0022-1767/91/1474-1352$02.00/0
`TUE JOUHNAL OF ~MMUNOLOGY
`Copyright 0 1991 by The American Association of Immunologists
`
`Val. 147. 1352-1359. No. 4. August 15. 1991
`Prfnted in U.S.A.
`
`REDUCED IMMUNOGENICITY AND IMPROVED PHARMACOKINETICS OF
`HUMANIZED ANTI-Tac IN CYNOMOLGUS MONKEYS
`
`R. LUKE,* PHILIP C. FAMILLETTI,'
`JOHN HAKIMI,'' RICHARD CHIZZONITE,' DAVID
`PASCAL BAILON," J O A. KONDAS,'
`LIN,' DAVID V. WEBER,"
`ROBERT S. PILSON,' PING
`CHERYL SPENCE," LISA J . MONDINI,' WEN-HUI TSIEN,' JAMES
`L. LEVIN," VON H. GALLATI,"
`AND WILLIAM R. BENJAMIN'
`LAURENCE KORN,+' THOMAS A. WALDMANN,** CARY QUEEN,+'
`From the Departments of "Imrnunopharmacology, 'Molecular Genetics, 'Drug Metabolism, 'Bioprocess Development, and
`"Protein Biochemistry, Roche Research Center, Hoffmann-La Roche, lnc., Nutley, NJ 071 10; 'TSl Mason Research Institute.
`Worchester, MA 01 608; ''Central Research, F. Hoffrnann-La Roche Ltd., 4002 Basel, Switzerland; *Protein Design Labs, Inc.,
`Mountain View, CA 94043; "Metabolism Branch, National Cancer Institute, National Institute of Health. Bethesda, MD 20892
`
`The anti-Tac mAb has been shown to bind to the
`p55 chain of the IL-PR, block IL-2 binding and in-
`hibit T cell proliferation. A humanized form of anti-
`Tac (HAT) has been constructed that retains the
`binding properties of murine anti-Tac (MAT). These
`two mAb were evaluated in cynomolgus monkeys to
`compare relative immunogenicity and pharmacoki-
`netic properties. Monkeys treated with HAT daily
`for 14 days exhibited anti-HAT
`antibody titers
`which were 5-
`to 10-fold lower than their MAT-
`treated counterparts and these
`antibodies devel-
`oped later than in the MAT-treated monkeys. Two
`of four monkeys receiving a single injection of MAT
`developed anti-MAT antibodies, whereas none of
`four monkeys developed antibodies after a single
`treatment with HAT. In monkeys injected with
`either HAT or MAT daily for 14 days, the anti-anti-
`body titers induced were inversely related to the
`amount of anti-Tac administered. Antibodies that
`developed against MAT were both anti-isotypic and
`anti-idiotypic, whereas those developed against
`HAT appeared to be predominantly anti-idiotypic.
`The pharmacokinetic properties, that is the half-life
`and area under the curve values, of HAT were also
`significantly different from those of MAT. The area
`under the curve values for HAT in naive monkeys
`were approximately twofold more than those for
`MAT, and the mean serum half-life of HAT was 214
`h, approximately four- to fivefold more than MAT.
`These pharmacokinetic values were reduced in
`monkeys previously sensitized with HAT or MAT
`suggesting that the presence of anti-antibodies al-
`tered these parameters.
`
`of at least two polypeptide chains that can independently
`bind IL-2: the p55, IL-2R a chain, or Tac peptide (2, 3),
`and the more recently discovered p75 or IL-2R p chain (4,
`5). Study of the p55 peptide was facilitated by the devel-
`opment of a mAb, MAT, which binds to human p55 (2).
`The Tac peptide is expressed on the surface of Ag- or
`mitogen-activated T cells but not on resting T cells. More-
`over, treatment of human T cells with MAT strongly
`inhibits their proliferative response to Ag or to IL-2 by
`preventing binding of IL-2 to p55 (3, 6).
`High levels of p55 are expressed on malignant cells of
`some lymphoid cancers such as adult T cell leukemia,
`cutaneous T cell lymphoma and Hodgkin's disease (1).
`Increased or abnormal IL-2R expression is also associated
`with many autoimmune conditions including rheumatoid
`arthritis, SLE, organ transplant rejection, and graft-vs-
`host disease (1). Hence, the IL-2R is a potentially useful
`and versatile therapeutic target. Agents that specifically
`eliminate Tac-expressing malignant cells or activated T
`cells involved in an autoimmune response could be effec-
`tive against those disorders without harming normal Tac-
`negative T cells. These agents would potentially be more
`selective than other immunosuppressants such as anti-
`bodies against the CD3 antigenic epitope (i.e., OKT3). In
`the case of autoimmune conditions, it might in fact only
`be necessary to suppress T cell proliferation by IL-2R
`blockade, without destroying the T cells, to achieve ther-
`apeutic benefit.
