Bispecific antibodies in cancer therapy.

Based upon in vitro and animal studies, a number of Phase I and II clinical trials have been initiated to test whether bispecific antibodies could redirect immune effectors against tumor cells in cancer patients. Recently, results from those trials showed beneficial effects in some patients but it is clear many problems remain to be solved. In addition, molecular engineering approaches are providing new and improved sources of clinically relevant bispecific antibodies.

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/ß4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

Yes-associated protein mediates immune reprogramming in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of inflammation and profound immune suppression. Here we identify Yes-associated protein (Yap) as a critical regulator of the immunosuppressive microenvironment in both mouse and human PDAC. Within Kras:p53 mutant pancreatic ductal cells, Yap drives the expression and secretion of multiple cytokines/chemokines, which in turn promote the differentiation and accumulation of myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Pancreas-specific knockout of Yap or antibody-mediated depletion of MDSCs promoted macrophage reprogramming, reactivation of T cells, apoptosis of Kras mutant neoplastic ductal cells and pancreatic regeneration after acute pancreatitis. In primary human PDAC, YAP expression levels strongly correlate with an MDSC gene signature, and high expression of YAP or MDSC-related genes predicts decreased survival in PDAC patients. These results reveal multifaceted roles of YAP in PDAC pathogenesis and underscore its promise as a therapeutic target for this deadly disease.

Phase I study of an anti-breast cancer immunotoxin by continuous infusion: report of a targeted toxic effect not predicted by animal studies.

260F9 Monoclonal antibody-recombinant ricin A chain, an immunotoxin reactive with approximately 50% of breast carcinomas, was given by continuous iv infusion at a dose of 50 micrograms/kg per day or 100 micrograms/kg per day. Five patients with refractory breast cancer received treatment for from 6 to 8 days. Severe toxic effects, including marked fluid overload and debilitating sensorimotor neuropathies, occurred in most patients. Immunoperoxidase studies suggested that 260F9 monoclonal antibody targeting of the Schwann cells may have induced demyelination and subsequent neuropathy. This is the first report of a targeted toxic effect due to an immunoconjugate.

Potentiation of tumor lysis by a bispecific antibody that binds to CA19-9 antigen and the Fc gamma receptor expressed by human large granular lymphocytes.

Murine monoclonal antibody therapy of human cancer rarely induces clinical responses. Antibody-induced cellular infiltrates rarely accumulate at sites of tumor, even in clinically responding lesions. Thus, the ability of these antibodies to promote host effector cell-mediated lysis of tumor via antibody-dependent cellular cytotoxicity (ADCC) has not been harnessed by existing treatment approaches. One potential explanation is that ADCC requires binding of antibody Fc domains to cellular Fc gamma receptors, and therapeutically administered murine antibodies must compete with vast excesses of human IgG for Fc gamma receptor occupancy. Chemically linked antibody heteroconjugates that bind selected target and effector cell structures via distinct Fab portions can mediate lysis of malignant cells in vitro in the presence of human serum. This approach addresses a potentially major obstacle to antibody therapy. Production of bispecific monoclonal antibodies with similar specificities and superior in vivo biodistribution characteristics would thus have potential clinical applications. We have prepared and purified a bispecific, monovalent monoclonal antibody and evaluated its in vitro effects. The IgG1-secreting hybridoma line 3G8 (alpha-human Fc gamma R III) was fused with the hybridoma line CA19-9, which produces an IgG1 antibody that binds to a glycoprotein shed by gastrointestinal cancers. Multiple clones with bispecific binding properties were identified. CA19-9 x 3G8 clonal supernatants and purified antibody, but not the parent antibodies, efficiently mediated specific in vitro lysis of cells of the SW948 line by human large granular lymphocytes (LGLs). Human serum-resistant target cell lysis augmentation at low effector:target ratios was seen using picogram amounts of antibody. In contrast, the IgG2 alpha variant of CA19-9, which also promotes ADCC by LGLs, was unable to augment lysis of SW948 cells when effectors were preincubated with human serum. This bispecific, monovalent monoclonal antibody is an efficient promoter of the anti-tumor effects of LGLs in physiological concentrations of human serum. In vivo models that evaluate treatment efficacy and promotion of inflammatory tumor infiltrates by bispecific monoclonal antibodies are required to assess the therapeutic potential of these novel constructs.

Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16.

Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)

Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity.

In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrate tumors rapidly and have rapid serum clearance, leading to excellent tumor:normal organ ratios. However, the absolute quantity of scFv retained in the tumor is low due to rapid serum clearance and monovalent scFv binding. We previously demonstrated that the presence of an additional binding site prolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affinity of each component scFv to the association between a dimeric scFv and its target antigen is largely unknown. Here, we have constructed bivalent diabody molecules from three affinity mutants of the human anti-ErbB2 (HER2/neu) scFv molecule C6.5 by shortening the peptide linker between the heavy (VH) and light (VL) chains variable domains from 15 to 5 amino acids. The shorter linker prevents intramolecular pairing of VH and VL, resulting in intermolecular pairing and creation of a dimeric Mr 50,000 molecule with two antigen-binding sites. The scFv used to create the diabodies span a 133-fold range of affinity for the same epitope of ErbB2 [133 nM (C6G98A), 25 nM (C6.5), and 1 nM (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on the immobilized ErbB2 extracellular domain. The association rate constants obtained for each diabody molecule were similar to that of the parental (component) scFv. However, the dissociation rate constants obtained for the bivalent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold for that of the C6ML3-9 diabody to 15-fold for the C6G98A diabody. This resulted in only a 22-fold difference in bivalent affinity, compared with a 133-fold difference in affinity for the respective scFv. Equilibrium-binding constants obtained by surface plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-37-fold more diabody was retained in tumor compared with the parental scFv monomers. This likely results from a higher apparent affinity, because of bivalent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitative tumor retention or tumor to blood ratios. In fact, the diabody constructed from the lowest affinity scFv exhibited the best tumor-targeting properties. We conclude that, above a threshold affinity, other factors regulate quantitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than does exhaustive scFv affinity maturation.

Divergent dose-related effects of gamma-interferon therapy on in vitro antibody-dependent cellular and nonspecific cytotoxicity by human peripheral blood monocytes.

Twenty-seven patients with advanced gastrointestinal malignancies received recombinant gamma-interferon (rIFN-gamma, Biogen) prior to treatment with the murine monoclonal antibody 17-1A (Centocor), which mediates human monocyte antibody-dependent cellular cytotoxicity (ADCC). rIFN-gamma was used because it enhances human monocyte Fc receptor expression, nonspecific monocyte cytotoxicity (NSMC) and ADCC in vitro. The study was designed to identify a rIFN-gamma dose with acceptable toxicities which enhanced NSMC and ADCC. Patients received one course of therapy consisting of rIFN-gamma by 4-h infusions daily for 4 days at doses ranging from 0.001 to 80.0 X 10(6) units/m2/d, followed by 400 mg of 17-1A on day 5. The maximally tolerated dose of rIFN-gamma in this study was 40 X 10(6) units/d. Significant toxicity was seen at the high (greater than 1 X 10(6) units) but not low (less than or equal to 1 X 10(6) units) dose levels. Monocytes were isolated from patients' peripheral blood at baseline and on Days 3 and 5 for cytotoxicity studies which measured 111-In release from SW1116 cells which bear the target antigen of 17-1A. Low dose rIFN-gamma enhanced NSMC by Day 5 as well as did high dose therapy. ADCC enhancement was seen with low dose therapy (% specific lysis on Day 5 = 23.5 +/- 6.4 SEM versus baseline of 9.6 +/- 3.3, P = 0.03), but not with high dose rIFN-gamma treatment. Total (i.e., NSMC + ADCC) monocyte cytotoxicity was equivalent in the low and high dose treatment groups, although ADCC contributed more to total values in the low dose group. These findings were particularly striking if monocytes were exposed to additional rIFN-gamma in vitro prior to incubation with labeled target cells. We conclude that low dose rIFN-gamma therapy is at least equivalent, and possibly superior to high doses in this setting. Furthermore, low dose therapy, supplemented by ex vivo incubation of purified monocytes with rIFN-gamma, may be an optimal treatment strategy for this cytokine.

