[CANCER RESEARCH 45, 879-885, February 1985)
`
`Human Anti-Murine lmmunoglobulin Responses in Patients Receiving
`Monoclonal Antibody Therapy 1
`Robert w. Schroff,2 Kenneth A. Foon, Shannon M. Beatty, Robert K. Oldham, and Alton C. Morgan, Jr.
`Biological Therapeutics Branch, Biological Response Modifiers Program, National cancer Institute, Frederick, Maryland 21701
`
`ABSTRACT
`
`Human anti-murine immunoglobulin responses were assessed
`in serum from three groups of patients receiving murine mono(cid:173)
`clonal antibody therapy. Each of the three patient groups re(cid:173)
`sponded differently. Chronic lymphocytic leukemia patients dem(cid:173)
`onstrated little or no preexisting murine immunoglobulin G-reac(cid:173)
`tive antiglobulin prior to treatment, while the cutaneous T -cell
`lymphoma and melanoma patients demonstrated preexisting
`antiglobulin levels in the same range as those demonstrated in
`healthy controls. None of 11 chronic lymphocytic leukemia pa(cid:173)
`tients receiving the T101 monoclonal antibody demonstrated an
`antiglobulin response, whereas all four of the cutaneous T-cell
`lymphoma patients receiving the same antibody developed in(cid:173)
`creased levels of antiglobulins. Three of nine malignant mela(cid:173)
`noma patients receiving the 9.2.27 monoclonal antibody showed
`an increase in antiglobulin titers. In patients developing antiglob(cid:173)
`ulin responses, the response was rapid, typically being detect(cid:173)
`able within 2 weeks. The antiglobulins were primarily immuno(cid:173)
`globulin G and, with the exception of a single melanoma patient
`in whom the response appeared to have a substantial 9.2.27-
`specific component (i.e., antiidiotype), were cross-reactive with
`most murine immunoglobulin G preparations tested. This pattern
`of results suggested that the antiglobulin was a secondary
`immune reaction with elevation of the levels of preexisting anti(cid:173)
`globulin which was cross-reactive with the mouse antibody ad(cid:173)
`ministered. While the presence of serum antiglobulin would be
`expected to present major complications to monoclonal antibody
`therapy, no clinical toxicity related to antiglobulin responses was
`observed in these patients, and no inhibition of antibody localiza(cid:173)
`tion on tumor cells was seen.
`
`INTRODUCTION
`
`Attempts at serotherapy of human tumors date back to the
`treatment of chronic myelogenous leukemia with antisera by
`Lindstrom (6) in 1927. However, due to the difficulty in obtaining
`large quantities of antisera of sufficient specificity, and the many
`side effects of crude antisera, this form of therapy has not come
`into general use. The development of monoclonal antibodies of
`defined specificity and unlimited availability has rekindled interest
`in the use of passively administered antibody as a form of cancer
`therapy (13).
`The development of host antibodies against passively admin(cid:173)
`istered immunoglobulin, with possible neutralization of the ad(cid:173)
`ministered immunoglobulin and anaphylactic or other immune
`' This project has been funded at least in part with Federal funds from the
`Department of Health and Human Services, under Contract N01-C0-2391 O with
`Program Resources, Inc.
`2 To whom requests for reprints should be addressed, at BRMP, NCl-FCRF,
`Bldg. 560, Room 31-93, Frederick, MD 21701.
`Received February 17, 1984; accepted November 2, 1984.
`
`reactions, has been viewed as a potential major complication to
`serotherapy. Recent reports of clinical trials with murine mono(cid:173)
`clonal antibodies have confirmed that human anti-mouse im(cid:173)
`munoglobulin antibodies may be induced (1, 2, 10, 14, 17). Miller
`et al. (10) reported development of anti-mouse immunoglobulin
`antibodies in 4 of 7 T -cell lymphoma patients treated with the
`anti-Leu-1 monoclonal antibody. In 3 of these 4 patients, the
`development of anti-mouse immunoglobulin antibodies appeared
`to contribute to tumor escape from therapy. Similarly, Dillman et
`al. (1) attributed the lack of response to therapy, in 2 of 4
`cutaneous T -cell lymphoma patients receiving the T101 mono(cid:173)
`clonal antibody, to the presence of human anti-mouse immuno(cid:173)
`globulin antibodies. Sears et al. (17) also reported the presence
`of human anti-mouse immunoglobulin antibodies in 9 of 18
`gastrointestinal tumor patients receiving the monoclonal antibody
`1083-17-1A. However, other studies did not report that human
`antiglobulin responses presented major problems in monoclonal
`antibody therapy (3, 7-9, 15). The relatively small number of
`reports in the literature of monoclonal antibody clinical trials, the
`variety of diseases treated, and the lack of uniformity in the
`design of these trials makes it difficult to draw general conclu(cid:173)
`sions as to the conditions under which host anti-mouse immuno(cid:173)
`globulin responses would be expected to develop.
