HIGHLIGHTS INTO HISTORICAL AND CURRENT IMMUNE INTERVENTIONS FOR CANCER

Abstract

Immunotherapy is an additional pillar when combined with traditional standards of care such as chemotherapy, radiotherapy, and surgery for cancer patients. It has revolutionized cancer treatment and rejuvenated the field of tumor immunology. Several types of immunotherapies, including adoptive cellular therapy (ACT) and checkpoint inhibitors (CPIs), can induce durable clinical responses. However, their efficacies vary, and only subsets of cancer patients benefit from their use. In this review, we address three goals: to provide insight into the history of these approaches, broaden our understanding of immune interventions, and discuss current and future approaches. We highlight how cancer immunotherapy has evolved and discuss how personalization of immune intervention may address present limitations.

Cancer immunotherapy is considered a recent medical achievement and in 2013 was selected as the “Breakthrough of the Year” by Science. While the breadth of immunotherapeutics has been rapidly expanding, to include the use of chimeric antigen receptor (CAR) T-cell therapy and immune checkpoint inhibitor (ICI) therapy, immunotherapy dates back over 3,000 years. The expansive history of immunotherapy, and related observations, have resulted in several approved immune therapeutics beyond the recent emphasis on CAR-T and ICI therapies. In addition to other classical forms of immune intervention, including human papillomavirus (HPV), hepatitis B, and the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) tuberculosis vaccines, immunotherapies have had a broad and durable impact on cancer therapy and prevention.

One classic example of immunotherapy was identified in 1976 with the use of intravesical administration of BCG in patients with bladder cancer; resulting in a 70% eradication rate and is now standard of care. However, a greater impact from the use of immunotherapy is documented by the prevention of HPV infections that are responsible for 98% of cervical cancer cases. In 2020, the World Health Organization (WHO) estimated that 341,831 women died from cervical cancer¹. However, administration of a single dose of a bivalent HPV vaccine was shown to be 97.5% effective in preventing HPV infections. These vaccines not only prevent cervical squamous cell carcinoma and adenocarcinoma, but also oropharyngeal, anal, vulvar, vaginal, and penile squamous cell carcinomas. The breadth, response and durability of these vaccines can be contrasted with CAR-T-cell therapies, which have significant barriers to their widespread use including logistics, manufacturing limitations, toxicity concerns, financial burden and lasting remissions observed in only 30 to 40% of responding patients. Another, recent immunotherapy focus are ICIs. ICIs are a class of antibodies that can increase the immune responses against cancer cells in patients. However, ICIs are only effective against tumors with a high mutational burden and are associated with a broad spectrum of toxicities requiring interruption of administration and/or administration corticosteroids; both of which limit immune therapy.

In summary, immune therapeutics have a broad impact worldwide, utilizing numerous mechanisms of action and when considered in their totality are more effective against a broader range of tumors than initially considered. These new cancer interventions have tremendous potential notability when multiple mechanisms of immune intervention are combined as well as with standard of care modalities.

Introduction

We tend to think that cancer immunotherapy is a recent medical achievement, providing an additional pillar for cancer therapy² that originated in the last few decades, but this is incorrect. In reality, forms of immunotherapy were noted 3,000 years ago in Egypt, and since based on anecdotal reports of tumors disappearing spontaneously or after an infection with concomitant high fever³. The first scientific attempts to modulate a patients’ immune response against cancer can be attributed to two German physicians, Fehleisen ⁴ and Busch⁵, who independently noticed significant tumor regression after erysipelas infection. In 1868, Busch intentionally infected a cancer patient with erysipelas, and following infection, documented that the patient’s tumor decreased in size. Fehleisen repeated this treatment in 1882, and obtained similar results; he eventually identified Streptococcus pyogenes as the causative agent of erysipelas⁴. The next advance in immune intervention was from William Coley, who is now generally accepted as the father of immunotherapy. Coley used the power of the immune system to treat cancer patients in the late 19th century. As an orthopedic surgeon, he operated on patients with bone sarcomas, and noticed that some patients developed postoperative wound infections, in association with a spontaneous regression of their unresected tumors. He followed up on this observation with a sarcoma patient that had a long-term regression after an erysipelas infection and began injecting heat-inactivated bacteria (“Coley’s toxins”) into patients with inoperable cancers⁶. He reported a significant number of regressions and cures in more than 1,000 patients, many of whom had sarcomas⁷.

The impact of Coley’s principles went largely unnoticed for several decades, requiring a greater knowledge of immunology, including the existence of T cells and an understanding of their role in immunity to understand a mechanism of action. It wasn’t until 1967 where the role of T-cells was recognized by Jacques Miller who published a report characterizing their function ⁸. Another critical milestone in our understanding of cancer immunology was reached in 1957, when Thomas and Burnet proposed the concept of cancer immunosurveillance⁹. The theory of cancer immunosurveillance suggested that lymphocytes could act as sentinels to identify and potentially eliminate cells transformed by mutations¹⁰. This concept is now incorporated as a component of cancer immunoediting, whereby immune surveillance system can “shape” the immunogenicity of tumor cells which are not initially eliminated¹¹. The theory of immune surveillance re-emerged in 1974, when Stutman demonstrated that nude mice with impaired T-cell function develop cancer more readily than wild type mice¹². Natural killer (NK) cells were identified soon after, providing an addition cellular, anti-tumor, and virus mediator^{13, 14}.

At the end of the twentieth century Schreiber, Dunn, Old and their teams proved that T-cells were able to provide anti-tumor surveillance and immune responses^{10, 11, 15, 16}. This was followed by the first identified human tumor antigen recognized by T lymphocytes in 1991¹⁷ and shortly later by the identification and cloning of the melanoma antigen, encoding the gene for melanoma antigen gene (MAGE), recognized by the cytotoxic T cells ¹⁷. Finally, in 1976, Coley’s principles were shown to be correct, when Morales et al.¹⁸ established the effectiveness of BCG for the treatment of superficial bladder cancer, building on the 1959 study by Old et al.¹⁹ and demonstrating the anti-tumor activity of BCG in murine models.

With these achievements, it is unexpected that the importance of modern immunotherapy was not formally recognized until 2018 when James Allison and Tasuku Honjo won the Nobel prize for their work on checkpoint molecules as therapeutic targets²⁰. Immune checkpoints protect healthy cells from damage by the immune system through both the activation and suppression of T cells²¹. Negative regulators of T cell activation act as ‘checkpoint molecules’ to regulate hyperactive T-cell responses and associated toxicity. However, tumor cells can express inhibitory receptors that decrease and/or block the function of tumor antigen-specific T cells. Ultimately, this prevents T cells from recognizing and killing cancer cells²². The successful development of CAR T-cells, vaccines, and oncolytic viruses have also rapidly advanced. Demonstrating significant therapeutic responses with some vaccine’s preventing the development of tumors.

In this review, we aim to provide a historical and biological perspective into the origin and clinical implementation of cancer immune interventions. This includes as overview of biological response modifiers, many of which are derived from synthetic epitopes identified from microbial ligands. We emphasize modalities that have received regulatory approval, discuss their efficacy and associated toxicities. We note, our emphasis extends beyond the therapies discussed in the popular press, such as ICIs and CAR-T cells to include interventions infrequently considered as immunotherapy but which have demonstrated broad efficacy and durable responses such as ACT using allogeneic stem cell transplantation (ASCT), lymphocyte infusions and tumor prophylaxis by viral vaccines. Because of the breadth and rapid emergence of novel immune intervention strategies we have limited this review to a discussion of approved and clinically relevant mediators and approaches. Finally, we summarize emerging therapies and strategies with promise within the field of cancer immunotherapy.

