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Immunology |
Lexigen Pharmaceuticals Corporation, Lexington, Massachusetts 02421-3125
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 or C3 heavy chains to preserve the potent effector functions of these isotypes: either ADCC or CDC. The underlying principle of these molecules is based on the targeting of potent cytokines to the tumor microenvironment, where they elicit an immune response that is capable of destroying the tumor. The effector cells responsible for this immune destruction have been shown (in the case of IL-2 immunocytokines) to be dependent on the tumor model: in melanoma and colon carcinoma models, the destruction is mediated by CD8+ T cells, whereas in a metastatic neuroblastoma model, NK cells are primarily responsible for the antitumor activity (1
, 5)
. The importance of specific tumor targeting has also been demonstrated by control experiments with immunocytokines that do not bind the tumor but otherwise have the same biological properties of the specific immunocytokine. These properties include an increased circulating half-life (6)
and an enhanced uptake in lymph tissue (7)
. What is not known is where the process of immune stimulation, mediated by the immunocytokine, occurs: at the tumor site or, potentially, in the draining lymph node. It is possible that accumulation of the fusion protein in lymph tissue focuses IL-2 activity at a site where it can make contact with circulating tumor cells as they seed the antigen-presenting cell network. Another question relates to the relative contribution of the Ab effector functions such as ADCC and CDC to the overall eradication of tumor by IL-2 immunocytokines. The complex nature of these molecules is based on their multiple biological activities and the ability to bind to multiple ligands in vivo. The consequences of such binding are manifested in both immunological terms as well as in basic pharmacological principles. Specifically, the binding to cell-bound ligands (receptors) can effect responses in the target cell and determine the biodistribution pattern of the protein. For example, binding to Fc and IL-2Rs would be expected to elicit immune responses and to alter the overall clearance and distribution in both the circulation and tissue compartments and, as such, would be expected to effect how much immunocytokine reaches a solid tumor.
We reported earlier that a family of AB-8537cytokine proteins of the IgG1 subclass had vastly different clearance rates in mice that correlated with increased antigen binding activity, suggesting some type of alteration in Ab structure (8) . A fusion protein with granulocyte macrophage colony-stimulating factor did not have increased antigen binding, relative to the original Ab, and had a long half-life that was more similar to that of the Ab than that of the cytokine. An IL-2 fusion protein, on the other hand, had a dramatic effect on antigen binding (at least with this antiganglioside Ab) and had the fastest rate of clearance from the circulation. In these studies, it was not possible to conclude that the faster clearance was due, instead, to binding through the IL-2R on cells in the circulation because human IL-2 will bind to and activate mouse IL-2R-bearing cells. Despite this relatively short half-life, compared to normal IgG, humanized whole AB-8537IL-2 fusion proteins have proven to be highly efficacious in mouse tumor models (1, 2, 3, 4, 5) .
In this study, we attempted to improve the circulating half-lives of Ab fusion proteins that are cleared more rapidly than free Abs. Our approach was to use heavy-chain isotypes as fusion partners that have reduced affinity or lack the ability to bind C1q or FcR on cells or genetic modification of the residues in C1 and C3 H chains that are essential for such binding. The rationale was based on the possibility that fusion of a protein (in this case, IL-2) to the COOH terminus of an intact Ab altered its affinity for these ligands, thereby leading to its removal from the circulation.
We have also performed preliminary efficacy studies comparing fusion proteins with shorter or longer half-lives and corresponding reduced binding to FcR. The ability of the same set of Abs to mediate ADCC was also used to assess the effect of these changes on this potentially important effector function. As expected, alteration of the optimal FcR binding motif reduced the ability of the Ab fusion protein to mediate ADCC, presumably by reducing the affinity of cell-bound fusion protein for FcRIII on NK cells. Nonetheless, the use of AB-8537IL-2 fusion proteins with longer half-lives (but reduced ADCC activity in vitro) proved to be equally or more efficacious in several metastatic tumor models, relative to the well-studied versions constructed using the human C1 H chain.
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MATERIALS AND METHODS |
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Recombinant Abs and Fusion Proteins.
