WO2023045859A1 - Cd38单克隆抗体及其应用 - Google Patents

Cd38单克隆抗体及其应用 Download PDF

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WO2023045859A1
WO2023045859A1 PCT/CN2022/119598 CN2022119598W WO2023045859A1 WO 2023045859 A1 WO2023045859 A1 WO 2023045859A1 CN 2022119598 W CN2022119598 W CN 2022119598W WO 2023045859 A1 WO2023045859 A1 WO 2023045859A1
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seq
amino acid
acid sequence
antigen
monoclonal antibody
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PCT/CN2022/119598
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English (en)
French (fr)
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马梵辛
李庆
黄莺
张贤达
陈鹏宇
杨周宁
刘爽
屈凤
陈春梅
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非同（成都）生物科技有限公司
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Priority to AU2022350815A priority Critical patent/AU2022350815A1/en
Publication of WO2023045859A1 publication Critical patent/WO2023045859A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present disclosure relates to a monoclonal antibody binding to CD38, especially a humanized monoclonal antibody against human CD38, an antigen-binding fragment thereof, a related pharmaceutical composition and its use for treating diseases.
  • CD38 is a type II transmembrane glycoprotein that plays a role in the regulation of cell migration, receptor-mediated adhesion through the interaction with CD31 or hyaluronic acid. CD38 also has extracellular enzyme activity, is involved in the production of nucleotide metabolites, and plays a role in controlling intracellular calcium storage.
  • CD38 is normally found on hematopoietic cells and is at low levels in solid tissues.
  • the expression of CD38 in hematopoietic cells depends on the differentiation and activation status of the cells. Under normal conditions, the expression level of CD38 in bone marrow and lymphocytes and some non-hematopoietic tissues is relatively low, and about 80% of resting NK cells and monocytes express CD38 at a low level, and various other blood cells The same is true for the types, including nodal germinal center lymphoblastoid cells, intrafollicular cells, dendritic cells, red blood cells, and platelets. In contrast, CD38 is also expressed on B cells, and particularly high levels of CD38 are expressed on normal plasma cells.
  • CD38 is expressed in a number of hematological malignancies such as Waldenström macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, NK cell leukemia, NK/T Lymphoma, plasma cell leukemia, chronic lymphocytic leukemia (CLL) and multiple myeloma (MM).
  • CD38 is particularly expressed at high levels in multiple myeloma (MM) cells, which makes CD38 a target for therapeutic antibodies targeting MM cell surface molecules.
  • CD38 antibodies may also have a therapeutic effect in other malignancies, such as small cell lung cancer, non-small cell lung cancer, lung bronchial epithelial carcinoma, breast cancer (evolved from malignant hyperplasia of the epithelial lining in breast ducts and lobules) ), tumors of the pancreas that arise from beta cells (insulinoma), tumors that develop from the epithelium in the intestine (such as adenocarcinoma and squamous cell carcinoma), prostate cancer, testicular seminoma, ovarian cancer and neuroblastoma.
  • malignancies such as small cell lung cancer, non-small cell lung cancer, lung bronchial epithelial carcinoma, breast cancer (evolved from malignant hyperplasia of the epithelial lining in breast ducts and lobules) ), tumors of the pancreas that arise from beta cells (insulinoma), tumors that develop from the epithelium in the intestine (such
  • CD38 expression is also associated with HIV infection.
  • Daratumumab (hereinafter referred to as “Dara” or “DARA” or “dara”) is the first CD38-targeting antibody that has been approved as a single drug or in combination for the treatment of multiple myeloma.
  • Sanofi's CD38 antibody Isatuximab (hereinafter referred to as "Isa”) has also recently been approved by the FDA for the treatment of relapsed/refractory multiple myeloma (R/R MM).
  • CD38 monoclonal antibodies that are undergoing clinical validation include TJ202/MOR202 (I-Mab/MorphoSys AG), SG301 (Shangjian Biologics), HLX15 (Henlius) and TAK-079 (Takeda). Therefore, it is necessary to provide more CD38 antibodies to meet clinical needs.
  • the present invention provides a monoclonal antibody binding to human CD38 or an antigen-binding fragment thereof, which comprises three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2, LCDR3, wherein: HCDR1
  • the amino acid sequence is shown in SEQ ID NO: 1 or a conservative substitution variant of one to three amino acids
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2 or a conservative substitution variant of one to three amino acids
  • HCDR3 The amino acid sequence is shown in SEQ ID NO: 6 or a conservative substitution variant of one to three amino acids
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 7 or a conservative substitution variant of one to three amino acids
  • LCDR2 The amino acid sequence is shown in SEQ ID NO: 9 or a conservative substitution variant of one to three amino acids
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11 or a conservative substitution variant of one to
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises amino acids as shown in SEQ ID NO: 12 or SEQ ID NO: 14 sequence or a variant thereof having at least 85% sequence identity; wherein VL comprises the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 21 or a variant thereof having at least 85% sequence identity.
  • the monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein (a) the heavy chain variable region VH comprises as shown in SEQ ID NO: 12 or a variant having at least 85% sequence identity thereof; the light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereof; or (b ) The heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant having at least 85% sequence identity therewith; the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 21 or a variant having at least 85% sequence identity therewith; or (c) the heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 14 or a variant having at least 85% sequence identity thereto; the light chain The variable region VL comprises the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • the Fc portion of the monoclonal antibody is modified to enhance binding to Fc ⁇ RIIIa (V) and/or Fc ⁇ RIIIa (F).
  • the Fc segment comprises the amino acid sequence set forth in SEQ ID NO: 27 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises, for example, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:
  • the heavy chain comprises, for example, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35 or its variant with at least 85% sequence identity body.
  • the antigen-binding fragment is selected from scFv, (scFv) 2 , Fab, Fab′, and F(ab′) 2 of a CD38 antibody. In some embodiments, the antigen-binding fragment forms part of a multispecific antibody, CAR-T, or BiTE.
  • the monoclonal antibody is of the IgG type. In some embodiments, the monoclonal antibody is of the IgGl, IgG2, IgG3 or IgG4 type. In some embodiments, the monoclonal antibody is of the IgG1 type. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof does not bind healthy human red blood cells. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of binding to healthy human PBMCs. In some embodiments, the affinity constant Kd of the monoclonal antibody or antigen-binding fragment thereof determined by Biacore is on the order of 10 ⁇ 9 M.
  • the monoclonal antibody or antigen-binding fragment thereof binds to a different epitope than daratumumab.
  • the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof, which competes with any of the above-mentioned monoclonal antibodies or antigen-binding fragments provided in the present disclosure for binding to human CD38.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that competes with FTL004-1, FTL004-6, or FTL004-7 or antigen-binding fragments thereof provided herein for binding to human CD38.
  • the present invention provides a pharmaceutical composition for treating CD38-positive cancer, which comprises the monoclonal antibody or antigen-binding fragment thereof described in the present disclosure, and a pharmaceutically acceptable carrier.
  • the cancer is a blood cancer.
  • the hematological cancer is selected from multiple myeloma (eg, relapsed/refractory multiple myeloma (R/R MM)), leukemia, or lymphoma.
  • the leukemia is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), young myelomonocytic Cellular leukemia (JML), adult T-cell lymphocytic leukemia (ATL) and plasma cell leukemia; said lymphoma is selected from the group consisting of small lymphocytic lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, follicular lymphoma, Mantle cell lymphoma, diffuse large cell B-cell lymphoma, and Burkitt lymphoma.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • JML young myelomonocytic Cellular leukemia
  • ATL adult T-
  • the cancer is a solid tumor.
  • the solid tumor is selected from the group consisting of melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophagus or gastrointestinal tract cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary tract cancer, endometrial cancer, cervical cancer, Lung adenocarcinoma, renal cell carcinoma (RCC), mesothelioma, nasopharyngeal carcinoma (NPC), esophagus and gastrointestinal tract.
  • NSCLC non-small cell lung cancer
  • NSCLC non-squamous NSCLC
  • colorectal cancer prostate cancer
  • gastric cancer ovarian cancer
  • liver cancer pancreatic cancer
  • the pharmaceutical composition further comprises a second therapeutic agent for the treatment of the same cancer.
  • the second therapeutic agent is a chemotherapeutic, radiotherapeutic, or biological agent.
  • the biologic is a monoclonal antibody, ADC, oncolytic virus, or CAR-T therapy.
  • the present disclosure also provides a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the present disclosure also provides a vector comprising the aforementioned nucleotide sequence.
  • the present disclosure also provides a non-human host cell comprising the aforementioned vector.
  • the present disclosure also provides cell lines that produce the monoclonal antibodies of the present disclosure or antigen-binding fragments thereof, recombinant expression vectors comprising the nucleotides of the present disclosure, and methods for producing antibodies by culturing antibody-producing cell lines.
  • the present disclosure also provides the application of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for treating CD38-positive cancer.
  • the present invention also provides a method for treating CD38-positive cancer in a subject, which comprises administering a therapeutically effective amount of any one of the monoclonal antibodies or antigen-binding fragments thereof of the present invention or the pharmaceutical composition of the present invention. Either one is administered to the subject. In some embodiments, the method further comprises administering the second therapeutic agent to the subject simultaneously or in any order.
  • Figure 1 Apoptotic activity of H2 antibody on Daudi, Ramos, Raji and SU-DHL-1 cells.
  • Figure 3 Elisa method to detect the cross-reactivity of antibodies to different species of CD38 antigenic proteins.
  • Figure 4 Apoptotic activity test of humanized antibody and positive control on Ramos and Raji cells.
  • FIG. 10 Tumor growth curves of different concentrations of FTL004-1, FTL004-7, FTL004-hu-1, Isa and FTL004-1+Dara.
  • FIG. 1 The tumor inhibition rate of different concentrations of FTL004-1, FTL004-7, FTL004-hu-1, Isa and FTL004-1+Dara.
  • Figure 14 Detection of CD38 antibody binding to H211 and SU-DHL-6 cells by flow cytometry.
  • FIG. 16 Flow cytometry detection of the binding reaction of CD38 antibody Dara and FTL004-1 antibody to normal human PBMC.
  • the term "patient” or “subject” refers to any organism to which a provided antibody, antigen-binding fragment thereof, or pharmaceutical composition is administered, for experimental, diagnostic, prophylactic, cosmetic and / or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates and/or humans). In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. A patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions (eg, cancer/tumor). In some embodiments, the patient is receiving or has received a therapy for the diagnosis and/or treatment of such a disease, disorder or condition.
  • animals eg, mammals such as mice, rats, rabbits, non-human primates and/or humans.
  • the subject is a human.
  • the subject suffers from or is susceptible to one or more disorders or conditions.
  • a patient may exhibit one or more symptoms of a disorder or condition
  • the term "comparable” means that two or more agents, entities, situations, effects, sets of conditions, etc., may not be identical to each other, but are similar enough to allow comparison therebetween (e.g., by level and/or or activities), from which conclusions can be reasonably drawn based on observed differences or similarities.
  • These comparable sets of conditions, actions, situations, individuals or populations are characterized by a number of generally consistent features and one or a few features that vary. Those of ordinary skill in the art will appreciate that in any given context, what degree of identity is required for two or more such agents, entities, situations, sets of conditions, actions or populations, etc., may be considered for quite.
  • composition or method described herein as “comprising” one or more of the described elements or steps is open-ended, meaning that the described elements or steps are required but may be within the scope of the described composition or method Add other components or steps. It is also to be understood that any composition or method described as “comprising” one or more of said elements or steps also describes a corresponding, more limited composition or method “consisting essentially of” said elements or steps, meaning The composition or method is intended to include the essential elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.
  • an epitope refers to a portion of an antigen to which an antibody or antigen-binding fragment binds.
  • an epitope may be a conformational epitope, ie, a portion of an antigen that is comprised in an antigen that is not covalently adjacent but that is close to each other in three-dimensional space when the antigen is in the relevant conformation.
  • a conformational epitope is an epitope comprised of noncontiguous amino acid residues contained in the extracellular domain of CD38.
  • the epitope may be a linear epitope, ie, an epitope comprising a sequence of amino acid residues contiguous in the primary structure in the extracellular domain of CD38.
  • Means for determining the exact sequence and/or in particular the amino acid residues of an epitope of CD38 are known in the literature and include competing binding of peptides from antigenic sequences to CD38 sequences from different species, truncation and/or inducing mutagenesis (for example by alanine scanning or other site-directed mutagenesis), phage display-based screening or (co)crystallographic techniques.
  • the term “antibody” refers to any form of antibody that exhibits a desired biological activity, such as inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling.
  • Antibody fragment and “antigen-binding fragment” refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable region (eg, one or more CDRs) of a parent antibody.
  • the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, each monoclonal antibody is directed against only a single determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations which usually comprise a number of different antibodies directed against a number of different determinants (epitopes).
  • the modifier "monoclonal” indicates the identity of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be prepared by any particular method.
  • monoclonal antibodies used in the present invention can be produced by hybridoma or recombinant DNA methods.
  • Monoclonal antibodies can include "chimeric" antibodies, humanized antibodies, or fully human antibodies.
  • antibodies form part of larger biomolecules, such as fusion proteins or antibody drug conjugates.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent-associated activity when the activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody for the target.
  • antibody fragments include, but are not limited to: Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as sc-Fv; Nanobodies; Domain antibodies; and multispecific antibodies (such as bispecific antibodies), CAR-T and BiTE formed from antibody fragments.
  • a "Fab fragment” consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab'fragment” contains one light chain and part of one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby the two heavy chains of the two Fab' fragments can Interchain disulfide bonds are formed to form F(ab') 2 molecules.
  • a "F(ab') 2 fragment” contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains.
  • the F(ab') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • An "Fv region” comprises variable regions from both heavy and light chains, but lacks constant regions.
  • single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides contain an additional polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Fc or “Fc fragment” or “Fc region” refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain, and in some cases, excluding part of the hinge.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminus of these domains.
