Evidence for a continual exchange of 5'-nucleotidase between the cell surface and cytoplasmic membranes in cultured rat fibroblasts

Approximately 40% of the 5'-nucleotidase activity in cultured rat embryo fibroblasts was patent, as judged by enzymatic assays comparing the activity of intact cells with detergent-solubilized cells. The patent activity was inhibited when cells were incubated with anti-5'-nucleotidase serum at 2 degrees C, whereas latent activity (calculated as the difference between total and patent activity) was not. Latent activity was inhibited by antibody when the antiserum was added directly to detergent-solubilized cells or when cells were cultured in the presence of antiserum for several hours. Patent activity was inhibited by antibody, and cells were returned to culture in antibody-free medium; after 12 hr, 30% of the total activity was expressed in intact cells and 60% of the anti-5'-nucleotidase, assayed by the binding of sheep antirabbit antibodies to intact cells, was lost from the cell surface, indicating an exchange of 5'-nucleotidase between the latent and patent compartments. Cytochemical studies showed that the patent activity was located on the cell surface and that latent activity was present in cytoplasmic vacuoles and vesicles, and in the Golgi complex. Over 30% of the anti-5'-nucleotidase internalized during 6 hr in culture returned to the cell surface after a further 9 hr, indicating a continual exchange of the enzyme between the cell surface and cytoplasmic membranes.     

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Antibody-drug conjugates: Intellectual property considerations

Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.     

Antibody-drug conjugates: Intellectual property considerations

Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.     

Plasmodium berghei infection in pregnant rats: effects on antibody response and course of infection in offspring.

The effects of primary, patent Plasmodium berghei infection in Sprague-Dawley rats during pregnancy upon the course of infection and the humoral antibody response to malaria in their offspring were examined. Malaria specific antibody determined by an indirect fluorescent antibody test correlated well with the parasitologic profiles of each experimental group. Utilization of foster mother groups indicated passive transfer of protective antibody through milk. Evidence for in utero sensitization by soluble malaria antigens was shown by an anamnestic-like antibody response during subsequent infection of offspring from infected mothers.     

全球人冠状病毒抗体领域研发现状与发展趋势

抗体在疾病的诊断、治疗和预防方面发挥着重要作用。随着2019新型冠状病毒(SARS-CoV-2)感染引起的肺炎不断传播,如何研发针对该病毒的抗体迫在眉睫。基于IncoPat数据库的专利信息和Cortellis数据库的药物信息,采用定量分析与定性调研相结合的方法,从申请趋势、技术分布、国家/地区分布、机构分布以及市场现状等维度对人冠状病毒抗体领域的专利进行态势分析。结果表明美国是专利技术拥有量最大的国家,中国是专利保护的重点国家。中国科学院等3家国内机构进入全球前十位。人冠状病毒抗体产品研发主要以SARS和MERS为主,部分MERS抗体已进入临床阶段。研究结果为2019冠状病毒病(COVID-19)的相关抗体研发提供数据参考与决策支撑。     

Babesia microti: Pathogenesis of parasite of human origin in the hamster

The pathogenesis of the disease in hamsters caused by the first human Babesia isolant, tentatively named Babesia microti, and the immunologic relationship of the organism to Babesia canis were studied. The patent phase of the disease was characterized by severe anemia and marked parasitemia which occurred between the 6th and 41st day following infection. An increase in total white cell count with a neutrophilia, eosinophilia, monocytosis, and lymphocytosis was observed during the patent phase. The patent phase was followed by development of a carrier state. This was demonstrated by relapse following splenectomy 113 days after infection. No statistically significant differences were observed between the serum profiles of infected and noninfected animals during the period monitored. A serologic relationship between B. microti and B. canis was revealed by the use of gel diffusion and indirect fluorescent antibody (IFA) tests. The IFA test was used to monitor serum antibody responses during the patent and carrier phases of the disease. Crossabsorption studies between B. canis and B. microti revealed that the two organisms possess common and specific antigens.     

The Cabilly patents: Status quo and relevance for antibody companies

This article discusses the status quo of the Cabilly patents, their scope of protection and the role these patents play for the therapeutic antibody industry in Europe and the US.Key words: antibody, expression, heavy, light, variable, patent, lifetime     

以PD-1 为靶点的抗肿瘤药物专利现状分析

PD-1(程序性死亡受体1)是一种重要的免疫抑制分子,其与癌症的治疗密切相关。目前FDA(美国食品药品监督管理局)已经批准了PD-1抑制剂抗体作为癌症治疗药,因此,PD-1具有极高的研究和应用价值。目前在专利领域,全球各大药企均已经开始专利布局,而在中国申请的专利也是日趋增多,对于我国企业来说,把握时机提前布局专利申请,有利于识别竞争对手,抢占市场。本文通过分析以PD-1为靶点的药物专利现状,特别是研究的重点领域,专利申请的国内重点布局等方面,为国内的申请人未来的研发方向和专利布局提供适当的指引。     

International intellectual property strategies for therapeutic antibodies

Therapeutic antibodies need international patent protection as their markets expand to include industrialized and emerging countries. Because international intellectual property strategies are frequently complex and costly, applicants require sound information as a basis for decisions regarding the countries in which to pursue patents. While the most important factor is the size of a given market, other factors should also be considered.Key words: antibody, patent, international, PCT, filing strategy     

