Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes.

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and kappa light (L) chain loci. Each of the two H and the two kappa chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique VH (5 versus 66) or Vkappa genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by VH and Vkappa gene complexity: (a) production of functional mu chains at the transition from the pre B-I to the pre B-II stage; (b) productive VkappaJkappa recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of mu chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.

Acquisition of mitogenic responsiveness by nonresponding lymphocytes upon insertion of appropriate membrane components.

The effect of insertion of plasma membrane components from lymphocytes responding to mitogens into the membranes of nonresponding cells using Sendai virus envelopes as vehicles was examined. T cells modified by B membranes were stimulated by lipopolysaccharide (LPS) to proliferate as well as to produce interleukin-2 activity. B cells modified by T membranes were stimulated by concanavalin A to proliferate and to produce interleukin-2 activity. B cells derived from C3H/HeJ LPS-nonresponder strain of mice, when modified by B membranes derived from the LPS-responder C3H/eb strain, acquired LPS responsiveness. These findings indicate that the inability of either T or B cells to respond to specific mitogens is due to the lack of suitable plasma membrane constituents and that by changing the membrane composition the lymphocytes can be endowed with new functions.

Monoclonal antibody therapy for prostate cancer.

Early detection of prostate cancer (PCa) and advances in hormonal and chemotherapy treatments have provided great clinical benefits to patients with early stages of the disease. However, a significant proportion of patients still progress to advanced, metastatic disease, for which no effective therapies are available. Therefore, there is a critical need for new treatment modalities, ideally targeted specifically to prostate cancer cells. The recent clinical and commercial successes of monoclonal antibodies (MAbs) have made them the most rapidly expanding class of therapeutics being developed for many disease indications, including cancer. PCa is well suited for antibody-based therapy due to the size and location of recurrent and metastatic tumors, and the lack of necessity to avoid targeting the normal prostate, a nonessential organ. These properties have fostered interest in the development and clinical evaluation of therapeutic MAbs directed to both well established and newly discovered targets in PCa. MAbs directed to established targets include those approved for other solid tumors, including anti-human epidermal growth factor receptor-2 (HER2) MAb trastuzumab, anti-epidermal growth factor receptor (EGFR) MAbs cetuximab and panitumumab, and the antivascular endothelial growth factor (VEGF) MAb bevacizumab. Genomics efforts have yielded a large number of novel, clinically relevant targets in PCa with the desirable expression profiling in tumor and normal tissues, and with an implicated role in tumor growth and spread. Growing efforts are directed to the development of naked or payload-conjugated therapeutic antibodies to these targets, and a variety of MAb products are currently progressing through preclinical and various stages of clinical development. The clinical experience with some of the commercialized MAb products points out specific challenges in conducting clinical trials with targeted therapy in PCa.

YAC vectors. Humanizing the mouse genome.

The ability to replace mouse genes with their human equivalents using "yeast artificial chromosome" technology provides a powerful new technique for studying the regulation and function of human genes.

A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy.

A fully human IgG2kappa monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and kappa light chain loci. The E7.6.3 MAb exhibits high affinity (KD = 5 x 10(-11) M) to the receptor, blocks completely the binding of both EGF and transforming growth factor alpha (TGF-a) to various EGFr-expressing human carcinoma cell lines, and abolishes EGF-dependent cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. The antibody (0.2 mg i.p. twice a week for 3 weeks) prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, the administration of E7.6.3 without concomitant chemotherapy results in complete eradication of established tumors as large as 1.2 cm3. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total E7.6.3 doses as low as 3 mg, administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice. No tumor recurrence was observed for more than 8 months after the last antibody injection, which further indicated complete tumor cell elimination by the antibody. The potency of E7.6.3 in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for the treatment of multiple EGFr-expressing human solid tumors, including those for which no effective chemotherapy is available. Being a fully human antibody, E7.6.3 is expected to exhibit minimal immunogenicity and a longer half-life as compared with mouse or mouse-derivatized MAbs, thus allowing repeated antibody administration, including in immunocompetent patients. These results suggest E7.6.3 as a good candidate for assessing the full therapeutic potential of anti-EGFr antibody in the therapy of multiple patient populations with EGFr-expressing solid tumors.

Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia.

We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.

Production of fully human antibodies by transgenic mice.

The ability to produce a diverse repertoire of fully human monoclonal antibodies may have significant applications to human therapy. One of the most promising approaches to the production of therapeutic human monoclonal antibodies is the creation of a mouse strain engineered to produce a large repertoire of human antibodies in the absence of mouse antibodies. Recently, such mice have been generated by introducing segments of human immunoglobulin loci into the germlines of mice deficient in mouse antibody production as a result of gene targeting. These mice produce significant levels of fully human antibodies with a diverse adult-like repertoire and, upon immunization with antigens, generate antigen-specific fully human monoclonal antibodies. Such strains of mice may provide the optimal source for producing human monoclonal antibodies with high affinity and specificity against a broad spectrum of antigens, including human antigens.

Specific lysis of antigenically irrelevant cells by cytotoxic T lymphocytes upon insertion of appropriate antigens into the target cell plasma membranes.

The importance of the membrane milieu to functional presentation of target cell (TC) antigens to cytotoxic T lymphocytes (CTL) was investigated by examining the interaction of CTL with TC plasma membrane (PM) fractions, in isolated form or integrated into antigenically irrelevant TC. Isolated ascitic vesicles, microsomes, and purified PM, containing serologically defined alloantigens that have been implicated as the relevant TC antigens, effectively, yet nonspecifically, inhibited the binding and lysis of TC by CTL. The same PM fractions, when inserted into antigenically irrelevant TC via vesicles containing Sendai virus components, rendered the TC susceptible to CTL-mediated lysis directed against the inserted antigens. These findings suggest that CTL interact specifically with TC determinants only when they are embedded in the proper membrane environment.

Early intrauterine development: I. The rate of growth of Caucasian embryos and fetuses between the 6th and 20th weeks of gestation.

Liberalization of abortion laws in various countries and states of Europe and America has offered an opportunity for the study of the correlations between menstrual age and the rate of bodily development of human embryos and fetuses. In several institutions where local bylaws mandated the consent of the patients requesting therapeutic abortion to pathological investigation of the products of conception, various parameters of growth were measured systematically. These included crown-rump and foot lengths and body weight. The analysis of the data indicated that in the embryosic stages of development the rate of growth is substantially slower than it had been assumed previously. In the more advanced fetal stages of development the findings supported the validity of the long-established standard of Streeter. Since the current study has been based on apparently normal gestations of healthy women, whereas earlier data had rested, to a great extent, on spontaneously aborted and extopic embryon and fetuses, it seems evident that pathological specimens are not suitable for the purpose of establishing reliable standards of normal intrauterine growth rate. The present data support the suggestion, based on various experimental and clinical observations, that pathological gestations often result from abnormal ovulations that occur at times other than the mid-cycle and that such conceptions are frequently followed by a bleeding episode that simulates menstruation. It is likely that the inclusion of a high number of such cases biased the results of earlier investigations concerning embryonic growth rate on account of the frequent incidence of erroneous menstrual histories. The material available did not permit the extension of the investigation beyond the 20th week of gestation. It appears, however, that the average growth rate in the mid-trimester may be slower than the data adopted by the American Academy of Pediatrics would indicate. The possible causes of some phenomena observed in the course of the investigation, such as unexplained scatter of growth-rate patterns at all developmental stages and discrepancies among various literary data, have been discussed in some detail. While a tentative attempt has been made to correlate the investigated parameters of fetal growth with biparietal diameters of the head, a measurement accessible to direct assessment by sonography during pregnancy, the necessity of improving these standards through a prospective study has been emphasized.

Early intrauterine development: II. The rate of growth in Black and Central American populations between 10 and 20 weeks' gestation.

