Immunol. Cell Bio!. (1990)68, 367-376
`
`Development of human anti-murine antibody (HAMA) response
`in patients
`
`Joe J. Tjandra, Lanny Ramadi and Ian F. C. McKenzie
`
`Research Centre for Cancer and Transplantation, Department of Pathology. University of
`Melbourne, Parkville. Victoria 3052. Australia
`
`(Submitted
`
`I June 1990. Accepted for publication 9 November 1990.)
`
`Summar>' Human anli-mouse antibody (HAMA) response was determined in the scrum of 67
`patients who received subcutaneously administered radiolabelled murine monoclonal antibodies
`(MoAb) (50 ^ig-3 mg) for immunolymphoseintigraphy and of 10 patients with advanced colorectal
`cancer who received murine MoAb-A'-acelyl melphalan (MoAb-ZV-AcMEL) conjugates (amount of
`MoAb ranged from 120 mg/m- body surface area to 1000 mg/m- body surfaee area) as therapy. A
`pre-existing low level of apparent human anti-mouse antibody reactivity could be deteeted in the
`serum of normal subjects and patients prior to administration of murine MoAb. Subcutaneous
`administration of low doses of murine MoAb. as used in immunolympboseintigraphy, was associated
`with a low incidence (4/67 or 6%) of elevated HAMA response; the use of F(ab')2 fragments was
`associated with the development of elevated HAMA response in one of three patients. By contrast,
`therapy with hepatic artery infusion of murine MoAb-A'-AcMEL conjugates in three repetitive daily
`doses (each infusion lasting 2 h)elieited elevated HAMA responses in 10/10(100%) patients, usually
`1-3 weeks after the start of therapy. The HAMA response of patients in the therapy group was higher
`than those in the immunolymphoseintigraphy study and the use of steroids did not prevent the
`development of the HAMA response. Further administration of MoAb-A-'-AcMEL conjugates to a
`patient, who had already developed HAMA, led to "serum siekness'-type symptoms and a transient
`reduction in the HAMA titres. The elevated HAMA response was polyclonal, containing increased
`levels of both immunoglobulin M and G (IgM and IgG) and was directed against mouse-specific
`determinants, the isotype (presumed to be the Fc portion), the F(ab')2 and the 'idiotype' of mouse
`immunoglobuiins.
`
`INTRODUCTION
`Murine monoclonal antibodies (MoAb) with
`specificity for tumour-associated antigens are
`increasingly being used as carrier molecules for
`radio-imaging agetits such as '^'I, ' "In, '^3|(i_
`4) and for therapeutic agents such as cytotoxic
`drugs (5-7). One problem with the use of murine
`MoAb in humans has been the development of
`the human anti-murine antibody (HAMA)
`response (8-11) which restricts repetitive dos-
`ing. However, reports differ on the incidence
`and nature of the development of HAMA
`response; the differences were probably related
`
`I. F. C. McKenzie, Research
`Correspondence:
`Centre for Cancer and Transplantation. Department
`of Pathology, University of Melbourne, Parkville, Vic.
`3052, Australia.
`Abbreviations used in this paper: " ' 1, iodine-131;
`HAMA, human anti-murine antibody; i,v., intra-
`venous; w/w, weight;weight; Ig, immunoglobulin;
`MoAb, monoelonal antibody/ies; PBS, phosphate buf-
`fered saline; s.c., subcutaneous{ly).
`
`to the different clinical programmes (amount of
`foreign protein administered, the route and
`number of treatments and the time interval
`between treatments), the immune status of the
`patients and the methodological differences in
`assay techniques for HAMA. In addition, the
`nature of HAMA response in cancer patients
`may be different from that elicited in transplant
`patients by mouse MoAb OKT3 and this is not
`surprising as 0KT3 has both stimulatory and
`suppressive effects on T cells (12).
`Radio-iodinated murine MoAb RCC-1 (reac-
`tive with breast cancer) had been administered
`subcutaneously (s.c.) for immunotymphoscinti-
`graphy, and when used together with cold iodine
`labelled 'blocking' Ly2l antibody (non-reactive
`with breast cancer), successful localization of
`axillary lymph node metastases from breast can-
`cer occurred in about 90% of cases (3). Various
`MoAb have also been conjugated to the A'-acetyl
`derivative of melphalan (/V-AcMEL) and shown
`to have in vitro and in vivo specificity and cyto-
`toxicity and specifically inhibit the growth of
`
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`368
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`J.J. TJANDRA Er.4L.