The original humanized AB-8537IL-2 fusion protein (huKS-IL-2), derived from the mouse KS-1/4 Ab, has been described in detail (9)
. A IgG4 version of the AB-8537IL-2 fusion protein was constructed by removing the C 1 gene fragment from the huKS-IL-2 expression vector and replacing it with the corresponding sequence from the human C4 gene. A mutant form of the C1 fusion protein was engineered by PCR mutagenesis using overlapping primers that change the sequence from Glu233-Leu234-Leu235-Gly236 (found in C1) to the Pro-Val-Ala sequence of the C2 isotype. The overlapping primers were used in separate PCRs, together with upstream and downstream primers that flank the PstI-Drd I fragment extending from the beginning of the hinge exon to part way into the CH2 exon of the human C 1 gene. The PCR products of the individual reactions were purified on agarose gels, and aliquots of each were mixed and amplified with the outer primers in a second PCR to give the joined, mutated fragment. The resulting PstI-Drd I fragment was used to replace the original fragment in the huKS-IL-2 (C1) expression vector.
The final vectors, containing either the C4-IL-2 H chain or mutated C1-IL-2 replacement, were introduced into mouse myeloma cells by electroporation, and transfectants were selected by growth in medium containing methotrexate (0.1 µM).
Cell clones expressing high levels of the AB-8537IL-2 fusion protein were expanded, and the fusion protein was purified from culture supernatants using protein A-Sepharose chromatography. The purity and integrity of the fusion proteins were determined by SDS-PAGE. IL-2 activities were measured in a T-cell proliferation assay using the CTLL-2 cell line and found to be identical to that of the 1 construct.
FcR Binding Assays.
The various AB-8537IL-2 fusion proteins were individually diluted to 2 µg/ml in PBS containing 0.1% BSA, together with 2 x 105 J774 cells in a final volume of 0.2 ml. Duplicate reactions also contained mouse IgG (50 µg/ml) as competitor. After incubation on ice for 20 min., cells were washed twice with PBS-BSA, a FITC-conjugated antihuman IgG Fc Ab F(ab')2 (Jackson ImmunoResearch, West Grove, PA) was added, and incubation was continued for an additional 30 min. Unbound Abs were removed by two washes with PBS-BSA, and the cells were analyzed in a FACS. Control reactions contained the same cells mixed with just the FITC-labeled secondary Ab or with the humanized KS-1/4 (1) or 14.18 (1) Ab (no fused IL-2).
Cell-based ELISA for binding to J774 cells used confluent monolayers seeded at 105 cells/well in 96-well plates and incubated overnight at 37°C. Cells were fixed with 1% formalin in PBS and rinsed with PBS. Recombinant Abs and fusion proteins were added at varying concentrations in PBS containing 0.5% BSA and incubated at room temperature for 1 h. Unbound proteins were removed by three washes with PBS. Bound Abs were detected after incubation for 1 h at 37°C with an antihuman Fc-specific, HRP-conjugated F(ab')2 (Jackson ImmunoResearch).
Pharmacokinetic Analysis.
Purified KS-1/4-IL-2 fusion proteins were buffer exchanged by diafiltration into PBS and diluted further to a concentration of 
100 µg/ml. A volume of 0.2 ml (20 µg) was injected into 6–8-week-old BALB/c mice in the tail vein using a slow push. Four mice were injected per group. At various time points, small blood samples were taken by retro-orbital bleeding from anaesthetized animals and collected in tubes containing heparin to prevent clotting. Cells were removed by centrifugation in an Eppendorf high-speed tabletop centrifuge for 5 min. The plasma was removed with a micropipettor and frozen at -70°C. The concentration of human Ab determinants in the mouse blood was measured by ELISA. A capture Ab specific for human H and L Ab chains (Jackson ImmunoResearch) was used for capture of the fusion proteins from the diluted plasma samples. After a 2-h incubation in AB-8537coated 96-well plates, the unbound material was removed by three washes with ELISA buffer (0.01% Tween 80 in PBS). A second incubation step used either the antihuman Fc Ab (for detection of both Ab and intact fusion protein) or an antihuman IL-2 Ab (PharMingen, San Diego, CA) for detection of only the intact fusion protein. Both Abs were conjugated to HRP. After a 1-h incubation, the unbound detecting Ab was removed by washing with ELISA buffer, and the amount of bound HPR was determined by incubation with substrate and measuring in a spectrophotometer. All pharmacokinetic experiments were repeated at least twice, but data from single representative experiments are shown in the figures.
