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Human mAbs produced by primary in-vitro immunization
Immunology Today, VoL 9, No. 1 I, 1988
35 Good, M.F., Berzofsky,J.A. and Miller, L.H. (1988)Annu.
Rev. Immunol. 6, 663 36 Berzofsky,J.A., Cease, K.B.. Cornette, J.L.etal. (1987) ImmunoL Rev. 98, 9 37 Townsend,A.R.M., Rothbard,J., Gotch, F.M. etaL (1986) Cell 44, 959 38 Maryanski,J.L., Pala, P., Corradin, G., Jordan, B.R.and Cerottini, J-C. (1986) Nature 324, 578 39 Carter, R., Miller, L.H., Rener,J. etal. (1984) Phil. Trans.R. Soc. London Ser. B 307, 201 Scherle, P.A.and Gerhard,W. (1986)J. Exp. Med. 164, "i114 41 Milich, D.R., McLachlan,A., Thornton, G.R. and Hughes,J.L.
(1987) Nature 329, 547 42 Lozner,E.C.,Sachs,D.H. and Shearer,G.M. (1974) J. Exp. Med. 139, 1204 43 Milich, D.R.and Chisari, F.V.(1982)J. ImmunoL 129, 320
Szmuness,W., Stevens,C.E.,Zang, E.A., Harley,E.J.and Kellner, A. (1981)Hepato/ogy 1,377 45 Berzofsky,J.A. (1978)J. Imrnunol. 120, 360 46 McLaughlin,G.L., Benedik,M.J. and Campbell, G.H. (1987) Am. J. Trop. Mecl. Hyg. 37, 258 47 Lawler,J. and Hynes,R.O.(1986)J. Cell. Biol. 103, 1635 Nossal, G.J.V.(1983) Annu. Rev. Immunol. 1,33 4g Gammon, G., Dunn, K., Shastri,N. etaL (1986)Nature 319, 413 $0 Pombo, D., Maloy, W.L., 13erzofsky,J.A. and Good, M.F. (1988)J. Immunol. 140, 3594 51 Etlinger, H., Felix,A.M., Gillessen,D. era/. (1988) J. ImrnunoL 140, 626 52 Lowell, G.H., Ballou,W.R., Smith, L.F.etal. (1988)J. Exp. Med. 167, 658 53 Guerin-Marchand,C., Druihle, P., Galey,B. etaL (1987) Nature 329, 164
man mAbsproducedby primary in- vitro immunization Despite the ease with which rodent monoclonal antibodies can now be produced, their clinical application is limited since they may elicit an anti-globulin response or a hypersensitivity reaction. Human monoclonal antibodies offer obvious advantages. However, as Carl Borrebaeck discusses here, the benefits of human monoclonal antibodies are undermined by the difficulties encountered in producing them. A more efficient method of in-vitro immunization of human B cells using the amino acid ester Leu-OMe may be the key to successful production of human monoclonal antibody-secreting hybridomas. Great expectations for human monoclonal antibodies as therapeutically valuable tools have often been expressed (reviewed in Refs 1-4). To date, however, the use of human monoclonal antibodies in in-vivo trials has been very limited s-7 and the full clinical potential of these antibodies remains to be further investigated. Areas where human monoclonal antibodies may prove useful include passive immunization against viral and bacterial diseases, elimination of drugs and toxins, diagnostic imaging c~f neoplasms, .targeting of drugs to tumors, and modulation of autoimmune conditions. The suggested but only partially verif:.ed advantages of human monoclonal antibodies are, first, they drastically reduce an anti-immunoglobulin (Ig) response when used in vivo. Second, their asparagine-linked carbohydrate sequences are more compatible with Fc receptors on human effector cells, compared with carbohydrate sequences of mouse antibodies. Finally, a different repertoire of antibody specificities can be obtained: human monoclonal antibodies may recognize epitopes not detected by foreign antibodies since xenogeneic immunizations predominantly give antibodies that react mainly with immunodominant structures, such as blood
Departmentof Biotechnology,Universityof Lund, PO Box 124, S-221 O0Lund,Sweden. (~ 1988, ElsevierSciencePublishersLid, UK 0167 4919/88/$0200
Carl A. K. Borrebaeck group substances, monomorphic framework epitopes on major histocompatibility complex (MHC) antigens etc. In contrast, human antibodies tend to recognize polymorphic MHC epitopes, tumor-associated antigens- 'altered self' antigens - and other determinants neglected by the murine immune system 8-11. This implies that completely new and more finely tuned specificities can be obtained with human monoclonal antibodies compared with their murine counterparts. This fact will prevent a more general application of recombinant DNA technology to antibodies 12 by combining the gene segments coding for the complement-determining regions (CDR1-3) from a mouse monoclonal antibody with segments coding for constant regions from human antibodies~3; the specificity of these chimeric antibodies will still be determined by the mouse immune system. However, antibody engineering will probably be very useful in changing the isotype, affinity and specificity of a human monoclonal antibody 14.
In-vitro immunization Human monoclonal antibodies have been produced against a Variety of antigens using lymphocytes from patients sensitized in vivo2. This dependence on in-vivo immunized lymphocytes severely limits the number of human monoclonal antibody specificities that can be obtained and, ethically, only a very limited number of immunogens can be used for deliberate immunizations of humans. In-vitro immunization (primary, antigenspecific activation of B cells in culture) would overcome this severe limitation in human hybridoma technology and allow the production of human monoclonal antibodies against therapeutically valuable antigens.
355
Immunology Today, Vol. 9, No. 1 I, 1988
Table 1. Humanmonoclonalantibodiesproducedbyprimaryimmunizationin culture Refs Antigen Lymphoid Dose Specific /('assa tissue (l~gml-~) efficient)'(M-~.)
(%)
Cells: SRBCb SRBC Allogeneiccells Peptides/proteins: Bombesin-tetanus toxoidconjugate Cardiacmyosin Acidphosphatase Ferritin HBsAG DNasel WT-49& Mann glycoproteins KLH PB1 Haptens: Digoxin Azophenylarsonate DNP EDTA TGAL
Tonsils PBL Spleen
O.IC 0.013c -
17-54 3 5-22
ND ND ND
Spleen Spleen Spleen Spleen Spleen Spleen
0.6 0.I 2 0.5 I
ld 18-32 33 3 20
ND 31 I-3 x 109 40 ND 22 ND 44 ND 44 ND 44
Spleen PBL PBL
1 1 1
5-7 ND 3.1-4.2 ND 3.5 ND
44 45 45
PBL Spleen Spleen Spleen -
I I-I0 1-10 0.01-I I-2
4.8 3 8 1.8 -
0.7 x 108 ND ND ND ND
46 41 42 47 48
0.5e -
-
ND ND
40 40
1
1.1-2.i ND
46
Tumor-associatedantigens: Carcinomas Lung(A549) Spleen Colon(LS174T) Spleen Melanoma p97 PBL
19 39 43
aKass:associationconstant;bSRBC:sheepred bloodcell; c%(vlv)suspensionof SRBC;%nti-bombesinspecificantibodies;eaffinity-purifiedcellsurfaceantigen, usingimmobilized89E5mousemonoclonalantibodies;ND: not determined.
