
Preferably:
the amino acid sequence of the variant 4 is shown as SEQ ID NO. 30;
the amino acid sequence of the variant 5 is shown as SEQ ID NO. 31;
the amino acid sequence of the variant 6 is shown as SEQ ID NO. 32 or SEQ ID NO. 33;
the amino acid sequence of the variant 7 is shown as SEQ ID NO. 34.
More preferably:
the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having amino acid sequences shown in SEQ ID NOS: 16-18 and SEQ ID NO:19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NOS: 26-29, respectively; or, the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having amino acid sequences shown in SEQ ID NOS: 20-22 and SEQ ID NO:19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NOS: 30-32 and SEQ ID NO:29, respectively; the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having amino acid sequences shown in SEQ ID NOS 23-25 and 19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NOS 26, 27, 33 and 34, respectively.
In a preferred embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO 35, SEQ ID NO 37 or SEQ ID NO 39, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in SEQ ID NO 35, SEQ ID NO 37 or SEQ ID NO 39; the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO 36, SEQ ID NO 38 or SEQ ID NO 40, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence as set forth in SEQ ID NO 36, SEQ ID NO 38 or SEQ ID NO 40.
In a preferred embodiment, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 35 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 36; or, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO:37 and the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 38; or, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:39 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 40.
Preferably, the SN38 antibody or antigen-binding fragment thereof comprises a murine or human antibody constant region or a mutation thereof; the murine antibody constant region comprises a heavy chain constant region of murine IgG1, igG2a, igG2b3, or IgG3 and a light chain constant region of the kappa or lambda type, and the human antibody constant region comprises a heavy chain constant region of human IgG1, igG2, igG3, or IgG4 and a light chain constant region of the kappa or lambda type; preferably, the mutation is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the constant region of the murine or human antibody.
In a preferred embodiment, the SN38 antibody or antigen-binding fragment thereof has a heavy chain constant region amino acid sequence as set forth in SEQ ID NO 41 at positions 124-459; the SN38 antibody or antigen-binding fragment thereof has a light chain constant region amino acid sequence as set forth in positions 110-209 of SEQ ID NO 42.
Preferably, the structure of the SN38 antibody or antigen-binding fragment thereof comprises an immunoglobulin, fab ', F (ab') 2 Fv, such as single chain Fv fragment (scFv).
In order to solve the above technical problems, the second technical solution provided by the present invention is: an isolated nucleic acid encoding an SN38 antibody or antigen-binding fragment thereof according to one of the claims.
Preferably, in the nucleotide sequence encoding the SN38 antibody or antigen-binding fragment thereof, the nucleotide sequence encoding the heavy chain variable region is shown as positions 1-369 of SEQ ID NO. 43, and the nucleotide sequence encoding the light chain variable region is shown as positions 1-327 of SEQ ID NO. 44; or the nucleotide sequence encoding the heavy chain variable region is shown as the 1 st-372 nd bits of SEQ ID NO. 47, and the nucleotide sequence encoding the light chain variable region is shown as the 1 st-327 th bits of SEQ ID NO. 48; the nucleotide sequence encoding the heavy chain variable region is shown as positions 1-363 of SEQ ID NO. 51, and the nucleotide sequence encoding the light chain variable region is shown as positions 1-327 of SEQ ID NO. 52.
In order to solve the technical problems, the third technical scheme provided by the invention is as follows: an expression vector comprising the isolated nucleic acid of claim two. Preferably, the origin vector of the expression vector is pcDNA3.1 (+), more preferably pcDNA3.1 (+) produced by Ensifier fundi (Shanghai) trading Limited.
In order to solve the above technical problems, the fourth technical solution provided by the present invention is: a transformant comprising the expression vector according to the third embodiment. Preferably, the transformant is a genetically modified lentivirus or immune effector cell. More preferably, the lentivirus is a lentivirus comprising a PLVX-EF1-CAR, lentiCRISPR v2-CAR, or pELNS-CAR, and the immune effector cell is a T lymphocyte, NK cell, or NKT cell.
In order to solve the above technical problems, the fifth technical solution provided by the present invention is: a test composition comprising an SN38 antibody or antigen-binding fragment thereof according to one of the claims, an isolated nucleic acid according to the second of the claims, an expression vector according to the third of the claims and/or a transformant according to the fourth of the claims.
In order to solve the technical problems, the sixth technical scheme provided by the invention is as follows: a kit comprising an SN38 antibody or antigen-binding fragment thereof according to claim one, an isolated nucleic acid according to claim two, an expression vector according to claim three, a transformant according to claim four, and/or a detection composition according to claim five.
Preferably, the kit further comprises (i) a means for administering the antibody or antigen-binding fragment thereof, the isolated nucleic acid, the expression vector, the transformant, or the detection composition; and/or (ii) instructions for use.
In order to solve the above technical problems, the seventh technical solution provided by the present invention is: a method of detecting SN38 in a sample by reacting with the sample using an SN38 antibody or antigen-binding fragment thereof according to claim one, an isolated nucleic acid according to claim two, an expression vector according to claim three, a transformant according to claim four, a detection composition according to claim five, and/or a kit according to claim six.
To solve the above technical problems, the eighth technical solution provided by the present invention is: use of an SN38 antibody or an antigen-binding fragment thereof according to claim one, an isolated nucleic acid according to claim two, an expression vector according to claim three, a transformant according to claim four, a detection composition according to claim five, or a kit according to claim six for detecting SN 38.
In addition, the invention also provides a ninth technical scheme: a pharmaceutical composition comprising an SN38 antibody or antigen-binding fragment thereof according to claim one, an isolated nucleic acid according to claim two, an expression vector according to claim three, and/or a transformant according to claim four.
Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
In addition, the invention also provides a tenth technical scheme: use of the SN38 antibody or antigen-binding fragment thereof according to claim one, the isolated nucleic acid according to claim two, the expression vector according to claim three, the transformant according to claim four, the pharmaceutical composition according to claim nine, or the kit according to claim six, for the manufacture of a medicament for the treatment of an SN 38-related disorder.
In addition, the invention also provides a technical scheme that: a method of treating an SN 38-associated disorder (e.g., side effects), the method comprising administering to a patient in need thereof a therapeutically effective amount of an SN38 antibody or antigen-binding fragment thereof according to one of the claims, an isolated nucleic acid according to the second of the claims, an expression vector according to the third of the claims, a transformant according to the fourth of the claims, a pharmaceutical composition according to the ninth of the claims.
Term(s)
In the present invention, the term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding, the definition of the 6 CDRs being provided by Kabat E.A. et al, (1991) Sequences of proteins of immunological interest, NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies to CDR1, CDR2, and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), and CDR1, CDR2, and CDR3 of the heavy chain variable domain (CDR H1, CDR H2, CDR H3, or H1, H2, H3).
In the present invention, the term "antibody" is used interchangeably to refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins (Ig) a, igD, igE, igG and IgM. The complete antibody forms a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in one line) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain", respectively. Each arm of Y comprises the variable region and the first constant region of a single heavy chain joined to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.
In the present invention, the "Fab" consists of one light chain and one heavy chain of CH1 and variable domains. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains. "Fab '" contains a light chain and a portion of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab's to form F (ab ') 2 A molecule. "F (ab') 2 A fragment "contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus F (ab') 2 The fragment consists of two fabs' held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains directed to a single arm of an antibody, but lacking a constant region.
In the present invention, the scFv (single chain antibody) can be a single chain antibody conventional in the art, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids. Wherein the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain [ see, e.g., bird et al, science 242, 423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA85:5879-5883 (1988)]. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art joints are made of repeating G 4 S amino acid sequence or a variant thereof. For example, a peptide having an amino acid sequence (G) 4 S) 4 Or (G) 4 S) 3 Linkers, but variants thereof may also be used.
As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of Pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical Press, london, chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. A Pharmaceutical formulation or Pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention having the desired purity may be prepared by mixing the antibody or antigen-binding fragment thereof of the invention with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient as is required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators and the like. The active ingredients are suitably present in combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies, or antigen-binding fragments thereof, of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
As known in the art, "nucleic acid" as used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.