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`human colon carcinomas xenografted in athy-
`mic mice (13,14). The MoAb-A'-AcMEL conju-
`gates have also been administered by means of
`hepatic artery
`infusion
`to patients with
`advanced colorectal cancer and have achieved
`biological responses in some patients (7).
`The aim of the study was to determine the nat-
`ure of the HAMA response in patients after
`regional administration of murine MoAb for
`immunolymphoscintigraphy (s.c. injection) or
`therapy (hepatic artery infusion).
`
`MATERIALS AND METHODS
`
`Murine monoclonal antibodies
`For imaging studies, murine MoAb 3E1 -2 immuno-
`globulin (IgM), RCC-1 (formerly called 17-1; IgG 2a)
`and Ly21 (IgG2a) were used. Antibody 3E1 2 was
`raised against fresh human breast carcinoma (15).
`RCC-1 was raised by immunizing inbred Biozzi mice
`with the MCF-7 breast cancer ceil line (16). The Ly2-1
`antibody is reactive with the murine Ly2l specificity
`but not witb human breast cancer (17). The Ly21 ami-
`body was used in the study as a "blocker' to reduce
`background non-specific uptake of '^'I-RCC-l (3).
`Murine MoAb used for therapy included 30-6 (IgG2b),
`which is reactive against a large number of colon car-
`cinoma cell lines (18), I-l and JGT (IgGl), which are
`both anti-CEA (carcinoembryonic antigen) (19). Irrel-
`evant murine MoAb used to evaluate the HAMA
`response included BCl (IgGi), BC2 (igGl), and BC3
`(IgM) which were directed against mucin-like glyco-
`proteins of breast (20); and polyclonal antibodies of
`monkey and sheep origin (produced in our labora-
`tory). MoAb 3E1 2 was purified from ascitic fluid (21)
`and the antibodies RCC-1 and Ly-21 were purified
`on Protein A-Sepharose (Pharmacia inc., Piscataway,
`New Jersey, USA as described previously (22); anti-
`bodies 30-6,1-1 and JGT by Protein A-Suparose (Phar-
`macia Inc., Piscataway, NJ). Purity was assessed by
`sodium dodecyl sulfate-polyacrylamide gel electro-
`phoresis. The F(ab')2 fragments were obtained from
`purified MoAb RCC-1 by pepsin digestion (23). Anti-
`bodies were aliquoted and stored at —70°C until
`used.
`
`Iodinalion of monoclonal antibodies
`Purified antibodies (3E1 2, RCC-1 or RCC-1
`F(ab')3) were radiolabelled with'^'I (Amersham Int.,
`UK) using the iodobead (24) or enzymobead reagent
`(Bio-Rad, Richmond, California, USA) (25). The puri-
`fied Ly2-1 antibody was labelled with non-radioactive
`sodium iodide or '^'l using the chloramine T method
`(26). Preparation of the iodinated antibodies has been
`previously described (3,21).
`
`Preparation of drug-antibody conjugates
`The A'-acetyl derivative of melphalan (A'-AcMEL)
`was prepared and conjugated to the antibodies (306,
`I-l and JGT) as described previously (14). The anti-
`body activity and cytotoxicity of the immunocon-
`
`jugates were ensured by resetting assay (27) and
`inhibition of DNA synthesis using [-"HJ-thymidine,
`respectively (14).
`
`Patient studies
`For diagnostic studies (immunolymphoscintigra-
`phy) to localize axillary lymph node metastases, 67
`patients with various breast conditions (benign and
`malignant) (Table 1) received a variety of iodinated
`preparations s.c. They included '^'I-labelled 3E1-2
`(50-200 Hg), '^'I-labelled RCC-1 (50-400 jig), '^ii.
`labelled Ly-21 (0 4-2 mg), '^iMabelled RCC-1 (04-1
`mg) together with'blocker'antibody Ly21 (2 mg) iod-
`inated with non-radioactive sodium iodide and '-^'I-
`labelled RCC-1 F(ab')2- To prevent thyroid uptake of
`free radioiodine, patients received potassium iodide (5
`mL of 16 54% w/v) and sodium perchlorate (400 mg)
`orally, 1 h before the s.c. injection; the potassium iod-
`ide was continued for 5 days after the injection.
