Antibody binding to human CD38, preparation method thereof, and use thereof
12509528 ยท 2025-12-30
Assignee
Inventors
- Wei Guo (Shanghai, CN)
- Jie Zhao (Shanghai, CN)
- Le Zhao (Shanghai, CN)
- Qingcheng Liu (Shanghai, CN)
- Jianhe Chen (Shanghai, CN)
- Haomin HUANG (Shanghai, CN)
- Zhenping ZHU (Shanghai, CN)
Cpc classification
G01N33/57484
PHYSICS
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C12N5/10
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
C12N5/10
CHEMISTRY; METALLURGY
Abstract
An antibody or an antigen-binding fragment thereof that binds to human CD38. The antibody or the antigen-binding fragment thereof can effectively bind to human CD38 and is applied to preparing a drug for treating diseases having strong CD38 expression such as multiple myeloma.
Claims
1. An antibody binding to human CD38 or antigen-binding fragment thereof, comprising: (a) heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 3, and (b) light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 5, the amino acid sequence of L-CDR3 is shown in SEQ ID NO: 6.
2. The antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, wherein the antibody is murine antibody, chimeric antibody or humanized antibody.
3. The antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment comprises a Fab fragment, a F(ab).sub.2 fragment, a Fv fragment.
4. The antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody binding to human CD38 or antigen-binding fragment thereof is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region of that is shown in SEQ ID NO: 8.
5. The antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody binding to human CD38 or antigen-binding fragment thereof is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of that is shown in SEQ ID NO: 10.
6. The antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain of the antibody binding to human CD38 or antigen-binding fragment thereof is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain of that is shown in SEQ ID NO: 12.
7. A nucleotide molecule encoding the antibody binding to human CD38 or antigen-binding fragment thereof of claim 1.
8. The nucleotide molecule of claim 7, wherein the nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region is shown in SEQ ID NO: 13, and the nucleotide sequence of that encoding the light chain variable region is shown in SEQ ID NO: 14.
9. The nucleotide molecule of claim 7, wherein the nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region is shown in SEQ ID NO: 15, and the nucleotide sequence of that encoding the light chain variable region is shown in SEQ ID NO: 16.
10. The nucleotide molecule of claim 7, wherein the nucleotide sequence of the nucleotide molecule encoding the heavy chain is shown in SEQ ID NO: 17, and the nucleotide sequence of that encoding the light chain is shown in SEQ ID NO: 18.
11. An expression vector comprising the nucleotide molecule of claim 7.
12. A host cell comprising the expression vector of claim 11.
13. A method for preparing the antibody binding to human CD38 or antigen-binding fragment thereof of claim 1, which comprises the following steps: a) culturing a host cell comprising an expression vector comprising a nucleotide molecule encoding the antibody binding to human CD38 or antigen-binding fragment thereof of claim 1 under conditions for expression to express the antibody binding to human CD38 or antigen-binding fragment thereof; b) isolating and purifying the antibody binding to human CD38 or antigen-binding fragment thereof.
14. A composition comprising the antibody binding to human CD38 or antigen-binding fragment thereof of claim 1 and a pharmaceutically acceptable carrier.
15. A CAR construct comprising a scFv, wherein the scFv comprises (i) a heavy chain variable region which comprises: heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the H-CDR3 is shown in SEQ ID NO: 3, and (ii) a light chain variable region which comprises: light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 5, the amino acid sequence of the L-CDR3 is shown in SEQ ID NO: 6.
16. A recombinant immune cell expressing exogenous CAR construct of claim 15.
17. An antibody-drug conjugate, comprising: (a) an antibody moiety, which comprises the antibody or antigen-binding fragment of claim 1; and (b) a coupling moiety coupled to the antibody moiety, wherein the coupling moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, and a combination thereof.
18. A method for in vitro detection of CD38 protein in a sample, which comprises the steps: (a) contacting the sample with the antibody or antigen-binding fragment thereof of claim 1 or an antibody-drug conjugate of the antibody or antigen-binding fragment thereof in vitro; (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD38 protein in the sample.
19. A method for treating CD38-related diseases, comprising: administering to the subject in need with the antibody binding to human CD38 or antigen-binding fragment thereof claim 1, a composition comprising the antibody binding to human CD38 or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, an antibody-drug conjugate of the antibody or antigen-binding fragment thereof, or combinations thereof.
20. The method of claim 19, wherein the CD38-related disease is selected from multiple myeloma, leukemia, B lymphocytoma, autoimmune disease, or a combination thereof.
21. A method for treating CD38-related diseases, comprising: administering to the subject in need with the recombinant immune cell of claim 16, wherein the recombinant immune cell is a CAR-T cell or a CAR-NK cell.
22. The method of claim 21, wherein the CD38-related disease is selected from multiple myeloma, leukemia, B lymphocytoma, autoimmune disease, or a combination thereof.
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
(17) Through extensive and intensive research, the present inventors obtained a CD38 antibody with high affinity and good biological activity after a large number of screenings, especially with excellent ADCC and CDC activity. The humanized CD38 antibody of the present invention has equivalent or even better activity than the commercially available CD38 antibody. In particular, in a mouse lymphoma model, the humanized CD38 antibody of the present invention can significantly prolong the survival time of the test animal. Therefore, the CD38 antibody of the present invention can be developed as an antitumor drug with superior curative effect. On this basis, the present invention has been completed.
The Terms
(18) As used herein, the term antibody (Ab) or immunoglobulin (IgG) refers to a heterotetrameric glycoprotein with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the numbers of disulfide bonds between heavy chains of different immunoglobulin isotypes are different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by constant regions, which consists of three domains CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is paired to the first constant region of the heavy chain, and the variable region of the light chain is paired to the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes. The light chain constant region includes (Kappa) or (Lambda). The heavy and light chains of the antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by the polypeptide disulfide bonds formed between the hinge regions.
(19) In the present invention, the terms Fab and Fc refer to papain may cleave the antibody into two identical Fab segments and one Fc segment. The Fab segment consists of VH and CH1 of the heavy chain of the antibody and VL and CL domains of the light chain. The Fc segment is fragment crystallizable (Fc), which consists of CH2 and CH3 domains of the antibody. The Fc segment has no antigen-binding activity and is the site of interaction between antibodies and effector molecules or cells.
