WO2024012583A1 - 人源化岩藻糖基化gm2ap抗体 - Google Patents

人源化岩藻糖基化gm2ap抗体 Download PDF

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WO2024012583A1
WO2024012583A1 PCT/CN2023/107554 CN2023107554W WO2024012583A1 WO 2024012583 A1 WO2024012583 A1 WO 2024012583A1 CN 2023107554 W CN2023107554 W CN 2023107554W WO 2024012583 A1 WO2024012583 A1 WO 2024012583A1
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seq
antibody
isolated antibody
cdr1
cdr2
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PCT/CN2023/107554
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French (fr)
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陈水田
林子文
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浩峰生物科技股份有限公司
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    • 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

Definitions

  • antibodies capable of binding to the glycosylated antigen GM2AP-GF. This application also provides pharmaceutical compositions containing the antibody, as well as therapeutic and diagnostic uses of the antibody.
  • Lung cancer ranks first as the leading cause of cancer-related death worldwide, accounting for 28% of all male cancer deaths and 26% of all female cancer deaths in the United States in 2013. Lung cancer can generally be divided into two main types: small cell lung cancer (SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC), accounting for 15% and 85% of lung cancers respectively.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • adenocarcinoma squamous cell carcinoma
  • large cell lung cancer Due to the lack of effective early detection tools and the inability to effectively treat late-stage cancer, the 5-year survival rate of lung cancer is only 15%. Therefore, early detection is necessary and beneficial for lung cancer patients to receive appropriate and effective treatment.
  • GM2-activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. After GM2AP is synthesized, it undergoes glycosylation, modification, and cleavage at the 32Ser position before it becomes the mature form. Mature GM2AP is a deglycosylated form of the glycoprotein with a molecular weight of 17.6 kDa.
  • GM2AP as a cofactor contains at least three functional features: a hydrophobic interaction region known as the ⁇ -cup structure, an oligosaccharide binding site, and a region that interacts with HexA.
  • GM2 ganglioside The aforementioned region that interacts with Hex A allows GM2 ganglioside to be broken down into GM3 by lysosomal hexosaminidase A (lysosomal ⁇ -hexminidase A, Hex A).
  • lysosomal hexosaminidase A lysosomal ⁇ -hexminidase A, Hex A
  • GM2AP can be recaptured into cells through a carbohydrate-independent mechanism in various cells, such as epidermal keratinocytes and fibroblasts.
  • Lack of functional GM2AP results in abnormal accumulation of GM2 gangliosides in the tissues of patients with AB GM2 gangliosidosis, a severe lysosomal storage disease.
  • Inherited GM2AP deficiency is also associated with changes in ganglioside content, as well as changes in tumor-associated ganglioside content that are associated with cancer progression.
  • Tumor-associated gangliosides are the result of proto-oncogene transformation and play a certain role in triggering invasion and metastasis. Tumor cells synthesize gangliosides and release them into their microenvironment, thereby increasing the content of tumor-associated gangliosides in the serum.
  • gangliosides are currently known to play regulatory roles in cell growth, adhesion, cell-cell interactions, and signaling.
  • the purpose of the present invention is to provide a novel antibody that has high affinity for glycosylated GM2AP antigen (glycosylated GM2AP, GM2AP-GF), but has high affinity for non-glycosylated GM2AP antigen (non-glycosylated GM2AP).
  • GM2AP glycosylated GM2AP antigen
  • GM2AP-GF glycosylated GM2AP antigen
  • non-glycosylated GM2AP antigen non-glycosylated GM2AP
  • This highly selective antibody is predicted to have a labeling or curative effect on lung cancer cells.
  • Another object of the present invention is to provide pharmaceutical compositions containing such antibodies, as well as therapeutic and diagnostic uses of such antibodies.
  • an isolated antibody comprising:
  • V H CDR1 comprising an amino acid sequence selected from SEQ ID NO: 9, 15, 21 or 27;
  • V H CDR2 comprising an amino acid sequence selected from SEQ ID NO: 10, 16, 22 or 28;
  • V H CDR3 comprising an amino acid sequence selected from SEQ ID NO: 11, 17, 23 or 29;
  • VL CDR1 comprising an amino acid sequence selected from SEQ ID NO: 12, 18, 24 or 30;
  • V L CDR2 comprising an amino acid sequence selected from SEQ ID NO: 13, 19, 25 or 31;
  • VL CDR3 comprising an amino acid sequence selected from SEQ ID NO: 14, 20, 26 or 32.
  • V H comprises a sequence having at least 90% identity to SEQ ID NO: 1, 3, 5 or 7
  • V L comprises a sequence having a sequence identical to SEQ ID NO: 2, 4, 6 or 8 sequences with at least 90% identity.
  • the present invention also provides a pharmaceutical composition, which contains the aforementioned isolated antibody and a pharmaceutically acceptable carrier.
  • the present invention provides an isolated polynucleotide encoding the aforementioned isolated antibody.
  • the polynucleotide comprises a first sequence encoding the VH of the antibody that is at least 90% identical to SEQ ID NO: 1, 3, 5, or 7; and a first sequence encoding the VL of the antibody A second sequence that is at least 90% identical to SEQ ID NO: 2, 4, 6 or 8.
  • the present invention also provides a vector comprising the aforementioned polynucleotide.
  • the present invention further provides a method for treating lung cancer, which includes: administering an effective dose of the aforementioned isolated antibody to an individual in need.
  • Figure 1 illustrates the results of antibody screening with the DCB phage library Dsyn2.1.
  • Each round is pre-cleaned with 100 ⁇ g/ml GM2AP to remove antibodies that bind to GM2AP.
  • the concentration of coating antigen in each round was 5 ⁇ g/ml (the experimental group was the same as the control group).
  • the histogram on the right side of each round represents GM2AP, and the histogram on the left side represents GM2AP-GF.
  • Figure 2 shows the specificity results of 13 anti-GM2AP-GF antibodies screened by ELISA analysis. For each antibody, the histogram on the right side represents GM2AP, and the histogram on the left side represents GM2AP-GF.
