CN112794911B - Humanized anti-folate receptor 1 antibody and application thereof - Google Patents
Humanized anti-folate receptor 1 antibody and application thereof Download PDFInfo
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- 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
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C12N2800/00—Nucleic acids vectors
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Abstract
The invention provides a humanized anti-folate receptor 1 antibody and application thereof. The invention discloses a humanized anti-Folate Receptor (FOLR1) antibody containing a unique sequence, which can specifically bind to FOLR1 and effectively inhibit FOLR1, and can be applied to inhibit diseases related to over-expression of FOLR1 or affected by the function of FOLR1, such as tumors.
Description
Technical Field
The invention belongs to the field of biotechnology and pharmacology, and particularly relates to a humanized anti-folate receptor 1 antibody and application thereof.
Background
Folate is an essential vitamin required for DNA synthesis and repair, as well as for cell division processes. Folate enters cells by binding to folate receptors on the cell surface, causing endocytosis of the receptors. The Folate Receptor is a transmembrane single-chain glycoprotein linked to glycosylated phosphatidylinositol, has high affinity for Folate, and contains three subtypes, namely, Folate Receptor α (FOLR1), Folate Receptor β (FOLR2), and Folate Receptor γ (FOLR 3). Since the folate receptor is rarely or not expressed in normal cells, but the tumor cells are more dependent on folate than normal somatic cells, the folate receptor on the tumor cells has obvious high expression, especially FOLR1, and is over-expressed in malignant tumors such as ovarian cancer, lung cancer, uterine cancer, pancreatic cancer and testicular cancer, so FOLR1 becomes one of the hot targets of anticancer drugs.
The developed medicine related to the FOLR1 antibody can specifically identify tumor cells, thereby achieving the effect of directly or indirectly killing tumors. Mov19 is a mouse monoclonal antibody against human FOLR1, isolated from hybridoma cells in 1987 (Int J Cancer. 1987 Mar 15;39(3): 297) 303), and its related amino acid sequence was disclosed in 1998 (Cancer Res. 1998 Mar 1;58(5): 991-. The invention carries out humanization transformation on the murine antibody on the basis of the prior art, mainly aims to reduce the immunogenicity of the murine antibody, reduce the occurrence probability of generating anti-drug antibodies in a patient body in the drug administration process, and further improve the stability of the murine antibody so that the murine antibody has better drug-forming property.
Disclosure of Invention
The invention aims to provide a humanized anti-folate receptor 1 antibody and application thereof.
In a first aspect of the present invention, there is provided an anti-folate receptor 1 antibody having a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 2.
In a preferred embodiment, the anti-folate receptor 1 antibody further comprises: an antibody having a heavy chain variable region amino acid sequence that is 85% or more (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) identical to a sequence shown in SEQ ID NO. 1 and having a light chain variable region amino acid sequence that is 85% or more (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) identical to a sequence shown in SEQ ID NO. 2, and having the same function as the antibody according to an embodiment of the present invention; preferably, the amino acids corresponding to the positions listed in table 1 in the heavy chain variable region/light chain variable region of the humanized antibody are conserved.
In another preferred embodiment, the anti-folate receptor 1 antibody is a humanized antibody.
In another aspect of the invention, there is provided a polynucleotide encoding said anti-folate receptor 1 antibody.
In another aspect of the invention, a construct (e.g., an expression vector) is provided comprising the polynucleotide.
In another aspect of the invention, there is provided an antibody expression system comprising said construct or genome having integrated therein an exogenous polynucleotide.
In another preferred embodiment, the antibody expression system is a cellular expression system.
In another aspect of the present invention, there is provided a method for preparing the anti-folate receptor 1 antibody, comprising: expressing said antibody using said antibody expression system under conditions suitable for expression of said antibody.
In a preferred embodiment, the preparation method further comprises: purifying and separating the antibody.
In another aspect of the present invention, there is provided an immunoconjugate comprising: the antibody as described above; and a functional molecule attached thereto (including but not limited to covalent attachment, coupling, attachment, adsorption).
In a preferred embodiment, the functional molecules include (but are not limited to): cytotoxins, radioisotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules that target surface markers of immune cells or detectable markers, extracellular hinge, transmembrane and intracellular signaling regions based on chimeric antigen receptor technology (CAR), or combinations thereof.
In another preferred embodiment, the molecule targeting a tumor surface marker is an antibody or ligand that binds to a tumor surface marker.
In another preferred embodiment, the tumor suppressor molecule is an anti-tumor cytokine or an anti-tumor toxin.