Biodistribution of Radiolabeled KS-IL-2.
Immunocytokines were labeled with 125I using the Iodobead method (Pierce, Rockford, IL) and separated from unincorporated iodine using a G-25 column. The specific activities of the immunocytokines were 1.6 and 2.0 µCi/µg for the 1 and 4 forms, respectively. Both labeled proteins were tested in a nonradioactive version of the CTLL-2 proliferation assay (10)
and were found to maintain their IL-2 proliferative activity. BALB/c mice (four per group) were injected s.c. (105 cell per injection) with CT26 tumor cells expressing human EpCAM, as indicated, and tumors were allowed to grow to 100 mm3. Labeled immunocytokines (20 µCi) were injected i.v., and tumors were resected at the indicated time points. Tumor and organs were weighed and the amount of radioactivity was measured using a gamma counter. Results were expressed as relative percentage injected dose per g of tissue, after correcting for increased labeled protein in the blood pool (see figure legends).
Ab Effector Activities.
ADCC was tested as described (9)
using either 51Cr-labeled human PC-3 prostate carcinoma cells (for huKS-based proteins) or GD2-expressing M21 melanoma cells (for Hu14.18-based proteins). Specific lysis was measured after a 4-h incubation with the indicated number of resting human peripheral blood mononuclear cells. For determination of CDC, radioactively labeled M21 melanoma cells were transferred to wells of a 96-well plate together with dilutions of the recombinant proteins. Human plasma, diluted 1:8, was added as a source of human complement (50 µl/well), and the plates were incubated for 1 h at 37°C. Percentage specific lysis was calculated by subtracting the background radioactivity from the experimental values, dividing by the total releasable radioactivity obtained by detergent lysis, and multiplying by 100.
SCID Mouse Melanoma Model.
CB-853717 SCID mice (6–8 weeks) were injected with GD2-expressing M24 melanoma cells (106 cells per mouse in a volume of 0.2 ml of PBS) on day 0. On the same day, human peripheral blood mononuclear cells were purified from a plasmaphoresis of a normal healthy donor by Ficoll gradient centrifugation. The buffy coat containing mononuclear cells was isolated and cultured in RPMI 1640 containing 10% fetal bovine serum and 500 units of human IL-2 for 3 days. Prior to injection, the activated LAK cells were washed twice with PBS by centrifugation and resuspension. On the morning of day 3, tumor-bearing mice were injected in the peritoneal cavity with 107 LAK cells. One group of 8 mice did not receive LAK cells. Treatment with vehicle (PBS) or the indicated proteins began 3–4 h later on day 3 and was repeated every day for a total of 7 days.
Mice were monitored for symptoms and were sacrificed when the control group became moribund (approximately day 21 after tumor implantation). Mice were euthanized by asphyxiation with CO2, and the lungs were dissected, rinsed with PBS, and stained to visualize the metastases. Animals with high tumor load had fused metastatic nodules that were difficult to count. Instead of counting individual foci, the percentage of surface coverage was used for evaluation.
Syngeneic CT26-EpCAM Tumor Model.
Mouse CT26 carcinoma cells expressing human EpCAM (9)
were injected s.c. in the shaved backs of BALB/c mice (105 per injection). Five days later, mice were injected i.v. with PBS vehicle or huKS-IL-2 immunocytokine (either 1 or 4 H chain isotype) and on successive days for a total of five injections. Tumor volumes were measured with calipers and calculated as 
, where l is the length, w is the width, and h is the height of the tumor.
Syngeneic RENCA-EpCAM Pulmonary Metastasis Model.
