VOLUME 43;; NO. 2 CNRéA a» PE 481-933..
`.
`February1985 :
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`
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`u n- -
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`PFIZER EX. 1106
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`Page 1
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`PFIZER EX. 1106
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`PFIZER EX. 1106
`Page 2
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`

`

`[CANCER RESEARCH 45. 879-885, February 19851
`
`:-tuman Anti-Murine Immunoglobulin Responses in Patients Receiving
`~VIonoclonal Antibody Therapy 1
`
`i lobert W. Schr.off,2 Kenneth A. Foon, Shannon M. Beatty, Robert K. Oldham, and Alton C. Morgan, Jr.
`
`,Jiological Therapeutics Branch, Biological Response Modiliers Program. National Cancer Institute, Frederick, Maryland 21701
`
`, BSTRACT
`
`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 chrqnic lymphocytic leukemia pa(cid:173)
`tients receiving the T1 01 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 NOt-C0-23910 with
`Program Resources, Inc.
`2 To whom requests for reprints should be addressed. at BRMP, NCI-FCRF,
`Bldg. 560, Room 31-93, Frederick, MD 21 701.
`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 at. (1 0) 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
`at. (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 at. (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 T1 01 in patients with CLL 3 and CTCL
`and the lgG2a monoclonal antibody 9.2.27 in patients with
`malignant melanoma (2, 3, 14). The T1 01 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 aM, 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 T1 01 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·
`neous T -cell lymphoma: ELISA. enzyme-linked immunosorbent assay.
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`879
`
`PFIZER EX. 1106
`Page 3
`
`

`

`HUMAN ANTI-MURINE 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 T1 01 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 T1 01 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% NaCI 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 T1 01 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 stQred 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 i>elyvinyl plates at
`100 ng of antibody per well and washed with 0.1 M tris (pH 8.3)-0.02%
`NaN3-0.5% 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 or JJ.) 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 JJ.g of protein bound to plates per ml of
`serum.
`For assays of antiglobulin specificity, plates were coated with T1 01
`Fab or 9.2.27 F(ab' )2 preparations (100 ngfwell); the mouse myeloma
`proteins MOPC-21 (lgG1 ), RPC-5 (lgG2a), UPC-1 0 (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 T1 01 or 9.2 .27 target antigen was assessed by performing
`the ELISA in the presence of a 1 000-fold-greater concentration (1 00 11g!
`well) of the soluble inhibitor murine lgG2a antibodies 9.2.27 , T1 01, D3,
`or RPC-5 as compared to the solid-phase target immunoglobulin.
`Assay for Mouse Immunoglobulin. 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 1 00 ngfwell and washed as above. Bound mouse immunoglobulin
`
`Table 1
`Summary of monoclonal antibody therapy
`
`No. of patients
`treated
`
`1
`2
`1
`2
`1
`1
`2
`
`Disease
`CLL
`CLL
`CLL
`CLL
`CLL
`CLL
`CLL
`
`CICL
`CTCL
`CTCL
`CTCL
`
`Melanoma
`Melanoma
`
`Total dose
`received
`(mg)
`
`6
`8
`50
`80
`150
`300
`400
`
`8
`66
`80
`162
`
`361
`860
`
`was detected with a goat anti-mouse immunoglobulin conjugated with
`alkaline phosphatase (Sigma) and compared against a standard curve of
`either T1 01 or 9.2.27 antibody.
`Assay for Human Serum Immunoglobulin. Serum lgG and lgM levels
`were determined by radial immunodiffusion utilizing Endoplate immuno(cid:173)
`globulin test kits obtained from Kallestad Laboratories, Austin , TX .
`Immunofluorescent 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 T1 01 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. Serurn
`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 inclfbated on plates coated
`with either the T1 01 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 1 000-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
`
`PFIZER EX. 1106
`Page 4
`
`