In another preferred embodiment, the anti-tumor toxin includes (but is not limited to): toxins acting on tubulin, such as Monomethyl auristatin (maytansinoid-related compounds and derivatives thereof), maytansinoid (maytansinoid) -related compounds and derivatives thereof, DNA-acting toxins, such as duocarmycin, calicheamicin, Pyrrobenzodiazepine (PBDs), SN-38, Dxd, and related compounds and derivatives thereof, which act on other functions in the cell, such as metabolism, transcription, translation, signal transduction, and the like.
In another preferred embodiment, the cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, or variants thereof.
In another preferred embodiment, the detectable label includes (but is not limited to): fluorescent markers, chromogenic markers.
In another preferred embodiment, the antibody that binds to a tumor surface marker is an antibody that recognizes an antigen other than folate receptor 1, including (but not limited to): EGFR, EGFRvIII, mesothelin, HER2, EphA2, Her3, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, ASGPR1 or CDH 16.
In another preferred embodiment, the intracellular signaling region includes (but is not limited to): CD3 zeta chain, fcepsilon RI gamma tyrosine activation motif, intracellular signaling regions of costimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, CD40, and the like.
In another preferred embodiment, the transmembrane region includes (but is not limited to): the transmembrane region of CD8 or CD 28.
In another preferred embodiment, the anti-tumor toxin is linked to the antibody via a linker, including but not limited to mc-vc-PAB (maleimidocaproyl valene-citrulline para-amino benzoyloxy).
In another aspect of the invention, there is provided a pharmaceutical composition (including a diagnostic composition, such as a diagnostic reagent) comprising: said antibody or said immunoconjugate.
In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In another aspect of the invention, there is provided a use of the antibody, the immunoconjugate or the pharmaceutical composition for the preparation of a formulation, kit or kit for the diagnosis or treatment of a tumor.
In a preferred embodiment, the tumor is a folate receptor 1 expressing tumor.
In another preferred embodiment, the tumor comprises: ovarian cancer, endometrial cancer, breast cancer, cervical cancer and the like.
In another aspect of the invention, a kit or kit is provided comprising said antibody, or said immunoconjugate, or said pharmaceutical composition.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
Figure 1 humanization of the Mov19 heavy chain variable region according to the invention.
FIG. 2 humanization of the Mov19 light chain variable region according to the invention.
Figure 3, affinity of the humanized Mov19 of the present invention for FOLR1 on ELISA.
Fig. 4, affinity of the humanized Mov19 at Octet for FOLR 1.
Figure 5, affinity of the humanized Mov19 of the present invention to FOLR1 on flow cytometry.
Figure 6, ADCC function of the humanized Mov19 of the present invention.
Figure 7, DLS results of the humanized Mov19 of the present invention.
Fig. 8, endocytic function of the humanized Mov19 of the present invention on tumor cells.
Figure 9, killing function of antibody conjugated drug of humanized Mov19 of the present invention against KB tumor cells.
FIG. 10, the killing function of the antibody conjugated drug of the humanized Mov19 of the present invention on T-47D tumor cells.
Detailed Description
The inventor of the invention has intensively studied and revealed that a humanized anti-Folate Receptor (FOLR1) antibody containing a unique sequence can specifically bind to FOLR1 and effectively inhibit FOLR1, and can be applied to inhibit diseases related to over-expression of FOLR1 or affected by the function of FOLR1, such as tumors.
When the existing anti-FOLR 1 antibody Mov19 is used as a murine antibody and developed into a medicament, the immunogenicity problem of the antibody can influence the efficacy of the medicament in a patient. The inventor carries out humanization treatment and optimization transformation based on the Mov19, and reduces the immunogenicity of the medicine in vivo as much as possible; and the humanized antibody is obviously superior to the original antibody in function and stability.
Term(s) for
As used herein, the term "antibody" or "immunoglobulin" is used herein as a generic term including full-length antibodies, single chain antibodies, and all portions, domains or fragments thereof (including but not limited to antigen binding domains or fragments). Furthermore, the term "sequence" as used herein (e.g. in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence" etc.) should generally be understood to include both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding the sequences, unless a more limited interpretation is required herein.
The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit single binding specificity and affinity for a particular epitope.
The term "humanized antibody" refers to a molecule having an antigen binding site substantially from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise the entire variable domain fused to a constant domain, or only Complementarity Determining Regions (CDRs) grafted to the appropriate framework regions in the variable domain. The antigen binding site may be wild-type or modified by one or more amino acid substitutions, for example to make it more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all of the CDR sequences. Other forms have one or more CDRs that have been altered relative to the original antibody.
"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. "sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
An "effective amount" of an agent refers to the amount necessary to effect a physiological change in the cell or tissue to which it is administered.
A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.
The term "pharmaceutical composition" refers to a formulation in a form such that the biological activity of the active ingredient contained therein is effective, and that is free of other ingredients having unacceptable toxicity to a subject to whom the composition will be administered.
"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
The term "treatment/prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of disease in a treated individual and may be a clinical intervention performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or delay the progression of a disorder.