............................... Murine monoclonal antibodies have been produced against more that 50 antigens using hybridomas derived from in-vitro immunized splenocytes (reviewed in Refs 15-18), indicating the general applicability of the production of mouse antibodies. In contrast, human B cells have defied major efforts to apply similar technology in the production of human monoclonal antibodies4.
356
Methodsof humanin-vitro imm~.:~.,;~tion Sheep red blood cells were the first immunogens to which human monoclonal antibodies were produced, using in-vitro immunization of tonsillar lymphocytes ~9. Although red blood cells have been useful for analysis of the B-cell response2o they may not be a representative immunogen. Studies with mouse B cells indicate that antibody production against soluble protein antigens involves carrier-specific and MHC-restricted helper T cells, which are not required for an antigen-specific activation of B cells against red blood cells21, !n-vitro immunization systems with sheep red blood cells as
immunogen could be used for the production of monoclonal antibodies only against red blood cell-bound haptens and not against soluble protein antigens. The in-vitro immune response against sheep red blood cells was also recently argued not to be a true primary antigenic stimulation 22. Nevertheless, the simple in-v.itro immunization system described by Strike eta/. 19 has been used to produce human monoclonal antibodies against human prostatic acid phosphatase using an allogeneic culture of spleen cells in the presence of pokeweed mitogen (PWM) and antigen 22. In-vitro stimulation (i.e. the induction of a secondary immune response in culture) has also been reported to require PWM. This has been used in the production of human monoclonal antibodies against several different viral antigens, using in-vivo presensitized patients23-27. At this stage of development, human in-vitro immunization experiments have generated several contradictory technical findings. Some investigators found human serum to be necessarywhereas other systems did not function without fetal calf serum19,20.28,29; the removal of CD8 + suppressor T cells or suppressor cells bearing type 2 histamine receptors before an in-vitro immunization has been reported to be essential28.29, whereas other investigators have found no effect when these cells were removed 3°,31. Cimetidine has been reported to have a beneficial effect on T-cell mediated suppression of antigen-specific B-cell activation 32, although this was later retracted 33. Ho and co-workers 3~ reported that cimetidine could not enhance an in-vitro antigen-specific response against tetanus toxoid. These opposing reports on human in-vitro immunization illustrate the complexity of the system for human monoclonal antibody production, compared with that of the mouse 4,17,34, and arise for a number of reasons. First, the source of B cells: the most readily available human lymphoid compartment is the peripheral blood. Compared to other sources obtained by surgery (e.g. spleens, tonsils and lymph nodes), peripheral blood lends itself easily ro repeated sampling. Peripheral blood has consistently performed poorly in the production of human monoclonal antibodies and has been considered to be a suboptimaB source of lymphoid cells28,35.36.This may be due to the failure to investigate the activation pathways of B cells with different origins, since, for example, the lymphokine requirements vary depending on the source of the lymphoid cells37. However, if this technology is to become generally available, ways must be found to produce human monoclonal antibodies using human peripheral blood lymphocytes. Second, the source of lymphokines varies: a number of polyclonal activators (PWM, phytohemagglutinin, concanavalin A, lipopolysaccharide, SAC) have been used to stimulate various lymphoid cells and the cell culture supernatants subsequently used as a source of lymphokines 17. In several in-vitro immunization/ stimulation procedures PWM has also been added directly to the cell culture medium 17.~9.22-27. Third, the ratio of T cells to B cells used varies from 1:0.2 to 1:5 and the pretreatment of the cell population used for in-vitro immunization differs !7,19,3~.38, Table ! lists the human monoclonal antibodies that have been produced against haptens, proteins and cells, using primary immunization of predominantly splenocytes in culture.
Immunology Today, Vol. 9, No. 11, 1988
The only valid criterion that shows that an in-vitro immunization has been obtained is that the monoclonal antibodies produced are the result of an antigendependent process and hence do not reflect the existence of naturally occurring antibodies49; a specific immunization has no effect on the repertoire of natural antibodies5°. This criterion is shown to be fulfilled by simply performing mock fusions, using B cells that have been in-vitro immunized in the absence of a specific antigen. As the number of reports on human in-vitro immunization is small, and the above criterion is not always shown to be fulfilled, it is difficult to evaluate the universal applicability of most of the systems 17~43,s~(Table 1).
I "PIaL(M.+) I
I IIe-OMc Ala-OMe Gly-O~ VaI-OMe
pBL(M. d~p,,,.~)1
I PS L ( . . + ) I
1 70% e~ove~; Leui lb, 63D3. & Mo2 ncg.cells
Reversible Ng-cytotoxicity[ _ (2-3 h duration) I
Strategy for improved in-vitro immunization of human B cells In an attempt to develop a general in-vitro immuniz-
ation system for human peripheral blood lymphocytes, the effect of different lymphokines, cell subpopulations38 and cellular regulatory circuits have been investigated ~8,34.4s.46. An in-vitro immunization system has been developed that uses human peripheral lymphocytes separated into accessory cells, B and T cells (at a ratio of 0.25:1:2) and is supported by lymphokines38. This combination of separated peripheral blood lymphocytes and lymphokines was necessary for antigen-specific B-cell activation but not sufficient to constitute a general in-vitro immunization system. Recently, the antigen-specific activation of peripheral B cells was shown to be drastically down-regulated by cytolytic cells such as large granular lymphocytes, cytotoxic and suppressive T cells45. This may explain the previous failure to use human peripheral lymphocytes as a source of immunocompetent B cells for hybridoma production. Normal peripheral B lymphocytes, without further subdivision, do not respond antigen-specifically when r, lttlJrprl in th~ n r p ~ . ~ n r ~ nT ~nt;n~n T~r C-~__7 H~yc38 However, if lysosome-rich subpopulations of human peripheral blood lymphocytes are removed by treatment with the lysosomotropic methyl ester of leucine (LeuOMe), the remaining cells respond antigen specifically during an in-vitro immunization 46. Human peripheral blood lymphocytes, without further subdivision, can in fact be directly immunized in vitro with even higher efficiency compared with separated B cells subsequently treated with Leu-OMe, thus eliminating several laborious cell separation steps45.46. .