CYTOKINES and BIOLOGIC RESPONSE MODIFIERS (BRMs)

Cytokines are small proteins secreted by immune cells with some approved for the treatment of cancer (Table 1). Cytokines have a crucial role in the signaling cross-talk between immune cells, as well as between immune cells and non-immune cells²³. The first cytokine to be discovered was interferon alpha, (IFN-α), also known as type I IFN, in 1957 by Isaacs and Lindenmann²⁴. IFNs comprise a large family of cytokines, among which IFN-α has a critical role in antitumor immunity²⁵. It has multifaceted roles in tumor control, including directly eradicating tumor cells by inducing their apoptosis and boosting effective antitumor immune responses through the stimulation of dendritic cell (DC) maturation and enhancing T-cell cytotoxicity²⁵. Clinical studies have demonstrated a therapeutic role for IFN-α at high levels in chronic myeloid leukemia and melanoma^{26, 27}. IFN alfa-2b (Intron A), an IFN that targets the IFNAR1/2 pathway, was approved by the US Food and Drug Administration (FDA) for the treatment of patients with Hairy cell leukemia, malignant melanoma, follicular lymphoma, and AIDS-related Kaposi’s sarcoma²⁸. Similar approvals were provided by the FDA for IFN alfa-2a (Roferon). The suboptimal pharmacodynamics of IFN alpha-2 stimulated the development of peginterferon alfa-2a (Pegasys) and peginterferon alfa-2b (Sylatron^®). Both covalent conjugates of recombinant IFN alfa-2 with monomethoxy-polyethylene glycol. The addition of PEG (polyethylene glycol) protects the IFN alfa-2 degradation, overcomes rapid renal clearance, improved stability, bioavailability and safety. This resulted in the US FDA approval of the pegylated IFNs in 2002 and 2011 for the adjuvant treatment of patients with stage III melanoma as evidenced by microscopic or gross nodal involvement for Sylatron²⁹ and HSB and HSC for Pegasys. Thus, PEG-Intron (Sylatron^®) provides a longer-acting formulation of IFN-α-2b, requiring subcutaneous (s.c.) injection once a week. As compared to IFN which is administered as an intravenous (i.v.) infusion daily for 5 days or weekly for 4 weeks, followed by a s.c. injection three times/week.

Table 1:

Food and Drug administration (FDA) approved cytokines for the treatment of neoplastic disease.

Cytokine	Approved neoplastic disease	Year
Interleukin-2 (aldesleukin; Proleukin)	Metastatic melanoma	1998
	metastatic kidney cancer	1992
rHuIL-2 & Danyelza® (Naxitamab-gqgk) & GM-CSF & isotretinoin	High-risk neuroblastoma	2020
Interferon alfa-2b (Intron A) #	Follicular lymphoma	1997
	Malignant melanoma	1995
	Genital warts	1988
	Hairy cell leukemia	1986
	AIDS-related Kaposi’s sarcoma	1988
IFN alpha-2a (Roferon) #	CML	1995
	Hairy cell leukemia,
	AIDS-related Kaposi’s sarcoma,
Peginterferon alfa-2b (Sylatron) #	Stage III melanoma as evidenced by microscopic or gross nodal involvement	2011
Pegasys (peginterferon alfa-2a)	Off label used for CML, ECD, MPNs, PV, MF, and systemic mastocytosis, as well as mycosis fungoides/Sezary syndrome, hairy cell leukemia, primary cutaneous CD30+ T-cell lymphoproliferative disorders, and adult T-cell leukemia/lymphoma.	2002
Alferon N (interferon alfa-N3)	R/R external condylomata acuminata (anogenital warts)	2020
Nadofaragene Firadenovec (rAd-IFNα/Syn3, (Adstiladrin))	High-risk BCG–unresponsive non-muscle invasive bladder cancer with carcinoma in-situ plus or minus papillary tumors	2022
Besremi (ropeginterferon alfa-2b-njft),	Polycythemia Vera	2021
Leukine Sargramostim (rhu GM-CSF)	Shorten time to neutrophil recovery and reduce incidence of severe and lifethreatening infections and infections resulting in death following induction chemotherapy in adult patients with AML	1991–1996
	Mobilization of hematopoietic progenitor cells into PB for leukapheresis and autologous transplantation in adult patients
	Acceleration of myeloid reconstitution following autologous BMT or PB progenitor cell transplantation
	MAcceleration of myeloid reconstitution following allogeneic BMT
	Treatment of delayed neutrophil recovery or graft failure after autologous or allogeneic BMT
Leukine Sargramostim (rhu GM-CSF)	Increase survival in patients acutely exposed to myelosuppressive doses of radiation	2018
Talimogene laherparepvec (T-VEC)	Herpes simplex 1 virus, genetically modified to express GM-CSF for the treatment of metastatic melanoma.	2015

^#While these cytokines have been approved by the FDA, they are no longer being manufactured for business reasons and thus are not currently used clinically. PB (peripheral blood), R/R (Relapsed/Refractory), AIDs (Acquired immunodeficiency disease), AML (acute myeloid leukemia), BCG (Bacillus Calmette Guérin), BMT (bone marrow transplantation), CML (chronic myeloid leukemia), AML (acute myeloid leukemia), GM-CSF (Granulocyte macrophage-colony stimulating factor), ECD (Erdheim-Chester disease), MPN (myeloproliferative neoplasms), PV (polycythemia vera), and MF (myelofibrosis),.

However, the manufacture of Roferon, Intron A, and Sylatron^® has been discontinued, not due to any safety, efficacy or quality, but based on business reasons. This observation maybe in association with the development of ICI based therapeutics^{30, 31}. However, Pegasys, in association with its FDA approval for the treatment of patients with chronic HSB and HSC is still used off label for hematologic malignances, polycythemia Vera, and relapse post allo SCT. The antitumor properties of IFN alfa-2b are counterbalanced by the induction of immunosuppressive membrane receptors including programmed cell death protein (PD)-1/PD-ligand (L)1, downregulation of IFN receptors, and loss of downstream signaling mediators. Thus, there is a rational for the evaluation of PEGylated IFNα in combination with pembrolizumab (anti-PD-1) which was studied in a phase 1b/2 study of anti-PD-1 treatment naive patients with stage IV metastatic mucosal or cutaneous melanoma (KEYNOTE-020, NCT02112032)³². The outcome of this study was an overall response rate (ORR) of 60.5%, including 1 complete response (CR) and 19 partial responses (PRs). The 24-month progression free survival (PFS) rate of 46% compared favorably to the 24-month PFS of ~30% reported in the KEYNOTE-006 study (NCT01866319) that evaluated pembrolizumab as monotherapy in a phase III trial³³. Further, in this study, CD8+ T cell infiltrates were significantly associated with clinical responses, in agreement with the expected mechanism of action of IFNs and immune checkpoint therapy. While promising, the ability of PEG-IFNα to potentiate the antitumor activity of pembrolizumab requires validation in randomized and controlled phase III clinical trial(s)³⁴. Despite the loss of recombinant IFN alphas to manufacturing considerations Alferon N (interferon alfa-N3) received FDA approval in 2020 for the treatment of patients with R/R external condylomata acuminata (anogenital warts)³⁵ Alferon N is a natural interferon product manufactured from pooled units of human leukocytes that have been stimulated by incomplete infection with the avian Sendai virus and incorporated multiple interferon-a subtypes. Besremi (ropeginterferon alfa-2b-njft), a novel mono-pegylated and extra-long-acting IFN³⁶, was also approved by the FDA in 2021 for the treatment of adult patients with polycythemia Vera³⁷. It is noted that both Alferon N and Besremi are also being studied off-label for myelodysplastic syndrome (MDS) and leukemia syndromes.

Another cytokine, Interleukin-2 (IL-2), was identified in 1976³⁸ and was shown to support the culture of T-cells in vitro. IL-2 was cloned in 1983³⁹ and was immediately studied in clinical trials demonstrating promising results including tumor shrinkage^{40, 41}. It has proven to be effective when administered in high doses to patients with metastatic melanomas and renal cell carcinomas (RCC) by enhancing the production of T-cells and activation of NK cells⁴². The US FDA approved the use of IL-2 as an immunotherapeutic treatment in 1991 for the treatment of metastatic RCC and in 1998 for metastatic melanoma⁴³. The clinical use of IL-2 immunotherapy has not been widely applied due to its short in vivo half-life, severe toxicity at therapeutic doses, and induction of immunosuppressive responses through regulatory T cell (Treg) expansion^{44, 45}. Many strategies for addressing these limitations of IL-2 have been implemented, such as fusing the Fc domains of immunoglobulins or PEG molecules to increase half-life⁴⁶. However, to date none of these engineered IL-2 moieties have received FDA approval⁴². Human granulocyte-macrophage colony stimulating factor (GM-CSF), is a 127-amino-acid glycoprotein originally isolated from culture medium of pneumocytes from lipopolysaccharide(LPS)-injected mice⁴⁷. GM-CSF was so named due to its ability to stimulate bone marrow (BM) cell proliferation in vitro, with the outgrowth of granulocytes and macrophages colonies ex vivo. GM-CSF is secreted by macrophages, fibroblasts, activated T-lymphocytes, NK, mast, endothelial and many tumor cells⁴⁸. It is a potent hematopoietic growth factor for the expansion and maturation of monocyte-macrophages, DCs and granulocytes from hematopoietic progenitor cells^{49, 50}. Further, GM-CSF has other important biological activities, including accelerating tissue repair and modulating host immunity to infection and cancer via the innate and adaptive immune systems. A finding documented in preclinical models and confirmed in some clinical studies.

