- Email: [email protected]
An ELISA for the detection of chimeric and human antibodies that contain κ light chains in nonhuman primate serum
Journal of Immunological Methods, 136 (1991) 143-146 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0022-1759/91/$03.50 ADONIS 002217599100070E
143
JIM 05838
Letter to the editors
An ELISA for the detection of chimeric and human antibodies that contain x light chains in nonhuman primate serum David Wunderlich and Brad Zerler Molecular Therapeutics, Inc., 400 Morgan Lane, W.. Haven, CT 06516, U.S.A. (Received 2 August 1990, revised received 30 October 1990, accepted 1 November 1990)
Dear Editors, Monoclonal antibodies are being used in passive immunotherapy of transplantation rejection (Thistlethwaite et al., 1988; Kirkman et al., 1989; Soulillou et al., 1990), malignant disease (Meeker et al., 1985; Khazaeli et al., 1988; Waldmann et al., 1988; Dyer et al., 1989), and autoimmune disorders (Kyle et al., 1989). Although the initial clinical success observed with rodent monoclonal antibody (mab) therapy is encouraging, the overall therapeutic effectiveness of mab therapy has been impeded by the host immune response against the rodent antibodies (Jaffers et al., 1983; Meeker et al., 1985; Khazaeli et al., 1988). To circumvent this obstacle investigators are using recombinant DNA techniques to join the genes encoding rodent variable regions to genes encoding human constant regions to make chimeric mouse/human antibodies (Morrison, 1985). The presence of the human constant region is expected to decrease the antigenicity associated with the original rodent antibodies (Hale et al., 1988; Lobuglia et al., 1989). Toxicity and pharmacological responses toward mab are usually determined in nonhuman primate model systems. In addition, many antigens targeted for therapeutic intervention, such as the interleukin-2 receptor (IL-2R), are species specific and can only be assayed for efficacy in primates (Shapiro et al., 1989). To avoid problems associated with human population studies, nonhuman
Correspondence to: D. Wunderlich, Molecular Therapeutics, Inc., 400 Morgan Lane, W. Haven, CT 06516, U.S.A.
primates such as cynomolgus monkeys are often used to analyze the therapeutic potential of antiIL-2R antibodies (Shapiro et al., 1989). Among other parameters serum titers of the administered antibodies have to be determined to establish biological efficacy. The titer of murine or rat mab can be assayed in nonhuman primates using the appropriate anti-rodent antibodies in the commonly used sandwich ELISA. However, there is considerable homology between human and nonhuman primate heavy chain constant regions, and chimeric or human antibodies can not be distinguished from nonhuman primate antibodies using a sandwich ELISA with anti-human IgG antibodies. Typically, antiserum to F(ab')2 is produced to quantitate exogenously administered chimeric or human mab in primate sera (LoBuglio et al., 1989). We have found that titers of human kappa light chain containing antibodies can easily be determined in nonhuman primate serum by a sandwich ELISA utilizing commercially available antihuman K constant region antibodies, thereby circumventing the need to develop anti-F(ab')2 antisera.
