PROTIDES OF THE
`
`BIOLOGICAL FLUIDS
`
`PROCEEDINGS OF THE THIRTY—SECOND
`COLLOQUIUM, 1984
`
`Edited by
`
`H. PEETERS
`
`Director
`
`Institute for Medical Biology
`Brussels, Belgium
`
`
`
`@
`
`PERGAMON PRESS
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`OXFORD - NEW YORK - TORONTO - SYDNEY - PARIS - FRANKFURT
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`IMMUNOGEN 2302, pg. 1
`Phigenix v. Immunogen
`|PR2014-00676
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`IMMUNOGEN 2302, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
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`First edition 1985
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`IMMUNOGEN 2302, pg. 2
`Phigenix v. Immunogen
`|PR2014-00676
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`IMMUNOGEN 2302, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`

`

`BJ.lnmum0mes
`
`
`
` This material may be protected by Copyright law (Title 17 U.S. Code)
`
`DRUG TARGETTING FOR 7
`
`NEUROBLASTOMA PATIENTS USING
`
`HUMAN POLYCLONAL ANTIBODIES
`
`
`G.MEUNOTJ.R.HOBBS*M.RADFORDF*K.B.COOKES
`A.hi EVANngNLIA.CASTELLO
`mulD.hL FORREST”
`
`JClwmicul Pathology and Paediatric Surgery qurlnwms, Weslmim‘rcr JWUIllUll School.
`London SW1. UK
`D'I’ptll‘llllé’lll of Child H(’(lllli. Southampton General Hm‘piml. Sou/lmmp/mi. UK
`””211 Clinim Pediatrim.
`la Univcrsini Ill Rama,
`lmly
`
`ABSTRACT
`
`Allogeneic antibody directed against neuroblastoma tumour cells was used as a
`carrier of daunorubicin and chlorambucil.
`7 children with neuroblastoma were
`treated by drug targetting alone, achieving three cures and one long term
`good response. Antibodies were bound to target cells to give a clinically
`safe treatment with a good quality of
`life. Better antibody production and a
`wider panel of drugs are needed to improve results.
`KEYWORDS
`
`Drug targetting; neuroblastoma; chemotherapy; antitumour antibody;
`immunotherapy.
`
`INTRODUCTION
`
`Despite the recent progress in chemotherapy the prognosis for neuroblastoma
`remains poor and only some
`7% of Evans Stage IV children will survive.
`Recently we have been interested in exploring the therapeutic potential of
`drug—targetting (10)
`a technique which has evolved from the original use of
`chlorambucil adsorbed to xenogeneic antibodies (2)
`to the use of
`toxins
`covalently coupled to relatively specific monoclonal antibodies (1,3,11,12L
`Westminster Hospital's clinical experience of drug—targetting, has been
`mainly with cases of advanced neuroblastoma over the age of
`tw0 years (4,7%
`In this report we summarise our clinical results using allogeneic antibodies
`as carriers of chlorambucil
`(CHL) or daunorubicin (DNRL
`
`ANTIBODY PRODUCTION
`
`raised in haploidentical well—informed Volunteers
`Allogeneic lgG was
`inoculated with irradiated neuroblastoma cells,
`following an ethically
`approved immunisation schedule (10).
`Specific antibody was detected on day
`14 by cytofluorimetry. Figure 1 shows that the plasma before immunisation
`evinced only a low degree of autofluorescence with the neuroblastoma cells
`used for the immunisation. The stronger intensity of
`the fluorescence on day
`14 indicates the presence of antibody binding to the tumour cells. This
`antibody did not bind to the lymphocytes of the tumour donor, so excluding
`the presence of anti—HLA antibodies. Purified gamma globulins were prepared
`from litre batches of plasma using the cold ethanol method (lO).The yield of
`the purification was IgG 57%,
`IgM 60%,
`IgA below 7%, Albumin below 2%. The
`high lgM recovery can be attributed to minor errors in the composition of
`the
`first precipitation buffer.