  • Figure 3 shows the results of antibody affinity analysis by ELISA on the final four selected antibodies.
  • the upper line segment represents GM2A-GF
  • the lower line segment represents GM2A.
  • antibody as used herein includes an intact antibody, as well as any antigen-binding fragment thereof (i.e., an "antigen-binding portion") or a single chain thereof.
  • Antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains bound to each other by disulfide bonds, or an antigen-binding site thereof.
  • Each heavy chain consists of a heavy chain variable region (herein abbreviated as VH ) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (herein abbreviated as VL ) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each V H and V L consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and typically the first component (Clq) of the complement system.
  • IMGT ImMunoGeneTics database
  • Kabat system in this disclosure means that according to Kabat (1991; Sequences of Proteins of Immunological Interest, 5th edit., NIH publication no. 91-3242 U.S. Department of Health and Human services), and Chothia (1987; J. Mol. Biol. 196, 901-917) standard for numbering amino acids.
  • This numbering system is widely used by those skilled in the art and is based on the sequence variability and three-dimensional loop portions of the variable domain sequences that are important for antigen-binding activity.
  • all amino acids in the light or heavy chain have well-defined positions; that is, the Kabat system applies to CDRs as well as framework regions.
  • the specific amino acid positions of any antibody can be numbered according to the Kabat system.
  • the rules for identifying CDR regions of VH and VL chains according to the Kabat system are shown at www.bioinf.org.uk/abs.
  • antigen-binding fragment refers to antibody fragments, such as diabodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific Sexual dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv diabody (bivalent diabody), formed from an antibody portion containing one or more CDRs A multispecific antibody, or any other antibody fragment that binds an antigen but does not contain a full-length or complete antibody structure.
  • the antigen-binding fragment may bind the same antigen as the original antibody or original antibody fragment (eg, original scFv).
  • an antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted to framework regions from one or more different human antibodies.
  • Fab has the structures of light chain and heavy chain variable regions, light chain constant region, and heavy chain first constant region (CH1), and has an antigen-binding position.
  • Fab' differs from Fab in that Fab' has a hinge region, including at least one cysteine residue at the C-terminus of the heavy chain CH1 domain.
  • F(ab') 2 is produced when the cysteine residues of the hinge region of Fab' are linked via disulfide bonds.
  • Fv is the smallest antibody fragment, having only heavy chain variable regions and light chain variable regions. Recombinant techniques for producing Fv fragments are known in the art.
  • Single chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other either directly or via a peptide linker sequence.
  • a double-chain Fv can have a structure in which the heavy chain variable region is linked to the light chain variable region via non-covalent bonds.
  • Single-chain Fvs can generally form a dimeric structure as in double-chain Fvs, in which the heavy chain variable domain is covalently bound to the light chain variable domain via a peptide linker, or the heavy and light chain variable domains are separated between each other. The C-termini are directly connected to each other.
  • the linker fragment may be a peptide linker fragment comprising from 1 to 100, or from 2 to 50 amino acids, and suitable sequences are known in the art.
  • Antigen-binding fragments can be obtained using proteases (eg, intact antibodies can be cleaved with papain to obtain Fab fragments, or pepsin can be used to obtain F(ab') 2 fragments), or can be prepared by genetic recombination techniques.
  • proteases eg, intact antibodies can be cleaved with papain to obtain Fab fragments, or pepsin can be used to obtain F(ab') 2 fragments
  • pepsin can be used to obtain F(ab') 2 fragments
  • isolated antibody for example, an isolated antibody that specifically binds GM2AP-GF, is cross-reactive to other antigens, such as GM2AP-GF molecules from other species. May also be reactive to the similar antigen GM2AP. Additionally, isolated antibodies can be substantially free of other cellular material, and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of homogeneous molecular composition.
  • a monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.
  • human antibody refers to an antibody in which both the framework and CDR regions of the variable region are derived from human immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human immunoglobulin sequences.
  • Human antibodies of the present disclosure may include amino acids not derived from those encoded by human immunoglobulin sequences (eg, mutations introduced via random or site-specific mutation in vitro or via somatic mutation in vivo). However, the term “human antibody” as used herein does not include antibodies in which CDR sequences derived from another mammalian species (eg, mouse) are grafted onto human framework sequences.
  • human monoclonal antibody refers to an antibody that exhibits a single binding specificity and whose variable region framework and CDR regions are derived from human immunoglobulin sequences.
  • recombinant human antibody includes all human antibodies prepared, expressed, produced or isolated via recombinant techniques, such as (a) isolated from animals (e.g. mice) transgenic or transchromosomal for human immunoglobulin genes Antibodies or fusionomas prepared therefrom (described further below), (b) antibodies isolated from host cells transformed for expression of human antibodies, such as transfectomas, (c) derived from recombinant, combinatorial human Antibodies isolated from antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other technique involving splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant techniques such as (a) isolated from animals (e.g. mice) transgenic or transchromosomal for human immunoglobulin genes Antibodies or fusionomas prepared therefrom (described further below), (b) antibodies isolated from host cells transformed for expression of human antibodies, such as transfectomas, (c) derived from recombinant, combinatorial human Antibodies isolated from
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human immunoglobulin sequences.
  • in vitro mutagenesis or, when using animals transfected with human Ig sequences, in vivo somatic mutagenesis
  • the VH and The VL region amino acid sequences although derived from and related to human germline VH and VL , may not naturally occur within the human antibody germline repertoire in vivo.
  • humanized antibody refers to antibodies produced by non-human species and contain protein sequences that have been modified to increase their similarity to naturally occurring human antibody variants. When using antibodies of non-human origin in humans, the humanization process is necessary to avoid undesirable immunogenic effects.
  • chimeric antibody refers to an antibody in which the antigen-binding regions (i.e., VH and VL ) from one species are fused to each other with constant regions from another species.