The calculation of sequence identity between sequences was performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Sequence comparisons between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48: 444-453) algorithms (available at http:// www.gcg.com) already integrated into the GAP program of the GCG software package, using either the Blossum 62 or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3,4, 5 or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www. GCG. Com), using NWSgapdna. CMP matrices and GAP weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3,4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix employing a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the PAM120 weighted residue table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithms that have been incorporated into the ALIGN program (version 2.0) ((1989) cabaos, 4. Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
As used herein, "expression vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of expression vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
The term "transformant" in the present invention includes cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as the cell selected or selected for in the originally transformed cell.
The numbers following the "variant 1", "variant 2", etc. variants of the invention have no special meaning and are intended to refer to the same terms only.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention discloses an SN38 antibody or an antigen binding fragment thereof and application thereof. Since there is no antibody against SN38 in the art, the SN38 antibodies of the present invention are all antibodies of a completely new sequence. The SN38 antibody has the capability of specifically recognizing the small molecule SN38, has higher affinity, and has wide prospect in clinical detection of the SN 38.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
EXAMPLE 1 preparation of SN38 immunogen
SN38 (Profibtechniques, inc., beijing) was coupled to KLH (Sigma-Aldrich, cat # H1757) and BSA (Sigma-Aldrich, cat # A1933), respectively, as immunogens.
KLH was coupled by first weighing 3mg of KLH, which was dissolved to 10mg/ml, 0.3ml total, with 0.1M sodium bicarbonate containing 5mM EDTA, pH 8.0. An amount of 2' iminothiolane hydrochloride (manufactured, cat # C100211) was then weighed out and dissolved in sterile water to 2mg/ml (14 mM). 0.25ml of 2 'iminothiolane hydrochloride was added to KLH, the number of KLH amino groups and the molar ratio of 2' iminothiolane hydrochloride were 1, and the reaction was carried out at room temperature for 2 hours. Dialyzed against 0.1M PBS containing 5mM EDTA, pH6.5 for 2h at room temperature. Then, 1.2mg of SN was weighed out and dissolved in 10mg/ml of dimethyl sulfoxide. After 2 hours, KLH-SH was taken out, and 1.2mg of SN38 was added thereto to conduct a reaction at room temperature for 2 hours. Dialyzed overnight against 1 XPBS at pH7.4 ℃ and exchanged once.
BSA coupling was performed by weighing 3mg BSA and dissolving it with 0.1M sodium bicarbonate containing 5mM EDTA at pH8.0 to 10mg/ml, total 0.3ml. An amount of 2' iminothiolane hydrochloride was then weighed out and dissolved in sterile water to 2mg/ml (14 mM). To BSA was added 0.255ml of 2' iminothiolane hydrochloride. The number of BSA amino groups and the molar ratio of 2' iminothiolane hydrochloride were 1. The reaction was carried out at room temperature for 2h. Dialyzed against 0.1M PBS containing 5mM EDTA, pH6.5 for 2h at room temperature. SN 38.3 mg was weighed out and dissolved in dimethyl sulfoxide to 10mg/ml. After 2 hours, BSA-SH was removed, and 1.3mg of SN38 was added thereto, followed by reaction at room temperature for 2 hours. Dialyzed overnight against 1 XPBS at pH7.4 ℃ and exchanged once.
Example 2 animal immunization
The immunization is divided into two groups, and different antigens are adopted for immunization respectively, 5 Balb/c mice (Viton Hua) in the A group are immunized by SN38-KLH, and 5 Balb/c mice in the B group are immunized by SN 38-BSA. Blood is collected 4 days before the start of immunization for standby, immunization is carried out once every 14 days for 4 rounds of immunization, each mouse is immunized by subcutaneous injection of 50 mu g of antigen, and blood collected 7 days after 3 rounds of immunization is subjected to indirect ELISA detection on the serum of the immunized animal so as to determine the level of immune response. Cell fusion was performed after the 4 th round of immunization.
The indirect ELISA method was used to detect serum titers 7 days after the 3 rd immunization. Respectively detecting SN38-KLH immune group and SN38-BSA immune group by using an enzyme label plate (Nunc, cat # 446442) with hydrophobic property, respectively coating the enzyme label plate strip with 1 mu g/ml of SN38-BSA and SN38-KLH, respectively using PBS (pH7.4) as coating liquid, respectively diluting serum by 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000 and 512000 times, adding the enzyme label plate strip to combine with small molecules, detecting IgG (Bethy Laboratories, cat # A90-231P) of enzyme-linked-bis-horseradish peroxidase-labeled goat anti-mouse Fc, and detecting OD (OD) by using the enzyme-labeled goat anti-mouse Fc 655nm The wavelength is the reference wavelength (Molecular Devices, model SpectraMax M2 e), OD 450nm The absorbance was measured, where the serum titers of the four numbered mice, 46, 47, 50 and 54, were all stronger. The results are shown in Table 1.
TABLE 1 serum titre
Figure BDA0003010900600000101
10 mouse sera were detected by the competitive ELISA method, and the sera of the SN38-KLH immunization group and the SN38-BSA immunization group were detected by respectively coating the enzyme-labeled plate strip with 0.1. Mu.g/ml SN38-BSA and SN 38-KLH. Wherein the coating solution is PBS (pH7.4), serum is diluted by 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000 and 512000 times respectively, and then added with 0 μ g/ml or 100 μ g/ml SN38 into an enzyme label plate, enzyme-linked dual anti-horseradish peroxidase labeled goat anti-mouse Fc IgG is detected, OD is detected 655nm Having a wavelength ofReference wavelength, OD 450nm The absorbance was measured and the results are shown in Table 2. Excessive SN38 micromolecule competition combination antiserum screening mice have stronger competition combination activity of the sera, and two mice 46 and 54 are selected for carrying out cell fusion experiments.
TABLE 2 mouse serum ELISA test values
Figure BDA0003010900600000111
Example 3 cell fusion and master clone screening:
cell fusion was performed by electrofusion for 2 rounds. The average fusion efficiency was about 2500 splenocytes capable of fusion to yield 1 hybridoma, and all cells from each round of fusion were plated in 96-well plates for expansion culture. Performing ELISA screening on a mother clone supernatant generated by the fusion cell, screening a positive mother clone by adopting an indirect ELISA method, coating an enzyme label plate strip with SN38-KLH of 1 mu G/ml for the mouse hybridoma cell mother clone immunized by SN38-BSA, respectively adding the mouse hybridoma cell mother clone immunized by SN38-BSA of 1C11, 1H3, 4F2, 10E9, 12E11, 13A1, 13H9, 14G7, 15B2 and 15G5 to be detected, combining the supernatant with the SN38-KLH, adding enzyme-linked bis (horseradish) peroxidase to mark IgG of the goat anti-mouse Fc, and increasing the antibody concentration when the OD value is higher.
SN38-KLH immunized mouse hybridoma cell clones are detected by coating enzyme-labeled laths with 1 microgram/ml SN38-BSA, SN38-KLH immunized mouse hybridoma cell clones 17C5, 19G10, 20H4, 21C11, 25A4, 26D12, 29D11, 29F7, 30D9 and 30F6 are added respectively to be detected, supernatants are combined with the SN38-BSA, enzyme-linked dual-anti-horseradish peroxidase is added to label IgG of goat anti-mouse Fc, and ELISA detection results of the mouse hybridoma cell clones are shown in Table 3.
TABLE 3 ELISA test results of SN38-immunized mouse hybridoma cell clones
Figure BDA0003010900600000112
Figure BDA0003010900600000121
The supernatants of positive parent clones were analyzed by indirect ELISA for competition ELISA. 0.5. Mu.g/ml SN38-BSA was coated with the parent clones tested for SN38-KLH immunization, and 1:6 times diluted cell supernatant and different concentrations of SN38 (0, 0.1, 1 and 10. Mu.g/ml), SN38-BSA coated on enzyme-labeled laths and added SN38 compete for binding to the antibody in the supernatant, the more SN38 added, the less SN38 bound to the coated SN38, after washing the plate, the addition of IgG of enzyme-linked di-anti-horseradish peroxidase labeled goat anti-mouse Fc for color development reaction, the lower the detected OD value, the better the specificity of the antibody to SN38, and the results are shown in Table 4. The parent clones immunized with SN38-BSA were examined in the same manner and the results are shown in Table 5.