Phase I trial of recombinant macrophage colony-stimulating factor and recombinant gamma-interferon: toxicity, monocytosis, and clinical effects.

Macrophage colony-stimulating factor (M-CSF) is a known inducer of proliferation and differentiation of cells of the mononuclear phagocyte lineage, and gamma-interferon (gamma-IFN) is a known activator of mononuclear phagocytes. In this Phase I clinical trial of combined therapy with M-CSF and gamma-IFN, 36 patients were treated with 14-day continuous infusions of M-CSF at doses ranging from 10 to 140 micrograms/kg/day. In all but five patients, gamma-IFN was administered by daily s.c. injection on days 8-14 of the M-CSF infusion at doses of 0.05 or 0.1 mg/m2/day. A total of 73 courses of M-CSF and 66 courses of gamma-IFN were administered. The maximally tolerated dose combination was 120 micrograms/kg/day M-CSF, 0.1 mg/m2/day gamma-IFN. The addition of gamma-IFN did not alter the maximally tolerated dose of M-CSF therapy, although some additional toxicities were noted with combined therapy. At the 140-micrograms/kg/day M-CSF dose level, grade 4 thrombocytopenia occurred in 2 of 3 patients, with a median platelet count nadir of 26,000/mm3 after 7-10 days of M-CSF infusion. At this dose level, there was one reversible grade 3 hepatic toxicity, and one grade 3 exacerbation of underlying chronic obstructive lung disease. Peripheral blood monocytosis was observed at all M-CSF dose levels exceeding 40 micrograms/kg/day, approaching 3-fold elevations at the 100-micrograms/kg/day M-CSF dose level. The induction of monocytosis was correlated with the development of thrombocytopenia. At the conclusion of therapy with 100 micrograms/kg/day M-CSF, 0.1 mg/m2/day gamma-IFN, 78% of peripheral blood monocytes expressed the low affinity Fc gamma receptor for aggregated immunoglobulin, Fc gamma RIII (CD16), and CD14 was expressed by only 36% of the cells. This phenotype has been shown previously to be associated with cellular activation. In contrast, 35% of monocytes from patients treated with M-CSF therapy alone at the same dose expressed CD16 and 88% expressed CD14. A partial clinical response was noted in a patient with metastatic renal cell carcinoma, and minor clinical responses were observed in patients with a diffuse/follicular lymphoma, metastatic renal cell carcinoma, and metastatic thymoma. At M-CSF doses exceeding 20 micrograms/kg/day within the maximally tolerated dose range, gamma-IFN did not modulate the ability of M-CSF to reliably induce peripheral blood monocytosis. This study shows that M-CSF and gamma-IFN therapy induces the proliferation and differentiation of circulating mononuclear phagocytes.

Phase I evaluation of combination therapy with interleukin 2 and gamma-interferon.

Recombinant interleukin 2 (IL-2) is a potent inducer of lymphokine-activated killer (LAK) activity directed against autologous and allogeneic tumors; these effects are mediated by CD3-negative, CD56-positive, and CD16-positive lymphocytes. Although IL-2 therapy has been associated with clinical responses, particularly in patients with renal cell carcinoma and melanoma, these responses have occurred with high, toxic doses of this cytokine. Since gamma-interferon (IFN-gamma) potentiates LAK activity in vitro and in animal models, we initiated a dose-escalating Phase I trial of IFN-gamma and IL-2 in patients with advanced cancer. Patients were treated three times weekly (Monday, Wednesday, and Friday) for 6 weeks with bolus injections of IL-2; each dose was preceded 2 h earlier by a s.c. injection of IFN-gamma. Patients were treated with IFN-gamma at 0.01, 0.05, 0.1, or 0.25 mg/m2/dose. At each IFN-gamma dose, cohorts of at least three patients were treated with IL-2 at 1, 2.5, 5.0, or 7.5 x 10(6) Cetus units/m2 dose. Patients with clinical responses continued therapy three times weekly, while those with stable disease at 6 weeks were then treated twice weekly. A total of 41 patients were treated, all with Eastern Cooperative Oncology Group performance status 0 or 1. All patients were evaluable for toxicity. Dose-limiting toxicities were cumulative fatigue and constitutional symptoms. One documented transmural myocardial infarct occurred. The maximally tolerated dose combination, based on analysis of IL-2 dose intensity, was 0.1 mg IFN-gamma/m2 and 7.5 x 10(6) Cetus units IL-2/m2 per dose. Two partial responses and two minor responses were observed. Treatment was not associated with dose-associated changes in peripheral blood lymphocyte phenotype, but there was a trend favoring IFN-gamma dose-associated rises in IL-2 induction of natural killer and LAK activity by treated patients' lymphocytes. Analysis of the cumulative effects of therapy on induction of natural killer and LAK activity by measurement of the median area under the curve of activation showed clear evidence of IFN-gamma and IL-2 dose-associated changes. The IL-2 dose effects on cell lysis were monotone, while the optimal IFN-gamma dose appeared to be 0.1 mg/m2/dose, with a bell-shaped dose-response curve described previously for other effects of this cytokine. Using this novel statistical method of evaluating the biological effects of treatment, the optimal biological dose was identical to the maximally tolerated dose.(ABSTRACT TRUNCATED AT 400 WORDS)