Chimeric mouse/human anti-IL-2R antibodies were expressed in Chinese hamster ovary (CHO) cells following cotransfection of two expression vectors containing DNA encoding the murine light and heavy chain variable regions coupled to the human kappa and IgG-1 heavy chain constant regions, respectively. Construction of the vectors and derivation of chimeric antibody producing cell lines will be detailed elsewhere (manuscript in preparation). Milligram quantities of chimeric anti-IL-2R antibodies were produced by a single
144
cell transfectant clone cultured in a Flo-Path disposable hollow fiber bioreactor system (Amicon, Danvers, MA). Immunoglobulin was purified from bioreactor harvests by protein G affinity chromatography (ImmunoPure immobilized protein G, Pierce, Rockford, IL). Normal cynomolgus monkey serum was kindly supplied by Dr. Robert Kirkman, Brigham and Women's Hospital, Boston, MA. For enzyme immunoassay (EIA) development, 1 ~tg/well sheep anti-human F(ab')2 antisera (Organon Teknika-Cappel, Malvern, PA), diluted in 0.015 M sodium carbonate, was adsorbed to the surface of Immunolon 1 EIA plates (Dynatech Laboratories, Chantilly, VA) by incubation overnight at 4 ° C . Nonspecific protein binding sites were blocked by incubation for 1 h at room temperature in 0.2 ml PBS supplemented with 0.05% Tween 20 and 1% BSA. Plates were then washed three times with PBS supplemented with 0.05% Tween 20. Cynomolgus monkey sera, 0.1 ml, with or without exogenously added chimeric antibodies, were added and incubated for 1 h at room temperature. Plates were washed as described above and incubated for 1 h at room temperature with 0.1 ml of the indicated dilution of either horse radish peroxidase (HRP) conjugated, affinity-purified, goat anti-human Fc fragment-specific antisera (Kirkegaard and Perry Laboratories, Gaithersburg, MD), or HRP-conjugated, affinitypurified, goat anti-human ~¢ light chain antisera (Fischer Scientific, Pittsburg, PA). HRP-labeled second antibodies were diluted in PBS/Tween-20. Plates were washed three times in P B S / T w e e n 20, and bound secondary antibodies were detected with the substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard and Perry Laboratories), Color development was stopped with 0.05 ml 8 N sulfuric acid and absorbance at 450 nanometers was determined in a V Max kinetic microplate reader (Molecular Devices, Palo Alto, CA). The binding of H R P conjugated anti-human Fc and anti-human ~¢ reagents to cynomolgus monkey sera is shown in Fig. 1. HRP-conjugated anti-human Fc secondary antibodies at a 1/1000 dilution produced high background signals at all dilutions of monkey sera. In contrast, very low background signals were observed with H R P conjugated antihuman K secondary antibodies at a 1/1000 dilu-
rT W
i.2-
LIJ O Z i.0<[ Z 0 o.8W ~3 Z
0.e.
CI3 CC 0 m
0.4.
O.2"
I
- -
1o
I too
CYNOMOLGUS
+-----
I
1ooo
MONKEY
1oooo
SERA
(I/dilution) Fig. l. Binding comparison of anti-human constant region antibodies to cynomolgus monkey serum. EIA plates were coated with sheep anti-human F(ab')2 antisera. Cynomolgus monkey sera was added to the plates at the indicated dilutions followed by either HRP conjugated anti-human Fc fragment specific antisera at a 1/1000 dilution (I), or HRP conjugated anti-human ~ specific antisera at a I/I000 dilution (O). tion. These results suggest that normal monkey immunoglobulin is bound by the primary anti-human F(ab')2 antibodies adsorbed to the EIA wells; this bound immunoglobulin is detected by H R P conjugated anti-human Fc specific antibodies, but not by anti-human ~ specific antibodies. In addition, chimeric antibodies at 10 ~ g / m l could not be detected in cynomolgus monkey sera at any of the indicated dilutions of HRP-anti-human Fc secondary antibodies (Fig. 2A), whereas the chimeric antibodies were detected in monkey sera by the H R P anti-human ~ secondary antibodies at dilutions extending through 1/16,000 (Fig. 2B). The inability of the H R P anti-human Fc antibodies to detect chimeric antibodies in monkey sera appears to be due to the high background signals observed with the reagent. The difference in back-
145 r
B
A t
4-
12
i o-
LQO o.e-[-
[130
Z
it
oe-
Z 0.6--
(_3
\
'
\
~D
i
O.4-
•
~ o~
t--~...11 5
1o
20
40
ao
16o
~°
\
0.2
10
320
20
40
80
HRP-ANTI-HUMAN
HRP-ANT T-HUMAN Fc ( I / d i l u t i o n X ~00)
(1/dilution
•
~
~60
320
KAPPA
X ~00)
Fig. 2. Detection of chimeric m o u s e / h u m a n antibodies in cynomolgus monkey sera with H R P conjugated anti-human constant region antisera. EIA plates were prepared as described in Fig. 1. Cynomolgus monkey sera was diluted 1 / 1 0 and added alone (e) or supplemented with 10 ~ g / m l chimeric antibodies (11) as described in the text. A: HRP-conjugated anti-human Fc specific antisera; B: HRP-conjugated anti-human K antisera.