`

`HUMAN ANTI-MURINE IMMUNOGLOBULIN RESPONSES
`
`Table 3
`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 .
`
`1600
`
`1400
`
`........
`1200 a,
`'6
`g
`1000 C/)
`Qi
`>
`800 ~
`
`0
`600 SF
`E
`400 2
`<ll
`Vl
`
`200
`
`280
`
`240 ........
`~
`200 Ol g
`
`T101 9.2.27
`CONTROLS
`
`CLL
`
`CTCL
`
`lolol
`
`120
`
`C/)
`160 Qj
`>
`<ll
`...J
`:::l:
`SF
`80 E
`:J
`....
`Q)
`Vl
`
`40
`
`........
`
`10 B
`~ Ol
`2:
`
`8
`
`6
`
`4
`
`C/)
`Qj
`>
`Q)
`...J
`
`.0
`0
`
`,!;; -s
`:Q> c <
`
`E 2
`:J
`....
`Q)
`Vl
`
`lolo l
`
`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
`IT1 01 for CLL and CTCL patients, 9.2.27 for melanoma (Mel ) patients] are shown
`(0 ). Serum immunoglobulin levels are indicated for comparison (•). 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).
`
`Table2
`Specific binding of human serum immunoglobulin in solid-phase ELISA lgG
`antiglobulin assay
`
`Serum specimen
`
`Melanoma Patient K. G.
`prior to 9.2.27 treat-
`ment
`
`Melanoma Patient K. G.
`following 9.2.27 treat-
`ment
`
`Normal control
`
`Solid-
`phase
`target
`antigen
`
`None•
`T101
`9.2.27
`
`None
`T101
`9.2.27
`
`None
`T1 01
`9.2.27
`
`Serum dilution
`
`1:10
`
`0.07b
`0.55
`0.47
`
`0.04
`0.56
`0.53
`
`0.08
`0.64
`0.70
`
`1:50
`
`0.00
`0.33
`0.31
`
`0.00
`0.43
`0.49
`
`0.00
`0.35
`0.37
`
`1:250
`
`1:1250
`
`0.00
`0.26
`0.24
`
`0.00
`0.42
`0.41
`
`0.00
`0.30
`0.25
`
`0.00
`0.14
`0.11
`
`0.00
`0.19
`0.20
`
`0.00
`0.10
`0.09
`
`• No target antigen or control protein bound to plates.
`b Mean of duplicate absorbance determinations at 405 nm.
`
`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 T1 01 or 9.2.27 antibodies as the target
`
`Monoclonal
`antibodies
`
`None
`9.2.27
`T101
`03
`APC-5
`
`Inhibitor
`
`% of inhibition
`
`Titer"
`70 ± 45b
`42 ± 30
`42 ± 5
`43 ± 36
`41 ± 9
`34 ± 27
`51 ± 10
`35 ± 36
`57 + 17
`: Reciprocal of the dilution yielding an absorbance at 405 nm of 0.3.
`Mean± S.D.
`
`antigens. In the T1 01 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 JLg/ml. Specimens from melanoma patients
`demonstrated somewhat higher levels of mouse lgG but, in all
`cases , were less than 25 JLg/ml. As depicted in Chart 2, CLL
`patients treated with T1 01 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 T1 01 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 th~ 4 CTCL patients were assessed for
`reactivity against whole T1 01 and a Fab fragment of T1 01 , 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 T1 01 .
`Sera from all 4 CTCL patients and the 3 melanoma patients
`demonstrated substantial reactivity with whole T1 01 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,
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`881
`
`PFIZER EX. 1106
`Page 5
`
`