  • amino acid modifications are made to the Fc region, eg, to alter binding to one or more FcyR receptors or FcRn receptors.
  • humanized antibody refers to an antibody comprising the CDRs of an antibody derived from a mammal other than human, and the framework regions (FR) and constant regions of a human antibody.
  • sequence “variant” refers to a sequence that differs from the shown sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.
  • a comparison of sequences and determination of % identity between two sequences can be accomplished using a mathematical algorithm.
  • “Variants having at least 85% sequence identity” refer to biological activity and function identical or substantially similar to the sequence shown (such as amino acid sequence) but having about 85%, about 90% sequence identity with the sequence shown A sequence that is about 95%, about 96%, about 97%, about 98%, or about 99% identical.
  • Constant substitutions refer to amino acid substitutions known to those skilled in the art that are made without generally altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially or substantially alter biological activity. "Not changed or not substantially changed” means that when measured by the same or similar method, one or more aspects have no more than about 20%, about 15%, about 10%, about 9%, compared to the object being compared. %, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • Constant substitution variant of one to three amino acids means that the variant has one to three (e.g., one to two, two to three, one, two or three, The same or different) amino acid substitutions are known to those skilled in the art, and such substitutions generally do not alter or do not substantially alter the biological activity of the resulting molecule.
  • each CDR may be replaced by a conservative substitution variant of one to three amino acids
  • each CDR is independently conservatively substituted by one to three amino acids.
  • LCDR2 may be replaced by one amino acid conservative substitution variant
  • HCDR2 may be replaced by two amino acid conservative substitution variants
  • HCDR1 and LCDR3 may not be substituted at all.
  • HCDR3 and LCDR3 are considered to occupy more important roles than other CDRs in antigen recognition, therefore, in the case of substitutions, the present disclosure prefers conservative substitutions for CDRs other than HCDR3 and LCDR3.
  • HCDR1, HCDR3 and LCDR3 are not substituted.
  • Preferred amino acid substitutions include, but are not limited to: (1) reducing proteolytic susceptibility, (2) reducing oxidation susceptibility, (3) altering binding affinity for forming protein complexes, (4) providing or modifying other physical properties of these analogs. Substitution of chemical or functional properties.
  • Analogs may include mutations in various sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in portions of the polypeptide other than the regions where intermolecular contacts are formed). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parental sequence (eg, amino acid substitutions should not tend to disrupt helices present in the parental sequence, or characterize other types of secondary structure that disrupt the parental sequence).
  • the binding domain of the monoclonal antibody or antigen-binding fragment thereof of the present invention may have a signal peptide, which is usually located at the N-terminal of the secreted protein and generally consists of 15-30 amino acids.
  • SRP signal recognition particle
  • protein synthesis is suspended or slowed down, the signal recognition particle carries the ribosome to the endoplasmic reticulum, and protein synthesis resumes.
  • the newly synthesized protein enters the cavity of the endoplasmic reticulum, and the signal peptide sequence is excised under the action of signal peptidase.
  • termination transit sequence exists at the C-terminus of the nascent peptide chain, it may not be excised by the signal peptidase, such as ovalbumin contains an internal signal peptide. Neither its precursor nor its mature form is cleaved by signal peptidases.
  • Specific binding when referring to a ligand/receptor, antibody/antigen or other binding pair, refers to a binding reaction that determines the presence or absence of said protein in a heterogeneous population of proteins and/or other biological agents. Thus, under specified conditions, a specific ligand/antigen binds to a specific receptor/antibody and does not bind in significant amounts to other proteins present in the sample. "Specific binding” means that the monoclonal antibody or antigen-binding fragment thereof of the present invention can specifically bind to at least two, three, four, five, six, seven, eight or More amino acid interactions.
  • the "specific binding" of an antibody is mainly characterized by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength).
  • Antibody binding epitopes can be determined by FACS, peptide spot epitope mapping, mass spectrometry or peptide ELISA.
  • Biacore method and/or ELISA method can measure the binding strength of an antibody to a specific epitope.
  • the signal-to-noise ratio is usually calculated as a representative measure of binding specificity. In such a signal-to-noise ratio, the signal represents the strength of the antibody binding to the target epitope, while the noise represents the strength of the antibody binding to other non-target epitopes.
  • the antibody under evaluation is considered to bind to the target epitope in a specific manner when the signal-to-noise ratio for the target epitope is about 50, ie "specifically binds".
  • An antigen-binding protein, including an antibody "specifically binds” an antigen if the antigen-binding protein, including an antibody, binds the antigen with high binding affinity as determined by the affinity constant (KD) value.
  • KD affinity constant
  • the affinity constant KD is lower than 10 ⁇ 9 M.
  • KD refers to the affinity constant for a particular antibody-antigen interaction.
  • “about” means that a value is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, depending in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation every practice in the art. Alternatively, “about” or “comprising essentially” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “comprising essentially” should be assumed to be within an acceptable error range for the specific value.
  • administering and “treating” are used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, it means the administration of an exogenous drug, therapeutic agent, diagnostic agent, or composition to the animal, human, subject, or Treatment, cells, tissues, organs or biological fluid contact.
  • administering can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting an agent with the cells and contacting the agent with a fluid, wherein the fluid contacts the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, for example, by reagents, diagnostics, binding compositions or by other cells.
  • the term “inhibiting” or “treating” includes delaying the development of symptoms associated with a disease and/or lessening the severity of those symptoms that are or are expected to develop with the disease. The term also includes alleviating pre-existing symptoms, preventing additional symptoms and alleviating or preventing the underlying cause of these symptoms. Accordingly, the term indicates that a beneficial outcome has been conferred on a vertebrate subject suffering from a disease.
  • the term "therapeutically effective amount” or “effective amount” refers to when the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 of the present invention is administered alone or in combination with another therapeutic agent to cells, When administered to a tissue or subject, an amount effective to prevent or slow down the disease or condition being treated.
  • a therapeutically effective dose further refers to an amount of the compound sufficient to cause relief of symptoms, such as treating, curing, preventing, or alleviating the associated medical condition, or increasing the rate of treatment, cure, prevention, or alleviation of the symptoms .
  • a therapeutically effective amount refers to that ingredient alone.
  • a therapeutically effective amount refers to a combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • a therapeutically effective amount will reduce symptoms by usually at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.
  • cancer malignant disease
  • tumor tumor-associated neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm phenotype characterized by apparently uncontrolled cell proliferation.
  • precancerous eg, benign
  • malignant premetastatic, metastatic, and non-metastatic cells.
  • the cancer of the invention may be associated with any CD38 expressing cancer.
  • the term "pharmaceutically acceptable" applied to a carrier, diluent or excipient for formulating a composition as disclosed herein means that the carrier, diluent or excipient must be compatible with The other ingredients of the composition are compatible and not deleterious to the recipient thereof.
  • a monoclonal antibody or antigen-binding fragment thereof binding to human CD38 of the present invention comprises three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2 , LCDR3, wherein: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1 or its conservative substitution variant of one to three amino acids; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2 or its conservative substitution variant of one to three amino acids The amino acid sequence of HCDR3 is shown in SEQ ID NO: 6 or its conservative substitution variant of one to three amino acids; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 7 or its one to three amino acids are conserved Sexual substitution variant; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9 or its conservative substitution variant of one to three amino acids; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11 or its one to three amino acid conservation Sexual substitution variant
  • a monoclonal antibody or antigen-binding fragment thereof binding to human CD38 of the present invention comprises three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2 , LCDR3, wherein: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1 or its conservative substitution variant of one to three amino acids; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2 or its conservative substitution variant of one to three amino acids
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 6; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 7 or a conservative substitution variant of one to three amino acids; the amino acid sequence of LCDR2 is shown in SEQ ID Shown in NO:9 or its conservative substitution variant of one to three amino acids; the amino acid sequence of LCDR3 is shown in SEQ ID NO:11.
  • a monoclonal antibody or antigen-binding fragment thereof binding to human CD38 of the present invention comprises three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2 , LCDR3, wherein: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2 or a conservative substitution variant of one to three amino acids; the amino acid sequence of HCDR3 is shown in SEQ ID Shown in NO:6; The amino acid sequence of LCDR1 is shown in SEQ ID NO:7 or its conservative substitution variant of one to three amino acids; The amino acid sequence of LCDR2 is shown in SEQ ID NO:9 or its one to three amino acids Conservative substitution variant; the amino acid sequence of LCDR3 is shown in SEQ ID NO:11.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2, LCDR3, wherein :
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:1;
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;
  • the amino acid sequence of HCDR3 is shown in Shown in SEQ ID NO:6;
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:7 or SEQ ID NO:8;
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:9 or SEQ ID NO:10;
  • the amino acid sequence of LCDR3 As shown in SEQ ID NO:11.
  • the complementarity determining region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: HCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HCDR2, whose amino acid sequence is shown in SEQ ID NO: 2; HCDR3, its amino acid sequence is shown in SEQ ID NO: 6; LCDR1, its amino acid sequence is shown in SEQ ID NO: 7; LCDR2, its amino acid sequence is shown in SEQ ID NO: 9; LCDR3, its amino acid sequence The sequence is shown in SEQ ID NO:11.
  • the complementarity determining region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: HCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HCDR2, whose amino acid sequence is shown in SEQ ID NO: 3; HCDR3, its amino acid sequence is shown in SEQ ID NO: 6; LCDR1, its amino acid sequence is shown in SEQ ID NO: 7; LCDR2, its amino acid sequence is shown in SEQ ID NO: 9; LCDR3, its amino acid sequence The sequence is shown in SEQ ID NO:11.
  • the complementarity determining region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: HCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HCDR2, whose amino acid sequence is shown in SEQ ID NO: 2; HCDR3, its amino acid sequence is shown in SEQ ID NO: 6; LCDR1, its amino acid sequence is shown in SEQ ID NO: 8; LCDR2, its amino acid sequence is shown in SEQ ID NO: 10; LCDR3, its amino acid sequence The sequence is shown in SEQ ID NO:11.
  • the complementarity determining region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: HCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HCDR2, whose amino acid sequence is shown in SEQ ID NO: 4; HCDR3, its amino acid sequence is shown in SEQ ID NO: 6; LCDR1, its amino acid sequence is shown in SEQ ID NO: 7; LCDR2, its amino acid sequence is shown in SEQ ID NO: 9; LCDR3, its amino acid sequence The sequence is shown in SEQ ID NO:11.
  • the complementarity determining region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: HCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HCDR2, whose amino acid sequence is shown in SEQ ID NO: 5; HCDR3, its amino acid sequence is shown in SEQ ID NO: 6; LCDR1, its amino acid sequence is shown in SEQ ID NO: 7; LCDR2, its amino acid sequence is shown in SEQ ID NO: 9; LCDR3, its amino acid sequence The sequence is shown in SEQ ID NO:11.
  • any of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is obtained according to the Kabat definition scheme.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to human CD38, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises HCDR1, HCDR2, HCDR3, and the light chain variable region (VL) comprises LCDR1, LCDR2, LCDR3, and said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are respectively combined with HCDR1, HCDR2 selected from any of the following groups , HCDR3, LCDR1, LCDR2, LCDR3 are the same:
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in any one of SEQ ID NO: 18, 19, 20 and 21 may be LCDR1, LCDR2 and LCDR3 of the variable region;
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain having an amino acid sequence as shown in any one of SEQ ID NO: 18, 19, 20 and 21 may be LCDR1, LCDR2 and LCDR3 of the variable region;
  • each set of CDRs is defined according to any one of the Kabat, Chothia, IMGT, Contact and AbM definition schemes. In a preferred embodiment of this aspect, wherein each set of CDRs is defined according to any one of the Chothia, IMGT, Contact and AbM definition schemes. In a preferred embodiment of this aspect, wherein each set of CDRs is defined according to the Kabat definition scheme. In a preferred embodiment of this aspect, wherein each set of CDRs is defined according to the Chothia definition scheme. In a preferred embodiment of this aspect, wherein each set of CDRs is defined according to the IMGT definition scheme. In a preferred implementation of this aspect, each set of CDRs is defined according to the Contact definition scheme. In a preferred embodiment of this aspect, wherein each set of CDRs is defined according to the AbM definition scheme.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 of the present invention comprises: (1) VH, which comprises such as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
  • VH which comprises such as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 13 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 13 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 20 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 21 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 14 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 15 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 14 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 15 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 14 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 20 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 14 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 21 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 16 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 17 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 18 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 16 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 17 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 16 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 20 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 16 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 21 or a variant having at least 85% sequence identity thereto.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: VH comprising the amino acid sequence shown in SEQ ID NO: 23 or having at least 85% sequence identity therewith and a VL comprising the amino acid sequence shown in SEQ ID NO: 24 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 25,
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:33.
  • the Fc portion of any of the monoclonal antibodies described above is modified to enhance binding to Fc ⁇ RIIIa(V) and/or Fc ⁇ RIIIa(F).
  • the Fc portion of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 27 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof binding to human CD38 of the present invention comprises: (1) a heavy chain, which comprises such as SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO:31 or a variant having at least 85% sequence identity thereto; and (2) a light chain comprising the amino acid shown in SEQ ID NO:34 or SEQ ID NO:35 sequence or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 28 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 34 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 28 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 29 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 34 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 34 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human CD38 comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 or a variant having at least 85% sequence identity thereto and a light chain comprising the amino acid sequence shown in SEQ ID NO: 34 or a variant having at least 85% sequence identity thereto.
  • the monoclonal antibody is of the IgG type. In some embodiments, the monoclonal antibody is of the IgG1 type.
  • the monoclonal antibody or antigen-binding fragment thereof does not bind healthy human red blood cells. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to healthy human PBMCs. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof does not bind healthy human erythrocytes and is capable of binding healthy human PBMCs. In some embodiments, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22 but does not bind healthy human erythrocytes.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22 but does not bind to healthy human red blood cells, and is combined with any of the monoclonal antibodies or antigen-binding fragments provided by the present disclosure. Antigen-binding fragments compete for binding to human CD38. In some embodiments, the disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22 but does not bind healthy human erythrocytes and competes with FTL004-1, FTL004-6, or FTL004-7 Binds to human CD38.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22, but does not bind healthy human erythrocytes, and competes with FTL004-1 for binding to human CD38.