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Effect of Leu-OMe
L-amino acid esters are known to act as lysosomotropic agents, having the ability to diffuse freely into the lysosomes of rat heart and liver cells, human monocytes, tissue macrophages and cytotytic cells52-s7. Inside the lysosomes the amino acid ester is rapidly metabolized to the free amino acid which, due to polarity, appears to diffuse out of the lysosomes at a much slower rate than the amino acid ester diffuses in. This leads to an accumulation of free amino acid within these organelles and a secondary swelling and rupture of the lysosomes. Thiele e t a / . 55 showed that L-leucine methyl ester (Leu-OMe) completely removed the accessory cells necessary for mitogen-induced proliferation of human T and B cells. In addition to the lysosomotropic effect of Leu-OMe on monocytes, this agent irreversibly removed natural killer (NK) activity from human peripheral mononuclear
Fig. 1. A summary of the effect of Leu-OMe on huma~ peripheralblood lymphoid cells. Monocytes(Me) condenseLeu-OMeto Leu-Leu-OMewhich inducesan irreversiblecytotoxicity to lysosome-richcells. Otherdipeptidessuchas Leu-Phe-OMe,Phe-Leu-OMe,VaI-Phe-OMeand VaI-Leu-OMecan also causeconcentration-dependentcytocoxicityalthough the production of corresponding dipeptides is very inefficient, as indicated by the hatched area. Mo2 defines monocytes; 63l)3 definesmononuclearphagocytes;Leu 1I ~ definesthe Fcreceptoron natural killer cellsand neutrophilicgranulocytes; OKM1definesmor.ocytes,null cellsand granulocytes. Adapted from Refs45,55,56,58,59.
cells, as assessed by lysis of K562 target cellss6.s8 or by ..................... tile expression of cell surface markers 45. This cytotoxic effect on cytolytic cells such as NK cells is not due to the lysosomotropic property of Leu-OMe, but is due to its , , ,~JI I~J~.y L ~ - U ~ I
IV~U
ly.}U~UI
I I01 L U I I U ~ ' I I~ r l L I U i ~ p l U U U L L
Leu-
Leu-OMes8. A number of different cells, all possessing cytolytic abilities, were shown to be irreversibly deleted following Leu-OMe treatment of periphera! blood mononuclear cells; this included cells such as mixed lymphocyte culture (MLC)-activated NK-like cells, cytotoxic T lymphocytes, and a subset of CD8 + T cells involved in the suppression of PWM-induced generation of immunoglobulin-secreting cells59. The irreversible effect on monocytes was also indicated to be mediated by the dipeptide. Despite the irreversible cytotoxic effects of Leu-OMe/Leu-Leu-OMe on cytolytic cells, these molecules had little or no effect on a number of other human cells:B cells, CD4 ÷ T cells, accessory cells (such as dendritic and vascular endothelial cells) and fibroblasts seem morphologically and functionally undisturbed by treatment with these agents45,55,56,sS.sg. In summary, the rate-limiting step in killing of macrophages, NK cells and CTL is the production of Leu-LeuOMe from Leu-OMe within the lysosomes of monocytes, rather than the lysosomotropic effect of Leu-OMe. The amino acid ester condensation is a product of lysosomal proteinase 1 (P. Lipsky, pers. commun.) which is present in freshly prepared monocytes. A schematic presentation of the action of Leu-OMe is summarized in Fig. 1. Production of human monodonal antibodies Leu-OMe-treated human peripheral blood lymphocytes may thus serve as the basis for a general in-vitro
357
Immunology Today, Vol. 9, No. 1 I, 1988
immunization system. Using this system, in-vitro immunized cells have been used in somatic cell hybridization, and antigen-specific human-human, humanmouse or human-(human-mouse) hybridomas have been successfully constructed 4s.46. Leu-OMe-treated peripheral lymphocytes have, for example, been immunized in culture against hemocyanin, digoxin, PB1 (a recombinant fragment of gp 120 from human immunodeficiency virus (HIV))6°, recombinant envelope peptides of HIV (M. Ohlin et al., pers. commun.) or p97 (a recombinant melanoma-associated antigen) 61. Human hybridomas secreting specific antibodies against these antigens were readily detected 46. No hybridoma secretiP.g antibodies specific for the immunogen were found if the antigen was omitted during the in-vitro immunization step. Several of these hybridomas have been reported to maintain a stable phenotype in culture for periods of more than 5-6 months.
358
This work is supported by grants from the Swedish Cancer Society, Nordisk Industrifond, the National Swedish Board for Technical Development, the Medical Faculty (University of Lund), Hesselmanska Stiftelsen, John and Augusta Persson Foundation, and Magnus Bervall Foundation.
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(1980) Proc. antigen presentation, T-cell help and immune response Natl Acad. ScL USA 77, 6841-6845 modifiers. The most important development will, how- 11 Effros, R.B., Hulette, C. M., Ettinger, R. eta/. (1986) ever, take place using antibody engineering. Although J. Immunol. 137, 1599-1603 the 'humanizing' of mouse monoclonal antibodies has its I:P Morrison, S. (1985) Science 229, 1202-1207 13 Jones,P.T.,Dear, P.H., Foote,J., Neuberger, M.S. and limitation regarding antibody specificity (see above), changing isotypes and regulating antibody affinities and Winter, G. (1986) Nature 321,522-525 specificities will be important technologies when applied 14 Roberts,S., Cheetham, J.C. and Rees,A.R. (1987) Nature 328, 731-734 to human monoclonal antibodies. To obtain the optimal 15 Borrebaeck, C.A.K. (1986) Trends Biotechnol. 4, 147-153 isotype and affinity for therapeutic applications we need 16 Borrebaeck, C.A.K. (1987)J. Pharm. Biomed. 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Immunol. 132, 1798-1803 depend on what application the antibody will be con- :~0 Hoffmann, M.K., Chun, M., Hirst, J.A. and Mittler, R.S. in In Vitro Immunization in Hybridoma Technology structed for, for example, imaging and tumor localization (1988) (Progress in Biotechnology Vol, 5) (Borrebaeck, C.A.K., ed.), require different qualities of the monoclonal antibody. 