(20) In the present invention, the term scFv refers to a single-chain antibody fragment (scFv), which is usually composed of heavy chain variable region and light chain variable region of the antibody, which are linked by a linking short peptide (linker) with 15-25 amino acids.
(21) In the present invention, the term variable means that certain parts of the variable region of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conserved part of the variable region is called the frame region (FR). The variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a -folded configuration, connected by the three CDRs that form the connecting loop, and in some cases may form a partly folded structure. The CDRs in each chain get close through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
(22) As used herein, the term frame region (FR) refers to amino acid sequences inserted between CDRs, i.e., those portions of the relatively conserved light and heavy chain variable regions of immunoglobulins between different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H. Accordingly, the light chain variable domain can thus be called (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FRR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can thus be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(FRR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or its derivatives, the derivative of the human antibody FR is substantially the same as the naturally occurring human antibody FR, i.e., sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.
(23) Knowing the amino acid sequences of the CDRs, those skilled in the art can easily determine the frame regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.
(24) As used herein, the term human frame region refers to a frame region that is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) as the naturally occurring human antibody frame region.
(25) As used herein, the term linker refers to one or more amino acid residues inserted into the immunoglobulin domain to provide sufficient mobility for the domain of light and heavy chains to fold into exchange for dual variable domain immunoglobulins. In the present invention, the preferred linker refers to the Linker1 and Linker2, wherein Linker1 connects VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect scFv with the heavy chain of another antibody.
(26) Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker may vary with the type of secondary structural element to be implemented in the linker.
(27) In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids replaced by amino acids with the same or similar properties to form a polypeptide. These conservatively variant polypeptides are preferably produced by amino acid substitution according to Table A.
(28) TABLE-US-00001 TABLE A Preferred Initial residue Representative substitution substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu
(29) In the present invention, the terms anti, binding, specific binding refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen targeted thereto. Typically, antibody binds to its antigen with an equilibrium dissociation constant (KD) of less than about 10.sup.7M, e.g., less than about 10.sup.8M, 10.sup.9M, 10.sup.10M, 10.sup.11M, or less. In the present invention, the term KD refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant is, the closer the antibody-antigen binding is and the higher the affinity between the antibody and the antigen is. For example, the binding affinity of an antibody to an antigen is determined using Surface Plasmon Resonance (SPR) in a BIACORE apparatus or the relative affinity of an antibody binding to an antigen is determined using ELISA.
(30) In the present invention, the term epitope refers to a peptide determinant specifically binding to an antibody. The epitope of the present invention is the region of the antigen bound by the antibody.
(31) The present invention also provides the polynucleotide molecule encoding the above-mentioned antibody or fragment thereof or fusion protein thereof. The polynucleotide of the present invention can be in a form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single stranded or double stranded. DNA can be the coding strand or the non-coding strand.
(32) Once a relevant sequence is obtained, recombination methods can be used to obtain the relevant sequence in large quantities. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
(33) The present invention further relates to vectors comprising said suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express protein.
(34) Pharmaceutical Composition and Application
(35) The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition comprising the antibody above mentioned, or the active fragment thereof, or the fusion protein thereof, and a pharmaceutically acceptable carrier. In general, these substances may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, pH is about 6-8, though the pH value may be varied depending on the nature of the substances to be formulated and the condition to be treated. The formulated pharmaceutical composition may be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intraluminal injection. In the present invention, the term pharmaceutical composition refers to the bispecific antibody of the present invention, may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition to exert efficacy more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while also protecting the protein multifunctional groups from degradation (including but not limited to coagulation, deamination or oxidation). The pharmaceutical composition according to the present invention comprises a safe and effective amount (e.g., 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the bispecific antibody above mentioned according to the present invention (or a conjugate thereof) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffers, glucose, water, glycerol, ethanol, and a combination thereof. Pharmaceutical preparations should correspond to the administration modes. The pharmaceutical composition according to the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. A pharmaceutical composition, for example, an injection and a solution, should be prepared under aseptic conditions. The dosage of active ingredient is therapeutically effective amount, for example from about 10 microgram per kilogram body weight to about 50 milligrams per kilogram body weight per day. In addition, the bispecific antibody of the present invention may also be used in combination with an additional therapeutic agent.
(36) When a pharmaceutical composition is used, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal, wherein the safe and effective amount is generally at least about 10 g per kilogram of body weight, and in most cases, no more than about 50 mg per kilogram of body weight, preferably, the amount is from about 10 g per kilogram of body weight to about 10 mg per kilogram of body weight. Of course, a specific amount should also depend on the factors such as administration route and physical conditions of a patient, which fall into the skills of skilled physicians.
(37) Antibody-Drug Conjugate (ADC)
(38) The present invention also provides an antibody-drug conjugate (ADC) based on the antibody according to the present invention.
(39) Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, and chemical conjugation is preferred. Preferably, the effector molecule is a therapeutically active drug. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small-molecule drug or a radionuclide.
(40) The antibody according to present invention and the effector molecule may be coupled by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that results in a linkage between an effector molecule and an antibody via a covalent bond, such as glutaraldehyde, etc. The coupling agent utilizing a carboxyl group may be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
(41) Certain residues on an antibody (such as Cys or Lys, etc.) are used to link a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymer) and therapeutic agents. An antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent. A functional agent (e.g., a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to an antibody. A functional agent can be linked to an antibody either directly or indirectly via a linker.
(42) Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC comprises a linker between a drug and an antibody. The linker can be a degradable or non-degradable linker. Typically, degradable linkers are easily degraded in an intracellular environment, for example, the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular protease (e.g., lysosomal protease or endosomal protease), or sugar linkers, for example, glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that are hydrolyzed at a pH of below 5.5, such as hydrazone linkers) and linkers that are degraded under reducing conditions (e.g. disulfide-bond linkers). A non-degradable linker typically releases a drug under conditions that the antibody is hydrolyzed by protease.
(43) Prior to linkage to an antibody, a linker has a reactive group capable of reacting with certain amino acid residues, and the linkage is achieved by the reactive group. A thiol-specific reactive group is preferred, and includes, for example, a maleimide compound, a halogenated (e.g. iodo-, bromo- or chloro-substituted) amide; a halogenated (e.g. iodo-, bromo- or chloro-substituted) ester; a halogenated (e.g. iodo-, bromo- or chloro-substituted) methyl ketone, a benzyl halide (e.g. iodide, bromide or chloride); vinyl sulfone, pyridyl disulfide; a mercury derivative such as 3,6-di-(mercurymethyl)dioxane, wherein the counter ion is CH3COO, Cl or NO3-; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide linked to an antibody via thiosuccimide.