GM-CSF was cloned in 1985⁵¹ with its DNA expressed in bacteria, yeast, and mammalian cells, allowing manufacture at scale of recombinant GM-CSF⁵². Indeed, Sargramostim [recombinant human (rhu GM-CSF)] was approved by the US FDA in 1991 to accelerate BM recovery in diverse settings of BM failure⁵³. Clinical studies using GM-CSF as a vaccine adjuvant or following intratumoral injection have shown T-cell stimulating activity, which has also been observed following systemic administration, in association with allogenic stem cell transplantation. However, in transplant trials, administration of GM-CSF was not found to enhance a T-cell immune response to allogeneic tumor cells or common HLA-restricted peptide antigens⁵⁴. The manufacture of the prostate vaccine, Sipuleucel-T (PROVENGE), also includes recombinant GM-CSF in the process and is the first (2010) and only therapeutic cancer vaccine to be approved by the U.S. FDA⁵⁵ in men who have metastatic, castration-resistant, prostate cancer with no or minimal symptoms. In these patients, Sipuleucel-T prolongs median survival by 4.1 months as compared to patients treated with a placebo. At 3 years, the proportion of patients alive in the vaccine group was 50% higher than that in the control group (31.7% versus 21.7%, respectively). The antigen targeted by this vaccine is prostate acid phosphatase (PAP), formulated as a fusion protein that combines recombinant PAP with recombinant GM-CSF. The PAP-GM-CSF fusion protein is incubated with autologous peripheral blood mononuclear cells (PBMC) obtained by leukapheresis. In this, therapeutic GM-CSF is used to activate antigen-presenting cells (APC) within the autologous PBMC product⁵⁶. Despite the observed clinical benefits, many cytokines are poorly tolerated, limiting further applications as monotherapies. Nonetheless, cytokines as monotherapy or in combination are still being investigated with other immunotherapies, including ACTs and CPIs, with the goal of increasing therapeutic outcomes. There are also at least two other approaches in which increased local levels of GM-CSF, adjacent to DCs, may facilitate the induction of antitumor T-cell responses. This includes the mixture of GM-CSF with antigen-loaded DC and intratumor injection of a cytolytic virus that secretes GM-CSF. A study by Andtbacka et al. in melanoma patients demonstrated that intra-lesional T-VEC therapy (a herpes simplex virus type 1–derived oncolytic immune therapy designed to selectively replicate within cancers and secrete GM-CSF to enhance systemic antitumor immune responses) resulted in tumor cell lysis and specific T-cell immune responses. This finding resulted in the FDA approval for the use of T-VEC in melanoma patients⁵⁷. Thus, the US FDA approved talimogene laherparepvec (T-VEC, also known as OncoVEX^GM-CSF) in 2015 for use in melanoma patients with injectable but non-resectable lesions in the skin and lymph nodes. Further, recombinant GM-CSF was also approved by the FDA in 2020 ⁵⁸ for administration in combination with an anti-GD2 monoclonal antibody (mAb) (Naxitamab) for the treatment of pediatric and adult patients with relapsed or refractory high risk neuroblastoma in the bone or BM demonstrating a partial response, minor response, or stable disease to prior therapy. ^{59, 60, 61}. Another therapeutic, Danyelza^® (naxitamab-gqgk), was documented to improve PFS and OS in subjects receiving GM-CSF-containing regimens compared to standard chemotherapy and historical controls, respectively^{59, 61}.

Generally defined, BRMs are drugs or approaches that modify the relationship between a tumor and a host’s immunity by modifying the host’s biological response to the tumor cells with resultant therapeutic effects⁶². BRMs include many agents such as mAbs, CPIs, cellular mediators (as part of ACT), and immune mediators (that include synthetically derived as well as ones purified and manufactured based on human and microbial products). All of which provide approaches whose mechanisms of action involve a patient’s own biological responses⁶³. These latter, non-specific immunotherapies (immunomodulators) have been long studied clinically⁶⁴ with several receiving regulatory approval as standard of care. In the following paragraphs, we discuss BCG, muramyl dipeptide (MDP), an engineered variant muramyl tripeptide phosphatidylethanolamine (MTP-PE), CpG 1018, Imiquimod, monophosphoryl lipid A (MPLA), and thymosin as examples of such BRMs (Table 2).

Table 2:

Biological response modifiers that have received regulatory approval as therapeutics, vaccine adjuvants by the US Food and Drug Agency (FDA) or European Medicines Agency (EMA) for Mifamuratide.

BRM	Disease approved	Approval Date
Mycobacterium bovis Bacillus Calmette-Guérin (BCG)	Bladder cancer	1990
N-acetyl-glucosamine-3yl-acetyl Lalanyl-D-isoglutamine (nor-MDP)	vaccine adjuvants
muramyl tripeptide phosphatidylethanolamine (MTPPE, Mifamurtide)	Osteosarcoma	2009
monophosphoryl lipid A (MPLA) as part of AS01 and AS04	zoster (Shingrix), HPV (Cervarix), HSB (Fendrix)	2009
thymosin - alpha-1 (Ta1)	margin-free (R0)-resected stage IA–IIIA non-small cell lung cancer (NSCLC)
Imiquimod	superficial cutaneous basal cell carcinoma	1997
CpG 1018 (Cytosine phosphoguanine)	HepB (HEPLISAV-B)	2017

HPV, human papillomavirus; HepB, hepatitis B

BCG was derived after 230 sub-cultures of the pathogen M. bovis. In 1921, Albert Calmette and Camille Guérin demonstrated that BCG was non-pathogenic in animals and protected against tuberculosis challenge in vaccinated animals. Providing the only commercially available vaccine against tuberculosis. Interestingly, an early report demonstrated that the administration of BCG decreased the frequency and number of bladder cancer recurrences ¹⁸ and exhibited antineoplastic activity against carcinoma in situ⁶⁵. Further validation of this observation led to the unsurprising approval of BCG as an immunotherapeutic in the treatment of bladder cancer (BC) patients in the 1970s. Since then, it has been a standard of care for high-risk, non-muscle-invasive bladder cancer (NMIBC) patients to limit recurrence and progression of the disease. In this treatment protocol, intravesical instillations of bacilli are applied weekly over the course of six weeks (“induction treatment”) after the transurethral resection of tumors visible at the lumen surface of the bladder. If the patient responds appropriately to the therapy, a “maintenance treatment” consisting of six-week periods of instillation every three months for one to three years is then used to obtain the optimum effect for avoiding recurrence and progression.

As BCG is a living organism, this creates challenges in manufacturing and understanding its mechanism of action. It has been long recognized that following instillation, BCG binds to urothelial cells by fibronectin and integrin receptors^{66, 67} resulting in a granulomatous inflammatory reaction with granulocyte, macrophage, and lymphocyte infiltration. This results in the induction of chemokines, cytokines including IL-1, IL2, IL-6, IL-8, IL-12, IL-18, IFN-γ, tumor necrosis factor (TNF)-α and GM-CSF^{68, 69, 70}. The role of T-cells was also established by the demonstration that athymic mice do not respond to BCG administration⁷¹. Myeloid cells, may also have a therapeutic role and have been documented to be directly cytotoxic to bladder cancer cells⁷², as have NK cells⁷³ and DCs⁷⁴.