  • the affinity constant Kd of the monoclonal antibody or antigen-binding fragment thereof determined by Biacore is on the order of 10 ⁇ 9 M. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof has an affinity constant Kd determined by Biacore of 1.42x10 -9 M to 2.78x10 -9 M.
  • the monoclonal antibody or antigen-binding fragment thereof binds to SEQ ID NO:22. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to a different epitope than isatuximab. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds a different epitope than daratumumab. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds a different epitope than isatuximab and daratumumab.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22 and that binds a different epitope than Isatuximab and Daratumumab. In some embodiments, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22, binds a different epitope than isatuximab and daratumumab, and does not bind healthy human erythrocytes.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that competes with any of the monoclonal antibodies or antigen-binding fragments provided herein for binding to human CD38.
  • a monoclonal antibody or antigen-binding fragment thereof provided herein competes for binding to human CD38 with any antibody selected from: H2, FTL004-hu-1, FTL004-hu-2, FTL004-hu-3 , FTL004-hu-4, FTL004-hu-5, FTL004-hu-6, FTL004-hu-7, FTL004-hu-8, FTL004-hu-9, FTL004-hu-10, FTL004-hu-11, FTL004 -hu-12, FTL004-hu-13, FTL004-hu-14, FTL004-hu-15, FTL004-hu-16, FTL004-hu-17, FTL004-hu
  • a monoclonal antibody or antigen-binding fragment thereof provided herein competes with FTL004-1, FTL004-6, or FTL004-7 for binding to human CD38. In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof provided herein compete with FTL004-1 for binding to human CD38.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22, binds to a different epitope than Isatuximab and Daratumumab, but does not bind to healthy human erythrocytes, and binds to the present Any such monoclonal antibody or antigen-binding fragment provided publicly competes for binding to human CD38.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22, binds to a different epitope than Isatuximab and Daratumumab, but does not bind to healthy human erythrocytes, and binds to FTL004 -1.
  • FTL004-6 or FTL004-7 competes for binding to human CD38.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 22, binds to a different epitope than Isatuximab and Daratumumab, but does not bind to healthy human erythrocytes, and binds to FTL004 -1 competes for binding to human CD38.
  • antibodies can be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) is removed, eg, by centrifugation or ultrafiltration. When the antibody is secreted into the culture medium, the supernatant from the expression system is usually first concentrated using commercially available protein concentration filters such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF can be used in any of the preceding steps to inhibit proteolysis and antibiotics can be used to prevent the growth of adventitious contaminants.
  • protein concentration filters such as Amicon or Millipore Pellicon ultrafiltration units.
  • Protease inhibitors such as PMSF can be used in any of the preceding steps to inhibit proteolysis and antibiotics can be used to prevent the growth of adventitious contaminants.
  • glycoproteins may be purified by adsorption of glycoproteins to a lectin substrate (e.g., a lectin affinity column) to remove fucose-containing glycoproteins from the preparation and thereby enrich for Set fucose-free glycoproteins.
  • a lectin substrate e.g., a lectin affinity column
  • the polynucleotide encoding the heavy chain and/or light chain is inserted into an expression vector so that the gene can be manipulated linked to transcription and translation sequences.
  • Expression vectors are all vectors with which the person skilled in the art knows how to conveniently ensure the expression of said heavy and/or light chain.
  • the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons and exosomes.
  • the viral vector is selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vaccinia viruses, baculoviruses, and lentiviruses.
  • retroviruses adenoviruses
  • adeno-associated viruses herpes simplex viruses
  • vaccinia viruses vaccinia viruses
  • baculoviruses lentiviruses.
  • the present disclosure also provides nucleotide sequences encoding the monoclonal antibodies or antigen-binding fragments thereof of the present invention.
  • the nucleotide molecule encodes the heavy chain and/or light chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • a single nucleic acid encodes the heavy chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and another nucleic acid molecule encodes the light chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides a polynucleotide encoding a polynucleotide selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, A polypeptide of the amino acid sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35.
  • the polypeptide encoded by the polynucleotide of the present invention is selected from SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 14, 18, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, and 35.
  • the invention is not limited to said polynucleotides per se, but also includes all polynucleotides showing at least 80% identity to said polynucleotides.
  • the present invention provides a vector comprising a nucleotide sequence of the present invention.
  • the vector contains nucleotides encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the nucleotides encode the heavy chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the nucleotides encode the light chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the present invention also provides vectors comprising polynucleotide molecules encoding their fusion proteins, modified antibodies, antibody fragments and probes.
  • polynucleotides of the invention and vectors comprising these molecules can be used to transform appropriate mammalian host cells. Transformation can be performed by any known method for introducing polynucleotides into cellular hosts known to those skilled in the art.
  • Another aspect of the present disclosure provides a pharmaceutical composition for treating CD38-positive cancer, which comprises any monoclonal antibody or antigen-binding fragment thereof described above in the present disclosure, and a pharmaceutically acceptable carrier.
  • “Pharmaceutical composition” means a pharmaceutical preparation for use in humans.
  • the pharmaceutical composition comprises the human CD38-binding monoclonal antibody or antigen-binding fragment thereof of the present invention and suitable formulations of carriers, stabilizers and/or excipients.
  • the invention provides pharmaceutical formulations comprising the monoclonal antibodies or antigen-binding fragments thereof of the invention.
  • the pharmaceutical composition of the present invention comprises the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier.
  • To prepare pharmaceutical or sterile compositions antibodies or antigen-binding fragments thereof are admixed with pharmaceutically acceptable carriers or excipients.
  • Formulations of therapeutic and diagnostic drugs in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.
  • Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with immunosuppressants can be determined in cell culture or in experimental animals by standard pharmaceutical methods, such as for determining the LD50 (lethal to 50% of a population dose) and ED50 (dose effective in 50% of the treated population) approach.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • Suitable routes of administration include parenteral (eg intramuscular, intravenous or subcutaneous) and oral administration.
  • Antibodies for use in pharmaceutical compositions or for practicing the methods of the invention may be administered in a variety of conventional ways, such as by oral ingestion, inhalation, topical application or transdermally, subcutaneously, intraperitoneally, parenterally, intraarterially or Intravenous injection.
  • the conjugate compound of the invention is administered intravenously.
  • the conjugate compound of the invention is administered subcutaneously.
  • one can administer the antibody in a local rather than systemic manner usually a depot or sustained release formulation, eg, by injecting the antibody directly into the site of action. Additionally, one can administer the antibody in a targeted drug delivery system.
  • Appropriate dosages are determined by the clinician, eg, using parameters or factors known or suspected in the art to affect therapy, or expected to affect therapy. Generally, dosages will be started slightly lower than optimum and thereafter increased by small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important diagnostic measures include measuring eg inflammatory symptoms or levels of inflammatory cytokines produced.
  • Antibodies, antibody fragments and cytokines can be provided by continuous infusion or by dosing at intervals (eg, one day, one week, or 1-7 times per week).
  • the dose may be given intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally or by inhalation.
  • a preferred dosage regimen is one that includes the maximum dose or frequency of dosing that avoids significant undesirable side effects.
  • the total weekly dose is usually at least 0.05 ⁇ g/kg body weight, more usually at least 0.2 ⁇ g/kg, most usually at least 0.5 ⁇ g/kg, typically at least 1 ⁇ g/kg, more typically at least 10 ⁇ g/kg, most typically is at least 109 ⁇ g/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg, ideally at least 10 mg/kg, more ideally at least 25 mg/kg, and Most ideally at least 50 mg/kg.
  • a small molecule therapeutic such as a peptidomimetic, natural product or organic chemical agent is approximately the same as that of an antibody or polypeptide.
  • the pharmaceutical composition of the present invention may also contain other medicaments, including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenic drugs or antimetabolite drugs, targeted tumor drugs, immunostimulators or immunomodulators or in combination with cytotoxic agents, cell Antibodies conjugated to growth inhibitory or other toxic drugs.
  • the pharmaceutical composition may also be administered in conjunction with other treatment modalities such as surgery, chemotherapy, and radiation.
  • Typical veterinary, experimental or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses and humans.
  • a human CD38-binding monoclonal antibody or antigen-binding fragment thereof of the invention may be used in combination with a second therapeutic agent for the treatment of the same cancer.
  • the second therapeutic agent and the monoclonal antibody or antigen-binding fragment thereof of the invention are administered at substantially the same time.
  • An individual will sometimes receive a second therapeutic agent concurrently with a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the second therapeutic agent or other agents typically administered to cancer patients and the monoclonal antibody or antigen-binding fragment thereof of the present invention can be combined into a pharmaceutical composition; in other specific embodiments, both Apply separately.
  • the second therapeutic agent is any agent that is advantageously combined with an anti-CD38 antibody.
  • agents that may be advantageously combined with anti-CD38 antibodies include, but are not limited to, other agents that inhibit CD38 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or interfere with CD38 upstream or downstream Reagents for signal transduction.
  • the second therapeutic agent may be a chemotherapeutic, radiotherapeutic, or biological agent.
  • the biological agent can be monoclonal antibody, ADC, oncolytic virus or CAR-T.
  • Another aspect of the present disclosure provides the use of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for treating CD38-positive cancer.
  • Another aspect of the present disclosure provides a method for treating CD38-positive cancer with a monoclonal antibody or an antigen-binding fragment thereof or a pharmaceutical composition of the present invention, the method comprising administering a therapeutically effective amount of a monoclonal antibody or an antigen-binding fragment thereof of the present invention Any one of or any one of the pharmaceutical compositions of the present invention is administered to a subject.
  • the CD3 positive cancer of the invention is a hematological cancer or a solid tumor.
  • the blood cancers include, but are not limited to, multiple myeloma (MM), leukemia (acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), young myelomonocytic leukemia (JML), adult T-cell lymphocytic leukemia (ATL), plasma cell leukemia), and lymphomas (small lymphocytic lymphoma, lymphoplasmacytic lymphoma lymphoma, marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell B-cell lymphoma, and Burkitt lymphoma).
  • the hematological cancer is multiple myeloma (MM).
  • the hematological cancer is multiple myeloma (MM).
  • the solid tumors include, but are not limited to, melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, gastric cancer , ovarian cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal tract cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary tract cancer, endometrial cancer, cervical cancer Carcinoma, lung adenocarcinoma, renal cell carcinoma (RCC) (eg, clear cell renal cell carcinoma or renal papillary cell carcinoma), mesothelioma, nasopharyngeal carcinoma (NPC), esophageal or gastrointestinal tract carcinoma, or any of these of metastatic disease.
  • the solid tumor is lung cancer.
  • Embodiment 1 Preparation and screening of hybridomas
  • CD38 monoclonal antibody was prepared by hybridoma technology, the recombined CD38 ectodomain was mixed with Freund's incomplete adjuvant in equal volume, and BALB/c mice were immunized by intramuscular injection. After three basic immunizations, the tail vein blood was collected, and the antibody titer in the serum of the mice was detected by ELISA, and the immunization was impacted three days before the fusion.
  • the immunized mouse spleens were fused with SP2/0 in batches, and the fused cells were resuspended in HAT-containing medium and transferred to a 96-well plate, marked according to the number, and placed in the Cultivate in a 37°C cell incubator, replace the medium at the right time and observe the growth regularly. After 10-12 days of cultivation, take the supernatant for ELISA detection.
  • the recombinant CD38 extracellular segment was purchased from Universal Biotech (Wuhan) Technology Co., Ltd., Cat. No. ATMP00140HU, and its amino acid sequence is shown in the table below.
  • Antibody clones that can bind to CD38-positive cells were screened by ELISA and flow cytometry, and hybridoma cells that could bind to the extracellular segment of CD38 with high affinity were screened by SPR technology.
  • the apoptosis-inducing ability of the screened hybridoma was detected by Annexin V/PI cell apoptosis kit, and the hybridoma H2 was screened out.
  • H2 showed apoptotic activity on Daudi, Ramos, Raji and SU-DHL-1 four CD38-positive cells, and the apoptotic activity on Daudi, Ramos and Raji cells was significantly superior Compared with the positive control Dara, the apoptotic activity on SU-DHL-1 cells was comparable to that of the positive control Dara.
  • the results of Biacore affinity test show that the affinity constant of the H2 antibody is comparable to that of the positive control Dara, indicating that it has comparable binding affinity with Dara.
  • the H2 monoclonal antibody was sequenced, and the sequence is shown in Table 3 below.
  • H2 monoclonal antibody After coating the antigen CD38, first add different concentrations of H2 monoclonal antibody to fully incubate, then add Isa and Dara above the saturation concentration, and detect the combination of Isa and Dara. If the site where the test antibody binds to the CD38 protein has a certain occlusion or overlap with Isa or Dara, then the binding activity of Isa or Dara will be reduced. The results indicated that the H2 antibody binds different epitopes to Isa or Dara ( Figure 2).
  • CD38 antigen proteins The cross-reactivity of antibodies to different species of CD38 antigen proteins was detected by Elisa method. Coat CD38 antigen (1 ⁇ g/ml, 50ml/well) of different species (human, cynomolgus, mouse, rat) and incubate overnight at 2-8°C. Antibody concentration: 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41 ⁇ g/ml.
  • 18 humanized sequences (marked as FLT004-hu-1 to FLT004-hu-18, the specific sequences are shown in Tables 4 to 6 below) were designed with H2 monoclonal antibody as the parent, and According to the designed humanized sequence, the target sequence was constructed, and HEK293E was transiently transfected to express the chimeric antibody.
  • the apoptotic activity of the humanized antibody was detected, and some results are shown in Figure 4.