A human tumor xenograft model of therapy with a bispecific monoclonal antibody targeting c-erbB-2 and CD16.

New strategies are required to clinically exploit the ability of monoclonal antibodies to target tumor for lysis by cellular effector mechanisms. In this report we examine the therapeutic effects of 2B1, a bispecific monoclonal antibody with specificity for the extracellular domain of the c-erbB-2 oncogene product and the human Fc gamma receptor, Fc gamma RIII (CD16), describe the characteristics and limitations of this model, and examine the mechanisms underlying the observed responses. The model uses SK-OV-3 human ovarian carcinoma xenografts in scid mice. These cells are susceptible to 2B1-directed lysis by human peripheral blood lymphocytes or lymphokine-activated killer cells, and maintain c-erbB-2 expression in vivo. 125I-labeled 2B1 selectively accumulates in tumor, with a peak of 10.5% injected dose/g of tumor 24 h following its i.v. injection. However, the selectivity of this binding is lessened by 2B1 accumulation in the lungs and other normal organs and persistence in the blood. This is caused by antibody binding to murine lung, colon, stomach, and skin expressing the epitope recognized by the anti-c-erbB-2 component of 2B1 in tumor-bearing, but not normal mice. In treatment studies using various permutations of antibody, human peripheral blood lymphocytes or lymphokine-activated killer cells and interleukin 2, cellular therapy alone had minimal effects on SK-OV-3 xenograft growth, but significantly improved when 2B1 treatment was incorporated. Median survivals increased from 80 +/- 3.5 days with no therapy to 131 +/- 7.3 days following therapy with 100 micrograms 2B1, interleukin 2, and human peripheral blood lymphocytes, with 70% of animals exhibiting no evidence of tumor at day 150. These effects were preserved when the cells were administered in human serum. In contrast, human serum abolished the antitumor effects of 520C9, which is the parent anti-c-erbB-2 antibody of 2B1. Thus 2B1-based therapy has therapeutic effects, without obvious toxicity, despite the targeting of this antibody to normal murine tissues. Since combinations of 2B1 and interleukin 2 may have antitumor properties, mechanisms other than bispecific monoclonal antibody-promoted conjugation of c-erbB-2 antigen-expressing tumor to CD16-expressing effector cells may be involved.

Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII.

