A novel immunization strategy using cytokine/chemokines induces new effective systemic immune responses, and frequent complete regressions of human metastatic melanoma

Abstract

Immune responses have been elicited by a variety of cancer vaccines, but seldom induce regressions of established cancers in humans. As a novel therapeutic immunization strategy, we tested the hypothesis that multiple cytokines/chemokines secreted early in secondary responses ex-vivo might mimic the secretory environment guiding new immune responses. The early development of immune responses is regulated by multiple cytokines/chemokines acting together, which at physiologic concentrations act locally in concert with antigen to have non-specific effects on adjacent cells, including the maturation of dendritic cells, homing and retention of T cells at the site of antigen, and the differentiation and expansion of T cell clones with appropriate receptors. We postulated that repeated injections into a metastasis of an exogenous chemokine/cytokine mixture might establish the environment of an immune response and allow circulating T cell clones to self- select for mutant neo-epitopes in the tumor and generate systemic immune responses. To test this idea we injected some metastases in patients with multiple cutaneous melanoma nodules while never injecting other control metastases in the same patient. New immune responses were identified by the development of dense lymphocytic infiltrates in never-injected metastases, and the frequent complete regression of never-injected metastases, a surprising observation. 70% of subjects developed dense infiltrates of cytotoxic CD8 cells in the center and margin of never-injected metastases; 38% of subjects had complete and often durable regressions of all metastases, without the use of check-point inhibitors, suggesting that, as a proof-of-principle, an immunization strategy can control advanced human metastatic melanoma.

Keywords: CD8 T cells; cancer vaccine; cytokines; immunotherapy; metastatic melanoma; tumor infiltrating lymphocytes.

Figure 1.

Rationale and design of procedures.

Figure 2.

Photographs of patients with cutaneous/subcutaneous metastases experiencing regressions after intralesional CKs. A. 70 year old female: large subcutaneous metastases undergoing rapid rejections. Only checked nodules injected. Dotted nodules are regressing. Collapsing lesion 12 was never injected. Some nodules marked for excision. B. 50 year old female: in transit metastases of left leg and inguinal scar, 4 positive lymph nodes, and no response to chemotherapy. Only circled area injected. Complete regressions and survived disease free for >5 years. Lost to follow up. C. 62 year old male: 6 lesions increased to 57+ small metastases on head and neck. Only lesions on right side injected. All lesions regressed: a 2 × 3 cm mass developed in neck and spontaneously regressed. Needle biopsy showed only residual pigment in melanophages. Became NED after 2 years; died of other causes at age 86, 24 years after entering the study. D. 55 year old male entered with disseminated subcutaneous metastases, face, chest, arms and leg; stage IV-M1a. All lesions including never-injected nodules on arm regressed by 6 months after entry.

Figure 3.

Ex-vivo measurements of (TIL) or blood lymphocytes (BL) to kill autologous melanoma cells. Cytotoxicity presented at increasing effector to melanoma target cell ratios. Each figure is representative of 6 to 10 experiments (A) Comparing the same number of lymphocytes, TIL are more lytic than BL. TIL•, BL ▪. (B) TIL preferentially kill autologous melanoma cells as compared to allogeneic cells, consistent with MHC restriction. Pt C TIL•, Pt M TIL▪ (C) Killing is MHC restricted and not due to NK cells. Pt C BL •, Pt C BL minus NK ▪, Pt F BL ▴, Pt F BL minus NK ▾, (D) Depletion of CD8 T cells abrogates the killing. BL •, BL minus CD8 T▪ (E) Repeated injection in 3 patients of CKs mixed with irradiated autologous melanoma cells over many weeks increases the killing ex-vivo.

Figure 4.

Chart of numbers of patients with regressing and recurrent metastases.

Figure 5.

Melanoma status of all patients ranked by the duration of follow up in months and years after entering. The time line for each patient is censored at death, initiation of chemotherapy, or when last examined. Red indicates no clinically evident disease (NED) by examination and chest x-ray for at least 8 weeks, and subsequent negative scans. Several patients had NED* when lost to follow up. Black indicates presence of metastatic melanoma. Note, many patients had prolonged periods of stable or slowly progressive disease. 4 patients who had NED for >2 years experienced a late relapse as indicated. A Patient was experiencing a rapid regression of many metastases after 27 weeks of CK injections (with dense lymphocytic infiltrates in never injected nodules) when she switched to a geographically closer physician, and began receiving injections of an allogeneic melanoma vaccine to enhance immune responses. Within 8 weeks after the last CKs and after 4 weekly injections of vaccine she demonstrated NED. Subsequent indicated recurrences were treated by irradiation or excision. B The recurrence of melanoma after 20 years of NED subsequently responded to Ipilimumab.

Figure 6.

A Kaplan-Meier plot of overall survival on study and after switching to chemotherapy. Tick marks indicate alive at end of study or when lost to follow-up.