  • Kassoc or " Ka” as used herein refers to the on-rate of a particular antibody-antigen interaction
  • Kdis or " Kd” as used herein refers to the off-rate of a particular antibody-antigen interaction.
  • K D herein refers to the dissociation constant, which is obtained from the ratio of K d to Ka (i.e., K d /K a ) and is expressed in molar concentration (M).
  • M molar concentration
  • an antibody "glycosylated modified GM2-activated protein” means that the binding constant K D of the antibody to GM2AP-GF is less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, and less than 1 ⁇ 10 - 9 M, less than 1 ⁇ 10 -10 M, less than 1 ⁇ 10 -11 M or lower.
  • the recovered phages were added to E. coli TGI bacterial solution for reaction, and then spread on 2YT-AG medium for culture. Scrape off the bacteria on the culture medium, add 2YT-AG culture medium to the bacterial solution, and culture until the OD600 is about 0.5. Add helper phage to the reaction. Centrifuge to remove the supernatant, add 2YT-AK medium to redissolve and culture. Centrifuge the supernatant, add PEG/NaCl, mix well, and centrifuge again. Remove the supernatant and redissolve the pellet in PBS to obtain the next round of phage library. Repeat the above steps for each round.
  • the insert DNA of each IgGl was prepared by PCR with specific primers designed for VH and VL on the phage.
  • the protein expression vector was reacted with restriction enzymes DraIII-HF and BsiWI-HF, and then purified by gel extraction kit. Add NheI-HF and MluI-HF and react.
  • the DNA ligation reaction is mediated by T4 DNA polymerase, and the ligated plasmid is transformed into the DH5 ⁇ host for isolation and amplification.
  • the DSyn2.1 human antibody library was used to screen anti-GM2AP-GF antibodies. After 3 to 4 rounds of screening, the results are shown in Figure 1. It was shown that in rounds 3c, 4Ca, and 4Cb, compared to the control group (antigen GM2AP), the phage group expressing single-chain antibodies had higher ELISA values for the experimental group (antigen GM2AP-GF), with OD 450 -OD 655 approx. It is 2.0 ⁇ 2.7, and there is a significant difference in the signals between the experimental group and the control group (antigen GM2AP). It is speculated that these phage groups may contain single-chain antibodies with affinity for GM2AP-GF.
  • the selected phage groups (3a, 3b, 3c and 4Ca) were subjected to a single phage ELISA assay to select phages that can recognize GM2AP-GF. A total of 288 strains were screened out, and 13 antibody sequences were obtained after sequencing, as shown in Tables 1-1 and 1-2 (all expressed in the Kabat system).
  • the 13 selected sequences were transformed into full-IgG1 antibodies. After small-scale expression and purification, ELISA specificity and affinity tests were performed. The results are shown in Figure 2. Among them, the OD 450 -OD 655 reading value of 6 antibodies (GF3B-2A1, GF3B-2A12, GF3B-A9, GF3B-2A7, GF3B-2A2, GF4Ca-E1) against GM2AP-GF is greater than 0.2, but GF4Ca-E1 has The OD. 450 -OD 655 reading value of GM2AP binding is 3, which is similar to the reading value of GM2AP-GF and has no specificity, so it is excluded.
  • GF3B-2A7 The expression level of antibody GF3B-2A7 is less than 3, and the lowest reading value in ELISA OD 450 -OD 655 is only 0.2, so it is also excluded. Finally, 4 antibodies were selected: GF3B-2A1, GF3B-2A12, GF3B-A9, and GF3B-2A2.
  • ELISA was used to conduct antibody affinity analysis, and the results are shown in Figure 3A-D.
  • the two antibodies GF3B-A9 ( Figure 3A) and GF3B-A12 ( Figure 3B) have high background values for GM2AP. It is speculated that they may recognize the position of GM2AP-GF close to GF, or recognize a small part of GM2AP.
  • the ELISA affinity analysis results in Figure 3A to D show that the four antibodies screened in this disclosure have higher affinity and lower KD value for the glycosylated GM2AP-GF antigen, while their affinity for the unglycated GM2AP antigen is lower. Poor or no affinity, high KD value or not detected. Therefore, these four antibodies are highly selective for GM2AP-GF. Generally speaking, a KD less than 10 -7 or 10 -8 indicates high affinity. In addition, the KD values of the four antibodies of the present disclosure for GM2AP-GF are all smaller than the KD values for GM2AP.
  • the disclosure further provides pharmaceutical compositions comprising the antibodies of the disclosure.
  • the composition is a pharmaceutical composition for treating lung cancer.
  • the lung cancer includes, but is not limited to, small cell lung cancer (SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC).
  • the pharmaceutical composition includes an antibody of the present disclosure and a carrier.
  • the antibody may be present in the composition in an amount effective to achieve the purpose of the composition.
  • the "effective amount” refers to the amount of active ingredient required to achieve the desired effect in an individual (eg, treatment of lung cancer), whether alone or in combination with one or more other active agents.
  • the effective amount will depend upon the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, and the nature of concurrent treatments, if any. , the specific route of administration, and similar factors within the health practitioner's knowledge and expertise).
  • Such antibody pharmaceutical compositions can be prepared by mixing the disclosed antibodies with the desired purity and one or more carriers.
  • such antibody pharmaceutical compositions can be prepared as aqueous solutions (see, for example, US Patent No. 6,171,586 and WO2006/044908), or as lyophilized preparations (see, for example, US Patent No. 6,267,958).
  • the carrier used for the antibody pharmaceutical composition of the present disclosure may be a pharmaceutically acceptable carrier, generally including but not limited to buffers, excipients, stabilizers, preservatives, solvents, dispersion media, coatings, antibacterial agents and antifungal agents agents, isotonic and absorption delaying agents, and physiologically compatible analogues.
  • the carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion).
  • Pharmaceutically acceptable carriers are generally non-toxic to the subjects receiving them at the doses and concentrations employed.
  • exemplary pharmaceutically acceptable carriers include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g.