Although BCG remains the standard of care in the treatment of high grade NMIBC, as a live organism it has potential for adverse effects. Although most patients tolerate intravesical BCG therapy, mild side effects are common and include regional bladder symptoms of dysuria and hematuria in up to 85% of patients^{75, 76}. Recurrence rates vary, depending on the patients in a study and the follow-up period, ranging from 11% with a median follow-up of 2.3 years⁷⁷ to 75% with a median follow-up of 3.7 years⁷⁸. Progression rates are more homogeneous, ranging from 7.7% to 33% ^{79, 80}. Cancer-specific mortality rates have been reported to range from 0 to 23%; however, are highest in trials with a longer follow-up. Overall mortality rates range from 0 to 61% and are markedly higher than the cancer-specific mortality, reflecting increased age of patients and the number of comorbidies⁸¹. Intravesical BCG was the first-line treatment for high-risk tumors and carcinoma in situ (CIS), but for patients who cannot tolerate BCG treatment, or who recur or progress during treatment, the recommended option is radical cystectomy. However, the advent of ICI and viral therapy has changed the therapy landscape for these BC patients, providing a chance for bladder preservation in the treatment of NMIBC. Durvalumab plus Oportuzumab monatox, Pembrolizumab, and Nadofaragene firadenovec (ADSTILADRINE) have shown CR rates of 41.6%, 40.6%, and 59.6% after 3 months⁸². Nadofaragene Firadenovec (rAd-IFNα/Syn3, a non-replicating adenovirus rAd-IFNα that transfers the human IFN α-2b gene into urothelial cells coupled with Syn3, a polyamide surfactant that enhances gene transfer and expression recently completed a phase III trial ⁸³. During the writing of this review the FDA approved nadofaragene firadenovec-vncg for the treatment of patients with high-risk Bacillus Calmette Guérin (BCG)–unresponsive non-muscle invasive bladder cancer with carcinoma in-situ plus or minus papillary tumors CS-003 (NCT02773849). The approval was based on a multicenter clinical trial in a population of 157 patients, of whom 98 had unresponsive BCG carcinoma in-situ with or without papillary tumors. Treatment with nadofaragene firadenovec was administered once every 3 months until 12 months, unacceptable toxicity, or recurrent disease. Patients without evidence of high-grade recurrence were permitted to continue the therapy every 3 months. The use of study drug resulted in a complete response (CR) rate of 51%. The median duration of response was 9.7 months, with 46% of patients maintaining their response for up to 1 year. In summary, CPIs and other immune modulatory agents now offer an increasing opportunity for bladder-preserving strategies, extending the impact of BCG. ICIs are discussed more extensively below in the section on Immune Checkpoint Inhibitor Therapy.

Many BRMs are derived from isolated and synthetized components from bacteria⁸⁴. One of the first synthetic BRMs identified was MDP (muramyl dipeptide), which is the minimal structural component capable of duplicating the activity of mycobacteria in Freund’s complete adjuvant⁸⁵. MDP was shown to be capable of activating monocytes and macrophages⁸⁶ and to have vaccine adjuvant activity⁸⁷. However, it was also found to be pyrogenic and arthrogenic, which was increased by coupling to synthetic carriers⁸⁸, limiting its clinical potential. Further, murine pharmacologic studies revealed it to be rapidly excreted, limiting its utility⁸⁹ and requiring administration as a water in oil emulsion⁹⁰. Several recent phase I studies with a N-acetyl-glucosamine-3yl-acetyl L-alanyl-D-isoglutamine (nor-MDP) adjuvant emulsified in Montanide ISA 720VG with HER-2 B-cell peptides demonstrated minimal toxicity (injection site reactions) and antibody responses⁹¹. A number of earlier developmental studies focused on the toxicity and pharmacologic limitations of MDP, and in the 1980s, liposome-encapsulated(L) MTP-PE, (Mifamurtide), a lipophilic synthetic analogue of MDP was identified, and shown to be capable of activating murine⁹² and human monocytes and macrophages⁹³. Subsequent, translational studies in osteosarcoma bearing dogs provided the evidence that L-MTP-PE had significant therapeutic activity for this tumor⁹⁴. These finding initiated a series of clinical trials with L-MTP-PE in osteosarcoma patients and documented that the optimum intravenous (i.v.) dosage was 1–4 mg/m2 twice weekly for 4 weeks⁹⁵, providing the basis for subsequent phase II and III trials. In 2009, based on a phase III trial, Mifamurtide received marketing authorization in the European Union (EU) by the European Medicines Agency (EMA), as adjuvant therapy with polychemotherapy for the postoperative treatment of patients (aged 2–30 years) with non-metastatic high-grade resectable osteosarcoma following complete macroscopic resection⁹⁶.

Another bacteria based BRM is monophosphoryl lipid A (MPLA). MPLA is a modified form of lipid A, the biologically active part of Gram-negative bacterial LPS endotoxin, and is a low-toxicity agonist of Toll like receptor-4 (TLR4). MPLA was created in the 1970s by Edgar Ribi, a scientist at the Rocky Mountain Laboratory in the United States. Ribi systematically manipulated the LPS structure by acid and base hydrolysis in order to develop a detoxified form of ‘Coleys Toxin’ for cancer treatment⁹⁷. In an early trial, the anti-tumor activity of MPL appeared to be unimpaired relative to its parental LPS, with no severe toxicity⁹⁸. MPLA was first authorized by the FDA as a component of adjuvant system 4 [AS04]⁹⁹. AS04 is an aluminum hydroxide semi-crystalline gel that is adsorbed hydrostatically with MPL and is used with several viral vaccines including the HPV vaccine Cervarix ¹⁰⁰ and the HBV vaccine Fendrix¹⁰¹. Thus, MPLA is the first TLR ligand to date, which achieved clinical and regulatory success as an adjuvant for preventive vaccines in healthy individuals.

Other adjuvant formulations are used that incorporate MPLA and lack alum completely, including the adjuvant system 1 [AS01]¹⁰². AS01 incorporates QS-21 as part of a liposomal complex in the varicella zoster vaccine Shingrix¹⁰³. Because MPLA is a highly purified derivative of the LPS component of the cell-wall of Salmonella enterica its success as an adjuvant is understood primarily in the context of its activity as a TLR4 agonist that directly activates dendritic cells¹⁰⁴. The TLR4-mediated activation of antigen-presenting and innate immune cells results in mild inflammatory conditions¹⁰⁵ and Th1 differentiation of T-cells ¹⁰⁶ providing a counterbalance to the Th2-differentiating properties of alum¹⁰⁷ via TLR4 immune stimulation and activation. Another adjuvant that was recently approved clinically is based on synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG, another TLR agonist. CpGs induce cells that express TLR9 to initiate an innate immune response with the production of Th1 and proinflammatory cytokines¹⁰⁸, resulting in the induction of cytotoxic T lymphocytes (CTLs) and IFN-γ secretion. It promotes an immunomodulatory effect of antigen, activates APCs, and speeds up the immune response. CpG facilitates the expression of MHC, CD40 and CD80 on plasmacytoid DCs and enhances antigen processing and presentation¹⁰⁹. HEPLISAV-B, a vaccine for hepatitis B approved by the FDA in 2017, is the first vaccine adjuvanted with CpG ODN¹¹⁰. In addition to the TLR agonists BCG and MPLA⁸⁴, the BRM imiquimod received US FDA regulatory approval for the treatment of dermatologic neoplastic and inflammatory diseases¹¹¹. Imiquimod (a small non-nucleoside imidazoquinoline) was recognized in the late 1980s to have therapeutic and prophylactic potential in animal models of cytomegalovirus (CMV)¹¹² and herpes simplex virus type 2 (HSV-2) infections¹¹³. Studies documented that Imiquimod was a potent inducer of immunostimulatory cytokines including IFNα, TNFα, IL-1β and IL-6¹¹⁴ in vivo, with antitumor effects.¹¹⁵ Imiquimod was extensively tested for the topical treatment of actinic keratosis (a precancerous lesion of the skin)^{116, 117}, basal cell carcinoma,^{118, 119} and genital and perianal warts (a common sexually transmitted disease caused by HPV)^{120, 121}. These and many other studies demonstrated that imiquimod (as a 5% cream) is safe, generally well tolerated, and efficient against multiple skin disorders. Resulting in imiquimods’ approval by the FDA for use in humans in 1997, as a countermeasure against genital and perianal warts. It was later, in 2002, Imiquimod was found to exert immunostimulatory and anticancer effects by binding to TLR7¹²². This observation led to another FDA approval in 2004 for the use of Imiquimod in humans against actinic keratosis and superficial basal cell carcinoma. Mechanistically, Imiquimod has been shown to stimulate the production of proinflammatory cytokines by acting as an adenosine receptor antagonist¹²³, as well as to promote the (CCL2-dependent) recruitment of plasmacytoid DCs into the tumor bed and their conversion into tumor-killing effector cells¹²⁴.

