Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

The complement system is a powerful tool of the innate immune system to eradicate pathogens. Both in vitro and in vivo evidence indicates that therapeutic anti-tumor monoclonal antibodies (mAbs) can activate the complement system by the classical pathway. However, the contribution of complement to the efficacy of mAbs is still debated, mainly due to the lack of convincing data in patients. A beneficial role for complement during mAb therapy is supported by the fact that cancer cells often upregulate complement-regulatory proteins (CRPs). Polymorphisms in various CRPs were previously associated with complement-mediated disorders.

In this review the role of complement in anti-tumor mAb therapy will be discussed with special emphasis on strategies aiming at modifying complement activity. In the future, clinical efficacy of mAbs with enhanced effector functions together with comprehensive analysis of polymorphisms in CRPs in mAb-treated patients will further clarify the role of complement in mAb therapy.     

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)₂ fragments, but not for full-length IgG or for closely-related F(ab’)₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)₂ fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)₂ fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.     

重组抗体工程及其在肿瘤靶向及癌症治疗中的应用

在过去的十几年中,重组抗体工程在基础研究、医学和药物生产上已经成为最有希望的领域之一。重组抗体及其片段在正在进行诊断和治疗的临床试验中占所有生物蛋白的30％以上。研究集中在抗体作为理想的癌症靶向试剂方面,最近由于FDA批准使用第一个工程化治疗抗体而使热度达到极点。过去的几年中,在设计、筛选及生产新型工程化抗体方面已经取得了重大进展。改革的筛选方法已经能够分离出高亲和力的癌－靶向及抗病毒的抗体,后能够抑制病毒用于基因治疗。癌症诊断和治疗的另一个策略是将重组抗体片段与放射性同位素、药物、毒素、酶以及生物传感器表面进行融合。双特异性抗体及相关融合蛋白也已经生产出来用于癌症的免疫治疗,在抗癌疫苗以及T细胞补充策略上有效地增强了人免疫应答。     

Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that^99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAbE48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (moouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into experession vectors containing the human 1 heavy-chain gene and the human k lightchain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9×10¹⁰ M^–1 and 1.6×10¹⁰ M^–1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost incative. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.     

抗CD52单克隆抗体ADCC转基因测活方法的建立

目的 建立转基因细胞法测定抗CD52单克隆抗体(单抗)的抗体依赖细胞介导的细胞毒性作用(antibody-dependentcell-mediatedcytotoxicity,ADCC)生物学活性检测方法。方法 ①以Ramos细胞系作为靶细胞,以Jurkat-hFcγRⅢa/FcεRIγ-NFAT转基因细胞系作为效应细胞,通过荧光素酶检测系统(Bright-GloTM luciferase Assay System)建立抗CD52单抗的ADCC生物学活性检测方法;②对靶细胞、量效范围、效靶比及诱导时间进行优化并进行方法学验证;③研究建立的方法应用于对不同抗CD52单抗的ADCC生物学活性检测。结果 建立抗CD52单抗的ADCC生物学活性检测方法,抗CD52单抗在该方法中存在量效关系,符合四参数方程:y=(A-D)/[1+(x/C)B]+D;经优化后确定抗体量效范围为起始质量浓度360μg/mL,4倍系列稀释9个稀释度;效靶比为3∶1,诱导时间为6.0h;该方法具有良好的专属性;4个不同稀释组回收率样品经3次测定,相对效价分别为(45.58±4.67)%、(71.61±9.45)%、(122.92±7.92)%和(149.94±14.58)%;对应的回收率分别为(91.16±9.34)%、(95.49±12.60)%、(98.34±6.34)%和(99.96±9.72)%,上述结果的变异系数(coefficientofvariation,CV)均小于15%;且该方法也适用于不同抗CD52单抗的ADCC效应评价。结论 利用转基因细胞法成功建立了抗CD52单抗ADCC生物学活性检测方法,该方法专属性强、准确性高、重复性好,可用于评价抗CD52单抗的ADCC生物学活性。     

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.     