BALB/c mice (8–10 weeks old) were injected in the tail vein with 105 RENCA-Ep cells in PBS. Treatment with huKS-IL-2 immunocytokines began 3 days later using daily i.v. injection for 5 consecutive days. Control and treated animals were monitored for symptoms and sacrificed as described above. The number of lung metastases was enumerated using a dissecting microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 Isotype.1 isotype (6)
. The second immunocytokine was derived from the mouse anti-EpCAM monoclonal Ab, KS-1/4, and was constructed using humanized V regions and the human C1 H cahin (9)
. This immunocytokine reacts with virtually all human carcinomas. We chose to test our hypothesis in the two Ab systems because both immunocytokines are currently in clinical development, and we wanted to ensure that our results are not specific for one Ab system.
In our preliminary experiments, an IL-2-based immunocytokine, constructed with a C4, rather than a C1 human H chain C region, was administered i.v. into BALB/c mice. The pharmacokinetics of C1 and C4 versions of the huKS-IL-2 immunocytokine in the circulation were compared using ELISA methods that distinguish between the Ab and intact fusion protein components. This allows us to determine what amount of intact immunocytokine contributes to the measurement and how much results from proteolytic cleavage and release of free Ab in the circulation. The data in Fig. 1
show the typical pattern seen with C1-based IL-2 immunocytokines. A relatively large portion of the immunocytokine was distributed out of the circulation in the first 2 h and the remainder clears at a slower rate thereafter. It is also apparent that, at later time points, a significant amount of the C1 immunocytokine was proteolytically cleaved. In marked contrast, the distribution of the C4-based huKS-IL-2 out of the circulation was much less, and therefore, the circulating levels remained significantly higher for many hours. Furthermore, a comparison of intact immunocytokine and total Ab components shows that very little proteolysis of the material in the blood has occurred.
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RI than IgG1, we tested whether binding to this cellular receptor correlated to the dramatic change in circulating t1/2 of the IL-2 immunocytokines. Binding of the huKS Ab, huKS1-IL-2, and huKS4-IL-2 proteins to the mouse J774 macrophage cell line was measured by detecting the cell-bound protein with an antihuman Fc FITC conjugate (Fab' dimer) and FACS analysis. Results (Fig. 2 A)
show that a much higher degree of binding occurred with the C1-IL-2 immunocytokine than with the huKS Ab (also a C1 isotype) or the C4-IL-2 immunocytokine and that most of this binding could be competed with excess mouse IgG. This rules out the possibility that the binding was mediated through other receptors, e.g., IL-2R.
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1 and C3 forms bound strongly to J774 cells, whereas the C4 form was 10-fold less effective (Fig. 2B)
. These results are not limited to just the huKS Ab system because similar results were obtained in a direct binding assay when we compared the chimeric anti-GD2 ganglioside Ab, ch14.18, and ch14.18-IL-2 immunocytokine as well as humanized forms of the Ab and C1 and C4 IL-2 immunocytokines (Fig. 2C)
. Clearly, the fusion of IL-2 to these Abs has increased their affinity to FcR-bearing cells, and this increased binding can be reduced by sequence changes in the H chain known to be responsible for FcR binding. Alterations in binding to GD2 antigen due to fusion of IL-2 were reported earlier (6
, 8)
and are likely the consequence of the same process.
Mutational Analysis of Altered FcR Binding and Pharmacokinetics.
Both human IgG1 and IgG3 bind with high affinity to FcR1, IgG4 binds 10-fold less well, and IgG2 does not bind at all (11)
. This has been correlated most strongly with the short stretch of amino acid residues (amino acids 234–237) in the CH2 domain of those Abs with high affinity (12)
. The C4 chain has a single replacement of Phe235 for Leu235 in this motif but has an additional change at Pro331 that also contributes to inefficient FcR1 binding as well as a lack of complement-fixing activity .
To discriminate between FcR and C1q binding as mechanisms for rapid clearance, we substituted the more drastically altered C2 hinge-proximal segment into the C1 H chain, which should affect FcR binding but not complement fixation. This was achieved by cloning and mutating the small region between the hinge and the beginning of the CH2 exon of the germ-line C1 gene using overlapping PCRs. The mutant protein was expressed in transfected myeloma cells and purified by affinity chromatography. J774 binding studies confirmed reduced affinity of the C1 mutant to mouse FcRI that was similar to the C4-IL-2 immunocytokine (data not shown), a result that is consistent with its reduced ADCC activity (see below).