A few BRMs, in addition to cytokines, have been identified based on peptide activity from organ extracts. Initial efforts to purify extracts from the thymus that could stimulate lymphopoiesis resulted in the development of the thymic preparation; thymosin fraction 5 (TF5). TF5 was shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities¹²⁵. Thymosin alpha 1 (Tα1) was the first biologically active polypeptide to be purified from TF5. Tα1 is an N-terminal acetylated acidic peptide of 28 amino acids ¹²⁶ that can restore lymphopoiesis in thymectomized mice and has shown immunostimulatory effects on T cells both in vitro and in vivo. In the context of cancer, Tα1 has been shown to increase the RFS and OS in both murine models and in humans with melanoma, lung cancer, and hepatocellular carcinoma ¹²⁷. The presumed mechanism of Tα1 bioactivity is its ability to stimulate anti-tumor immune responses by increasing the number and cytotoxic function of T-cells, as well as restore NK cell activity¹²⁸. Interestingly, the immune-stimulatory effects of Tα1 have been shown to be more effective in combination with chemotherapy, since Tα1 by itself may also stimulate myeloid suppressor cells and inhibit anti-tumor immune responses¹²⁹. Phase III clinical trials using Tα1 have recently been completed with encouraging results. In one study, Tα1 treatment was investigated on the long-term survival of 5,746 margin-free (R0)-resected stage IA–IIIA non-small cell lung cancer (NSCLC) patients¹³⁰. In this study patients were divided into a Tα1 group combined with standard of care and a control group who did not receive Ta1. A propensity score matching (PSM) analysis was performed to reduce bias, resulting in 1,027 pairs of patients. Following PSM, the baseline clinicopathological characteristics were similar between the two cohorts. In this study, the 5-year DFS and OS rates were significantly higher in the Tα1 group compared with the control group. Further, a multivariable analysis showed that Tα1 treatment was independently associated with an improved prognosis. Further, a subgroup analyses documented that Tα1 therapy improved DFS and/or OS in all subgroups independent of age, sex, Charlson Comorbidity Index (CCI), smoking status, and pathological tumor-node-metastasis (TNM) stage. These observation were most notable in patients with non-squamous cell NSCLC and without targeted therapy.

In another recent phase II study in locally advanced NSCLC (LANSCLC) patients Tα1 was assessed for the management of radiation pneumonitis (RP) in patients given concurrent chemoradiotherapy (CCRT)¹³¹. In this phase II, single-arm trial patients with unresectable LANSCLC received definitive CCRT and weekly Tα1 from the start of CCRT until 2 months after CCRT. Patients received 51 Gy in 17 daily fractions or 40 Gy in 10 daily fractions in the first course followed by a re-evaluation and those patients without disease progression had an adaptive plan of 15 Gy in 5 daily fractions or 24 Gy in 6 daily fractions as a boost. Concurrent chemotherapy consisted of weekly docetaxel (25 mg/m2) and nedaplatin (25 mg/m2) during radiation therapy. The primary endpoint in this study was the incidence of Grade (G) ≥2 RP. Secondary endpoints included the incidence of late pulmonary fibrosis, total lymphocyte count (TLC), and serum C-reactive protein (CRP) levels. TLC and CRP data were collected at baseline, 2 to 3 weeks during CCRT, the end of CCRT, and 2 and 6 months after CCRT. Patients treated with CCRT but without Tα1 intervention during the same period were selected as the control group by propensity score matching. Sixty-nine patients were enrolled in the study with an additional 69 patients selected as the control group. The incidence of G≥2 RP was significantly lower in the study group compared with control cases. G1 late pulmonary fibrosis occurred in 3.7% of patients in the control group and did not significantly differ compared with no event in the study group. Compared with the control group, the incidence of G3 to G4 lymphopenia was significantly lower in the Tα1 group, and the median TLC nadir was significantly higher in the Tα1 group. Further, the proportion of patients with maximum CRP ≥100 mg/L was significantly lower in the Tα1 group. Together, these two studies support the potential utility of Tα1 administration in combination with cytotoxic therapy for the treatment of NSCLC patients and to include patients with locally advanced diseases.

VIRAL ONCOGENE VACCINES

Prophylactic vaccines against the hepatitis B virus (HBV) and HPV have been effective in lowering the incidence of hepatocellular carcinoma and HPV associated tumors, respectively¹³². Following approval in 1981 by the US FDA, the anti-HBV vaccine was the first preventative cancer vaccine approved for use in the clinical settings¹³². The HBV vaccine comprising recombinant HBV surface antigen (HBsAg) confer lifetime immunity¹³³ significantly reducing hepatitis-associated HCC globally^{133, 134}.

High risk (HR)-HPV infections are a precursor for precancerous lesions, including premalignant glandular or squamous intraepithelial lesions of the cervix, also known as cervical intraepithelial neoplasia (CIN), and if untreated, can progress to invasive neoplastic lesions of the cervix¹³⁵. A similar progression occurs for other HPV driven tumors at other sites such as the anus, head and neck, penis, vagina, and vulva. Since 1974, the carcinogenic role of HPV has been associated with the transforming potential of the E6 and E7 proteins of HPV 16 and 18 ¹³⁶. The role of HPV in causing other cancers such as head and neck and anal cancer has been documented more recently^{136, 137}.

HPV is a frequent sexually transmitted infection with over 80% of men and women infected with HPV at least once before the age of 50¹³⁵. This contributes to cervical cancer risk; however, when effective cervical cancer screening and treatment are available, the incidence of cervical cancer is low. In contrast, if screening is not routinely undertaken, which is common in low-income countries, the incidence of cervical cancer is high and is the fourth most frequent cancer in women worldwide. These infections are responsible for 604,127 cases of cervical cancer and 341,831 deaths in 2020, with some 90% of the cases occurring in low-middle-income countries¹.

In contrast to cervical cancers, about 70% of oropharyngeal cancers are now HPV positive such that 30,000 new diagnoses a year are attributed to HPV. It is noted that these patients have a better prognosis than the HPV negative cancers in association with a higher response rate to treatment¹³⁸. In contrast to cervical and oropharyngeal cancers, vaginal and vulvar cancers as well as penile cancers are rare^{1, 139}. Penile cancer is diagnosed in less than 0.001 % of men in high income countries with 50% of the cases related to HPV¹⁴⁰. In addition to screening and treatment of pre-malignant lesions, HPV-related malignancies can be prevented using prophylactic vaccines, which will be discussed. Five HPV vaccines are currently licensed (Table 3). All the vaccines are based on virus like particles (VLP) that are self-assembled L1 capsid proteins resembling the HPV particle but are without HPV nucleic acids. These vaccines are not infectious and induce a strong immune response, with a 10X increased antibody titer compared to the levels occurring following a natural infection. Antibody titers peak 30 days after the last dose and plateau after 18–24 months. Long term follow-up has shown that the vaccine-induced HPV antibody response is durable, with seropositivity >15 years post-vaccination^{141, 142}. Countries with an established vaccination program are reporting on HPV vaccine effectiveness and herd protection^{143, 144}. Ten years after the initiation of HPV vaccination, it has been reported that vaccination reduces cervical HPV infections by 66–90%, genital warts in men and women by 31–93%, and CIN2/3 by 31%–85% that varies based on the age at vaccination^{143, 144, 145}. Further, national registries in Denmark, Sweden and UK have confirmed the effectiveness of HPV vaccination based on the prevention of invasive cervical disease with a risk reduction ranging from 53 to 87%^{144, 146}. Similar to cervical cancer the frequency of oropharyngeal cancers caused by HPV is high ranging from 22 to over 70% depending on the country reporting^{147, 148}. Oropharyngeal cancers predominantly affect men with an incidence that is significantly increasing in older men, while declining in men younger than 45. This observation can be explained due to the association with the HPV vaccination-era^{147, 148}. Recent publications have reported an 80% decrease in oral HPV infection in men after the introduction of vaccination in the US, which is consistent with data from clinical trials^{149, 150}. In summary, multiple clinical trials¹⁵¹ have demonstrated that HPV vaccination can prevent both HPV associated cervical and oral cancers. The incidence of HPV positivity among females aged 14–19 decreased by 88% and by 81% in females 20–24 years old¹⁵². Two of the currently approved vaccines, Gardasil (Merck) and Cervarix (GlaxoSmithKline), provide effective protection against chronic HPV infection and also prevent cervical intra-epithelial neoplasia (CIN), adenocarcinoma in situ and cervical cancer¹⁵².

Table 3:

HPV, human papillomavirus vaccines that have received regulatory approval.