TABLE 4 ELISA test values of positive SN38-KLH immunoparent clone supernatants
Figure BDA0003010900600000122
TABLE 5 ELISA test values of supernatants of positive SN38-BSA immunoparent clones
Figure BDA0003010900600000123
The culture supernatants of clone Nos. 17C5, 25A4, 1H3, 13H9 and 14G7 tested under the same conditions, had stronger signal values, and had better competitive effects (lower signal value, i.e., OD) after adding SN38 with concentration of 0.01. Mu.g/ml and 0.1. Mu.g/ml for competition 450nm /OD 655nm Less than 0.4), performing frozen strain storage to perform subcloning.
Example 4 subclone screening
Positive parent clones were subcloned by limiting dilution to ensure that monoclonal cells were eventually taken. The subcloning stage was performed for 3 rounds, using indirect and competitive ELISA for subcloning screening, and 2 stable daughter clones were selected for amplification and cryopreservation per parent clone. 5ml of supernatant was collected from each subclone before cryopreservation, and all subtypes were identified using a subclass kit (Sigma-Aldrich, cat # 11493027001).
Performing indirect ELISA positive screening on the subclone supernatant, immunizing by SN38-BSA, coating an enzyme-labeled plate strip by using 1 mu g/ml SN38-KLH, coating the enzyme-labeled plate strip by using 1 mu g/ml SN38-KLH (SN 38-KLH) immunized by using SN38-KLH (SN 38-BSA), adding the subclone supernatant to be detected, combining with a small molecule coated on the enzyme-labeled plate strip, and performing display reaction by using IgG of enzyme-linked dual-anti-horseradish peroxidase-labeled goat anti-mouse Fc. The higher the OD, the higher the antibody concentration, the results are shown in Table 6.
TABLE 6 measurement of antibody concentration in supernatant of subcloned cells
Figure BDA0003010900600000131
The culture supernatant of the subclone cells is analyzed by competitive ELISA, 0.5 mu g/ml SN38-BSA is coated with the subclone for detecting SN38-KLH immunity, 300 times of diluted supernatants of the subclone cells 17C5-1, 17C5-2, 25A4-1 and 25A4-2 and different concentrations of SN38 (0, 0.01 and 0.1 mu g/ml) are added, enzyme-linked anti-horseradish peroxidase labeled goat anti-mouse Fc IgG is added for color reaction, and the specificity of the antibody to the micromolecule is better when the detection OD value is lower. The SN38-BSA immunized subclones 1H3-1, 1H3-2, 13H9-1, 13H9-2, 14G7-1 and 14G7-2 were examined in the same manner, and the results are shown in Table 7.
TABLE 7 specific detection of antibodies in the supernatants of subcloned cells
Figure BDA0003010900600000132
Figure BDA0003010900600000141
Example 5 monoclonal antibody identification
Monoclonal antibodies 1H3-1, 13H9-2 and 14G7-1 were produced, and the antibodies were purified by Protein A/G affinity chromatography (Cytiva, cat No. 29049104) and stored in Phosphate Buffered Saline (PBS) by dialysis. The titer detection was performed by polyacrylamide gel electrophoresis (SDS-PAGE), indirect ELISA, competitive ELISA, and NanoDrop2000 (Thermo) for antibody concentration.
The monoclonal antibody titer is detected by adopting an indirect ELISA method. Using a hydrophobic enzyme label plate, coating an enzyme label plate strip by SN38-KLH with the concentration of 1 mu g/ml, using PBS (pH7.4) as coating liquid, respectively diluting antibodies by 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000 and 512000 times, adding the enzyme label plate strip to combine with the SN38-KLH, performing an enzyme-linked secondary anti-horseradish peroxidase labeled goat anti-mouse Fc IgG to perform a display reaction, and performing OD (optical density) display reaction 655nm Wavelength is the reference wavelength, OD 450nm When the absorbance of the sample is measured, the titers of 1H3-1, 13H9-2 and 14G7-1 reach 512000, and the results are shown in Table 8.
TABLE 8 monoclonal antibody titer test values for indirect ELISA
Figure BDA0003010900600000142
Competitive ELISA detection: coating SN38-KLH at 0.5 mu G/ml, adding purified 1H3-1, 13H9-2 and 14G7-1 antibodies (the concentration range of the antibodies is 5000-0.005 ng/ml) and SN38 (0, 0.01 and 0.1 mu G/ml) at different concentrations, allowing the coated SN38-KLH and the added SN38 to compete for binding of the antibodies, wherein the more small molecular weight is added and the less SN38 is bound, adding anti-horseradish peroxidase labeled goat anti-mouse Fc IgG after washing the plate, and the lower the OD value is, the better the specificity of the antibodies to SN38 is. The result of competitive binding of SN38 monoclonal antibody 1H3-1 is in line with the expectation, the binding force of the monoclonal antibody and antigen is reduced along with the increase of SN38 dosage, and the specificity of the monoclonal antibody to SN38 is strong.
TABLE 9 specificity test values of 1H3-1 monoclonal antibody
Figure BDA0003010900600000143
Figure BDA0003010900600000151
TABLE 10 13H9-2 monoclonal antibody specificity test values
Figure BDA0003010900600000152
TABLE 11 specificity test values of 14G7-1 mAb
Figure BDA0003010900600000153
EXAMPLE 6 monoclonal antibody sequence identification
1H3-1, 13H9-2 and 14G7-1 hybridoma cells are recovered, and the number of the hybridoma cell sediment cells collected freshly is 5 multiplied by 10 6 The above hybridoma cell lysate was used to fully lyse 5X 10 cells using TriZol Reagent 6 Total RNA is extracted from the hybridoma cells which are collected freshly, the concentration is not lower than 100 ng/mu l, the total volume is 20 mu l, the total volume is 5 mu g, and an RNA band is clear and has no obvious degradation when detected by agarose gel electrophoresis. Performing reverse transcription to obtain cDNA, amplifying variable region sequences of heavy chains and light chains, and cloning to a target vector for sequencing light and heavy chains. The results are shown in the tables below.