`The Biological Therapeutics Branch of the National Cancer
`Institute has recently completed Phase I clinical trials with the
`lgG2a monoclonal antibody T101 in patients with CLL 3 and CTCL
`and the lgG2a monoclonal antibody 9.2.27 in patients with
`malignant melanoma (2, 3, 14). The T101 antibody recognizes
`the T65 antigen present on the cell surface of both normal and
`malignant T-cells, as well as some B-cell cancers, including CLL
`(16). The 9.2.27 antibody recognizes a M, 250,000 glycoprotein(cid:173)
`proteoglycan associated with melanoma (11). In this paper, the
`host anti-mouse immunoglobulin responses observed during
`these trials are summarized, with a comparison of the differences
`and similarities in the responses elicited within the 3 disease
`groups, and an analysis of the specificity of the detected anti(cid:173)
`bodies.
`
`MATERIALS AND METHODS
`
`Patients. Patients considered for the clinical trial with T101 were
`adults with histologically confirmed diagnosis of CLL or CTCL. Patients
`with malignant melanoma were considered as candidates for treatment
`with the 9.2.27 antibody. Patients received no radiation or immunosup(cid:173)
`pressive drugs for at least 4 weeks prior to entry into these trials. Prior
`to treatment, all patients were fully ambulatory and had no serious
`unrelated disease, and their tumor cells were positive for reactivity with
`the antibody to be used in therapy. The mean and range of age of each
`patient population was: CLL, 59, 43 to 81; CTCL, 56, 42 to 68; mela(cid:173)
`noma, 48, 23 to 72 years.
`3 The abbreviations used are: CLL, chronic lymphocytic leukemia; CTCL, cuta(cid:173)
`neous T-<:ell lymphoma; ELISA, enzyme-linked immunosorbent assay.
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`879
`
`1 of 7
`
`BI Exhibit 1106
`
`

`

`HUMAN ANTl-MUAINE IMMUNOGLOBULIN RESPONSES
`
`The control population used in this study consisted of 11 healthy
`individuals with no history of cancers and no previous therapy with
`murine-derived agents and ranged in age from 20 to 45 years, with a
`mean of 31 years.
`Study Plan. Patients were treated with either T101 or 9.2.27 mono(cid:173)
`clonal antibody. Details of the design and clinical findings of each trial
`have been reported elsewhere (3, 14). Briefly, patients with CLL or CTCL
`received T101 antibody i.v. at fixed-dose levels of 1, 10, 50, or 100 mg.
`Patients were treated twice weekly for 4 weeks. Initially, patients received
`the total dose of antibody in 100 ml of 0.9% NaCl solution (saline) with
`5% human albumin over 2 hr. Due to pulmonary toxicity associated with
`the rapid rate of infusion, this was later amended so that antibody was
`administered at a rate of no more than 1 to 2 mg of T101 antibody per
`hr. Melanoma patients received the 9.2.27 antibody by i.v. infusion in
`100 ml of saline with 5% human serum albumin over 2 hr. Each patient
`received single doses of antibody twice weekly on an escalating dose
`schedule of 1, 10, 50, 100, and 200 mg or 10, 50, 100, 200, and 500
`mg. A summary of the number of patients treated and the amount of
`antibody administered is presented in Table 1.
`Assay for Human Anti-Mouse Antibody. Sera used in all assays
`were separated from peripheral blood and stored at -20° until use.
`Antiglobulins in dilutions of serum were measured using solid-phase
`T101 or 9.2.27 antibodies dried at 37° overnight onto polyvinyl plates at
`100 ng of antibody per well and washed with 0.1 M tris (pH 8.3)-0.02%
`NaN:r0.5% Tween 20 (Sigma Chemical Co., St. Louis, MO). Dilutions of
`serum were incubated on the plates at room temperature for 45 min.
`Bound human immunoglobulin was detected with heavy chain-specific
`('Y orµ) goat anti-human immunoglobulin conjugated with alkaline phos(cid:173)
`phatase (Sigma) during a 45-min incubation. For comparison, standard
`curves were generated against solid-phase human lgM myeloma proteins
`or pooled normal human lgG (Cappel Laboratories, Cochranville, PA),
`and antiglobulin expressed as µg of protein bound to plates per ml of
`serum.