The term "detectable label" refers to a marker that can be attached to an antibody for determining the presence or absence and amount of a particular target in a subject to be tested. The "detectable label" may be, but is not limited to: enzymes, fluorescent labels, nuclides, quantum dots, colloidal gold, and the like. More particularly, for example, it may be selected from: horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase.
In the invention, the tumor is a tumor expressing a folate receptor 1; preferably, the tumor includes, but is not limited to: ovarian cancer, endometrial cancer, breast cancer, cervical cancer, and the like, or combinations thereof.
Antibodies
The invention provides an anti-FOLR 1 antibody, wherein the anti-FOLR 1 antibody has a heavy chain variable region of an amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region of an amino acid sequence shown in SEQ ID NO. 2. The invention also includes: an antibody having a heavy chain variable region amino acid sequence that is 85% or more (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) identical to a sequence shown in SEQ ID NO. 1 and having a light chain variable region amino acid sequence that is 85% or more (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) identical to a sequence shown in SEQ ID NO. 2, and having the same function as the antibody according to an embodiment of the present invention; preferably, the amino acids corresponding to the positions listed in table 1 in the heavy chain variable region/light chain variable region of the humanized antibody are conserved.
The antigen binding properties of antibodies are generally determined by 3 CDR regions, which are arranged in an ordered arrangement with FR regions that do not directly participate in the binding reaction. These CDRs form a loop structure, and the β sheets formed by the FRs in between are spatially adjacent to each other, constituting the antigen binding site of the antibody. CDR regions are sequences of proteins of immunological interest, and the antibodies of the invention include sequence modifications to the CDR regions as well as the framework regions.
The anti-FOLR 1 antibody provided by the invention can be efficiently and specifically combined with human FOLR1, can efficiently block the action of FOLR1, and can efficiently carry another substance coupled with the FOLR to target cells.
The invention also includes a fusion protein of an anti-FOLR 1 antibody comprising a first domain of an antibody according to the invention and a second domain for extending half-life in vivo and/or having binding effect on effector cells. The fusion protein can be a binding molecule that is capable of specifically binding to a cell expressing FOLR 1.
The second domain, a fragment for extending in vivo half-life may include serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin (such as an antibody against serum albumin), a polyethylene glycol-liposome complex, and the like. The fragment having a binding effect to effector cells in the second domain may include an immunoglobulin Fc region or the like, preferably selected from human immunoglobulin Fc regions. Included in the human immunoglobulin Fc region are mutations that alter Fc-mediated effector functions, including a combination of one or more of CDC activity, ADCC activity, ADCP activity. The immunoglobulin may be selected from the group consisting of IgG, IgA1, IgA2, IgD, IgE, IgM, and the like, and in particular the IgG may be selected from the group consisting of one or more of the IgG1, IgG2, IgG3, or IgG4 subtypes, and the like. Inclusion of an immunoglobulin Fc region in an antibody fusion protein can allow the fusion protein to form dimers while extending the in vivo half-life of the fusion protein and increasing Fc-mediated associated activity. In a specific embodiment of the invention, the immunoglobulin Fc region may be the Fc region of human IgG1, more specifically may be a wild-type IgG1 Fc sequence, which may be introduced with mutations that alter Fc-mediated effector functions, e.g., a) mutations that alter Fc-mediated CDC activity; b) a mutation that alters Fc-mediated ADCC activity; a mutation that alters Fc-mediated ADCP activity. Such mutations are described in the following documents: leonard G Presta, Current Opinion in Immunology 2008, 20: 460-.
In the fusion protein of the anti-FOLR 1 antibody provided by the invention, a connecting peptide can be arranged between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain consisting of alanine (a) and/or serine (S) and/or glycine (G), and may be 3 to 30 amino acids in length, preferably 3 to 9, 9 to 12, 12 to 16, 16 to 20, and in another embodiment of the present invention, may be 8 or 15 in length.
In a specific embodiment of the invention, the inventors humanize murine antibody Mov19, removing the murine sequences outside the CDRs of the antibody. Although the humanized Mov19 has no difference in affinity with the murine Mov19, the humanized Mov19 is significantly superior to the murine Mov19 in functions such as thermostability, ADCC (ADCC) and endocytosis. The inventor couples the humanized Mov19 with Monomethyl auristatin (MMAE) to prepare an antibody coupling medicament, and the result shows that the antibody coupling medicament has remarkable killing effect on tumor cells.
The invention also provides an isolated polynucleotide encoding an antibody of the invention, or encoding a fusion protein as described, which polynucleotide may be RNA, DNA, cDNA or the like. Methods for providing such isolated polynucleotides will be known to those skilled in the art and may be, for example, prepared by automated DNA synthesis and/or recombinant DNA techniques, etc., or may be isolated from a suitable natural source.