  • octadecyldimethylbenzyl ammonium chloride hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butylphenol or benzyl alcohol; alkyl parabens such as p-hydroxybenzoate Methyl benzoate or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 amino acids) peptides; proteins, Such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides and disaccharides and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mann
  • Pharmaceutically acceptable carriers for the antibody pharmaceutical compositions of the present disclosure may also include interstitial drug dispersions, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP) (see, e.g., U.S. Pat. Publication No. 2005/0260186 and 2006/0104968), such as human soluble PH-20 hyaluronidase glycoprotein (such as rHuPH20 or Baxter International, Inc.).
  • interstitial drug dispersions such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP) (see, e.g., U.S. Pat. Publication No. 2005/0260186 and 2006/0104968), such as human soluble PH-20 hyaluronidase glycoprotein (such as rHuPH20 or Baxter International, Inc.).
  • composition may also include pharmaceutically acceptable excipients such as disintegrants, binders, lubricants, preservatives, or combinations thereof.

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Abstract

提供了能够结合糖基化抗原GM2AP-GF的抗体。还提供了包含此抗体的药物组合物，以及此类抗体的治疗与诊断用途。

Description

人源化岩藻糖基化GM2AP抗体 技术领域
本文公开了能够结合糖基化抗原GM2AP-GF的抗体。本申请还提供包含此抗体的药物组合物，以及此抗体的治疗与诊断用途。
 背景技术
肺癌在全世界癌症相关死亡原因中排名第一，占2013年美国所有男性癌症死亡人数的28％，所有女性癌症死亡人数的26％。一般可将肺癌分为两个主要类型：小细胞肺癌(small cell lung cancer，SCLC)及非小细胞肺癌(non-small cell lung cancer，NSCLC)，分别占肺癌的15％及85％。NSCLC由三种主要组织学亚型组成：腺癌、鳞状细胞癌及大细胞肺癌。由于缺乏有效早期检测的工具，对于末期癌症又无法有效治疗，肺癌的5年存活率只有15％。因此，对肺癌患者来说，要接受适当而具有成效的治疗，早期检测是必要且有利的。
GM2活化蛋白质(GM2-activator protein，GM2AP)是一种小的单体蛋白质，包含用于Asn连接糖基化的单一位置。GM2AP合成出来之后在32Ser位置经过糖基化、修饰、切割后，才成为成熟形式。成熟的GM2AP是一种去糖基化形式(deglycosylated form)的糖蛋白质，其分子量为17.6kDa。作为辅助因子的GM2AP包含至少三项功能性特征：被称为β-cup结构的疏水性作用区域、寡糖结合位置以及与HexA相互作用的区域。前述与Hex A相互作用的区域可使GM2神经节苷脂(ganglioside)通过溶酶体氨基已糖酯酶A(lysosomal β-hexminidase A，Hex A)分解成GM3。然而，所合成的GM2AP只有三分之一被分泌出去。可通过多种细胞(如表皮角质细胞和纤维母细胞)中某种与碳水化合物无关的机制(carbohydrate-independent mechanism)将GM2AP再捕捉到细胞内。缺乏功能性的GM2AP会使GM2神经节苷脂在罹患AB型GM2神经节苷脂症(gangliosidosis，一种严重的溶小体贮积疾病)的患者的组织中产生异常堆积。遗传性GM2AP缺乏也与神经节苷脂含量的变化、以及与癌症病程相关的肿瘤相关神经节苷脂(tumor-associated ganglioside)含量的变化有关。肿瘤相关神经节苷脂是原癌基因转变的结果，在引发侵袭及转移的过程中发挥一定的作用。肿瘤细胞会合成神经节苷脂，并将之释放到其微环境中，而使血清中肿瘤相关神经节苷脂的含量增加。此外，目前已知神经节苷脂在细胞生长、粘附、细胞-细胞相互反应及信号传导方面都发挥调控的作用。
发明内容
本发明的目的是提供一种新颖的抗体，其对糖修饰(糖基化)的GM2AP抗原(glycosylated GM2AP,GM2AP-GF)具有高亲和力，但对无糖基修饰的GM2AP抗原(non-glycosylated GM2AP,GM2AP)无亲和力或仅具低亲和力，亦即具有高选择性。此具有高选择性的抗体预测对肺癌细胞有标记或治愈效果。本发明的另一目的是提供包含此抗体的药物组合物，以及此类抗体的治疗与诊断用途。
为了达成上述目的，本发明提供一种分离的抗体，包含：