单克隆抗体仿制药物的结构分析策略

市场高达数千亿美元的单克隆抗体药物即将结束专利保护期,这对发展中国家产业升级,提高国民医疗水平,都是一次难得的战略机遇。然而,单抗类蛋白仿制药的研发与质控工作难度巨大,必须使用液相色谱-质谱联用技术进行分析。针对单抗仿制药结构必须进行的分析项目(氨基酸序列表达正确性、糖基化修饰形态相似性、以及高级结构统一致性)的液质分析方法进行了系统介绍。此外还就分析工作效率及质控过程的法规依从问题予以讨论。     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guardians at the gate: Biosimilar and patent reform legislation could fundamentally change the guards for therapeutic monoclonal antibodies—Part 2

Patent protection and FDA exclusivities are the two principal forms of protection available to companies that develop therapeutic monoclonal antibodies. Propo-sed changes to both forms of protection are currently being debated in the United States Congress. Specifically, Congress is presently debating both biosimilar and patent reform legislations. Although no bill has yet passed, it is expected that patent reform legislation should pass this year. It is less likely that a biosimilar bill will pass this year. However, when legislations are enacted, the changes will significantly impact the business of therapeutic monoclonal antibodies.Key words: patent reform, biosimilar legislation, therapeutic antibody, Eshoo, Waxman, S 615, HR 1260     

Patenting antibodies in Europe

As a complement to the series of articles beginning in this issue on obtaining, maintaining and enforcing antibody patents in the United States, this article provides a comparison of the requirements for patenting antibodies in the United States and Europe and provides examples illustrating the application of European patent law to antibodies.     

Lyophilized veins as arterial interposition allografts

Venous allografts were evaluated in two models. Lyophilized allograft veins used as interposition grafts in the infrarenal aorta of the canine were studied and found to be patent at 1 year. Pathologic examination of the grafts revealed mild intimal hyperplasia and persistence of the basic structure of the lyophilized vessel. The ability of venous tissue to elicit an antibody response when transplanted into an allogeneic recipient was studied in the rat using the lymphocyte cytotoxicity assay. Fresh and Me₂SO-cryoprotected frozen veins produced circulating antibody when used as interposition grafts in the infrarenal aorta of the rat. Lyophilized and noncryoprotected frozen veins did not induce measurable antibody. Lyophilized allograft veins are a nonimmunogenic vascular graft material with acceptable long-term patency.     

Action of a thymic cytokine TsIF in reversing the autoimmune disease state of the MRL/1pr mouse

T suppressor cells differentiate from bone marrow precursors when cocultured with thymic epithelium, a thymic-derived cytokine TsIF, or mixture of both. (TsIF is a trademark of Ventrex Laboratories, Inc., Portland, ME, and is the subject of a U.S. patent by Ventrex Laboratories, Inc., Portland, ME.) These cells, when transplanted into the lupus-rheumatoid arthritis-prone mouse, prevent acquisition of disease as assessed by lack of both antinuclear antibody, rheumatoid factor, and survival beyond mean time for MRL/lpr mice. When TsIF is administered directly into these lupus-rheumatoid arthritis-prone mice, an equivalent sparing effect is manifested.     

Correlation between protective antibody response and patent infection with Hymenolepis nana in mice

Correlation between protective antibody response and patent infection with Hymenolepis nana in mice. International Journal for Parasitology16: 197–203. Mice inoculated with mouse-derived cysticercoids of Hymenolepis nana, as well as with eggs, produced IgG and IgE antibodies that were detected by double diffusion (DD) and passive cutaneous anaphylaxis (PCA), respectively. When mice inoculated with eggs (day 0) were challenged with eggs (day 66), all were resistant to the challenge (assessed by the failure of cysticercoid recovery in the intestinal tissue) and produced protective antibodies evidenced by passive transfer, as well as IgG and IgE isotypes. When mice inoculated with eggs (day 0) were treated with a highly efficacious cestocide, praziquantel on day 6 at the beginning of the lumen phase, all were also resistant to the egg challenge on day 66, however, IgE, IgG, and protective antibodies were not detected. When mice treated with praziquantel before patent infection were repeatedly challenged with high doses of eggs, some of them produced IgG and IgE antibodies. From these results, it is suggested that (1) the production of protective antibody is a secondary response after patency (which may be ascribed to eggs released from mature worms), and (2) mice initially given eggs are highly resistant to egg challenge showing that an effector mechanism of acquired resistance to egg challenge may be expressed without high titres of protective antibody, at least in the serum.     

The lone inventor: low success rates and common errors associated with pro-se patent applications

A pro-se patent applicant is an inventor who chooses to represent himself while pursuing ("prosecuting") a patent application. To the author's knowledge, this paper is the first empirical study addressing how applications filed by pro-se inventors fare compared to applications in which inventors were represented by patent attorneys or agents. The prosecution history of 500 patent applications filed at the United States Patent and Trademark Office were analyzed: inventors were represented by a patent professional for 250 of the applications ("represented applications") but not in the other 250 ("pro-se applications"). 76% of the pro-se applications became abandoned (not issuing as a patent), as compared to 35% of the represented applications. Further, among applications that issued as patents, pro-se patents' claims appear to be narrower and therefore of less value than claims in the represented patent set. Case-specific data suggests that a substantial portion of pro-se applicants unintentionally abandon their applications, terminate the examination process relatively early, and/or fail to take advantage of interview opportunities that may resolve issues stalling allowance of the application.