Long term response in a patient with neoplastic meningitis secondary to melanoma treated with (131)I-radiolabeled antichondroitin proteoglycan sulfate Mel-14 F(ab')(2): a case study.

Even with novel chemotherapeutic agents and external beam radiation therapy, the prognosis of neoplastic meningitis secondary to malignant melanoma is still dismal. The authors report a case study of a 46-year-old white female who presented with progressive hearing loss, severe headaches, nausea, vomiting, and a rapid decline in neurologic status. She was referred to Duke University Medical Center after conventional chemotherapy for malignant melanoma failed. She was enrolled in a Phase I trial of (131)I-labeled monoclonal antibody Mel-14 F(ab')(2) fragment administered intrathecally. Within a year after her treatment, she recovered, having a normal neurologic exam except for residual bilateral hearing loss. The authors discuss dosimetry, preclinical, and clinical studies conducted with Mel-14 F(ab')(2) and introduce a potentially promising therapy option in the treatment of neoplastic meningitis in patients with malignant melanoma. Currently, the patient remains neurologically normal except for a mild bilateral hearing loss more than 4 years after treatment and has no radiographic evidence of neoplastic meningitis.

Dextran, gelatin, and hydroxyethyl starch do not affect permeability for albumin in cat skeletal muscle.

OBJECTIVE: To evaluate the effects of the three commercially available colloid solutions, 6% dextran 70, 6% hydroxyethyl starch (HES) 200/0.5, and 3.5% urea-linked gelatin on permeability for human albumin in a skeletal muscle in vivo model by evaluating their effects on the reflection coefficient for albumin. DESIGN: Controlled laboratory study. SETTING: University research laboratory. SUBJECTS: Eighteen adult cats. INTERVENTIONS: The autoperfused and denervated calf muscles of the cat hindlimb were placed in a plethysmograph. The transvascular fluid absorption induced by an increase in the colloid osmotic pressure following a fixed intravenous bolus of human albumin was analyzed, first before start of, and then during an intra-arterial infusion to, the muscle preparation of the synthetic colloid to be analyzed. Capillary filtration coefficient as a measure of microvascular fluid permeability (conductance) was analyzed before and after start of the synthetic colloid. MEASUREMENTS AND MAIN RESULTS: Arterial blood flow, arterial and venous blood pressures, total vascular resistance, tissue volume changes, capillary filtration coefficient, and plasma volume were measured before and during the colloid infusion. According to the Starling fluid equilibrium, the ratio between the reflection coefficients for albumin on two occasions (before and after infusion of the synthetic colloid) can be calculated from the maximum osmotic absorption rates induced by a fixed intravenous bolus infusion of albumin and from the capillary filtration coefficients. Obtained data were adjusted for different plasma volume at the two occasions. We found that none of the three synthetic colloids analyzed had any significant effect on the reflection coefficient for albumin. CONCLUSION: An effect on albumin microvascular permeability of the synthetic colloids dextran 70, HES 200/0.5, and urea-linked gelatin could not be shown by a method analyzing their effect on the reflection coefficient for albumin.

Increased binding affinity enhances targeting of glioma xenografts by EGFRvIII-specific scFv.

Combinatorial variation of CDR3 of V(H) and V(L), followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma-bearing athymic mice. MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K(d) = 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impact in vivo glioma-specific targeting and immunotherapy.

Phase I trial results of iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant gliomas.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Identifying potential tumor markers and antigens by database mining and rapid expression screening.

Genes expressed specifically in malignant tissue may have potential as therapeutic targets but have been difficult to locate for most cancers. The information hidden within certain public databases can reveal RNA transcripts specifically expressed in transformed tissue. To be useful, database information must be verified and a more complete pattern of tissue expression must be demonstrated. We tested database mining plus rapid screening by fluorescent-PCR expression comparison (F-PEC) as an approach to locate candidate brain tumor antigens. Cancer Genome Anatomy Project (CGAP) data was mined for genes highly expressed in glioblastoma multiforme. From 13 mined genes, seven showed potential as possible tumor markers or antigens as determined by further expression profiling. Now that large-scale expression information is readily available for many of the commonly occurring cancers, other candidate tumor markers or antigens could be located and evaluated with this approach.