VH CDR1，其包含选自于SEQ ID NO:9、15、21或27的氨基酸序列；
VH CDR2，其包含选自于SEQ ID NO:10、16、22或28的氨基酸序列；
VH CDR3，其包含选自于SEQ ID NO:11、17、23或29的氨基酸序列；
VL CDR1，其包含选自于SEQ ID NO:12、18、24或30的氨基酸序列；
VL CDR2，其包含选自于SEQ ID NO:13、19、25或31的氨基酸序列；以及
VL CDR3，其包含选自于SEQ ID NO:14、20、26或32的氨基酸序列。
本发明还提供一种分离的抗体，其中VH包含具有与SEQ ID NO:1、3、5或7至少90％同一性的序列；以及VL包含具有与SEQ ID NO:2、4、6或8至少90％同一性的序列。
本发明又提供一种药物组合物，其包含前述分离的抗体及药学上可接受的载体。
接着，本发明提供一种分离的多核苷酸，其编码前述的经分离的抗体。在某些实施例中，该多核苷酸包含编码抗体的VH的第一序列，其具有与SEQ ID NO:1、3、5或7至少90％的同一性；以及编码抗体的VL的第二序列，其具有与SEQ ID NO:2、4、6或8至少90％的同一性。
本发明还提供一种包含前述的多核苷酸的载体。
本发明更提供一种治疗肺癌的方法，包括：给予有需要的个体有效剂量的前述分离的抗体。
 附图说明
图1说明了以DCB噬菌体库Dsyn2.1进行抗体筛选的结果。每轮先经过100μg/ml GM2AP进行预清洁，去除与GM2AP结合的抗体。每轮涂覆(coating)抗原的浓度为5μg/ml(实验组与对照组相同)。各轮右侧的直方图表示GM2AP，左侧的直方图表示GM2AP-GF。
图2为以ELISA分析筛选出来的13种抗GM2AP-GF抗体特异性的结果。各抗体中右侧的直方图表示GM2AP，左侧的直方图表示GM2AP-GF。
图3为最后选出的4株抗体，其以ELISA进行抗体亲和力分析的结果。图3A-D中，上方的线段表示GM2A-GF，下方的线段表示GM2A。
 具体实施方式
对于本文和所附权利要求中的描述，单数形式“一(a)”和“一个(an)”包括复数指示物，除非上下文另有明确指示。因此，例如，“一蛋白质”包括多于一种蛋白质，且“一化合物”指多于一种化合物。“包含(comprise)”、“包含(comprises)”、“包含(comprising)”、“包括(include)”、“包括(includes)”、“包括(including)”的使用是可互换的，而非限制性的。更应理解的是，各具体实施例的描述中，使用术语“包括(comprising)”的情况下，本领域技术人员将理解，在一些特定情况下，可以使用语言“基本上由......组成”或“由......组成”替代。
当提供一定范围的数值，除非上下文另有明确规定，否则应当理解，该数值区间的整数以及该数值区间的每个整数的十分之一，介于该范围的上限与下限之间，以及在该范围内的任何其他陈述值或中间值，都涵盖在本公开内。这些较小范围的上限和下限可以独立地包括在该较小范围内，同时也包含在本公开内，受限于所述范围内的任何特别排除的限制。当陈述的范围包括该限制的一或两者时，排除这些限制的(i)或(ii)的范围也包括在本公开中。例如，“1至50”包括“2至25”、“5至20”、“25至50”、“1至10”等。
本公开中引用的所有文献、专利、专利申请和其他文件，皆完整并入本文以作为参考数据，其内容如同每一独立文献、专利、专利申请或其他文件分别指出，皆并入本文以作为参考目的。
应当理解的是，包括附图，以上一般性描述和以下详细描述仅是示范性和解释性的，并非用于限制本公开。
本文描述中使用的技术和科学术语将具有本领域技术人员通常理解的含义，除非另有明确定义。
本文中术语“抗体”包括完整抗体，以及其任一抗原结合片段(即“抗原结合部分”)或其单链。“抗体”指包含通过二硫键相互结合的至少两条重(H)链与两条轻(L)链，或其抗原结合部位的糖蛋白。每条重链由重链可变区(于此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(于此缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步细分为称作互补决定区(CDR)的高度变异区，并穿插有更保守性的区域，称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)和典型的补体系统的第一成分(C1q)。
框架区与CDR的内容依据Kabat等人(请见Kabat et al.,Sequences of Proteins of Immunological Interest,U.S.Department of Health and Human Services,1991)，以及ImMunoGeneTics数据库(IMGT)(请见Lefranc,Nucleic Acids Res 29:207-9,2001；以及在线imgt.cines.fr/IMGT_vquest/vquest？livret＝0&Option＝humanIg)定义。
“Kabat系统”在本公开中表示根据Kabat(1991；Sequences of Proteins of Immunological Interest,5th edit.,NIH publication no.91-3242U.S.Department of Health and Human services)，以及Chothia(1987；J.Mol.Biol.196,901-917)方式编号氨基酸的标准。此编号系统被本领域技术人员广泛使用，其以对抗原结合活性相当重要的可变结构域序列的序列变异性和三维环部分为基础。在Kabat系统中，轻链或重链的所有氨基酸都具有明确的位置；即Kabat系统适用于CDR以及框架区。任何抗体的特定氨基酸位置可根据Kabat系统进行编号。根据Kabat系统识别VH和VL链的CDR区的规则显示于www.bioinf.org.uk/abs.。