  • the results showed that the apoptosis-inducing activity of FTL004-hu-1/6/7 was the best, and it was superior to the positive control Isa in both Ramos and Raji cells; the apoptosis-inducing activity of FTL004-hu-13
  • the apoptotic activity was next, and showed comparable apoptosis-inducing activity to the positive control Isa on Ramos cells, and superior to the apoptosis-inducing activity of the positive control Isa on Raji cells.
  • the activity of the humanized antibody was detected by ELISA, and the results are shown in Figure 5.
  • a comprehensive comparison of the absorbance curve and the EC50 value shows that FTL004-hu-1/3/5/6/7/9 (respectively corresponding to the humanized-1/ 3/5/6/7/9) had better activity than the positive control Isa; FTL004-hu-11/12/13/15/17 (corresponding to humanized-11/12/13/15/ 17) is next in activity.
  • Oxidation stability Some samples with better activity were selected for oxidation stability test. The results are shown in the table below. It can be seen that the peak content of SEC fragments increased after 0.1% H 2 O 2 treatment for 6 hours and 24 hours. The results are shown in Table 9. Comprehensive comparison: Antibody FTL004-hu-1/2/6/7/13 has the best oxidation stability, and the increase of fragment peaks is small; Antibody FTL004-hu-5//9/11/15/17 is more stable Poor, fragment peaks increase more.
  • FTL004-hu-1/6/7 antibody has better activity and physical and chemical properties. All three were used for subsequent Fc mutation engineering.
  • mice NSG mice, 3-5 males per group; cell line: SU-DHL-6, subcutaneously transplanted (1 ⁇ 10 7 mice/0.2ml/mouse).
  • Dosage and frequency 0, 2.5, 10 mg/kg; Q2Wx8 (administration of 6 times in the low dose group); indicators: tumor volume, tumor weight and weight of liver and kidney organs.
  • the tumor inhibition rates of the experimental groups were as follows: Dara group was 12.96%; %; 29.63% for FTL004-hu-6; 42.59% for FTL004-hu-7. It can be seen that the tumor inhibition rates of FTL004-hu-1/6/7 were significantly better than the positive controls Dara and Isa. Through gross observation and weighing of liver and kidney ( Figure 8), it was found that Dara, Isa and FTL004-hu-1/6/7 had no obvious liver and kidney toxicity.
  • the Fc segment of an antibody interacts with many Fc receptors and ligands, and produces a series of biological effects.
  • the Fc receptor family - Fc ⁇ R plays a key role.
  • the formation of Fc/Fc ⁇ R complexes can effectively recruit corresponding effector cells, leading to intracellular signal transduction and a series of subsequent important immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis and Cytotoxic attack, etc.
  • Antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CDC) can be increased through Fc fragment engineering.
  • the Fc segment of the above-mentioned FTL004-hu-1/6/7 antibody was modified, and the modified FTL004-hu-1/6/7 antibody was named FTL004-1/6/7 respectively.
  • the 19th serine residue in the Fc segment was replaced with an aspartic acid residue (S19D)
  • the 112th isoleucine residue was replaced with a glutamic acid residue (I112E)
  • the 136th aspartic acid residue was replaced with a glutamic acid residue amino acid residue (D136E)
  • 138-position leucine residue was replaced by methionine residue (L138M)
  • glycine and lysine residues at positions 226 and 227 were deleted, and named as FTL004-1/6 /7
  • the N-terminus of the antibody sequence further includes a signal peptide as shown in SEQ ID NO: 32 (the specific sequence is shown in Table 12 below), and the activity and physical and chemical properties of the antibody before and after modification are compared.
  • the Biacore affinity test was carried out on the antibodies before and after Fc modification, and the results showed that there was no significant change in the binding affinity of the antibody after Fc mutation to the antigen.
  • the activity of the antibody inducing apoptosis of Romas, Daudi, Raji and SU-DHL-6 cells before and after Fc mutation was detected by the same method as above, and the results are shown in Table 14 below.
  • the antibodies before and after the Fc mutation had a certain ability to induce apoptosis on the four CD38 positive cells, and all of them were better than the positive control antibodies Dara and Isa.
  • Binding of antibodies to Fc gamma receptors following Fc mutation is detected using methods known in the art. The detection results are shown in Table 15 below, indicating that the antibody after Fc segment mutation shows increased binding to Fc ⁇ RIIIa (V) and Fc ⁇ RIIIa (F).
  • mice NSG mice, 3 in each group, male; cell line: SU-DHL-6, subcutaneously transplanted (1 ⁇ 107/0.2ml/mouse).
  • Dosage and frequency of administration 10, 30mg/kg; Q2Wx8; indicators: tumor volume, tumor weight and weight of liver and kidney organs.
  • 10mg/kg and 30mg/kg of FTL004-1, FTL004-7, FTL004-hu-1, 30mg/kg of Isa and FTL004-1+Dara both 10mg/kg were administered intraperitoneally (Q2Wx8).
  • the tumor inhibition rates of the experimental groups were as follows: 7.407% for the Isa 30mg/kg group; 7.407% for the FTL004-1 10mg/kg group ; FTL004-1 30mg/kg group was 18.518%; FTL004-7 10mg/kg group was 33.333%; FTL004-7 30mg/kg group was 18.518%; FTL004-hu-1 10mg/kg group was 25.925%; FTL004-hu -1 30mg/kg group was -18.518%; FTL004-hu-1 10mg/kg+Dara 10mg/kg group was -7.407%. Frozen tissue sections of the above tumor tissues were used to stain the positive tissues with the FTL004-1 antibody, and it can be seen that the FTL004 antibody was weakly positive ( FIG. 12 ).
  • NCG mice NCG mice, 4 in each group, male; cell line: NCI-H211, subcutaneously transplanted (1 ⁇ 10 7 /0.28ml/mouse).
  • Dosage and frequency of administration 30mg/kg (FTL004-1 and Isatuximab); Q2Wx8; indicators: tumor volume, tumor weight and liver and kidney organ weight.
  • Example 14 Binding experiment of CD38 antibody to erythrocytes and PBMC
  • Flow cytometry was used to detect the binding of CD38 antibody to rhesus monkey red blood cells.
  • Dara and Isa did not bind to monkey red blood cells, and FTL004-1 antibody had a certain binding EC50 value to monkey red blood cells. The value was about 2-5 ⁇ g/ml.
  • Flow cytometry was used to detect the binding of CD38 antibody to normal human red blood cells. It can be seen from Figure 15 that Dara has a high binding to healthy (normal) human red blood cells. Isa also has a certain binding to normal human red blood cells, but the binding is relatively weak. , FTL004-1 antibody does not bind to normal human erythrocytes. Flow cytometry was used to detect the binding of CD38 antibody to PBMC of healthy people. It can be seen from Figure 16 that both Dara and FTL004-1 antibodies (corresponding to 004-1 in Figure 16) had positive binding reactions to PBMC of normal people.

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Abstract

提供了结合至人CD38的单克隆抗体, 其抗原结合片段, 其药物组合物以及其在治疗CD38阳性的癌症中的应用。

Description

CD38单克隆抗体及其应用 技术领域
本公开涉及结合至CD38的单克隆抗体，特别是抗人CD38的人源化单克隆抗体，其抗原结合片段以及相关的药物组合物及治疗疾病的用途。
背景技术
CD38是一种II型跨膜糖蛋白，通过与CD31或透明质酸的相互作用在细胞迁移、受体介导的粘附的调节中发挥作用。CD38还具有胞外酶活性，并参与核苷酸代谢物的产生，和控制细胞内钙储存中发挥作用。
CD38通常在造血细胞上发现，在实体组织中处于低水平。CD38在造血细胞中的表达取决于细胞的分化和活化状态。在正常条件下，CD38在骨髓和淋巴细胞以及一些非造血组织中的表达水平相对较低，大约80％的静息NK细胞和单核细胞均以较低的水平表达CD38，各种其它血液细胞类型也是如此，包括淋巴结生发中心淋巴母细胞、滤泡内细胞、树突状细胞、红细胞和血小板。相比之下，CD38也在B细胞上表达，而在正常浆细胞表达特别高水平的CD38。
CD38在大量血液恶性肿瘤中表达，例如华氏巨球蛋白血症、原发性全身性淀粉样变性、套细胞淋巴瘤、急性成淋巴细胞性白血病、急性髓样白血病、NK细胞白血病、NK/T细胞淋巴瘤、浆细胞白血病、慢性淋巴细胞性白血病(CLL)和多发性骨髓瘤(MM)。CD38特别是在多发性骨髓瘤(MM)细胞中具有高水平的表达，这使得CD38成为靶向MM细胞表面分子的治疗性抗体的靶点。