`Anti-IL-2R antibodies have been effective in animal
`models as well as in early human trials. In vivo admin-
`istration of anti-IL-2R antibodies greatly prolonged sur-
`vival of heart allografts in mice and rats (7, 8) and alle-
`viated insulitis in nonobese diabetic mice and lupus ne-
`phritis in NZB X NZW mice (9). MAT itself was highly
`effective in prolonging survival of allografts in cynomol-
`gus monkeys (1 0) with improved efficacy observed with
`HAT (1 1). In phase I clinical trials for kidney transplan-
`Received for publication December 19, 1990.
`tation, prophylactic administration of MAT significantly
`Accepted for publication May 30, 1991.
`reduced the incidence of rejection episodes, without as-
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`sociated toxicity (1 2). Another anti-IL-2R antibody was
`aduertlsement in accordance with 18 U.S.C. Section 1734 solely to indi-
`also effective in this setting (13). Treatment with MAT
`cate this fact.
`' Address correspondence and reprint requests
`to Dr. John Hakimi.
`induced temporary partial or complete remission in 7 of
`Hoffmann-LaRoche, Department of Immunopharmacology, 340 Kings-
`20 patients with adult T cell leukemia (14) (T. A. Wald-
`land St.. Nutley. N J 071 10.
`mann, unpublished observations).
`Abbreviations used in this paper: IL-2R. IL-2 complex; MAT. mouse
`anti-Tac: Tac. p55 subunit of the human IL-2R; sIL-2R. soluble rIL-2R;
`Several major problems limit
`the effectiveness of a
`HAT, humanlzed anti-Tac: HRP. horseradish peroxidase: AUC. area under
`murine mAb such as MAT when used in human patients.
`the curve.
`1352
`
`The cellular receptor for IL-2 plays an important role
`in regulation of immune function (1). The IL-2R2 consists
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`PHARMACOKINETICS OF HUMANIZED ANTI-TAC
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`Group
`
`1353
`obtained from Dr. F. Khan, Bloprocess Development, Hoffmann-La
`The mouse antibody is immunogenic in humans and
`Roche Inc.. Nutley, NJ.
`provokes a neutralizing antibody response, and may not
`HRP-labeled IL-2. HAT and MAT were prepared using a modifi-
`be as efficient as a human antibody at recruiting human
`cation of a previously described method (29). A total of 20 mg of
`IN) in 6 ml of
`HRP, grade 1 (Boehringer-Mannheim. Indianapolis,
`immune effector functions. In addition, mouse antibodies
`distilled water was activated by adding 1 .O ml of 0.1 M NaI04 for 20
`have a much shorter circulating half-life in humans than
`min at room temperature (20-25°C) and subsequently quenched with
`do natural human antibodies (1 5).
`1.0 ml of 0.5 M ethylene glycol. The activated HRP was dialyzed
`Problems associated with the therapeutic use of murine
`
`against 5 mM sodium acetate buffer, pH 4.5, and brought up to a
`final volume of IO ml. Five mg of protein were dialyzed against 0.1
`antibodies have been partially addressed by the genetic
`M NaHC03, pH 8.0, and added to the activated HRP and diluted with
`construction of chimeric antibodies, which combine the
`IO ml of 0.5 M sodium carbonate buffer, pH 9.5. After 2 h at room
`V region binding domain of a mouse antibody with hu-
`temperature. 3 ml of 0.1 M NaBH, were added and incubated in the
`dark for 4 to 6 h at 4°C. The HRP-conjugated proteins were dialyzed
`man antibody C regions (16). However, because chimeric
`against 0.1 M sodium phosphate buffer, pH 6.5, and then diluted
`antibodies retain the whole mouse V region, they may
`with a n equal volume of 0.2 M sodium phosphate buffer, 20 mg/ml
`still be immunogenic. Data on the treatment of human
`BSA. 1 mglml Thimersol. and 2 mg/ml phenol.
`patients with chimeric antibodies are only beginning to
`Monkeys and experimental protocol. Elght groups of four 4 to 6
`kg cynomolgus monkeys (two males and two females: Mason Re-
`accumulate (15. 17).