TABLE 12 HCDR amino acid sequence and numbering
HCDRs 1H3-1 13H9-2 14G7-1
SEQ ID NO: 1 4 7
HCDR1 GYTFTTYW GYTFTTAG GYTFTGYW
SEQ ID NO: 2 5 8
HCDR2 INPSNGRT INTHSGVP INPGNGRT
SEQ ID NO: 3 6 9
HCDR3 ATSGPYYSYDVAWFAY ARASSSLYRYDQAWFTY ARPLDGYYETWFSY
TABLE 13 LCDR amino acid sequence and numbering
LCDRs 1H3-1 13H9-2 14G7-1
SEQ ID NO: 10 12 14
LCDR1 TGAVTTRNF TGAVTTSNY TGAVTTSNF
SEQ ID NO:
LCDR2 GTN GTY GTN
SEQ ID NO: 11 13 15
LCDR3 ALWYTTHWV ALWHSNHWV ALWYRNHWV
TABLE 14 HFR amino acid sequences and numbering
Figure BDA0003010900600000161
TABLE 15 LFR amino acid sequences and numbering
Figure BDA0003010900600000162
Figure BDA0003010900600000171
TABLE 16 light and heavy chain variable region amino acid sequences and numbering of each anti-SN 38 monoclonal antibody
Figure BDA0003010900600000172
1H3-1 heavy chain nucleic acid sequence (SEQ ID NO: 43):
CAGGTCCAACTACAGCAGCCTGGGGCTGAACTGGTGAGGCCTGGGGCTTCAGTGAAACTGTCCTGCAAGGATTCTGGCTACACTTTTACCACCTACTGGATGCACTGGGTGATGCAGAGGCCTGGACAAGGTCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCGGACTAACTACAATGAGAGGTTCAAGAACAAGGCCACAGTGACTGTAGACAAATCCTCCACCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAACATCAGGGCCCTACTATAGTTACGACGTGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTTGTCACTGTCTCTGCAgccaaaacaacacccccatcagtctatccactggcccctgggtgtggagatacaactggttcctctgtgactctgggatgcctggtcaagggctacttccctgagtcagtgactgtgacttggaactctggatccctgtccagcagtgtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcagtgactgtcccctccagcacctggccaagtcagaccgtcacctgcagcgttgctcacccagccagcagcaccacggtggacaaaaaacttgagcccagcgggcccatttcaacaatcaacccctgtcctccatgcaaggagtgtcacaaatgcccagctcctaacctcgagggtggaccatccgtcttcatcttccctccaaatatcaaggatgtactcatgatctccctgacacccaaggtcacgtgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactatccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgcaaggtcaacaacaaagacctcccatcacccatcgagagaaccatctcaaaaattaaagggctagtcagagctccacaagtatacatcttgccgccaccagcagagcagttgtccagggaagatgtcagtctcacttgcctggtcgtgggcttcaaccctggagacatcagtgtggagtggaccagcaatgggcatacagaggagaactacaaggacaccgcaccagtcctggactctgacggttcttacttcatatacagcaagctcgatataaaaacaagcaagtgggagaaaacagattccttctcatgcaacgtgagacacgagggtctgaaaaattactacctgaagaagaccatctcccggtctccgggtaaa
1H3-1 light chain nucleic acid sequence (SEQ ID NO: 44):
CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACCAGGAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACGGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACACCACCCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAggtcagcccaagtccactcccactctcaccgtgtttccaccttcctctgaggagctcaaggaaaacaaagccacactggtgtgtctgatttccaacttttccccgagtggtgtgacagtggcctggaaggcaaatggtacacctatcacccagggtgtggacacttcaaatcccaccaaagagggcaacaagttcatggccagcagcttcctacatttgacatcggaccagtggagatctcacaacagttttacctgtcaagttacacatgaaggggacactgtggagaagagtctgtctcctgcagaatgtctc
13H9-2 heavy chain nucleic acid sequence (SEQ ID NO: 47):
CAGATCCATCTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGGCAGTCAGGATCTCCTGCAAGGCTTCTGGGTATACTTTCACAACTGCTGGAATGCAGTGGGTGCAAAAGATGCCAGGAAAGGGTTTGAGGTGGATTGGCTGGATAAACACCCACTCTGGAGTGCCAAGATATGCAGAGGACTTCGCGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCACTATTGTTTATTTACAAATAAGCAACCTCAAATATGAGGACACGGCTACGTATTTCTGTGCGAGAGCCTCCTCCTCCCTCTATAGATACGACCAGGCCTGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACGGTCTCTGCAgccaaaacaacacccccatcagtctatccactggcccctgggtgtggagatacaactggttcctctgtgactctgggatgcctggtcaagggctacttccctgagtcagtgactgtgacttggaactctggatccctgtccagcagtgtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcagtgactgtcccctccagcacctggccaagtcagaccgtcacctgcagcgttgctcacccagccagcagcaccacggtggacaaaaaacttgagcccagcgggcccatttcaacaatcaacccctgtcctccatgcaaggagtgtcacaaatgcccagctcctaacctcgagggtggaccatccgtcttcatcttccctccaaatatcaaggatgtactcatgatctccctgacacccaaggtcacgtgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactatccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgcaaggtcaacaacaaagacctcccatcacccatcgagagaaccatctcaaaaattaaagggctagtcagagctccacaagtatacatcttgccgccaccagcagagcagttgtccagggaagatgtcagtctcacttgcctggtcgtgggcttcaaccctggagacatcagtgtggagtggaccagcaatgggcatacagaggagaactacaaggacaccgcaccagtcctggactctgacggttcttacttcatatacagcaagctcgatataaaaacaagcaagtgggagaaaacagattccttctcatgcaacgtgagacacgagggtctgaaaaattactacctgaagaagaccatctcccggtctccgggtaaa
13H9-2 light chain nucleic acid sequence (SEQ ID NO: 48):
CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAATACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGCTCTAATAGGTGGTACCTACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGTCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTGTGGCACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAggtcagcccaagtccactcccactctcaccgtgtttccaccttcctctgaggagctcaaggaaaacaaagccacactggtgtgtctgatttccaacttttccccgagtggtgtgacagtggcctggaaggcaaatggtacacctatcacccagggtgtggacacttcaaatcccaccaaagagggcaacaagttcatggccagcagcttcctacatttgacatcggaccagtggagatctcacaacagttttacctgtcaagttacacatgaaggggacactgtggagaagagtctgtctcctgcagaatgtctc
14G7-1 heavy chain nucleic acid sequence (SEQ ID NO: 51):
CAGGTCCGACTGCAGCAGCCTGGGGCTGAACTGGTGACACCTGGGGCTTCAGTGAAGCTGTCCTGTAAGGCTTCTGGCTACACCTTCACCGGCTACTGGATGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTGGCAACGGTCGTACTAACTACATTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACCCCTTGATGGTTATTACGAGACCTGGTTTTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAgccaaaacaacacccccatcagtctatccactggcccctgggtgtggagatacaactggttcctctgtgactctgggatgcctggtcaagggctacttccctgagtcagtgactgtgacttggaactctggatccctgtccagcagtgtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcagtgactgtcccctccagcacctggccaagtcagaccgtcacctgcagcgttgctcacccagccagcagcaccacggtggacaaaaaacttgagcccagcgggcccatttcaacaatcaacccctgtcctccatgcaaggagtgtcacaaatgcccagctcctaacctcgagggtggaccatccgtcttcatcttccctccaaatatcaaggatgtactcatgatctccctgacacccaaggtcacgtgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactatccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgcaaggtcaacaacaaagacctcccatcacccatcgagagaaccatctcaaaaattaaagggctagtcagagctccacaagtatacatcttgccgccaccagcagagcagttgtccagggaagatgtcagtctcacttgcctggtcgtgggcttcaaccctggagacatcagtgtggagtggaccagcaatgggcatacagaggagaactacaaggacaccgcaccagtcctggactctgacggttcttacttcatatacagcaagctcgatataaaaacaagcaagtgggagaaaacagattccttctcatgcaacgtgagacacgagggtctgaaaaattactacctgaagaagaccatctcccggtctccgggtaaa
14G7-1 light chain nucleic acid sequence (SEQ ID NO: 52):
CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGCGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAGTATATTTCTGTGCTCTCTGGTACAGAAACCACTGGGTGTTCGGTGGAGGAACCGAACTGACTGTCCTAggtcagcccaagtccactcccactctcaccgtgtttccaccttcctctgaggagctcaaggaaaacaaagccacactggtgtgtctgatttccaacttttccccgagtggtgtgacagtggcctggaaggcaaatggtacacctatcacccagggtgtggacacttcaaatcccaccaaagagggcaacaagttcatggccagcagcttcctacatttgacatcggaccagtggagatctcacaacagttttacctgtcaagttacacatgaaggggacactgtggagaagagtctgtctcctgcagaatgtctc
note: in the nucleic acid sequence, the upper case is a variable region and the lower case is a constant region
Example 7 antibody affinity assay
The affinity constants (KD) of the antibodies 1H3-1, 13H9-2 and 14G7-1 to SN38 were determined using surface plasmon resonance (GE, model Biacore T200) analysis. Immobilization of SN38-KLH onto the CM5 chip surface by means of covalent coupling via amino groups (GE Healthcare), dilution of the antibodies 1H3-1, 13H9-2 and 14G7-1 to different concentrations with buffers (10 mM HEPES,150mM NaCl,3mM EDTA,0.05% Tween 20, pH 7.4): 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM and 100nM, injected into the chip respectively, with a flow rate of 30 μ L/min, sample binding time of 300s, and dissociation time of 600s. After each dissociation of binding, the chip surface was regenerated with 50mM hydrochloric acid for 60s at a flow rate of 100. Mu.L/min.