139-162, ElsevierScience Publishers Furthermore, the immortalization step needs to be im- 21 Marrack, P., Graham, S.D.Jr, Kushnir, E. eta/. (1982) proved to ensure the construction of stable hybridomas Immunol. Rev. 63, 33-49 that enable us to produce large amounts of antibodies in :~2 Yamaura, N., Makino, M., Walsh, L.J., Bruce, A.W. and air-lift fermentors or in culture equipment based on Choe, B-K. (1985)J. Immunol. Meth. 84, 105.-116 hollow fiber/ceramic core units. This might be achieved :~3 Masuho0Y., Sugano,T., Matsumoto, Y., Sawada, 5. and by using transfection of oncogenic DNA62, Epstein-Barr Tomibe, K. (1986)Biochem. Biophys. Res. Commun. 135, virus (EBV) transformation 63, polyethylene glycol (PEG) 495-500 fusions using improved parental cell lines, or combi- 24 Matsumoto, Y., Sugano,T., Miyamoto, C. and Masuho, Y. nations of these technologies. Transfection of oncogenic (1986) Biochem. Biophys. Res. Commun. 137, 272-280 25 Maeda, T., Eda, Y., Nishiyama,K. etal. (1986) Hybridoma 5, DNA is an especially attractive approach since it obviates 33-41 the dependence on low-frequency PEG fusions. :~6 Colucci, G., Kohtz, D. S. and Waksal, S.D. (1986)Liver6, 145-152 Conclusions 27 Sugano,T., Matsumoto, Y., Miyamoto, C. and Masuho, Y. Dorfman ~ stressed in 1985 that in-vitro immunization (1987) Eur. J. Immunol. 17, 359-364 was the major obstacle in the area of human antibody 28 Lagace,J. and Brodeur, B.R.(1985)J. Immunol. Meth. 85, 127-136 development. This is now changing as the requirements for lymphokines and certain cell subpopulations during 29 Cavagnaro,J. and Osband, M.E. 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Immunology Today, Vol. 9, No. 77, 1988
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Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp. 267-276, ElsevierScience Publishers 48 Jacot-Guillarmod, H. (1988) in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp. 295-302, ElsevierScience Publishers 49 Ternynck,T. and Avrameas, S. (1986)Irnmunol. Rev. 94, 99-112 50 Gearing, A.J.H., Bird, C.R., Callus, M. and Thorpe, R. (1986) I-lybridorna 5, 243-247 51 Engleman,E.G., Foung, S.K.H., Larrick,J. and Raubitschek, A. (eds)(1985) Human Hybridomas and Monoclonal Antibodies, Plenum Press 52 Goldman, R. and Kaplan,A. (1973) Biochim. Biophys. Acta 318, 205-216 53 Reeves,J.P.(1979) J. Biol. Chem. 254, 8914-8921 54 Reeves,J.P., Decker, R.S.,Crie, J.S.and Wildenthal, K. (1981) Proc. Natl Acad. Sci. USA 78, 4426-4429 55 Thiele, D.L., Kurosaka, M. and Lopsky, P.E.(1983) J. Immunol. 131, 2282-2290 56 Thiele, D.L. and Lipsky,P.E.(1985) J. hnmunol. 134, 786-793 57 Shau, H. and Golub, S. (1985)J. Imrnunol. 134, 1136-1141 58 Thiele, D.L. and Lipsky,P.E.(1985) Proc. NatlAcad. Sci. USA 82, 2468-2472 59 Thiele, D.L. and Lipsky,P.E.(1986)J. Immunol. 136, 1038-1048 60 Puttney, S.D., Matthews, T.J., Robey,W.G., etal. (1986) Science 234, 1392-1395 61 Rose,T.M., Plowman, G.D., Teplow, D.B. etal. (1986) Proc. Natl Acad. Sci. USA 83, 1261-1265 62 Jonak, Z.L, Owen, J.A. and Machy, P. (1988)in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology I/ol. 5) (Borrebaeck, C.A.K., ed.), pp. 163-192, ElsevierScience Publishers 63 Kozbor, D. (1988) in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp. 193-208, ElsevierScience Publishers
to some extent a personal viewpoint and there are arguably several important developments after 1975 by Q.N Myrvik and R.S. Weiser, Lea and that have been omitted. Febiger, 1988. $34.50 (x + 601 pages) ISBN The next two chapters cover the 0 8721 10870 biology of bacteria and viruses, detailing the morphology of each; their This general softbacktextbook with biochemical composition and, with an eye-catching cover systematically respect to the bacteria, detailing deals with the field of bacteriology some important biochemical pathand mycology in a concise and com- ways, the physiology of their growth prehensive manner. It is 601 pages and giving a taxonomy based largely long, which includes a glossary and upon the organisms' shape and an index. The contents are divided metabolism. I am not sure why the into 40 chapters, each containing a authors have decided to devote one list of references pertaining to that chapter to viral structure in a book chapter. The book is supplemented covering bacteriology and mycology, throughout by line diagrams and particularly when the book does not black and white photographs. cover other aspects of virology or The first chapter is an historical infection by viruses. overview of the subject with a list The following five chapters deal several pages long of important with bacterial genetics, sterilization disinfection; antimicrobial milestones, starting in 1546 with and Fracastoro and ending in 1975 with chemotherapy; the normal microbial Kohler and Milstein. This sort of list is flora and the pathogenesis of dis1988, ElsevierSciencePublishersLtd, UK 0167- 4919/88/$02.00
ease of these infectious agents. The bacterial genetics chapter gives a brief resume of the main elements of genetic organization (such as transposons and plasmids) and genetic control, and the main methods of genetic exchange between bacteria. It does not go into any detail about the mechanism of recombination or repair. In the chapter on disinfection and sterilization, the authors cover physical and chemical methods of sterilization and list a number of disinfectants. British readers should be aware that several of the tradenames used are, of course, American. In the following chapter, the authors go into a little detail on the structure of a number of the most commonly used antimicrobial agents. They approach the subject by discussing the antimicrobial agents in terms of their site of action. The chapter does not cover
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35 Good, M.F., Berzofsky,J.A. and Miller, L.H. (1988)Annu.
Rev. Immunol. 6, 663 36 Berzofsky,J.A., Cease, K.B.. Cornette, J.L.etal. (1987) ImmunoL Rev. 98, 9 37 Townsend,A.R.M., Rothbard,J., Gotch, F.M. etaL (1986) Cell 44, 959 38 Maryanski,J.L., Pala, P., Corradin, G., Jordan, B.R.and Cerottini, J-C. (1986) Nature 324, 578 39 Carter, R., Miller, L.H., Rener,J. etal. (1984) Phil. Trans.R. Soc. London Ser. B 307, 201 Scherle, P.A.and Gerhard,W. (1986)J. Exp. Med. 164, "i114 41 Milich, D.R., McLachlan,A., Thornton, G.R. and Hughes,J.L.