`search Institute. Worchester. MA] were treated daily on days 1
`To further reduce the immunogenicity of murine anti-
`through 14
`(Table I). Groups 1 and 5 received PBS as a vehicle
`bodies, Winter and colleagues (18-21) constructed "hu-
`control. Monkeys in groups 2. 3, and 4 received HAT at doses of
`0.05, 0.5, or 5.0 mg/kg. respectively. and groups 6. 7, and 8 received
`manized antibodies, in which only the minimum neces-
`MAT at doses of 0.05, 0.5, or 5.0 mglkg. respectively. On day 42.
`sary parts of the mouse antibody, the CDR, were com-
`groups 1 to 4 and groups 5 to 6 received a single 5 mglkg dose of
`bined with human V region frameworks and C regions.
`HAT or MAT, respectively. Test
`samples were administered via
`Based on this approach, we have recently constructed a
`venous catheters surgically placed in the femoral vein attached to a
`vascular port. Samples were administered as single bolus injections
`humanized anti-Tac antibody (22). The humanized anti-
`within several seconds. Blood samples were obtained by venipunc-
`Tac antibody (HAT) retains several key mouse framework
`ture throughout the 55-day study. Monkeys were tranquilized with
`residues, predicted by computer modeling, which are re-
`intramuscular ketamine HC1 before administration of test samples
`and collection of serum samples.
`quired to maintain high affinity binding for p55. In ad-
`Immunosorbant assays. To measure the serum levels of monkey
`dition, the humanized antibody mediates antibody-de-
`antibodies against HAT or MAT, Nunc-lmmuno MaxiSorp (Nunc,
`pendent cellular cytotoxicity against T cell leukemia cells
`Naperville, IL] wells were coated with 100 ng of either HAT or MAT
`in 200 pI of PBS overnight (20-24 h) at 4°C. To each well, 100 pl of
`(23). Previously, it was demonstrated in cynomolgus mon-
`1% fatty acid and globulin-free BSA (Sigma Chemical Co., St. Louis,
`keys with cardiac allografts that HAT appeared less im-
`MO) in PBS were added for 1 h at room temperature, followed by
`munogenic than MAT (1 1). In this study, cynomolgus
`washing with PBS containing 0.05% Tween 20. Wells were incu-
`monkeys were given MAT and HAT to further evaluate
`bated with 200 pl of goat standards or test samples, plus 50 pl of
`HRP-HAT or HRP-MAT at a final dilution of 1/4000 overnight at
`the relative immunogenicity and pharmacokinetics of the
`4°C. Samples were diluted
`in 25 mM sodium phosphate, 75 mM
`two mAb. To provide a stringent test of HAT, we applied
`NaCl. 0.05% Tween 20, 0.01% BSA. 50 pglml phenol red, pH 7.4.
`a dosing schedule of frequent injections that would reveal
`The initial concentration of the unknowns in the assay was 1/3 with
`subsequent threefold dilutions. The plates were washed and then
`any immunogenicity.
`developed with 1 mM 2,2'-azinobis (3-ethylbenzthiazoline-sulfonic
`acid) (Sigma) in 0.1 M citrate buffer. 0.03% Hz02, pH 4.2, for 30 min.
`The absorbance at 405 nm was determined with a Vmax Kinetic
`Microplate reader (Molecular Devices, Menlo Park, CA). The color
`intensity is directly proportional to the antibody concentration in
`the serum samples. The
`relative concentrations of anti-HAT and
`anti-MAT antibodies in the monkey serum samples were calculated
`from a goat antibody standard curve titrated on each plate. The
`values expressed are apparent antibody levels, because the detection
`of antibodies in this assay is dependent on concentration, affinity,
`and presence of blocking agents such as anti-Tac and slL-2R. The
`assay primarily detects free monkey antibodies: however, some an-
`tibody from antibody-anti-Tac complexes would be detected if a
`reequilibrium of the antibody interactions was established in the
`wells during the overnight incubation.
`Serum concentrations of HAT and MAT were determined in an
`IL-2 immunosorbant receptor assay (27). Plates were coated with 16
`ng of sIL-2R in 200 pl of PBS overnight at 4°C and then blocked with
`1 % BSA as described above. Wells were washed and incubated with
`200 pl of sample overnight at 4°C. Typically, the initial serum in the
`assay was diluted 1/10 with subsequent 1/2 dilutions. The initial
`sample concentration varied depending on which treatment group
`
`MATERIALS A N D METHODS
`Cells. MAT was produced in tissue culture a s described previously
`(2). HAT was produced from SP2/0 cells transfected with the genes
`encoding for the H and L chains of the humanized antibody (22,23).