此外，也有一些报道显示，CD38抗体可能在其他恶性肿瘤中也有治疗作用，例如小细胞肺癌、非小细胞肺癌、肺支气管上皮癌，乳腺癌(由乳腺导管和小叶中的上皮衬里的恶性增生演变而来)、由β细胞演变而来的胰腺肿瘤(胰岛素瘤)、由肠中的上皮演变而来的肿瘤(如腺癌和鳞状细胞癌)、前列腺癌、睾丸精原细胞瘤、卵巢癌和神经母细胞瘤。很多研究也提示了CD38在自身免疫中的作用，例如在Graves病和甲状腺炎、1型和2型糖尿病、以及在哮喘期间的气道平滑肌细胞的炎症。此外，CD38表达也与HIV感染相关。
Daratumumab(下文简称“Dara”或“DARA”或“dara”)是第一个CD38靶向抗体，已被批准作为单一药物或联合用药治疗多发性骨髓瘤。Sanofi公司的CD38抗体Isatuximab(下文简称“Isa”)近期也获FDA批准用于治疗复发性/难治性多发 性骨髓瘤(R/R MM)。正在进行临床验证的CD38单抗还包括TJ202/MOR202(天境生物/MorphoSys AG)、SG301(尚健生物)、HLX15(复宏汉霖)以及TAK-079(Takeda)。因此，有必要提供更多的CD38抗体以满足临床需求。
发明内容
本发明提供一种结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示或其一至三个氨基酸的保守性取代变体；HCDR2的氨基酸序列如SEQ ID NO:2所示或其一至三个氨基酸的保守性取代变体；HCDR3的氨基酸序列如SEQ ID NO:6所示或其一至三个氨基酸的保守性取代变体；LCDR1的氨基酸序列如SEQ ID NO:7所示或其一至三个氨基酸的保守性取代变体；LCDR2的氨基酸序列如SEQ ID NO:9所示或其一至三个氨基酸的保守性取代变体；LCDR3的氨基酸序列如SEQ ID NO:11所示或其一至三个氨基酸的保守性取代变体。
在一些实施方案中，所述单克隆抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中VH包含如SEQ ID NO:12或SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；其中VL包含如SEQ ID NO:18或SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述单克隆抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中(a)重链可变区VH包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体；或者(b)重链可变区VH包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体；或者(c)重链可变区VH包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述单克隆抗体的Fc段被修饰以增强与FcγRIIIa(V)和/或FcγRIIIa(F)的结合。在一些实施方式中，Fc段包含如SEQ ID NO:27所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述单克隆抗体或其抗原结合片段包含重链和轻链，其中重链包含如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30或SEQ ID NO:31所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34或SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述抗原结合片段选自CD38抗体的scFv、(scFv) 2、Fab、Fab′和F(ab′) 2。在一些实施方式中，所述抗原结合片段构成多特异性抗体、CAR-T或BiTE的一部分。
在一些实施方式中，所述单克隆抗体是IgG型。在一些实施方式中，所述单克隆抗体是IgG1、IgG2、IgG3或IgG4型。在一些实施方式中，所述单克隆抗体是IgG1型。在一些实施方式中，所述单克隆抗体或其抗原结合片段不结合健康人红细胞。在一些实施方式中，所述单克隆抗体或其抗原结合片段能与健康人PBMC结合。在一些实施方式中，所述单克隆抗体或其抗原结合片段通过Biacore测定的亲和力常数Kd为10 -9M级别。或其抗原结合片段与Isatuximab结合不同的抗原表位。在一些实施方式中，所述单克隆抗体或其抗原结合片段与Daratumumab结合不同的抗原表位。
另一方面，本公开提供一种单克隆抗体或其抗原结合片段，其与本公开提供的上述任一单克隆抗体或抗原结合片段竞争结合至人CD38。在一些实施方式中，本公开提供一种单克隆抗体或其抗原结合片段，其与本公开提供的FTL004-1、FTL004-6或FTL004-7或它们的抗原结合片段竞争结合至人CD38。
另一方面，本发明提供一种用于治疗CD38阳性癌症的药物组合物，其包含本公开所述的单克隆抗体或其抗原结合片段，以及药学上可接受的载体。在一些实施方式中，其中所述癌症是血液癌。在一些实施方式中，所述血液癌选自多发性骨髓瘤(例如复发性/难治性多发性骨髓瘤(R/R MM))、白血病或淋巴瘤。在一些实施方式中所述白血病选自急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓细胞性白血病(CML)、年轻型骨髓单核细胞性白血病(JML)、成人T细胞淋巴性白血病(ATL)和浆细胞白血病；所述淋巴瘤选自小淋巴细胞淋巴瘤、淋巴浆细胞淋巴瘤、边缘区淋巴瘤、滤泡型淋巴瘤、套细胞淋巴瘤、弥漫性大细胞B细胞淋巴瘤和伯基特淋巴瘤。
在一些实施方式中，所述癌症是实体瘤。在一些实施方式中，所述实体瘤选 自黑素瘤、肺癌、鳞状非小细胞肺癌(NSCLC)、非鳞状NSCLC、结肠直肠癌、前列腺癌、去势抵抗性前列腺癌、胃癌、卵巢癌、肝癌、胰腺癌、甲状腺癌、头颈部鳞状细胞癌、食道或胃肠道癌、乳腺癌、输卵管癌、脑癌、尿道癌、泌尿生殖道癌、子宫内膜癌、宫颈癌、肺腺癌、肾细胞癌(RCC)、间皮瘤、鼻咽癌(NPC)、食道癌和胃肠道癌。
在一些实施方式中，所述药物组合物进一步包含用于治疗相同癌症的第二治疗剂。在一些实施方式中，所述第二治疗剂是化疗剂、放疗剂或生物制剂。在一些实施方式中，所述生物制剂是单克隆抗体、ADC、溶瘤病毒或CAR-T疗法。
另一方面，本公开还提供一种编码本发明所述的单克隆抗体或其抗原结合片段的核苷酸序列。另一方面，本公开还提供一种包含前述核苷酸序列的载体。另一方面，本公开还提供一种包含前述载体的非人类的宿主细胞。此外，本公开还提供生成本公开的单克隆抗体或其抗原结合片段的细胞系，包含本公开的核苷酸的重组表达载体，以及通过培养抗体生产细胞系来制备抗体的方法。
另一方面，本公开还提供本发明所述的单克隆抗体或其抗原结合片段或药物组合物在制备用于治疗CD38阳性癌症的药物中的应用。
另一方面，本发明还提供一种治疗对象中的CD38阳性癌症的方法，其包括将治疗有效量的本发明单克隆抗体或其抗原结合片段中的任何一个或本发明的药物组合物中的任何一个施用至该对象。在一些实施方案中，所述方法还包含同时或按任何次序向所述对象施用所述第二治疗剂。
附图说明
图1.H2抗体在Daudi、Ramos、Raji和SU-DHL-1细胞上的细胞凋亡活性。
图2.Elisa法检测H2抗体与Isa及Dara竞争结合抗原表位的鉴定。
图3.Elisa法检测抗体对不同种属的CD38抗原蛋白的交叉反应。
图4.人源化抗体与阳性对照在Ramos和Raji两种细胞上的凋亡活性测试。
图5.ELISA法检测人源化CD38抗体的活性。
图6.人源化抗体FTL004-hu-1/6/7的肿瘤生长曲线。
图7.人源化抗体FTL004-hu-1/6/7的抑瘤率。
图8.人源化抗体FTL004-hu-1/6/7的肝肾毒性。
图9.Elisa法检测人源化抗体FTL004-1/6/7的活性变化。
图10.不同浓度的FTL004-1、FTL004-7、FTL004-hu-1、Isa以及FTL004-1+Dara的肿瘤生长曲线。
图11.不同浓度的FTL004-1、FTL004-7、FTL004-hu-1、Isa以及FTL004-1+Dara的抑瘤率。
图12.免疫组化检测肿瘤组织CD38的表达。
图13.CD38抗体对小细胞肺癌H211细胞的体内杀伤效果。
图14.流式细胞术检测CD38抗体对H211、SU-DHL-6细胞的结合。
图15.流式细胞术检测CD38抗体对正常人红细胞的结合反应。
图16.流式细胞术检测CD38抗体Dara和FTL004-1抗体对正常人的PBMC的结合反应。
具体实施方式
定义
如本文所使用，术语“患者”或“对象”是指被施用或可被施用所提供的抗体、其抗原结合片段或药物组合物的任何生物体，以用于实验、诊断、预防、化妆和/或治疗目的。典型的对象包括动物(例如哺乳动物，如小鼠、大鼠、兔、非人灵长类动物和/或人)。在一些实施方案中，对象是人。在一些实施方案中，对象患有或易患一种或多种病症或病况。患者可表现出病症或病况的一种或多种症状，或可能已被诊断患有一种或多种病症或病况(如癌症/肿瘤)。在一些实施方案中，患者正在接受或已接受用于诊断和/或治疗此类疾病、病症或病况的某种疗法。
如本文所使用，术语“相当的”是指两种或更多种药剂、实体、情况、作用、条件集合等彼此可能不一致，但具有足够相似性以允许在其间进行比较(例如通过水平和/或活性进行比较)，由此可基于所观察到的差异或相似性而合理地得出结论。这些相当的条件集合、作用、情况、个体或群体是由多个大体上一致的特征以及一个或少数变化的特征表征。本领域的普通技术人员应理解，在上下文中，在任何给定情况中两种或更多种此类药剂、实体、情况、条件集合、作用或群体等需要什么样的同一性程度可以被视为相当的。
本文中被描述为“包含”一个或多个所述元件或步骤的组合物或方法是开放式的，意味着所述元件或步骤是必需的，但是可以在所述组合物或方法的范围内添加其它元件或步骤。还应理解，被描述为“包含”一个或多个所述元件或步骤的任 何组合物或方法也描述“基本上由所述元件或步骤组成”的相应、更有限的组合物或方法，意味着所述组合物或方法包括所述必需元件或步骤，并且还可以包括不会实质上影响所述组合物或方法的基本和新颖特征的额外元件或步骤。
如本文所使用，术语“表位”是指抗体或抗原结合片段所结合的抗原的一部分。在一些实施方案中，表位可能是构象表位，即包含在抗原中虽未共价相邻但当该抗原呈相关构象时在三维空间中彼此接近的该抗原中的部分。例如，对于CD38，构象表位是包含在CD38胞外域中的、由不相邻的多个氨基酸残基构成的表位。在一些实施方案中，表位可能是线性表位，即包含CD38胞外域中在一级结构上相邻的一段氨基酸残基序列的表位。用于测定CD38的表位的确切序列和/或特别是氨基酸残基的方式是文献中已知的，包括与来自抗原序列的肽竞争结合至来自不同物种的CD38序列、截短和/或诱变(例如通过丙氨酸扫描或其它定点诱变)、基于噬菌体展示的筛选或(共)晶体学技术。
如本文中使用的，术语“抗体”是指表现所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。“抗体片段”和“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母抗体的抗原结合区或可变区(例如一个或多个CDR)。在一些实施方式中，抗体是单克隆抗体。在另一些实施方式中，抗体是多克隆抗体。本文所用术语“单克隆抗体”是指从基本上同种抗体群中获得的抗体，即除了可能少量存在的可能的天然突变体外，构成所述群的各个抗体是一致的。单克隆抗体具有高度特异性，可针对单个的抗原位点。此外，与通常包括针对多个不同的决定簇(表位)的多种不同抗体的常规(多克隆)抗体制备物相反，每种单克隆抗体仅针对抗原上的单个决定簇。修饰语“单克隆”表示从基本上同种抗体群获得的抗体的特性，不能理解为需要通过任何特定方法来制备所述抗体。例如，用于本发明的单克隆抗体可通过杂交瘤或重组DNA方法制备。单克隆抗体可以包括“嵌合”抗体、人源化抗体或全人源抗体。在一些实施方式中，抗体构成更大的生物分子的一部分，例如融合蛋白或抗体药物偶联物。抗体片段保留母抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。
因此，如本文使用的，抗体片段的例子包括但不限于：Fab、Fab′、F(ab′) 2和 Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；纳米抗体；结构域抗体；和由抗体片段形成的多特异性抗体(如双特异性抗体)、CAR-T和BiTE等。“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′) 2分子。“F(ab′) 2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab′) 2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单个多肽链中。一般而言，Fv多肽在VH和VL结构域之间包含额外的多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。
“Fc”或“Fc段”或“Fc区”是指多肽，其包含抗体的恒定区，排除第一恒定区免疫球蛋白域，并且在一些状况下，排除铰链的部分。因此，Fc是指IgA、IgD及IgG的最后两个恒定区免疫球蛋白域，IgE和IgM的最后三个恒定区免疫球蛋白域，以及这些结构域的柔性铰链N-末端。在一些实施方案中，对Fc区进行氨基酸修饰例如来改变与一个或多个FcγR受体或FcRn受体的结合。
如本文所用，术语“人源化的抗体”是指包含衍生自除人以外的哺乳动物的抗体的CDR、以及人抗体的框架区(FR)和恒定区的抗体。
如本文中使用的，序列“变体”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列。
本文所用的两个序列之间的“％同一性”是指所述序列共有的等同位置的数目的函数(即％同源性＝等同位置数/总位置数x 100)，其中会考虑到空位数目及各空位长度，所述空位需要在进行两个序列最佳比对时引入。序列比较和两个序列之间％同一性的确定可用数学算法来完成。
“具有至少85％序列同一性的变体”是指在生物学活性和功能上与所示序列(如氨基酸序列)相同或实质性相似但序列上与所示序列具有约85％、约90％、约95％、约96％、约97％、约98％或约99％同一性的序列。
“保守性取代”是指本领域技术人员已知的氨基酸取代，进行这种取代通常 不改变所得到的分子的生物学活性。一般而言，本领域技术人员公认在多肽非必需区的单个氨基酸取代基本上不改变或不实质性改变生物学活性。“不改变或不实质性改变”是指当以相同或相似方法测定时，与所比较的对象相比，一个或多个方面具有不超过约20％、约15％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％或约1％的差异。
“一个至三个氨基酸的保守性取代变体”是指与所示序列相比，变体具有一个至三个(例如一个至两个、两个至三个、一个、两个或三个，相同或不同的)本领域技术人员已知的氨基酸取代，并且进行这种取代通常不改变或不实质性改变所得到的分子的生物学活性。当每个CDR都可能被一个至三个氨基酸的保守性取代变体替代时，每个CDR独立地被一个至三个氨基酸保守性取代。例如，LCDR2被一个氨基酸保守性取代变体替代，HCDR2可被两个氨基酸保守性取代变体替代，HCDR1和LCDR3可不被任何取代。当有多个CDR涉及保守性取代时，所有取代总体上不改变或不实质性改变抗体分子的生物学活性。通常，HCDR3和LCDR3被认为在抗原识别中占据比其他CDR更重要的作用，因此，在取代的情况下，本公开优选对HCDR3和LCDR3以外的CDR进行保守性取代。在一些实施方式中，HCDR1、HCDR3和LCDR3不被取代。优选的氨基酸取代包括但不限于：(1)降低蛋白质水解敏感度，(2)降低氧化敏感度，(3)改变形成蛋白质复合物的结合亲和力，(4)提供或修饰这些类似物的其他物理化学或功能性能的取代。类似物可以包括除了天然存在的肽序列之外的各种序列的突变。例如，可以在天然存在的序列中(优选在形成分子间接触的区域之外的多肽部分中)进行单个或多个氨基酸取代(优选保守氨基酸取代)。保守氨基酸取代不应该实质上改变亲代序列的结构特征(例如氨基酸替代不应该倾向破坏在亲代序列中存在的螺旋，或者表征破坏亲代序列的其他二级结构类型)。
可以预期的是，本发明的单克隆抗体或其抗原结合片段的结合结构域可以带有信号肽，其通常位于分泌蛋白的N端，一般由15～30个氨基酸组成。当信号肽序列合成后，被信号识别颗粒(SRP)所识别，蛋白质合成暂停或减缓，信号识别颗粒将核糖体携带至内质网上，蛋白质合成重新开始。在信号肽的引导下，新合成的蛋白质进入内质网腔，而信号肽序列则在信号肽酶的作用下被切除。如终止转运序列存在于新生肽链的C端，也可以不被信号肽酶切除，如卵清蛋白含有内部信号肽。 它的前体与成熟形式都没有被信号肽酶切除的过程。
当提及配体/受体、抗体/抗原或其它结合对时，“特异性”结合是指在蛋白和/或其它生物试剂的异质群体中确定是否存在所述蛋白的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其它蛋白结合。“特异性结合”是指本发明的单克隆抗体或其抗原结合片段能够特异性地与各人靶标分子的至少两个、三个、四个、五个、六个、七个、八个或更多氨基酸相互作用。抗体的“特异性结合”主要由两个参数来表征：定性参数(结合表位或抗体结合位置)和定量参数(结合亲和力或结合强度)。抗体结合表位可通过FACS法、肽点表位作图法、质谱法或肽ELISA法测定。Biacore法和/或ELISA法可测定抗体与特定表位的结合强度。通常将信噪比作为结合特异性的代表性测定计算方法。在这样的信噪比中，信号代表抗体结合至目标表位的强度，而噪声代表抗体与其他非目标表位结合的强度。优选地，对于目标表位的信噪比为约50时可以认为所评估的抗体以特异性方式结合至目标表位，即“特异性结合”。如果抗原结合蛋白(包括抗体)以如通过亲和力常数(KD)值确定的高结合亲和力与抗原结合，则抗原结合蛋白(包括抗体)与抗原“特异性地结合”。在一些实施方式中，所述亲和力常数KD低于10 -9M。如本文使用的术语“KD”是指特定抗体-抗原相互作用的亲和力常数。