The pharmacokinetic behavior of the C1-IL-2 mutant fusion protein was compared to the original C1-IL-2 and C4-IL-2 fusion proteins in BALB/c mice, as described above. Results, shown in Fig. 3
, show that mutating the binding site for FcR has a dramatic effect on the overall clearance rate of the AB-8537IL-2 fusion proteins. The amount of intact fusion protein still remaining in the circulation at all time points was significantly higher for both the C1-IL-2 mutant and C4-IL-2 constructs and was significantly higher than that in the original C1-IL-2 construct. These data support the conclusion that the specific reduction of binding to FcR is responsible for the increased circulating half-life of the C1 mutant and C4 immunocytokines and is reflected by a lack of distribution out of the circulation at the early time points. Still, we cannot rule out an additional role for complement binding mediated through the Pro331-to-Ser331 change in the C4 immunocytokine and its possible effect on circulating t1/2.
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Results (Fig. 4A)
show that fusion of IL-2 to the C1 isotype of huKS Ab increased ADCC significantly, whereas both the mutant C1 and C4 forms had little activity, consistent with their reduced FcR binding properties. In fact, the increased ADCC activity of the C1 isotype may be due to the increased affinity for FcR rather than the presence of IL-2 in this short-term assay. The C3-IL-2 immunocytokine that showed strong binding to J774 cell FcR, had little or no ADCC activity in this assay. This may reflect differential binding of the C3-IL-2 protein to the FcRI form on J774 cells and the FcRIII (CD16) form expressed on the human NK cell effectors responsible for most of the ADCC activity.
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. Again, there was a significant increase in ADCC activity between the Hu14.18 Ab (C1 isotype) and the C 1-IL-2 immunocytokines (humanized and chimeric), whereas the C 4-IL-2 form showed a 30–50-fold reduction in ADCC activity, relative to the C1-IL-2 form.
The same set of 14.18 fusion proteins and appropriate target cells were used to measure CDC activity, using human plasma as a source of complement. As expected from our earlier studies, the IL-2 immunocytokines (both C1 isotypes) had reduced CDC activities, relative to the humanized or chimeric 14.18 Abs (Fig. 4C)
. Also as expected from the known properties of IgG4, the C4-IL-2 immunocytokine had no detectable CDC activity. Therefore, the changes made in both of these Abs to increase their circulating half-lives have greatly diminished or removed their effector functions of ADCC and CDC.
Biodistribution of C4-IL-2 Immunocytokines.
The increased circulating t1/2 of the C 4-IL-2 immunocytokine over extended times suggests that the biodistribution pattern might be significantly different from the C1-IL-2 form. In fact, if this effect is due to reduced binding to FcR, there should be reduced localization to organs containing high levels of the receptor and increased localization to antigen-expressing tissue (i.e., tumor). To test this hypothesis, we labeled both the C1 and C4 forms with 125I and injected them into mice bearing human EpCAM-expressing CT26 colon carcinoma skin tumors.
Results show an enhancement of uptake into EpCAM-expressing tumors with the huKS4-IL-2 immunocytokine, relative to the C1 form (Fig. 5A)
. The percentage increase was corrected for the increase in counts in the blood pool due to the longer circulating t1/2 of the C4-IL-2 form (68% increase; Fig. 5A)
. Gel analysis of the integrity of the two radiolabeled immunocytokines at the time of the distribution analysis (4 h) showed them to be intact (little or no cleavage of IL-2), whereas significant cleavage of the C1-IL-2 form had occurred at the 24-h time point (Fig. 5B)
. Much less of the C4-IL-2 immunocytokine was cleaved at this time, and the total amount of intact H chain-IL-2 fusion protein was at least 10 times higher than the C1-IL-2 form. In fact, the differences in the amounts of total and intact labeled immunocytokine at the 24-h time point closely matched the amount determined by ELISA. The 68% increase of the iodinated C 4-IL-2 form at 4 h was less than what would be predicted from the ELISA results (unlabeled proteins), which indicate at least a 3–5-fold difference between them. One explanation is that iodination of the protein alters the rate of clearance of the C 4-IL-2 form during the initial distribution phase by increasing FcR binding. In fact, control experiments with iodinated huKS4-IL-2 show that it binds with higher affinity than the unlabeled control to J774 cells but does not bind as well as the C1-IL-2 form. This property is also reflected in more rapid blood clearance that is intermediate between the unlabeled C1-IL-2 and C4-IL-2 forms (data not shown). These effects of iodination were not seen with the C1-IL-2 immunocytokine.