(44) A drug may be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, an antibody is linked to a drug via a linker, and the drug has a functional group that can form a bond with the linker. For example, a drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group that can form a bond with a linker. When a drug is directly linked to a linker, the drug has a reactive group before being linked to an antibody.
(45) Useful drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, a drug-linker can be used to form an ADC in a simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive moiety of a linker in the first step, and then the functional group on the linker reacts with a drug in the subsequent step, so as to form an ADC.
(46) In general, the functional group on a linker is selected so that it can specifically react with the suitable reactive group on a drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with the reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1,3-dipolar cycloaddition between the azide and alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reacting with hydrazides and alkoxyamines), phosphines (suitable for reacting with azides); isocyanates and isothiocyanates (suitable for reacting with amines and alcohols); and activated esters, for example, N-hydroxysuccinimide esters (suitable for reacting with amines and alcohols). These and other linkage strategies, for example, those described in Bioconjugation Technology (2nd Edition (Elsevier)), are well known to those skilled in the art. Those skilled in the art could understand that when a complementary pair of reactive functional groups is selected for a selective reaction between a drug moiety and a linker, each member of the complementary pair can be used for the linker, and can also be used for the drug.
(47) The present invention further provides a method for preparing an ADC, which may further comprise: under conditions sufficient to form an antibody-drug conjugate (ADC), combining an antibody to a drug-linker compound.
(48) In certain embodiments, the method of the present invention comprises: under conditions sufficient to form an antibody-linker conjugate, combining an antibody to a bifunctional linker compound. In these embodiments, the method of the present invention further comprises: under conditions sufficient to covalently link the drug moiety to the antibody via a linker, combining the antibody-linker conjugate to the drug moiety.
(49) In some embodiments, an antibody-drug conjugate (ADC) has a formula as follows:
(50) ##STR00001## wherein, Ab is an antibody, LU is a linker; D is a drug; and the subscript p is a value selected from 1 to 8.
(51) The following embodiments are further descriptions of the present invention and should not be understood as limitations of the present invention. Examples do not include a detailed description of traditional methods, such as those methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those with ordinary skills in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. Unless otherwise stated, percentages and parts are calculated by weight.
(52) The experimental materials and sources used in the following embodiments and the preparation methods of the experimental reagents are described below.
(53) Experimental Materials:
(54) CHO-S Cells: purchased from Thermo Fisher Scientific. Recombinant cell line CHOS-CD38: Human full-length CD38 was stably transfected into CHO-S cells to obtain a monoclonal cell line expressing CD38 stably by clone screening. Raji cells: purchased from ATCC, CCL-86. Mouse myeloma cells SP2/0: purchased from ATCC, catalog number CRL-1581. Balb/c mice: purchased from Shanghai Lingchang Biotechnology Co., Ltd. CB-17 SCID mice: purchased from Shanghai Lingchang Biotechnology Co., Ltd. Ramos Cells: purchased from ATCC, catalog number CRL-1596. Daudi cells: purchased from ATCC, catalog number CCL-213. DND-41 cells: purchased from Fenghui Biotechnology. Human peripheral blood mononuclear cell PBMC: purchased from Ausails Biotechnology (Shanghai) Co., Ltd. Reverse Transcription Kit: purchased from Takara. Goat anti-mouse secondary antibody: purchased from Millipore, catalog number AP181P. Donkey anti-mouse PE fluorescent secondary antibody: purchased from Jackson, catalog number 715-116-150. Goat anti-human PE fluorescent secondary antibody: purchased from Jackson, catalog number 109-115-098 F16 Black Maxisorp Plate: purchased from Nunc, catalog number 475515.
Experimental Reagents: PBS buffer: Sangon Biotech (Shanghai) Co., Ltd., catalog number B548117-0500. SFM Medium: purchased from Thermo Fisher Scientific, Inc., catalog number 12045-076. TMB: purchased from BD Company, catalog number 555214. NGD: purchased from sigma, catalog number N5131-25MG. Bovine serum albumin (BSA): purchased from Fetal Bovine Serum. -mercaptoethanol, fetal bovine serum, glutamine, sodium pyruvate, MEM-NEAA, 1% Penicillin-streptomycin were all purchased from Gibco. Phenol-free RPMI-1640: purchased from Gibco, catalog number 11835055. CytoTox 96 Non-Radioactive Cytotoxicity Assay: purchased from Promega, catalog number: G1780. HAT: purchased from Sigma-Alhrich, catalog number H0262-10VL. CCK-8: purchased from Tongren Chemical, catalog number CK04. Trizol: purchased from Thermo Fisher Scientific, catalog number 15596018.
Experimental Instruments: Electrofusion Instrument: purchased from BTX. Flow cytometry (CytoFLEX Cytometer System): purchased from Beckman Coulter.
