PD-L1-Independent Mechanisms Control the Resistance of Melanoma to CD4+ T Cell Adoptive Immunotherapy.

Immunotherapy is becoming the standard of care for melanoma. However, resistance to therapy is a major problem. Previously, we showed that tumor-specific, cytotoxic CD4+ T cells from tyrosinase-related protein 1 transgenic mice could overcome secondary resistance to recurring melanoma when anti-programmed cell death 1 ligand (PD-L1) checkpoint blockade was combined with either anti-lymphocyte-activated gene 3 (LAG-3) Abs or depletion of tumor-specific regulatory T (Treg) cells. In this study, we show that PD-L1 expressed by the host, not B16 melanoma, plays a major role in the early stages of exhaustion or primary resistance. We observed durable regression of melanoma in tumor-bearing PD-L1-/-RAG-/- mice with transfer of naive tumor-specific CD4+ T cells. However, exhausted tumor-specific CD4+ T cells, which included tumor-specific Treg cells, failed to maintain durable regression of tumors in PD-L1-/-RAG-/- mice unless tumor-specific Treg cells were eliminated, showing nonredundant pathways of resistance to immunotherapy were present. Translating these findings to a clinically relevant model of cancer immunotherapy, we unexpectedly showed that anti-PD-L1 checkpoint blockade mildly improved immunotherapy with tumor-specific CD4+ T cells and irradiation in wild-type mice. Instead, anti-LAG-3 checkpoint blockade, in combination with tumor-specific CD4+ T cells and irradiation, overcame primary resistance and treated established tumors resulting in fewer recurrences. Because LAG-3 negatively regulates effector T cell function and activates Treg cells, LAG-3 blockade may be more beneficial in overcoming primary resistance in combination immunotherapies using adoptive cellular therapy and irradiation than blockade of PD-L1.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NKIL-12) produced high amounts of IFNγ. The addition of a low number of A-NKIL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NKIL-12 cells. OT-I-CTLs and A-NKIL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NKIL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs.

Restoring immune function of tumor-specific CD4+ T cells during recurrence of melanoma.

Recurrent solid malignancies are often refractory to standard therapies. Although adoptive T cell transfer may benefit select individuals, the majority of patients succumb to their disease. To address this important clinical dilemma, we developed a mouse melanoma model in which initial regression of advanced disease was followed by tumor recurrence. During recurrence, Foxp3(+) tumor-specific CD4(+) T cells became PD-1(+) and represented >60% of the tumor-specific CD4(+) T cells in the host. Concomitantly, tumor-specific CD4(+) T effector cells showed traits of chronic exhaustion, as evidenced by their high expression of the PD-1, TIM-3, 2B4, TIGIT, and LAG-3 inhibitory molecules. Although blockade of the PD-1/PD-L1 pathway with anti-PD-L1 Abs or depletion of tumor-specific regulatory T cells (Tregs) alone failed to reverse tumor recurrence, the combination of PD-L1 blockade with tumor-specific Treg depletion effectively mediated disease regression. Furthermore, blockade with a combination of anti-PD-L1 and anti-LAG-3 Abs overcame the requirement to deplete tumor-specific Tregs. In contrast, successful treatment of primary melanoma with adoptive cell therapy required only Treg depletion or Ab therapy, underscoring the differences in the characteristics of treatment between primary and relapsing cancer. These data highlight the need for preclinical development of combined immunotherapy approaches specifically targeting recurrent disease.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.

Cytokine gene therapy using adenovirally transduced, tumor-seeking activated natural killer cells.

We previously demonstrated that adoptively transferred, interleukin (IL)-2-activated natural killer (A-NK) cells are effective in reducing B16 lung tumors in tumor-bearing animals. This effect depends on high and often toxic doses of IL-2 to support the survival and antitumor functions of the transferred A-NK cells. We hypothesized that A-NK cells transduced to express pro-NK cell cytokines would become less dependent on high and potentially toxic amounts of IL-2. Here, we demonstrate that A-NK cells adenovirally transduced to express mIL-12 survive well and function efficiently in mice bearing B16 lung tumors when supported with low, nontoxic doses of IL-2. The intratumoral survival of nontransduced "bystander'' A-NK cells also increased when they were coinjected with IL-12 gene-transduced A-NK cells. The enhanced survival of exogenously delivered, IL-12 gene-transduced A-NK cells resulted in greater antitumor responsiveness. This led to a 7- to 10-day increase in median survival time compared with tumor-bearing mice receiving mock-transduced A-NK cells. These data show that the presence of IL-12 around tumor-infiltrating A-NK cells enhances their antitumor activity while reducing their requirement for systemically administered IL-2.

Depletion of B220+NK1.1+ cells enhances the rejection of established melanoma by tumor-specific CD4+ T cells.

Five-year survival rates for patients diagnosed with metastatic melanoma are less than 5%. Adoptive cell transfer (ACT) has achieved an objective response of 50% by Response Evaluation Criteria in Solid Tumors (RECIST) in this patient population. For ACT to be maximally effective, the host must first be lymphodepleted. It is hypothesized that lymphodepletion may remove regulatory elements and cytokine sinks, or increase the activation and availability of antigen presenting cells (APCs). We use an in vivo model to study the ACT of tumor-associated antigen (TAA)-specific CD4+ T cells (TRP-1 cells). We have discovered that depletion of NK1.1+ cells enhances the rejection of established melanoma tumors by adoptively transferred TRP-1 CD4+ T cells. NK1.1+ cell depletion increases the number of CD4+ T cells, the serum concentration of pro-inflammatory cytokines, autoimmune vitiligo, host survival and prevented recurrence after ACT. Because multiple cells express NK1.1, we targeted different NK1.1+ cell populations using antibodies specific for NK cells, pre-mNK cells, and innate lymphoid cells (ILCs). Our data suggests that NK1.1+B220+ pre-mNK cells (also known as interferon-producing killer dendritic cells; IKDCs) are an important inhibitor of the CD4+ T cell response to melanoma. Understanding this mechanism may help design new immunotherapies to modulate the activity of pre-mNKs in the face of an antitumor immune response and inhibit their suppression of adoptively transferred T cells.