BackgroundT 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.MethodsPatients 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 1010 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.ResultsFourteen 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 111Indium-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.ConclusionThese 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.

Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.

We report a clinical study of the pharmacokinetics, toxicity, and biological activity of intravenously and intramuscularly administered recombinant γ-interferon (rIFN-γ) consisting of 143 amino acids. Ten patients with metastatic cancer were given rIFN-γ at doses of 0.01–2.5 mg/m² by alternating intramuscular and intravenous bolus injections with a minimum intervening period of 72 h. After intravenous administration, rIFN-γ was cleared monoexponentially with a short half-life of 25–35 min as determined by bioassay and enzyme immunoassay. After intramuscular injection, a longer half-life of 227–462 min was measured by enzyme immunoassay. Serum titers were detected by bioassay only at high doses, suggesting partial loss of antiviral activity at the intramuscular site. However, other biological effects were retained as evidenced by fever, chills, and fatigue after both routes of administration and granulocytopenia after intramuscular but not intravenous doses. Two of 10 patients showed objective evidence of tumor regression. These data suggest that further studies with intramuscular as well as prolonged intravenous infusions of rIFN-γ are indicated.

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In the context of the central role of the alveolar macrophage in host defense of the respiratory epithelial surface, and the ability ofIFN-'y to activate mononuclear phagocytes, we have evaluated strategies to use rIFN-'y to activate human alveolar macrophages in vivo. To accomplish this, rIFN-,y was administered to nonsmoking normals, the amounts of IFN--y quantified in serum and respiratory epithelial lining fluid (ELF) and the status of IFN-'y related activation of blood monocytes and alveolar macrophages was evaluated by quantifying the expression of mRNA transcripts of IP-10, a gene induced specifically by IFN-'y. Systemic administration (subcutaneous) of maximally tolerated amounts of rIFN-'y (250 gg) was followed by detectable levels of IFN-'y in serum but not ELF, the expression of IP-10 transcripts in blood monocytes but not alveolar macrophages, and multiple systemic adverse effects. To circumvent the inability of systemic administration to reach respiratory ELF and activate alveolar macrophages, rIFN-y (250-1,000 jig) was inhaled as an aerosol once daily for 3 d. Strikingly, while IFN-y was not detected in serum it was detectable in respiratory ELF in a dose-dependent fashion. Further, alveolar macrophages, but not blood monocytes, expressed IP-10 mRNA transcripts and, importantly, inhalation of aerosolized rIFN-'y was not associated with local or systemic adverse effects. Thus, it is feasible to use rIFN-,y to activate alveolar macrophages by targeting the cytokine directly to the lung. These data suggest a potential strategy for targeted cytokine therapy, without systemic side effects, to augment respiratory tract defenses in individuals at risk for or with lung infection. (J. Clin. Invest. 1991. 88:297-302.)

A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-7), 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/m 2 of IFN (1 mg~> 10 x 106 units of IFN) escalating to 10 mg/m 2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chils, fatigue, anorexia, and granulocytopenia. The influenzalike symptoms were very similar to those encountered with IFN-a but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48-72 h following administration of rIFN-7. Absolute granulocyte counts only rarely dropped below 1000 mm 3. 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 rain. 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.

Interferon gamma (IFN-gamma) is a lymphokine with potent in vitro effects on cell growth and immune function. We have investigated the effects of rIFN-gamma (sp act approximately 2 X 10(7) U/mg, purity greater than 99%) in 16 evaluable patients with advanced malignancy in a phase 1 trial. Patients were treated with six-hour intravenous (IV) infusions daily, five days a week for 2 weeks. After a 2-week rest period, the IV treatment cycle was repeated. Responders were maintained on repeated IV treatment cycles or daily intramuscular (IM) injections. Patients were entered at fixed dose levels of 0.1, 0.5, or 1.0 mg/m2/d. The maximum safely tolerated dose was 0.5 mg/m2. The most common side effects were constitutional symptoms, including fever, chills, fatigue, and myalgias. Reversible and transient increases in hepatic transaminase and decrease in granulocyte counts were seen. Treatment was associated with a dose-dependent increase in serum levels of beta 2 microglobulin. Partial responses (PRs) were observed in one patient with Hodgkin's disease and one patient with chronic lymphocytic leukemia. Fairly constant levels of serum IFN were found at four and six hours during infusion, followed by a rapid decline within one to two hours. We conclude that rIFN-gamma can be safely administered by a six-hour IV infusion and that it can induce in vivo some of the biologic effects reported in in vitro studies.

Recombinant interferon y (rIFN-y) 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 H.O2 secretory capacity of monocytes from cancer patients receiving intravenous rIFN-'yat 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 H202. Monocytes from 11 of the 13 subjects (85%) studied through 20 treatment cycles responded to rIFN-y with elevation in H202 secretion in .67% of the tests conducted >1 hr after the start of treatment. Five of the five subjects tested had monocytes with diminished H202 secretory capacity when tested immediately after a 1-hr infusion of rIRN-y, 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 H202 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 H202 secretory capacity, the ability of rIFN-y to activate mononuclear phagocytes is manifest upon its administration to patients with advanced malignancy. These findings raise the possibility that rIFN-y may activate mononuclear phagocytes in man. An opportunity to begin testing this hypothesis arose with the initiation of phase I trials of rIFN-y in cancer patients (10) and the development of a quantitative assay for monocyte H202 secretion suitable for serial measuremens on a few milliliters of blood (11). The results provide evidence for the activation of human monocytes after the systemic administration of lymphokine. MATERIALS AND METHODSStudy Population and Design. Adults with advanced malignancies refractory to conventional therapy received doses of 0.1, 0.5, or 1.0 mg/mi2 of body area rIFN-y by continuous intravenous infusion in 5% (wt/vol) dextrose in water over a 6-hr period each day for 5 days each wk in 2-wk cycles. After a 2-wk rest, the treatment cycle was repeated. Monocyte function was studied immediately before the first infusion of each cycle, and at one or more of the following times: at the end of the first 6-hr infusion, or immediately before the infusions given on the 2nd, 3rd, 4th, or 5th day. In all, 98 assays (each with 3-10 replicates) were carried out on cells from 7 subjects. The same doses ofrIFN-y were administered in a second phase I trial over 1 hr on alternate days three times per wk. In this case, monocyte function was studied immediately prior to the first infusion, and at one or more of the following times: immediately after the first infusion, and on the ...