(55) Antibody Sequences of the present invention are as follows:
(56) TABLE-US-00002 SEQID Name Sequence NO. H-CDR1of TYWMQ 1 CD38antibody H-CDR2of AIYPGDGDITYNQKFKG 2 CD38antibody H-CDR3of EGYYYGGALDY 3 CD38antibody L-CDR1of TASSSVSSSYLH 4 CD38antibody L-CDR2of GTSNLAS 5 CD38antibody L-CDR3of HRYHRSPWT 6 CD38antibody CD38antibody QVQLQQSGAELARPGASVKLSCKASGYTENTY 7 heavychain WMQWVKQRPGQGLEWIGAIYPGDGDITYNQKF variableregion KGKATLTADKSSNTAYMHLSSLASEDSAVYYCA 50G12-VH REGYYYGGALDYWGQGTSVTVSS CD38antibody QIFLTQSPAIMSASLGERVTMTCTASSSVSSSYLH 8 lightchain WYQQKPGSPPKLWMYGTSNLASGVPPRFSGSGS variableregion GTSYSLTISSMEAEDAATYYCHRYHRSPWTFGG 50G12-VL GTKLEIK CD38 QVQLVQSGAEVKKPGASVKVSCKASGYTFNTY 9 humanized WMQWVRQAPGQGLEWMGAIYPGDGDITYNQK antibodyheavy FKGRVTLTADKSTSTVYMELSSLRSEDTAVYYCA chainvariable REGYYYGGALDYWGQGTLVTVSS region50G12- Hu-VH CD38 DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLH 10 humanized WYQQKPGKAPKLWMYGTSNLASGVPSRFSGSG antibodylight SGTDYTLTISSLQPEDFATYYCHRYHRSPWTFGQ chainvariable GTKVEIK region50G12- Hu-VL CD38 QVQLVQSGAEVKKPGASVKVSCKASGYTENTY 11 humanized WMQWVRQAPGQGLEWMGAIYPGDGDITYNQK antibodyheavy FKGRVTLTADKSTSTVYMELSSLRSEDTAVYYCA chain50G12- REGYYYGGALDYWGQGTLVTVSSASTKGPSVFP Hu-HC LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CD38 DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLH 12 humanized WYQQKPGKAPKLWMYGTSNLASGVPSRFSGSG antibodylight SGTDYTLTISSLQPEDFATYYCHRYHRSPWTFGQ chain50G12- GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL Hu-LC LNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 50G12-VH caggttcagctccagcagtctggggctgagctggcaagacctggggcctc 13 heavychain agtgaagttg60tcctgcaaggcttctggctacacctttaatacctattggatg variableregion cagtgggtaaaacagaggcctggacagggtctggaatggattggggctatt nucleotide tatcctggagatggtgatattacatataatcagaagtttaagggcaaggccac sequence attgactgcagataaatcttccaacacagcctacatgcacctcagcagcttgg catctgaggactcagcggtctattactgtgcaagagagggatattattacggc ggggctttggactactggggtcaaggaacctcagtcaccgtctcctca 50G12-VL caaatttttctcacccagtctccagcaatcatgtctgcatctctaggggaacgg 14 lightchain gtcaccatgacctgcactgccagctcaagtgtgagttcaagctacttgcactg variableregion gtaccagcagaagccaggatccccccccaaactctggatgtatggcacatc nucleotide caacctggcttctggagtcccacctcgcttcagtggcagtgggtctgggacc sequence tcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattac tgccaccggtatcatcgttccccgtggacgttcggtggaggcaccaagctg gaaatcaaa 50G12-Hu-VH caggtgcagctcgtgcagtccggcgctgaggtgaagaagcccggcgcctc 15 heavychain cgtgaaggtgtcctgcaaggcctccggctacaccttcaacacctattggatg variableregion caatgggtgaggcaggcccccggccagggcctggagtggatgggcgcc nucleotide atctaccccggcgatggcgacatcacctacaaccagaagtttaagggcagg sequence gtgaccctgacagctgataaatctacatctactgtgtacatggagttatcttctc tgagatctgaggatacagctgtgtactattgtgctagagagggatactattatg gcggagccctggattattggggacagggaacactggtgacagtgtcttct 50G12-Hu-LC gatatccagatgacccagtctccttcttccctgtccgcttctgtgggagataga 16 lightchain gtgacaattacatgtaccgcttcttcttctgtgtcttcttcttacctgcattggtatc variableregion agcagaagcctggcaaggctcctaaactgtggatgtatggaacatctaatct nucleotide ggcttctggcgtgccttctagattttctggctctggatctggcaccgattacac sequence actgaccatctctagcctgcagcctgaggattttgccacatactactgtcaca gatatcacagatctccttggacctttggccagggcaccaaggtggagatcaa g 50G12-Hu-HC caggtgcagctcgtgcagtccggcgctgaggtgaagaagcccggcgcctc 17 heavychain cgtgaaggtgtcctgcaaggcctccggctacaccttcaacacctattggatg nucleotide caatgggtgaggcaggcccccggccagggcctggagtggatgggcgcc sequence atctaccccggcgatggcgacatcacctacaaccagaagtttaagggcagg gtgaccctgacagctgataaatctacatctactgtgtacatggagttatcttctc tgagatctgaggatacagctgtgtactattgtgctagagagggatactattatg gcggagccctggattattggggacagggaacactggtgacagtgtcttctg cgagcaccaagggaccttccgtgtttcccctcgcccccagctccaaaagca ccagcggcggaacagctgctctcggctgtctcgtcaaggattacttccccga gcccgtgaccgtgagctggaacagcggagccctgacaagcggcgtccac accttccctgctgtcctacagtcctccggactgtacagcctgagcagcgtggt gacagtccctagcagctccctgggcacccagacatatatttgcaacgtgaat cacaagcccagcaacaccaaggtcgataagaaggtggagcctaagtcctg cgacaagacccacacatgtcccccctgtcccgctcctgaactgctgggagg cccttccgtgttcctgttcccccctaagcccaaggacaccctgatgatttcca ggacacccgaggtgacctgtgtggtggtggacgtcagccacgaggacccc gaggtgaaattcaactggtacgtcgatggcgtggaggtgcacaacgctaag accaagcccagggaggagcagtacaattccacctacagggtggtgtccgt gctgaccgtcctccatcaggactggctgaacggcaaagagtataagtgcaa ggtgagcaacaaggccctccctgctcccatcgagaagaccatcagcaaag ccaagggccagcccagggaacctcaagtctataccctgcctcccagcagg gaggagatgaccaagaaccaagtgagcctcacatgcctcgtcaagggcttc tatccttccgatattgccgtcgagtgggagtccaacggacagcccgagaac aactacaagacaacaccccccgtgctcgattccgatggcagcttcttcctgta ctccaagctgaccgtggacaagtccagatggcaacaaggcaacgtcttcag ttgcagcgtcatgcatgaggccctccacaaccactacacccagaagagcct ctccctgagccctggaaag 50G12-Hu-HC gatatccagatgacccagtctccttcttccctgtccgcttctgtgggagataga 18 lightchain gtgacaattacatgtaccgcttcttcttctgtgtcttcttcttacctgcattggtatc nucleotide agcagaagcctggcaaggctcctaaactgtggatgtatggaacatctaatct sequence ggcttctggcgtgccttctagattttctggctctggatctggcaccgattacac actgaccatctctagcctgcagcctgaggattttgccacatactactgtcaca gatatcacagatctccttggacctttggccagggcaccaaggtggagatcaa gagaaccgtcgccgctcccagcgtcttcatcttcccccccagcgatgagca gctgaagagcggaaccgccagcgtggtgtgcctgctgaacaacttctaccc cagggaggccaaggtgcaatggaaggtggacaacgccctacagagcgg caactcccaggagagcgtgaccgagcaggacagcaaggatagcacctac agcctgagcagcaccctcaccctgagcaaggccgactacgagaagcaca aggtgtacgcctgcgaggtgacccatcagggcctgagcagccctgtgacc aagagcttcaacaggggcgagtgc Daratumumab EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAM 19 heavychain SWVRQAPGKGLEWVSAISGSGGGTYYADSVKG variableregion RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKD aminoacid KILWFGEPVFDYWGQGTLVTVSS sequence Daratumumab EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW 20 lightchain YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT variableregion DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTK aminoacid VEIK sequence Isatuximab QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYW 21 heavychain MQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQ variableregion GKATLTADKSSKTVYMHLSSLASEDSAVYYCAR aminoacid GDYYGSNSLDYWGQGTSVTVSS sequence Isatuximab DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVA 22 lightchain WYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGA variableregion GTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGG aminoacid TKLEIK sequence
Example 1 Preparation of a Positive Control Antibody
(57) The heavy chain and light chain variable region amino acid sequences of Daratumumab described in examples of the present invention are from the WHO Drug Information, Vol. 24, No. 1, 2010, i.e., SEQ ID NO: 19 and 20 of the present invention.