Construct and antibody expression system
The invention also provides a construct comprising an isolated polynucleotide of the invention. The construction method of the construct should be known to those skilled in the art, and for example, the construct may be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc., and more particularly, may be constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector of the present invention is generally referred to various commercially available expression vectors and the like well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or other vectors. The vector may also include one or more control sequences operably linked to the polynucleotide sequence, which may include a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the amino acid sequence to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequences may also include a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is linked to the 3' terminus of the nucleotide sequence encoding the polypeptide, and any terminator which is functional in the host cell of choice may be used in the present invention.
In general, suitable vectors will comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, these promoters may be lac or trp promoters including, but not limited to, E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, methanol oxidase promoter of Pichia pastoris and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. The marker gene may be used to provide a phenotypic trait for selection of transformed host cells, and may include, but is not limited to, dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, tetracycline or ampicillin resistance for E.coli, and the like, for example. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence which, if inserted into the vector, will enhance transcription, the enhancer being a cis-acting element of DNA, usually about 10 to 300 base pairs, acting on the promoter to enhance transcription of the gene.
The invention also provides an antibody expression system, which comprises the construct or the exogenous polynucleotide integrated in the genome. Any cell suitable for expression of an expression vector may be used as a host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, and specifically including but not limited to E.coli, Streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf 9; CHO, COS, HEK293 cells, or Bowes melanoma cells. Methods for constructing such expression systems will be known to those skilled in the art and may be, for example, combinations including, but not limited to, one or more of microinjection, particle gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.
Immunoconjugates
The invention also provides an immunoconjugate comprising an antibody of the invention, or a fusion protein of the invention. The immunoconjugate typically further comprises a functional molecule attached (including but not limited to covalent attachment, coupling, attachment, adsorption) to the antibody live fusion protein, which functional molecule may be a molecule including but not limited to a detectable label, a cytotoxin, a radioisotope, a biologically active protein, a targeting tumor surface marker, a tumor suppressor molecule, a targeting immune cell surface marker, or the like, or combinations thereof.
The method of preparing the immunoconjugates will be known to those skilled in the art, for example, the antibodies and/or fusion proteins can be linked to functional molecules either directly or through a spacer of suitable length, either by chemical cross-linking or by genetically engineered fusion expression, to obtain the immunoconjugates.
For therapeutic purposes, it may be appropriate to combine therapeutic effector groups, e.g. radioactive groups, i.e. groups consisting of radioisotopes or radionuclides (e.g. radionuclides)3H、14C、15N、33P、35S、90Y、99Tc、111ln、123l、125l、131l、201TI、213Bi), toxins or cytotoxic groups such as cytostatic agents.
The immunoconjugate may comprise an antibody or fusion protein of the invention and a detectable label. Such detectable labels include, but are not limited to: fluorescent markers, chromogenic markers, protein tags; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, e.g., immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels are well known to those skilled in the art for detection/analysis/diagnostic techniques and/or methods known in the art.
The antibodies or fusion proteins of the invention can be coupled to a labeling group (labeled polypeptide) which can then be used, for example, for diagnosisThe purpose is broken. Suitable labeling groups may be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (e.g. fluorescent proteins such as GFP, RFP etc., dyes, rhodamine, fluorescein and its derivatives such as FITC, cyanine dyes), enzyme groups (e.g. horseradish peroxidase, alkaline phosphatase, beta-galactosidase), chemiluminescent groups, biotin groups, metal particles (e.g. gold particles), magnetic particles (e.g. with a magnetic containing magnetite (Fe) (Fe;)3O4) And/or maghemite (Fe)2O3) Core of (b), a predetermined polypeptide group, etc.
The immunoconjugate may comprise an antibody or fusion protein of the invention and a molecule that targets a surface marker of an immune cell. The molecules of the surface marker of the targeted immune cells can identify the immune cells, carry the antibody of the invention to reach the immune cells, and simultaneously, the antibody of the invention can target the immune cells to tumor cells, thereby utilizing the killing effect of the antibody of the invention and simultaneously triggering the immune cells to specifically kill the tumors.
The immunoconjugate may comprise an antibody or fusion protein of the invention and at least one tumor surface marker-targeting or tumor-inhibiting molecule. The tumor-inhibiting molecule may be an anti-tumor cytokine or an anti-tumor toxin. For example, the cytokine may be, but is not limited to: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, and the like. The tumor surface marker targeting molecules can, for example, act synergistically with the antibodies of the invention to more precisely target tumor cells.