`Cells were optimized for antibody secretion by limiting dilution clon-
`ing. Production of HAT was performed in a 3-liter continuous per-
`fusion bioreactor (Bellco Biotechnology, Vineland, NJ) equipped with
`a glass cylinder matrix as previously described (24, 25). The cells
`were grown at 37°C in Iscoves's modified Dulbecco's medium (JRH
`Biosciences, Lenexam, KS) supplemented with 5% FCS (JRH Bios-
`ciences), 100 U/ml penicillin G, 100 pglml streptomycin, and 25 mM
`HEPES buffer. pH 6.9 to 7.0. During the production phase of the
`fermentation, days 9 to 83. the medium flow rate was maintained at
`416 ml/h and the conditioned medium contained approximately 8
`mglliter of HAT.
`Proteins. HAT and MAT were purified on separate IL-2R affinity
`chromatography columns with capacities of 125 and 300 mg. re-
`spectively (26). Briefly, purified recombinant sIL-2R (27) was im-
`mobilized on NuGel P-AF Poly-N-hydroxysuccinimide (Separation
`Industries. Metuchin, NJ). Antibodies eluted and concentrated from
`the receptor column were
`further purified on
`two serially linked
`Sephacryl S-300 columns (60 X 1 1.3 cm. Pharmacia Fine Chemicals,
`Piscataway, N J ] in Dulbecco's PBS (Whittaker Bioproducts, Walkers-
`ville, MD). All purification steps were carried out at 4°C. and buffers
`were prepared with ultra pure water (Hydro, Research Triangle Park,
`NC). The final products were sterilized
`through a 0.2
`Corning
`filter (Corning Glass Works, Corning, NY) and found to contain less
`than 10 endotoxin units/mg (28). Purity was determined by SDS-
`PAGE under reducing and nonreducing conditions and found to be
`more than 99%.
`Anti-HAT and anti-MAT standards were prepared by immunizing
`goats with the respective proteins in CFA. The goat IgG standards
`were isolated on protein A-Sepharose CL-4b (Pharmacia) and affinity
`purified on HAT or MAT AffiCel- 10 affinity columns (Bio-Rad. Rich-
`mond. CA). Purified human rIL-2 expressed in Escherichia coli was
`
`TABLE I
`Immunogenicity study treatment qrouvs
`Challenge Dose
`Daily Dose
`Response
`Days 1 to 14
`Day 42
`
`Vehicle control
`HAT. 5 mg/kg None
`1
`HAT. 0.05 mg/kg HAT. 5 makg None
`2
`HAT. 0.5 mg/kg
`HAT. 5 mg/kg None
`3
`HAT. 5.0 mg/kg
`4
`HAT, 5 mg/kg Anaphylaxis
`
`Vehicle control
`MAT, 5 mg/kg None
`5
`MAT. 0.05 mg/kg MAT, 5 mg/kg Anaphylaxis
`6
`7 MAT. 0.5 mg/kg
`None"
`MAT. 5.0 mg/kg
`None"
`8
`a Monkeys not challenged with MAT due to the anaphylaxis observed
`in group 6.
`
`114
`
`414
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`PHARMACOKINETICS OF HUMANIZED ANTI-TAC
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`IMMUNOGENICITY AND
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`1354
`was studied. Without washing the samples from the wells, 50 rl of
`HRP-IL-2 was added to a final dilution of 1/2000. After 3 h at room
`temperature, wells were washed and developed as described above.
`The color intensity is inversely proportional to the anti-Tac concen-
`tration in the samples. HAT and MAT concentrations in the serum
`were calculated from a standard curve of purified HAT and MAT
`titrated on each plate. In this assay, only the anti-Tac available to
`the
`bind to the slL-2R would be detected. A s discussed above for
`immunogenicity ELISA. a new equilibrium of the antibody complexes
`in the serum could occur In the wells during the 24 h incubation.
`Pharrnacokfnetfcs. The AUC and tllz values for HAT and MAT
`were estimated to reflect the total body burden of the antibody within
`the Intravascular pool as well as the serum die-away curve, respec-
`tively. Serum concentrations of the antibodies were plotted vs time
`on a log-linear graph and the AUC values were calculated by trape-
`zoidal rule (30). The apparent elimination t,,z from a single dosing
`was estimated by linear regression analysis of the terminal portion
`of the curve from a minimum of four data points.