`For assays of antiglobulin specificity, plates were coated with T101
`Fab or 9.2.27 F(ab')2 preparations (100 ng/well); the mouse myetoma
`proteins MOPC-21 (lgG1), RPC-5 (lgG2a), UPC-10 (lgG2a), MOPC-141
`(lgG2b), and FLOPC-21 (lgG3) (Litton Bionetics, Kensington, MD); mouse
`lgG (Sigma); mouse lgM (Pel Freeze Biologicals, Rogers, AR); rabbit lgG
`(Dako, Denmark); or whole T101 or 9.2.27 antibodies. Inhibition of
`binding to T101 or 9.2.27 target antigen was assessed by performing
`the ELISA in the presence of a 1000-fold-greater concentration (100 µg/
`well) of the soluble inhibitor murine lgG2a antibodies 9.2.27, T101, D3,
`or RPC-5 as compared to the solid-phase target immunoglobulin.
`Assay for Mouse lmmunoglobulin. Murine immunoglobulin in dilu(cid:173)
`tions of serum was assayed using affinity-purified goat anti-mouse
`immunoglobulin (KPL, Gaithersburg, MD) adsorbed onto polyvinyl plates
`at 100 ng/well and washed as above. Bound mouse immunoglobulin
`
`Table 1
`Summary of monoclonal antibody therapy
`Total dose
`received
`(mg)
`6
`8
`50
`80
`150
`300
`400
`
`Disease
`CLL
`CLL
`CLL
`CLL
`CLL
`CLL
`CLL
`
`No. of patients
`treated
`1
`2
`1
`2
`1
`1
`2
`
`CTCL
`CTCL
`CTCL
`CTCL
`
`Melanoma
`Melanoma
`
`8
`66
`80
`162
`
`361
`860
`
`7
`2
`
`was detected with a goat anti-mouse immunoglobulin conjugated with
`alkaline phosphatase (Sigma) and compared against a standard curve of
`either T101 or 9.2.27 antibody.
`Assay for Human Serum lmmunoglobulin. Serum lgG and lgM levels
`were determined by radial immunodiffusion utilizing Endoplate immuno(cid:173)
`globulin test kits obtained from Kallestad Laboratories, Austin, TX.
`lmmunofluorescent Staining of Melanoma Specimens. Tumor cells
`were prepared as single-cell suspensions by teasing tissues which were
`obtained from skin lesions. To assess in vivo localization of the murine
`9.2.27 antibody, the cell suspensions were incubated with fluorescein
`isothiocyanate-conjugated goat anti-mouse lgG (Tago, Inc., Burlingame,
`CA) for 30 min at 4°. The cells were then washed by centrifugation and
`analyzed on a Cytofluorograf 50H (Ortho Diagnostic Systems, West(cid:173)
`wood, MA). A similar goat antibody directed against mouse lgM (Tago)
`was used as a negative control, and incubation in the presence of excess
`9.2.27 antibody served as a positive control. All biopsy specimens were
`obtained 24 hr following infusion of the 9.2.27 antibody.
`Statistical Evaluation. Serum antiglobulin levels for a given patient
`were considered significantly increased at antiglobulin levels greater than
`2 S.D.s above the mean of the healthy control group.
`
`RESULTS
`
`Development of Antiglobulin Responses. In order to deter(cid:173)
`mine the level of mouse-reactive antiglobulins which could be
`detected in healthy individuals by our ELISA, antiglobulin levels
`were assessed in 11 normal donors. As illustrated in Chart 1,
`the control population demonstrated detectable levels of lgG and
`lgM antiglobulin reactive with both the T101 and 9.2.27 antibod(cid:173)
`ies. These preexisting antiglobulin levels in the CLL patients prior
`to therapy were significantly lower (p < 0.005 by Student's t
`test) than those demonstrated by the healthy controls. Serum
`immunoglobulin levels were determined on the same specimens.
`Both serum lgG and lgM levels were significantly lower in the
`CLL group as compared to the control group. However, CLL
`serum immunoglobulin levels were roughly one half that of con(cid:173)
`trols, while CLL antiglobulin levels were less than one tenth that
`of control antiglobulin levels.