2B1 is a bispecific murine monoclonal antibody (BsMAb) with specificity for the c-erbB-2 and Fc gamma RIII extracellular domains. This BsMAb promotes the targeted lysis of malignant cells overexpressing the c-erbB-2 gene product of the HER2/neu proto-oncogene by human natural killer cells and mononuclear phagocytes expressing the Fc gamma RIII A isoform. In a Phase I clinical trial of 2B1, 15 patients with c-erbB-2-overexpressing tumors were treated with 1 h i.v. infusions of 2B1 on days 1, 4, 5, 6, 7, and 8 of a single course of treatment. Three patients were treated with daily doses of 1.0 mg/m2, while six patients each were treated with 2.5 mg/m2 and 5.0 mg/m2, respectively. The principal non-dose-limiting transient toxicities were fevers, rigors, nausea, vomiting, and leukopenia. Thrombocytopenia was dose limiting at the 5.0 mg/m2 dose level in two patients who had received extensive prior myelosuppressive chemotherapy. Murine antibody was detectable in serum following 2B1 administration, and its bispecific binding properties were retained. The pharmacokinetics of this murine antibody were variable and best described by nonlinear kinetics with an average t 1/2 of 20 h. Murine antibody bound extensively to all neutrophils and to a proportion of monocytes and lymphocytes. The initial 2B1 treatment induced more than 100-fold increases in circulating levels of tumor necrosis factor-alpha, interleukin 6, and interleukin 8 and lesser rises in granulocyte-monocyte colony-stimulating factor and IFN-gamma. Brisk human anti-mouse antibody responses were induced in 14 of 15 patients. Several minor clinical responses were observed, with reductions in the thickness of chest wall disease in one patient with disseminated breast cancer. Resolution of pleural effusions and ascites, respectively, were noted in two patients with metastatic colon cancer, and one of two liver metastases resolved in a patient with metastatic colon cancer. Treatment with 2B1 BsMAb has potent immunological consequences. The maximum tolerated dose and Phase II daily dose for patients with extensive prior myelosuppressive chemotherapy was 2.5 mg/m2. Continued dose escalation is required to identify the maximally tolerated dose for patients who have been less heavily pretreated.

Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies.

Mr 25,000 single-chain Fv (scFv) molecules are rapidly eliminated from the circulation of immunodeficient mice, yielding highly specific retention of small quantities of scFv in human tumor xenografts. We postulated that the specific retention of scFv in tumor could be enhanced by engineering significant increases in the affinity of the scFv for its target antigens. Affinity mutants of the human anti-HER2/neu (c-erbB-2) scFv C6.5 were generated by site-directed mutagenesis, which target the same antigenic epitope with a 320-fold range in affinity (3.2 x 10(-7) to 1.0 x 10(-9) M). In vitro, the Kd of each scFv correlated closely with the duration of its retention on the surface of human ovarian carcinoma SK-OV-3 cells overexpressing HER2/neu. In biodistribution studies performed in scid mice bearing established SK-OV-3 tumors, the degree and specificity of tumor localization increased significantly with increasing affinity. At 24 h after injection, tumor retention of the highest affinity scFv was 7-fold greater than that of a mutant with 320-fold lower affinity for HER2/neu. Because the rapid renal clearance of scFv may blunt the impact of improved affinity on tumor targeting, the distributions were also assayed in the absence of renal clearance (e.g., in mice rendered surgically anephric). In this model, the peak tumor retentions of the two higher affinity scFv approximated that reported previously for IgG targeting the same SK-OV-3 tumors in scid mice with intact kidneys. In contrast, the mutant with the lowest affinity for HER2/neu failed to accumulate in tumor, indicating the presence of an affinity threshold that must be exceeded for active in vivo tumor uptake. These results indicate that affinity can significantly impact the in vivo tumor-specific retention of scFv molecules.

High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules.

Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.

Phase I evaluation of an anti-breast carcinoma monoclonal antibody 260F9-recombinant ricin A chain immunoconjugate.