`For multiple dose pharmacokinetics, the maximum serum concen-
`trations and time to reach maximum serum concentrations were
`obtained visually from the serum concentration-time graphs. The
`apparent tllz after multiple dosing was approximated from a mini-
`mum of three serum concentration-time points obtained after the
`final dose.
`
`RESULTS
`Study design and clinical observations. A cynomol-
`gus monkey study was designed to evaluate the relative
`immunogenicity and pharmacokinetic properties of MAT
`and HAT. A schematic representation of the study design
`is shown in Figure 1 and details of the treatment groups
`are described in Table I. During the study, the monkeys
`remained behaviorally and clinically normal with
`the
`following exceptions. On day 42 one female monkey in
`group 4 exhibited an apparent anaphylactic
`response
`posttreatment with 5 mg/kg of HAT. This monkey was
`treated with epinephrine, dexamethasone, Benadryl, and
`was hydrated with saline. The
`monkey gradually im-
`proved and by day 44 appeared normal. All four monkeys
`in group 6 that received 0.05 mg/kg/day MAT initially,
`also exhibited an apparent anaphylactic response post-
`treatment with 5 mg/kg MAT. The monkeys responded
`to epinephrine and fluids. The animals in the MAT treat-
`ment groups 7 and 8 were not challenged on day 42. In
`various ELISA systems, no increase in total monkey IgE
`was observed, nor was the presence of Ag-specific anti-
`anti-Tac IgE detected (data not shown). The cause of this
`anaphylactic response remains unknown.
`Immunogenicity characterization. Monkey antiglob-
`ulin levels (i.e., antibodies to HAT and MAT) were evalu-
`ated in an Ag-bridging ELISA, which can be used to detect
`antibodies of various species and isotypes using the same
`reagents. Affinity-purified goat anti-HAT and goat anti-
`MAT antibody standards were similarly detected in the
`range of 100 to 1000 ng/ml in their respective assays
`(data not shown).
`The time-dependent development of antibodies in in-
`dividual monkeys is shown for MAT in Figure 2 and for
`
`TREA rMmr
`14 DAYS
`11111*,1111"11
`
`CHALLENGE
`DAY 4 2
`
`1
`
`10
`
`2 0
`
`3 0
`
`4 0
`
`5 0
`
`BLOOD COlLECTlON DAYS
`
`/ \
`DAY .z
`P-T
`26,6,1,*...8
`12,145. h,
`
`Figure 1. Immunogenicity and pharmacokinetic study design for eval-
`uation of anti-Tac antibodies in cynomolgus monkeys. See Table
`I for
`additional detail.
`
`226
`
`160
`
`76
`
`0
`
`aoo I B. Group 7
`
`.- E 8 0 0 a
`
`226
`
`160
`
`76
`
`0
`0
`
`6
`
`26
`
`
`
`
`
`10 20
`
`
`
`16
`
`SO
`
`5 6
`
`40
`
`46
`
`Time (day)
`Figure 2. Time-dependent development of anti-MAT antibodies in in-
`dividual monkeys administered A. 0.05, B, 0.50. C. 5.0 mg/kg/day MAT
`for 14 days. Anti-MAT concentrations weredetermined in an ELISA using
`an affinity purified goat anti-MAT antibody as a standard.
`
`HAT in Figure 3 (note differences in the ordinate scales).
`Prebleed sera from all 32 monkeys and sera from day 0
`to 42 from control monkeys in groups 1 and 5 showed no
`activity in the ELISA. In the MAT-treated groups, 9 of 12
`monkeys developed antibodies during the initial 14 day
`treatment period, usually by day 12. In contrast, anti-
`HAT antibodies in all but one of the 12 HAT-treated
`monkeys were not detected until at least 5 to 10 days
`after the final dose of HAT was administered. In addition,
`the HAT-treated monkeys showed dramatically lower
`serum antiglobulin concentrations than the MAT-treated
`groups.