The immunoconjugate may also be a Chimeric Antigen Receptor (CAR) that is expressible in immune cells. The "immune cell" and "immune effector cell" are used interchangeably and include: t lymphocytes, NK cells, NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises, connected in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises an antibody or fusion protein of the invention. The design of transmembrane and intracellular signaling regions based on CAR technology is well known in the art: for example, the transmembrane region is the transmembrane region of molecules such as CD8 and CD28, the intracellular signaling region is the intracellular signaling region of the continuous and costimulatory signaling molecules CD28, CD27, CD137, CD134, MyD88 and CD40 activated by the Immunoreceptor Tyrosine Activation Motif (ITAM) CD3 zeta chain or Fc epsilon RI gamma tyrosine. More specifically, for T lymphocytes, those in which the intracellular signaling region comprises only ITAMs are first generation CAR T lymphocytes, in which the chimeric antigen receptor portions are linked as follows: scFv-TM-ITAM, which can provoke cytotoxic effects against tumors; second generation CAR T lymphocytes incorporate the intracellular signaling regions of CD28 or CD137 (also known as 4-1BB) in which portions of the chimeric antigen receptor are linked as follows: scFv-TM-CD 28-ITAM or scFv-TM-/CD 137-ITAM; B7/CD28 or 4-1BBL/CD137 costimulation effect generated in an intracellular signal region causes the continuous proliferation of T lymphocytes, and can improve the level of cytokines such as IL-2 and IFN-gamma secreted by the T lymphocytes, and simultaneously improve the survival cycle and the anti-tumor effect of CAR T in vivo; third generation CAR T lymphocytes, in which the chimeric antigen receptor portions are linked as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM further improve the survival cycle of CAR T in vivo and its anti-tumor effect.
According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present invention may be an extracellular binding region, a transmembrane region and an intracellular signaling region, which are linked in this order, including: the antibody or fusion protein of the invention, CD8, and CD3 ζ; the antibodies or fusion proteins of the invention, CD8, CD137, and CD3 ζ; the antibody or fusion protein of the invention, the transmembrane region of the CD28 molecule (CD28a), the intracellular signaling region of the CD28 molecule (CD28b), and CD3 ζ; or an antibody or fusion protein of the invention, a transmembrane region of a CD28 molecule, an intracellular signaling region of a CD28 molecule, CD137 and CD3 ζ.
The chimeric antigen receptor is expressed on the surface of an immune effector cell, and the immune effector cell can have highly specific cytotoxicity on tumor cells expressing FOLR1 while utilizing the killing effect of the antibody.
As a preferred mode of the present invention, the antibody of the present invention can be linked to a tumor-inhibiting molecule, preferably an anti-tumor toxin, including a toxin that acts on tubulin, such as Monomethyl auristatin (monomethylauristatin) related compounds and derivatives thereof, maytansinoid (maytansinoid) related compounds and derivatives thereof; DNA-acting toxins such as duocarmycin, calicheamicin, Pyrrolobinadiazepines (PBDs), SN-38, Dxd and related compounds and derivatives thereof; and related compounds and derivatives thereof which act on other functions within the cell, such as metabolism, transcription, translation, signal transduction, and the like. The invention also includes analogs, isomers, precursors, etc. of these small molecule compounds as toxins.
Pharmaceutical composition and kit
The invention also provides a pharmaceutical composition comprising an anti-FOLR 1 antibody of the invention, or a fusion protein of an anti-FOLR 1 antibody of the invention, or an immunoconjugate of the invention.
Various pharmaceutically acceptable carriers in the art may also be included in the pharmaceutical composition. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and specifically may include, but are not limited to: buffers such as acetate, Tris, phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; chlorhexidine, benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum proteins, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; tonicity adjusting agents such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants, such as polysorbates; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEE, PLURONICS or polyethylene glycol (PEG). Pharmaceutical formulations for in vivo administration are generally sterile and methods for achieving sterility of the pharmaceutical formulations will be known to those skilled in the art and may be achieved, for example, by filtration through sterile filtration membranes. The pharmaceutical composition of the present invention may be in various dosage forms including, but not limited to, tablets, injections, lyophilizates, and the like.
The amount of the fusion protein and immunoconjugate in the pharmaceutical composition is generally an effective amount, which corresponds to an amount of the active ingredient that can be determined depending on the subject to be treated and the particular mode of administration. For example, the fusion protein and the immunoconjugate may be present in an amount ranging from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99% by total mass of the pharmaceutical composition.
The fusion proteins, immunoconjugates and pharmaceutical compositions of the invention can be administered as single active ingredients or in combination therapy, i.e. in combination with other agents. For example, the combination therapy may be the fusion protein, immunoconjugate, pharmaceutical composition in combination with at least one other anti-neoplastic agent. As another example, the combination therapy may be the use of the fusion protein, immunoconjugate, pharmaceutical composition in combination with antibodies targeting other tumor-specific antigens such as, but not limited to: a combination of one or more of an anti-EGFR antibody, an anti-VEGF antibody, an anti-HER 2 antibody, or an anti-Claudin 18a2 antibody, and the like, said inhibitor preferably may be a monoclonal antibody.
