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*
Medarex, Inc., Annandale, NJ 08801;
Department of Physiology, Dartmouth Medical School, Lebanon, NH 08801; and
Department of Immunology and Medarex Europe, University Hospital Utrecht, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R leads to enhanced Ag-specific responses in vitro and in vivo.
However, the ability to overcome immunologic nonresponsiveness by
targeting poorly immunogenic Ags to FcR has not been investigated.
To address this question in a simple model, we immunized transgenic
mice expressing human CD64 (FcRI) and their nontransgenic
littermates with Fab' derived from the murine anti-human CD64 mAb
m22. The m22 Fab' served as both the targeting molecule and the Ag. We
found that only CD64-expressing mice developed anti-Id titers to
m22. Furthermore, chemically linked multimers of m22 Fab', which
mediated efficient internalization of the human CD64, were
significantly more potent than monomeric m22 F(ab')2 at
inducing anti-Id responses. In all cases, the humoral responses
were specific for m22 Id and did not react with other murine IgG1 Fab'
fragments. Chemical addition of a second murine Fab' (520C9
anti-human HER2/neu) to m22 Fab' multimers demonstrated that IgG1
and IgG2a anti-Id titers could be generated to 520C9 only in the
CD64-expressing mice. These results show that targeting to CD64 can
overcome immunological nonresponsiveness to a weak immunogen.
Therefore, targeting to CD64 may be an effective method to enhance the
activity of nonimmunogenic tumor vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R)
expressed by APCs can potentiate activation of cultured T cells
(2, 3, 4, 5, 6, 7, 8, 9) and promote immune responses when targeted in vivo
(10, 11). Three classes of leukocyte FcR have been
defined molecularly, FcRI (CD64), FcRII (CD32), and FcRIII
(CD16), and their relative contributions to beneficial and pathogenic
mechanisms are being elucidated (12, 13).
Using bispecific Abs that simultaneously bind Ag and FcRII/FcRIII
on the surface of APCs, Snider and Segal demonstrated the ability of
these FcRs to greatly enhance Ag processing and presentation to T
cells in vitro (3) and Ag-specific Ab responses in vivo
(10). More recently, Wernersson et al. (14)
have shown that IgG enhancement of Ag-specific Ab responses is
drastically reduced in FcR -chain knockout mice that do not express
functional FcRI or FcRIII, implicating one or both of these
FcR in the enhancing effect. When mice that were deficient in
FcRIII were tested in this system, Ab enhancement of the immune
response was comparable to that in wild-type mice. This result
suggested that FcRI was sufficient to mediate the enhanced response.
Collectively these studies have clearly demonstrated that targeting
model immunogenic Ags to FcR on APCs, and particularly CD64, results
in enhanced immune responses. However, it is not clear whether this
approach would effectively induce immune responses to weakly
immunogenic tumor Ags.
Id determinants of the Igs expressed by B cell tumors can serve as tumor-specific Ags, but are weakly immunogenic. Induction of anti-Id responses usually requires CFA, cytokines such as GM-CSF, and/or conjugation to highly immunogenic carrier proteins such as keyhole limpet hemocyanin (15, 16, 17, 18). Therefore, murine Ab Id can serve as an appropriate model of a poorly immunogenic tumor Ag in mice. Previously, Heijnen and co-workers have reported on a transgenic mouse model in which the human CD64 transgene is expressed and regulated in a pattern consistent with the expression and regulation of CD64 on human APCs (11, 19). With this model they demonstrated enhance IgG responses to a CD64-targeted humanized Ab. In this report we extend those studies by investigating the humoral responses to murine idiotypic Ags targeted to CD64.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F(ab')2 of the anti-FcRI mAb m22
(IgG1) (20) and the murine anti-human HER2/neu 520C9
(21) were prepared by pepsin digestion and purified by
flowing through a protein A column followed by Superdex 200 (Pharmacia,
Piscataway, NJ) gel filtration chromatography. To make multimeric
F(ab')3+ molecules, m22
F(ab')2 was treated with a 20-fold molar excess
of sulfo-succinimidyl
4-N-maleimidomethyl-cyclohexane-1-carboxylate (Pierce,
Rockford, IL). Separately, m22 F(ab')2 or 520C9
F(ab')2 was reduced to Fab' with 2-ME amine. The
Fab' were then added to m22 F(ab')2-maleimide and
incubated for 2 h at ambient temperature. Purification of the
multimers was performed by Superdex 200 gel filtration chromatography.