`The antibody titer developed to HAT as well as MAT
`was in general inversely related to the protein dose ad-
`ministered. In group 4 which was treated with 5 mg/kg/
`day HAT, only one monkey had detectable antibodies by
`only HAT-treated monkey that
`day 42. This was the
`exhibited an anaphylactic response upon challenge with
`HAT on day 42 (Table I), even though monkeys from
`other groups had apparently higher serum antibody lev-
`els on day 42. All monkeys in group 6 that received 0.05
`mg/kg/day MAT exhibited an anaphylactic response
`upon rechallenge on day 42. Monkeys in groups 7 and 8
`were not challenged (Table I). Thus, four monkeys treated
`
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`IMMUNOGENICITY AND PHARMACOKINETICS
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`OF HUMANIZED ANTI-TAC
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`1355
`
`80 -
`. A. Group 2
`
`sot
`40 t
`
`3
`
`Y
`
`B. Group 3
`
`a l-
`I
`O
`+
`>,
`W 0
`n .-
`r
`a } C. Group 4
`
`Y 8 0 I
`
`"1
`
`40
`
`0
`
`Y
`
`Y
`
`/+-+
`A , *
`Time (day)
`Figure 3. Time-dependent development of anti-HAT antibodies in in-
`dividual monkeys administered A, 0.05. 8, 0.50, C, 5.0 mgkgfday HAT
`for 14 days. Anti-HAT concentrations were determined in an ELISA using
`an affinity purified goat anti-HAT antibody as a standard.
`
`..
`
`"
`
`:ontrol 16. 0.05 mglko C. 0.5 mglkg D. 5.0 mglkg
`loooo A. Control 6. 0.05 mglko C. 0.5 mglkg D. 5.0 mglkg
`.2 E a, 1000;
`0 -
`0 2
`
`100
`
`U
`
`10 r
`
`1
`
`0.s .
`.
`
`.
`.
`
`42
`42
`
`55
`55
`
`.
`.
`5s
`5s
`6 s
`4 2
`6 s
`4 2
`Time (day)
`
`.
`.
`
`4 2
`4 2
`
`66
`66
`
`.
`.
`
`Figure 4. Primary and secondary immune responses to HAT and MAT.
`Anti-HAT (0) and anti-MAT (A) antibody concentrations from individual
`monkeys on day 42 before high-dose challenge and on day 55. Data in A
`represents the anti-MAT response on day 55 in two naive monkeys from
`group 5. No anti-HAT antibodies developed in any monkeys from group
`1 . Before challenge animals received multiple doses of E. 0.05. C. 0.50.
`D. 5.0 mg/kg/day of anti-Tac antibody for 14 days. Monkeys dosed with
`MAT in C and D were not rechailenged with MAT on day 42 (data not
`shown).
`
`9. Anti-MAT
`
`,A-A/' P
`
`-,O--O
`
`100
`
`80
`
`60
`
`c 100
`Q) 0
`i
`7 5
`
`50
`
`2 5
`
`a
`
`with MAT developed an anaphylactic response at a dose
`100-fold lower than the individual high dose HAT-treated
`monkey.
`A comparison of day 42 (prechallenge) and day 55
`serum antiglobulin levels from all challenged monkeys
`in groups 1 to 6 is shown in Figure 4. A primary immune
`response to the single treatment with MAT was observed
`Concentration (ng/ml)
`5, although the same
`in two naive monkeys in group
`treatment with HAT to group 1 monkeys resulted in no
`Figure 5. Characterization of anti-HAT and anti-MAT responses in
`monkeys on day 35. A shows the anti-MAT from a monkey in group 6
`antibodies (Fig. 4A). A secondary immune response was
`and B shows the anti-HAT response from a monkey in group 2. A fixed
`observed in all animals previously treated with antibody.
`amount of antiserum was incubated In the presence of various concen-
`No secondary response was observed in groups 7 and 8,
`trations of HAT (0). MAT (A), sIL-2R (0), human IgG (V). or mouse IgG (0).
`
`These data are representative of the data obtained from all of the monkeys
`because they were not challenged. The greatest second-
`with antiglobulin antibodles on day 35.
`ary responses were observed in the monkeys receiving
`either 0.05 mg/kg MAT or HAT (Fig. 4B).
`The specificity (i.e., anti-Id or anti-isotype) of the anti-
`HAT and anti-MAT responses was determined in a com-
`petitive ELISA assay (Fig. 5). Inhibition of antibody bind-
`ing in the ELISA by HAT, MAT, as well a s sIL-2R indi-
`cates the presence of anti-CDR or anti-idiotypic antibod-
`ies, because these proteins specifically compete for or
`block recognition of the CDR regions of anti-Tac. Com-
`petition by irrelevant human and mouse
`IgG proteins
`indicates the presence of anti-isotypic antibodies. The
`
`goat anti-HAT and anti-MAT antibodies were partially
`inhibited by all of the competitors (data not shown],
`indicating that HAT and MAT administered to goats in-
`duced both a n anti-idiotypic and anti-isotypic response.