`To substantiate that the assay used was in fact detecting
`human anti-mouse immunoglobulin antibody, 2 control experi(cid:173)
`ments were performed. To demonstrate that the binding of
`human immunoglobulin to the ELISA plate was not nonspecific,
`control and patient specimens were incubated on plates coated
`with either the T101 or 9.2.27 antibodies, or left uncoated. Table
`2 demonstrates that binding did not occur in the absence of
`mouse immunoglobulin on the plates and that binding was
`roughly equivalent irrespective of the antibody used to coat the
`plates. To further substantiate that the preexisting human anti(cid:173)
`body activity was indeed reactive with mouse immunoglobulin,
`we performed the ELISA for human anti-mouse immunoglobulin
`activity in the presence of a 1000-fold-greater concentration of
`a variety of murine lgG2a preparations. As indicated in Table 3,
`roughly 50% of the activity could be inhibited in such a manner.
`The percentage of inhibition represents the decrease in titer due
`to the presence of the inhibitor immunoglobulin. While there was
`substantial variability between titers of antiglobulin in the 5
`individuals examined, the percentage of inhibition in each case
`was quite similar, as indicated by the relatively low S.D. The
`inhibition was not restricted to the mouse immunoglobulin prep(cid:173)
`aration used as the solid-phase antigen. The remaining 50% of
`the activity is most likely attributable to the weak affinity of
`antiglobulins for soluble immunoglobulin (12) as compared to the
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`880
`
`2 of 7
`
`BI Exhibit 1106
`
`

`

`HUMAN ANTl-MURINE IMMUNOGLOBULIN RESPONSES
`
`6
`
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`.......
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`
`CLL
`
`CTCL
`
`T101 9.2.27
`CONTROLS
`Chart 1 . Antiglobulin and serum immunoglobulin levels in healthy controls and
`patients prior to therapy. Serum lgG (A) or lgM (8) antibody levels to both T101
`and 9.2.27 antibodies in controls and the appropriate treatment antibody in patients
`(T101 for CLL and CTCL patients. 9.2.27 for melanoma (Mel) patients] are shown
`(fill). Serum immunoglobulin levels are indicated for comparison (II). Columns, mean
`of each group; bars, S.D. The number of individuals in each group was: control,
`11; CLL, 11; CTCL, 4; melanoma, 9. ', levels in patient groups which were
`significantly lower than those of the appropriate control group (p < 0.005 as
`determined by the Student t test).
`
`Serum dilution
`
`Table3
`Inhibition of 9.2.27-reactive human antiglobulin activity In normal human serum
`with murine lgG2a
`Values represent the mean of 5 healthy control specimens.
`Inhibitor
`
`Monoclonal
`antibodies
`70 ± 45b
`None
`42 ± 5
`42 ± 30
`9.2.27
`~±9
`43±~
`T1~
`51 ± 10
`34 ± 27
`03
`57 ± 17
`35 ± ~
`RPC-5
`: Reciprocal of the dilution yielding an absorbance at 405 nm of 0.3.
`Mean± S.D.
`
`% of inhibition
`
`antigens. In the T101 trial, serum specimens were obtained
`before the third, fifth, and seventh doses. These specimens were
`obtained immediately prior to doses in order to minimize the
`possibility of circulating free mouse lgG being present in the
`specimen. To confirm that serum mouse lgG levels were low,
`mouse lgG levels were quantitated in all serum samples. Speci(cid:173)
`mens from CLL and CTCL patients all demonstrated mouse lgG
`levels of less than 1 µg/ml. Specimens from melanoma patients
`demonstrated somewhat higher levels of mouse lgG but, in all
`cases, were less than 25 µg/ml. As depicted in Chart 2, CLL
`patients treated with T101 failed to develop detectable antiglob(cid:173)
`ulin levels over the period of therapy. In contrast, while CTCL
`patients demonstrated rather low antiglobulin levels prior to
`receiving T101 antibody, a significant increase in lgG levels of
`antiglobulin developed over the course of therapy in all 4 patients
`(Chart 3). Three of these 4 patients also demonstrated rises in
`lgM antiglobulin levels over the course of therapy, but not to the
`same magnitude as lgG responses.
`Of the 9 melanoma patients in the 9.2.27 trial, 3 developed
`significant levels of lgG antiglobulin (Chart 4). These same 3
`patients demonstrated lower, but yet significant, levels of lgM
`antiglobulin during the course of therapy. All 3 individuals who
`developed antiglobulin levels received a total of 361 mg of 9.2.27
`antibody.
`Specificity of Antiglobulin Response. In order to determine
`the specificity of the antiglobulin responses elicited, sera from
`patients who demonstrated significant elevations in antiglobulin
`levels were tested against a variety of immunoglobulins (Table
`4). Specimens from the 4 CTCL patients were assessed for
`reactivity against whole T101 and a Fab fragment of T101, as
`well as 5 lgG murine myeloma proteins, the lgG and lgM com(cid:173)
`ponents of normal mouse serum, and a rabbit lgG preparation.