Four women with metastatic breast cancer were treated with monoclonal antibody 260F9-recombinant ricin A chain, a ricin A chain immunoconjugate (IC) which targets a Mr 55,000 antigen expressed by human mammary carcinomas. Patients were treated by daily, 1-h i.v. injections for 6 to 8 consecutive days. Two patients were treated with 10 micrograms/kg daily and the two others were treated with 50 micrograms/kg daily. The trial was suspended after four patients had been treated because patients treated with a continuous infusion schedule with this IC had developed significant neurological toxicity at doses similar to those used in this study. The half-life of the IC showed a t 1/2 alpha of approximately 1.8 h, a t 1/2 beta of approximately 8.3 h, and a peak concentration of about 200 ng/ml, at the lower dose level, and showed a t 1/2 alpha of approximately 2.5 h, t 1/2 beta of about 10.4 h, and a peak concentration of 500 and 850 ng/ml for the two patients at the higher dose level. All four patients developed evidence of a human anticonjugate antibody response within 16 days of the onset of therapy. The treatment was associated with significant toxicity, manifested by a syndrome consisting of weight gain, edema, hypoalbuminemia, and dyspnea. Similar symptoms were observed in patients treated by continuous infusions of the IC. This clinical syndrome, seen at doses of IC which were insufficient to saturate antigen-expressing malignant tumor deposits in this trial, has been seen in other IC therapy trials and in clinical trials using the cytokine interleukin 2. To investigate a possible mechanism responsible for this toxicity, human monocytes were incubated with varying concentrations of IC. There was detectable binding of IC to human monocytes at IC concentrations which were achieved clinically in this trial. Furthermore, the binding appeared to be abrogated by preincubation of the monocytes with pooled human immunoglobulin, thus suggesting that binding occurs via Fc gamma receptor-mediated mechanisms. Binding was not affected if different linkers between recombinant ricin A chain and the antibody were used or if a different antibody moiety was used in the IC preparation. Chemically linked dimers of MOPC-21 bound to human monocytes at least as well as the ICs; this binding was not abrogated by preincubation with pooled human immunoglobulin. Since the IC preparations used in this clinical trial contained small percentages of dimers and/or multimers, the clinical toxicity syndromes which we observed may be related to this series of observations.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody delivery and effector cell activation in a phase II trial of recombinant gamma-interferon and the murine monoclonal antibody CO17-1A in advanced colorectal carcinoma.

Murine monoclonal antibodies of the immunoglobulin G2a isotype interact with human effector cells to mediate antibody-dependent cellular cytotoxicity (ADCC) directed against malignant cells which express antigens recognized by these antibodies. gamma-Interferon enhances these effects in vitro. In a Phase I trial of murine monoclonal antibody CO17-1A and recombinant gamma-interferon (rIFN-gamma), we demonstrated that low doses of rIFN-gamma were superior to high doses in augmenting ADCC mediated by treated patients' monocytes. These results formed the basis for a Phase II trial of CO17-1A combined with low dose rIFN-gamma. Nineteen patients with metastatic colorectal carcinoma were treated with four consecutive daily infusions of 1.0 X 10(6) IU/m2 rIFN-gamma, with 150 mg of CO17-1A administered on days 2, 3, and 4. Therapy was tolerated well. Peripheral blood mononuclear cells were purified from patient samples obtained at baseline and at 1, 4, or 24 h following the start of the first rIFN-gamma infusion and were tested for their ability to lyse 111In-labeled cells of the SW1116 colorectal line. Enhancement of monocyte ADCC was seen by 24 h, while lymphocyte ADCC and natural killer activity directed against K562 cells were enhanced to a lesser extent. Nonspecific lysis of SW1116 cells by effectors was not seen at the time points examined. While CO17-1A antigen expression was observed in most biopsies, 131I-labeled CO17-1A imaged positively in less than one-half of the organs known to harbor metastases, and therapeutic antibody delivery was not always demonstrated by immunoperoxidase staining techniques of tissue obtained following therapy. In antigen-positive lesions, tissue pO2 levels appear to identify lesions which would image positively. No objective responses were seen. Our findings suggest that prolonged therapy with low doses of rIFN-gamma potentiates ADCC but that physiological obstacles to therapeutic antibody delivery are significant. In order to evaluate the validity of this therapeutic approach, measures to enhance antibody delivery are needed, starting with systematic evaluations of therapy with escalating doses of CO17-1A combined with low dose rIFN-gamma therapy.

Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.

The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.

Binding characteristics and antitumor properties of 1A10 bispecific antibody recognizing gp40 and human transferrin receptor.