The invention also provides a detection kit comprising the antibody, fusion protein or immunoconjugate of the invention. The kit can also comprise the following components according to needs: containers, controls (negative or positive controls), buffers, adjuvants, etc., which can be selected by one skilled in the art as appropriate. Instructions for use may also be included in the kit to facilitate handling by those skilled in the art.
The invention further provides a detection method for detecting the FOLR1 protein by using the antibody, which comprises but is not limited to qualitative detection, quantitative detection and positioning detection. Specifically, the detection methods include, but are not limited to, immunofluorescence assays, immunohistochemistry, radioimmunoassays, and the like.
A method of detecting the presence or absence of FOLR1 protein in a sample can comprise: contacting the sample with an antibody of the invention; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of FOLR1 protein in the sample. The sample may be a cell and/or tissue sample; the sample may be fixed or dissolved in a medium; detecting the level of FOLR1 protein in the fixed or solubilized sample. In some embodiments, the test object may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is further conjugated with a fluorescent dye, chemical, polypeptide, enzyme, isotope, tag, or the like that is detectable or can be detected by other reagents.
Use of
The invention also provides the use of an antibody, fusion protein, immunoconjugate or pharmaceutical composition of the invention in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease associated with a cell expressing (or overexpressing) FOLR 1.
The "therapeutically effective amount" of the fusion proteins, immunoconjugates, pharmaceutical compositions provided herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or prevention of injury or disability due to disease affliction. For example, for the treatment of a FOLR 1-associated tumor (including, e.g., ovarian cancer, endometrial cancer, breast cancer, cervical cancer, etc.), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can be assessed by examining the ability to inhibit cell growth, which can be measured in vitro by assays well known to those skilled in the art. A therapeutically effective amount of the fusion protein, immunoconjugate, pharmaceutical composition is generally capable of reducing tumor size, or otherwise alleviating a symptom in a subject. One skilled in the art can select an appropriate therapeutically effective amount depending on the circumstances, for example, the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected. The prescription of treatment (e.g., determination of dosage, etc.) may be determined by a physician, and factors that are generally considered include, but are not limited to, the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Typically, but not necessarily, a "prophylactically effective amount" is typically lower than a "therapeutically effective amount" since the prophylactic dose is administered to the subject prior to the onset of the disease or at an early stage of the disease.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature.
Example 1 modification of the amino acid sequence of antibodies
The amino acid sequences of the variable regions of the heavy and light chains of Mov19 were from the NCBI database with the amino acid sequence of the heavy chain variable region at GenBank accession no: CAA68252, light chain variable region amino acid sequence GenBank accession No.: CAA 68253.
The inventors have performed an analysis of the variable regions of the Mov19 heavy and light chains using the IMGT method, wherein the CDR1, CDR2 and CDR3 regions that are determinative of antigen binding are shown in FIG. 1 and FIG. 2 as the dashed boxes labeled corresponding to Mov19_ VH and Mov19_ VL.
Through comprehensive analysis, the inventors carried out sequence modification on the heavy chain and the light chain of Mov19, which are mainly shown in Table 1.
TABLE 1
After the sequence transformation, the inventor obtains a humanized Mov19 monoclonal antibody, wherein the heavy chain variable region sequence of the monoclonal antibody is as follows:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYFMNWVRQAPGQGLEWIGRIHPYDGDTFYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRYDGSRAMDYWGQGTTVTVSS(SEQ ID NO: 1)
the light chain variable region sequence is as follows:
DIVLTQSPASLAVSPGQRATITCRASQSVSFAGTSLMHWYQQKPGQPPKLLIYRASNLEAGVPARFSGSGSGTDFTLTINPVEANDAANYYCQQSREYPYTFGGGTKLEIK(SEQ ID NO: 2)
the present inventors performed codon optimization of coding nucleic acids corresponding to the amino acid sequences of the heavy and light chain variable regions of Mov19 and humanized Mov19, respectively, and performed gene synthesis.
Meanwhile, the present inventors synthesized nucleic acids encoding the heavy chain constant region (GenBank accession No.: AXN93653.1) and the light chain constant region (GenBank accession No.: AWK57456.1) of human antibodies into full-length genes of Mov19 and humanized Mov19 heavy and light chains, respectively.
Then, the present inventors constructed the full-length gene on pCGS3(Merck) expression vector, transfected it into expihho cells for expression, and purified to obtain chimeric Mov19 and humanized Mov19 antibody proteins.
Example 2 affinity determination of humanized Mov19
1. ELISA detection
First, the present inventors tested the affinity of Mov19 and humanized Mov19 for FOLR1 using ELISA.