在本发明中，“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
当用“施用”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“施用”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“施用”和“治疗”还意味着例如通过试剂、诊 断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
如本文中所使用的，术语“抑制”或“治疗”包括延缓与疾病有关的症状的发展和/或减轻所述疾病将要或预期发展的这些症状的严重程度。所述术语还包括减缓已有症状、防止另外的症状和减缓或防止这些症状的潜在原因。因此，所述术语表示已将有益结果赋予患有疾病的脊椎动物对象。
如本文中所使用的，术语“治疗有效量”或“有效量”是指当将本发明的结合至人CD38的单克隆抗体或其抗原结合片段单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。
术语“癌症”、“恶性疾病”、“肿瘤”、“瘤”及“癌”在本文中可互换地使用，意思指展现相对较异常、不受控制和/或自主性生长，使其展现以明显失去控制的细胞增殖为特征的异常生长表型的细胞。一般来说，本申请中用于检测或治疗的所关注细胞包括癌变前(例如良性)、恶性、转移前、转移性以及非转移性细胞。本发明的癌症可与任何表达CD38的癌症相关。
如本文所使用，应用于配制如本文所公开的组合物的运载体、稀释剂或赋形剂的术语“药学上可接受”意思是指，所述运载体、稀释剂或赋形剂必须与该组合物的其它成分相容并且对其接受者无害。
抗CD38单克隆抗体
在本发明的一个方面，本发明的一种结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示或其一至三个氨基酸的保守性取代变体；HCDR2的氨基酸序列如SEQ ID NO:2所示或其一至三个氨基酸的保守性取代变体；HCDR3的氨基酸序列如SEQ ID NO:6所示或其一至三个氨基酸的保守性取代变体；LCDR1的氨基酸序列如SEQ ID NO: 7所示或其一至三个氨基酸的保守性取代变体；LCDR2的氨基酸序列如SEQ ID NO:9所示或其一至三个氨基酸的保守性取代变体；LCDR3的氨基酸序列如SEQ ID NO:11所示或其一至三个氨基酸的保守性取代变体。
在本发明的一个方面，本发明的一种结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示或其一至三个氨基酸的保守性取代变体；HCDR2的氨基酸序列如SEQ ID NO:2所示或其一至三个氨基酸的保守性取代变体；HCDR3的氨基酸序列如SEQ ID NO:6所示；LCDR1的氨基酸序列如SEQ ID NO:7所示或其一至三个氨基酸的保守性取代变体；LCDR2的氨基酸序列如SEQ ID NO:9所示或其一至三个氨基酸的保守性取代变体；LCDR3的氨基酸序列如SEQ ID NO:11所示。
在本发明的一个方面，本发明的一种结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示；HCDR2的氨基酸序列如SEQ ID NO:2所示或其一至三个氨基酸的保守性取代变体；HCDR3的氨基酸序列如SEQ ID NO:6所示；LCDR1的氨基酸序列如SEQ ID NO:7所示或其一至三个氨基酸的保守性取代变体；LCDR2的氨基酸序列如SEQ ID NO:9所示或其一至三个氨基酸的保守性取代变体；LCDR3的氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示；HCDR2的氨基酸序列如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5所示；HCDR3的氨基酸序列如SEQ ID NO:6所示；LCDR1的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8所示；LCDR2的氨基酸序列如SEQ ID NO:9或SEQ ID NO:10所示；LCDR3的氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的互补决定区包含：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:2所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1， 其氨基酸序列如SEQ ID NO:7所示；LCDR2，其氨基酸序列如SEQ ID NO:9所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的互补决定区包含：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:3所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:7所示；LCDR2，其氨基酸序列如SEQ ID NO:9所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的互补决定区包含：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:2所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:8所示；LCDR2，其氨基酸序列如SEQ ID NO:10所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的互补决定区包含：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:4所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:7所示；LCDR2，其氨基酸序列如SEQ ID NO:9所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的互补决定区包含：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:5所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:7所示；LCDR2，其氨基酸序列如SEQ ID NO:9所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
在上述的任一实施方式中，任一所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3根据Kabat定义方案得到。
在本发明的另一个方面，本公开提供结合人CD38的单克隆抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区(VH)包含HCDR1、HCDR2、HCDR3，轻链可变区(VL)包含LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3分别与选自以下任何一组的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3相同：
(a)氨基酸序列如SEQ ID NO:12所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18、19、20和21中任一个所示的轻链可变区的LCDR1、LCDR2和LCDR3；
(b)氨基酸序列如SEQ ID NO:13所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18或19所示的轻链可变区的LCDR1、LCDR2和LCDR3；
(c)氨基酸序列如SEQ ID NO:14所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18、20和21中任一个所示的轻链可变区的LCDR1、LCDR2和LCDR3；
(d)氨基酸序列如SEQ ID NO:15所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18所示的轻链可变区的LCDR1、LCDR2和LCDR3；
(e)氨基酸序列如SEQ ID NO:16所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18、19、20和21中任一个所示的轻链可变区的LCDR1、LCDR2和LCDR3；
(f)氨基酸序列如SEQ ID NO:17所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:18或19所示的轻链可变区的LCDR1、LCDR2和LCDR3；以及
(g)氨基酸序列如SEQ ID NO:23所示的重链可变区的HCDR1、HCDR2和HCDR3，和氨基酸序列如SEQ ID NO:24所示的轻链可变区的LCDR1、LCDR2和LCDR3。
在上述本发明的另一个方面，其中各组CDR根据Kabat、Chothia、IMGT、Contact和AbM定义方案中的任何一个定义。在该方面的优选的实施方式中，其中各组CDR根据Chothia、IMGT、Contact和AbM定义方案中的任何一个定义。在该方面的优选的实施方式中，其中各组CDR根据Kabat定义方案定义。在该方面的优选的实施方式中，其中各组CDR根据Chothia定义方案定义。在该方面的优选的实施方式中，其中各组CDR根据IMGT定义方案定义。在该方面的优选的实施方式中，其中各组CDR根据Contact定义方案定义。在该方面的优选的实施方式中，其中各组CDR根据AbM定义方案定义。
在本发明的另一个方面，本发明的结合人CD38的单克隆抗体或其抗原结合片段包含：(1)VH，其包含选自如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及(2)VL，其包含选自如SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20或SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:13所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:13所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:20所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:21所示的氨基酸序列或与其具有 至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:15所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:15所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:20所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:16所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:17所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:16所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:17所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:19所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:16所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:20所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:16所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方式中，所述结合人CD38的单克隆抗体或其抗原结合片段的可变区包含：VH，其包含如SEQ ID NO:23所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及VL，其包含如SEQ ID NO:24所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含重链恒定区和轻链恒定区，其中，重链恒定区包含如SEQ ID NO:25所示的氨基酸序列，轻链恒定区包含如SEQ ID NO:33所示的氨基酸序列。
在一些实施方案中，以上所述任一单克隆抗体Fc段被修饰以增强与FcγRIIIa(V)和/或FcγRIIIa(F)的结合。在一些实施方案中，所述单克隆抗体的Fc段包 含如SEQ ID NO:27所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在本发明的另一个方面，本发明的结合人CD38的单克隆抗体或其抗原结合片段包含：(1)重链，其包含如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30或SEQ ID NO:31所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及(2)轻链，其包含如SEQ ID NO:34或SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:28所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:28所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:29所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:30所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:30所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体。
在一些实施方案中，所述结合人CD38的单克隆抗体或其抗原结合片段包含：重链，其包含如SEQ ID NO:31所示的氨基酸序列或与其具有至少85％序列同一性的变体；以及轻链，其包含如SEQ ID NO:34所示的氨基酸序列或与其具有至 少85％序列同一性的变体。
在一些实施方案中，所述单克隆抗体是IgG型。在一些实施方案中，所述单克隆抗体是IgG1型。
在一些实施方案中，所述单克隆抗体或其抗原结合片段不结合健康人红细胞。在一些实施方案中，所述单克隆抗体或其抗原结合片段能与健康人PBMC结合。在一些实施方案中，所述单克隆抗体或其抗原结合片段不结合健康人红细胞并且能与健康人PBMC结合。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，但不结合健康人红细胞。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，但不结合健康人红细胞，并且与本公开提供的任一所述单克隆抗体或抗原结合片段竞争结合至人CD38。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，但不结合健康人红细胞，并且与FTL004-1、FTL004-6或FTL004-7竞争结合至人CD38。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，但不结合健康人红细胞，并且与FTL004-1竞争结合至人CD38。
在一些实施方案中，所述单克隆抗体或其抗原结合片段通过Biacore测定的亲和力常数Kd为10 -9M级别。在一些实施方案中，所述单克隆抗体或其抗原结合片段通过Biacore测定的亲和力常数Kd为1.42x10 -9M至2.78x10 -9M。
在一些实施方案中，所述单克隆抗体或其抗原结合片段结合至SEQ ID NO:22。在一些实施方案中，所述单克隆抗体或其抗原结合片段与Isatuximab结合不同的抗原表位。在一些实施方案中，所述单克隆抗体或其抗原结合片段与Daratumumab结合不同的抗原表位。在一些实施方案中，所述单克隆抗体或其抗原结合片段与Isatuximab和Daratumumab结合不同的抗原表位。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，并且与Isatuximab和Daratumumab结合不同的抗原表位。在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，与Isatuximab和Daratumumab结合不同的抗原表位，并且不结合健康人红细胞。