本文中术语“抗原结合片段”是指抗体片段，例如双抗体、Fab、Fab'、F(ab’)2、Fv片段、二硫键稳定化Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv’)、二硫键稳定化双抗体(ds diabody)、单链抗体分子(scFv)、scFv双体(双价双抗体)、由包含一或多个CDR的抗体部分形成的多特异性抗体，或结合抗原但不包含全长或完整抗体结构的任何其他抗体片段。抗原结合片段可结合与原始抗体或原始抗体片段(如原始scFv)相同的抗原。在某些具体实施例中，抗原结合片段可包含来自特定人类抗体的一个或多个CDR，其与来自一个或多个不同人类抗体的框架区嫁接。
在上述抗原结合片段中，Fab具有轻链和重链可变区、轻链恒定区、重链第一恒定区(CH1)的结构，并具有一个抗原结合位置。Fab’不同于Fab之处在于Fab’具有一铰链区(hinge region)，在重链CH1结构域的C端包括至少一个半胱氨酸残基。当Fab'的铰链区的半胱氨酸残基经由二硫键连接时，会产生F(ab’)2。
Fv是最小的抗体片段，仅具有重链可变区和轻链可变区。用于产生Fv片段的重组技术在本领域中是已知的。“单链Fv抗体”或“scFv”是指由轻链可变区和重链可变区组成的改造抗体，该轻链可变区和重链可变区直接或经由肽连接序列彼此连接。双链Fv可具有其中重链可变区经由非共价键与轻链可变区连接的结构。单链Fv通常可以形成如在双链Fv中的二聚体结构，其中重链可变区经由肽连接片段共价结合至轻链可变区，或是重链和轻链可变区在其C-端彼此直接连接。连接片段可为包括1至100，或2至50个氨基酸的肽连接片段，且其合适序列在本领域中是已知的。
抗原结合片段可使用蛋白酶获得(例如，可用木瓜蛋白酶切割完整抗体以获得Fab片段，或可用胃蛋白酶切割以获得F(ab’)2片段)，或可通过基因重组技术来制备。
“分离的抗体”，例如特异性结合GM2AP-GF的分离的抗体对其他抗原，如来自其他物种的GM2AP-GF分子，具有交叉反应性。也可能对类似抗原GM2AP具有反应性。此外，分离的抗体可实质上不含其他细胞材料，和/或化学物质。
本文中术语“单克隆抗体”或“单克隆抗体组合物”指均质分子组成的抗体分子制剂。单克隆抗体组合物展现出对特定表位的单一结合特异性与亲和力。
本文中术语“人类抗体”指其可变区的框架区和CDR区均衍生自人类免疫球蛋白序列的抗体。此外，如果该抗体含有恒定区，该恒定区也衍生自人类免疫球蛋白序列。本公开的人类抗体可包括非衍生自人类免疫球蛋白序列编码的氨基酸(例如经由体外随机或位点特异性突变或经由体内体细胞突变引入的突变)。然而，本文中术语“人类抗体”并不包括衍生自另一种哺乳动物物种(如小鼠)的CDR序列移植到人类框架序列上的抗体。
术语“人类单克隆抗体”是指表现出单一结合特异性，且其可变区的框架区和CDR区均衍生自人类免疫球蛋白序列的抗体。
本文中术语“重组人类抗体”包括所有经由重组技术制备、表达、产生或分离的人类抗体，例如(a)从用于人免疫球蛋白基因的转基因或转染色体的动物(例如小鼠)分离的抗体或由其制备的融合瘤(下面进一步描述)，(b)从宿主细胞分离的抗体，该宿主细胞经转化而用于表达人类抗体，例如转染瘤，(c)由重组、组合性人类抗体库分离的抗体，以及(d)以涉及将人类免疫球蛋白基因序列剪接至其他DNA序列的任何其他技术制备、表达、产生或分离的抗体。此种重组人类抗体具有可变区，其中框架区和CDR区衍生自人类免疫球蛋白序列。然而，在某些具体实施例中，可对于此类重组人类抗体进行体外突变(或者当使用人类Ig序列转染的动物时，进行体内体细胞突变)，且因此，重组人类抗体的VH和VL区氨基酸序列，虽然衍生自且相关于人类种系VH和VL，可能非天然存在于体内人类抗体种系库内。
术语“人源化抗体”指由非人类物种所产生的抗体，并包含已被修饰以增加它们与人类天然产生的抗体变异物的相似性的蛋白质序列。当在人类中使用非人类来源的抗体时，人源化过程是必要的，以避免不希望的免疫原性效应。相较之下，本文中术语“嵌合抗体”指经由将来自某一物种的抗原结合区(即VH和VL)与来自另一物种的恒定区互相融合的抗体。
本文中术语“Kassoc”或“Ka”指特定抗体-抗原相互作用的结合速率，本文中术语“Kdis”或“Kd”指特定抗体-抗原相互作用的解离速率。术语“KD”于此表示解离常数，其由Kd与Ka的比率(即Kd/Ka)获得，并以摩尔浓度(M)表示。可使用本领域中已建立的方法来测定抗体的KD值。
如本文中所述，抗体“糖基化修饰的GM2活化蛋白”指该抗体与GM2AP-GF的结合常数KD小于1×10-7M、小于1×10-8M、小于1×10-9M、小于1×10-10M、小于1×10-11M或更低。
实施例一人源化糖基化GM2AP抗体的制备
产生及选择对糖基化GM2AP抗原具有特异性的人源化抗体
[根据细则26改正 28.07.2023]
此实例展现对糖基化GM2AP具有特异性的人源化抗体的产生。简言之，以GM2AP-GF以及GM2AP抗原蛋白，自中国台湾生物技术开发中心(Development Center for Biotechnology,DCB)拥有的全合成人源抗体库DSyn 2.1进行抗体筛选(bio-panning)。
1.筛选
用缓冲液稀释GM2AP-GF及GM2AP抗原后，转移至ELISA板，于4℃下反应1晚。以PBS洗涤，加入MPBS与噬菌体反应，之后进行显色与回收。
将回收的噬菌体加入大肠杆菌TGI菌液中反应，铺接在2YT-AG培养基上培养。刮去培养基上的细菌，加入2YT-AG培养基至菌液中，培养至OD600约0.5。加入辅助噬菌体反应。离心除去上清液，加入2YT-AK培养基重新溶解并培养。离心取上清液，加入PEG/NaCl混匀，然后再次离心。去除上清液，用PBS重新溶解沉淀，得到下一轮的噬菌体库。每轮重复上述步骤。
2.用于筛选的噬菌体ELSIA
挑取细菌至96孔板中培养(每孔含2YT-AG培养基)。取菌液加入2YT-A培养基中再次培养。加入辅助噬菌体反应。添加2YT-AK培养基再次培育(噬菌体库)。同时将GM2AP-GF和GM2AP以缓冲液稀释后转移至ELISA板反应。用PBS洗涤后，加入MPBS、噬菌体库反应。用PBST洗涤后，添加噬菌体偶联缓冲液反应。用PBST洗涤后，加入TMB反应。加入终止缓冲液后，以OD450测量。
3.将人类IgG1基因复制到蛋白质表达载体
以专为噬菌体上的VH和VL设计的特异性引物(specific primer)，通过PCR制备各IgGl的插入DNA。蛋白表达载体与限制酶DraIII-HF和BsiWI-HF反应，然后通过凝胶提取试剂盒纯化。添加NheI-HF和MluI-HF并反应。DNA连接反应由T4DNA聚合酶介导，将连接的质粒转化至DH5α宿主中进行分离和扩增。