TABLE 17 measurement of affinity
Cloning ka(1/Ms) kd(1/s) KD(M)
1H3-1 1.28E+05 3.90E-04 3.06E-09
13H9-2 1.28E+05 1.35E-04 1.06E-09
14G7-1 2.78E+05 3.42E-03 1.23E-08
The results of the measurement of the affinity constants of antibodies 1H3-1, 13H9-2 and 14G7-1 to SN38 are shown in Table 17.
SEQUENCE LISTING
<110> Shanghai Compound Dangjiang biomedical corporation
<120> SN38 antibody or antigen-binding fragment thereof, and use thereof
<130> P20016924C
<160> 52
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 HCDR1
<400> 1
Gly Tyr Thr Phe Thr Thr Tyr Trp
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 HCDR2
<400> 2
Ile Asn Pro Ser Asn Gly Arg Thr
1 5
<210> 3
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 HCDR3
<400> 3
Ala Thr Ser Gly Pro Tyr Tyr Ser Tyr Asp Val Ala Trp Phe Ala Tyr
1 5 10 15
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 HCDR1
<400> 4
Gly Tyr Thr Phe Thr Thr Ala Gly
1 5
<210> 5
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 HCDR2
<400> 5
Ile Asn Thr His Ser Gly Val Pro
1 5
<210> 6
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 HCDR3
<400> 6
Ala Arg Ala Ser Ser Ser Leu Tyr Arg Tyr Asp Gln Ala Trp Phe Thr
1 5 10 15
Tyr
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 HCDR1
<400> 7
Gly Tyr Thr Phe Thr Gly Tyr Trp
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 HCDR2
<400> 8
Ile Asn Pro Gly Asn Gly Arg Thr
1 5
<210> 9
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 HCDR3
<400> 9
Ala Arg Pro Leu Asp Gly Tyr Tyr Glu Thr Trp Phe Ser Tyr
1 5 10
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 LCDR1
<400> 10
Thr Gly Ala Val Thr Thr Arg Asn Phe
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 LCDR3
<400> 11
Ala Leu Trp Tyr Thr Thr His Trp Val
1 5
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 LCDR1
<400> 12
Thr Gly Ala Val Thr Thr Ser Asn Tyr
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 LCDR3
<400> 13
Ala Leu Trp His Ser Asn His Trp Val
1 5
<210> 14
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 LCDR1
<400> 14
Thr Gly Ala Val Thr Thr Ser Asn Phe
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 LCDR3
<400> 15
Ala Leu Trp Tyr Arg Asn His Trp Val
1 5
<210> 16
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VH FR1
<400> 16
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Asp Ser
20 25
<210> 17
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VH FR2
<400> 17
Met His Trp Val Met Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
1 5 10 15
Glu
<210> 18
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VH FR3
<400> 18
Asn Tyr Asn Glu Arg Phe Lys Asn Lys Ala Thr Val Thr Val Asp Lys
1 5 10 15
Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
20 25 30
Ser Ala Val Tyr Tyr Cys
35
<210> 19
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VH FR4
<400> 19
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
1 5 10
<210> 20
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VH FR1
<400> 20
Gln Ile His Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Ala Val Arg Ile Ser Cys Lys Ala Ser
20 25
<210> 21
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VH FR2
<400> 21
Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Leu Arg Trp Ile Gly
1 5 10 15
Trp
<210> 22
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VH FR3
<400> 22
Arg Tyr Ala Glu Asp Phe Ala Gly Arg Phe Ala Phe Ser Leu Glu Thr
1 5 10 15
Ser Ala Thr Ile Val Tyr Leu Gln Ile Ser Asn Leu Lys Tyr Glu Asp
20 25 30
Thr Ala Thr Tyr Phe Cys
35
<210> 23
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VH FR1
<400> 23
Gln Val Arg Leu Gln Gln Pro Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser
20 25
<210> 24
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VH FR2
<400> 24
Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
1 5 10 15
Glu
<210> 25
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VH FR3
<400> 25
Asn Tyr Ile Glu Lys Phe Lys Thr Lys Ala Thr Leu Thr Val Asp Lys
1 5 10 15
Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
20 25 30
Ser Ala Val Tyr Tyr Cys
35
<210> 26
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 and 14G7-1 VL FR1
<400> 26
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser
20 25
<210> 27
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 and 14G7-1 VL FR2
<400> 27
Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile
1 5 10 15
Gly
<210> 28
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VL FR3
<400> 28
Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly
1 5 10 15
Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala
20 25 30
Ile Tyr Phe Cys
35
<210> 29
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 and 13H9-2 VL FR4
<400> 29
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
1 5 10
<210> 30
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VL FR1
<400> 30
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Asn
20 25
<210> 31
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VL FR2
<400> 31
Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Ala Leu Ile
1 5 10 15
Gly
<210> 32
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VL FR3
<400> 32
Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly
1 5 10 15
Asp Lys Ser Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala
20 25 30
Ile Tyr Phe Cys
35
<210> 33
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VL FR3
<400> 33
Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly
1 5 10 15
Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala
20 25 30
Val Tyr Phe Cys
35
<210> 34
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VL FR4
<400> 34
Phe Gly Gly Gly Thr Glu Leu Thr Val Leu
1 5 10
<210> 35
<211> 123
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VH amino acid sequence
<400> 35
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Asp Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met His Trp Val Met Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Arg Phe
50 55 60
Lys Asn Lys Ala Thr Val Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Gly Pro Tyr Tyr Ser Tyr Asp Val Ala Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 36
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 VL amino acid sequence
<400> 36
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Arg
20 25 30
Asn Phe Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly
35 40 45
Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Thr Thr
85 90 95
His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 37
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VH amino acid sequence
<400> 37
Gln Ile His Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Ala Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala
20 25 30
Gly Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Leu Arg Trp Ile
35 40 45
Gly Trp Ile Asn Thr His Ser Gly Val Pro Arg Tyr Ala Glu Asp Phe
50 55 60
Ala Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Ile Val Tyr
65 70 75 80
Leu Gln Ile Ser Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Ala Ser Ser Ser Leu Tyr Arg Tyr Asp Gln Ala Trp Phe Thr
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 38
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 VL amino acid sequence
<400> 38
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Asn Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Ala
35 40 45
Leu Ile Gly Gly Thr Tyr Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ser Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp His Ser Asn
85 90 95
His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 39
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VH amino acid sequence
<400> 39
Gln Val Arg Leu Gln Gln Pro Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Gly Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Leu Asp Gly Tyr Tyr Glu Thr Trp Phe Ser Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 40
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 VL amino acid sequence
<400> 40
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Phe Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly
35 40 45
Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Val Tyr Phe Cys Ala Leu Trp Tyr Arg Asn
85 90 95
His Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu
100 105
<210> 41
<211> 459
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 heavy chain amino acid sequence
<400> 41
