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Background: T cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s., Methods: Patients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 10 10 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72., Results: Fourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111 Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed., Conclusion: These findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity.

A new, genetically transmitted retrovirus has been isolated from the Old World monkey Colobus polykomos. This virus, designated CPC-1, is readily transmitted to both feline and human cells in culture. Nucleic acid hybridization studies reveal that there are 50-70 copies of the CPC-1 genome in colobus cellular DNA. Related virogene sequences can be detected in the DNA of all other Old World monkeys, as well as in the DNA of at least one ape species, the chimpanzee, indicating that this virus has been genetically transmitted in primates for 30-40 million years. CPC-1 is partially related to the type C virus previously isolated from stumptail monkeys (MAC-1). These two viruses have nucleic acid sequence homology, antigenic crossreactivity in their major viral structural protein, and a very similar host range in vitro. CPC-1 and MAC-1 therefore belong to the same class of genetically transmitted primate type C viruses and, as such, represent the first example in primates of analogous endogenous retroviruses isolated from two distantly related species.

Urine from nude mice contains epidermal growth factor (EGF) and a minor acid-stable component with an apparent molecular weight of 20,000, which competes with EGF for binding to EGF membrane receptors and which promotes colony formation by normal rat kidney cells in soft agar. The levels of this Mr 20,000 urine-derived growth factor are increased approximately 4- to 10-fold in nude mice bearing tumors following s.c. injection of cultured human tumor cells. Following removal of the primary tumor, the concentration of this factor is reduced to basal levels, and thus, elevated levels of this growth factor appear to be dependent on tumor burden. The Mr 20,000 urinary component is separable into four EGF competing activities by high-performance liquid chromatography; the major species is immunologically related to mouse submaxillary gland EGF and therefore appears to be of host origin. However, in addition to elevated levels of host growth factor, urine from tumor-bearing mice also contains transforming growth factor activity in amounts comparable to that released by the tumor cells in culture. The tumor-derived urinary transforming growth factor activity is immunologically unrelated to EGF but is immunoreactive with an antiserum to transforming growth factor-alpha. We propose that the nude mouse may be a useful model to examine the role of both host- and tumor-derived growth factors in tumorigenesis and the usefulness of these factors as biological markers of response to therapy and tumor progression.

Tumor necrosis factor is a cytokine derived from activated macrophages. This agent is cytostatic and cytolytic against transformed human cell lines in vitro and has in vivo activity against a variety of murine tumors. We report a clinical study of the pharmacokinetics, toxicity, and biological activity of i.v. and i.m. administered recombinant human tumor necrosis factor (rTNF). Twenty patients with metastatic cancer were given rTNF in doses ranging from 1 to 200 micrograms/m2 by alternating i.m. and i.v. bolus injections with a minimal intervening period of 72 h. Each patient received a maximum of eight treatments given twice weekly over a 4-week period. With i.v. bolus administration, serum concentrations of rTNF were detected by enzyme-linked immunosorbent assay at doses of 25 micrograms/m2 or greater. The clearance of rTNF in the serum was described by a monoexponential equation with a half-life calculated to be 14-18 min. After i.m. administration, serum concentrations of rTNF were consistently detected by enzyme-linked immunosorbent assay at doses of 150 micrograms/m2 or greater. Peak concentrations were observed within 2 h and rTNF was occasionally detected, at the lower limit of sensitivity of the assay, at 24 h postinjection. rTNF was well tolerated clinically in this dose range, and there was evidence of antitumor effect.

