Contributions to Oncology
`Beitrage zur Onkologie
`Vol. 43
`
`Series Editors
`H Huber. Innsbruck; W. QueifJer, Mannheim
`
`KARGER
`
`Basel · MUnchen . PAris · London · New York · New Delhi · Bangkok · Singapore · Tokyo · Sydney
`
`IMMUNOGEN 2074, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
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`

`
`Antibodies as Carriers
`of Cytotoxicity
`
`Volume Editors
`H.-H. Sedlacek, G. Seemann, D. Hoffmann, 1. Czech, P. Lorenz,
`C. Kolar, K. Bosslet, Marburg
`
`19 figures and 47 tables, 1992
`
`KARGER
`
`Basel· MOnchen . Paris· London · New York · New DOlhi . Bangkok · Singapore · Tokyo ' Sydney
`
`IMMUNOGEN 2074, pg. 2
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`Sedlacek / Seemann / Hoffmann / Czech / Lorenz / Kolar / Bosslet
`
`102
`
`Antibody Conjugates
`
`As has already been discussed in detail [834] the localization rate of a
`specific antibody to a solid tumor is very low, irrespective whether the
`antibody is applied i. v., i. p. or by other routes (tables 26, 27 and 29). Facing
`this fact and based on an average localization rate of .01 % MAb/g tumor, it
`was calculated whether radioactive compounds, toxins or cytostatics linked
`to the antibody might have a chance to kill a tumor (1 g weight) without
`causing life-threatening toxicities to the patient [834]. The result of this cal(cid:173)
`culation is summarized in table 35.
`We supposed that 1 g tumor contains 10 9 tumor cells, each cell expos(cid:173)
`ing 10 6 epitopes. We took into consideration that 80Gy of 131 1 or 90y are
`necessary to kill 1 g tumor. Moreover, that ten molecules of Ricin are
`
`Table 35. Dose calculation for systemic therapy with radionuclides or toxins linked to monoclonal antibodies
`
`in vitro
`Dose needed for
`killing (> 99 %) 109
`tumor ceJls
`(1 g tumor)
`
`in vivo
`Localization rate
`(%/g tumor)
`
`Dose of conjugate
`needed for therapy
`of 1 g tumor
`
`Maximum tolerated
`dose (MID)
`
`MTD leads to a
`local dose of
`
`1311
`
`90y
`
`Ricin
`
`Doxorubicin
`
`pure toxins
`
`80 Gy
`
`80 Gy
`
`1010 molecules
`
`1017 molecules
`
`MAb
`conjugates
`
`80 Gy
`
`80 Gy
`
`1011 molecules
`(Ricin A: MAb = 1:1 ;
`cytotox. activity of
`conjugate 0.1 of Ricin)
`
`1016 molecules
`(Dox. : MAb = 10:1;
`cytotox. activity of
`conjugate = Dox)
`
`0.031lg
`
`MAb
`conjugate
`
`0.01
`
`0.01
`
`0.01
`
`40-80 GBq
`
`20 GBq
`
`300 llg
`
`3 mg
`
`0.01
`
`30 g
`
`4-8 GBq
`
`2GBq
`
`«
`
`IOO lig
`
`«
`
`5 g
`
`8,0 Gy
`tumor
`2.7 Gy
`liver
`2,2 Gy
`kidney
`2,3 Gy
`spleen
`bone marrow 1,3 Gy
`
`8 Gy
`6Gy
`52 Gy
`38 Gy
`<2 Gy
`
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`
`Antibodies as Carriers of Cytotoxicity
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`103
`
`necessary to kill one tumor cell. Thus, 10 10 Ricin molecules are necessary to
`kill 10 9 tumor cells. In case one Ricin A molecule (MW 30kD) is bound to
`one antibody molecule, we consequently nee~ 10 10 antibody conjugate
`molecules to kill 10 9 tumor cells, provided the molar cytotoxicity of the
`conjugate and of Ricin are the same. Mostly the cytotoxic activity of the
`conjugate is less (factor of 10 or more) than the one of Ricin. In case cyto(cid:173)
`toxicity is reduced by a factor of 10, we need about 10 11 antibody Ricin A
`conjugates to kill 1 g tumor. This corresponds to about .03 f1.g of the anti(cid:173)
`body conjugate for 1 g tumor. Bearing in mind the localization rate of .01 %
`MAb/g tumor and supposing that the conjugate has the same localization
`rate as known for the MAb alone, we have to inject into a tumor patient
`about 300 f1.g of the Ricin A conjugate to have .03 f1.g of it localized at the
`tumor site. 300 f1.g of this Ricin A conjugates seem to be a dose far beyond
`the maximum tolerated dose.
`In case of Doxorubicin our measurements revealed that we need about
`1 x 10 8 molecules to kill a sensitive tumor cell [834]. For 1 g tumor (10 9 cells)
`we consequently need at least 10 17 molecules of Doxorubicin (MW 550 D).