(58) The heavy chain and light chain variable region amino acid sequences of Isatuximab described in examples of the present invention are from WHO Drug Information, Vol. 29, No. 3, 2015, i.e., SEQ ID NO: 21 and 22 of the present invention.
(59) The DNA sequences of above-mentioned heavy and light chain variable regions were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized Daratumumab heavy chain variable region gene was linked to the human IgG1 heavy chain constant region gene to obtain a full-length heavy chain gene, named Daratumumab-HC-IgG1. The Daratumumab light chain variable region gene was linked to the human Kappa chain constant region gene to obtain a full-length light chain gene, named Daratumumab-LC. Daratumumab-HC-IgG1 and Daratumumab-LC genes were constructed into pcDNA3.4 expression vectors, and the obtained heavy and light chain expression vectors were transferred into HEK293F cells to express antibodies by PEI transfection, and HEK293F cells were cultured with Free Style 293 Expression Medium. The transfected HEK293F cells were cultured in a CO.sub.2 shaking incubator for 5 days, then the cell supernatant was collected by centrifugation, and the antibody in the supernatant was purified by Protein A affinity chromatography, and the antibody obtained was named Daratumumab. In addition, antibody Isatuximab was obtained with similar experimental methods.
(60) Human CD38 extracellular segment sequence information is from https://www.uniprot.org/uniprot/P28907. The DNA of CD38 extracellular segment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The recombinant gene was constructed into the pcDNA3.4 expression vector. The recombinant protein expressed by His tag fusion was purified by metal chelation affinity chromatography column for one-step purification of the recombinant protein in the cultured supernatant. Fc tag fusion-expressed recombinant proteins were purified in one step using Protein A/G affinity chromatography columns. The obtained protein was named CD38-His/CD38-Fc.
Example 2 Antigen-Immuned Animals and Preparation and Screening of Hybridomas
(61) Step 1: Antigen-Immunized Mice
(62) Balb/c mice were immunized by routine intraperitoneal injection with recombinant overexpressed cell line CHOS-CD38 or tumor cell line Raji. On the first day, Balb/c mice were injected intraperitoneally with 100 l of Freund's complete adjuvant. On the second day, Balb/c mice were intraperitoneally immunized with recombinant cell line CHOS-CD38 or Raji cells, 5*10.sup.6 cells per mouse. On the 14th day, Balb/c mice were intraperitoneally injected for boosting immunization with recombinant cell line CHOS-CD38 or Raji cells, 5*10.sup.6 cells per mouse, and on the 36th day, mice were immunized with recombinant cell line CHOS-CD38 or Raji cells as before. Three weeks later, CD38-His antigen protein was injected intraperitoneally for stimulation. After 3-4 days, mouse spleens were taken for cell fusion experiment.
(63) Step 2: Preparation and Screening of Hybridomas
(64) 3-4 days after the last immunization of mice, mouse spleen cells and mouse myeloma cells SP2/0 were fused by electrofusion apparatus using conventional hybridoma technique protocols. The fused cells were uniformly suspended in complete medium, which consisting of 1:1 mixture of RPMI1640 and DMEM F12 medium and adding 1% Glutamine, 1% Sodium pyruvate, 1% MEM-NEAA (minimum basic mediumnon-essential amino acid solution), 1% Penicillin-streptomycin, 50 M of -mercaptoethanol and 20% FBS (fetal bovine serum). The fused cells were divided into 36 96-well plates at 10.sup.5 cells/100/well and cultured overnight. The next day, 100 l of complete medium containing 2HAT were added to each well, so that the culture medium in the 96-well plate was 200 l/well (containing 1HAT). After 7-12 days, the supernatant was harvested and hybridoma wells with positive CD38 binding activity were screened by Cell based ELISA.
(65) Wherein, the Cell based ELISA method for screening hybridoma wells with positive CD38 binding activity was as follows: the recombinant cell line CHOS-CD38 was diluted to 2*10.sup.6 cells/ml with PBS buffer, and was added into cell culture plate at 100 l/well, and cultured overnight at 37 C. On the next day, the supernatant was removed, 100 l/well cell fixative was added and fixed at room temperature for one hour, and then the supernatant was removed and 5% skim milk powder was added to block for two hours at 37 C. The plate was washed with PBST once for use. The collected hybridoma supernatant was added into the blocked plate sequentially, 100 l/well, and the plate was placed at 37 C. for 1h. The plate was washed with PBST for 3 times, and the HRP-labeled goat anti-mouse IgG was added and the plate placed at 37 C. for 30 min. After washing the plate with PBST for 5 times, the plate was patted on the absorbent paper to remove residual droplets as far as possible. 100 l TMB was added to each well, and the plate was placed at room temperature (205 C.) for 5 minutes away from light; 50 2M H.sub.2SO.sub.4 terminating solution was added to each well to terminate the substrate reaction. OD values were read at 450 nm on the microplate reader to analyze the binding ability of the antibody to target antigen CD38. A total of 30 hybridoma cell lines were obtained through screening. The 30 hybridoma cell lines obtained through screening were expanded in serum-containing complete medium, and centrifuged to replace the medium with serum-free medium SFM medium so that the cell density was 1-210.sup.7/ml. The cells were cultured at 8% CO.sub.2 and 37 C. for 1 week. The supernatant was obtained by centrifugation and purified by Protein G affinity chromatography. Three strains of monoclonal antibody against human CD38 were obtained, named 50G12, 279D11, 153F11, and 50G12 was the best active antibody.