`Serum from all MAT-treated monkeys was completely
`inhibited with excess
`MAT, demonstrating that the
`ELISA assay is specific for MAT (Fig. 5A). HAT and sIL-
`2R were the next most effective inhibitors followed by
`mouse IgG. Thus, the monkey response to MAT was a
`mixture of anti-isotypic and anti-idiotypic antibodies
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`IMMUNOGENICITY AND PHARMACOKINETICS OF HUMANIZED ANTI-TAC
`
`similar to the goat anti-MAT and anti-HAT responses.
`Complete inhibition of monkey anti-HAT antibodies was
`achieved with HAT. again demonstrating assay specific-
`ity (Fig. 5B). Human and mouse IgG had little effect
`indicating that the anti-HAT response in monkeys is not
`an anti-isotypic response. MAT and sIL-2R were almost
`as effective as HAT in inhibiting anti-HAT binding. Thus,
`the monkey anti-HAT antibodies are directed toward de-
`terminants shared by MAT and HAT, and blocked by sIL-
`2R, Le., toward the CDR regions. This supports our con-
`clusion that the anti-HAT response is anti-idiotypic in
`monkeys.
`Pharmacokinetic characterization. The pharmacoki-
`netic characteristics of HAT and MAT were determined
`in a competitive immunosorbant receptor assay. In this
`assay, both proteins inhibited IL-2 binding within twofold
`of each other. The detection limit was in the range of 125
`to 500 ng/ml. Serum concentrations of HAT and MAT
`were measurable only in monkeys receiving doses of 0.5
`and 5 mg/kg/day of antibody (Figs. 6 and 7, respectively,
`note the differences of the ordinate scales). In general,
`HAT concentrations were increased over the dosing
`period, suggesting that equilibrium was not achieved with
`receptor sites or the extravascular space. The mean max-
`imum concentrations after dosing with 0.5 and 5 mg/kg/
`day of HAT for 14 days were 57 k 20 (mean f SD) and
`726 k 1 15 pg/ml, respectively. In contrast, maximum
`concentrations of 0.5 and 5.0 mg/kg/day of MAT were
`26 f 9 and 31 1 k 57 pg/ml, respectively, but occurring
`at approximately 7 to 9 days after the initiation of ther-
`apy. The mean time course of decline or tl12 values of
`HAT from the serum after 14 days of dosing were highly
`
`100 I
`
`Group 7
`
`B. Group 3
`
`f
`*
`100
`0
`2 75
`a, >
`a, -
`E
`2
`$ 25
`
`5 0
`
`5
`
`10 15
`
`20 25
`
`3 0 3 5 40 45
`
`0
`0
`
`Time (day)
`Figure 6. Serum concentration profile of A, MAT or E, HAT in individ-
`ual monkeys receiving 0.50 mg/kg/day of antibodies for 14 days. Anti-
`MAT and anti-HAT concentrations were determined in a competitive IL-
`2 immunosorbant receptor assay using the respective purified anti-Tac
`mAb as the standard.
`
`1000
`(A. Grow 8
`
`k 500 t
`
`[B. Group 4
`
`750
`
`1000
`'i;
`-
`>
`a, -
`E
`
`500
`
`v) 250
`
`0
`0
`
`5
`
`10 15 20 25 3 0 35 40 45
`
`Time (day)
`Figure 7. Serum concentration profile of A, MAT or B, HAT in individ-
`ual monkeys receiving 5.0 mg/kg/day of antibodies for 1 4 days. See Figure
`6 for additional details.
`
`variable, ranging from approximately 47 to 432 h, and
`independent of dose. The t,/2 of MAT was not calculated
`due to the rapid decline of serum concentrations even
`during the 14-day dosing regimen.
`In control naive animals, the serum concentration-time
`profiles of HAT were significantly different from the
`profiles with MAT (Table 11). The individual AUC and t1/2
`values after a single i.v. dose of 5 mg/kg of HAT or MAT
`to control monkeys in groups 1 and 5, respectively, on
`day 42 are shown in Table 11. The mean AUC was ap-
`proximately twofold more in the HAT-treated control
`monkeys when compared to the MAT-treated control
`counterparts, 26,657 k 6237 vs 1 1,442 f 3563 pg. h/ml,
`respectively. A four- to fivefold difference was observed
`in the mean tIl2 values between HAT and MAT (213.6 f
`58.8 and 47.8 k 9.04 h, respectively) (Fig. 8).