4.人类IgG1抗体的瞬时表达(transient expression)
将纯化的表达质粒溶解在NaCl溶液中。将聚乙烯亚胺(PEI)溶解在NaCl溶液中。混合两种溶液，并在室温下静置。将混合液放入预先装有Freestyle293细胞培养物的125ml摇瓶中，培养4次后补充新鲜培养基。将转染的细胞放入轨道振荡培养箱中培养7～8天后，收集细胞培养上清液离心并过滤。
5.纯化人类IgG1抗体，并以SDS-PAGE检查蛋白质质量
6.酶联免疫吸附试验ELISA
用缓冲液将抗原稀释至4μg/ml。将100μl溶液添加至96孔ELISA板后，放入4℃冷藏过夜。96孔ELISA板用300μl PBS洗涤3次，然后加入300μl脱脂牛奶-磷酸盐缓冲液(MPBS)，37℃反应2小时。将每种抗体通过3倍系列稀释液，获得12个点(第1个点是10-7M)。连续添加100μl的系列稀释样品于96孔板中，并在37℃下反应90分钟。用300μl PBS洗涤3次，加入100μl偶联缓冲液，于37℃下反应1小时。用300μl PBS洗涤3次后，加入100μl TMB，37℃反应10分钟。最后添加100μl终止缓冲液并测量OD450与OD655。
实施例二 GM2AP-GF与GM2APELISA亲和力测试
利用DSyn2.1人源抗体库筛选抗GM2AP-GF的抗体，经过3～4轮的筛选后，结果如图1所示。显示在第3c、4Ca、4Cb轮中，相较于对照组(抗原GM2AP)，表现单链抗体的噬菌体群对实验组(抗原GM2AP-GF)有较高的ELISA值，OD450-OD655约为2.0～2.7，且实验组与对照组(抗原GM2AP)信号有明显差异，推测这些噬菌体群中可能含有对GM2AP-GF具亲和力的单链抗体。
将筛选出的噬菌体群(3a,3b,3c及4Ca)进行单一噬菌体ELISA assay试验，以挑选出可识别GM2AP-GF的噬菌体。总共筛选出288株，经测序后获得13种抗体序列，如表1-1、1-2所示(皆以Kabat系统表示)。
表1-1重链(Heavy chain)
表1-2轻链(Light chain)
选出的13种序列转构建成full-IgG1形式的抗体，经小量表达纯化后，进行ELISA的特异性和亲和力测试，结果如图2。其中有6株抗体(GF3B-2A1、GF3B-2A12、GF3B-A9、GF3B-2A7、GF3B-2A2、GF4Ca-E1)对GM2AP-GF的OD450-OD655阅读值大于0.2，但GF4Ca-E1对GM2AP结合的OD.450-OD655阅读值为3，与其对GM2AP-GF的阅读值相近，没有特异性，故排除。抗体GF3B-2A7表达量小于3，且在ELISA OD450-OD655的阅读值最低只有0.2，故也排除。最终选出4株抗体：GF3B-2A1、GF3B-2A12、GF3B-A9、GF3B-2A2。
最后使用ELISA进行抗体亲和力分析，结果如图3A～D所示。GF3B-A9(图3A)和GF3B-A12(图3B)两株抗体对GM2AP有较高的背景值，推测可能识别GM2AP-GF靠GF的位置，或是识别GM2AP的一小部分位置。
图3A～D的ELISA亲和力分析结果显示，本公开筛选出的4株抗体，对于糖基化的GM2AP-GF抗原具有较高的亲和力，KD值较低，而对未糖化的GM2AP抗原的亲和力较差或无亲和力，KD值较高或未检出。因此，此4株抗体对GM2AP-GF具高选择性。一般而言，KD小于10-7或10-8即表示具有高亲和力。此外，本公开的4株抗体对GM2AP-GF的KD值皆小于对GM2AP的KD值。
由于此4株抗体对GM2AP-GF具高选择性，根据文献Potprommanee L,Ma HT,Chen CH,Aobchey P,Boonyapranai K,et al.(2013)Human Urinary GM2-activator Protein as a Potential Biomarker for Lung Cancer.J Proteomics Bioinform 6:264-270.的内容，可预期这些抗体具有对癌化细胞来源的GM2AP蛋白有特异性，可作为生物标记或治疗用途。
实施例三抗体的药物组合物
本公开更提供包含本公开抗体的药物组合物。在具体实施例中，该组合物为用于治疗肺癌的药物组合物。该肺癌包括但不限于小细胞肺癌(small cell lung cancer，SCLC)及非小细胞肺癌(non-small cell lung cancer，NSCLC)。
在具体实施例中，该药物组合物包含本公开的抗体与载体。该抗体可以有效量存在于该组合物中以用于达到该组合物的目的。本文中使用的该“有效量”指在个体中达到所希望的效果(如治疗肺癌)所需的活性成分的量，不论是单独或与一种或多种其他活性剂组合。如本领域技术人员所知，有效量根据所治疗的特定病症、病况的严重程度、个体患者参数(包括年龄、身体状况、体型、性别和体重、治疗持续时间、并行治疗的性质(若有)、特定的给药途径以及健康从业者的知识和专业中的类似因素)而变化。这些因素对于本领域技术人员是公知的，并且可以用不超过常规的实验来解决。通常优选使用单一成分或其组合的最大剂量，即根据合理的医学判断的最高安全剂量。然而，本领域技术人员应理解的是，出于医疗因素、心理因素或实际上任何其他原因，患者可能坚持较低剂量或可耐受剂量。
此种抗体药物组合物可通过将具有所需纯度的本公开抗体与一种或多种载体混合而制备。典型地，此类抗体药物组合物可制备为水性溶液(请见如美国专利号6,171,586与WO2006/044908)，或冻干制剂(请见美国专利号6,267,958)。
用于本公开抗体药物组合物的载体可为药学上可接受的载体，一般包括但不限于缓冲剂、赋形剂、稳定剂、防腐剂、溶剂、分散介质、包衣、抗菌剂和抗真菌剂、等渗剂和吸收延迟剂，以及生理上相容的类似物。载体最好是应适用于静脉内、肌肉内、皮下、非经肠胃、脊柱或表皮施用(例如通过注射或输注)。药学上可接受的载体通常在所使用的剂量和浓度下，对于接受它们的受试者而言是无毒的。