`For therapy, nine patients with extensive colorectal
`hepatic metastases (119 also had pulmonary metas-
`tases) and one patient who had a curative resection of
`the Duke's C colon cancer received between 120
`mg/m^ and 1000 mg/m^ body surface area of MoAb
`(274-1696 mg) (I-l and/or JGT and/or 30 6) conju-
`gated with between 5 mg/m^ body surface area to 20
`mg/m^ body surface area of jV-AcMEL (Table 2). The
`MoAb selected for conjugation with A-AcMEL were,
`where possible, individually chosen for each patient,
`based on the binding of the particular antibody (I-1,
`JGT, 30 6) to sections of the primary colon cancer
`tissue as assessed by immunoperoxidase staining. In
`general, MoAb was selected only if it stained >50% of
`the carcinoma cells on the sections. None of the
`patients received any other form of treatment for 4
`weeks before and 8 weeks after treatment with the
`i m m unoconj ugates.
`The immunoconjugate was administered via hep-
`atic artery infusion over 2 h per day for 2 days (7). All
`patients received the immunoconjugates
`in three
`equal doses (r = 0 h, ? = 24 h, ? = 48 h). Patients had
`prophylactic intravenous (i.v.) dexamethasone 8 mg
`just before each infusion of the immunoconjugate and
`oral prednisolone 10 mg daily for 7 days after com-
`pletion of infusion.
`the Medical
`Both studies were approved by
`Research Board of the Royal Melbourne Hospital and
`written informed consent was obtained from every
`patient. Blood samples were obtained from patients
`before, during and after the injection of MoAb conju-
`gates to detect human anti-murine immunoglobulin
`(HAMA) response. Normal serum samples (n = 20)
`were also obtained from apparently healthy blood
`donors. Ail samples were aliquoted and stored at
`-7O'C.
`
`Human anti-murine antibody (HAMA) response
`Human antibodies against the murine MoAb were
`measured by an enzyme-linked immunosorbcnt assay
`(ELISA), modified from that described previously (9).
`Ninety-six well flexible polyvinylchloride (PVC)
`plates (Costar, Cambridge, Massachusetts, USA) were
`
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`HUMAN ANTI-MURINE lMMUNOGLOBULIN RESPONSES
`
`369
`
`coated with 100 nL/well of various purified murine
`MoAb (5^g/mL) in a 01 M carbonate buifer. pH 9-6
`and non-specific binding blocked with 1% bovine
`serum aibumin/PBS (phosphate buffered saline) pH
`7-6. Serial dilutions of patients' sera and pooled nor-
`mal human serum (50 tiL/well) in PBS/0 05% Tween
`20 were added to the antibody coated wells and incu-
`bated for 16 h at 4°C. Plates were then washed with
`PBS/0 05% Tween 20 and then reacted with 50
`nUwell of a 1:600 dilution of sheep anti-human
`immunogiobulin conjugated to horseradish peroxi-
`dase (Amersham International, UK) for 3 h at 37°C.
`The colour reaction was developed using 50 (iL of
`0-03% ABTS [2.2'-azino-di- (3-ethylbenthiazoline)
`sulfonate] {Amersham International. UK) and 0 02%
`hydrogen peroxide (BDH Chemicals, Poole, UK) and
`read with an ELISA plate reader (Titertek Multiscan
`MC) at a wavelength of 405 nm. In some cases, the IgM
`and IgG components ofthe HAMA response were sep-
`arately measured by using phosphatase labelled affin-
`ity purified goat anti-human IgM or IgG {Kirkegaard
`and Parry, Maryland) respectively and the colour reac-
`tion developed with alkaline phosphatase substrate.
`Results were expressed as the absorbance value of
`patient serum compared with control serum (pooled
`normal human serum from 20 apparently healthy
`blood donors) and a positive test was defined as one in
`which the absorbance was equal to or greater than
`twice the absorbance of pooled normal human serum.
`The HAMA titre was determined by obtaining the
`inverse of the highest serum dilution which gave a
`positive test result, and was arbitrarily graded as weak
`(titre 100-<1600), moderate (titre 1600-6400) or
`strong (titre > 64(>0). The background was too high at
`serum dilution less than 1/100; thus, within the sensi-
`tivity ofthe assay, HAMA titre of less than 100 eannot
`be determined accurately.