The application of the growth-rate standards, extablished for Caucasian embryos and fetuses in a previous report, to Black and Central American racial groups has been investigated. Comparison between menstrual age and crown-to-rump length indicated differences in the 10 to 15 weeks' gestation range. However, growth rates for the same groups were practically identical between the 15th and 20th weeks of pregnancy. This finding suggests that the actual rate of growth is closely similar in the respective ethnic groups and that apparent discrepancies reflect erroneous, or purposefully false, menstrual histories rather than dissimilar growth patterns. Largely identical rates of development were suggested by the crown-rump length to foot length to body weight interrelations among the various racial groups. A moderate, but rather predictable, deviation from the earlier established standards was noted in the crown-rump length versus foot length ratios of Black American fetuses, providing the only exception to what appears to be a practically identical rate of growth for the investigated ethnic groups in the first half of gestation. The evaluation of the results was extended to involve the effect of educational and social factors on currently available data of embryonic and fetal growth. It is suggested that heretofore unconsidered factors may affect the validity of widely quoted standards of intrauterine growth.

Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs.

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Sex ratio of the stillborn fetuses and neonates dying in the first week.

We have studied the autopsy findings of 220 stillborn fetuses and 319 neonates who died during the first week of life and were born between January 1, 1964 and December 31, 1983. An attempt was made in the course of this review to determine the sex ratio in various weight groups and aetiological categories. In almost all weight groups and all pathogenetic categories, the number of males was higher than that of females, providing an overall sex ratio of 147 of the stillborn fetuses and neonates dying during the first week.

The adolescent childbirth rate in Hungary.

The rate of childbirth of adolescents in Hungary is reviewed. The rate of childbirth increased among teenagers during the last half century.

The abnormalities of the presentation in twin pregnancy and perinatal mortality.

The author conducted a retrospective review of the obstetric patient material in his department between January 1, 1975 and December 31, 1992, concerning the clinical implications associated with various lies and presentations of twins. The material included 329 twins (1.07% of all births). Breech, or transverse lies complicated 186 (56.53%) twin gestations. The frequency of premature newborns was among 186 malpresented twin pairs 85 (45.7%) and 101 (54.3%) were mature. The percentage of malpresented twin fetuses increased with maternal age and parity. Abnormal presentations were more frequent (78.49%) among B than A fetuses (54.84%). Perinatal mortality was more frequent among B than A fetuses and more frequent between malpresented twin pairs than the vertex-vertex presented twins.

Sex ratio of spontaneously aborted fetuses and delivered neonates in second trimester.

The author investigated in patient material from 12 years 251 cases involving spontaneous abortion and delivery in the second trimester. The material included 28 sets of twins and one set of triplets, thus the total number of fetuses and neonates was 281. The sex ratio was 136.1, higher than that documented natural sex ratio in the second trimester or at term. The ratio approaches that experienced in cases of perinatal death.

Medico-legal aspects of brachial plexus injury: the obstetrician's point of view.

The author reviews the obstetric literature with regard to factors predisposing to or predicting the occurrence of brachial plexus injury in the newborn. Based on the evaluated data, it is concluded that, whereas there are identifiable predisposing factors for shoulder dystocia, to which the occurrence of brachial plexus injury is widely attributed, the predictive power of the various identifiable factors is generally low. The writer also quotes literary data which suggest that injury to the brachial plexus may occur in utero before labor and, thus, unrelated to arrest of the shoulders during the process of delivery. The contemporary literature contains diverse and often contradictory opinions which do not provide clear-cut guidelines for the practicing obstetrician for the prevention of brachial plexus injury. In some parts of the world a disproportionately large number of malpractice claims against obstetricians derive from such injuries. Therefore, the formulation of a consensus concerning the definition of shoulder dystocia, the identification of preventive measures to be utilized to avoid its occurrence and the required clinical management in case of arrest of the shoulders are unresolved problems that the medical profession needs to address.

Infanticide: new medical considerations.

A relationship between disturbed hormonal and metabolic equilibrium during puerperium and ensuing mental and emotional imbalance has been postulated for a long time. In England and Wales this consideration has resulted in the crime of infanticide being reduced from murder to manslaughter. Recent data indicate that the therapeutic and even routine administration by physicians of powerful vasoactive agents during puerperium, and particularly that of ergot derivatives, is probably the most frequent factor leading to 'pure puerperal psychosis'. It is proposed, therefore, that this psychosis is in fact, in most instances, a form of ergotism and its signs and symptoms and consequences, including coincidental infanticide, themselves are actually manifestations of acute ergot poisoning. It is suggested that based on this information a thorough re-evaluation of the relevant legal concepts is necessary.