Glioma-associated antigen expression in oligodendroglial neoplasms. Tenascin and epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR), its variant, EGFRvIII, and tenascin are glioma-associated antigens that are hyperexpressed by neoplastic glial cells relative to normal brain, making them attractive antigenic targets for immunotherapy. Preliminary surveys indicate that oligodendroglial tumors also produce these proteins, although the exact patterns and degrees of reactivity are not known. In this study we examined the immunoreactivity of tenascin among 50 oligodendroglial tumors, including 25 well-differentiated oligodendrogliomas (WDOs) and 12 glioblastomas (GBMs) exhibiting high proportions of oligodendroglia-like cells. We used well-characterized immunoreagents with defined specificities against the target antigens on formalin-fixed, paraffin-embedded archival tissue. The tumors were graded according to WHO guidelines. Immunoreactivity was reported on a 1-3 scale according to staining intensity multiplied by a 1-3 distribution scale distribution within tumor as focal (1), multifocal (2), and diffuse (3) for both the parenchymal and the perivascular components. Although there is considerable overlap in antigen production among the grades of tumor, this study establishes the production of tenascin and wild-type EGFR (but not EGFR vIII) in oligodendroglial neoplasms and supports the concept that antigen production increases with tumor grade.

Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors.

The epidermal growth factor receptor (EGFR) is often amplified and rearranged structurally in tumors of the brain, breast, lung, and ovary. The most common mutation, EGFRvIII, is characterized by an in-frame deletion of 801 base pairs, resulting in the generation of a novel tumor-specific epitope at the fusion junction. A murine homologue of the human EGFRvIII mutation was created, and an IgG2a murine mAb, Y10, was generated that recognizes the human and murine equivalents of this tumor-specific antigen. In vitro, Y10 was found to inhibit DNA synthesis and cellular proliferation and to induce autonomous, complement-mediated, and antibody-dependent cell-mediated cytotoxicity. Systemic treatment with i.p. Y10 of s.c. B16 melanomas transfected to express stably the murine EGFRvIII led to long-term survival in all mice treated (n = 20; P < 0.001). Similar therapy with i.p. Y10 failed to increase median survival of mice with EGFRvIII-expressing B16 melanomas in the brain; however, treatment with a single intratumoral injection of Y10 increased median survival by an average 286%, with 26% long-term survivors (n = 117; P < 0.001). The mechanism of action of Y10 in vivo was shown to be independent of complement, granulocytes, natural killer cells, and T lymphocytes through in vivo complement and cell subset depletions. Treatment with Y10 in Fc receptor knockout mice demonstrated the mechanism of Y10 to be Fc receptor-dependent. These data indicate that an unarmed, tumor-specific mAb may be an effective immunotherapy against human tumors and potentially other pathologic processes in the "immunologically privileged" central nervous system.

EGFRvIII as a promising target for antibody-based brain tumor therapy.

Cell surface receptors are attractive candidates for targeted therapy of cancer. Growth factors and their receptors play important roles in the regulation of cell division, development, and differentiation. Among those, the epidermal growth factor receptor (EGFR) was the first identified to be amplified and/or rearranged in malignant gliomas. The most common rearranged form, EGFR type III variant (EGFRvIII), has a deletion in its extracellular domain that results in the formation of a new, tumor-specific target found in glioblastoma multiforme, as well as in breast, ovarian, prostate, and lung carcinomas. Monoclonal antibodies have been developed with specific activity against this mutant receptor. These antibodies are internalized into the cell after receptor binding. Specific antibodies, either unarmed or armed with cytotoxic agents, including radioisotopes and toxins, have shown a promising role for EGFRvIII as a target for brain tumor therapy.

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro.

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.

Monoclonal antibody therapy of human gliomas: current status and future approaches.