`Specimens from the 3 melanoma patients who demonstrated
`antiglobulin responses were tested against a similar panel, with
`the exception that the F(ab')2 fragment of 9.2.27 was substituted
`for the Fab fragment of T101.
`Sera from all 4 CTCL patients and the 3 melanoma patients
`demonstrated substantial reactivity with whole T101 or 9.2.27,
`most murine myeloma proteins of the different lgG subclasses,
`and mouse lgG (Table 4). Little or no reactivity was observed
`against the Fab or F(ab')2 fragments or to mouse lgM. These
`results suggest that the antiglobulin response elicited in these
`patients was directed to determinants common to murine lgG
`and was not specific for either the T101 or 9.2.27 antibody.
`Further, the lack of reactivity to Fab or F(ab')2 fragments sug(cid:173)
`gests that the reactivity is directed against determinants on the
`Fe region of the immunoglobulin molecule and not determinants,
`
`Table2
`Specific binding of human serum immunoglobulin in solid-phase ELISA lgG
`antig/obulin assay
`Solid-
`phase
`target
`antigen
`None8
`T101
`9.2.27
`
`Serum specimen
`Melanoma Patient K. G.
`prior to 9.2.27 treat-
`ment
`
`1:10
`o.oT'
`0.55
`0.47
`
`1:50
`0.00
`0.33
`0.31
`
`1:250
`0.00
`0.26
`0.24
`
`1:1250
`0.00
`0.14
`0.11
`
`Melanoma Patient K. G.
`following 9.2.27 treat-
`ment
`
`None
`T101
`9.2.27
`
`0.04
`0.56
`0.53
`
`0.00
`0.43
`0.49
`
`Normal control
`
`0.00
`0.08
`None
`0.35
`0.64
`T101
`0.37
`0.70
`9.2.27
`• No target antigen or control protein bound to plates.
`b Mean of duplicate absorbance determinations at 405 nm.
`
`0.00
`0.42
`0.41
`
`0.00
`0.30
`0.25
`
`0.00
`0.19
`0.20
`
`0.00
`0.10
`0.09
`
`solid-phase immunoglobulin, or to nonspecific interactions (4)
`such as Fe-Fe interactions between the human immunoglobulin
`and solid-phase murine immunoglobulin.
`Antiglobulin levels were assessed in patients over the period
`of treatment with either T101 or 9.2.27 antibodies as the target
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`881
`
`3 of 7
`
`BI Exhibit 1106
`
`

`

`HUMAN ANTl-M~INE IMMUNOGLOBWN RESPONSES
`
`A
`
`••••••••• ••••••••• •••••••• • •••••••
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`3
`Dose Number
`Dose Number
`Chart 2. Antiglobulin levels in CLL patients. Serum lgG (A) or lgM (8) antibody levels to the T101 antibody are indicated. Points, determinations on serun specimens
`from 9 patients. obtained either prior to therapy (Dose 0) or immedlately preceding the indicated dose.
`
`0
`
`7
`
`A
`
`B
`
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`
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`~
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`•
`•
`
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`7
`
`3
`5
`5
`3
`Dose Number
`Dose Number
`Chart 3. Antiglobulin levels in CTCL patients. Serum lgG (A) or lgM (8) antibody levels to the T101 antibody are indicated. Points, determinations on serun specimens
`from patients receiving total T101 doses of 8 (....._.), 66 (•· • · ·•). 80 (._--e), or 162 (..._...) mg. Specimens were obtained either prior to therapy (Dose OJ or
`imme<iately prior to the indicated dose.
`
`0
`
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`·---------------------.. -------· __..
`.. .,..... .. ~ ,.-.~~
`.................... /,.. .
`
`200
`100
`50
`10
`200
`100
`50
`10
`9.2.27 Antibody Dose (mg)
`9.2.27 Antibody Dose (mg)
`Chart 4. Antiglobuin levels in melanoma patients. Serum lgG (A) or lgM (8) antibody levels to the 9.2.27 antibody are indicated. Points, determinations on serun
`specimens from 9 patients obtained either prior to therapy (Pre) or immedlately preceding the indicated dose. Points from the 3 patients with significant (>2 S.D. above
`mean of normal controls) responses are comected to indicate the ~ of the response.