FOLR1-His protein (Acro) was adsorbed on an ELISA plate, and after blocking skim milk, antibodies to Mov19 and humanized Mov19 were added in a gradient and incubated at room temperature for 1 hour, and then unbound antibodies were washed off. Then, a secondary HRP-labeled goat anti-human Fc antibody (Jackson) was added, and after 1 hour of incubation at room temperature, the unbound secondary antibody was washed away. Finally, TMB is used for developing color, and an absorption value at 450nm is recorded by a microplate reader.
By analysis, the results are shown in fig. 3, and the affinity of humanized Mov19 for FOLR1 is similar to murine Mov19, indicating that the affinity of Mov19 for FOLR1 is not changed by the humanization procedure of the present invention.
2. Octet detection
Next, the inventors tested the affinity of Mov19 and humanized Mov19 to FOLR1 using Octet.
The results are shown in FIG. 4, where Mov19 had a KD of 3.11pM and humanized Mov19 had a KD of 3.44pM, indicating that the affinities of these two to FOLR1 are very similar. From the values of Ka and Kdis, the two are also very similar, indicating that the humanization procedure of the present invention did not alter the affinity of Mov19 for FOLR 1.
3. Flow cytometry detection
Next, the inventors examined the affinity of antibodies to Mov19 and humanized Mov19 for FOLR1 on the cell membrane using flow cytometry.
SK-OV-3 cell lines highly expressing FOLR1 were selected, and gradient diluted antibodies Mov19 and humanized antibodies Mov19 were added, and after incubation at 4 ℃ for 1 hour, unbound antibodies were washed away. Then, a secondary goat anti-human Fc antibody (Jackson) labeled with PE was added, and after incubation at 4 ℃ for 1 hour, the unbound secondary antibody was washed away. And finally detecting by using a flow cytometer.
By analysis, the results are shown in fig. 5, and the affinity of humanized Mov19 for FOLR1 on tumor cells is similar to murine Mov19, indicating that the humanization procedure of the present invention does not affect the affinity of Mov19 for FOLR1 on tumor cells.
Example 3 detection of antibody-dependent cell-mediated cytotoxicity (ADCC)
In this example, the inventors tested the ADCC function of Mov19 and humanized Mov19 against tumor cells.
A KB cell line highly expressing FOLR1 is taken as a target cell, 10000 cells per well are inoculated into a 96-well culture plate, and the culture is carried out overnight at 37 ℃. Mov19 and humanized Mov19 were diluted in a gradient and added to 96 well plates with target cells and incubated at 37 ℃ for half an hour. NK cells were isolated from human PBMC cells, and 10000 cells per well were added to a 96-well plate containing target cells and antibodies, and incubated at 37 ℃ for 20-24 hours. Finally, the antibody was tested for ADCC function using the LDH kit (Invitrogen).
The results are shown in figure 6, where both Mov19 and humanized Mov19 have significant ADCC function against tumor cells, with EC50 of Mov19 at 15.2pM and EC50 of humanized Mov19 at 4.811 pM.
The above results demonstrate that the humanized Mov19 has significantly stronger ADCC function.
Example 4 detection of antibody thermostability
In this example, the thermostability of antibodies to Mov19 and humanized Mov19 was tested using Dynamic Light Scattering (DLS) method.
Specifically, the present inventors examined light scattering when Mov19 and humanized Mov19 antibody were heated from 25 ℃ to 85 ℃ using a dls (wyatt) instrument, thereby obtaining values of polymerization temperature Tagg for Mov19 and humanized Mov 19.
As shown in FIG. 7, the Tagg value of Mov19 was 66.54 ℃ while that of humanized Mov19 was 78.85 ℃.
The above results demonstrate that the humanized Mov19 antibody has significantly higher thermostability.
Example 5 detection of the endocytosis Capacity of the antibodies
In this example, the ability of antibodies to Mov19 and humanized Mov19 to bind to FOLR1 on the cell membrane to undergo endocytosis was examined.
The inventor mixes 5 mug/ml of Mov19 and humanized Mov19 antibody with SK-OV-3 cell strain respectively, incubates for 1 hour at 4 ℃, washes away unbound antibody, then puts the cells into a 37 ℃ incubator for incubation, takes out samples at different time points of 0h, 0.5h, 1h and 2h respectively, and puts the samples at 4 ℃ for placement. After all time points samples were taken, a secondary goat anti-human Fc antibody (Jackson) labeled with PE was added to the samples and after 1 hour of incubation at 4 ℃ unbound secondary antibody was washed away. And finally detecting by using a flow cytometer.
Through analysis, the results are shown in fig. 8, the humanized Mov19 is endocytosed faster than murine Mov19, 10% of the antibody is obviously endocytosed at 0.5h, and the antibody which is endocytosed at 1h is also obviously better than murine Mov19, which indicates that the endocytosing capacity of the humanized Mov19 is better than that of murine Mov 19.