The bispecific murine monoclonal antibody (MAb) 1A10 has specificity for the human transferrin receptor (TfR) and the human tumor-associated antigen gp40. This antibody, therefore, functions as an "antigen fork" by binding to two distinct antigens on the same malignant cell. Highly purified 1A10 inhibits the growth of cells coexpressing high levels of human TfR and the tumor-associated antigen gp40 by binding to both target antigens. In SW948 cells, the majority of 1A10 binding is via its gp40 specificity, and half-maximal inhibition of cell growth by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay requires 20-30-micrograms/ml concentrations of 1A10. The binding of 1A10 correlates with growth inhibition in the cell lines HT-29, SK-OV-3, OVCAR-2, and OVCAR-3. The growth of OVCAR-10 cells, which express little gp40 and TfR, is not inhibited by 1A10. However, SK-BR-3 cells, which express abundant gp40 and extremely high levels of TfR, are insensitive to the effects of 1A10. In some cell lines, combined exposure to 1A10 and the iron chelator deferoxamine mesylate has synergistic antiproliferative effects. A single i.p. dose of 600 micrograms 1A10 is sufficient to achieve an estimated tumor concentration of at least 30 micrograms/ml for 7 days in C.B17/Icr-scid mice bearing SW948 human tumor xenografts. Treatment of scid mice bearing day 2 or day 4 SW948 xenografts with single or multiple 1A10 doses inhibits tumor growth in a dose-related fashion. Antitumor effects are not seen with therapy using either parental antibody of 1A10. The antiproliferative properties of 1A10 in tumor cells overexpressing gp40 and TfR suggest avenues for the development of new bispecific antibody-promoted treatment strategies.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv.

The in vivo properties of monovalent and divalent single-chain Fv (sFv)-based molecules with the specificity of the anti-c-erbB-2 monoclonal antibody 741F8 were examined in scid mice bearing SK-OV-3 tumor xenografts. 741F8 sFv monomers exhibited rapid, biphasic clearance from blood, while a slightly slower clearance was observed with the divalent 741F8 (sFv')2 comprising a pair of 741F8 sFv' with a C-terminal Gly4Cys joined by a disulfide bond. Following i.v. injection, the 741F8 sFv monomer was selectively retained in c-erbB-2-overexpressing SK-OV-3 tumor, with excellent tumor:normal organ ratios uniformly exceeding 10:1 by 24 h. The specificity of this effect was demonstrated by the lack of retention of the anti-digoxin 26-10 sFv monomer, as evaluated by biodistribution studies, gamma camera imaging, and cryomacroautoradiography studies. The specificity index (741F8 sFv retention/26-10 sFv retention) of 741F8 monomer binding, measured by the percentage of injected dose per g of tissue, was 13.2:1 for tumor, and 0.8 to 2.1 for all tested normal organs, with specificity indices for tumor:organ ratios ranging from 7.0 (kidneys) to 16.7 (intestines). Comparing divalent 741F8 (sFv')2 with the 26-10 (sFv')2, similar patterns emerged, with specificity indices for retention in tumor of 16.9 for the Gly4Cys-linked (sFv')2. These data demonstrate that, following their i.v. administration, both monovalent and divalent forms of 741F8 sFv are specifically retained by SK-OV-3 tumors. This antigen-specific binding, in conjunction with the 26-10 sFv controls, precludes the possibility that passive diffusion and pooling in the tumor interstitium contributes significantly to long-term tumor localization. 741F8 (sFv')2 species with peptide spacers exhibited divalent binding and increased retention in tumors as compared with 741F8 sFv monomers. Since the blood retention of the (sFv')2 is slightly more prolonged than that of the monomer, it was necessary to demonstrate that the increased tumor localization of the peptide-linked (sFv')2 was due to its divalent nature. The significantly greater localization of the divalent bismalimidohexane-linked 741F8 (sFv')2 as compared with a monovalent 741F8 Fab fragment of approximately the same size suggests that the increased avidity of the (sFv')2 is a factor in its improved tumor retention. This is the first report of successful specific in vivo targeting of tumors by divalent forms of sFv molecules. The improved retention of specific divalent (sFv')2 by tumors may have important consequences for targeted diagnostic or therapeutic strategies.

Acquisition of estrogen independence induces TOB1-related mechanisms supporting breast cancer cell proliferation.

Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, a systems biology approach was used. A library of small interfering RNAs targeting an estrogen receptor (ER)- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent ER-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mammalian target of rapmycin (mTOR) inhibitors in estrogen-independent cells but not in estrogen-dependent cells. Phosphoproteomic profiles from reverse-phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with ER-positive breast cancers.