To make the m22 x 520C9 F(ab')3+ multimers,
equimolar amounts of m22 Fab' and 520C9 Fab' were added to the m22
F(ab')2-maleimide. The control Abs m32.2,
anti-human CD64 (murine IgG1), A77; anti-human CD89 (murine
IgG1), H22; and anti-human CD64 (humanized IgG1), were purified
from supernatant of hybridomas or transfected myeloma cells.
Modulation of CD64
Peritoneal macrophages were elicited in human CD64 transgenic and nontransgenic littermates with 1 ml of 3% Brewer’s thioglycolate (Difco, Detroit, MI) for 4 days before recovery by peritoneal lavage. The macrophages were incubated in polypropylene microtiter plates with m22 F(ab')2 or m22 F(ab')3+ at varying concentrations for 2 h at 37°C (active group) or at 4°C (control group). Cells were harvested after being placed on ice for 15 min and then washed and incubated on ice with 32.2 anti-CD64 Ab conjugated to FITC, which binds to a site on CD64 that is distinct from the m22 epitope (20). Samples were washed, and fluorescence was measured using a FACScalibur (Becton Dickinson, San Jose, CA).
Immunization of mice
The mice employed for these studies were 5- to 12-wk-old females from the human CD64-transgenic FVB/N line 1852 previously described by Heinen et al. (11, 19). We used F1 mice from heterozygous mating such that approximately 50% of each litter expressed human CD64. For each experiment human CD64-expressing mice were matched with their nontransgenic littermates as controls. All immunizations were performed i.p. using the Ribi Adjuvant System (Sigma, St. Louis, MO) in a final volume of 200 µl/mouse. Mice were immunized with 25 µg of Ag three or four times at approximately 2-wk intervals. Blood was drawn for analysis 1 wk after each immunization.
Analysis of anti-Id responses
Anti-Id responses were evaluated by sandwich immunoassays. Microtiter plates were coated with F(ab')2 from relevant or control Abs. Nonspecific adsorption was blocked with 5% BSA in PBS. Samples of plasma from mice were diluted in PBS and incubated on the plates for 1–2 h at 37°C. After extensive washing, the plates were incubated with either goat anti-murine IgG Fc-specific or isotype-specific alkaline phosphatase-conjugated probes. The alkaline phosphatase substrate, p-nitrophenylphosphate, was added to the plates, and absorbance was read at 405–650 nm. Titers were based on the highest dilution that gave a significant value (>50% increase in OD) above nonimmune pooled FVB/N sera. Pooled sera from immunized mice were tested for inhibition of PE-labeled m22 or m32.2 binding to CD64 by flow cytometry. U-937 cells were incubated with m22-PE (0.05 µg/ml) or m32.2-PE (0.1 µg/ml) in the presence of varying dilutions of serum from immunized mice for 75 min at 4°C. The cells were washed and analyzed on a FACScalibur instrument with CellQuest software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Id of the surface Ig expressed in B cell lymphoma have been
well established as a unique target for effective immunotherapy in
animal models (15, 16, 17, 18), and anti-Id Abs can induce
prolonged remissions in non-Hodgkin’s lymphoma (22).
Idiotypic determinants contain unique sequences, yet they are poorly
immunogenic and do not mediate significant protection against a tumor
challenge when administered as unmodified vaccines (17, 18). However, Id-based vaccines can induce protective responses
in animal models when linked to immunogenic carrier proteins
(15, 16, 17, 18) and have recently been shown to induce
anti-tumor responses in patients with follicular lymphoma when
combined with GM-CSF (23). We reasoned that the Id
of the anti-CD64 Ab m22 (m22-Id) could serve as a model of a weakly
immunogenic tumor Ag while providing specific targeting to the human
FcRI (CD64) on APCs in transgenic mice. Furthermore, mAb 22
efficiently binds CD64 in vivo due to its specificity for an epitope
that is distinct from the Fc binding site.
Transgenic mice expressing human CD64 as well as their nontransgenic
littermates were immunized with F(ab')2 of the
m22. The mice were bled 7 days after receiving biweekly immunizations
of 25 µg of m22 F(ab')2 mixed in Ribi adjuvant.
After the fourth immunization, three of the six transgenic mice, but
none of the six nontransgenic littermates, exhibited significant m22-Id
specific IgG titers (Fig. 1
). This
anti-serum was m22-Id specific and did not react with other
murine F(ab')2 Ab fragments (data not shown). The
m22-Id was not immunogenic in normal mice; however, targeting to CD64
partially overcame this immunologic nonresponsiveness.