ground signals observed with the anti-human heavy and light chain secondary antibodies probably reflects high and low protein conservation between human and monkey heavy and light chain constant regions, respectively. This EIA procedure can detect as little as 0.1 #g chimeric antibodies in cynomolgus monkey
°
\
Z 7
sera (Fig. 3). Chimeric antibodies, 5 - 5 0 #g, were added to 1.0 ml of monkey sera and logarithmic dilutions of these sera were assayed for the presence of antibodies and compared to monkey sera without added antibodies. All initial concentrations of chimeric antibodies were detectable, and titration curves were linear through a 1 / 1 0 0 0 dilu-
o.,-
0 if? o.3-
tlJ 0 Z
<
~
\
o.e-
0 m rn o,t-
1o
too
1ooo
:toooo
CYNOMOLGUS MONKEY SERA (l/di lution)
o.os
o.lo
1,oo
so.oo
I 1oo.oo
C H I M E R I C ANTIBODY [ug/mI)
Fig. 3. Sensitivity of anti-human g EL1SA, A: EIA plates were prepared as described in Fig. 1. Chimeric antibodies, 50 #g (O), 10/~g (!1), 5 ~g (A), were added to 1 ml of monkey sera. These stocks were serially diluted as indicated and added to the plates. The open symbol represents monkey sera without added chimeric antibodies. B: data from A plotted as chimeric antibody concentration at each serial dilution vs. absorbance at 450 nm.
146
tion of sera (Fig. 3A). In addition, there was good correlation between the chimeric antibody concentrations determined in each titration curve (Fig.
3B). As chimeric and human mab therapy becomes common, the ability to analyze serum titers and clearance rates of the administered protein in nonhuman primate model systems will become increasingly important to establish biological efficacy. In contrast to developing anti-F(ab')2 antibodies for use in quantitation assays, the procedure outlined in this report is quickly performed and uses commercially available reagents. In addition, the described assay can detect as little as 0.1 /xg of chimeric or human antibodies in nonhuman primate sera.
References Dyer, M.J.S., Hale, G., Hayhoe, F.G.J. and Waldmann, H. (1989) Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of antibody isotype. Blood 73, 1431-1439. Hale, G., Clark, M.R., Marcus, R., Winter, G., Dyer, M.J.S., Phillips, J.M., Riechmann, L. and Waldmann, H. (1988) Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-IH. Lancet i, 1394-1399. Jaffers, G.J., Colvin, R.B., Cosimi, A.B., Giorgi, J.V., Goldstein, G., Fuller, T.C., Kurnick, J.T., Lillehei, C. and Russell, P.S. (1983) The human response to murine OKT3 monoclonal antibody. Transplant. Proc. 15, 646-648. Khazaeli, M.B., Saleh, M.N., Wheeler, R.H., Huster, W.J., Holden, H., Carrano, R. and LoBulgio, A.F. (1988) Phase I trial of multiple large doses of murine monoclonal antibody CO17-1A. II. Pharmokinetics and immune response. J. Natl. Cancer Inst. 80, 937-942. Kirkman, R.L., Shapiro, M,E., Carpenter, C.B., Milford, E.L.,
Ramos, E.L., Tilney, N.L., Waldmann, T.A., Zimmerman, C.E. and Strom, T.B. (1989) Early experience with anti-Tat in clinical renal transplantation. Transplant. Proc. 21, 1766-1768. Kyle, V., Coughlan, R.J., Tighe, H., Waldman, H. and Hazleman, B.L. (1989) Beneficial effect of monoclonal antibody to interleukin 2 receptor on activated T cells in rheumatoid arthritis. Ann. Rheum. Dis. 48, 428-429. LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., Rogers, K., Harvey, E.B., Sun, L., Ghrayeb, J. and Khazaeli, M.B. (1989) Mouse/murine