`Supposed we can conjugate 10 Doxorubicin molecules to one antibody
`molecule and provided the cytotoxicity of Doxorubicin is not impaired by
`the conjugation process we need about 10 16 molecules of the Doxorubicin
`antibody conjugate to kill 1 g tumor. This corresponds to about 3 mg of the
`antibody conjugate for 1 g tumor. Thus, 30 g of the conjugate has to be
`injected into the tumor patient to achieve a local accumulation at the tumor
`site of 3 mgt g tumor.
`Also, this dose seems to us beyond the maximum tolerated dose. Fur(cid:173)
`thermore, the costs to produce such an amount of the conjugate are con(cid:173)
`siderable. In case of J3l I or 90y labeled to monoclonal antibodies we can
`make a similar calculations: About 80 Gy of radioactivity are needed at the
`tumor site to sterilize 1 g of tumor. Considering the localization rate of
`.01 % of the given antibody/g tumor, we can only reach about 8 Gy at the
`tumor site when we apply the maximum tolerated dose of 131 I conjugated
`antibody (4 - 8 GBq) or of 90 Y conjugated antibody (2 GBq). Thus, the local(cid:173)
`ization rate is too low by a factor of 10 to allow systemic radioimmuno(cid:173)
`therapy of solid tumors.
`Consequently, we predicted [834] that the therapy of solid tumors by
`the systemic application of MAb conjugated with radionuclides, toxins or
`cytostatics is not possible due to the general toxicity of the compound [834].
`Whether the localization rate of the conjugate can sufficiently (i. e. by a
`factor of 10) be increased through the additional application of drugs (table
`
`IMMUNOGEN 2074, pg. 4
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`
`Sedlacek/ Seemann / Hoffmann / Czech / Lorenz / Kolar / Bosslet
`
`104
`
`32,33) is very questionable. As has been pointed out, the best manipulation
`increased the localization by a factor of 2 - 4, which seems to be insufficient
`to allow any direct radioimmuno- or chemoimmunotherapy with the use of
`MAb conjugates.
`The clinical data with radionuclides, conjugated to antibodies now
`available, have been reviewed by several authors recently. Dykes et al. [236]
`came to the conclusion that as long as tumor uptake of antibodies remains
`in the region of .005 % of injected dose per g of tumor, tissue radioimmuno(cid:173)
`therapy cannot be useful. To achieve predictable therapeutic benefit, a sub(cid:173)
`stantial improvement in uptake ratios is essential and to obtain tumor
`killing at least ten times the current values must be achieved. Kramer et al.
`[508] came to the same conclusion.
`The objection against radionuclide antibody conjugates in the treat(cid:173)
`ment of solid tumors can also be applied to antibody conjugates with toxins
`or cytostatics.
`The most important studies in this field are summarized in table 36. It
`is evident that in all studies listed [508] the therapeutic effect is either zero
`or questionable, despite the fact that the maximal tolerated doses have
`been given.
`In contrast to solid tumors, lymphomas and leukemias seem to offer
`better possibilities for radioimmuno- or chemoimmunotherapy. Targeting
`may be particularly effective in those hematologic malignancies where cir(cid:173)
`culating malignant cells may be better exposed to i. v. injected antibody
`conjugates. Thus, the localization rate of antibody conjugates should be
`significantly higher in leukemia than in solid tumors.
`Indeed, as listed in table 37, cytotoxic antibody conjugates were ther(cid:173)
`apeutically effective in a considerable number of studies. Whether this
`therapeutic effectiveness is superior to the one of other treatment modali(cid:173)
`ties has to be elaborated in the future especially with respect to the side
`effects induced by the different therapeutic regiments [535, 809].
`One of the main potential problems of monophasic treatment of
`tumors with naked antibodies or with antibody toxin conjugates is the
`selection of resistant tumor cells treated with monoclonal antibody not
`exposing the respective specific antigen. Inxenografted tumors treated with
`monoclonal antibody vincaalkaloid immunoconjugates progression of
`such resistant tumors after initial regression could indeed be observed
`[920,921]. The same could be found with 90y labeled anti CEA antibodies
`[258, 259, 448, 449] in mice and with 131 I labeled antibodies in man [252-
`254,933].
`
`IMMUNOGEN 2074, pg. 5
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`
`Antibodies as Carriers of Cytotoxicity
`
`105
`
`Table 36. Clinical trials for systemic treatment of solid tumors with immunotoxins or
`immunoconjugates
`
`Tumor
`
`Immunotoxin Dose
`
`n CR PR MR Toxicities
`
`Reference
`
`Melanoma MEL-RA
`
`0.01-1 mg/kg 22 ?
`(4x-5x)
`
`?
`
`?
`
`Melanoma MEL-RA
`
`0.4 mg/kg
`(5 x)
`
`46
`
`?