Seventeen well-characterized human lung cancer cell lines were examined for the presence of specific membrane receptors for epidermal growth factor (EGF) and nerve growth factor (NGF) as well as for the production of diffusible factors capable of stimulating soft agar growth. These cell lines represented all four major histological types of human lung cancer including small cell carcinoma of the lung (SCCL) and the three types of non-SCCL (epidermoid, large cell, and adenocarcinoma). The SCCL lines included three lines referred to as "converters" because they had lost SCCL morphological and biochemical properties during prolonged passage in vitro. Specific receptors for EGF and NGF were detected by measuring the binding of 125I-radiolabeled growth factor to the cell surface. These assays revealed that EGF receptors are found on five of six non-SCCL cell lines and are not found on any of the SCCL lines. In contract, NGF binding was detected at low levels on three of eight SCCL lines and on all three SCCL converters but was not observed for non-SCCL lines. Thus, SCCL and SCCL converter cell lines are distinguished from non-SCCL by the pattern of membrane receptors for EGF and NGF. Such differences may ultimately prove useful as biological markers for the different histological types of lung cancer. Moreover, the majority of SCCL cells and all of the non-SCCL cells tested were found to produce diffusible growth factors which can stimulate soft agar growth of nontransformed normal rat kidney fibroblasts. Although some correlation between soft agar growth factor production and the absence of EGF receptors may exist for SCCL cells, the production of transforming growth factors appears to be a general property of human lung cancer cells in vitro and is independent of EGF receptor expression.

We report a clinical study of the pharmacokinetics, toxicity, and biological activity of i.v.- and i.m.-administered recombinant gamma-interferon (rIFN-gamma) consisting of 143 amino acids. Ten patients with metastatic cancer were given rIFN-gamma at doses of 0.01 to 2.5 mg/sq m by alternating i.m. and i.v. bolus injections with a minimum intervening period of 72 h. After i.v. administration, rIFN-gamma was cleared monoexponentially with a short half-life of 25 to 35 min as determined by bioassay and enzyme immunoassay. After i.m. injection, a longer half-life of 227 to 462 min was measured by enzyme immunoassay. Serum titers were detected by bioassay only at high doses, suggesting partial loss of antiviral activity at the i.m. site. However, other biological effects were retained as evidenced by fever, chills, and fatigue after both routes of administration and granulocytopenia after i.m., but not i.v., doses. Two of ten patients showed objective evidence of tumor regression. These data suggest that further studies with i.m. as well as prolonged i.v. infusions of rIFN-gamma are indicated.

Recombinant interferon gamma (rIFN-gamma) activates macrophage antimicrobial and antitumor functions and related metabolic processes, including secretion of reactive oxygen intermediates in mice and in cultured mouse and human macrophages. To look for similar actions in man, we monitored the H2O2 secretory capacity of monocytes from cancer patients receiving intravenous rIFN-gamma at 0.1, 0.5, or 1.0 mg/m2 of body area over 6 hr daily or over 1 hr on alternate days. Monocytes taken just before the first infusion served as controls and were comparable to normal donor monocytes in secretion of H2O2. Monocytes from 11 of the 13 subjects (85%) studied through 20 treatment cycles responded to rIFN-gamma with elevation in H2O2 secretion in greater than or equal to 67% of the tests conducted greater than 1 hr after the start of treatment. Five of the five subjects tested had monocytes with diminished H2O2 secretory capacity when tested immediately after a 1-hr infusion of rIRN-gamma, at which time the amount of adherent mononuclear cell protein recovered from the blood averaged only 24% of the control. At all other times tested (from 6 hr to 5 days after infusion) combined results for all subjects showed enhancement of H2O2 releasing capacity. Statistically significant mean increases ranged from 1.4- to 2.8-fold above the control and included the sets in which monocytes collected 24 hr following a single infusion were assayed the same day or the next. By the criterion of enhanced H2O2 secretory capacity, the ability of rIFN-gamma to activate mononuclear phagocytes is manifest upon its administration to patients with advanced malignancy.