Example 3 Binding Activity of Murine Antibody 50G12 to Human CD38-Fc Protein
(66) The binding ability of murine antibody to human CD38-Fc protein was determined by indirect enzyme-linked immunosorbent assay (ELISA). The specific methods were as follows: CD38-Fc protein was diluted in coating solution (50 mM carbonate coating buffer, pH 9.6) to 1 g/ml to coat plate at 4 C. overnight. Then the plate was blocked with 5% skim milk and incubated at 37 C. for 2 hours. After washing the plate with PBST for 3 times, the anti-human CD38 antibody 50Ga12 protein prepared in the laboratory was diluted by gradient with 1% BSA buffer and added into the pre-coated CD38-FC plate with 100 l/well, and incubated at 37 C. for 1 hour. The plate was washed with PBST for 3 times, and the HRP-labeled goat anti-mouse IgG was added and placed at 37 C. for 30 min. After washing the plate with PBST for 3 times, the plate was patted on the absorbent paper to remove residual droplets as far as possible. 100 l TMB was added to each well, and then the plate was placed at room temperature (205 C.) away from light for 5 min. Then 50 l 2M H.sub.2SO.sub.4 terminating solution was added to each well to terminate the substrate reaction. OD values were read at 450 nm on the microplate reader to analyze the binding ability of the tested antibody to the target antigen human CD38-Fc. The results are shown in
(67) As shown in
Example 4 the Ability of Murine Antibodies to Inhibit CD38 Enzyme Activity
(68) CD38 is an enzyme that catalyzes the conversion of nicotinamide guanine dinucleotide (NGD) into cyclic GDP-ribose, which is capable of stimulating fluorescence. The inhibitory effects of murine antibodies 50G12, 279D11, 153F11 and control antibodies Daratumumab, Isatuximab and IgG control on CD38 enzyme activity were determined by fluorescence method. The specific methods were as follows:
(69) The murine antibodies 50G12, 279D11, 153F11 to be tested and the control antibodies Daratumumab, Isatuximab and IgG control were diluted to 600 g/ml with Tris-Hcl, and a gradient dilution was performed by 3 times with a total of 10 wells. CD38-His antigen was diluted to 5 g/ml with Tris-Hcl, and the antigen and sample antibody were added to the reaction plate in equal volumes with 50 l each. The plate was shaken for 10 min, and incubated at 37 C. for 30 min. The background wells were set: (1) diluent well: 200 l diluent; (2) NGD wells: 100 l diluent; (3) antigen well: 50 l antigen, 150 l diluent. After incubation, the NGD was diluted with Tris-Hcl to 250 g/ml, 100 l per well, except for diluent and antigen wells. The plate was shaken for 5 min, and incubated at 37 C. for 90 min. The plate was read at Ex:300, EM:410 with a multifunction microplate reader and data were collected and processed. The results are shown in
(70) As shown in
Example 5 the Binding Ability of Murine Antibodies to Recombinant High-Expression Cell Line
(71) CHOS-CD38 Cells and Tumor Cell Line DND-41 Cells
(72) Flow cytometry was used to detect the binding activity of murine antibodies to recombinant high-expression cell line CHOS-CD38 cells and tumor cell line DND-41 cells. The specific methods were as follows:
(73) CHOS-CD38 and DND-41 cells were collected, centrifuged to remove the cell culture medium, and the cells were washed twice with PBS buffer. The cells were counted and diluted with 1% BSA FACS buffer to 2*10.sup.6 cells/ml. The cells were spread into round-bottom 96-well plate for use. The antibody to be tested was diluted with 1% BSA buffer for 8 gradients and added to the round-bottom plate with the above cells. The plate was incubated at 4 C. for 1 hour. After centrifugation, the supernatant was removed and the plate was washed 3 times with 1% BSA FACS buffer. 100 l donkey anti-mouse PE fluorescent secondary antibody or goat anti-human PE fluorescent secondary antibody was added to each well at a ratio of 1:300 (see the instructions for fluorescent secondary antibody for details), and the plate was incubated at 4 C. for 1 hour. The samples were washed for 3 times with 1% BSA FACS buffer and then re-suspended with 1% BSA FACS buffer at 200 l/well. FACScalibur BD was used to analyze the samples. The results of antibody binding to cells are shown in
(74) It can be seen from
Example 6: Murine Antibody CDC Activity Assay
(75) When a specific antibody binds to the corresponding antigen on the cell membrane surface, it can activate the classical complement pathway, thereby forming a membrane attack complex to lyse target cells, which is called CDC action. CDC activity was measured on CHOS-CD38, Raji, DND-41, Ramos, and Daudi cells with the following specific methods:
(76) Anti-human CD38 monoclonal antibody was diluted to the initial concentration of 20 g/ml using cell culture medium as buffer, and then diluted with a 3-fold concentration gradient to obtain a total of 8 concentrations of dilution. Target cells expressing CD38 (Daudi cells, etc.) were counted and resuspended to 3*10.sup.5 cells/ml. 100 l of anti-human CD38 monoclonal antibody of various concentration dilutions and 80 l of high-expression CD38 target cells were pre-incubated for 15 min, then 20 l of 50% fresh human serum (donated by volunteers) was added and mixed. Positive control well was target cells alone added with serum, while the negative control hole was cell-free medium. The cells were incubated in the incubator for 12-18h. After adding 20 l CCK-8 for 4h, microplate reader was used to measure the absorption value at 450 nm, and the killing rate was calculated according to the reading at 450 nm. The formula for calculating the killing rate is:
Killing rate (killing %)=(positive control absorption valueexperimental group absorption value)/(positive control absorption valuenegative control absorption value)*100.