`
`500
`
`Pre
`
`500
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`882
`
`4 of 7
`
`BI Exhibit 1106
`
`

`

`HUMAN ANTl-MURINE IMMUNOGLOBll..IN RESPONSES
`
`Table4
`Specificity of posttherapy antiglobulin rnponses
`Values represent µg/ml of serum of human lgG ar lgM antiglobulin reactive with
`the indicated target antigen. Specimens were obtained immediately prior to the
`final dose of antibody.
`
`CTCL patients
`
`H.B. W.F.
`
`J. T.
`
`J. s.
`
`Melanoma patients
`M.F. c.s. C.J.S.
`
`71.4
`4.9
`
`91.2
`12.8
`
`61.2
`14.4
`
`63.0
`14.4
`
`40.0 157.5 107.1
`43.7
`58.0
`44.1
`
`Antibody
`preparation
`T101
`lgG
`lgM
`T101 Fab
`lgG
`lgM
`9.2.27
`lgG
`lgM
`9.2.27 F(ab')2
`lgG
`lgM
`MOPC-21 ('Y1)
`lgG
`lgM
`RPC-5('Y2a)
`lgG
`lgM
`UPC-10('Y2a)
`lgG
`lgM
`MOPC-141 (1'2b)
`lgG
`lgM
`FLOPC-21 (1'3)
`lgG
`lgM
`MouselgG
`lgG
`lgM
`Mouse lgM
`lgG
`lgM
`Rabbit lgG
`lgG
`lgM
`•ND, not done.
`
`1.9
`0
`
`1.9
`0.5
`
`2.7
`1.2
`
`111.8
`1.6
`
`55.9
`6.4
`
`75.0
`6.3
`
`ND
`ND
`ND
`ND
`150.9 n.9
`2.3
`8.2
`150.9 n.9
`2.3
`8.2
`
`ND
`ND
`
`61.3
`7.5
`
`61.3
`7.5
`
`184.2 104.2 104.2
`4.8
`1.2
`5.7
`
`165.2 180.4 148.0
`11.1
`2.2
`9.1
`
`185.6 142.4 142.4
`1.9
`3.4
`3.6
`
`1.5
`2.7
`
`17.9
`6.8
`
`ND
`ND
`
`51.4
`5.3
`
`51.4
`5.3
`
`19.8
`2.6
`
`24.9
`15.2
`
`2.8
`3.3
`
`Ncf
`ND
`
`ND
`ND
`
`ND
`ND
`
`25.1 129.4 108.7
`27.8 189.0
`29.1
`
`1.9 109.5
`4.7 125.7
`
`127.6 236.9
`14.8 167.3
`
`1.9
`12.0
`
`20.8
`36.4
`
`127.6 236.9 100.8
`14.8 167.3
`22.2
`
`85.8 256.7 158.6
`12.8
`53.4
`13.4
`
`116.2 173.3
`70.4 162.8
`
`159.1 166.7
`182.7 228.8
`
`29.2
`0.7
`
`33.1
`6.2
`
`41.7
`6.7
`
`38.2
`8.5
`
`108.4 297.5
`103.2· 213.5
`
`1.1
`0
`
`1.1
`0.9
`
`0
`0
`
`0
`0.8
`
`0
`0.5
`
`1.1
`1.6
`
`1.5
`0.7
`
`1.7
`3.7
`
`1.9
`1.0
`
`82.2
`13.5
`
`3.1
`29.2
`
`66.5
`59.9
`
`73.4
`12.8
`
`50.1
`12.0
`
`62.9
`75.2
`
`1.5
`1.2
`
`12.1
`17.7
`
`100 -
`
`H!I..
`
`W~.
`
`J.T:
`
`BO
`
`:
`
`I
`
`I
`I'
`I
`
`fl
`r
`
`••CC
`
`20
`
`o
`
`ii
`I ''·
`'
`' If
`fll 11 C D o a C D ••co •BCD
`Inhibitor
`Chart 5. Inhibition of human serum lgG antiglobulin activity with soluble murine
`lgG2a monodonal antibody preparations. Specimens obtained prior to antibody
`therapy (II) ar immediately prior to the final dose of antibody (II) were assessed by
`solid-phase ELISA for antiglobulin activity against the antibody used in therapy
`(T101 or 9.2.27). Inhibition of binding of serum antibody to the solid-phase antigen
`was assessed following addition of a 1000-fold excess of the following lgG2a
`murine monodonal antibodies: 9.2.27 (A); T101 (8); 03 (C); ar RPC-5 (D). Results
`are expressed as the percentage of Inhibition of titer in the presence of each
`blocking antibody as compared to the titer in the absence of a blocking antibody.