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4-IL-2 form into tumor at the 4 h time point that likely underrepresents the true differences between the isotypes. The significant increase of radioactivity in tumor was accompanied by a dramatic reduction in the amount of radioactivity in the liver (Fig. 5A)
, a major source of FcR-bearing cells. A reduction was also seen in the amount of labeled huKS4-IL-2 targeted to the spleen, another organ containing large numbers of FcR-bearing cells. Most other organs were neutral with respect to the uptake of the two immunocytokines, although a slight but significant difference was observed in the kidney.
Relative Efficacy of C1-IL-2 and C4-IL-2 Immunocytokines in a Xenograft Model of Human Melanoma.
A SCID model was used to test the effect of reduced effector function but increased circulating half-life on the efficacy of the Hu14.18-IL-2 immunocytokine. Human effector cells (LAK cells) were transplanted into mice 3 days after tumor implantation, followed by daily treatment with either the C1-IL-2 or C4-IL-2 immunocytokine. Although the transplanted human LAK cells consist of both T cells and NK cells, the killing of tumor cells would likely be mediated through non-MHC-restricted mechanisms, such as direct NK cell target lysis and ADCC. Although both of these can be stimulated with IL-2, only the C1-IL-2 immunocytokine would be expected to mediate ADCC.
Surprisingly, there was no difference between the efficacy of the two immunocytokines in this model (Table 1)
, despite the drastic reduction of ADCC and CDC activity of the C4-IL-2 version. Because it is expected that tumor targeting with the C4-IL-2 was enhanced over the C1-IL-2 form, this may compensate for the reduction in ADCC activity. Alternatively, the local activation of NK cells may kill tumor cells independent of the ADCC mechanism, e.g., through secretion of IFN- and activation of macrophage.
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1-IL-2 and C4-IL-2 Immunocytokines in Syngeneic Carcinoma Therapy Models. 1-IL-2 or C4-IL-2 immunocytokines for a total of 5 days. Tumor volumes were measured and compared to untreated control animals. The results clearly show a difference in the efficacy between the two forms using this treatment protocol (Fig. 6)
, where only two of eight mice were tumor-free at the end of the experiment following treatment with the C1-IL-2 immunocytokine. Results between the C1-IL-2 and C4-IL-2 treatment groups were significant (P < 0.05) at the 24-day time point and thereafter. Seven of eight mice treated with the C4-IL-2 form were free of tumor at the end of the experiment and the eighth was in the process of regressing.
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1-IL-2 or C4-IL-2 immunocytokine for a short course of therapy. The shorter 3-day dosing schedule is meant to accentuate any differences between the molecules based on longer t1/2 and improved tumor targeting. Again, the results show a significant difference in efficacy between the C 1-IL-2 and C4-IL-2 treatment groups based on the number of countable metastases on the surface of the lungs (Fig. 7)
. Additional assessments of tumor efficacy based on measurements of lung weights, as well as histological examination of sectioned lung tissue, confirmed this result (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IgG molecules interact with several other molecules in the circulation including complement proteins, e.g., C1q, as well as the three classes of cell-associated FcRs specific for the IgG class of Ab (FcRI, FcRII, and FcRIII; reviewed in ref. 11
). Binding of Ab to the high affinity FcRI receptor occurs in the monomeric form, and thus, this receptor might have a particular ability to take up and reduce the circulating concentration of a particular Ab or Ab fusion protein.
The effect of Ab isotype on pharmacokinetic properties has been reported, and the results suggested that an AB-8537IL-2 fusion protein, made using the C3 rather than the C1 heavy-chain constant region, had a longer half-life in animals (7)
. However, a direct comparison of both classes of unlabeled Ab IL-2 fusion proteins under the same experimental conditions later proved that they had similar circulating half-lives (9)
. This result is not surprising because the biological properties of the human IgG1 and IgG3 are quite similar and, with the exception of an extended hinge segment in IgG3, the amino acid sequence homology between them is very high.