(77) The results are shown in
Example 7 Humanization of Murine Anti-Human CD38 Monoclonal Antibody
(78) Step 1: Determination of the Variable Region Sequence of Murine Anti-Human CD38 Monoclonal Antibody
(79) Total RNA was extracted from a 50G12 hybridoma monoclonal cell line using Trizol, and mRNA was reverse-transcribed into cDNA using a reverse transcription kit. 50G12 light chain variable region gene and heavy chain variable region gene were amplified by PCR using the combined primers reported in the literature (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dubel, the sequences of the combined primers are from page 323), and then PCR products were cloned into pMD18-T vector. The variable region gene sequences were sequenced and analyzed. The sequence information of the 50G12 variable regions of murine antibody was as follows: the total length of the heavy chain variable region gene sequence is 357 bp, encoding 119 amino acid residues, wherein the nucleotide sequence is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 7. The total length of the light chain variable region gene sequence is 321 bp, encoding 107 amino acid residues, wherein the nucleotide sequence is shown in SEQ ID NO: 14, and the amino acid sequence is shown in SEQ ID NO: 8.
(80) Step 2: Humanization of Murine Monoclonal Antibody Against Human CD38
(81) The amino acid sequences of the heavy chain variable region and light chain variable region of 50G12 murine antibody were analyzed. The complementarity determinant regions (CDR) and frame regions (FR) of the heavy chain and light chain of 50G12 murine antibody were determined according to the Kabat rule. The amino acid sequences of the heavy chain CDR of 50G12 murine antibody are H-CDR1: SEQ ID NO: 1, H-CDR2: SEQ ID NO: 2 and H-CDR3: SEQ ID NO: 3, and the amino acid sequences of the light chain CDR are L-CDR1: SEQ ID NO: 4, L-CDR2: SEQ ID NO: 5 and L-CDR3: SEQ ID NO: 6.
(82) The homology of the heavy chain variable region of murine 50G12 monoclonal antibody with the sequence of human IgG germ line was compared on https://www.ncbi.nlm.nih.gov/igblast/. IGHV1-46*01 was selected as heavy chain CDR transplantation template, and the heavy chain CDR of murine antibody 50G12 was transplanted into the IGHV1-46*01 skeleton region. WGQGTLVTVSS was added as the fourth frame region after H-CDR3 to obtain the heavy chain variable region sequence of CDR transplantation. Similarly, the homology of the light chain variable region of murine antibody 50G12 with the sequence human IgG germ line was compared. IGKV1-39*01 was selected as the light chain CDR transplantation template, and the light chain CDR of murine antibody 50G12 was transplanted into the skeleton region of IGKV1-39*01. FGQGTKVEIK was added after L-CDR3 as the fourth frame region to obtain the light chain variable region sequence of CDR transplantation. Based on the variable region of CDR transplantation, some amino acid sites in the frame region were performed reverse mutation. Reverse mutation is the mutation of some amino acids (amino acids that are important for maintaining the structure and affinity of the antibody) in the frame region of the CDR transplantation variable region into amino acids in the corresponding position of the frame region of the mouse origin.
(83) During mutation, amino acid sequence was encoded by Kabat, and the location of site was indicated by Kabat code. Preferably, for CDR transplantation heavy chain variable region, according to the Kabat code, T at position 30 was mutated to N, M at position 69 was mutated to L, R at position 71 was mutated to A, and T at position 73 was mutated to K. For CDR transplantation light chain variable region, L at position 47 was mutated to W, I at position 48 was mutated to M, and F at position 71 was mutated to Y. The above heavy chain variable region and light chain variable region with mutation sites were defined as humanized heavy chain variable region and light chain variable region, named 50G12-Hu-VH and 50G12-Hu-VL, respectively. The amino acid sequence of 50G12-Hu-VH is SEQ ID NO: 9. The amino acid sequence of 50G12-hu-VL is SEQ ID NO: 10.
(84) The DNA encoding the humanized heavy chain and light chain variable region was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthetic humanized heavy chain variable region DNA was connected with the human IgG1 heavy chain constant region DNA to obtain the full-length humanized heavy chain DNA, named 50G12-Hu-HC. The human heavy chain variable region DNA sequence is shown in SEQ ID NO: 15, and the full-length humanized heavy chain DNA sequence is shown in SEQ ID NO: 17. The human light chain variable region DNA was connected with the human Kappa chain constant region DNA to obtain the full-length human light chain DNA, named 50G12-Hu-LC. The DNA sequence of human light chain variable region is shown in SEQ ID NO: 16, and the full-length human light chain DNA sequence is shown in SEQ ID NO: 18. The 50G12-Hu-HC and 50G12-Hu-LC genes were constructed into pcDNA3.4 expression vectors, respectively, and the antibody was expressed and purified using the method described in the above examples. Wherein, its heavy chain amino acid sequence is shown in SEQ ID NO: 11, and the light chain amino acid sequence is shown in SEQ ID NO: 12, and the obtained antibody is named 50G12-Humanized.
(85) In addition, the heavy chain variable region of murine antibody 50G12 was connected with the constant region of human IgG1 heavy chain to obtain a chimeric heavy chain gene, named 50G12-Chi-HC. The light chain variable region of murine 50G12 was connected with the human Kappa chain constant region to obtain a chimeric light chain gene, named 50G12-Chi-LC. The 50G12-Chi-HC and 50G12-Chi-LC genes were constructed into pcDNA3.4 expression vectors, respectively, and the antibody was expressed and purified using the method described in the above examples. The antibody obtained is named 50G12-Chimeric.
Example 8 the Binding Ability of 50G12-Humanized to CD38
(86) Flow cytometry was used to detect the binding capacity of 50G12-Chimeric and 50G12-Humanized to CD38 on the surface of Daudi cells. The specific methods were as follows:
(87) After Daudi cells were counted, PBS solution containing 1% BSA was used to inoculate the cells into 96-well round-bottom culture plates with 210.sup.5 cells per well. 50 l anti-CD38 antibody in gradient dilution with PBS solution was added to the 96-well plate. The plate was incubated at room temperature for 1 h, then it was centrifuged to discard the supernatant. Then the cells was washed with PBS twice. FITC-labeled goat anti-human (Fc-Specific) antibody was added (1:1000 diluted with PBS containing 1% BSA) and the plate was incubated at room temperature for half an hour. The cells was centrifuged and washed, then the Mean Fluorescence Intensity (MFI) of the FITC channel was detected by flow cytometry. The flow cytometer software was used to process the experimental data and the mean fluorescence intensity was calculated. The GraphPad Prism6 was used for data analysis and mapping, and EC50 was calculated.