Animals suffering from malignancy or chronic infection develop characteristic metabolic abnormalities, including a well-defined hypertriglyceridemic state. These abnormalities have been attributed to release of one or more mediators from activated macrophages. We report that cancer patients receiving RIFN-gamma, a potent macrophage activator, at doses of greater than or equal to 0.25 mg/m2/d i.m. show marked increases in triglyceride but not in cholesterol levels (pretreatment triglyceride level of 180 +/- 190 mg/dl [mean +/- SD] vs. a day-14 level of 370 +/- 242 mg/dl, n = 23, p less than 0.001 by the paired t test). This hypertriglyceridemia was characterized by an increase in very low-density lipoproteins and a decrease in plasma post-heparin lipase activity, consistent with defective triglyceride clearance (mean pretreatment lipase level of 2.1 mumol/ml/h vs. a day-14 level of 1.2 mumol/ml/h, n = 6, p = 0.02 by the paired t test). rIFN-gamma did not directly inhibit lipoprotein lipase enzymatic activity in vitro. Other possible mechanisms of action, such as suppression of lipase by an rIFN-gamma-induced mediator released from activated macrophages, or a direct effect of interferon on lipase biosynthesis, require further investigation. Our observations provide evidence that factors produced by the immune system can regulate lipid metabolism in man.

Reverse-phase high-performance liquid chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)-dependent beta-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent alpha-TGF-like activity. All urine samples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF. Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD) acetonitrile Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique Mr 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase competitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguishing HPLC profiles of urinary TGF from cancer patients versus that from normal individuals.

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B-derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.

A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-gamma), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m2 of IFN (1 mg greater than or equal to 10 X 10(6) units of IFN) escalating to 10 mg/m2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chills, fatigue, anorexia, and granulocytopenia. The influenza-like symptoms were very similar to those encountered with IFN-alpha but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48-72 h following administration of rIFN-gamma. Absolute granulocyte counts only rarely dropped below 1000 mm3. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4-8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.

Human melanoma cells were examined in an indirect membrane immunofluorescence assay for surface nerve growth factor (NGF) and NGF receptors. This assay revealed that human melanoma cells have various levels of NGF and NGF receptors on the plasma membrane, whereas a variety of human sarcoma and carcinoma tumor cells and normal human fibroblasts are negative. Surface NGF could be detected on melanoma cells with a rabbit antiserum directed to NGF at titers as high as 1:64; prior adsorption of this antibody with mouse 2.5S NGF resulted in a loss of fluorescence. The melanoma cells were positive whether or not they were grown in the presence of fetal calf serum. NGF production by human melanomas is a previously unrecognized property of this differentiated cell type. Although other cells in culture have been shown to produce NGF, the association of NGF production with the presence of NGF receptors on the cell surface is rare among tumor cells, and may represent an opportunity for "autostimulation" of melanoma cells by this growth factor.

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.

Highly purified human lymphoblastoid interferon (HLBI) derived from virus-stimulated Namalwa cells was administered by 6-h IV infusion or IM injection to 40 patients with a variety of disseminated malignancies refractory to standard therapy. Each patient received doses escalating from 0.1 to 50 X 10(6) U for up to 5 weeks. Extensive monitoring for clinical effect, toxicity, and pharmacokinetics has revealed higher peak serum interferon levels and somewhat more pronounced systemic toxicity for the IV than for the IM route of administration. Objective evidence of tumor regression was observed in two patients receiving HLBI IV.

Mus musculus SC-1 cells were infected with M7 baboon type C virus. The progeny of this infection included viral pseudotypes that contained M7 helper virus and endogenous 30S retrovirus-associated sequences derived from SC-1 cells (RAS). The RAS sequences are unrelated by nucleic acid hybridization criteria to previously described types of murine retroviruses and do not code for known murine viral structural proteins. The RAS genome is present in multiple copies in the DNA of laboratory (M. musculus) and Asian (M. caroli and M. cervicolor) mice, is expressed in the RNA of uninfected mouse cells, and can be efficiently rescued by type C, but not type B, viruses. RAS is closely related to 30S virus-associated RNA in NIH/3T3 and BALB/c JLSV-9 cells and may be analogous to the defective 30S RNA sequences found in rats.