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Asp Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met His Trp Val Met Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Arg Phe
50 55 60
Lys Asn Lys Ala Thr Val Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Gly Pro Tyr Tyr Ser Tyr Asp Val Ala Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
115 120 125
Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser
130 135 140
Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val
145 150 155 160
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe
165 170 175
Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr
180 185 190
Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala
195 200 205
His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly
210 215 220
Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys
225 230 235 240
Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro
245 250 255
Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr
260 265 270
Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser
275 280 285
Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His
290 295 300
Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Ala Leu Pro Ile
305 310 315 320
Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn
325 330 335
Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys
340 345 350
Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu
355 360 365
Gln Leu Ser Arg Glu Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe
370 375 380
Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu
385 390 395 400
Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr
405 410 415
Phe Ile Tyr Ser Lys Leu Asp Ile Lys Thr Ser Lys Trp Glu Lys Thr
420 425 430
Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr
435 440 445
Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys
450 455
<210> 42
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> 1H3-1 light chain amino acid sequence
<400> 42
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Arg
20 25 30
Asn Phe Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly
35 40 45
Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Thr Thr
85 90 95
His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ser Thr Pro Thr Leu Thr Val Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Lys Glu Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asn Phe Ser Pro
130 135 140
Ser Gly Val Thr Val Ala Trp Lys Ala Asn Gly Thr Pro Ile Thr Gln
145 150 155 160
Gly Val Asp Thr Ser Asn Pro Thr Lys Glu Gly Asn Lys Phe Met Ala
165 170 175
Ser Ser Phe Leu His Leu Thr Ser Asp Gln Trp Arg Ser His Asn Ser
180 185 190
Phe Thr Cys Gln Val Thr His Glu Gly Asp Thr Val Glu Lys Ser Leu
195 200 205
Ser Pro Ala Glu Cys Leu
210
<210> 43
<211> 1377
<212> DNA
<213> Artificial Sequence
<220>
<223> 1H3-1 heavy chain nucleic acid sequence
<400> 43
caggtccaac tacagcagcc tggggctgaa ctggtgaggc ctggggcttc agtgaaactg 60
tcctgcaagg attctggcta cacttttacc acctactgga tgcactgggt gatgcagagg 120
cctggacaag gtcttgagtg gattggagag attaatccta gcaacggtcg gactaactac 180
aatgagaggt tcaagaacaa ggccacagtg actgtagaca aatcctccac cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aacatcaggg 300
ccctactata gttacgacgt ggcctggttt gcttactggg gccaagggac tcttgtcact 360
gtctctgcag ccaaaacaac acccccatca gtctatccac tggcccctgg gtgtggagat 420
acaactggtt cctctgtgac tctgggatgc ctggtcaagg gctacttccc tgagtcagtg 480
actgtgactt ggaactctgg atccctgtcc agcagtgtgc acaccttccc agctctcctg 540
cagtctggac tctacactat gagcagctca gtgactgtcc cctccagcac ctggccaagt 600
cagaccgtca cctgcagcgt tgctcaccca gccagcagca ccacggtgga caaaaaactt 660
gagcccagcg ggcccatttc aacaatcaac ccctgtcctc catgcaagga gtgtcacaaa 720
tgcccagctc ctaacctcga gggtggacca tccgtcttca tcttccctcc aaatatcaag 780
gatgtactca tgatctccct gacacccaag gtcacgtgtg tggtggtgga tgtgagcgag 840
gatgacccag acgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 900
acacaaaccc atagagagga ttacaacagt actatccggg tggtcagtgc cctccccatc 960
cagcaccagg actggatgag tggcaaggag ttcaaatgca aggtcaacaa caaagacctc 1020
ccatcaccca tcgagagaac catctcaaaa attaaagggc tagtcagagc tccacaagta 1080
tacatcttgc cgccaccagc agagcagttg tccagggaag atgtcagtct cacttgcctg 1140
gtcgtgggct tcaaccctgg agacatcagt gtggagtgga ccagcaatgg gcatacagag 1200
gagaactaca aggacaccgc accagtcctg gactctgacg gttcttactt catatacagc 1260
aagctcgata taaaaacaag caagtgggag aaaacagatt ccttctcatg caacgtgaga 1320
cacgagggtc tgaaaaatta ctacctgaag aagaccatct cccggtctcc gggtaaa 1377
<210> 44
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> 1H3-1 light chain nucleic acid sequence
<400> 44
caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60
acttgtcgct caagtactgg ggctgttaca accaggaact ttgccaactg ggtccaagaa 120
aaaccagatc atttattcac gggtctaata ggtggtacca acaaccgagc tccaggtgtt 180
cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca 240
cagactgagg atgaggcaat atatttctgt gctctatggt acaccaccca ttgggtgttc 300
ggtggaggaa ccaaactgac tgtcctaggt cagcccaagt ccactcccac tctcaccgtg 360
tttccacctt cctctgagga gctcaaggaa aacaaagcca cactggtgtg tctgatttcc 420
aacttttccc cgagtggtgt gacagtggcc tggaaggcaa atggtacacc tatcacccag 480
ggtgtggaca cttcaaatcc caccaaagag ggcaacaagt tcatggccag cagcttccta 540
catttgacat cggaccagtg gagatctcac aacagtttta cctgtcaagt tacacatgaa 600
ggggacactg tggagaagag tctgtctcct gcagaatgtc tc 642
<210> 45
<211> 460
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 heavy chain amino acid sequence
<400> 45
Gln Ile His Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Ala Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala
20 25 30
Gly Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Leu Arg Trp Ile
35 40 45
Gly Trp Ile Asn Thr His Ser Gly Val Pro Arg Tyr Ala Glu Asp Phe
50 55 60
Ala Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Ile Val Tyr
65 70 75 80
Leu Gln Ile Ser Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Ala Ser Ser Ser Leu Tyr Arg Tyr Asp Gln Ala Trp Phe Thr
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr
115 120 125
Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly
130 135 140
Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser
145 150 155 160
Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr
165 170 175
Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val
180 185 190
Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val
195 200 205
Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser
210 215 220
Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His
225 230 235 240
Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe
245 250 255
Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val
260 265 270
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
275 280 285
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
290 295 300
His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Ala Leu Pro
305 310 315 320
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
325 330 335
Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile
340 345 350
Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala
355 360 365
Glu Gln Leu Ser Arg Glu Asp Val Ser Leu Thr Cys Leu Val Val Gly
370 375 380
Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr
385 390 395 400
Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser
405 410 415
Tyr Phe Ile Tyr Ser Lys Leu Asp Ile Lys Thr Ser Lys Trp Glu Lys
420 425 430
Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr
435 440 445
Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys
450 455 460
<210> 46
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> 13H9-2 light chain amino acid sequence
<400> 46
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Asn Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Ala
35 40 45
Leu Ile Gly Gly Thr Tyr Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ser Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp His Ser Asn
85 90 95
His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ser Thr Pro Thr Leu Thr Val Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Lys Glu Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asn Phe Ser Pro
130 135 140
Ser Gly Val Thr Val Ala Trp Lys Ala Asn Gly Thr Pro Ile Thr Gln
145 150 155 160
Gly Val Asp Thr Ser Asn Pro Thr Lys Glu Gly Asn Lys Phe Met Ala
165 170 175
Ser Ser Phe Leu His Leu Thr Ser Asp Gln Trp Arg Ser His Asn Ser
180 185 190
Phe Thr Cys Gln Val Thr His Glu Gly Asp Thr Val Glu Lys Ser Leu
195 200 205
Ser Pro Ala Glu Cys Leu
210
<210> 47
<211> 1380
<212> DNA
<213> Artificial Sequence