The development of immunotherapeutic protocols for the treatment of human CNS neoplasia over the past two decades has been impressive. Several crucial aspects have been defined, characterized, and in many cases, optimized (Wikstrand CJ, Zalutsky MR, Bigner DD: In: Liau LM, Bigner DD (eds) Brain Tumor Immunotherapy. Humana Press (in press), 2000). Specific Mabs or constructs reacting with targetable antigens are currently available and in clinical trial. In addition, additional antigens currently under study (angiogenesis-related markers, developmentally associated antigens for medulloblastoma such as L1, and the identification of new targets by SAGE, just in its infancy, will provide a veritable library of available targets for therapy. The molecular engineering and affinity maturation techniques being applied to Mab fragment optimization have already been rapidly effective in generating a variety of Mab constructs of appropriate affinity for clinical trial; as new targets are defined, and experience is accrued with the various constructs currently and prospectively available, the optimal targeting of a multitude of antigens will be possible.

Iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with recurrent malignant gliomas: phase I trial results.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine 131 (131I)-labeled 81C6 monoclonal antibody (mAb) in brain tumor patients with surgically created resection cavities (SCRCs) and to identify any objective responses to this treatment. METHODS: In this phase I trial, eligible patients were treated with a single injection of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating dosages of 131I (starting dose of 20 mCi with a 20-mCi escalation in subsequent cohorts) administered through an Ommaya reservoir in the SCRC. Patients were followed up for toxicity and response until death or for a minimum of 1 year after treatment. The SCRC patients, who were previously irradiated, were followed up without additional treatment unless progressive disease was identified. RESULTS: We administered 36 treatments of 131I doses up to 120 mCi to 34 previously irradiated patients with recurrent or metastatic brain tumors. Dose-limiting toxicity was reached at 120 mCi and was limited to neurologic or hematologic toxicity. None of the patients treated with less than 120 mCi developed significant neurologic toxicity; one patient developed major hematologic toxicity (MHT). The estimated median survival for patients with glioblastoma multiforme (GBM) and for all patients was 56 and 60 weeks, respectively. CONCLUSION: The MTD for administration of 131I-labeled 81C6 into the SCRCs of previously irradiated patients with recurrent primary or metastatic brain tumors was 100 mCi. The dose-limiting toxicity was neurologic toxicity. We are encouraged by the minimal toxicity and survival in this phase I trial. Radiolabeled mAbs may improve the current therapy for brain tumor patients.

The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target.

Any immunotherapeutic approach to cancer cell eradication is based upon the specific recognition of neoplastic cells and the sparing of surrounding normal tissue; perhaps nowhere is this distinction more important than within the central nervous system, due to the diffuse infiltrative nature of primary glial tumor cell growth. Whether ultimate effect moieties are immunoglobulins, fragments and/or their constructs with drugs, toxins, radionuclides, or immune cells, the specificity of effector: cell surface marker is crucial. This review describes the identification, immunologic characterization, and biologic behavior of a transmembrane tumor-specific altered growth factor receptor molecule which may well serve as a mediator of multiple immunotherapeutic approaches: the class III variant of the epidermal growth factor receptor, EGFRvIII.

Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII.