(1987) Nature 329, 547 42 Lozner,E.C.,Sachs,D.H. and Shearer,G.M. (1974) J. Exp. Med. 139, 1204 43 Milich, D.R.and Chisari, F.V.(1982)J. ImmunoL 129, 320
Szmuness,W., Stevens,C.E.,Zang, E.A., Harley,E.J.and Kellner, A. (1981)Hepato/ogy 1,377 45 Berzofsky,J.A. (1978)J. Imrnunol. 120, 360 46 McLaughlin,G.L., Benedik,M.J. and Campbell, G.H. (1987) Am. J. Trop. Mecl. Hyg. 37, 258 47 Lawler,J. and Hynes,R.O.(1986)J. Cell. Biol. 103, 1635 Nossal, G.J.V.(1983) Annu. Rev. Immunol. 1,33 4g Gammon, G., Dunn, K., Shastri,N. etaL (1986)Nature 319, 413 $0 Pombo, D., Maloy, W.L., 13erzofsky,J.A. and Good, M.F. (1988)J. Immunol. 140, 3594 51 Etlinger, H., Felix,A.M., Gillessen,D. era/. (1988) J. ImrnunoL 140, 626 52 Lowell, G.H., Ballou,W.R., Smith, L.F.etal. (1988)J. Exp. Med. 167, 658 53 Guerin-Marchand,C., Druihle, P., Galey,B. etaL (1987) Nature 329, 164
man mAbsproducedby primary in- vitro immunization Despite the ease with which rodent monoclonal antibodies can now be produced, their clinical application is limited since they may elicit an anti-globulin response or a hypersensitivity reaction. Human monoclonal antibodies offer obvious advantages. However, as Carl Borrebaeck discusses here, the benefits of human monoclonal antibodies are undermined by the difficulties encountered in producing them. A more efficient method of in-vitro immunization of human B cells using the amino acid ester Leu-OMe may be the key to successful production of human monoclonal antibody-secreting hybridomas. Great expectations for human monoclonal antibodies as therapeutically valuable tools have often been expressed (reviewed in Refs 1-4). To date, however, the use of human monoclonal antibodies in in-vivo trials has been very limited s-7 and the full clinical potential of these antibodies remains to be further investigated. Areas where human monoclonal antibodies may prove useful include passive immunization against viral and bacterial diseases, elimination of drugs and toxins, diagnostic imaging c~f neoplasms, .targeting of drugs to tumors, and modulation of autoimmune conditions. The suggested but only partially verif:.ed advantages of human monoclonal antibodies are, first, they drastically reduce an anti-immunoglobulin (Ig) response when used in vivo. Second, their asparagine-linked carbohydrate sequences are more compatible with Fc receptors on human effector cells, compared with carbohydrate sequences of mouse antibodies. Finally, a different repertoire of antibody specificities can be obtained: human monoclonal antibodies may recognize epitopes not detected by foreign antibodies since xenogeneic immunizations predominantly give antibodies that react mainly with immunodominant structures, such as blood
Departmentof Biotechnology,Universityof Lund, PO Box 124, S-221 O0Lund,Sweden. (~ 1988, ElsevierSciencePublishersLid, UK 0167 4919/88/$0200
Carl A. K. Borrebaeck group substances, monomorphic framework epitopes on major histocompatibility complex (MHC) antigens etc. In contrast, human antibodies tend to recognize polymorphic MHC epitopes, tumor-associated antigens- 'altered self' antigens - and other determinants neglected by the murine immune system 8-11. This implies that completely new and more finely tuned specificities can be obtained with human monoclonal antibodies compared with their murine counterparts. This fact will prevent a more general application of recombinant DNA technology to antibodies 12 by combining the gene segments coding for the complement-determining regions (CDR1-3) from a mouse monoclonal antibody with segments coding for constant regions from human antibodies~3; the specificity of these chimeric antibodies will still be determined by the mouse immune system. However, antibody engineering will probably be very useful in changing the isotype, affinity and specificity of a human monoclonal antibody 14.
In-vitro immunization Human monoclonal antibodies have been produced against a Variety of antigens using lymphocytes from patients sensitized in vivo2. This dependence on in-vivo immunized lymphocytes severely limits the number of human monoclonal antibody specificities that can be obtained and, ethically, only a very limited number of immunogens can be used for deliberate immunizations of humans. In-vitro immunization (primary, antigenspecific activation of B cells in culture) would overcome this severe limitation in human hybridoma technology and allow the production of human monoclonal antibodies against therapeutically valuable antigens.
355
Immunology Today, Vol. 9, No. 1 I, 1988
Table 1. Humanmonoclonalantibodiesproducedbyprimaryimmunizationin culture Refs Antigen Lymphoid Dose Specific /('assa tissue (l~gml-~) efficient)'(M-~.)
(%)
Cells: SRBCb SRBC Allogeneiccells Peptides/proteins: Bombesin-tetanus toxoidconjugate Cardiacmyosin Acidphosphatase Ferritin HBsAG DNasel WT-49& Mann glycoproteins KLH PB1 Haptens: Digoxin Azophenylarsonate DNP EDTA TGAL
Tonsils PBL Spleen
O.IC 0.013c -
17-54 3 5-22
ND ND ND
Spleen Spleen Spleen Spleen Spleen Spleen
0.6 0.I 2 0.5 I
ld 18-32 33 3 20
ND 31 I-3 x 109 40 ND 22 ND 44 ND 44 ND 44
Spleen PBL PBL
1 1 1
5-7 ND 3.1-4.2 ND 3.5 ND
44 45 45
PBL Spleen Spleen Spleen -
I I-I0 1-10 0.01-I I-2
4.8 3 8 1.8 -
0.7 x 108 ND ND ND ND
46 41 42 47 48
0.5e -
-
ND ND
40 40
1
1.1-2.i ND
46
Tumor-associatedantigens: Carcinomas Lung(A549) Spleen Colon(LS174T) Spleen Melanoma p97 PBL
19 39 43
aKass:associationconstant;bSRBC:sheepred bloodcell; c%(vlv)suspensionof SRBC;%nti-bombesinspecificantibodies;eaffinity-purifiedcellsurfaceantigen, usingimmobilized89E5mousemonoclonalantibodies;ND: not determined.
............................... Murine monoclonal antibodies have been produced against more that 50 antigens using hybridomas derived from in-vitro immunized splenocytes (reviewed in Refs 15-18), indicating the general applicability of the production of mouse antibodies. In contrast, human B cells have defied major efforts to apply similar technology in the production of human monoclonal antibodies4.