<220>
<223> 13H9-2 heavy chain nucleic acid sequence
<400> 47
cagatccatc tggtgcagtc tggacctgag ctgaagaagc ctggagaggc agtcaggatc 60
tcctgcaagg cttctgggta tactttcaca actgctggaa tgcagtgggt gcaaaagatg 120
ccaggaaagg gtttgaggtg gattggctgg ataaacaccc actctggagt gccaagatat 180
gcagaggact tcgcgggacg gtttgccttc tctttggaaa cctctgccac tattgtttat 240
ttacaaataa gcaacctcaa atatgaggac acggctacgt atttctgtgc gagagcctcc 300
tcctccctct atagatacga ccaggcctgg tttacttact ggggccaagg gactctggtc 360
acggtctctg cagccaaaac aacaccccca tcagtctatc cactggcccc tgggtgtgga 420
gatacaactg gttcctctgt gactctggga tgcctggtca agggctactt ccctgagtca 480
gtgactgtga cttggaactc tggatccctg tccagcagtg tgcacacctt cccagctctc 540
ctgcagtctg gactctacac tatgagcagc tcagtgactg tcccctccag cacctggcca 600
agtcagaccg tcacctgcag cgttgctcac ccagccagca gcaccacggt ggacaaaaaa 660
cttgagccca gcgggcccat ttcaacaatc aacccctgtc ctccatgcaa ggagtgtcac 720
aaatgcccag ctcctaacct cgagggtgga ccatccgtct tcatcttccc tccaaatatc 780
aaggatgtac tcatgatctc cctgacaccc aaggtcacgt gtgtggtggt ggatgtgagc 840
gaggatgacc cagacgtcca gatcagctgg tttgtgaaca acgtggaagt acacacagct 900
cagacacaaa cccatagaga ggattacaac agtactatcc gggtggtcag tgccctcccc 960
atccagcacc aggactggat gagtggcaag gagttcaaat gcaaggtcaa caacaaagac 1020
ctcccatcac ccatcgagag aaccatctca aaaattaaag ggctagtcag agctccacaa 1080
gtatacatct tgccgccacc agcagagcag ttgtccaggg aagatgtcag tctcacttgc 1140
ctggtcgtgg gcttcaaccc tggagacatc agtgtggagt ggaccagcaa tgggcataca 1200
gaggagaact acaaggacac cgcaccagtc ctggactctg acggttctta cttcatatac 1260
agcaagctcg atataaaaac aagcaagtgg gagaaaacag attccttctc atgcaacgtg 1320
agacacgagg gtctgaaaaa ttactacctg aagaagacca tctcccggtc tccgggtaaa 1380
<210> 48
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> 13H9-2 light chain nucleic acid sequence
<400> 48
caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60
acttgtcgct caaatactgg ggctgttaca actagtaact atgccaactg ggtccaagaa 120
aaaccagatc atttattcac tgctctaata ggtggtacct acaaccgagc tccaggtgtt 180
cctgccagat tctcaggctc cctgattgga gacaagtctg ccctcaccat cacaggggca 240
cagactgagg atgaggcaat atatttctgt gctctgtggc acagcaacca ttgggtgttc 300
ggtggaggaa ccaaactgac tgtcctaggt cagcccaagt ccactcccac tctcaccgtg 360
tttccacctt cctctgagga gctcaaggaa aacaaagcca cactggtgtg tctgatttcc 420
aacttttccc cgagtggtgt gacagtggcc tggaaggcaa atggtacacc tatcacccag 480
ggtgtggaca cttcaaatcc caccaaagag ggcaacaagt tcatggccag cagcttccta 540
catttgacat cggaccagtg gagatctcac aacagtttta cctgtcaagt tacacatgaa 600
ggggacactg tggagaagag tctgtctcct gcagaatgtc tc 642
<210> 49
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 heavy chain amino acid sequence
<400> 49
Gln Val Arg Leu Gln Gln Pro Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Gly Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Leu Asp Gly Tyr Tyr Glu Thr Trp Phe Ser Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala
165 170 175
Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro
180 185 190
Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro
195 200 205
Ala Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile
210 215 220
Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro
225 230 235 240
Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn
245 250 255
Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val
260 265 270
Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
275 280 285
Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
290 295 300
Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Ala Leu Pro Ile Gln His
305 310 315 320
Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
325 330 335
Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu
340 345 350
Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu
355 360 365
Ser Arg Glu Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro
370 375 380
Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn
385 390 395 400
Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile
405 410 415
Tyr Ser Lys Leu Asp Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser
420 425 430
Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys
435 440 445
Lys Thr Ile Ser Arg Ser Pro Gly Lys
450 455
<210> 50
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> 14G7-1 light chain amino acid sequence
<400> 50
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Phe Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly
35 40 45
Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Thr Glu Asp Glu Ala Val Tyr Phe Cys Ala Leu Trp Tyr Arg Asn
85 90 95
His Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ser Thr Pro Thr Leu Thr Val Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Lys Glu Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asn Phe Ser Pro
130 135 140
Ser Gly Val Thr Val Ala Trp Lys Ala Asn Gly Thr Pro Ile Thr Gln
145 150 155 160
Gly Val Asp Thr Ser Asn Pro Thr Lys Glu Gly Asn Lys Phe Met Ala
165 170 175
Ser Ser Phe Leu His Leu Thr Ser Asp Gln Trp Arg Ser His Asn Ser
180 185 190
Phe Thr Cys Gln Val Thr His Glu Gly Asp Thr Val Glu Lys Ser Leu
195 200 205
Ser Pro Ala Glu Cys Leu
210
<210> 51
<211> 1371
<212> DNA
<213> Artificial Sequence
<220>
<223> 14G7-1 heavy chain nucleic acid sequence
<400> 51
caggtccgac tgcagcagcc tggggctgaa ctggtgacac ctggggcttc agtgaagctg 60
tcctgtaagg cttctggcta caccttcacc ggctactgga tgcactgggt gaggcagagg 120
cctggacaag gccttgagtg gattggagag attaatcctg gcaacggtcg tactaactac 180
attgagaagt tcaagaccaa ggccacactg actgtagaca aatcctccaa cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aagacccctt 300
gatggttatt acgagacctg gttttcttac tggggccaag ggactctggt cactgtctct 360
gcagccaaaa caacaccccc atcagtctat ccactggccc ctgggtgtgg agatacaact 420
ggttcctctg tgactctggg atgcctggtc aagggctact tccctgagtc agtgactgtg 480
acttggaact ctggatccct gtccagcagt gtgcacacct tcccagctct cctgcagtct 540
ggactctaca ctatgagcag ctcagtgact gtcccctcca gcacctggcc aagtcagacc 600
gtcacctgca gcgttgctca cccagccagc agcaccacgg tggacaaaaa acttgagccc 660
agcgggccca tttcaacaat caacccctgt cctccatgca aggagtgtca caaatgccca 720
gctcctaacc tcgagggtgg accatccgtc ttcatcttcc ctccaaatat caaggatgta 780
ctcatgatct ccctgacacc caaggtcacg tgtgtggtgg tggatgtgag cgaggatgac 840
ccagacgtcc agatcagctg gtttgtgaac aacgtggaag tacacacagc tcagacacaa 900
acccatagag aggattacaa cagtactatc cgggtggtca gtgccctccc catccagcac 960
caggactgga tgagtggcaa ggagttcaaa tgcaaggtca acaacaaaga cctcccatca 1020
cccatcgaga gaaccatctc aaaaattaaa gggctagtca gagctccaca agtatacatc 1080
ttgccgccac cagcagagca gttgtccagg gaagatgtca gtctcacttg cctggtcgtg 1140
ggcttcaacc ctggagacat cagtgtggag tggaccagca atgggcatac agaggagaac 1200
tacaaggaca ccgcaccagt cctggactct gacggttctt acttcatata cagcaagctc 1260
gatataaaaa caagcaagtg ggagaaaaca gattccttct catgcaacgt gagacacgag 1320
ggtctgaaaa attactacct gaagaagacc atctcccggt ctccgggtaa a 1371
<210> 52
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> 14G7-1 light chain nucleic acid sequence
<400> 52
caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60
acttgtcgct caagtactgg ggctgttaca actagtaact ttgccaactg ggtccaagaa 120
aaaccagatc atttattcac tggtctaata ggtggtacca acaaccgcgc tccaggtgtt 180
cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca 240
cagactgagg atgaggcagt atatttctgt gctctctggt acagaaacca ctgggtgttc 300
ggtggaggaa ccgaactgac tgtcctaggt cagcccaagt ccactcccac tctcaccgtg 360
tttccacctt cctctgagga gctcaaggaa aacaaagcca cactggtgtg tctgatttcc 420
aacttttccc cgagtggtgt gacagtggcc tggaaggcaa atggtacacc tatcacccag 480
ggtgtggaca cttcaaatcc caccaaagag ggcaacaagt tcatggccag cagcttccta 540
catttgacat cggaccagtg gagatctcac aacagtttta cctgtcaagt tacacatgaa 600
ggggacactg tggagaagag tctgtctcct gcagaatgtc tc 642

Claims (14)

1. An SN38 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and/or a light chain variable region;
the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 1 or a variant 1 thereof; the HCDR2 comprises an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NO. 2, SEQ ID NO. 5 and SEQ ID NO. 8; the HCDR3 comprises an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 9;
the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 14 or a variant 2 thereof; the LCDR2 comprises an amino acid sequence as set forth in GTN or GTY; the amino acid sequence of the LCDR3 comprises the amino acid sequence shown as SEQ ID NO. 15 or variant 3 thereof;
the variant has at least the function of the SN38 antibody or antigen-binding fragment thereof.