(88) As shown in
Example 9 50G12-Humanized Inhibiting CD38 Cyclase Activity
(89) The inhibitory effect of 50G12-Humanized on CD38 cyclase activity was determined by fluorescence method. The specific methods were as follows:
(90) 50 mM MES buffer solution with PH6.5 was prepared, and 200 M niacinamide guanine dinucleotide (NGD) solution was prepared with MES buffer solution. CD38-His was diluted to 2 g/ml with MES buffer, and then anti-CD38 antibody with a final concentration of 10 g/ml was added. F16 Black Maxisorp Plate was added with 50 L NGD solution, followed by 50 L solution containing CD38-His and anti-CD38 antibodies. The Relative Fluorescence Unit (RFU) was measured in kinetic mode using multifunction microplate reader SpectraMax M5, and the excitation and emission wavelengths were set at 300 nm and 410 nm, respectively. The GraphPad Prism6 was used for data analysis and mapping.
(91) The results are shown in
Example 10 50G12-Humanized ADCC Activity Determination
(92) The Fab segment of antibody binds to antigen epitopes on cell surface, and the Fc segment binds to Fc receptors on the surface of effector cells (NK cells, macrophages, etc.), which can mediate effector cells to directly kill target cells, that is the effect of ADCC. The ADCC activity of the anti-CD38 antibody was determined here. The specific methods were as follows:
(93) The phenol red-free PMI-1640 was added with 2% fetal bovine serum. Target cells Daudi and human peripheral blood mononuclear cells (PBMC) were mixed in a ratio of 1:25 with the medium and were inoculated into a round-bottom 96-well plate with 150 L/well. Finally, each well contained 210.sup.4 Daudi cells and 510.sup.5 PBMCs. 50 L gradient diluted anti-CD38 antibody was added. The plate was incubated overnight in a 5% CO.sub.2 cell incubator at 37 C. 50PL culture-supernatant was taken, and 50 L CytoTox 96 Non-Radioactive Cytotoxicity Assay Reaction solution was added. After 30 min, termination solution was added to terminate the reaction, and OD490 was read by microplate reader. The GraphPad Prism6 was used for data analysis and mapping, and EC50 was calculated.
(94) As shown in
Example 11 50G12-Humanized CDC Activity Determination
(95) The CDC activity of 50G12-Humanized was determined in Daudi and DND-41 cell models. The detailed experimental methods were as follows: anti-human CD38 monoclonal antibody was diluted to the initial concentration of 20 g/ml using cell culture medium as buffer, and then diluted with a 3-fold concentration gradient to obtain a total of 8 concentrations of dilution. Target cells expressing CD38 (Daudi cells, etc.) were counted and resuspended to 3*10.sup.5 cells/ml. 100 l of anti-human CD38 monoclonal antibody of various concentration dilutions and 80 l of high-expression CD38 target cells were pre-incubated for 15 min, then 20 l of 50% fresh human serum (donated by volunteers) was added and mixed. Positive control well was target cell alone added with serum, while the negative control well was cell-free medium. The cells were incubated in the incubator for 12-18h. After adding 20 l CCK-8 for 4h, Microplate reader was used to measure the absorption value at 450 nm, and the killing rate was calculated according to the reading at 450 nm. The formula for calculating the killing rate is:
Killing rate (killing %)=(positive control absorption valueexperimental group absorption value)/(positive control absorption valuenegative control absorption value)*100.
(96) The experimental results are shown in
Example 12 The Ability of 50G12-Humanized to Induce Apoptosis
(97) Anti-CD38 antibodies bind to the corresponding antigens on the cell membrane surface to induce apoptosis (Deckert J, Wetzel M, Bartle L M, et al. SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+ Hematologic Malignancies[J]. Clinical Cancer Research, 2014, 20(17): 4574-4583). In apoptotic cells, the membrane phosphatidylserine (PS) is transferred from the inside to the outside of the cell membrane, exposing PS to the external cellular environment. Annexin V is a 35-36 KDa calcium ion dependent phospholipid binding protein with high affinity for PS and can bind to exposed PS. The apoptosis-inducing activity of the anti-CD38 antibody was determined using the FITC-labeled Annexin V apoptosis assay kit. The specific methods were as follows:
(98) 2% fetal bovine serum was added to RPMI-1640. Daudi cells were inoculated into 96-well plates with 110.sup.5 cells/150 L per well. 50 L gradient diluted anti-CD38 antibody was added. The plate was incubated at 37 C. in 5% CO.sub.2 cell incubator for 24h. Apoptotic cells were stained with FITC-labeled Annexin V apoptosis detection kit. The cells was centrifuged and washed, then the mean fluorescence intensity of the FITC channel was detected by flow cytometry. The flow cytometer software was used to process the experimental data and calculate the proportion of stained cells in the total cells. The GraphPad Prism6 was used for data analysis and mapping, and EC50 was calculated.
(99) As shown in
Example 13 In Vivo Efficacy Evaluation of 50G12-Humanized
(100) The in vivo antitumor activity of anti-CD38 humanized antibody 50G12-Humanized was verified in a human Ramos lymphoma cell line CB-17 SCID mouse xenograft model. The specific methods were as follows:
(101) Ramos cells were cultured in vitro, the cell concentration was adjusted to 510.sup.7 cells/ml after harvest, and 200 l cell suspension per mouse was inoculated into female CB-17 SCID mice by tail vein injection to establish a xenograft model. On day 7 after inoculation, mice were randomly divided into control group, Daratumumab treatment group, Isatuximab treatment group, and 50G12-Humanized treatment group with 10 mice in each group. Treatment was started at a dose of 40 mg/kg antibody twice a week for three weeks. The survival time of tumor-bearing mice was observed. The animal humanitarian endpoint is that unilateral hind limb or bilateral hind limb paralysis of tumor-bearing mice, or weight loss of more than 20%, or inability to eat and drink freely due to severe physical condition of tumor-bearing mice. The animal was euthanized and the survival time was recorded.
(102) The results are shown in