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Since Ags generally require internalization before being processed
and subsequently presented to T cells, chemically synthesized multimers
of the M22 Fab' were generated to induce more efficient internalization
of Ag-CD64 complexes. To make the multimers, m22 Fab' were linked
to m22 F(ab')2 using the bifunctional linker
succinimidyl4-N-maleimidomethyl-cyclohexane-1-carboxylate.The
data in Fig. 2A depict a
nonreducing SDS-PAGE gel comparing m22 F(ab')2
(lane 1) and the m22 Fab' multimer (designated m22
F(ab')3+; lane 2). The multimer
consisted of several molecular species representing
F(ab')3, F(ab')4,
F(ab')5, F(ab')6, and some
higher m.w. forms.
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m22 F(ab')3+ elicits potent anti-Id IgG responses in human CD64 transgenic mice
We next examined the ability of m22 F(ab')3+
multimers to generate an m22 anti-Id response in human CD64
transgenic and nontransgenic littermates. Using the same protocol as
that employed for F(ab')2 immunizations, a
markedly stronger anti-Id response was observed (Fig. 3A). After only three
immunizations with the multimer, high titers of m22-specific IgG were
generated in all the CD64 transgenic mice (n = 12), and
no significant anti-Id IgG was generated in any of the
nontransgenic littermates (n = 10). Contrary to what we
observed with the F(ab')2 immunizations, most of
the transgenic mice developed anti-Id responses following just two
immunizations of multimers (data not shown). To determine whether the
enhanced efficiency of the multimer to generate anti-Id responses
was due to an increased persistence in vivo, we immunized human CD64
transgenic mice with various doses of the multimer. Surprisingly, we
found that three immunizations with as little as 0.25 µg/dose of the
multimer were sufficient to generate a significant anti-Id response
(Fig. 3B).
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The specificity of the anti-Id response is demonstrated in
Fig. 4. Pooled sera from immunized
transgenic mice reacted specifically with m22
F(ab')2 and not with isotype- and
allotype-matched control F(ab')2 fragments (Fig. 4A). In addition, the sera from immune transgenic mice bound
to F(ab')2 of the humanized 22 (H22), which
contains only the complementarity-determining regions of m22
(27). Sera from immunized nontransgenic mice did not bind
to any of the F(ab')2 (data not shown).
Similarly, immune sera from the human CD64-expressing mice efficiently
blocked m22 binding to CD64 on U-937 cells in a specific manner (Fig. 4B). The same sera did not interfere with m32.2 binding to
CD64, and sera from nontransgenic immunized mice did not inhibit m22
binding. The data clearly show that the immune serum was m22-Id
specific and blocked the ability of m22 to bind CD64.
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To determine whether an immune response could be generated against
a different Fab' by targeting to human CD64, we generated multimers of
the m22 Fab' linked to the Fab' of the murine anti-HER2/neu mAb,
520C9 (21). One millimole each of 520C9 and m22 Fab' were
chemically coupled to 1 mmol of m22 F(ab')2.
Similar to the m22 F(ab')3+, these multimers
predominantly contained species of F(ab')3 to
F(ab')6 with an estimated 3:1 ratio of m22 Fab'
to 520C9 Fab'. Human CD64 transgenic and nontransgenic littermates were
immunized with three 25-µg doses of 22-520C9
F(ab')3+ and assessed for both 520C9 and m22
anti-Id IgG titers. The data in Fig. 5 show that seven of eight immunized CD64
transgenic mice developed high levels of IgG specific for both 520C9-Id
and m22-Id. None of the seven nontransgenic mice developed measurable
titers to either 520C9-Id or m22-Id. These data demonstrate that
targeting a weakly immunogenic tumor Ag to CD64, such as lymphoma Id,
in a manner that results in internalization by
CD64+ APCs leads to a potent immune response to
the Ag. Interestingly, in individual mice the anti-520C9-Id titers
were generally 1 log greater than the anti-m22-Id titers.