`The pharmacokinetic profiles in the multiple-dosed
`groups were significantly altered. Only four MAT-treated
`monkeys were rechallenged on day 42 due to the observed
`anaphylactic response. Three of the monkeys had no
`detectable serum MAT levels, whereas in the fourth mon-
`key, levels were detectable but not within the quantita-
`tion limits of our assay governed by the standard curve
`(data not shown). Clearly, the elimination of MAT
`from
`group 6 animals that were treated with 0.05 mg/kg/day
`MAT was significantly enhanced compared to group 5
`animals that had not received MAT previously. Kinetic
`parameters of all but two of the HAT-treated monkeys in
`groups 2 to 4 were estimated (Table 11). The AUC values
`in groups 2 and 3 were lower than those in naive animals
`[group 1). In animals treated with 5 mg/kg/day (group 4),
`the tlI2 and AUC values were higher than the values
`obtained for group 2 and 3 monkeys. One monkey in
`group 4 had greater values than the naive group animals.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1037 Page 5 of 8
`
`

`
`IMMUNOGENICITY AND PHARMACOKINETICS OF HUMANIZED ANTI-TAC
`
`1357
`
`TABLE I1
`Individual AUC (pg. hlmll and 1% (hJ values in monkeys given single i.u. dose of 5 mg/kg of HAT or MAT on day 42
`Group 5
`Group 4
`Group 3
`Group 2
`Group 1
`
`HAT Controls
`tn
`AUC
`259
`35,251
`270
`22.140
`167
`21,961
`200
`27,276
`NE, Not evaluable
`
`HAT 0.05 mg/kg
`tn
`AUC
`19.9
`8.004
`NE"
`NE
`26.9
`6,705
`9.7
`2,848
`
`HAT 0.5 mg/kg
`AUC
`4,883
`5.489
`2,050
`677
`
`tu,
`34.5
`26.7
`18.8
`8.9
`
`HAT 5 mg/kg
`AUC
`tn
`115
`29,485
`318
`99.870
`28
`6,850
`NE
`NE
`
`MAT Controls
`AUC
`9.325
`8,424
`16,388
`11,633
`
`tu,
`53.1
`38.1
`57.6
`42.6
`
`a
`l-
`I
`r
`
`' 'I
`
`0
`
`5 0 100 1 5 0 2 0 0 2 5 0
`
`Time (hour)
`Figure 8. HAT (0) and MAT (0) serum concentration in naive monkeys
`administered a single bolus of 5 makg anti-Tac. See Figure 7 for addi-
`tional details.
`
`b '
`
`100000 i
`
`A
`
`B
`
`10
`
`2 0
`
`30
`
`40
`
`5 0
`
`6 0
`
`0
`
`This may be explained by elevated serum concentrations
`
`of HAT on day 42 from the multiple dosing regimen (Fig.
`6). In addition, the tl12 values of each HAT-treated animal
`after rechallenge were significantly reduced compared to
`values after steady-state dosing (data not shown).
`An association between anti-HAT serum concentra-
`tions and either AUC or tl12 values was observed (Fig. 9).
`Anti-HAT
`(ug/ml)
`These pharmacokinetic parameters were inversely re-
`Figure 9. Associations between anti-HAT concentrations vs AUC (A)
`lated to the serum anti-HAT concentrations, indicating
`and half-life (B) values after a single 5 mg/kg rechallenge dose on day 42
`3 (0, four animals), and group 4 [..
`that elevated antibody levels contribute to the accelerated
`in monkeys in group 1 (0, four animals). group 2 (0. three animals), group
`three animals). See Table 11.
`elimination of HAT. Correlation of the kinetic parameters
`and antibody levels for the
`HAT-treated monkeys in
`monkeys on day 42 was sufficient to evoke a n antibody
`groups 2 and 3 appeared similar. The group
`4 values
`response within 13 days (Fig. 4). MAT was similar to
`appeared more typical of the parameters measured for
`other murine antibodies studied in primates (31, 32) in
`the control group 1 monkeys suggesting that antibody
`being recognized as a foreign Ag and inducing both anti-
`effects on HAT pharmacokinetics were similar between
`idiotypic and anti-isotypic responses.
`groups. Taken together, the survival of HAT was mean-
`HAT clearly proved to be less immunogenic than MAT.
`ingfully longer than that of MAT in naive monkeys. Fur-
`Anti-HAT antibody titers were 5- to 10-fold lower in their
`thermore, the development of antibodies to the adminis-
`respective dosing groups, and th