各种此类药学上可接受的载体在本领域中为已知(请见如Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))。用于本公开抗体组合物中的示范性药学上可接受的载体包括但不限于：缓冲剂，例如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；六甲铵氯化物；苯扎氯铵；苄索氯铵；苯酚、丁基酚或苯甲醇；对羟基苯甲酸烷基酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；以及间甲酚)；低分子量(少于约10个氨基酸)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；糖类如蔗糖、甘露糖醇、海藻糖或山梨糖醇；形成盐的反离子如钠；金属络合物(如Zn-蛋白质络合物)；和/或非离子表面活性剂如聚乙二醇(PEG)。
用于本公开抗体药物组合物的药学上可接受的载体尚可包括间质药物分散剂(interstitial drug dispersion)，例如可溶性中性-活性透明质酸酶糖蛋白(sHASEGP)(请见如美国专利公开第2005/0260186号与第2006/0104968号)，如人类可溶性PH-20透明质酸酶糖蛋白(如rHuPH20或Baxter International,Inc.)。
该组合物还可包含药学上可接受的赋形剂，如崩解剂、粘合剂、润滑剂、防腐剂或其组合。

Claims (20)

  1. 一种分离的抗体，其包含：
    VH CDR1，其包含选自于SEQ ID NO:9、15、21或27的氨基酸序列；
    VH CDR2，其包含选自于SEQ ID NO:10、16、22或28的氨基酸序列；
    VH CDR3，其包含选自于SEQ ID NO:11、17、23或29的氨基酸序列；
    VL CDR1，其包含选自于SEQ ID NO:12、18、24或30的氨基酸序列；
    VL CDR2，其包含选自于SEQ ID NO:13、19、25或31的氨基酸序列；以及
    VL CDR3，其包含选自于SEQ ID NO:14、20、26或32的氨基酸序列。
  2. 如权利要求1所述的分离的抗体，其中该VH CDR1、VH CDR2、及VH CDR3分别为SEQ ID NO:9、SEQ ID NO:10及SEQ ID NO:11；其中该VL CDR1、VL CDR2及VL CDR3分别为SEQ ID NO:12、SEQ ID NO:13及SEQ ID NO:14。
  3. 如权利要求1所述的分离的抗体，其中该VH CDR1、VH CDR2及VH CDR3分别为SEQ ID NO:15、SEQ ID NO:16及SEQ ID NO:17；其中该VL CDR1、VL CDR2及VL CDR3分别为SEQ ID NO:18、SEQ ID NO:19及SEQ ID NO:20。
  4. 如权利要求1所述的分离的抗体，其中该VH CDR1、VH CDR2及VH CDR3分别为SEQ ID NO:21、SEQ ID NO:22及SEQ ID NO:23；其中该VL CDR1、VL CDR2及VL CDR3分别为SEQ ID NO:24、SEQ ID NO:25及SEQ ID NO:26。
  5. 如权利要求1所述的分离的抗体，其中该VH CDR1、VH CDR2及VH CDR3分别为SEQ ID NO:27、SEQ ID NO:28及SEQ ID NO:29；其中该VL CDR1、VL CDR2及VL CDR3分别为SEQ ID NO:30、SEQ ID NO:31及SEQ ID NO:32。
  6. 如权利要求1所述的分离的抗体，其中VH包含具有与SEQ ID NO:1、3、5或7至少90％同一性的序列；以及VL包含具有与SEQ ID NO:2、4、6或8至少90％同一性的序列。
  7. 如权利要求1至6中任一项所述的分离的抗体，其中该抗体为人源化抗体。
  8. 如权利要求1至6中任一项所述的分离的抗体，其中该抗体为全长抗体或其抗原结合片段。
  9. 如权利要求8所述的分离的抗体，其中该抗体为全长抗体，其为 IgG分子。
  10. 如权利要求1-6中任一项所述的分离的抗体，其中该抗体特异性地结合糖基化修饰的GM2活化蛋白(GM2-activator protein，GM2AP-GF)。
  11. 如权利要求10所述的分离的抗体，其与糖基化修饰的GM2活化蛋白的结合亲和力KD小于1×10-7M、1×10-8M、小于1×10-9M、小于1×10-10M或更低。
  12. 如权利要求10所述的分离的抗体，该抗体与GM2活化蛋白的结合亲和力KD1大于该抗体与糖基化修饰的GM2活化蛋白的结合亲和力KD2。
  13. 如权利要求12所述的分离的抗体，其中该结合亲和力KD1、KD2以酶联免疫吸附分析法(ELISA)测量。
  14. 如权利要求1所述的分离的抗体，其中该抗体进一步与可检测标记、免疫粘附分子、成像剂、治疗剂或细胞毒性剂缀合。
  15. 如权利要求14所述的分离的抗体，其中该成像剂选自于：放射线标记、酶、荧光标记、发光标记、生物发光标记、磁性标记及生物素。
  16. 如权利要求14所述的分离的抗体，其中该治疗剂或细胞毒性剂选自于：抗代谢物、烷化剂、抗生素、生长因子、细胞激素、抗血管生成剂、抗有丝分裂剂、蒽环类、毒素及细胞凋亡剂。
  17. 一种药物组合物，其包含如权利要求1至16中任一项所述的分离的抗体及药学上可接受的载体。
  18. 如权利要求17所述的药物组合物，其中该药学上可接受的载体包含缓冲剂、界面活性剂、盐、氨基酸、抗氧化剂、糖衍生物或其组合。
  19. 一种分离的多核苷酸，其编码如权利要求1至6中任一项所述的抗体；该多核苷酸包含编码抗体的VH的第一序列，其具有与SEQ ID NO:1、3、5或7至少90％的同一性；以及编码抗体的VL的第二序列，其具有与SEQ ID NO:2、4、6或8至少90％的同一性。
  20. 一种载体，包含如权利要求19所述的分离的多核苷酸。
PCT/CN2023/107554 2022-07-14 2023-07-14 人源化岩藻糖基化gm2ap抗体 WO2024012583A1 (zh)

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