在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其与本公开提供的任一所述单克隆抗体或抗原结合片段竞争结合至人CD38。在一些实施 方案中，本公开提供的单克隆抗体或其抗原结合片段与选自以下任一抗体竞争结合至人CD38：H2、FTL004-hu-1、FTL004-hu-2、FTL004-hu-3、FTL004-hu-4、FTL004-hu-5、FTL004-hu-6、FTL004-hu-7、FTL004-hu-8、FTL004-hu-9、FTL004-hu-10、FTL004-hu-11、FTL004-hu-12、FTL004-hu-13、FTL004-hu-14、FTL004-hu-15、FTL004-hu-16、FTL004-hu-17、FTL004-hu-18、FTL004-1、FTL004-6和FTL004-7。在一些实施方案中，本公开提供的单克隆抗体或其抗原结合片段与FTL004-1、FTL004-6或FTL004-7竞争结合至人CD38。在一些实施方案中，本公开提供的单克隆抗体或其抗原结合片段与FTL004-1竞争结合至人CD38。
在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，与Isatuximab和Daratumumab结合不同的抗原表位，但不结合健康人红细胞，并且与本公开提供的任一所述单克隆抗体或抗原结合片段竞争结合至人CD38。
在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，与Isatuximab和Daratumumab结合不同的抗原表位，但不结合健康人红细胞，并且与FTL004-1、FTL004-6或FTL004-7竞争结合至人CD38。
在一些实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，其结合至SEQ ID NO:22，与Isatuximab和Daratumumab结合不同的抗原表位，但不结合健康人红细胞，并且与FTL004-1竞争结合至人CD38。
当使用重组技术时，抗体可产生在细胞内、周质空间或直接分泌到培养基中。若抗体在细胞内产生，则作为第一步，例如通过离心或超滤去除颗粒碎片(宿主细胞或裂解的片段)。当抗体分泌到培养基中时，通常首先用市售的蛋白质浓缩过滤器(例如Amicon或Millipore Pellicon超滤单元)浓缩来自所述表达系统的上清液。可在任何前述步骤中使用蛋白酶抑制剂(例如PMSF)以抑制蛋白水解，可使用抗生素以防止外来污染物生长。
根据待回收的抗体，还可利用其它蛋白质纯化技术，例如离子交换柱上分级分离、乙醇沉淀、反相HPLC、硅胶色谱法、阴离子或阳离子交换树脂(例如聚天冬氨酸柱)色谱法、色谱聚焦法、SDS-PAGE和硫酸铵沉淀。在一个实施方式中，糖蛋白可通过以下方法来纯化：使糖蛋白吸附到凝集素基底上(例如凝集素亲和柱)，以从制备物中去除含岩藻糖的糖蛋白并由此富集无岩藻糖的糖蛋白。
为了表达本发明所述的单克隆抗体或其抗原结合片段的重链和/或轻链，编码所述重链和/或轻链的多核苷酸被插入到表达载体中，以便基因被可操作地连接到转录和翻译序列。表达载体是本领域技术人员能够知道如何便利确保所述重链和/或轻链表达的所有载体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。本领域技术人员将意识到编码重链和轻链的多核苷酸能够被克隆到不同的载体中，或者在相同的载体中。在优选的实施方式中，所述多核苷酸被克隆在相同的载体中。
因此，本公开还提供了编码本发明所述的单克隆抗体或其抗原结合片段的核苷酸序列。在一个实施方式中，核苷酸分子编码本发明所述的单克隆抗体或其抗原结合片段的重链和/或轻链。在优选的实施方式中，单条核酸编码抗本发明所述的单克隆抗体或其抗原结合片段的重链，另一条核酸分子编码本发明所述的单克隆抗体或其抗原结合片段的轻链。
本发明的另一个方面提供多核苷酸，其编码具有选自SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34和35的氨基酸序列的多肽。在优选的实施方式中，本发明的多核苷酸编码的多肽选自SEQ ID NO：1、2、6、7、8、9、10、11、12、14、18、21、23、24、25、26、27、28、29、30、31、33、34和35。本发明不限于所述多核苷酸本身，还包括所有显示与所述多核苷酸至少80％同一性的多核苷酸。
本发明提供了含有本发明的核苷酸序列的载体。在一个实施方式中，该载体含有编码本发明所述的单克隆抗体或其抗原结合片段的核苷酸。在另一个实施方式中，所述核苷酸编码本发明所述的单克隆抗体或其抗原结合片段的重链。在另一个实施方式中，所述核苷酸编码本发明所述的单克隆抗体或其抗原结合片段的轻链。本发明还提供了包含编码其融合蛋白、修饰的抗体、抗体片段及探针的多核苷酸分子的载体。
本发明的多核苷酸和包含这些分子的载体能够被用于转化适当的哺乳动物宿主细胞。可以通过本领域技术人员公知的将多核苷酸引入细胞宿主的任何已知方 法进行转化。
药物组合物
本公开的另一个方面提供用于治疗CD38阳性癌症的药物组合物，其包含本公开以上所述任一单克隆抗体或其抗原结合片段，以及药学上可接受的载体。
“药物组合物”是指用于人的药物制剂。该药物组合物包含本发明的结合人CD38的单克隆抗体或其抗原结合片段以及载体、稳定剂和/或赋形剂的合适制剂。本发明提供包含本发明的单克隆抗体或其抗原结合片段的药物制剂。在一些实施方案中，本发明的药物组合物包含本发明的单克隆抗体或其抗原结合片段，以及药学上可接受的载体。为了制备药物组合物或无菌组合物，让抗体或其抗原结合片段与可药用载体或赋形剂混合。可通过与生理学上可接受的载体、赋形剂或稳定剂混合，来制备呈例如冻干粉、浆液、水溶液剂或混悬剂形式的治疗及诊断药物的制剂。
可通过标准药物方法，在细胞培养物或实验动物中测定单独给予或与免疫抑制剂联合给予的抗体组合物的毒性和治疗功效，所述方法例如为用于测定LD50(使群体的50％致死的剂量)和ED50(有效治疗群体的50％的剂量)的方法。毒性和治疗效果之间的剂量比是治疗指数，可表示为LD50与ED50之比。从这些细胞培养物测定及动物研究中获得的数据可用于调配用于人的剂量范围。所述化合物的剂量优选在包括毒性极少或无毒性的ED50的循环浓度范围内。可根据采用的剂型及所用的给药途径，使剂量在该范围内变化。
合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)及口服给药。可按多种常规方式给予用于药物组合物或用于实践本发明方法的抗体，这些方法例如有经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。在一个实施方式中，静脉内给予本发明的结合化合物。在另一个实施方式中，皮下给予本发明的结合化合物。或者，人们可以以局部而非全身方式(通常为长效制剂或缓释制剂)给予抗体，例如经由将抗体直接注射到作用位点。此外，人们可以在靶向药物递送系统中给予抗体。
由临床医生例如用本领域已知或怀疑影响治疗或预期影响治疗的参数或因子来测定合适的剂量。通常，开始剂量比最佳剂量稍低，此后少量增加直到达到相对于任何不良副作用所要的或最佳的作用效果。重要的诊断测量包括测量例如炎性症状或所产生的炎性细胞因子的水平。
可通过连续输注或通过以一定间隔(例如一天、一周或每周1-7次)给药来提供抗体、抗体片段和细胞因子。可通过静脉内、皮下、腹膜内、经皮肤、局部、经口、经鼻、经直肠、肌内、大脑内、脊柱内或通过吸入来提供剂量。优选剂量方案是包括避免显著的不合乎需要的副作用的最大剂量或给药频率的方案。周总剂量通常为至少0.05μg/kg体重，更通常为至少0.2μg/kg，最通常为至少0.5μg/kg，典型地为至少1μg/kg，更典型地为至少10μg/kg，最典型地为至少109μg/kg，优选为至少0.2mg/kg，更优选为至少1.0mg/kg，最优选为至少2.0mg/kg，理想地为至少10mg/kg，更理想地为至少25mg/kg，而最理想地为至少50mg/kg。基于摩尔/kg计算，小分子治疗剂例如肽模拟物、天然产物或有机化学药剂的所需剂量与抗体或多肽的剂量接近相同。
本发明药物组合物还可以含有其它药剂，包括但不限于细胞毒剂、细胞生长抑制剂、抗血管形成药物或抗代谢药物、靶向肿瘤药物、免疫刺激剂或免疫调节剂或与细胞毒剂、细胞生长抑制剂或其它毒性药物缀合的抗体。也可与其它治疗形式(例如手术、化疗及放射)一起施用所述药物组合物。典型的兽医、实验或研究对象包括猴、狗、猫、大鼠、小鼠、兔、豚鼠、马和人。
具体地，本发明的结合人CD38的单克隆抗体或其抗原结合片段可与用于治疗相同癌症的第二治疗剂组合使用。在具体实施例中，第二治疗剂及本发明的单克隆抗体或其抗原结合片段于实质上相同的时间施用。个体有时会同时使用第二治疗剂及本发明的单克隆抗体或其抗原结合片段。在一种实施方式中，该第二治疗剂或其他典型施用至癌症病患的药剂及本发明的单克隆抗体或其抗原结合片段可组合成药物组合物；在其他具体实施例中，两者分别施用。
第二治疗剂是有利地与抗CD38抗体组合的任一试剂。可有利地与抗CD38抗体组合的示例性试剂包括但不限于抑制CD38活性的其他试剂(包括其他抗体或其抗原结合片段、肽抑制剂、小分子拮抗剂等)和/或干扰CD38上游或下游信号转导的试剂。所述第二治疗剂可以是化疗剂、放疗剂或生物制剂。所述生物制剂可以是单克隆抗体、ADC、溶瘤病毒或CAR-T。
治疗方法及应用
本公开的另一方面提供了本发明的单克隆抗体或其抗原结合片段或药物组合物在制备用于治疗CD38阳性癌症的药物中的应用。
本公开的另一方面提供了用本发明的单克隆抗体或其抗原结合片段或药物组合物治疗CD38阳性癌症的方法，所述方法包括将治疗有效量的本发明单克隆抗体或其抗原结合片段中的任何一个或本发明的药物组合物中的任何一个施用至对象。
在一些实施方案中，本发明的CD3阳性癌症是血液癌症或实体瘤。在一些实施方案中，所述血液癌症包括但不限于多发性骨髓瘤(MM)、白血病(急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓细胞性白血病(CML)、年轻型骨髓单核细胞性白血病(JML)、成人T细胞淋巴性白血病(ATL)、浆细胞白血病)、以及淋巴瘤(小淋巴细胞淋巴瘤、淋巴浆细胞淋巴瘤、边缘区淋巴瘤、滤泡型淋巴瘤、套细胞淋巴瘤、弥漫性大细胞B细胞淋巴瘤和伯基特淋巴瘤)。在一些实施方案中，所述血液癌症是多发性骨髓瘤(MM)。在一些实施方案中，所述血液癌症是复发性/难治性多发性骨髓瘤(R/R MM)。
在一些实施方案中，所述实体瘤包括但不限于黑素瘤、肺癌、鳞状非小细胞肺癌(NSCLC)、非鳞状NSCLC、结肠直肠癌、前列腺癌、去势抵抗性前列腺癌、胃癌、卵巢癌、肝癌、胰腺癌、甲状腺癌、头颈部鳞状细胞癌、食道或胃肠道癌、乳腺癌、输卵管癌、脑癌、尿道癌、泌尿生殖道癌、子宫内膜癌、宫颈癌、肺腺癌、肾细胞癌(RCC)(例如、肾透明细胞癌或肾乳头状细胞癌)、间皮瘤、鼻咽癌(NPC)、食道癌或胃肠道癌或其任何一种的转移病变。在一些实施方案中，所述实体瘤是肺癌。
实施例
实施例1.杂交瘤的制备及筛选
采用杂交瘤技术制备CD38单克隆抗体，用重组的CD38胞外域与弗氏不完全佐剂等体积混合后采用肌肉注射的方式免疫BALB/c小鼠。经过三次基础免疫后采取尾静脉血，用ELISA检测小鼠血清中抗体滴度，融合前三天冲击免疫。首先按照杂交瘤细胞融合的实验步骤，分批将免疫的小鼠脾脏分别与SP2/0融合后，用含有HAT的培养基重悬融合细胞并转移到96孔板，按照编号做好标记，在37℃细胞培养箱培养，适时更换培养基定期观察生长情况，培养10-12天后，取上清进行ELISA检测。重组的CD38胞外段购自普健生物(武汉)科技有限公司，货号ATMP00140HU，其氨基酸序列如下表所示。
表1.重组的CD38胞外段氨基酸序列
Figure PCTCN2022119598-appb-000001
经过ELISA、流式细胞术筛选能与CD38阳性细胞结合的抗体克隆，最后用SPR技术筛选出能与CD38胞外段以高亲和力结合的杂交瘤细胞。通过Annexin V/PI细胞凋亡试剂盒检测检测筛选出的杂交瘤的诱导细胞凋亡的能力，筛选出杂交瘤H2。
如图1所示，H2在Daudi、Ramos、Raji和SU-DHL-1四种CD38阳性的细胞上均显示出细胞凋亡活性，其中在Daudi、Ramos和Raji细胞上的细胞凋亡活性显著优于阳性对照Dara，在SU-DHL-1细胞上的细胞凋亡活性与阳性对照Dara相当。Biacore亲和力测试结果(表2)表明，H2抗体与阳性对照Dara的亲和力常数相当，说明其与Dara具有相当的结合亲和力。对H2单抗进行测序，序列如下表3所示。
表2.H2单抗的亲和力测试
样品名称 KD(M) Ka(1/Ms) Kd(1/s)
Dara 1.22×10 -8 2.97×10 5 3.63×10 -3
H2 1.46×10 -8 6.61×10 4 9.69×10 -4
表3.H2抗体的互补决定区序列和可变区序列
Figure PCTCN2022119598-appb-000002
实施例2.Elisa检测抗原表位竞争占位
包被抗原CD38后，先加入不同浓度的H2单抗充分孵育后，加入饱和浓度以上的Isa及Dara，检测Isa及Dara的结合。如果测试抗体与CD38蛋白结合的位点与Isa或Dara有一定的遮挡或重叠，那么Isa或Dara的结合活性会降低。结果表明H2抗体与Isa或Dara结合不同的抗原表位(图2)。
实施例3.CD38抗体与不同种属抗原交叉反应
利用Elisa法检测抗体对不同种属的CD38抗原蛋白的交叉反应。包被不同种属(人(human)、食蟹猴(cynomolgus)、小鼠(mouse)、大鼠(rat))CD38抗原(1μg/ml，50ml/well)，2～8℃过夜孵育。抗体浓度：300、100、33.3、11.1、3.7、1.23、0.41μg/ml。
Elisa结果显示(图3)：Dara抗体与人源抗原结合，不与食蟹猴、小鼠、大鼠源抗原结合；H2抗体(Hybridoma-2)与人源与猴源抗原有结合，不与小鼠、大鼠源抗原结合。
实施例4.H2抗体人源化及嵌合抗体的设计、载体构建及表达
首先使用鼠源抗体的序列在人源框架库中搜索排名靠前的序列作为候选序列，然后将人源序列同源建模到鼠源抗体的骨架上，保留鼠源CDR的序列，然后计算同源模型的能量，判断人源化抗体的稳定性。人源化CDR突变体采用相同的策略，不同之处在于替换鼠源CDR序列为突变体序列，并且保留抗原的结构。根据人源化设计改造，以H2单抗为母本，设计了18株人源化序列(标记为FLT004-hu-1至FLT004-hu-18，具体序列如下表4至6所示)，并根据设计的人源化序列，对目的序列进行构建，HEK293E瞬时转染表达嵌合抗体。
表4.人源化后抗体的CDR序列
Figure PCTCN2022119598-appb-000003
Figure PCTCN2022119598-appb-000004
表5.人源化后抗体的可变区序列
Figure PCTCN2022119598-appb-000005
表6.人源化后抗体的恒定区序列
Figure PCTCN2022119598-appb-000006
实施例5.人源化抗体Biacore亲和力检测
通过Biacore检测人源化抗体的亲和力，结果如下表7所示。其中，FTL004-hu-1/3/5/6/7/11/13的亲和力常数与阳性对照Dara的亲和力常数相当，说明其与Dara具有相当的结合亲和力。
表7.人源化后抗体的亲和力分析
Figure PCTCN2022119598-appb-000007
Figure PCTCN2022119598-appb-000008
实施例6.人源化抗体凋亡活性检测
对人源化抗体凋亡活性进行检测，部分结果如图4所示。结果显示FTL004-hu-1/6/7的诱导细胞凋亡活性最好，且在Ramos和Raji两种细胞上均显示出优于阳性对照Isa的诱导细胞凋亡活性；FTL004-hu-13的凋亡活性次之，且在Ramos细胞上显示出与阳性对照Isa相当的诱导细胞凋亡活性，在Raji细胞上显示出优于阳性对照Isa的诱导细胞凋亡活性。
通过ELISA检测人源化抗体的活性，结果如图5所示，综合比较吸光度曲线及EC50值可知，FTL004-hu-1/3/5/6/7/9(分别对应图中humanized-1/3/5/6/7/9)的活性较好，均优于阳性对照Isa；FTL004-hu-11/12/13/15/17(分别对应图中humanized-11/12/13/15/17)的活性次之。
实施例7.抗体稳定性考察
高温稳定性：分别将人源化抗体样品在-20℃和40℃放置1周后，分析样品中聚体、单体和片段的百分比，结果如下表所示。部分抗体在40℃放置1周后SEC纯度未出现明显变化，结合初始产物纯度分析，认为FTL004-hu-1/2/5/6/7(分别对应表8中的FTL004-hum-1/2/5/6/7)产物初始纯度及稳定性均较好，或在细胞内轻重链的表达、组装及分泌上有优势。结果如表8所示。
表8.人源化后抗体的高温稳定性分析
Figure PCTCN2022119598-appb-000009
氧化稳定性：挑选部分活性较好的样品进行氧化稳定性试验，结果如下表所示，可以看出：0.1％H 2O 2作用6h与24h，SEC片段峰含量均有增加。结果如表9所示。综合比较：FTL004-hu-1/2/6/7/13号抗体氧化稳定性最好，片段段峰增加较小；FTL004-hu-5//9/11/15/17号抗体稳定性较差，片段峰增加较多。
表9.人源化后抗体的氧化稳定性分析
Figure PCTCN2022119598-appb-000010