`
`RESULTS
`
`Imaging studies
`A low level of apparent anti-murine immunogio-
`bulin activity can be measured in the serum of
`normal individuals (NHS) (Fig. I, NHS) and
`also in the pre-immune setum of patients. These
`levels were detected both in patients who later
`produced an elevated HAMA response and in
`those who did not produce such a response. A
`HAMA response was considered positive when
`the absorbance of the serum was equal to or
`greater than twice the absorbance of pooled
`normal sera (Fig. 1). Table 1 sumtnarizes the
`measurements of antibody to murine antibodies
`in 67 patients with various breast conditions
`(benign and malignant) prior to subcutaneous
`injection of murine antibodies for
`imaging
`studies and 2 weeks-2 months after injection.
`Four of 67 patients (6%) showed a positive
`HAMA response {Table I), which developed
`
`3
`
`102
`
`103
`
`10*
`
`106
`
`Serum diiution"'
`
`Fig. 1. Level of pre-exist ing anti-murine antibody in
`a representative pooled normal human serum (HI)
`compared with the elevated HAMA response in a
`patient (•)(Table 2; Patient 7) who had received thera-
`peutic administration of murine MoAb-A'-AcMEL
`conjugates 4 weeks previously.
`
`within 2 months of exposure. None of. the
`patients had a strong response (titre > 6400) and
`in the limited number of patients studied, the
`intensity of the response did not relate to the
`amount (50 |ig-3 mg) of murine antibody
`injected. In addition, none of the patients
`developed any elinical side effects from
`the
`injection that could be related to the develop-
`ment of a positive HAMA response. Thus doses
`of up to 3 mg of murine MoAb given s.c. did not
`give rise to an elevated HAMA response in most
`patients.
`
`Therapy studies
`Table 2 summarizes the HAMA response in ten
`patients with advanced colorectal cancer prior
`to and after therapy with MoAb-A'-acetyl mel-
`phalan conjugates (amount of antibody: 120
`mg/m^-lOOO mg/m2)
`via hepatic
`artery
`infusion. All the patients had three doses of tbe
`injection, 24 h apart and received prophylactic
`steroid therapy 1 h before and for 7 days after
`therapy. All the ten patients (100%) developed
`elevated HAMA responses; the intensity of the
`response did not correlate with the amount of
`murine antibodies received (Table 2) or with the
`pre-itnmune level of anti-murine immunogiobu-
`lin activity. The elevated HAMA
`response
`
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`370
`
`J.J.
`
`Table 1. Summary of HAMA response in patients receiving murine MoAb for imaging studies.
`
`Number of patients with HAMA
`response (titre)
`
`Time from
`administration
`of Ab
`
`No. of
`patients
`studied
`
`Nil
`« 1 00
`
`)
`
`Weak
`(IOO-<IOOO)
`
`Moderate
`(1000-6400)
`
`Strong
`(>6400)
`
`0000000000
`
`000101000I
`
`0 100000000
`
`12
`11
`17
`16
`32
`31
`
`333 2
`
`12
`
`17
`
`32
`
`3 3
`
`02
`
`02
`
`02
`
`02
`
`02
`
` weeks-2 months
`
` weeks-2 months
`
` weeks-2 months
`
` weeks-2 months
`
` weeks-2 months
`
`MoAb administered
`(amount)
`
`3E1-2 (50-200 (ig)
`
`RCC-1 (50-400 tig)
`
`RCC-1 (400tig-lmg)
`+Ly-2l{2mg)
`Ly-2 1 {400jig-2mg)
`
`RCC-1 F(ab')2 (400 ^g)
`
`Table 2. Summary of therapy studies.
`
`Patient
`1
`
`MoAb administered
`
`Amount of Ab^
`
`I-l
`
`120mg/mMRXl)
`
`Time from administration
`of antibody (weeks)
`
`I-l, JGT
`
`160mg/m2(RX2)
`
`HAMA response
`(titre)
`<too
`1-5X105
`1 0X105
`1 0X105
`1-9X105
`1-5X105
`<100
`5-0X104
`2-5X10''
`<100
`2-0X10''
`l ^ X I O -"
`<100
`<100
`3-2X103
`<100
`1-0X105
`5-0X10^
`<I00
`5-0X10^
`5-OXlOt
`<100
`4-0X105
`2-0X105
`<100
`1-0X105
`<100
`3-2X103
`3-2X103
`<100
`5-OXIO'*
`^When multiple antibodies were used for drug conjugation, the final preparation of the immunoconjugates had,
`where possible, equal proportions of eaeh MoAb. Doses were expressed in amount of MoAb/surface area of the
`patient. RXl, first course of treatment; RX2, second course of treatment, given to Patient 1.