Modification of monoclonal antibody carbohydrates by oxidation,conjugation, or deoxymannojirimycin does not interfere with antibody effector functions

Site-specific attachment of metal chelators or cytotoxic agents to the carbohydrate region of monoclonal antibodies results in clinically useful immunoconjugates [Doerr et al. (1991) Ann Surg 214: 118, Wynant et al. (1991) Prostate 18: 229]. Since the capacity of monoclonal antibodies (mAb) to mediate tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may accentuate the therapeutic effectiveness of immunoconjugates, we determined whether site-specific modification of mAb carbohydrates interfered with these functions. The chemical modifications examined consisted of periodate oxidation and subsequent conjugation to either a peptide linker/chelator (GYK-DTPA) or a cytotoxic drug (doxorubicin adipic dihydrazide). mAb-associated carbohydrates were also modified metabolically by incubating hybridoma cells in the presence of a glucosidase inhibitor deoxymannojirimycin to produce high-mannose antibody. All four forms (unaltered, oxidized, conjugated and high-mannose) of murine mAb OVB-3 mediated tumor cell lysis via CDC. Similarly, equivalent ADCC was observed with native and conjugated forms of mAb OVB-3 and EGFR.1. ADCC was achieved with different murine effector cells such as naive (NS), poly (I^*C)- and lipopolysaccharide-stimulated (SS) spleen cells, orCorynebacterium-parvum-elicited peritoneal cells (PEC). All murine effector cell types mediated tumor cell lysis but differed in potency such that PEC>SS>NS. Excellent ADCC activity was also demonstrable by human peripheral blood mononuclear cells with OVB-3-GYK-DTPA and high-mannose OVB-3 mAb. ADCC activity was detectable in vivo: both native and conjugated OVB-3 inhibited growth of OVCAR-3 xenografts in nude mice primed withC. parvum. In conclusion, modification of mAb carbohydrates did not compromise their in vivo or in vitro biological functions. Therefore, combination therapy using immunomodulators to enhance the effector functions of site-specific immunoconjugates could be seriously contemplated.     

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: A case study

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.     

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2^?/? mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.     

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2^−/− mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO? and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Inhibitory monoclonal antibody targeting ADAM17 expressed on cancer cells

ADAM17 is upregulated in many cancers and in turn activates signaling pathways, including EGFR/ErbB, as well as those underlying resistance to targeted anti-EGFR therapies. Due to its central role in oncogenic pathways and drug resistance mechanisms, specific and efficacious monoclonal antibodies against ADAM17 could be useful for a broad patient population with solid tumors. Hence, we describe here an inhibitory anti-ADAM17 monoclonal antibody, named D8P1C1, that preferentially recognizes ADAM17 on cancer cells. D8P1C1 inhibits the catalytic activity of ADAM17 in a fluorescence-based peptide cleavage assay, as well as the proliferation of a range of cancer cell lines, including breast, ovarian, glioma, colon and the lung adenocarcinoma. In mouse models of triple-negative breast cancer and ovarian cancer, treatment with the mAb results in 78% and 45% tumor growth inhibition, respectively. Negative staining electron microscopy analysis of the ADAM17 ectodomain in complex with D8P1C1 reveals that the mAb binds the ADAM17 protease domain, consistent with its ability to inhibit the ADAM17 catalytic activity. Collectively, our results demonstrate the therapeutic potential of the D8P1C1 mAb to treat solid tumors.     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

The role of monoclonal antibody A7 as a drug modifier in cancer therapy

An anticancer antibiotic, neocarzinostatin (NCS), was covalently conjugated to the murine monoclonal antibody A7 (mAb A7), which recognizes the glycoprotein on the cell surface of human colon cancer. The biological and pharmacological properties of the conjugate (A7-NCS) were examined and compared with those of unconjugated NCS. A7-NCS exhibited a strong binding and cytotoxicity to the cell and an antigen-specific tumor accumulation. Significant tumoricidal effects in vivo were observed in the antigen-positive tumor-bearing mice treated with A7-NCS, whereas NCS mixed with mAb A7 and NCS alone were relatively ineffective. In the antigennegative tumor, the tumoricidal effect of A7-NCS was lower than in the antigen-positive tumor. The NCS concentration in blood and tumor were significantly elevated by conjugation to mAb A7. The NCS localization in tumor was higher in the antigen-positive tumor than in the antigen-negative tumor. Death due to acute toxicity was observed at a dose of 20 units (U) NCS in mice treated with unconjugated NCS, whereas toxicity was seen with a much higher dose of NCS (100 U) if the drug was conjugated to the mAb. These findings show that mAb A7 confers more favorable pharmacological properties on an anticancer drug, making it potentially more useful for cancer chemotherapy.This work has been supported in part by a grant-in-aid for cancer research from the Ministry of Education and for the comprehensive 10-year strategy for cancer control from the Ministry of Health and Welfare, Japan     

Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody

Summary A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [³H]thymidine ([³H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by⁵¹Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10⁶. Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30 000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of kerationcytes and certain tumor cells on collagen. This research was supported by a grant from the Elsa U. Pardee Foundation, a Training Grant from the National Institutes of Health, Bethesda, MD, and the Psoriasis Research Institute. Part of this work has appeared as an abstract in Fed. Proc. 43:1929, Abst. #2994, 1984.     