`
`CONJUGATION
`
`the antibodies were covalently
`After testing for sterility and pyrogenicity,
`coupled to DNR or CHL (8) by the carbodiimide method,
`to give 3 moles DNR
`PBF—O
`
`M3
`
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`Z114
`
`IMMUNOTOXINS
`
`removed by dialysis
`(70%), was
`free drug,
`bound per mole antibody. Residual
`The coupling did not
`impair
`the ability of
`the antibodies to bind to
`neuroblastoma cells, as shown in figure 1 where the intrinsic fluorescence of
`DNR contributes 7.6%;
`the non—specific fluorescence was 0.27% for cells
`alone,
`0.96% for normal human serum,
`1.23% for cells + DNR and 6.41% for
`cells +
`IgG/DNR conjugate without FTIC 2nd antibody. Fluorescence increased
`to 68.9% for cells +
`IgG + 2nd FTIC Ab and to 76.5% for cells +
`IgG/DNR
`conjugate + 2nd FTIC Ab. All conjugates contained IgG polymers on SDS—PAGE
`but, most
`importantly,
`the drug—antibody conjugates retained the capacity to
`kill neuroblastoma cells (8)
`
`PATIENTS & PROTOCOL
`from children affected
`Patients were chosen, as in our previous reports (4),
`by neurohlastoma,
`see table 1. Each intravenous dose was 1 mg DNR/Kg/dose or
`0.5 mg CHL/Kg/dose, coupled to gamma globulin, usually at a frequency of
`two
`DNR and/or one CHL dose per week.
`The treatment was continued up to 1 year,
`reaching a total DNR dose of 35—50 mg/Kg of body weight, over double that
`permitted for free DNR.
`
`The results are shown in Table I. Catecholamine reduction was observed in all
`7 patients (6). The non—stage IV children present no evidence of disease
`three years after diagnosis. Masses were reduced in 3 out of
`4 stage IV
`patients, according to X—ray, CT scan and ultrasound measurements.
`Bone
`deposits healed in 2 out of 7 children.
`In one case a bilateral chylothorax
`was resolved using intrapleural conjugate injections (9%
`
`Side effects were originally confined to minor bone marrow depression in the
`first 5 patients, attributed to unbound drug. For the last two patients the
`conjugates were dialysed before injection,
`to remove the free drug, and no
`bone marrow depression occurred. No heart
`toxicity was seen, either
`clinically or by ECG and ultrasound analyses, despite the high total doses of
`DNR achieved. No serious immunological and/or toxic effects were observed.
`
`PRELIMINARY LOCALISATION
`is seen in
`The ability of the conjugates to bind to tumour cells 'in vivo‘
`figure 2, which shows
`immunoperoxidase staining of a malignant cell
`from an
`infiltrated bone marrow aspirate from patient 1, using anti—human IgG. The
`suggested presence of anti—tumour antibody doesn't elucidate the origin of
`the human IgG. Preliminary experiments of
`radioimmunolocalisation
`autoradiography using nude mouse xenografts and 'in vivo' on patients, also
`indicate that the lZS—I—antibodies are indeed able to localise to the tumour
`cells.
`
`DISCUSSION
`The original use of xenogeneic antibodies to deliver toxic substances to
`tumours,
`(Mathe (5)
`and Ghose (2)),
`employed 'one—shot' doses hoping to
`eliminate the tumours in the hosts and have,
`in general, failed to produce
`cures. Xenogeneic antibodies usually produce a host response with subsequent
`neutralisation of the treatment. To avoid such problems our work has used
`human antibodies aiming for long—term drug targetting.
`
`Previous reports (4,6,7) have presented our results in other neuroblastoma
`patients. Here we confirm that allogeneic antibodies can be used to localise
`DNR and CHL onto tumour cells in human patients, producing measurable tumour
`effects and minimal side effects (e.g.
`no hair
`loss was observed%
`Furthermore we
`showed that
`3 drug molecules bound per molecule of antibody
`preserves the ability of that antibody to bind to the target, figure 1, and
`kill.
`The prolonged response, as indicated by the continued fall in VMA for
`10 days after a single dose in contrast to a rise again after only 3 days
`with free drug,
`indicates that conjugate therapy may not be cell cycle
`dependent. Higher binding ratios produce protein denaturation, affecting
`turnover and antigenicity. Even our low drug antibody ratios form protein
`polymers. Since these may stimulate absorption into the cell (possibly by
`phagocytosis) we
`intend to evaluate the role of
`these aggregates before
`trying new coupling methods. More clinical evidence of localisation is needed
`but
`the 3 cures achieved in patients 2,3 and 4 and the good response of
`
`IMMUNOGEN 2302, pg. 4
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`

`DRUG TARGETTING USING HUMAN POLYCLONAL ANTIBODIES
`
`AIS
`
`patient 7 show that allogeneic antibody can be used successfully for drug
`targetting on neuroblastoma. A wider panel of antitumour agents and a better
`antibody production technique will benefit
`the patients.