综合上述结果，初步认为FTL004-hu-1/6/7号抗体理化性质较优，对三者进行综合稳定性考察(结果如下表10所示)。1号人源化抗体稳定性最佳，对氧化敏感，对高温酸碱冻融等较稳定。
表10.人源化后抗体的综合稳定性分析
Figure PCTCN2022119598-appb-000011
综上，认为FTL004-hu-1/6/7号抗体活性及理化性质较优。将三者用于后续的Fc突变改造。
实施例8.人源化抗体的体内药效学
通过之前的筛选结果，挑选出亲和力高、活性好、稳定性好的三株人源化抗体FTL004-hu-1/6/7进行进一步动物药效学检测。动物：NSG小鼠，每组3～5只， 雄性；细胞系：SU-DHL-6，皮下移植(1×10 7个/0.2ml/只)。药剂量和频率：0、2.5、10mg/kg；Q2Wx8(低剂量组共给药6次)；指标：肿瘤体积、瘤重及肝肾脏器重量。
由肿瘤生长曲线(图6)可知：10mg/kg的FTL004-hu-1腹腔注射(Q2Wx8)明显抑制SU-DHL-6荷瘤小鼠肿瘤生长，效果明显优于同剂量的Dara且优于同剂量的Isa；10mg/kg的FTL004-hu-6和7抑瘤效果虽不及FTL004-hu-1，但仍优于同剂量的Dara和Isa。给药27天实验终点时，将肿瘤解剖称重，计算抑瘤率(图7)，实验组抑瘤率分别如下：Dara组12.96％；Isa组为2.78％；FTL004-hu-1组为59.26％；FTL004-hu-6为29.63％；FTL004-hu-7为42.59％。由此可知，；FTL004-hu-1/6/7的抑瘤率均明显优于阳性对照Dara和Isa。通过大体观察和对肝脏、肾脏进行称重(图8)，发现Dara、Isa及FTL004-hu-1/6/7均无明显肝肾毒性。
实施例9.抗体Fc段改造
抗体的Fc段同许多Fc受体以及配体相互作用，并产生一系列的生物学效应。对于IgG Fc段来说，Fc受体家族-FcγR起着关键作用。Fc/FcγR复合体的形成可以有效地募集相应的效应细胞，从而导致细胞内的信号转导以及后续一系列重要的免疫反应，诸如炎症介质的释放、B细胞活化、胞吞作用、吞噬作用和细胞毒性攻击等。通过Fc段工程改造可以增加抗体依赖细胞介导的细胞毒性作用(ADCC)、抗体依赖性细胞介导的细胞吞噬作用(ADCP)及补体介导的细胞毒性作用(CDC)等作用。
根据本领域已知的方法对上述FTL004-hu-1/6/7号抗体进行Fc段改造，FTL004-hu-1/6/7号抗体改造后分别命名为FTL004-1/6/7，将Fc段第19位丝氨酸残基取代为天冬氨酸残基(S19D)、112位异亮氨酸残基取代为谷氨酸残基(I112E)、136位天冬氨酸残基取代为谷氨酸残基(D136E)、138位亮氨酸残基取代为蛋氨酸残基(L138M)，并删除226位和227位的甘氨酸残基和赖氨酸残基，并命名为FTL004-1/6/7，抗体序列的N端进一步包含如SEQ ID NO：32所示的信号肽(其具体序列如下表12所示)，比较抗体改造前后的活性及理化性质差别。
表11.Fc段改造前后的序列
Figure PCTCN2022119598-appb-000012
Figure PCTCN2022119598-appb-000013
表12.信号肽的序列
MDPKGSLSWRILLFLSLAFELSYG SEQ ID NO：32
表13.人源化抗体Fc段改造前后的抗体序列
Figure PCTCN2022119598-appb-000014
Figure PCTCN2022119598-appb-000015
对Fc改造前后的抗体进行Biacore亲和力测试，结果显示，Fc突变后的抗体与抗原结合亲和力无明显变化。通过以上所述相同方法检测Fc突变前后抗体诱导Romas、Daudi、Raji和SU-DHL-6细胞凋亡的活性，结果如下表14所示。Fc突变前后抗体在4种CD38阳性细胞上都有一定的诱导凋亡能力，且都优于阳性对照抗体Dara和Isa。
表14.人源化抗体Fc段改造前后的诱导细胞凋亡活性
Figure PCTCN2022119598-appb-000016
通过ELISA检测Fc突变后抗体的活性，由图9可知，FTL004-1/6/7的ELISA活性相近，其中FTL004-1活性稍高于FTL004-6/7。与FTL004-hu-1/6/7相比，活性无明显变化。
使用本领域已知的方法检测Fc突变后抗体与Fcγ受体的结合。检测结果如下表15所示，表明Fc段突变后的抗体表现出与FcγRIIIa(V)、FcγRIIIa(F)的结合增加。
表15.人源化抗体Fc段改造后的与Fcγ受体的结合
Figure PCTCN2022119598-appb-000017
实施例10.Fc改造后抗体的体内药效学
动物：NSG小鼠，每组3只，雄性；细胞系：SU-DHL-6，皮下移植(1×107个/0.2ml/只)。给药剂量和频率：10、30mg/kg；Q2Wx8；指标：肿瘤体积、瘤重及肝肾脏器重量。10mg/kg和30mg/kg的FTL004-1、FTL004-7、FTL004-hu-1、30mg/kg的Isa以及FTL004-1+Dara(均为10mg/kg)腹腔注射(Q2Wx8)给药。
结果见图10，从第4次给药后开始直至第8次给药结束，均可抑制SU-DHL-6荷瘤小鼠肿瘤生长，FTL004-1低高剂量有一定剂量相关性。给药29天实验终点时，将肿瘤解剖称重，计算抑瘤率(图11)，实验组抑瘤率分别如下：Isa 30mg/kg组为7.407％；FTL004-1 10mg/kg组为7.407％；FTL004-1 30mg/kg组为18.518％；FTL004-7 10mg/kg组为33.333％；FTL004-7 30mg/kg组为18.518％；FTL004-hu-1 10mg/kg组为25.925％；FTL004-hu-1 30mg/kg组为-18.518％；FTL004-hu-1 10mg/kg+Dara 10mg/kg组为-7.407％。将上述肿瘤组织进行冰冻组织切片，用FTL004-1抗体进行阳性组织的染色，可以看到FTL004抗体为弱阳性反应(图12)。
实施例11.CD38抗体诱导CD38阳性实体瘤细胞的凋亡
经对多种实体肿瘤细胞系的筛选，得到CD38强阳性的小细胞型肺癌H211细胞，中等阳性的非小细胞型肺癌A549细胞，弱阳性的非小细胞型肺癌H157细胞、前列腺癌细胞DU145细胞。
在CD38阳性的实体肿瘤细胞上进行凋亡实验。由下表16可知，在CD38表达丰度较低的DU145、H157细胞上，各个抗体组均未观察到明显的细胞凋亡情况，而对于CD38高表达的小细胞型肺癌H211细胞，不同抗体显示出不同的诱导凋亡能力，相对而言，样品FTL004-1诱导凋亡能力更佳。
表16.CD38抗体诱导CD38阳性癌细胞的凋亡率
Figure PCTCN2022119598-appb-000018
实施例12.CD38抗体对小细胞肺癌H211细胞的体内杀伤效果
动物：NCG小鼠，每组4只，雄性；细胞系：NCI-H211，皮下移植(1×10 7个/0.28ml/只)。给药剂量和频率：30mg/kg(FTL004-1和Isatuximab)；Q2Wx8；指标：肿瘤体积、瘤重及肝肾脏器重量。
实验结果见图13，可见30mg/kg的FTL004-1和Isa每周静脉注射给药2次，持续8次，均有抑制NCI-H211皮下瘤的生长的趋势，且同剂量的FTL004-1抑瘤效果稍好于Isa。
实施例13.CD38抗体对H211、SU-DHL-6细胞的结合实验
利用流式细胞术，检测不同浓度梯度下Dara、Isa、FTL004-1抗体，对CD38阳性细胞H211、SU-DHL-6细胞的结合并确认其结合EC50。
结果表明(图14)，FTL004-1抗体(对应图14中的004-1)与Isa抗体EC50值相近，Dara值较高，尤其在H211细胞上，EC50＝1.940μg/ml。
实施例14.CD38抗体对红细胞和PBMC的结合实验
利用流式细胞术，检测CD38抗体对恒河猴红细胞的结合，Dara、Isa对猴红细胞不结合，FTL004-1抗体对猴红细有一定结合EC50值在2-5μg/ml左右。利用流式细胞术，检测CD38抗体对正常人红细胞的结合，由图15可知，Dara对健康(正常)人红细胞有较高的结合Isa也对正常人红细胞有一定的结合，但结合相对较弱，FTL004-1抗体对正常人红细胞不结合。利用流式细胞术，检测CD38抗体对健康人PBMC的结合，由图16可知，Dara和FTL004-1抗体(对应图16中的004-1)对正常人的PBMC都有阳性结合反应。
应该理解的是，尽管已经通过优选实施方式和任选的特征具体公开了本发明，但是本领域技术人员可以对本文所公开的本发明进行修改、改进和变化，这些修改、改进和变化被认为在本发明的范围内。在此提供的材料、方法和实施例是优选的实施方式的代表和示例性的，并且不旨在作为对本发明范围的限制。

Claims (26)

  1. 一种结合人CD38的单克隆抗体或其抗原结合片段，其包含三个重链互补决定区HCDR1、HCDR2、HCDR3和三个轻链互补决定区LCDR1、LCDR2、LCDR3，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示或其一至三个氨基酸的保守性取代变体；HCDR2的氨基酸序列如SEQ ID NO:2所示或其一至三个氨基酸的保守性取代变体；HCDR3的氨基酸序列如SEQ ID NO:6所示或其一至三个氨基酸的保守性取代变体；LCDR1的氨基酸序列如SEQ ID NO:7所示或其一至三个氨基酸的保守性取代变体；LCDR2的氨基酸序列如SEQ ID NO:9所示或其一至三个氨基酸的保守性取代变体；LCDR3的氨基酸序列如SEQ ID NO:11所示或其一至三个氨基酸的保守性取代变体。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中：HCDR1的氨基酸序列如SEQ ID NO:1所示；HCDR2的氨基酸序列如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5所示；HCDR3的氨基酸序列如SEQ ID NO:6所示；LCDR1的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8所示；LCDR2的氨基酸序列如SEQ ID NO:9或SEQ ID NO:10所示；LCDR3的氨基酸序列如SEQ ID NO:11所示。
  3. 根据权利要求1或2所述的单克隆抗体或其抗原结合片段，其中：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:2所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:7所示；LCDR2，其氨基酸序列如SEQ ID NO:9所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
  4. 根据权利要求1或2所述的单克隆抗体或其抗原结合片段，其中：HCDR1，其氨基酸序列如SEQ ID NO:1所示；HCDR2，其氨基酸序列如SEQ ID NO:2所示；HCDR3，其氨基酸序列如SEQ ID NO:6所示；LCDR1，其氨基酸序列如SEQ ID NO:8所示；LCDR2，其氨基酸序列如SEQ ID NO:10所示；LCDR3，其氨基酸序列如SEQ ID NO:11所示。
  5. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中重链可变区VH包含如SEQ ID NO:12或SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:18或SEQ ID NO: 21所示的氨基酸序列或与其具有至少85％序列同一性的变体。
  6. 根据权利要求5所述的单克隆抗体或其抗原结合片段，其中
    (a)重链可变区VH包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体；
    (b)重链可变区VH包含如SEQ ID NO:12所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:21所示的氨基酸序列或与其具有至少85％序列同一性的变体；或者
    (c)重链可变区VH包含如SEQ ID NO:14所示的氨基酸序列或与其具有至少85％序列同一性的变体；轻链可变区VL包含如SEQ ID NO:18所示的氨基酸序列或与其具有至少85％序列同一性的变体。
  7. 根据权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段，其Fc段被修饰以增强与FcγRIIIa(V)和/或FcγRIIIa(F)的结合。
  8. 根据权利要求7所述的单克隆抗体或其抗原结合片段，其Fc段包含如SEQ ID NO:27所示的氨基酸序列或与其具有至少85％序列同一性的变体。
  9. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中重链包含如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30或SEQ ID NO:31所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34或SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体。
  10. 根据权利要求9所述的单克隆抗体或其抗原结合片段，其中
    (a)重链包含如SEQ ID NO:28所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体；
    (b)重链包含如SEQ ID NO:28所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体；
    (c)重链包含如SEQ ID NO:29所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体；
    (d)重链包含如SEQ ID NO:30所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体；
    (e)重链包含如SEQ ID NO:30所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:35所示的氨基酸序列或与其具有至少85％序列同一性的变体；或者
    (f)重链包含如SEQ ID NO:31所示的氨基酸序列或与其具有至少85％序列同一性的变体，轻链包含如SEQ ID NO:34所示的氨基酸序列或与其具有至少85％序列同一性的变体；。
  11. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中所述抗原结合片段选自Fv、scFv、(scFv) 2、Fab、Fab′和F(ab′) 2。
  12. 根据权利要求1至10任一项所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是IgG1型。
  13. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其不结合健康人红细胞。
  14. 根据权利要求1至13任一项所述的单克隆抗体或其抗原结合片段，其通过Biacore测定的亲和力常数Kd为10 -9M级别。
  15. 根据权利要求1至14任一项所述的单克隆抗体或其抗原结合片段，其与Isatuximab结合不同的抗原表位。
  16. 根据权利要求1至15任一项所述的单克隆抗体或其抗原结合片段，其与Daratumumab结合不同的抗原表位。
  17. 一种单克隆抗体或其抗原结合片段，其与如权利要求1至16任一项所述的单克隆抗体或抗原结合片段竞争结合至人CD38。
  18. 一种用于治疗CD38阳性癌症的药物组合物，其包含权利要求1至17任一项所述的单克隆抗体或其抗原结合片段，以及药学上可接受的载体。
  19. 根据权利要求18所述的药物组合物，其中所述癌症是血液癌。
  20. 根据权利要求19所述的药物组合物，其中所述血液癌选自多发性骨髓瘤、白血病或淋巴瘤。
  21. 根据权利要求20所述的药物组合物，其中所述白血病选自急性淋巴细 胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓细胞性白血病(CML)、年轻型骨髓单核细胞性白血病(JML)、成人T细胞淋巴性白血病(ATL)和浆细胞白血病；所述淋巴瘤选自小淋巴细胞淋巴瘤、淋巴浆细胞淋巴瘤、边缘区淋巴瘤、滤泡型淋巴瘤、套细胞淋巴瘤、弥漫性大细胞B细胞淋巴瘤和伯基特淋巴瘤。
  22. 根据权利要求18所述的药物组合物，其中所述癌症是实体瘤。
  23. 根据权利要求22所述的药物组合物，其中所述实体瘤选自黑素瘤、肺癌、鳞状非小细胞肺癌(NSCLC)、非鳞状NSCLC、结肠直肠癌、前列腺癌、去势抵抗性前列腺癌、胃癌、卵巢癌、肝癌、胰腺癌、甲状腺癌、头颈部鳞状细胞癌、食道或胃肠道癌、乳腺癌、输卵管癌、脑癌、尿道癌、泌尿生殖道癌、子宫内膜癌、宫颈癌、肺腺癌、肾细胞癌(RCC)、间皮瘤、鼻咽癌(NPC)、食道癌和胃肠道癌。
  24. 根据权利要求18所述的药物组合物，其进一步包含用于治疗相同癌症的第二治疗剂。
  25. 根据权利要求24所述的药物组合物，其中所述第二治疗剂是化疗剂、放疗剂或生物制剂。
  26. 根据权利要求25所述的药物组合物，其中所述生物制剂是单克隆抗体、ADC、溶瘤病毒或CAR-T。
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