`
`30-6, I-l, JGT
`
`980 mg/m^
`
`I-l
`
`250 mg/m2
`
`30-6, I-l, JGT
`
`340 mg/m^
`
`I-l
`
`380 mg/m^
`
`1-1, JGT
`
`500 mg/m3
`
`30-6
`
`440 mg/m^
`
`30-6, 1-1, JGT
`
`1000mg/m2
`
`'
`
`I-l, JGT
`
`I-l, JGT
`
`820 mg/m^
`
`2 3 4 5 6 7 8 9 1
`
`0
`
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`HUMAN ANTI-MURINE iMMUNOGLOBULlN RESPONSES
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`
`developed in 1/10 patients as early as Day 5 after
`the first administration of infusion, but most
`patients (7/10) developed elevated HAMA
`response (as defined earlier) between I and 3
`weeks after the exposure to the foreign protein
`(Fig. 2). The peak response occurred 14-28 days
`following exposure in 7/8 cases; in two other
`patients, serum beyond this period was not
`available. In 2/3 patients who were followed for
`up to 9 months, the elevation of HAMA
`response persisted but there was a gradual fall in
`the titres. Five of the patients (Table 2; Patient 1
`RX2. patients 7, 8, 9 and 10) developed fever
`and this appeared to correlate with the amount
`of the immunoconjugates received and was not
`related to the intensity of the HAMA response.
`One patient (Patient 1) received 120 mg/m- of
`MoAb and developed an elevated HAMA
`response (Fig. 3). Two months later a further
`dose (160 mg/m^ MoAb) was given and
`symptoms suggestive of a Type III hypersensi-
`tivity 'serum sickness' reaction developed but
`was self limiting. During the second course of
`treatment {Fig. 3, RX2). there was an initial
`reduction of HAMA titre which might indicate
`immune complex formation, followed by a
`boost of the HAMA response, as from Day 10
`onwards.
`
`Specificity of HAMA response
`To determine whether the HAMA response was
`directed against common determinants on the
`constant domain of mouse immunoglobuiins or
`whether it was specific for each MoAb. the bind-
`ing of patients' sera to microtitre wells contain-
`ing relevant MoAb (i.e.. the same as that injected
`into patients), or other murine MoAb ('irrel-
`evant') of the same or different isotypes as that
`of the relevant antibody were assayed as before.
`The results of a representative patient (Table 2;
`Patient 1) are shown. As shown in Fig. 4(a). sera
`from Patient 1 in the therapy studies, who had a
`positive HAMA response, contained antibodies
`that bound well to both the relevant (injected:
`I-I) and the irrelevant murine MoAb (3E1-2,
`BC2). irrespective of antibody subclass. Similar
`results were obtained when the serum samples of
`other patients who developed a positive HAMA
`response were tested.
`
`This indicates tbat there is a major reaction to
`common determinants of all mouse immunoglo-
`buiins; such a reaction could be with the kappa
`light chain, but this was not specifically meas-
`ured.
`
`300 000
`
`200 000
`
`100 000
`
`2 3-
`
`2-
`
`1-
`
`Rx1 W2 W4 W8 0x2 W2 W4
`
`Time (weeks)
`
`Time (weeks)
`
`Fig. 2. Time in weeks after initiation of MoAb-A'-
`AcMEL treaimcnl wben elevated HAMA responses
`were first detected. Week 1 refers to days 1-7 after the
`start of treatment; Week 2 refers to days 8-14. and so
`on.
`
`Fig. 3. Development of elevated HAMA response in
`Patient I (Table 2) after the first treatment (RX 1) of
`MoAb-A'-AcMEL with time. Changes in levels of free
`circulating HAMA during (RX2) and after second
`administration of MoAb-A'-AcMEL were also illus-
`trated. The time was expressed in weeks (W) from the
`time of respective treatment.