chimeric monoclonal antibody in man: kinetics and immune response. Proc. Natl. Acad. Sci. U.S.A. 86, 4220-4224. Meeker, T.C., Lowder, J., Maloney, D.G., Miller, R.A., Thielemans, K., Warnke, R. and Levy, R. (1985) A clinical trial of anti-idiotype therapy for B cell malignancy. Blood 65, 1349 1363. Morrison, S.L. (1985) Transfectomas provide novel chimeric antibodies. Science 229, 1202-1207. Shapiro, M.E., Reed, M.H., Strom, T.B., Carpenter, C.B., Milford, E i . and Kirkman, R.L. (1989) The role of a primate model of renal transplantation in the development of new monoclonal antibodies. Am. J. Kidney Dis. 5, 58-60. Soulillou, J.-P., Cantarovich, D., Le Mauff, B., Giral, M., Robillard, N., Hourmant, M., Hirn, M. and Jacques, Y. (1990) Randomized control trial of a monoclonal antibody against the interleukin-2 receptor (33B3.1) as compared with rabbit antithymocyte globulin for prophylaxis against rejection of renal allografts. New Engl. J. Med. 322, 11751182. Thistlethwaite, J.R., Stuart, J.K., Mayes, J.T., Gaber, A.O., Woodle, S., Buckingham, M.R. and Stuart, F.P. (1988) Monitoring and complications of monoclonal therapy. Complications and monitoring of OKT3 therapy. Am. J. Kidney Dis. 11, 112-119. Waldmann, T.A., Goldman, C.K., Bongiovanni, K.F., Sharrow. S.O., Davey, M.P., Cease, K.B., Greenberg, S.J. and Longo, D.L. (1988) Therapy of patients with human T-cell lymphotrophic virus l-induced adult T-cell leukemia with anti-tac, a monoclonal antibody to the receptor for interleukin-2. Blood 72, 1805-1816.
143
JIM 05838
Letter to the editors
An ELISA for the detection of chimeric and human antibodies that contain x light chains in nonhuman primate serum David Wunderlich and Brad Zerler Molecular Therapeutics, Inc., 400 Morgan Lane, W.. Haven, CT 06516, U.S.A. (Received 2 August 1990, revised received 30 October 1990, accepted 1 November 1990)
Dear Editors, Monoclonal antibodies are being used in passive immunotherapy of transplantation rejection (Thistlethwaite et al., 1988; Kirkman et al., 1989; Soulillou et al., 1990), malignant disease (Meeker et al., 1985; Khazaeli et al., 1988; Waldmann et al., 1988; Dyer et al., 1989), and autoimmune disorders (Kyle et al., 1989). Although the initial clinical success observed with rodent monoclonal antibody (mab) therapy is encouraging, the overall therapeutic effectiveness of mab therapy has been impeded by the host immune response against the rodent antibodies (Jaffers et al., 1983; Meeker et al., 1985; Khazaeli et al., 1988). To circumvent this obstacle investigators are using recombinant DNA techniques to join the genes encoding rodent variable regions to genes encoding human constant regions to make chimeric mouse/human antibodies (Morrison, 1985). The presence of the human constant region is expected to decrease the antigenicity associated with the original rodent antibodies (Hale et al., 1988; Lobuglia et al., 1989). Toxicity and pharmacological responses toward mab are usually determined in nonhuman primate model systems. In addition, many antigens targeted for therapeutic intervention, such as the interleukin-2 receptor (IL-2R), are species specific and can only be assayed for efficacy in primates (Shapiro et al., 1989). To avoid problems associated with human population studies, nonhuman
Correspondence to: D. Wunderlich, Molecular Therapeutics, Inc., 400 Morgan Lane, W. Haven, CT 06516, U.S.A.