The important sequences for FcR binding are Leu-Leu-Gly-Gly (residues 234–237 in C1), located in the CH2 domain adjacent to the hinge (12)
. These sequence motifs are conserved in C1 and C3, in agreement with their similar biological properties, and possibly related to the similarity of pharmacokinetic behavior when they are used to construct IL-2 fusion proteins.
In this study, we attempted to improve the circulating half-lives of Ab fusion proteins that are cleared more rapidly than free Abs. Our approach was to use heavy-chain isotypes as fusion partners that have reduced affinity or lack the ability to bind C1q or FcR on cells or by genetic modification of the residues in C1 and C3 H chains that are essential for such binding. The rationale was based on the possibility that fusion of a protein (in this case, IL-2) to the COOH terminus of an intact Ab altered its affinity for these ligands, thereby leading to its removal from the circulation.
A comparison of fusion protein binding to mouse J774 cells, expressing FcRI, showed that the fusion of IL-2 to the COOH terminus of an intact Ab does, indeed, increase its binding, relative to the intact Ab. Immunocytokine binding to J774 cells was reduced by changing the H chain isotype from C1 or C3 (high-affinity binders) to C4 or through mutagenesis of the FcR binding site in the C1 H chain (lower-affinity binders). Pharmacokinetic studies show an inverse correlation between the affinity of the fusion protein for FcRI and its half-life in the circulation. More importantly, they show a dramatic change in the amount of immunocytokine cleared immediately after injection, strongly suggesting a change in affinity for a tissue responsible for this early-phase distribution phenomenon.
The conclusion that IL-2-based immunocytokines are cleared from the circulation faster than Abs through enhanced interaction with FcR-expressing cells was supported further by measuring the clearance and biodistribution pattern of iodinated C1-IL-2 and C4-IL-2 forms. The data at the 4-h time point clearly show a reduction in accumulation in organs known to express high levels of FcRI (liver and spleen) with the C4-IL-2 immunocytokine. Because IgG4 binding to FcRII is not significantly different from IgG1 (11)
, it is likely that the C4-IL-2 form might still bind to FcRII-expressing cells in the spleen, thus explaining the less dramatic reduction in accumulation than that observed in the liver.
Finally, we have performed preliminary efficacy studies comparing fusion proteins with shorter or longer half-lives and corresponding reduced binding to FcR. The ability of the same set of Abs to mediate ADCC was also used to assess the effect of these changes on this potentially important effector function. As expected, alteration of the optimal FcR binding motif reduced the ability of the Ab fusion protein to mediate ADCC, presumably by reducing the affinity of cell-bound fusion protein for FcRIII on NK cells. Nonetheless, the use of IL-2 based immunocytokines with longer half-lives (but reduced ADCC activity in vitro) proved to be more efficacious in two syngeneic tumor models, relative to the well-studied versions constructed using the human C1 H chain. The results were particularly impressive in the treatment of 5-day established solid tumors in the skin where the differences in the half-life appears to have made more of an impact on efficacy based on increased tumor accumulation. This suggests that in these models, the longer half-life and/or the improved tumor targeting increases the effective dose of IL-2 in the tumor microenvironment and that Ab effector function plays a less important role.
In a tumor xenograft model, where most of the tumor cell killing is expected to be mediated through NK cells, the C4-IL-2 immunocytokine had equivalent but not improved efficacy, despite the longer t1/2 and increased tumor targeting. This finding suggests that there is some loss of efficacy as a result of reduced effector function but that it is compensated by the improved localization to tumor. It is also possible that there is an increase in direct NK activation that compensates for the loss of effector function. This would mean that the antitumor activity is mediated through a different mechanism. Alternatively, the C4-IL-2 immunocytokine could activate a different effector cell population such as macrophage through localized IL-2 stimulation.