`
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`The binding of sera of patients, before and
`after immunization, to polyclonal antibodies of
`monkey and sheep origin coated on the plate was
`also assessed in a direct binding assay. Data
`from a representative patient (Table 2. Patient
`
`(a)
`
`Pro-treat ment
`Posl-treatment
`
`1-1
`
`Monk
`BC2
`3E1,2
`Targei preparations
`
`Sheep
`
`Ly2.1
`
`Fig. 4. (a) Reaction of HAMA in the serum of a
`representative patient {Table 2: Patient 1) with various
`murine MoAb, relevant (I-l:IgGl) and irrelevant
`(3EI-2: IgM, BC2: IgGl) and to polyclona! antibodies
`from monkey (Monk) and sheep. The various anti-
`bodies were coated on the plate and solid phase bind-
`ing assays performed similar to HAMA assay; a
`1:2000 dilution ofserum was used. The binding of pre-
`existing HAMA in the same patient to the various anti-
`bodies was also shown.
`
`Inhibition of HAMA activity by different murine
`(b)
`MoAb preparations. Serum of Patient I was incubated
`with relevant MoAb (I-l, JGT; both IgGl) or irrel-
`evant murine MoAb of different
`isotypes (30-6:
`IgG2b. BC2: IgGl, BC3: IgM, Ly 2 1: IgG2a) in dif-
`ferent concentrations (250 jjg/mL or I |jg/mL) and
`tested in HAMA assays by measuring the binding of
`the reaction mixture to MoAb I-l coated plate. Only
`the results ofa representative patient (Patient 1) are
`shown, for clarity. Results were compared to the bind-
`ing of the same respective serum sample in HAMA
`assay in the absence of a blocking antibody (con-
`trol).
`
`I) are presented in Fig. 4(a). There was detect-
`able antibody response towards immunoglobu-
`lin preparations of monkey and sheep in the
`'pre-immune' serum but the elevated antibody
`response after therapy was directed predomi-
`nantly against determinants unique to murine
`immunoglobulin preparations. Similar results
`were obtained when the sera of other patients
`who had a positive HAMA response were
`tested.
`A competitive inhibition ELISA, performed
`on immune serum from Patient 1 showed that
`MoAb I-l and JGT (relevant MoAb) competed
`for !-l binding sites more efficiently than the
`irrelevant murine MoAb of the same (IgGl:
`BCD or different (IgG2a: Ly-21; lgG2b:30-6;
`IgM: BC3) isotypes (Fig. 4b). However, all the
`murine MoAb inhibited the binding of human
`anti-murine antibody
`to murine antibody
`(coated on the plate) to some extent. Similar
`results were obtained using sera from other
`patients with positive HAMA response in both
`the therapy and the imaging studies (data not
`shown). In these inhibition studies., which are
`analogous to an absorption test, it was clear that
`the best inhibition by irrelevant antibody in the
`case of Patient I (Fig. 4b) was by antibody BC2
`that was ofthe IgG 1 isotype (same isotype as the
`administered antibodies) and this inhibited
`better than irrelevant IgG2a, IgG2b and IgM
`antibodies. It was concluded that part of the
`HAMA response was anti-isotype specific, pre-
`sumably to the Fc region ofthe IgGl immuno-
`globulin. The fact that inhibition was best using
`the relevant (administered) antibody M or JGT,
`indicated that some ofthe immune response was
`specific to the administered antibodies. This lat-
`ter response is called, in various studies, *anti-
`idiotypic' (10.28) but we have not conducted the
`appropriate studies to determine whether this
`component ofthe HAMA response is to the anti-
`gen binding site or to other sites on the murine
`antibody molecules. Thus the HAMA response
`in the patients studied had three components: (i)
`anti-mouse
`immunoglobulin
`(mouse-specific
`determinants: heavy and/or light chains); (ii)
`anti-isotype; and (iii) antibody response specific
`to the administered mouse immunoglobulin
`(loosely termed 'anti-idiotype' in this study).
`
`In addition, the IgM and IgG components of
`the anti-murine immunoglobulin activity could
`be separately measured in the 'pre-immune'
`human serum (Fig. 5) and each component was
`increased with the development of elevated
`HAMA response. It is clear that the HAMA
`response was essentially polycional and con-
`tained increased levels of IgM as well as IgG.