`ACKNOWLEDGMENTS
`to C.N.R. Progetto Finalizzato 'Controllo Crescita
`The Authors are grateful
`Neoplastica'
`for financial support,
`to the staff of Robert Mond Ward,
`Westminster Childrens Hospital for their care of
`the patients and to the
`staff of
`the Edmund Fane Laboratory for their expert advice and help.
`REFERENCES
`1. Embleton, M.J., G.F. Rowland, R.G. Simmonds, E. Jacobs, C.H. Marsden and
`R.W. Baldwin (1983). Br. J. Cancer,47, 43—49.
`2. Ghose, T., S.T. Novell, A. Guclu, D. Cameron, D. Bodurtha and A.S.
`McDonald (1972). Br. Med. J., 3,
`495—499.
`3. Gregoriadis, G. (1981). Lancet, 2, 241—246.
`H. Peeters
`In:
`4. Hobbs,
`J.R., G. Melino and P. Riches (1984).
`ed.,Protides of Biological Fluids, Pergamon, Oxford, 21, 783—786.
`
`5. Mathe', G., T. Lee and .1. Bernard (1958). C. R. Acad. Sci., 246,
`1626—
`1628.
`
`6. Melino, G.
`(1985).
`In: H.Peeters ed., Protides of_ Biological Fluids,
`Pergamon, Oxford,
`_3_2_,
`in press.
`7. Melino, G., P. Elliott, K.B. Cooke, A.M. Evans,
`J.R. Hobbs (1984).
`Proceedings 20th ASCO Meeting, Toronto, Canada, C—l79.
`
`8. Melino, G., P. Hazarika, P. Elliott,
`J.R. Hobbs and K.B. Cooke (1982).
`Biochem. Soc. Trans., 10, 505.
`9. Melino, G., Z.B. Tabara and J.R. Hobbs (1984).J. Pediatr. Surg.,
`in press.
`10. Con, C.J., M. Apsey, H. Buckleton, K.B. Cooke,
`I. Hanham, P. Hazarika,
`J.R. Hobbs
`and B. McLeod (1975). Behring. Inst. Mitt., 56,
`228—235.
`11. Raso, V., J. Ritz, M. Basala and S.F. Schlossman (1982). Cancer Res., 42,
`457—464.
`12. Rowland, G.F., J.R.F. Corvalan, C.A. Axton, V.A. Gore, C.H. Marsden, W.
`Smith
`and R.G.
`Simmonds
`(1984).
`In:
`H. Peeters
`ed.,
`Protides of Biological Fluids, Pergamon, Oxford, 81, 783—786.
`
`
`TABLE 1'. Patients details
`
`Pts.
`
`age
`
`sex
`
`stage
`
`‘in vitro'
`killing
`
`clin.resp.
`
`survival
`since diagnosis
`(m0)
`
`4.
`P
`+
`IV
`111
`2
`1
`46.+
`NED
`+
`111*
`f
`2
`2
`44.+
`NED
`+
`111*
`f
`7
`3
`32.+
`NED
`NT
`11*
`f
`2
`4
`12.
`P
`NT
`IV
`f
`5
`5
`3.
`N
`NT
`1V
`m
`8
`6
`14.+
`G
`+
`IV
`f
`4
`7
`tumour after surgery.
`*= Lymph node deposits and macroscopic residual
`clin.resp:— Good: over 50%, Partial: below 50%, None: below 25%.
`NED: no evidence of disease
`
`IMMUNOGEN 2302, pg. 5
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`

`4W
`
`IMMUNOTOXINS
`
`——— IMMUNE SERUH PRE-CDNJUGRTION
`"""’IHHUNE SERUM POST-CONJUORTION
`“" PRE-IHNUNISRTION SERUM
`
`EELLCflNT
`
`FLUORESCENCE INTENSITY
`
`FIGURE 1
`
`Staining of neuroblastoma cells.
`
`
`
`
`7"”
`
`FIGURE 2
`
`Immunoperoxidase staining of a neuroblastoma cell.
`
`IMMUNOGEN 2302, pg. 6
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