`
`Melanoma MEL-RA
`
`Colon carc. Co791-RA
`
`Colon carc. Co791-RA
`
`Breast carc. 260F9-RA
`
`0.4 mg/kg
`(5 x)
`
`0.02-02
`mg/kg (5 x)
`
`0.05-02
`mg/kg (5 x)
`
`0.01-0.05
`mg/kg (6-8x)
`
`6 0
`
`0
`
`0
`
`17 0
`
`0
`
`7 0
`
`0
`
`0
`
`4 0
`
`0
`
`0
`
`Breast carc. 260F9-RA
`
`0.05-0.1
`mg/kg (6-8x)
`
`5 0
`
`0
`
`0
`
`Colon carc. B72.3_9Oy
`
`2-10 mg/m
`
`27 0
`
`0
`
`0
`
`hepatotox.
`edema
`weight gain
`
`hepatotox.
`edema
`weight gain
`
`hepatotox.
`edema
`neuropathy
`
`hepatotox.
`edema
`weight gain
`
`hepatotox.
`edema
`weight gain
`neuropathy
`
`fever
`thrombocytop.
`leukopenia
`
`Colon care. A7-NCS
`
`15-90 mg
`(lx-2x)
`
`41 0
`
`0
`
`3
`
`0
`
`Colon carc. VDS-MAb
`
`0.11 g/m
`
`24 0
`
`0
`
`0
`
`gastrointest.
`toxicity
`
`thrombo-
`cytopenia
`
`Various
`
`Mitomycin
`MAb
`
`Various
`
`ADM-Mab
`
`2.3 g
`(total)
`
`}
`
`2-3 g
`(total)
`
`43
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`anemia
`
`[877]
`
`[878]
`
`[123]
`
`[123]
`
`[878]
`
`[1014]
`
`[356]
`
`[392]
`
`[944]
`
`[809]
`
`[676]
`
`Colon carc. pseudomonas
`Exotoxin-MAb
`
`5 g/kg
`(Lp.)
`
`0
`
`0
`
`0
`
`hepatotoxicity
`
`[809]
`
`RA = Ricin A; NCS = Neocarcinostatin; VDS = Vindesin; ADM = Adriamycin
`
`IMMUNOGEN 2074, pg. 6
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`
`Sedlacek / Seemann / Hoffmann / Czech / Lorenz / Kolar / Bosslet
`
`106
`
`Table 37. Clinical trials for systemic treatment of leukemia with immunotoxins or immuno-
`conjugates
`
`Leukemia
`
`Immunotoxin Dose
`
`n
`
`CR PR MR Toxicities
`
`Reference
`
`B-CLL
`
`anti CD5-RA
`
`CLL
`
`anti CD5-RA
`
`T-ALL
`
`anti CDS-RA
`
`anti B4-RA
`
`7-14 mg/m2
`(6x)
`3 mg/m2
`(8 x)
`10 mg/m2
`(2x)
`
`160 {.lg/kg
`(5 x)
`
`5 0
`
`4 0
`
`6 0
`
`25
`
`anti B4-RA
`
`90-280 {.lg/kg
`
`18
`
`anti B4-RA
`
`160-400 {.lg/kg 36 2
`
`NHLlNonT-
`ALL/-B-CLL
`
`NHL-NonT-
`ALLlB-CLL
`
`NHL-CLLI
`B-cell
`
`0
`
`0
`
`0
`
`2
`
`3
`
`4
`
`0
`
`0
`
`0
`
`0
`
`0
`
`[427)
`
`[427)
`
`[523)
`
`10
`
`hepatotox.
`
`[646)
`
`4
`
`hepatotox.
`
`[646)
`
`10
`
`hepatotox.
`thromhocytop.
`oedema
`
`[89)
`
`[338)
`
`B-cell/NHL
`
`LL-2-1311
`
`3 mg/kg
`(30 mci)
`
`5 0
`
`2
`
`2
`
`0
`
`Depending on the individual tumor type such resistant regrowing
`tumor cells might indeed regain their specific antigen again [259], but,
`nevertheless, we have to face selection of resistant cells as a considerable
`problem of monophasic treatment of tumor with immunoconjugates once
`this treatment will show sufficient clinical activity.
`
`Maneuvers to Overcome Immunogenicity
`
`Murine antibodies are xenogeneic for and immunogenic to man.
`Parenteral application to patients of murine antibodies for diagnostic or
`therapeutic purposes consequently leads to a human immune response.
`This immune response prominates to be an antibody response, which can
`be measured in the serum of the patient but can also be assayed in the
`supernatants of antibody producing lymphocytes, isolated from the pa(cid:173)
`tients and cultured in vitro after infection with the EBV [94, 95, 506]
`(table 38). Human anti-mouse antibodies (HAMA) have been detected
`even two days after murine MAb application [823 - 825] but can mostly be
`found within five to ten days of MAb administration [508]. However, occur-
`
`IMMUNOGEN 2074, pg. 7
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