The potential of therapeutic targeting of tumor cell surface epidermal growth factor receptors (EGFRs) by modified ligands or specific antibodies has been limited by the normal tissue distribution of the receptor. The identification and characterization of a variant of this receptor, EGFRvIII, which is not expressed in normal tissues but has been described in gliomas, non-small cell lung carcinomas, and breast carcinomas, has provided a highly specific, internalizing target for antibody-mediated approaches. To determine the feasibility of immunotargeting EGFRvIII, we have assessed the qualitative distribution and quantitative expression at both the population and cellular levels of EGFRvIII in 21 biopsy samples of human gliomas by indirect analytical and quantitative flow cytometry and by immunohistochemical assay of frozen and formalin-fixed tissue. Consistent with previous reports, 50% of gliomas tested (1 of 2 anaplastic astrocytomas, 7 of 12 glioblastoma multiforme, and 2 of 6 oligodendrogliomas) expressed EGFRvIII, as determined by a minimum of 2 separate assays. Minimum estimates of the proportion of positive tumor cells in these populations ranged from 37-86%; in four of five cases in which quantitation of the EGFRvIII density/cell was performed, values of 2.7-6.8 x 10(5) were obtained with monoclonal antibody (mAb) L8A4 (EGFRvIII specific), levels consistent with successful in vivo immunotargeting. Confocal microscopic analysis confirmed that the subcellular localization of EGFRvIII was identical to that described for EGFR: predominant cell membrane expression, with some perinuclear distribution suggestive of localization to the Golgi region. Neither EGFR nor EGFRvIII was found within the nucleus. This study establishes for the first time that approximately 50% of human glioma biopsies contain cell populations expressing a sufficient number of membrane-expressed EGFRvIIIs to mediate specific anti-EGFRvIII mAb localization. Coupled with previous demonstrations of the rapid internalization of specific mAb-EGFRvIII complexes and the susceptibility of the targeted cells to isotope or toxin-mediated cytotoxicity, this study establishes the validity of targeting EGFRvIII for therapy of mutant receptor-positive gliomas, breast carcinomas, and non-small cell lung carcinomas.

Receptor dimerization is not a factor in the signalling activity of a transforming variant epidermal growth factor receptor (EGFRvIII).

The type-III deletion variant of the epidermal growth factor receptor (EGFRvIII) is frequently found in glioblastomas and other malignant human tumours. Although EGFRvIII confers ligand-independent oncogenic transformation of cell lines, the mechanism by which it promotes aberrant cellular proliferation is unknown. Using cell lines expressing comparable numbers of either wild-type receptor (EGFRwt) or EGFRvIII, we compared several parameters of receptor activation: dimerization, tyrosine phosphorylation and activation of intracellular signalling proteins. Like activated EGFRwt, EGFRvIII was phosphorylated and bound constitutively to the Shc adapter protein. Indeed, EGFRvIII-associated Shc had a higher phosphotyrosine content than Shc associated with stimulated EGFRwt. EGFRwt dimerized in response to either EGF or transforming growth factor alpha. Higher cross-linker concentrations and incubation at higher temperatures (37 degrees C) allowed detection of EGFRwt dimers even in the absence of exogenous ligand. In contrast, EGFRvIII failed to dimerize under any conditions studied. Moreover, neither mitogen-activated protein kinase nor phospholipase Cgamma were phosphorylated in EGFRvIII-expressing cells. We conclude that the deletion of 267 amino acids from the 621-amino-acid N-terminal domain of EGFR does not result simply in a constitutively activated receptor, but alters the spectrum of signalling cascades utilized. Furthermore the ligand-independent transforming activity of EGFRvIII is independent of receptor dimerization.

Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate.

Monoclonal antibody (mAb) L8A4, specific for the tumor-associated mutant epidermal growth factor receptor variant III (EGFRvII), is internalized and degraded after cell binding. Four paired-label experiments were performed in athymic mice bearing EGFRvIII-positive xenografts to determine the suitability of N-succinimidyl 3-iodo-5-pyridinecarboxylate (SIPC) for labeling this internalizing mAb. In mice with HC2 20 d2 xenografts, tumor uptake reached a maximum of 32.7 +/- 2.0% injected dose/g when labeled using SIPC, a value significantly higher (P < 0.05, paired t test) than that observed when L8A4 was labeled using lodogen (24.4 +/- 2.2% injected dose/g). The specificity of mAb uptake in HC2 20 d2 and U87MG(delta)EGFR xenografts was measured in separate experiments by coadministration of L8A4 and nonspecific, isotype-matched P3X63Ag8 mAb, both radioiodinated using SIPC. Tumor localization indices were approximately 10 or more by 72 h, a degree of specificity 3-4 times higher than that reported previously when labeling was performed using the tyramine cellobiose (TCB) method. In a final study directly comparing L8A4 labeled using SIPC and TCB, similar tumor levels were obtained (SIPC, 33.7 +/- 6.1% injected dose/g at 24 h; TCB, 37.8 +/- 6.7% injected dose/g at 24 h); however, tumor-to-tissue ratios for the liver, spleen, and kidneys were 3 times higher with SIPC at later time points. These results suggest that SIPC is a promising method for labeling this anti-EGFRvIII mAb and possibly other mAbs that internalize after binding.