Clinical significance of autologous tumor killing (ATK) activity and its induction therapy in human cancer

The activity of blood lymphocytes to kill autologous freshly isolated tumor cells tested at the time of surgery predicts a favorable clinical course in patients who have primary localized solid tumor and receive curative operation. The strong correlation of autologous tumor killing (ATK) activity with disease-free interval and total survival indicates that ATK activity is a meaningful prognostic indicator and provides evidence for immunological control of tumor growth and metastasis. Although there is no direct evidence that ATK lymphocytes play a critical role in regression of tumor and prevention of tumor regrowth, the lack of ATK activity in patients who relapsed and died may not result from other factors related to their poor performance status, immune functions and tumor characteristics. Clinical trials with ATK induction therapy resulted in an improvement of the clinical outcome in patients who naturally have no such potential. The data indicate that the presence of both natural and induced ATK activity is strongly associated with long-term survival. In addition, adoptive transfer of BRM-induced ATK effector cells resulted in prolongation of survival time even in patients with documented metastatic tumors. Thus, considerable emphasis should be placed on a strategy that induces ATK activityin vivo. Such an approach may provide a new focus for cancer immunotherapy.Abbreviations ATK Autologous tumor killing - BRM biological response modifiers - AIDS acquired immune deficiency syndrome - NK natural killer - LGL large granular lymphocytes - TIL tumor-infiltrating lymphocytes - MHC major histocompatibility complex - TCR T cell antigen receptor - LAK lymphokine-activated killer - IL Interleukin - IFN interferon - TNF tumor necrosis factor - ATKF autologous tumor killing factor - LFA-1 leukocyte function-associated antigen 1 - ICAM-1 intercellular adhesion molecule 1 - mAb monoclonal antibodies     

肿瘤导向治疗的最新临床研究进展

20余年来,肿瘤的导向治疗研究经历了高潮和低潮的多次反复,终于在近几年取得了突破性进展,以Ritux-imab、Tractuzumab和DAB39IL-2为代表的抗肿瘤导向治疗制剂巳经通过FDA批准上市,展示出肿瘤导向治疗的光明前景。     

Site-specific antibody drug conjugates for cancer therapy

Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors—target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris^®) and ado-trastuzumab emtansine (Kadcyla^®). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0–8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.     

Biological significance of autologous tumor-killing activity and its induction therapy

Conclusion The overall results presented in this review demonstrate that positive ATK activity at the time of surgery predicts a favorable clinical course in patients who have primary localized solid tumor and receive curative operation. The strong correlation of ATK activity with disease-free interval and total survival (a) indicates that ATK activity is a meaningful prognostic indicator and (b) provides evidence for immunological control of tumor growth and metastasis. According to these data, it is unlikely that cancer patients who remain tumor-free after 5 years of follow-up will develop recurrence or die from the disease. Although there is no direct evidence that ATK effector cells play a critical role in regression of tumor and prevention of tumor regrowth, the lack of ATK activity in patients who relapsed and died after surgery may not result from factors related to their poor performance status since no differences have been observed in background factors between ATK-positive and-negative groups. The prognostic value of ATK activity in patients with documented metastatic tumors has not been established yet. In this respect, however, the induction of ATK activity by BRM has positively correlated with prolonged survival time, while such a correlation is not observed with other parameters such as NK cells or LAK cell activity.Based on the possible biological significance of ATK activity, clinical trials have been conducted to determine whether the induction of ATK activity before surgery by administration of BRM could improve the clinical outcome in patients who naturally have no such potential. The preliminary data indicate that the presence of both natural and induced ATK activity is strongly associated with longterm survival. Thus, considerable emphasis should be placed on a strategy that induces ATK activity in vivo. Such an approach may provide a new focus for cancer immunotherapy.     

抗盐酸克伦特罗单克隆抗体的固定及活性测定

采用响应面法对抗盐酸克伦特罗抗体的固定化条件进行研究。结果表明：当固定温度4℃,固定方式振荡,抗体稀释倍数5．325×105,料液比0．948mL／,固定时间11．372h时,固定化抗体活力最高,即HRP酶标IgG底物显色反应的OD可达1．4978。优化后的实验值与模型预测值（1．5013）基本相符。