`
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`
`(a)
`
`(b)
`
`103
`
`io<
`
`Serum dilution"'
`
`103
`
`,o4
`
`105
`
`106
`
`Serum ditulion-'
`
`103
`
`10*
`
`10^
`
`10^
`
`10^
`
`Serum dilution"'
`
`10*
`103
`Serum dtlulion-'
`
`lO"
`
`(b)
`
`0.6
`
`05-
`
`0.4-
`
`0 3-
`
`0.2-
`
`0 1-
`
`0.0
`10^
`
`Fig. 5. Levels of (a) IgG and (b) IgM HAMA detected
`in the serum of a representative patient (Table 2:
`Patient 6) before (a) and after (•) therapeutic injection
`of MoAb-JV-AcMEL conjugates.
`
`HAMA response to Fc and Ftah'): of mouse
`immunoglobulin
`in
`the relative contribution
`To determine
`patients' sera of antibodies to Fc and F(ab')2
`portions of the injected tnurine immunoglobu-
`lin. ELISA plates were set up with whole IgG
`MoAb, or with equimolar amounts of the F(ab')2
`fragments of the same immunoglobulin; and the
`binding of patietits" sera to these different prep-
`arations was assayed using serum samples with
`an elevated HAMA response. In a representative
`patient (Fig.6), the serum displayed elevated
`responses to both the whole murine immuno-
`globulins and the F(ab'); fragments. The reac-
`tions to the whole IgG of both relevant (Fig. 6a)
`and irrelevant (Fig. 6b) antibodies were greater
`than to F(ab')2 fragments, although this could
`
`Fig. 6. Elevated HAMA responses made by a repre-
`sentative patient (from imaging studies) to whole IgG
`(•) and F(ab')2 (•) of (a) relevant (RCC-1) and (b) irrel-
`evant (Ly-2-1) murine MoAb: both were oflgG2a sub-
`class. Relevant antibody refers to the administered
`antibody.
`
`reflect differences in the amount of antigen
`bound. The pre-immune sera also displayed a
`similar pattern of react ion. although the binding
`was less. Thus the HAMA response was directed
`against both the Fc and F(ab')2 of mouse
`immunoglobulin.
`
`DISCUSSION
`The HAMA respranses in patients who received
`murine MoAb for diagnostic studies or for ther-
`apy were examined in this study. Immunolym-
`phoscintigraphy. using subcutaneously adminis-
`tered antibodies in amounts ranging from 50
`|ig-3 mg was clinically safe (3.21) and was
`associated with a low incidence (4/67) of
`
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`374
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`J.J.
`
`HAMA, as measured by the ELISA system used
`in the study. In addition, the degree of response
`tends to be mild to moderate (HAMA litres 100-
`6400). as defined in the study. This is in contrast
`to the higher incidence of HAMA observed with
`some studies of immunoscintigraphy which
`employed larger doses (016-20 mg) of murine
`MoAb injected intravenously (29) or indeed
`with the larger amounts of antibody given intra-
`arterially in this study. The difference in im-
`mune response may be because the dose used
`was small (50 ^g-3 mg) and was injected subcu-
`taneously rather than intravenously. A similar
`lack of elevated HAMA response had been
`reported by others who used small doses (250
`(jg) of murine antibodies injected intravenously
`(8). However, there are other possible expla-
`nations, such as '-^'I-MoAb being taken up in B
`cells which may be inactivated by the 'hot' anti-
`gen, although such an inactivation did not occur
`after intravenous use of radiolabelled antibodies
`(29). Another major difference for consideration
`is that the melphalan-antibody conjugate could
`be more immunogenic than either MoAb or' ^' I-
`MoAb used in the imaging procedure; we have
`no information on whether this is the ease.
`However, the low incidence of HAMA response
`in imaging is of practical importance as it indi-
`cates that such diagnostic procedures can be
`repeated in most patients and do not preclude
`future therapy with murine MoAb.
`
`By contrast, hepatic artery infusion of murine
`MoAb-.V-acetyl melphalan conjugates in three
`repetitive doses over 48 h elicited prompt and
`dramatic
`immune response to the murine
`immunoglobulins in al! ten patients. An elevated
`HAMA response was noted in a patient as early
`as the fifth day following exposure to the
`foreign protein; however, the peak values of
`HAMA response usually occurred 14-28 days
`following exposure. The degree of HAMA
`response did not correlate with the amount of
`murine antibodies received within the dose
`range of 120-1000 mg/m^ (Table 2). and there
`was no correlation between (he degree of
`immune response in the patients and the clinical
`response (7). The high mcidence of elevated
`HAMA response in this group of patients could
`be related to the larger doses used, its repeated
`(three doses) daily administration and the route
`of adminstration by hepatic artery infusion.