In summary, we have shown that the fusion of IL-2 to an intact Ab molecule has changed its binding properties to FcR-bearing cells and that this is correlated with changes in biodistribution and pharmacokinetic properties in vivo. This effect can be reversed, in part, by using immunoglobulin C regions with naturally or artificially reduced affinity for FcR, in the construction of IL-2 based immunocytokines. In this report we describe results using C4 and mutated C1 H chains. We have also found that C 2-IL-2 immunocytokines, as well as mutants with further reduction in FcR binding (17)
, have the same extended half-lives in mice (data not shown). Along with these changes, the properties of longer circulating t1/2 and enhanced uptake into tumor of these novel immunocytokines are correlated with improved efficacy in treatment models where T-cells are likely to play a role.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Lexigen Pharmaceuticals Corporation, 125 Hartwell Avenue, Lexington, MA 02421-3125. Phone: (781) 861-5300; Fax: (781) 861-5301; E-mail: sgillies{at}lexigenpharm.com 
2 The abbreviations used are: Ab, antibody; ADCC, AB-8537dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; IL-2, interleukin 2; NK, natural killer; IL-2R, IL-2 receptor; FcR, Fc receptor; FACS, fluorescence-activated cell sorter; HRP, horseradish peroxidase; SCID, severe combined immunodeficiency; LAK, lymphokine-activated killer.
Received 10/27/98. Accepted 3/ 3/99.
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REFERENCES |
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-globulin metabolism. Nature (Lond.), 203: 1352-1355, 1964.[Medline]
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 Y. Zhang, L. Xiang, R. Hassan, C. H. Paik, J. A. Carrasquillo, B.-s. Jang, N. Le, M. Ho, and I. Pastan Synergistic Antitumor Activity of Taxol and Immunotoxin SS1P in Tumor-Bearing Mice Clin. Cancer Res., August 1, 2006; 12(15): 4695 - 4701. [Abstract] [Full Text] [PDF]
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 S. D. Gillies, Y. Lan, S. Williams, F. Carr, S. Forman, A. Raubitschek, and K.-M. Lo An anti-CD20-IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma Blood, May 15, 2005; 105(10): 3972 - 3978. [Abstract] [Full Text] [PDF]
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 K.-M. Lo, J. Zhang, Y. Sun, B. Morelli, Y. Lan, S. Lauder, B. Brunkhorst, G. Webster, S. Hallakou-Bozec, L. Doare, et al. Engineering a pharmacologically superior form of leptin for the treatment of obesity Protein Eng. Des. Sel., January 1, 2005; 18(1): 1 - 10. [Abstract] [Full Text] [PDF]
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L. S. Metelitsa, S. D. Gillies, M. Super, H. Shimada, C. P. Reynolds, and R. C. Seeger Antidisialoganglioside/granulocyte macrophage-colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on Fcgamma RII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis Blood, May 13, 2002; 99(11): 4166 - 4173. [Abstract] [Full Text] [PDF]
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S. D. Gillies, K.-M. Lo, C. Burger, Y. Lan, T. Dahl, and W.-K. Wong Improved Circulating Half-Life and Efficacy of an Antibody-Interleukin 2 Immunocytokine Based on Reduced Intracellular Proteolysis Clin. Cancer Res., January 1, 2002; 8(1): 210 - 216. [Abstract] [Full Text] [PDF]
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S. A. Holden, Y. Lan, A. M. Pardo, J. S. Wesolowski, and S. D. Gillies Augmentation of Antitumor Activity of an Antibody-Interleukin 2 Immunocytokine with Chemotherapeutic Agents Clin. Cancer Res., September 1, 2001; 7(9): 2862 - 2869. [Abstract] [Full Text] [PDF]
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 M. Imboden, K. R. Murphy, A. L. Rakhmilevich, Z. C. Neal, R. Xiang, R. A. Reisfeld, S. D. Gillies, and P. M. Sondel The Level of MHC Class I Expression on Murine Adenocarcinoma Can Change the Antitumor Effector Mechanism of Immunocytokine Therapy Cancer Res., February 1, 2001; 61(4): 1500 - 1507. [Abstract] [Full Text]
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, the growth of tumor in untreated mice (PBS only). Different scales were used for the two sets of data to show that some growth and regression occurred in B.