primates such as cynomolgus monkeys are often used to analyze the therapeutic potential of antiIL-2R antibodies (Shapiro et al., 1989). Among other parameters serum titers of the administered antibodies have to be determined to establish biological efficacy. The titer of murine or rat mab can be assayed in nonhuman primates using the appropriate anti-rodent antibodies in the commonly used sandwich ELISA. However, there is considerable homology between human and nonhuman primate heavy chain constant regions, and chimeric or human antibodies can not be distinguished from nonhuman primate antibodies using a sandwich ELISA with anti-human IgG antibodies. Typically, antiserum to F(ab')2 is produced to quantitate exogenously administered chimeric or human mab in primate sera (LoBuglio et al., 1989). We have found that titers of human kappa light chain containing antibodies can easily be determined in nonhuman primate serum by a sandwich ELISA utilizing commercially available antihuman K constant region antibodies, thereby circumventing the need to develop anti-F(ab')2 antisera.
Chimeric mouse/human anti-IL-2R antibodies were expressed in Chinese hamster ovary (CHO) cells following cotransfection of two expression vectors containing DNA encoding the murine light and heavy chain variable regions coupled to the human kappa and IgG-1 heavy chain constant regions, respectively. Construction of the vectors and derivation of chimeric antibody producing cell lines will be detailed elsewhere (manuscript in preparation). Milligram quantities of chimeric anti-IL-2R antibodies were produced by a single
144
cell transfectant clone cultured in a Flo-Path disposable hollow fiber bioreactor system (Amicon, Danvers, MA). Immunoglobulin was purified from bioreactor harvests by protein G affinity chromatography (ImmunoPure immobilized protein G, Pierce, Rockford, IL). Normal cynomolgus monkey serum was kindly supplied by Dr. Robert Kirkman, Brigham and Women's Hospital, Boston, MA. For enzyme immunoassay (EIA) development, 1 ~tg/well sheep anti-human F(ab')2 antisera (Organon Teknika-Cappel, Malvern, PA), diluted in 0.015 M sodium carbonate, was adsorbed to the surface of Immunolon 1 EIA plates (Dynatech Laboratories, Chantilly, VA) by incubation overnight at 4 ° C . Nonspecific protein binding sites were blocked by incubation for 1 h at room temperature in 0.2 ml PBS supplemented with 0.05% Tween 20 and 1% BSA. Plates were then washed three times with PBS supplemented with 0.05% Tween 20. Cynomolgus monkey sera, 0.1 ml, with or without exogenously added chimeric antibodies, were added and incubated for 1 h at room temperature. Plates were washed as described above and incubated for 1 h at room temperature with 0.1 ml of the indicated dilution of either horse radish peroxidase (HRP) conjugated, affinity-purified, goat anti-human Fc fragment-specific antisera (Kirkegaard and Perry Laboratories, Gaithersburg, MD), or HRP-conjugated, affinitypurified, goat anti-human ~¢ light chain antisera (Fischer Scientific, Pittsburg, PA). HRP-labeled second antibodies were diluted in PBS/Tween-20. Plates were washed three times in P B S / T w e e n 20, and bound secondary antibodies were detected with the substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard and Perry Laboratories), Color development was stopped with 0.05 ml 8 N sulfuric acid and absorbance at 450 nanometers was determined in a V Max kinetic microplate reader (Molecular Devices, Palo Alto, CA). The binding of H R P conjugated anti-human Fc and anti-human ~¢ reagents to cynomolgus monkey sera is shown in Fig. 1. HRP-conjugated anti-human Fc secondary antibodies at a 1/1000 dilution produced high background signals at all dilutions of monkey sera. In contrast, very low background signals were observed with H R P conjugated antihuman K secondary antibodies at a 1/1000 dilu-
rT W
i.2-
LIJ O Z i.0<[ Z 0 o.8W ~3 Z
0.e.
CI3 CC 0 m
0.4.