356
Methodsof humanin-vitro imm~.:~.,;~tion Sheep red blood cells were the first immunogens to which human monoclonal antibodies were produced, using in-vitro immunization of tonsillar lymphocytes ~9. Although red blood cells have been useful for analysis of the B-cell response2o they may not be a representative immunogen. Studies with mouse B cells indicate that antibody production against soluble protein antigens involves carrier-specific and MHC-restricted helper T cells, which are not required for an antigen-specific activation of B cells against red blood cells21, !n-vitro immunization systems with sheep red blood cells as
immunogen could be used for the production of monoclonal antibodies only against red blood cell-bound haptens and not against soluble protein antigens. The in-vitro immune response against sheep red blood cells was also recently argued not to be a true primary antigenic stimulation 22. Nevertheless, the simple in-v.itro immunization system described by Strike eta/. 19 has been used to produce human monoclonal antibodies against human prostatic acid phosphatase using an allogeneic culture of spleen cells in the presence of pokeweed mitogen (PWM) and antigen 22. In-vitro stimulation (i.e. the induction of a secondary immune response in culture) has also been reported to require PWM. This has been used in the production of human monoclonal antibodies against several different viral antigens, using in-vivo presensitized patients23-27. At this stage of development, human in-vitro immunization experiments have generated several contradictory technical findings. Some investigators found human serum to be necessarywhereas other systems did not function without fetal calf serum19,20.28,29; the removal of CD8 + suppressor T cells or suppressor cells bearing type 2 histamine receptors before an in-vitro immunization has been reported to be essential28.29, whereas other investigators have found no effect when these cells were removed 3°,31. Cimetidine has been reported to have a beneficial effect on T-cell mediated suppression of antigen-specific B-cell activation 32, although this was later retracted 33. Ho and co-workers 3~ reported that cimetidine could not enhance an in-vitro antigen-specific response against tetanus toxoid. These opposing reports on human in-vitro immunization illustrate the complexity of the system for human monoclonal antibody production, compared with that of the mouse 4,17,34, and arise for a number of reasons. First, the source of B cells: the most readily available human lymphoid compartment is the peripheral blood. Compared to other sources obtained by surgery (e.g. spleens, tonsils and lymph nodes), peripheral blood lends itself easily ro repeated sampling. Peripheral blood has consistently performed poorly in the production of human monoclonal antibodies and has been considered to be a suboptimaB source of lymphoid cells28,35.36.This may be due to the failure to investigate the activation pathways of B cells with different origins, since, for example, the lymphokine requirements vary depending on the source of the lymphoid cells37. However, if this technology is to become generally available, ways must be found to produce human monoclonal antibodies using human peripheral blood lymphocytes. Second, the source of lymphokines varies: a number of polyclonal activators (PWM, phytohemagglutinin, concanavalin A, lipopolysaccharide, SAC) have been used to stimulate various lymphoid cells and the cell culture supernatants subsequently used as a source of lymphokines 17. In several in-vitro immunization/ stimulation procedures PWM has also been added directly to the cell culture medium 17.~9.22-27. Third, the ratio of T cells to B cells used varies from 1:0.2 to 1:5 and the pretreatment of the cell population used for in-vitro immunization differs !7,19,3~.38, Table ! lists the human monoclonal antibodies that have been produced against haptens, proteins and cells, using primary immunization of predominantly splenocytes in culture.
Immunology Today, Vol. 9, No. 11, 1988
The only valid criterion that shows that an in-vitro immunization has been obtained is that the monoclonal antibodies produced are the result of an antigendependent process and hence do not reflect the existence of naturally occurring antibodies49; a specific immunization has no effect on the repertoire of natural antibodies5°. This criterion is shown to be fulfilled by simply performing mock fusions, using B cells that have been in-vitro immunized in the absence of a specific antigen. As the number of reports on human in-vitro immunization is small, and the above criterion is not always shown to be fulfilled, it is difficult to evaluate the universal applicability of most of the systems 17~43,s~(Table 1).
I "PIaL(M.+) I
I IIe-OMc Ala-OMe Gly-O~ VaI-OMe
pBL(M. d~p,,,.~)1
I PS L ( . . + ) I
1 70% e~ove~; Leui lb, 63D3. & Mo2 ncg.cells
Reversible Ng-cytotoxicity[ _ (2-3 h duration) I
Strategy for improved in-vitro immunization of human B cells In an attempt to develop a general in-vitro immuniz-
ation system for human peripheral blood lymphocytes, the effect of different lymphokines, cell subpopulations38 and cellular regulatory circuits have been investigated ~8,34.4s.46. An in-vitro immunization system has been developed that uses human peripheral lymphocytes separated into accessory cells, B and T cells (at a ratio of 0.25:1:2) and is supported by lymphokines38. This combination of separated peripheral blood lymphocytes and lymphokines was necessary for antigen-specific B-cell activation but not sufficient to constitute a general in-vitro immunization system. Recently, the antigen-specific activation of peripheral B cells was shown to be drastically down-regulated by cytolytic cells such as large granular lymphocytes, cytotoxic and suppressive T cells45. This may explain the previous failure to use human peripheral lymphocytes as a source of immunocompetent B cells for hybridoma production. Normal peripheral B lymphocytes, without further subdivision, do not respond antigen-specifically when r, lttlJrprl in th~ n r p ~ . ~ n r ~ nT ~nt;n~n T~r C-~__7 H~yc38 However, if lysosome-rich subpopulations of human peripheral blood lymphocytes are removed by treatment with the lysosomotropic methyl ester of leucine (LeuOMe), the remaining cells respond antigen specifically during an in-vitro immunization 46. Human peripheral blood lymphocytes, without further subdivision, can in fact be directly immunized in vitro with even higher efficiency compared with separated B cells subsequently treated with Leu-OMe, thus eliminating several laborious cell separation steps45.46. .
.
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Effect of Leu-OMe
L-amino acid esters are known to act as lysosomotropic agents, having the ability to diffuse freely into the lysosomes of rat heart and liver cells, human monocytes, tissue macrophages and cytotytic cells52-s7. Inside the lysosomes the amino acid ester is rapidly metabolized to the free amino acid which, due to polarity, appears to diffuse out of the lysosomes at a much slower rate than the amino acid ester diffuses in. This leads to an accumulation of free amino acid within these organelles and a secondary swelling and rupture of the lysosomes. Thiele e t a / . 55 showed that L-leucine methyl ester (Leu-OMe) completely removed the accessory cells necessary for mitogen-induced proliferation of human T and B cells. In addition to the lysosomotropic effect of Leu-OMe on monocytes, this agent irreversibly removed natural killer (NK) activity from human peripheral mononuclear
Fig. 1. A summary of the effect of Leu-OMe on huma~ peripheralblood lymphoid cells. Monocytes(Me) condenseLeu-OMeto Leu-Leu-OMewhich inducesan irreversiblecytotoxicity to lysosome-richcells. Otherdipeptidessuchas Leu-Phe-OMe,Phe-Leu-OMe,VaI-Phe-OMeand VaI-Leu-OMecan also causeconcentration-dependentcytocoxicityalthough the production of corresponding dipeptides is very inefficient, as indicated by the hatched area. Mo2 defines monocytes; 63l)3 definesmononuclearphagocytes;Leu 1I ~ definesthe Fcreceptoron natural killer cellsand neutrophilicgranulocytes; OKM1definesmor.ocytes,null cellsand granulocytes. Adapted from Refs45,55,56,58,59.