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The quality of the immune response to a given Ag, particularly a
tumor-associated or viral Ag, can be as important as the quantity of
the response. To address these issues, we determined the duration and
isotype specificity of the 520C9 anti-Id response in transgenic
mice immunized with 22-520C9 F(ab')3+. Anti-Id
responses were determined in the same mice at 1 wk, 4 mo, or 7 mo after
the third immunization with 22-520C9 F(ab')3+
(Fig. 6A). These data
illustrate that the mice maintained significant 520C9 Id-specific IgG
titers for at least 7 mo, demonstrating a lasting response to the
immunization. In addition, both a strong IgG1 as well as a strong IgG2a
Id-specific titer were elicited, suggesting that targeting to CD64 can
activate both a Th1 and a Th2 response (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with reports on the lack of immunogenicity of idiotypic
determinants (15, 16, 17, 18), we found that chemically coupled
Fab' of the murine anti-human CD64 mAb m22 and anti-human
HER2/neu mAb 520C9 did not elicit measurable IgG humoral responses in
nontransgenic mice. In fact, immunization of transgenic mice with whole
520C9 mAb employing the same protocol also failed to elicit significant
anti-Id titers to 520C9 (data not shown). However, strong
anti-Id titers to both the coupled fragments were induced
by targeting to APCs in the human CD64 transgenic mice. Importantly,
the development of anti-Id IgG to the targeting moiety (m22) did
not impede the ability to elicit 520C9-specific anti-Id responses.
The transgene-expressed human CD64 is predominantly responsible for the
observed effects, since the Fc portion of both m22 and 520C9 was
removed to eliminate the potential interactions with murine FcRs.
However, given that the mice were immunized three times, it is
plausible that immune complexes formed between the Ag and anti-Id
IgG may have been taken up and processed by murine FcRs.
Although strong Id-specific responses were found in the immunized transgenic mice, due to technical limitations these studies were unable to determine whether Ab responses to constant domains of m22 or 520C9 Fab' were elicited. Any Abs to murine Fab' constant domains would bind the large excess of murine IgG in serum and not be detected unless overwhelming responses were elicited. None of the immune mice displayed symptoms of adverse reactions up to 7 mo postimmunization.
Comparison of the anti-Id responses from m22
F(ab')2 vs m22 F(ab')3+
immunized transgenic mice suggests that CD64 cross-linking is important
for eliciting more rapid and consistent responses. Polyvalent binding
of the m22 F(ab')3+ multimers to CD64 improved
the efficiency of eliciting anti-Id responses, most likely by
enhanced internalization of Ag:receptor complexes. The multimeric form
of m22 Fab' also may have enhanced immune responses by mechanisms other
than superior internalization. Cross-linking of FcRs has profound
effects on macrophage and dendritic cell activity, resulting in the
secretion of cytokines and up-regulation of costimulatory and adhesion
molecules (28, 29, 30). Although we were unable to demonstrate
consistent up-regulation of CD40, CD80, or CD54 on peritoneal
macrophages treated with m22 F(ab')3+ (data not
shown), it is feasible that activation of macrophages or dendritic
cells via CD64 cross-linking contributed to potent vaccine effects of
the multimer.
Previously, several investigators have shown that developing immunity specific for the idiotypic determinants expressed by surface Ig on B cell lymphoma cells can lead to potent tumor cell-specific immunity (15, 16, 17, 18, 22). Our experiments demonstrated that a specific anti-520C9 Id response can be readily elicited by directing the 520C9-Id to human CD64, and we are currently developing a syngeneic tumor model in human CD64 transgenic mice to determine whether this approach can be used as a viable tumor vaccine. The high levels and persistence of the anti-Id IgG responses indicate that these vaccines may have therapeutic effects in an appropriate model. In addition, both IgG1 and IgG2a anti-Id isotypes were elicited, suggesting that CD64-targeted vaccines can enhance both humoral (Th2) and cellular (Th1) immunity to the Id.
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Footnotes |
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Received for publication June 2, 2000. Accepted for publication September 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor (monocyte/macrophage)-specific immunogens. J. Immunol. 149:3477.[Abstract]
RI-targeted fusion proteins result in efficient presentation by human monocytes of antigenic and antagonist T cell epitopes. J. Clin. Invest. 98:2001.[Medline]
RI (CD64) expressed on human blood dendritic cells. J. Immunol. 158:3090.[Abstract]
RIa (CD64) ligand binding chain triggers major histocompatibility complex class II antigen presentation independently of its associated FcR -chain. Blood 94:808.RI/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. Invest. 97:331.[Medline]
chain-deficient mice and increased in FcRII-deficient mice. J. Immunol. 163:618.RI/CD64 transgenic model for in vivo analysis of (bispecific) antibody therapeutics. J. Hematother. 4:351.[Medline]
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RI) monoclonal antibody. J. Immunol. 155:4996.[Abstract]
RI and FcRII induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-FcR interactions: functional activation of FcRII by treatment with proteases or neuraminidase. J. Immunol. 144:1304.[Abstract]
receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class 1-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371.This article has been cited by other articles:
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