Vaccine	Target	Adjuvant	Expression system	Manufacturer	Approved
Gardasil	HPV 6, 11, 16, 18	Amorphous aluminum hydroxyphosphate sulfate (AAHS)	Saccharomyces cerevisiae	Merck	2006
Cervarix	HPV 16, 18	Adjuvant System 04 (AS04)	Baculovirus	Glaxo-SmithKline	2007
Gardasil9	HPV 6, 11, 16, 18, 31, 33 45, 52 and 58	Amorphous aluminum hydroxyphosphate sulfate (AHHS)	Saccharomyces cerevisiae	Merck	2015
Cecolin	HPV 16, 18	Aluminum hydroxide	Escherichia Coli	Xiamen Innovax	2019
HOV2	HPV 16, 18	NA	Pichia pastoris	Walvax	2022

Thus, cervical cancer and other HPV associated tumors are a serious public health challenge and remain one of the most common cancers in women with a high mortality rate despite existing preventative, screening, and treatment approaches. Since HPV was recognized as an oncogenic virus preventative HPV vaccines have made great progress. However, people already infected with HPV require an effective treatment that would ensure long-term survival. Currently, clinical trials investigating HPV therapeutic vaccines have shown promise with the induction of a T-cell mediated immune response, and potential to induce the regression of cervical lesions and viral eradication. As discussed above the initial two prophylactic vaccines, Gardasil (introduced in 2006) and Cervarix (in use since 2007), are highly effective in preventing new infections with oncogenic HPV-16 and -18, as well as low-risk HPV-6 and -11 by antibodies against HPV capsid proteins^{153, 154} that unfortunately are rarely present in HPV-transformed cells. Thus, the millions of patients who have persistent, oncogenic HPV infection or HPV associated malignancies do not benefit from these existing vaccines^{135, 155}. Approximately 90% of deaths from cervical cancer occurred in low- and middle-income countries due to a lack of vaccine prevention, early diagnosis, and effective screening¹. In 2018, WHO started a new campaign to decrease the incidence rate of cervical cancer, with the aim to eventually eradicate the disease¹⁵⁶. Ideally, if all countries meet the objectives of the campaign by 2030, it is suggested that the number of new cases would decrease by 40% ¹. However, it is now estimated that the annual number of new cases of cervical cancer will increase to 700,000, and the number of deaths will reach 400,000 by 2030¹⁵⁶. This increase is associated with the uneven screening and vaccination that occurs mainly in high-income countries ¹⁵⁶. Although preventative measures are expected to eliminate cervical cancer in the long term, a short-term solution is critically needed for those already infected.

Thus, there is at present an emphasis on the development of vaccines that have effective activity for the treatment of for precancerous cervical lesions and cervical cancer. One of these nucleic-acid vaccine candidates; VGX-3100¹⁵⁷ recently completed a phase III clinical trial in patients with HPV-related, high-grade squamous intraepithelial lesions. This vaccine encodes for HPV 16 and 18 antigen E6+E7, was well-tolerated and found to elicit cell-mediated immune responses¹⁵⁷. In an earlier phase II, randomised, double-blind, placebo controlled clinical trial in women with HPV-16 or HPV-18-positive CIN2/3, both histopathological regression and viral clearance were reported to be significantly greater after intramuscular (i.m.) vaccination with VGX-3100 followed by electroporation as compared with placebo treated patients. However, the clinical efficacy, was modest with the suggestion to have potential to help prevent malignancies in patients living with persistent, oncogenic HPV infections but who did not benefit from prophylactic vaccines¹⁵⁸. The phase III trial (NCT03185013) with VGX-3100 (REVEAL1) was completed April 7, 2021; however, the primary end points (histopathological regression and virologic clearance of HPV16 and/or HPV18 at week 36) for the intent-to-treat population were not met. A second randomized, phase III trial (NCT03721978) has completed recruitment with the enrollment of 203 patients and is expected to have results reported in the 4Q22/1Q23¹⁵⁹.

Currently, despite treatment of locally advanced cervical cancer with chemoradiation, patients have a high recurrence rate and a poor 5-year survival rate, estimated at 50% and 70%, respectively¹⁵⁹. To address this challenge, the MEDI0457 vaccine, composed of the VGX-3100 plasmid coupled with an IL-12 expression plasmid to promote T-cell function, has been evaluated clinically based on PFS at 12 months with an estimated 88.9% PFS rate¹⁶⁰. Taken together these studies support the potential for the use of DNA therapeutic vaccines to induce or boost immune responses against HPV infections. However, improvements to the efficacy of DNA therapeutic vaccines and their implementation into treatment regimens in combination with conservative surgical treatment are needed to assess outcomes.

VACCINES IN ONCOLOGY

As previously mentioned, sipuleucel-T was approved by the U.S FDA in April 2010 for the treatment of symptomatic metastatic castration resistant prostate cancer (mCRPC)⁵⁵. Sipuleucel-T is an autologous vaccine made by activating PBMCs obtained by leukapheresis with a fusion protein formed from a prostate antigen (prostatic acid phosphatase (PAP)) as the immunogen and GM-CSF^{56, 161}. The Sipuleucel-T vaccination results in PAP-specific T cells capable of cytotoxicity against PAP-expressing prostate cancer cells^{55, 162}. A pivotal phase III clinical trial (IMPACT) of Sipuleucel-T in men with mCRPC with minimal or no symptoms, was reported to prolong median survival time (MST) by 4.1 months compared with patients treated with placebo. In this trial, the proportion of patients in the vaccine group who were alive at 3 years was 50% higher than that in the control group (31.7% versus 21.7%, respectively); providing a slight, but significant, improvement in the median overall survival (OS)¹⁶³ in mCRPC. The recommendation was that sipuleucel-T be used as initial therapy for asymptomatic or minimally symptomatic patients with mCRPC, at a time when the cancer disease burden is lower and immune function potentially higher. It was also noted that the usual outcome biomarkers (decline in PSA and improvement in bone or CT scans) are not observed with Sipuleucel-T intervention¹⁶⁴. Therefore, the benefit to the individual patient cannot be ascertained using currently available testing methods¹⁶⁴. However, the complexity and high expense of producing Sipuleucel-T have limited its widespread use¹⁶⁵. A recent study (PROCEED) confirmed data from the IMPACT study with a report of additional security and tolerability data. A highlight in this trial is the PFS between the end of Sipuleucel-T and following treatment administered in these patients, which would translate into a significant clinical benefit¹⁶⁶. Combination treatment with a phase Ib study using Sipuleucel-T and atezolizumab (anti PD-L1) in metastatic castration resistant prostate cancer provides a rational therapeutic strategy have documented safety and suggest that the combination may be beneficial, based on correlative immune testing¹⁶⁷.

ONCOLYTIC IMMUNOTHERAPY

Oncolytic viral therapy is a newly emerging form of cancer therapeutics involving both immunotherapy and biological therapies of cancer based on the use of existing biological agents to treat cancer. Genetically modified viruses have been developed that lack virulence, but are still able to infect and lyse cancer cells¹⁶⁸. Lysed and dying cancer cells can release antigens and cytokines that further recruit immune cells, stimulating immune attack and an overall inflammatory response at the tumor site^{169, 170, 171}.

The first oncolytic virus (Table 1), talimogene laherparepvec (T-VEC), was approved by the FDA in 2015 for the treatment of metastatic melanoma¹⁶⁸. T-VEC is a herpes simplex 1 virus, genetically modified to express GM-CSF, a cytokine that chemoattracts and expands many types of immune cells^{169, 170, 171}. Injections are given directly into the tumor site, especially in metastases and those regions which cannot be removed surgically. Viral infection and replication induces lysis providing one targeted mechanisms of action involved in the viral-induced destruction of cancer cells, which undergo further attack by an immune system stimulated by tumor antigens released by lytic destruction¹⁷¹ In the phase III study for T-VAC a randomized open-label phase III trial with unresectable stage IIIB–IVM1c melanoma patients randomized 2:1 to receive intratumoral T-VAC or subcutaneous recombinant GM-CSF. Outcomes included OS, durable response rate (DRR), ORR, CRs, and safety were reported¹⁷². The intent to treat population included 436 patients with 295 allocated to T-VAC and 141 to GM-CSF. The median OS was 23.3 months and 18.9 months in the T-VAC and GM-CSF arms, respectively¹⁷². The DRR was 19.0 and 1.4% ORR was 31.5 and 6.4%. A CR was achieved in 16.9% in T-VAC patients and 0.7% in the GM-CSF arm. In the T-VAC treated patients, the median time to CR was 8.6 months; but a median CR duration was not reached¹⁷³.