`

`HUMAN ANTI-MURINE IMMUNOGLOBULIN RESPONSES
`
`A
`
`B
`
`175
`
`0'>
`
`:t 150
`3 ., 125
`
`(/)
`
`100
`
`75
`
`50
`
`c
`0
`.,
`a.
`C/)
`a::
`-~
`-s
`..0
`0
`:2> 25
`c
`
`<(
`
`• •••••••••••••••••••••••••••••••••
`
`......... ......... ........ ....... .
`
`175
`
`0'>
`
`:t 150
`3 ., 125
`
`C/)
`
`100
`
`75
`
`50
`
`c
`0
`.,
`5r
`a::
`.£
`-s
`..0
`0
`:2> 25
`c
`
`<(
`
`0
`
`7 .. -
`
`5
`3
`5
`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 serum specimens
`from 9 patients-obtained either prior to therapy (Dose 0) or immediately preceding the indicated dose.
`
`0
`
`7
`
`A
`
`B
`
`t50
`
`Ol
`
`:t 175
`3 t25
`G> .,
`c
`0 a. .,
`
`100
`
`75
`
`I)
`D:
`,!:
`~ 50
`.,Q
`0
`:2> 25
`c <(
`
`.--...
`
`t75
`
`Ol
`
`:t t50
`3 125
`G> .,
`c
`0 a. .,
`I)
`D:
`c
`'5
`.,Q
`0
`~ 25
`c <(
`
`100
`
`75
`
`50
`
`.. --------.... ~' .
`
`....... ............
`- __.- z~=-=-•
`
`....
`
`-
`
`0
`
`3
`5
`Dose Number
`
`7
`
`0
`
`5
`3
`Dose Number
`
`7
`
`Chart 3. Antiglobulin leveis in CTCL patients. Serum lgG (A) or lgM (8) antibody levels to the T1 01 antibody are indicated. Points , determinations on serum specimens
`from patients receiving total T101 doses of 8 (._____....), 66 (e .. . ·• ). 80 (e ---e), or 162 (e -__.) mg. Specimens were obtained either prior to therapy (Dose 0) or
`immediately prior to the indicated dose.
`
`A
`
`B
`
`.--... 175
`
`:t 150
`3 125
`.,
`c
`0 a.
`.,
`.!: -s
`..0
`0
`;Q> 25
`c
`
`0'>
`
`C/)
`
`C/)
`
`0::
`
`<(
`
`100
`
`75
`
`50
`
`175
`
`0'.
`
`:t 150
`3 125
`.,
`C/) c
`0
`a.
`1/) .,
`.£ -s
`..0
`0
`:2> 25
`c <(
`
`100
`
`D:
`
`75
`
`50
`
`. ---- --- -- -- --- -------.. -
`-·
`.. ~-;:~-. ...... ..::-:~::-,,. . ..
`~------
`
`Pre
`
`100
`50
`10
`200
`9.2.27 Antibody Dose (mg)
`
`500
`
`,.
`
`..
`
`Pre
`
`200
`100
`50
`10
`9.2.27 Antibody Dose (mg)
`
`500
`
`Chart4. Antiglobulin levels in melanoma patients. Serum lgG (A) or lgM (8) antibody levels to the 9.2.27 antibody are indicated. Points , determinations on serum
`specimens from 9 patients obtained either prior to therapy (Pre) or immediately preceding the indicated dose. Points from the 3 patients with significant (>2 S.D. above
`mean of normal controls) responses are connected to indicate the progression of the response .
`
`CANCER RESEARCH VOL. 45 FEBRUARY 1985
`882
`
`PFIZER EX. 1106
`Page 6
`
`

`

`Antibody
`preparation
`
`T101
`lgG
`lgM
`T101 Fab
`lgG
`lgM
`9. 2. 27
`lgG
`lgM
`9 2. 27 F(ab' ).
`lgG
`lgM
`rAOPC-21 ('Y 1)
`lgG
`lgM
`RPC-5 (1·2a)
`lgG
`lgM
`UPC-1 0 ('Y2a)
`lgG
`lgM
`MOPC-141 (-y 2b)
`lgG
`lgM
`FLOPC-21 ('Y3)
`lgG
`lgM
`ouse lgG
`lgG
`lgM
`Mouse lgM
`lgG
`lgM
`Rabbit lgG
`lgG
`lgM
`• NO. 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
`
`NO
`NO
`
`NO
`NO
`
`NO
`NO
`
`150.9
`2.3
`
`77.9
`8.2
`
`61 .3
`7.5
`
`77 .9
`82
`
`150.9
`2.3
`
`61.3
`7.5
`;
`184.2 104.2 104.2
`5.7
`4.8
`1.2
`
`165.2 180.4 148.0
`11 .1
`2.2
`9.1
`
`185.6 142.4 142.4
`3.6
`1.9
`3.4
`
`1.5
`2.7
`
`17.9
`6.8
`
`NO
`NO
`
`51 .4
`5.3
`
`51 .4
`5.3
`
`19.8
`2.6
`
`24.9
`15.2
`
`2.8
`3.3
`
`No•
`NO
`
`NO
`NO
`
`NO
`NO
`
`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
`13.4
`12.8
`53.4
`
`116.2 173.3
`70.4 162.8
`
`166.7
`159.1
`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
`
`68.5
`59.9
`
`73.4
`12.8
`
`50.1
`12.0
`
`62.9
`75.2
`
`1.5
`1.2
`
`12.1
`17.7
`
`H. B.
`
`W.F.
`
`J.T.
`
`J.S.
`
`c.s.
`
`100
`
`BO
`
`§ 60
`:E
`f
`~ ~ 0
`(,)
`~
`Q.
`
`20
`
`Inhibitor
`Chart 5. Inhibition of human serum lgG antiglobulin activity with soluble murine
`lgG2a monoclonal antibody preparations. Specimens obtained prior to antibody
`therapy (D) or immediately prior to the final dose of antibody (II) were assessed by
`solid-phase ELISA for antiglobulin activity against the antibody used in therapy
`(T1 01 or 9.2.27). Inhibition of binding of serum antibody to the solid-phase antigen
`was assessed following addition of a 1 000-fold excess of the following lgG2a
`murine monoclonal antibodies: 9.2.27 (A); T101 (8); 03 (C); or RPC-5 (0). 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