2. The SN38 antibody or antigen-binding fragment thereof of claim 1, wherein variant 1 is obtained after mutation at position 6, 7, and/or 8 of the amino acid sequence set forth in SEQ ID NO. 1, wherein the mutation at position 6 is preferably T6G, the mutation at position 7 is preferably Y7A, and the mutation at position 8 is preferably W8G;
the variant 2 is obtained after mutation at 7 th position and/or 9 th position of the amino acid sequence shown in SEQ ID NO. 14, wherein the mutation at the 7 th position is preferably S7R, and the mutation at the 9 th position is preferably F8Y;
the variant 3 is obtained after mutation at the 4 th position, the 5 th position and/or the 6 th position of the amino acid sequence shown in SEQ ID NO. 15, wherein the mutation at the 4 th position is preferably Y4H, the mutation at the 5 th position is preferably R5S or R5T, and the mutation at the 6 th position is preferably N6T;
preferably:
the amino acid sequence of the variant 1 is shown as SEQ ID NO. 4 or SEQ ID NO. 7;
the amino acid sequence of the variant 2 is shown as SEQ ID NO. 10 or SEQ ID NO. 12;
the amino acid sequence of the variant 3 is shown as SEQ ID NO. 11 or SEQ ID NO. 13.
3. The SN38 antibody or antigen-binding fragment thereof of claim 1, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOS: 1-3, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 4-6, respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 7-9, respectively;
and/or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 10, GTN and SEQ ID NO. 11 respectively; or LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 12, GTY and SEQ ID NO. 13 respectively; or LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 14, GTN, SEQ ID NO. 15 respectively;
preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO 1-3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO 10, GTN, SEQ ID NO 11, respectively; or
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 4-6 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 12, GTY and SEQ ID NO. 13 respectively;
the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO 7-9 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO 14, GTN and SEQ ID NO 15 respectively.
4. The SN38 antibody or antigen-binding fragment thereof of any of claims 1-3, wherein the heavy chain variable region comprises HFR1, HFR2, HFR3, and HFR4, wherein the amino acid sequences of HFR1, HFR2, HFR3, and HFR4 are set forth in SEQ ID NOS 16-18 and SEQ ID NO 19, respectively; or the amino acid sequences of the HFR1, the HFR2, the HFR3 and the HFR4 are respectively shown in SEQ ID NO. 20-22 and SEQ ID NO. 19; or the amino acid sequences of the HFR1, the HFR2, the HFR3 and the HFR4 are respectively shown in SEQ ID NO. 23-25 and SEQ ID NO. 19;
and/or, the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4, wherein the LFR1 comprises the amino acid sequence shown in SEQ ID NO:26 or variant 4 thereof; the LFR2 comprises an amino acid sequence as shown in SEQ ID NO. 27 or variant 5 thereof; the LFR3 comprises an amino acid sequence as shown in SEQ ID NO. 28 or variant 6 thereof; the LFR4 comprises the amino acid sequence shown as SEQ ID NO. 29 or variant 7 thereof;
preferably:
the amino acid sequence of the variant 4 is shown as SEQ ID NO. 30;
the amino acid sequence of the variant 5 is shown as SEQ ID NO. 31;
the amino acid sequence of the variant 6 is shown as SEQ ID NO. 32 or SEQ ID NO. 33;
the amino acid sequence of the variant 7 is shown as SEQ ID NO. 34;
more preferably:
the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having the amino acid sequences shown in SEQ ID Nos. 16-18 and 19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID Nos. 26-29, respectively; or, the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having the amino acid sequences shown in SEQ ID Nos. 20-22 and 19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID Nos. 30-32 and 29, respectively; the heavy chain variable region comprises HFR1, HFR2, HFR3 and HFR4 having amino acid sequences shown in SEQ ID NOS 23-25 and 19, respectively, and the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NOS 26, 27, 33 and 34, respectively.
5. The SN38 antibody or antigen-binding fragment thereof of any of claims 1-4, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO 35, SEQ ID NO 37, or SEQ ID NO 39, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence set forth in SEQ ID NO 35, SEQ ID NO 37, or SEQ ID NO 39; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO 36, SEQ ID NO 38 or SEQ ID NO 40, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO 36, SEQ ID NO 38 or SEQ ID NO 40;
preferably, the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO. 35 and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO. 36; or, the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO. 37 and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO. 38; or, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:39 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 40.
6. The SN38 antibody or antigen-binding fragment thereof of any of claims 1-5, wherein the SN38 antibody or antigen-binding fragment thereof comprises a murine or human antibody constant region or a mutation thereof; the murine antibody constant region comprises a heavy chain constant region of murine IgG1, igG2a, igG2b3, or IgG3 and a light chain constant region of the kappa or lambda type, and the human antibody constant region comprises a heavy chain constant region of human IgG1, igG2, igG3, or IgG4 and a light chain constant region of the kappa or lambda type;
preferably, the mutation is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the constant region of the murine or human antibody;
more preferably, the SN38 antibody or antigen-binding fragment thereof has a heavy chain constant region amino acid sequence as set forth in SEQ ID NO 41 at positions 124-459; the SN38 antibody or antigen-binding fragment thereof has a light chain constant region amino acid sequence as set forth in positions 110-209 of SEQ ID NO 42.
7. As claimed inThe SN38 antibody or antigen-binding fragment thereof of any of claims 1-6, wherein the SN38 antibody or antigen-binding fragment thereof structurally comprises an immunoglobulin, fab ', F (ab') 2 Fv, for example, a single-chain Fv fragment.
8. An isolated nucleic acid encoding the SN38 antibody or antigen-binding fragment thereof of any one of claims 1-7;
preferably, in the nucleotide sequence encoding the SN38 antibody or antigen-binding fragment thereof, the nucleotide sequence encoding the heavy chain variable region is shown as positions 1-369 of SEQ ID NO. 43, and the nucleotide sequence encoding the light chain variable region is shown as positions 1-327 of SEQ ID NO. 44; or the nucleotide sequence encoding the heavy chain variable region is shown as the 1 st-372 nd bits of SEQ ID NO. 47, and the nucleotide sequence encoding the light chain variable region is shown as the 1 st-327 th bits of SEQ ID NO. 48; the nucleotide sequence encoding the heavy chain variable region is shown as positions 1-363 of SEQ ID NO. 51, and the nucleotide sequence encoding the light chain variable region is shown as positions 1-327 of SEQ ID NO. 52.
9. An expression vector comprising the isolated nucleic acid of claim 8; preferably, the starting vector of the expression vector is pcDNA3.1 (+).
10. A transformant comprising the expression vector of claim 9; preferably, the transformant is a genetically modified lentivirus or immune effector cell; more preferably, the lentivirus is a lentivirus comprising a PLVX-EF1-CAR, lentiCRISPR v2-CAR, or pELNS-CAR, and the immune effector cell is a T lymphocyte, NK cell, or NKT cell.
11. A test composition comprising the SN38 antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 8, the expression vector of claim 9, and/or the transformant of claim 10.
12. A kit comprising the SN38 antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 8, the expression vector of claim 9, the transformant of claim 10, and/or the detection composition of claim 11;
preferably, the kit further comprises (i) a means for administering the antibody or antigen-binding fragment thereof, the isolated nucleic acid, the expression vector, the transformant, or the detection composition; and/or (ii) instructions for use.
13. A method of detecting SN38 in a sample, the method comprising reacting with the sample using the SN38 antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 8, the expression vector of claim 9, the transformant of claim 10, the detection composition of claim 11, and/or the kit of claim 12.
14. Use of the SN38 antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 8, the expression vector of claim 9, the transformant of claim 10, the detection composition of claim 11, or the kit of claim 12 for detecting SN 38.
CN202110375203.9A 2021-04-08 2021-04-08 SN38 antibody or antigen binding fragment thereof and application thereof Pending CN115197323A (en)

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