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line.

The human glioma D-54MG cell line grown in vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3'-isoLM1, an alteration that was dose dependent, with the highest dose (1 x 10(8)) resulting in a phenotype that was most like that of the inoculum. After one passage in vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grown in vitro than in those grown in vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors.

Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: phase I trial results.

We aimed to determine the maximum tolerated dose (MTD) of 131I-labeled 81C6 in patients with leptomeningeal neoplasms or brain tumor resection cavities with subarachnoid communication and to identify any objective responses. 81C6 is a murine IgG monoclonal antibody that reacts with tenascin in gliomas/carcinomas but does not react with normal adult brain. 131I-labeled 81C6 delivers intrathecal (IT) radiation to these neoplasms. This study was a Phase I trial in which patients were treated with a single IT dose of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating doses of 131I (starting dose, 40 mCi; 20 mCi escalations) on 10 mg 81C6. MTD is defined as the highest dose resulting in serious toxicity in no more than two of six patients. Serious toxicity is defined as grade III/IV nonhematological toxicity or major hematological toxicity. We treated 31 patients (8 pediatric and 23 adult). Eighteen had glioblastoma multiforme. Patients were treated with 131I doses from 40 to 100 mCi. Hematological toxicity was dose limiting and correlated with the administered 131I dose. No grade III/IV nonhematological toxicities were encountered. A partial response occurred in 1 patient and disease stabilization occurred in 13 (42%) of 31 patients. Twelve patients are alive (median follow-up, > 320 days); five are progression free >409 days median posttreatment. The MTD of a single IT administration of 131I-labeled 81C6 in adults is 80 mCi 131I-labeled 81C6. The MTD in pediatric patients was not reached at 131I doses up to 40 mCi normalized for body surface area.

Tumor antigens in astrocytic gliomas.

Gliomas affect 15,000 to 17,000 Americans every year and carry a dismal prognosis. The potential of immunologically mediated diagnosis and therapy, although greatly enhanced since the advent of monoclonal antibodies, has not been fully realized due to significant problems, most especially the challenge of identifying antigenic molecules specific to glial tumors. Other problematic issues include antigen-associated factors such as heterogeneity, modulation, shedding, and cross-reactivity with normal cells, and factors associated with therapeutic agent delivery, typically variable tumor perfusion and unfavorable diffusional forces in tumor microenvironment. An understanding of these problems called for the delineation of operationally specific antigens (tumor-associated antigens not expressed by the normal central nervous system) combined with the use of compartmental therapeutic approaches to increase the specificity of therapy. Numerous antigens have been identified and are classified as extracellular/matrix-associated, membrane-associated, and intracellular antigens. Nevertheless, only a few have been demonstrated to be of significant therapeutic and diagnostic utility. These few include the extracellular matrix-associated antigens tenascin and GP 240, defined by the monoclonal antibodies 81C6 and Mel-14, both of which are now in Phase I clinical trials, and membrane-associated ganglioside molecules, primarily 3', 6'-isoLD1, defined by the antibody DMAb-22. Recent identification of the overexpression of a deletion variant of the epidermal growth factor receptor (EGFRvIII) in up to 50% of the more malignant glial tumors and the subsequent creation of monoclonal antibodies that are specific to this molecule and do not recognize the wild-type EGFR provide the most exciting development yet in the design of specific antiglioma immunoconjugates. In addition, the tumor-specific nature of EGFRvIII combined with improved knowledge of immune mechanisms, especially in the context of the central nervous system, will facilitate the design of highly selective cell-mediated therapeutic approaches with a view toward obtaining tumor-specific immunity.