Example 6 lethality assay of tumor cells
In this example, humanized Mov19 antibody was prepared as an antibody conjugate drug and the killing effect on tumor cells was examined.
The inventor firstly incubates humanized Mov19 with TCEP (sigma), opens disulfide bonds among chains, then adds mc-vc-PAB-MMAE (medchexpress) for coupling, and finally purifies by a desalting column to remove redundant small molecule drugs, and the obtained antibody coupling drug is named humanized Mov 19-vc-MMAE. The KB cell line or T-47D cell line was inoculated into 96-well cell plates at 5000 cells per well and cultured overnight at 37 ℃. The humanized Mov19-vc-MMAE was diluted in a gradient with IgG-vc-MMAE as a negative control, added to 96-well plates containing KB cells or T-47D cells, respectively, and cultured at 37 ℃ for 5 days. The cell viability is detected by CellTiter-glo (Promega), so that the killing effect of the antibody coupling drug on the cells is obtained.
As shown in FIG. 9, it was shown that the humanized Mov19-vc-MMAE had a significant killing effect on KB cells expressing FLOR1 with an IC50 of 1.243nM, whereas the negative control IgG-vc-MMAE and the small molecule drug vc-MMAE alone had a certain killing effect on tumor cells at very high concentrations.
As shown in FIG. 10, it was shown that the humanized Mov19-vc-MMAE also had significant killing effect on FLOR1 expressing T-47D cells, while the negative control IgG-vc-MMAE had a certain killing effect on tumor cells at very high concentration.
In conclusion, the humanized Mov19 antibody prepared into the antibody conjugate drug can have a remarkable killing effect on the tumor cells expressing FLOR1, and the drug effect on treating the tumor patients with high FLOR1 expression is proved.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Claims (16)
1. An anti-folate receptor 1 antibody, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
2. The anti-folate receptor 1 antibody of claim 1, wherein said anti-folate receptor 1 antibody is a humanized antibody.
3. A polynucleotide encoding the anti-folate receptor 1 antibody of claim 1 or 2.
4. A construct comprising the polynucleotide of claim 3.
5. An antibody expression system comprising the construct or genome of claim 4 having integrated therein an exogenous polynucleotide of claim 3.
6. A method of making the anti-folate receptor 1 antibody of claim 1, comprising: expressing the antibody using the antibody expression system of claim 5 under conditions suitable for expression of the antibody.
7. An immunoconjugate, comprising: the antibody of claim 1; and a functional molecule attached thereto.
8. The immunoconjugate of claim 7, wherein the functional molecule comprises: cytotoxins, radioisotopes, biologically active proteins, molecules targeting tumor surface markers, molecules inhibiting tumors, molecules targeting surface markers of immune cells or detectable markers, extracellular hinge, transmembrane and intracellular signaling regions based on chimeric antigen receptor technology, or combinations thereof.
9. The immunoconjugate of claim 8, wherein the molecule targeting a tumor surface marker is an antibody or ligand that binds a tumor surface marker; or
The tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin.
10. The immunoconjugate of claim 8, wherein the detectable label comprises: fluorescent markers, chromogenic markers; or
The intracellular signaling region comprises: CD3 zeta chain, fcepsilon RI gamma tyrosine activation motif, intracellular signaling regions of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, CD 40; or
The transmembrane region comprises: the transmembrane region of CD8 or CD 28.
11. The immunoconjugate of claim 9, wherein the anti-tumor toxin comprises: toxins that act on tubulin; a toxin or derivative thereof which acts on DNA; a compound or derivative thereof that acts on intracellular metabolism, transcription, translation or signal transduction; or
The anti-tumor cytokines comprise: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, or variants thereof; or
The antibody that binds to a tumor surface marker is an antibody that recognizes an antigen other than folate receptor 1, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, Her3, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, ASGPR1 or CDH 16.
12. The immunoconjugate of claim 11, wherein the anti-tumor toxin is linked to the antibody of claim 1 via a linker comprising mc-vc-PAB; or
The tubulin-acting toxins include: monomethyl auristatin compounds or derivatives thereof, maytansinoid compounds or derivatives thereof.
13. A pharmaceutical composition, comprising: the antibody of claim 1 or the immunoconjugate of any one of claims 7 to 12.
14. Use of the antibody of claim 1, the immunoconjugate of any one of claims 7 to 12 or the pharmaceutical composition of claim 13 for the preparation of a formulation, kit or kit for the diagnosis or treatment of tumors.
15. The use according to claim 14, wherein the tumour is a tumour expressing folate receptor 1.
16. A kit or kit comprising an antibody according to claim 1, or an immunoconjugate according to any one of claims 7 to 12, or a pharmaceutical composition according to claim 13.
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