O.2"
I
- -
1o
I too
CYNOMOLGUS
+-----
I
1ooo
MONKEY
1oooo
SERA
(I/dilution) Fig. l. Binding comparison of anti-human constant region antibodies to cynomolgus monkey serum. EIA plates were coated with sheep anti-human F(ab')2 antisera. Cynomolgus monkey sera was added to the plates at the indicated dilutions followed by either HRP conjugated anti-human Fc fragment specific antisera at a 1/1000 dilution (I), or HRP conjugated anti-human ~ specific antisera at a I/I000 dilution (O). tion. These results suggest that normal monkey immunoglobulin is bound by the primary anti-human F(ab')2 antibodies adsorbed to the EIA wells; this bound immunoglobulin is detected by H R P conjugated anti-human Fc specific antibodies, but not by anti-human ~ specific antibodies. In addition, chimeric antibodies at 10 ~ g / m l could not be detected in cynomolgus monkey sera at any of the indicated dilutions of HRP-anti-human Fc secondary antibodies (Fig. 2A), whereas the chimeric antibodies were detected in monkey sera by the H R P anti-human ~ secondary antibodies at dilutions extending through 1/16,000 (Fig. 2B). The inability of the H R P anti-human Fc antibodies to detect chimeric antibodies in monkey sera appears to be due to the high background signals observed with the reagent. The difference in back-
145 r
B
A t
4-
12
i o-
LQO o.e-[-
[130
Z
it
oe-
Z 0.6--
(_3
\
'
\
~D
i
O.4-
•
~ o~
t--~...11 5
1o
20
40
ao
16o
~°
\
0.2
10
320
20
40
80
HRP-ANTI-HUMAN
HRP-ANT T-HUMAN Fc ( I / d i l u t i o n X ~00)
(1/dilution
•
~
~60
320
KAPPA
X ~00)
Fig. 2. Detection of chimeric m o u s e / h u m a n antibodies in cynomolgus monkey sera with H R P conjugated anti-human constant region antisera. EIA plates were prepared as described in Fig. 1. Cynomolgus monkey sera was diluted 1 / 1 0 and added alone (e) or supplemented with 10 ~ g / m l chimeric antibodies (11) as described in the text. A: HRP-conjugated anti-human Fc specific antisera; B: HRP-conjugated anti-human K antisera.
ground signals observed with the anti-human heavy and light chain secondary antibodies probably reflects high and low protein conservation between human and monkey heavy and light chain constant regions, respectively. This EIA procedure can detect as little as 0.1 #g chimeric antibodies in cynomolgus monkey
°
\
Z 7
sera (Fig. 3). Chimeric antibodies, 5 - 5 0 #g, were added to 1.0 ml of monkey sera and logarithmic dilutions of these sera were assayed for the presence of antibodies and compared to monkey sera without added antibodies. All initial concentrations of chimeric antibodies were detectable, and titration curves were linear through a 1 / 1 0 0 0 dilu-
o.,-
0 if? o.3-
tlJ 0 Z
<
~
\
o.e-
0 m rn o,t-
1o
too
1ooo
:toooo
CYNOMOLGUS MONKEY SERA (l/di lution)
o.os
o.lo
1,oo
so.oo
I 1oo.oo
C H I M E R I C ANTIBODY [ug/mI)
Fig. 3. Sensitivity of anti-human g EL1SA, A: EIA plates were prepared as described in Fig. 1. Chimeric antibodies, 50 #g (O), 10/~g (!1), 5 ~g (A), were added to 1 ml of monkey sera. These stocks were serially diluted as indicated and added to the plates. The open symbol represents monkey sera without added chimeric antibodies. B: data from A plotted as chimeric antibody concentration at each serial dilution vs. absorbance at 450 nm.
146
tion of sera (Fig. 3A). In addition, there was good correlation between the chimeric antibody concentrations determined in each titration curve (Fig.