cells, as assessed by lysis of K562 target cellss6.s8 or by ..................... tile expression of cell surface markers 45. This cytotoxic effect on cytolytic cells such as NK cells is not due to the lysosomotropic property of Leu-OMe, but is due to its , , ,~JI I~J~.y L ~ - U ~ I
IV~U
ly.}U~UI
I I01 L U I I U ~ ' I I~ r l L I U i ~ p l U U U L L
Leu-
Leu-OMes8. A number of different cells, all possessing cytolytic abilities, were shown to be irreversibly deleted following Leu-OMe treatment of periphera! blood mononuclear cells; this included cells such as mixed lymphocyte culture (MLC)-activated NK-like cells, cytotoxic T lymphocytes, and a subset of CD8 + T cells involved in the suppression of PWM-induced generation of immunoglobulin-secreting cells59. The irreversible effect on monocytes was also indicated to be mediated by the dipeptide. Despite the irreversible cytotoxic effects of Leu-OMe/Leu-Leu-OMe on cytolytic cells, these molecules had little or no effect on a number of other human cells:B cells, CD4 ÷ T cells, accessory cells (such as dendritic and vascular endothelial cells) and fibroblasts seem morphologically and functionally undisturbed by treatment with these agents45,55,56,sS.sg. In summary, the rate-limiting step in killing of macrophages, NK cells and CTL is the production of Leu-LeuOMe from Leu-OMe within the lysosomes of monocytes, rather than the lysosomotropic effect of Leu-OMe. The amino acid ester condensation is a product of lysosomal proteinase 1 (P. Lipsky, pers. commun.) which is present in freshly prepared monocytes. A schematic presentation of the action of Leu-OMe is summarized in Fig. 1. Production of human monodonal antibodies Leu-OMe-treated human peripheral blood lymphocytes may thus serve as the basis for a general in-vitro
357
Immunology Today, Vol. 9, No. 1 I, 1988
immunization system. Using this system, in-vitro immunized cells have been used in somatic cell hybridization, and antigen-specific human-human, humanmouse or human-(human-mouse) hybridomas have been successfully constructed 4s.46. Leu-OMe-treated peripheral lymphocytes have, for example, been immunized in culture against hemocyanin, digoxin, PB1 (a recombinant fragment of gp 120 from human immunodeficiency virus (HIV))6°, recombinant envelope peptides of HIV (M. Ohlin et al., pers. commun.) or p97 (a recombinant melanoma-associated antigen) 61. Human hybridomas secreting specific antibodies against these antigens were readily detected 46. No hybridoma secretiP.g antibodies specific for the immunogen were found if the antigen was omitted during the in-vitro immunization step. Several of these hybridomas have been reported to maintain a stable phenotype in culture for periods of more than 5-6 months.
358
This work is supported by grants from the Swedish Cancer Society, Nordisk Industrifond, the National Swedish Board for Technical Development, the Medical Faculty (University of Lund), Hesselmanska Stiftelsen, John and Augusta Persson Foundation, and Magnus Bervall Foundation.
References 1 Dorfman, N.A. (1985) J. Biol. ResponseModif.4, 213-239 2 Larrick,J.W. and Bourla,JM. (1986)J. Biol. Resp. Modif. 5, 379-393 3 Thompson, K.M. (1988)lmmunol. Today9, 113-117 4 James, K. and Bell, G.T. (1987)J. Immunol. Meth. 100, 5-40 5 Phillips,J., Sikora, K. and Watson, J.V. (1982) Lancetii, 1214-1215 6 Burnett, K.G., Hayden,J.M., Hemandez, R. etal. (1987)in Human Hybridomas. Diagnostic and Therapeutic Applications (Immunology Series, Vol. 30)(Strelkauskas, A.J., ed.), pp. 253-265, Marcel DekkerInc. 1 Irie, R.F.and Morton, D.L (1986) Proc. NatlAcad. Sci. USA 83, 8694-8698 8 Kozbor, D. and Roder,J.C.(1983)Immunol. Today4, 72-79 Futuredevelopments 9 Borup-Christensen;P., Erb, K., Jensenius,J.C., Nielsen,B. and Some parameters involved in an optimal human in- Svehag, S.E.(1986) Int. J. Cancer37, 683-688 vitro immunization remain to be investigated, such as 10 Schlom,J., Wunderlich, D. and Teramoto, Y.A. (1980) Proc. antigen presentation, T-cell help and immune response Natl Acad. ScL USA 77, 6841-6845 modifiers. The most important development will, how- 11 Effros, R.B., Hulette, C. M., Ettinger, R. eta/. (1986) ever, take place using antibody engineering. Although J. Immunol. 137, 1599-1603 the 'humanizing' of mouse monoclonal antibodies has its I:P Morrison, S. (1985) Science 229, 1202-1207 13 Jones,P.T.,Dear, P.H., Foote,J., Neuberger, M.S. and limitation regarding antibody specificity (see above), changing isotypes and regulating antibody affinities and Winter, G. (1986) Nature 321,522-525 specificities will be important technologies when applied 14 Roberts,S., Cheetham, J.C. and Rees,A.R. (1987) Nature 328, 731-734 to human monoclonal antibodies. To obtain the optimal 15 Borrebaeck, C.A.K. (1986) Trends Biotechnol. 4, 147-153 isotype and affinity for therapeutic applications we need 16 Borrebaeck, C.A.K. (1987)J. Pharm. Biomed. Anal, 5, to 'tailor make' specific changes to preformed human 783-792 monoclonal antibodies produced by in-vitro immuniza- 11 Borrebaeck, C.A.K. (1988) in In Vitro Immunization in tion. Affinity/specificity and isotype can be changed by Hybridoma Technology (Progressin Biotechnology Vol. 5) site-directed mutagenesis ~4, construction of chimeric (Borrebaeck, C.A.K., ed.), ElsevierScience Publishers human antibodies ~2, or long term in-vitro culture of 18 Borrebaeck, C.A.K.Adv. Drug Delivew Rev. (in press) 19 Strike, L.E., Devens, B.H. and Lundak, R.L.(1984) normal B cells activated antigen specifically. The optimal requirements of a human antibody will J. Immunol. 132, 1798-1803 depend on what application the antibody will be con- :~0 Hoffmann, M.K., Chun, M., Hirst, J.A. and Mittler, R.S. in In Vitro Immunization in Hybridoma Technology structed for, for example, imaging and tumor localization (1988) (Progress in Biotechnology Vol, 5) (Borrebaeck, C.A.K., ed.), require different qualities of the monoclonal antibody. 139-162, ElsevierScience Publishers Furthermore, the immortalization step needs to be im- 21 Marrack, P., Graham, S.D.Jr, Kushnir, E. eta/. (1982) proved to ensure the construction of stable hybridomas Immunol. Rev. 63, 33-49 that enable us to produce large amounts of antibodies in :~2 Yamaura, N., Makino, M., Walsh, L.J., Bruce, A.W. and air-lift fermentors or in culture equipment based on Choe, B-K. (1985)J. Immunol. Meth. 84, 105.