`The patient Initials corresponding to those in Table 4 are indicated over each
`distribution.
`
`sented in Table 4, indicate that this patient developed an anti(cid:173)
`globulin response which, although not completely specific for the
`9.2.27 antibody, consisted of a substantial component which
`appears to be specific for the 9.2.27 antibody.
`In order to determine the specificity of the preexisting antiglob(cid:173)
`ulin in these patients, pretreatment sera from 3 of the CTCL
`patients and the 3 melanoma patients with elevated antiglobulin
`levels during therapy were examined for reactivity against the
`panel of mouse and rabbit immunoglobulin preparations indicated
`in Table 4. These analyses demonstrated that the specificity of
`preexisting antiglobulins was very broad, consistent with the
`broad specificity of posttherapy antiglobulins in these patients.
`Data from a representative CTCL patient are presented in Chart
`6. This particular patient demonstrated detectable lgG antiglob(cid:173)
`ulin to many, but not all, murine immunoglobulin preparations
`examined and lgM reactivity against all murine immunoglobulin
`preparations. Elevated antiglobulin responses posttherapy con(cid:173)
`sisted of lgG antibodies.
`Effect of Antlglobulin Responses upon Therapy and In Vivo
`Localization of Antibody. Clinical responses in these Phase I
`trials were either transient or undetectable (2, 3, 14). The CLL
`patients all demonstrated transient decreases in leukemia counts
`but failed to demonstrate lasting effects following cessation of
`therapy. The CTCL patients had minor regressions of skin lesions
`that continued throughout the course of therapy regardless of
`antiglobulin responses. None of the 9 melanoma patients dem(cid:173)
`onstrated any regression of cutaneous nodules during the course
`of therapy. However, in vivo localization of the 9.2.27 antibody
`was detected in biopsy specimens removed during the course
`of therapy. The presence of antiglobulin responses did not
`appear to affect in vivo localization in the 3 patients with sub(cid:173)
`stantial antiglobulin levels. For example, Chart 7 compares in
`vivo binding of the treatment antibody as detected by immuno(cid:173)
`fluorescence and flow cytometry to the level of serum lgG
`antiglobulin in one patient. Serum antiglobulin levels as high as
`
`such as the antibody-combining site or idiotype, which reside in
`the Fab region. Interestingly, the 3 melanoma patients showed
`reactivity with rabbit lgG, indicating that the antiglobulin response
`in these patients was not mouse specific. The antiglobulin re(cid:173)
`sponse in the CTCL patients, however, appeared to be specific
`for mouse lgG.
`One melanoma patient, Patient C. S., demonstrated substan(cid:173)
`tial reactivity against the 9.2.27 F(ab')2 fragment as well as the
`whole 9.2.27 antibody (Table 4). However, this reactivity was
`not restricted to the 9.2.27 antibody but was also present against
`the murine myeloma proteins and the mouse and rabbit lgG
`preparations. To further investigate the potential of a 9.2.27 anti(cid:173)
`icliotypic component in the antiglobulin response of this and the
`other patients, a series of blocking studies similar to those
`described in Table 3 was performed. The results of these studies
`are presented in Chart 5. Due to insufficient quantities of serum,
`the 2 melanoma patients other than Patient C. S., who developed
`significant elevations in antiglobulin levels, could not be examined
`in this manner. With the exception of Patient C. S., the antiglob(cid:173)
`ulin response was largely inhibitable with all of the murine lgG2a
`preparations used, with no evidence of an anti-idiotypic compo(cid:173)
`nent to the antiglobulin response. Patient C. S. demonstrated
`over 80% inhibition in the presence of soluble 9.2.27 antibody,
`as compared to roughly 30% inhibition with the other prepara(cid:173)
`tions. These data, in combination with the binding studies pre-
`
`CANCER RESEARCH VOL 45 FEBRUARY 1985
`883
`
`5 of 7
`
`BI Exhibit 1106
`
`

`

`HUMAN ANTl-MURINE IMMl.JllOGLOBULIN RESPONSES
`
`Illa A
`
`I
`
`Le;ena
`
`g;;!ll'iii;-~
`1!11 11'\Ni -T~y
`
`0 .1
`
`.. . ..,
`
`•::1
`~.!'
`::I
`
`_ ......
`......... . ......
`;::;
`- .. ~~ ~.!::
`• ~ : u
`0
`... .!::
`
`-
`
`u .. . ..,
`on...,. • ;::;
`.......... . ......
`• -
`_ ......
`.... .. o-
`;::;
`.......
`•
`on.-..