3B). As chimeric and human mab therapy becomes common, the ability to analyze serum titers and clearance rates of the administered protein in nonhuman primate model systems will become increasingly important to establish biological efficacy. In contrast to developing anti-F(ab')2 antibodies for use in quantitation assays, the procedure outlined in this report is quickly performed and uses commercially available reagents. In addition, the described assay can detect as little as 0.1 /xg of chimeric or human antibodies in nonhuman primate sera.
References Dyer, M.J.S., Hale, G., Hayhoe, F.G.J. and Waldmann, H. (1989) Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of antibody isotype. Blood 73, 1431-1439. Hale, G., Clark, M.R., Marcus, R., Winter, G., Dyer, M.J.S., Phillips, J.M., Riechmann, L. and Waldmann, H. (1988) Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-IH. Lancet i, 1394-1399. Jaffers, G.J., Colvin, R.B., Cosimi, A.B., Giorgi, J.V., Goldstein, G., Fuller, T.C., Kurnick, J.T., Lillehei, C. and Russell, P.S. (1983) The human response to murine OKT3 monoclonal antibody. Transplant. Proc. 15, 646-648. Khazaeli, M.B., Saleh, M.N., Wheeler, R.H., Huster, W.J., Holden, H., Carrano, R. and LoBulgio, A.F. (1988) Phase I trial of multiple large doses of murine monoclonal antibody CO17-1A. II. Pharmokinetics and immune response. J. Natl. Cancer Inst. 80, 937-942. Kirkman, R.L., Shapiro, M,E., Carpenter, C.B., Milford, E.L.,
Ramos, E.L., Tilney, N.L., Waldmann, T.A., Zimmerman, C.E. and Strom, T.B. (1989) Early experience with anti-Tat in clinical renal transplantation. Transplant. Proc. 21, 1766-1768. Kyle, V., Coughlan, R.J., Tighe, H., Waldman, H. and Hazleman, B.L. (1989) Beneficial effect of monoclonal antibody to interleukin 2 receptor on activated T cells in rheumatoid arthritis. Ann. Rheum. Dis. 48, 428-429. LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., Rogers, K., Harvey, E.B., Sun, L., Ghrayeb, J. and Khazaeli, M.B. (1989) Mouse/murine chimeric monoclonal antibody in man: kinetics and immune response. Proc. Natl. Acad. Sci. U.S.A. 86, 4220-4224. Meeker, T.C., Lowder, J., Maloney, D.G., Miller, R.A., Thielemans, K., Warnke, R. and Levy, R. (1985) A clinical trial of anti-idiotype therapy for B cell malignancy. Blood 65, 1349 1363. Morrison, S.L. (1985) Transfectomas provide novel chimeric antibodies. Science 229, 1202-1207. Shapiro, M.E., Reed, M.H., Strom, T.B., Carpenter, C.B., Milford, E i . and Kirkman, R.L. (1989) The role of a primate model of renal transplantation in the development of new monoclonal antibodies. Am. J. Kidney Dis. 5, 58-60. Soulillou, J.-P., Cantarovich, D., Le Mauff, B., Giral, M., Robillard, N., Hourmant, M., Hirn, M. and Jacques, Y. (1990) Randomized control trial of a monoclonal antibody against the interleukin-2 receptor (33B3.1) as compared with rabbit antithymocyte globulin for prophylaxis against rejection of renal allografts. New Engl. J. Med. 322, 11751182. Thistlethwaite, J.R., Stuart, J.K., Mayes, J.T., Gaber, A.O., Woodle, S., Buckingham, M.R. and Stuart, F.P. (1988) Monitoring and complications of monoclonal therapy. Complications and monitoring of OKT3 therapy. Am. J. Kidney Dis. 11, 112-119. Waldmann, T.A., Goldman, C.K., Bongiovanni, K.F., Sharrow. S.O., Davey, M.P., Cease, K.B., Greenberg, S.J. and Longo, D.L. (1988) Therapy of patients with human T-cell lymphotrophic virus l-induced adult T-cell leukemia with anti-tac, a monoclonal antibody to the receptor for interleukin-2. Blood 72, 1805-1816.