-116 hollow fiber/ceramic core units. This might be achieved :~3 Masuho0Y., Sugano,T., Matsumoto, Y., Sawada, 5. and by using transfection of oncogenic DNA62, Epstein-Barr Tomibe, K. (1986)Biochem. Biophys. Res. Commun. 135, virus (EBV) transformation 63, polyethylene glycol (PEG) 495-500 fusions using improved parental cell lines, or combi- 24 Matsumoto, Y., Sugano,T., Miyamoto, C. and Masuho, Y. nations of these technologies. Transfection of oncogenic (1986) Biochem. Biophys. Res. Commun. 137, 272-280 25 Maeda, T., Eda, Y., Nishiyama,K. etal. (1986) Hybridoma 5, DNA is an especially attractive approach since it obviates 33-41 the dependence on low-frequency PEG fusions. :~6 Colucci, G., Kohtz, D. S. and Waksal, S.D. (1986)Liver6, 145-152 Conclusions 27 Sugano,T., Matsumoto, Y., Miyamoto, C. and Masuho, Y. Dorfman ~ stressed in 1985 that in-vitro immunization (1987) Eur. J. Immunol. 17, 359-364 was the major obstacle in the area of human antibody 28 Lagace,J. and Brodeur, B.R.(1985)J. Immunol. Meth. 85, 127-136 development. This is now changing as the requirements for lymphokines and certain cell subpopulations during 29 Cavagnaro,J. and Osband, M.E. (1983) BioTechhiques 1, optimal antigen-specific activation of human B cells 30-36 30 Hoffmann, M.K. and Hirst, J.A. (1985)in Human become more apparent. The down-regulatory effect of and Monoclonal Antibodies (Engleman,E.G., lysosome-rich cytolytic cells on human lymphoid cells is Hybridomas Foung, S.K.H., Larrick,J. and Raubitschek,A., eds), pp. very pronounced, and their removal seems to pave the 277-289, Plenum Press way for a general human in-vitro immunization system 31 Ho, M-K.0 Rand, N., Murray,J., Kato, K. and Rabin, H. using the easily accessible peripheral blood B lympho- (1985)J. Immunol. 135, 3831-3838 cyte. 32 Olsson, L. and Brains, P. (1985)in Human Hybridomas and
Immunology Today, Vol. 9, No. 77, 1988
MonoclonalAntibodies (Engleman,E.G., Foung, S.K.H., Larrick, J. and Raubitschek, A., eds), pp. 227-244, Plenum Press 33 Brams,P. (1988) in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp.37-58, ElsevierScience Publishers 34 Borrebaeck, C.A.K. (1988) in In Vitro Immunization in Hybridorna Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp. 209-230, ElsevierScience Publishers 35 Olsson,L., Kronstr~m, H., Cambon-De Mo~zon, A. etal. (1983)J. Immunol. Meth. 61, 17-32 36 Ho, M-K. (1987)in Human Hybridomas. Diagnostic and Therapeutic Applications (Immunology Series Vol. 30) (Strelkauskas, A.J., ed.), pp. 23-38, Marcel Dekker Inc. 37 Jelinek,D.F. and Lipsky,P.E.(1987)Adv. Immunol. 40, 1-59
38 Danielsson,L., M611er,S.A. and Borrebaeck, C.A.K. (1987) Immunology 61, 51-55 39 Terashima, M., Shimada, S., Komatsu, H. and Osawa, T. (1987) Immunol. Lett. 15, 89-93 Ho, M-K. (1988) in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck, C.A.K., ed.), pp. 247-256, ElsevierScience Publishers 41 Wasserman, R.L., Budens, R.D. and Thaxton~.E.S.(1986) J. Immunol. Meth. 93, 275-283 42 Bieber, M. and Teng, N.N.H. (1987)in Human Hybridomas. Diagnostic and Therapeutic Applications (Immunology Series Vol. 30) (Strelkauskas,A.J., ed.), pp. 39--46, Ma:rcelDekker Inc. 43 Hulette, C.M., Effros, B. and Walford, R.L.(1987! Tissue Antigens 30, 25-33 Pollock, B.J.and d'Apice, A.J.F.(1988)in In Vitro Immunization in Hybridoma Technology (Progress in Biotechnology Vol. 5) (Borrebaeck,C.A.K., ed.), pp. 277-284, ElsevierSciencePublishers 45 Borrebaeck,C.A.K., Danielsson,L. and M611er,S.A. (1987) Biochem. Biophys. Res. Commun. 148, 941-946 46 Borrebaeck,C.A.K., Danielsson,L. and M611er,S.A. (1988) Proc. Nat/ Acad. Sci. USA 85, 3995-3999 47 McRoberts,N., Burnett, K.G. and Boerner, P. (1988)in In
Fundamentalsof Medical Bacteriologyand Mycology
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to some extent a personal viewpoint and there are arguably several important developments after 1975 by Q.N Myrvik and R.S. Weiser, Lea and that have been omitted. Febiger, 1988. $34.50 (x + 601 pages) ISBN The next two chapters cover the 0 8721 10870 biology of bacteria and viruses, detailing the morphology of each; their This general softbacktextbook with biochemical composition and, with an eye-catching cover systematically respect to the bacteria, detailing deals with the field of bacteriology some important biochemical pathand mycology in a concise and com- ways, the physiology of their growth prehensive manner. It is 601 pages and giving a taxonomy based largely long, which includes a glossary and upon the organisms' shape and an index. The contents are divided metabolism. I am not sure why the into 40 chapters, each containing a authors have decided to devote one list of references pertaining to that chapter to viral structure in a book chapter. The book is supplemented covering bacteriology and mycology, throughout by line diagrams and particularly when the book does not black and white photographs. cover other aspects of virology or The first chapter is an historical infection by viruses. overview of the subject with a list The following five chapters deal several pages long of important with bacterial genetics, sterilization disinfection; antimicrobial milestones, starting in 1546 with and Fracastoro and ending in 1975 with chemotherapy; the normal microbial Kohler and Milstein. This sort of list is flora and the pathogenesis of dis1988, ElsevierSciencePublishersLtd, UK 0167- 4919/88/$02.00
ease of these infectious agents. The bacterial genetics chapter gives a brief resume of the main elements of genetic organization (such as transposons and plasmids) and genetic control, and the main methods of genetic exchange between bacteria. It does not go into any detail about the mechanism of recombination or repair. In the chapter on disinfection and sterilization, the authors cover physical and chemical methods of sterilization and list a number of disinfectants. British readers should be aware that several of the tradenames used are, of course, American. In the following chapter, the authors go into a little detail on the structure of a number of the most commonly used antimicrobial agents. They approach the subject by discussing the antimicrobial agents in terms of their site of action. The chapter does not cover
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