`......
`:.;..,
`:.;..,
`·~
`I
`u
`a
`•
`I
`I
`•
`0
`• ~:: u
`..
`u
`.. u
`-
`..
`.. u
`u
`... ~ 0
`=:!:: ~~
`"ca
`... .!:;
`~.£ "!P...
`ILP...
`o-
`0-
`.... ...
`&:~ ~.!::
`.... ...
`""
`""
`o~ ::I
`::I
`::I
`::I
`::II
`Chart 6. Specificity of pretherapy and posttherapy antiglobulins in a CTCL patient. Saun lgG (A} and lgM (8) antiglobulin levels were assessed before and after
`therapy with the T101 antibody. The target preparation and its subtype if monoclonal are indicated on the llbscissa. Absence of a bar indicates an l.lldetectable level of
`antiglobulin (<0.1 ,.g/ml).
`
`.....
`
`..
`-.......
`
`;::;
`•
`r ......
`~.!t
`ILP...
`o~ ::I
`
`• •::1
`~.!t
`::I
`
`Legend
`1121 t.2.27 Binding
`-
`Antlglobulln
`
`100
`
`ao
`
`0
`~
`.,!;
`,,
`Cl so
`.!:
`.!:
`ID
`~ •O
`l'l
`ai
`~ 20
`
`100
`
`60
`
`c
`0
`Q.
`
`........
`~
`ao ci
`..
`::I.
`._,
`..
`..
`..
`•O a:: s
`:s
`20 t
`
`<
`
`0
`
`0
`
`0
`
`200
`100
`50
`10
`9.2.27 Antibody Dose (mg)
`Chart 7. In vivo localization of 9.2.27 antibody on melanoma cells in the pres(cid:173)
`ence of circulating antiglobulin. Binding of the 9.2.27 antibody was assessed in
`biopsy specimens taken prior to therapy and then at 24-hr intervals following the
`indicated dose in a single patient. Saun specimens for antiglobulin determinations
`were obtained immediately prior to the indicated dose, and the values indicate ,.g
`of human lgG per ml reactive with the 9.2.27 antibody. Paired serum and biopsy
`specimens were not obtained at the 10- and~ periods.
`
`175 µg/ml did not inhibit localization of 9.2.27 antibody to mela(cid:173)
`noma cells in vivo.
`
`DISCUSSION
`
`The development of host antiglobulin responses represents a
`potential obstacle to effective monoclonal antibody therapy. Such
`a response would be expected to result in immune complex
`formation, possibly inducing serum sickness or renal toxicity, or
`interfering with the efficacy of treatment, either by inhibiting
`binding of the administered antibody to tumor cells or by increas(cid:173)
`ing the removal of antibody by the reticuloenclothelial system.
`Examples of antiglobulins inhibiting the binding of monoclonal
`antibodies to tumor cells have been reported (1, 10)., In order to
`better characterize the development of host antiglobulin re(cid:173)
`sponses in patients receiving monoclonal antibody therapy. we
`examined human anti-murine immunoglobulin levels in 3 different
`
`patient populations receiving 2 different antibody preparations.
`This paper represents, to our knowledge, the first detailed inves(cid:173)
`tigation of antiglobulin responses of multiple patient populations
`receiving different murine monoclonal antibodies.
`The CLL patients demonstrated little or no detectable levels
`of antiglobulin prior to therapy. Conversely, the CTCL and mel(cid:173)
`anoma patient groups and the normal control group had meas(cid:173)
`urable levels of preexisting mouse lgG-reactive antiglobulin.
`While the antiglobulin levels in the CTCL or melanoma patient
`groups were not significantly increased or decreased as com(cid:173)
`pared to the control group, the 3 melanoma patients who devel(cid:173)
`oped increased levels of antiglobulin following therapy demon(cid:173)
`strated significantly elevated antiglobulin levels of either lgG or
`lgM class prior to therapy.
`All 3 patient populations received murine lgG2a antibodies.
`Although the treatment schedule and total dose levels varied
`among the patient groups, similar dose levels were administered
`to each group, and the period of therapy (2.5 to 4 weeks) was
`comparable. Each of the 3 patient groups responded differently.
`Within those patients receiving T101 antibody therapy, 0 of 10
`CLL patients as opposed to all 4 of 4 CTCL patients developed
`increased levels of antiglobulins. Three of the 9 melanoma pa(cid:173)
`tients receiving the 9.2.27 antibody developed increased levels
`of antiglobulins.
`The individuals who developed significant elevations in anti(cid:173)
`globulins did so very r