`This is in accord with the high incidence of
`HAMA (17/18 patients) that was detected after
`repeated daily i.v. infusions of an IgGl antibody
`in patients with pancreatic cancer (28). It is of
`interest that the frequency of HAMA had been
`noted to be lower (50%) with single infusions of
`
`murine antibody 17-1A (IgG2a) (30). Although
`others had reported a reduction of this anti-
`murine response when patients received high
`initial doses of MoAb 17-1A (700 mg) by i.v.
`infusions, and had attributed that to induction
`of immune tolerance (31), this was not the
`experience of this study. The repeated daily
`administrations of the immunoconjugates via
`hepatic artery infusion before the development
`of detectable elevated HAMA response were
`well tolerated; by contrast, injection of the
`immunoconjugates in the presence of an ele-
`vated HAMA response was associated with an
`initial reduction in the HAMA titres which
`probably corresponded to immune complex for-
`mation and with clinical symptoms suggestive of
`a Type III hypersensitivity 'serum sickness'
`reaction, followed by a boost in the HAMA
`response (Fig. 3). The presence of an elevated
`HAMA response therefore precludes any further
`exposure to murine immunoglobulins. Where
`increased anti-murine antibody levels were
`detected after exposure to murine antibodies
`either for imaging or for therapy, the HAMA
`response was polyclonal. containing increased
`levels of both IgM and IgG and was directed
`against mouse-specific determinants, the iso-
`type (presumed to be the Fc portion), the
`F(ab')2. as well as the 'idiotype' (i.e. anti-indi-
`vidual injected antibody) of mouse immuno-
`globulin. The contradictory reports on the nat-
`ure of the HAMA response, especially with
`regard to the "anti-idiotypic' component could
`be related to the different assay systems used to
`evaluate the response (8,10.32.33).
`
`The low level of human anti-murine antibody
`response detected in the serum of normal sub-
`jects and in patients prior to administration of
`murine MoAb, is in accord with other studies
`(8-10) and there is evidence that this at least in
`part reflects rheumatoid factor activity in nor-
`mal human sera and no! a pre-existing specific
`antibody for murine immunoglobulin (34). It
`appeared that a significant component of the
`elevated HAMA response in patients given
`murine MoAb represented secondary response
`to antigenic determinants common to mouse
`immunoglobulins and some unknown immu-
`nogen, to which low levels of sensitization had
`already occurred. However, the predominant
`component ofthe elevated HAMA response was
`directed against mouse-speeifie determinants
`(Fig. 4a) and it is generally conceded (35) that
`the antibody response developed by patients was
`specifically related to the species from which the
`administered antibody was derived.
`The findings of this study have several clinical
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`PETITIONER'S EXHIBITS
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`HUMAN ANTI-MURINE lMMUNOGLOBULIN RESPONSES
`
`375
`
`implications. First, as the human anti-murine
`antibody response was polyclonal and was
`directed against the antigenic determinants on
`the constant, Fc, as well as the F(ab')2 portion of
`the mouse immunogiobulin moiecuie. the use of
`F(ab')2 fragments would not confer a real advan-
`tage over intact IgG, as illustrated by the ele-
`vated HAMA response in 1/3 patients given
`F(ab')2 in imaging studies (Table 1). Second, the
`administration of murine antibodies subcu-
`taneously in doses up to 3 mg was associated
`with a low incidence (4/67 or 6%) or elevated
`HAMA response and thus would not, in most
`cases, preclude subsequent exposure to murine
`
`antibodies. Third, repeated (three doses) daily
`administrations of murine antibodies over 48 h
`in doses up to 1000 mg/m^ by hepatic artery
`infusion was not associated with untoward eom-
`plieation. provided there was no prior immu-
`nization
`to murine
`immunoglobulins
`(7).
`Finally, the use of steroids in the peri-therapy
`period did not prevent the development of'posi-
`tive' HAMA response.
`
`Acknowledgements The authors thank Marie Pica,
`and Geoff Pietersz for their assistance in the prep-
`aration of this manuscript and Toula Athanasiadis and
`Janet Cameron for secretarial assistance.
`
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