Immune checkpoint inhibitor (ICI) therapy such as PD-1 inhibitors, have limited response rates in some cancer patients, especially those with poor immune infiltration in tumor lesions¹⁷⁴. However, oncolytic viruses are believed to have potential to enhance antitumor activity of ICIs by inducing systemic immunity and clinical studies have shown that T-VEC can promote immune infiltration such that the tumor microenvironment (TME) can turn from ‘cold’ to ‘hot’, promoting the efficacy of ICI¹⁷⁵. Early results from phase I-II clinical trials have shown benefits of combining T-VEC and ICI in advanced melanoma. Whether combined with the PD-1 inhibitor pembrolizumab or the cytotoxic T-lymphocyte associated protein (CTLA)-4 inhibitor ipilimumab, the responses to combined therapy have been found to be significantly improved^{169, 175}. However, a phase III clinical trial of T-VEC combined with pembrolizumab did not significantly improve PFS or OS compared with the control group in unresectable stage IIIB–IVM1c melanoma¹⁶⁹. However, subgroup analysis suggested that melanoma patients with normal baseline levels of Lactate dehydrogenase (LDH) and low tumor burden may benefit from this combination therapy. It should be emphasized that despite the promise of T-VAC acquired immunity, immunity specific to the virus has potential to limit repeat therapy in the same patient¹⁷⁰.

IMMUNE CHECKPOINT INHIBITOR (ICI) THERAPY

Activation signals from the T-cell receptor (TCR) and sustained co-stimulatory signals (such as signals from CD28, a co-stimulatory receptor expressed on T cells^{176, 177, 178}) are required for optimal T cell activation and proliferation. However, chronic activation of the TCR and co-stimulatory molecules can compromise effector T cell function resulting in decreased anti-tumor activity^{179, 180, 181}, often identified as T-cell exhaustion. Thus, optimizing T cell co-stimulatory receptor expression is critical to limit T-cell exhaustion and maintain functional cytotoxic T-cell activity. Phenotypically, exhausted T cells have upregulated expression of several inhibitory receptors, including CTLA4, PD-1¹⁸², lymphocyte activation gene 3 (LAG-3)¹⁸³, T cell immunoglobulin and mucin-domain containing 3 (TIM3)¹⁸⁴, T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT)¹⁸⁵, V-domain immunoglobulin suppressor of T cell activation (VISTA)¹⁸⁶, and B and T cell lymphocyte attenuator (BTLA)¹⁸⁷. These proteins are currently all active targets of investigational cancer immunotherapies¹⁸⁸.

Monoclonal Abs that have received FDA regulatory approval have targeted several co-stimulatory molecules, commonly referred to as ICI, and include antibodies to CTLA-4, LAG-3, PD-1, and its ligand, PD-L1¹⁸⁹ (Table 4). Use of these ICIs aids in the restoration of T-cell responses for therapeutic intent. As mentioned previously, immune checkpoints are an approach to prevent excessive immune responses that are toxic to healthy cells in the host. Immune checkpoint signaling within T-cells is initiated when the cognate ligand interacts with tumor cells or suppressive immune cells (typically myeloid cells) resulting in the suppression of immune effector function, reduced cytotoxic function, and suppressive cytokine profiles. ICIs block checkpoint proteins from binding to their appropriate ligands, preventing the “off” signal for T-cell function. Ipilimumab was the first FDA-approved ICI to block the checkpoint inhibitor, CTLA-4^{190, 191}. Ipilimumab is an IgG1 mAb, that was approved in 2011 by the US FDA to treat patients with metastatic melanoma¹⁹². Monotherapy with ipilimumab was reported to improve OS rates¹⁹³ and durable objective response in patients with advanced melanoma¹⁹⁴. In addition to ipilimumab monotherapy, a phase III trial in patients with advanced melanoma (NCT01844505), in combination with nivolumab (PD-1 targeting ICI) resulted in longer PFS and a higher ORR¹⁹². The nivolumab–ipilimumab combination (ipi/nivo) resulted in an OS of 58% at 3 years, with significantly lower PFS with monotherapies of nivolumab at 52% and ipilimumab at 34%¹⁹⁵.

Table 4.

FDA approval history of checkpoint inhibitors targeting solid tumors.

Trade Name	Target antigen	Tumor	FDA approval	Indication
Dostarlimab + Ctx	PD-L1	biliary tract cancer	2022	advanced or metastatic biliary tract cancer
Pembrolizumab	PD1	bladder CA	2020	second line therapy for invasive bladdar cancer
Pembrolizumab	PD1	Cervical	2018	Recurrent or metastatic cervical cancer patients with progression on or after chemotherapy whose tumors express PD-L1
Pembrolizumab + CTx	PD1	Cervical	2021	second line therapy for Cervical cancer with PD-L1
Nivolumab	PD1	CRC	2017	Treatment of patients 12 years and older with dMMR and MSI-H metastatic CRC that has progressed following RX with Fluoropyrimidine, Oxaliplatin, and Irinotecan
Nivolumab + Ipilimumab	PD1, CTLA-4	CRC	2018	Metastatic CRC with MSI-H or dMMR
Pembrolizumab	PD1	CRC	2017	Unresectable or metastatic, MSI-H CRC
Pembrolizumab	PD1	CRC	2020	first line therapy MSI-H or dMMR Colorectal Cancer
Dostarlimab	PD-L1	endometrial cancer	2021	second line therapy dMMR endometrial cancer
Pembrolizumab + lenvatinib	PD1	Endometrial carcinoma	2021	second line therapy
Nivolumab	PD1	ESCC	2020	second line therapy
Pembrolizumab	PD1	ESCC	2019	second line therapy
Nivolumab	PD1	Eso or GEJ cancer	2021	adjuvant therapy
Pembrolizumab + CTx	PD1	Eso or GEJ carcinoma	2021	first line therapy
Pembrolizumab	PD1	Gastric/gastroesophageal junction	2017	Recurrent locally advanced or metastatic, gastric or gastroesophageal junction tumors express PD-L1
Pembrolizumab + trastuzumab + CTx	PD1	GC or GEJ cancer	2021	HER2+ first line therapy
Nivolumab + CTx	PD1	GC, GEJ, and Eso adenocarcinoma	2021	first line therapy
Nivolumab + Ipilimumab	PD1 + CTLA-4	HCC	2018	Intermediate or poor risk advanced hepatocellular carcinoma without prior treatment
Pembrolizumab	PD1	HCC	2018	Hepatocellular carcinoma previously treated with Sorafenib
Nivolumab	PD1	HCC	2017	Hepatocellular carcinoma previously treated with Sorafenib
Nivolumab + ipilimumab	PD1 + CTLA-4	HCC	2020	HCC that has failed sorafenib
Atezolizumab + bevacizumab	PD-L1	HCC	2020	first line therapy
Dostarlimab +tremelimumab	PD-L1 + CTLA-4	HCC	2022	unresectable HCC
Pembrolizumab	PD1	HNSCC	2016	Recurrent or metastatic HNSCC with progression on or after platinum-containing chem
Nivolumab	PD1	HNSCC	2016	Advanced HNSCC with progression on/after a platinum-based therapy
Pembrolizumab	PD1	HNSCC	2020	HNSCC
Pembrolizumab	PD1	Lymphoma	2017	Refractory classical Hodgkin lymphoma patients, or those who have relapsed after three or more prior lines of therapy
Nivolumab	PD1	Lymphoma	2016	Recurrent Hodgkin lymphoma following auto HSCT and post-TX Brentuximab Vedotin
Pembrolizumab	PD1	Lymphoma	2018	Refractory primary mediastinal large B-cell lymphoma patients, or who have relapsed after two or more prior lines of therapy
Nivolumab + ipilimumab	PD1, CTLA-4	Malignant pleural mesothelioma	2020	first line therapy
Pembrolizumab	PD1	Markel cell CA	2018	Recurrent locally advanced or metastatic Merkel cell carcinoma
Avelumab	PD-L1	Markel cell CA	2017	Metastatic Merkel cell carcinoma
Ipilimumab	CTLA-4	Melanoma	2015	Adjuvant treatment of cutaneous melanoma patients with pathologic involvement of regional lymph nodes of more than 1 mm who have undergone complete resection
Ipilimumab	CTLA-4	Melanoma	2011	Unresectable or metastatic melanoma with previous systematic treatment previously
Pembrolizumab	PD1	Melanoma	2014	Unresectable or metastatic melanoma and disease progression following Ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor
Pembrolizumab	PD1	Melanoma	2015	Unresectable or metastatic melanoma
Nivolumab + Ipilimumab	PD1, CTLA-4	Melanoma	2015	BRAF V600 wild-type, unresectable or metastatic melanoma
Nivolumab	PD1	Melanoma	2014	Unresectable or metastatic melanoma and disease progression following Ipilimumab and, if BRAF V600 positive, a BRAF inhibitor
Pembrolizumab	PD1	Melanoma	2019	Melanoma with LN involvement following complete resection
Nivolumab	PD1	Melanoma	2017	Adjuvant treatment of advanced melanoma
relatlimab + Nivolumab	PD1 + LAG-3	Melanoma	2022	Unresectable metastatic melanoma
Atezolizumab + CTx	PD-L1	Melanoma	2020	First line therapy BRAF V600E melanoma
Atezolizumab	PD-L1	Non SqCC NSCLC	2018	Metastatic disease with no EGFR or ALK genomic tumor mutation
Pembrolizumab + CTx	PD1	Non SqCC NSCLC	2017	Previously untreated metastatic non-Sq NSCLC
Pembrolizumab CTx	PD1	Non SqCC NSCLC	2018	Metastatic, disease with no EGFR or ALK genomic tumor mutation
Atezolizumab + CTx	PD-L1	Non SqCC NSCLC	2019	First line therapy
Pembrolizumab	PD1	NSCLC	2019	First-line Rx for stage III NSCLC patients who are not candidates for resection, definitive chemoradiation or metastatic NSCLC. Tumors must have no EGFR or ALK aberrations and be PD-L1+
Atezolizumab	PD-L1	NSCLC	2016	Metastatic NSCLC patients whose disease progressed during or following platinum-containing chemo
Nivolumab	PD1	NSCLC	2015	Metastatic NSCLC with progression or after platinum
Pembrolizumab	PD1	NSCLC	2016	Metastatic NSCLC patients whose tumors express PD-1
Durvalumab	PD-L1	NSCLC	2018	Unresectable stage III NSCLC whose disease has not progressed following concurrent platinum and radio RX
Pembrolizumab	PD1	NSCLC	2015	Unresectable stage III NSCLC whose tumors express PDL1
Atezolizumab + CTx	PD-L1	NSCLC	2019	Metastatic NSCLC patients whose tumors express PD-L1
Nivolumab + ipilimumab	PD1 + CTLA-4	NSCLC	2020	NSCLC expressing PD-L1 >1%
Nivolumab + ipilimumab	PD1 + CTLA-4	NSCLC	2020	First line therapy
Atezolizumab	PD-L1	NSCLC	2020	NSCLC + high PD-L1 first line therapy
Atezolizumab	PD-L1	NSCLC	2021	Adjuvant therapy
Dostarlimab + Ctx	PD-L1	NSCLC	2022	Metastatic NSCLC
Nivolumab	PD1	RCC	2015	Advanced RCC in patient with prior anti-angiogenic RX
Pembrolizumab + CTx	PD1	RCC	2019	First line therapy for RCC
Avelumab + CTx	PD-L1	RCC	2019	First line therapy for RCC
Nivolumab + cabozantinib	PD1	RCC	2021	first line therapy
Pembrolizumab + lenvatinib	PD1	RCC	2021	first line therapy
Pembrolizumab	PD1	RCC	2021	adjuvant therapy
Cemiplimab-rwlc	PD1	SCC	2018	Metastatic or locally advanced cutaneous squamous cell carcinoma patients who are not candidates for curative surgery or curative radiation
Pembrolizumab	PD1	SCCHN	2019	First line therapy for SCCHN
Nivolumab	PD1	SCLC	2018	Progressive metastatic small cell lung cancer with progression after platinum
Durvalumab + CTx	PD-L1	SCLC	2020	First line therapy
Pembrolizumab	PD1	SCLC	2019	third line therapy
Dostarlimab	PD-L1	solid tumors	2021	second line therapy dMMR solid tumors
Pembrolizumab + CTx	PD1	Sq NSCLC	2018	Metastatic squamous non-small cell lung cancer
Nivolumab	PD1	Sq NSCLC	2015	Metastatic squamous NSCLC with progression on or after platinum
Atezolizumab	PD-L1	TNBC	2019	Unresectable locally advanced or metastatic TNBC patients whose tumors express PD-L1
Pembrolizumab + CTx	PD1	TNBC	2020	TNBC with PD-L1
Pembrolizumab + CTx	PD1	TNBC	2021	neoadj/adj therapy
Nivolumab	PD1	urothelial	2017	Locally advanced or metastatic urothelial carcinoma patients who have disease progression during or after platinum Rx
Durvalumab	PD-L1	urothelial	2017	Locally advanced or metastatic urothelial carcinoma patients who have disease progression during or after platinum Rx
Atezolizumab	PD-L1	urothelial	2016	Locally advanced or metastatic urothelial carcinoma patients who have disease progression during or after platinum Rx
Avelumab	PD-L1	urothelial	2017	Locally advanced or metastatic urothelial carcinoma patients who have disease progression during or after platinum Rx
Pembrolizumab	PD1	urothelial	2017	Esophageal/Gastroesophageal Junction Cancer
Avelumab	PD-L1	urothelial	2020	first line therapy
Nivolumab	PD1	urothelial carcinoma	2021	adjuvant therapy for high-risk urothelial carcinoma

Neoadj, neoadjuvant; adj, adjuvant; NSCLC, non-small cell lung cancer; PD-L1, programmed death-ligand-1; LN, lymph nodes; SqCC, squamous cell carcinoma; Non-SqCC, non-squamous cell carcinoma; RCC, renal cell carcinoma; TNBC, triple negative breast cancer; HCC, hepatocellular carcinoma; Eso, Esophageal, GC, gastric cancer; GEJ, gastroesophageal junction, ESCC, esophageal squamous cell carcinoma; CTx, chemotherapy; SCLC, small cell lung cancer; SCCHN, squamous cell carcinoma, head and neck; MSI-H, microsatellite instability high; dMMR, mismatch repair deficient.

Thus, Ipilimumab was also approved by the US FDA for use in combination with nivolumab (ipi/nivo) for adjuvant therapy of patients and as a monotherapy for patients with unresectable or metastatic melanoma. Furthermore, ipi/nivo therapy was successfully used to treat patients with other tumors, including hepatocellular carcinoma (HCC)¹⁹⁶, non-small cell lung cancer (NSCLC)¹⁹⁷, unresectable malignant pleural mesothelioma¹⁹⁸, advanced RCC¹⁹⁹, unresectable or metastatic esophageal squamous cell carcinoma (ESCC), and metastatic colorectal carcinoma (CRC) with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR)²⁰⁰. In summary, combination therapy with ipi/nivo has been shown to be superior to monotherapy with ipilimumab or nivolumab based on therapeutic efficacy but to have a higher rate of immune-related adverse events (irAEs) limiting its clinical use¹⁹⁵.

PD-1 is expressed on activated T cells, B cells, and myeloid cells²⁰¹, and its two ligands, PD-L1 and PD-L2, are expressed on tumor cells and myeloid derived suppressor cells (MDSCs)^{202, 203}. The PD-1/PD-L1 axis results in immunosuppression by inducing apoptosis of activated T-cells, T-cell exhaustion and anergy, enhancing T-reg immunosuppressive function, limiting T cell proliferation, and reducing T cell activation and IL-2 production²⁰². Blockade of PD-1 or PD-L1 interactions with ICIs inhibits the signaling of T cells, their ability to kill cancer cells and create an immune response. CTLA-4, as mentioned previously, is expressed on activated T-cells, and regulates T cell proliferation as an early immune response. This contrasts to PD-1, which suppresses T-cells function as a late immune response, primarily in peripheral tissues¹⁸².

Nivolumab, pembrolizumab, as well as Cemiplimab are fully human IgG4 anti-PD1 mAbs. Recently, in patients with platinum-resistant R/M head and neck squamous cell carcinomas (HNSCC), nivolumab monotherapy was randomized and compared with second-line single agents (docetaxel, methotrexate, or cetuximab) in the phase III trial CHECKMATE 141. This phase III trial was composed of 361 patients, with response rates (RRs) consistently higher in the nivolumab cohort than in the other cohort studied. In the nivolumab cohort there were 6 CRs and 26 PRs with a median OS of 7.5 months with nivolumab versus 5.1 months in the control cohort. Further, patients receiving nivolumab had a significantly lower mortality risk (30%) and the estimated PFS was 9.9% in patients treated by medical decision versus 19.7% in the patients that received nivolumab. These data demonstrate the benefit of nivolumab to patients over a longer time and support a significantly improved quality of life²⁰⁴. In addition, grade 3 or 4 adverse events (AEs) were reduced from 35.1% to 13.1% with nivolumab administration²⁰⁵. Together, these studies support the use of nivolumab as a monotherapy for relapsed/metastatic (R/M) HNSCC patients with disease progression or following platinum-based therapy.

Besides the use of nivolumab monotherapy in patients with HNSCC, it is also indicated for patients with unresectable or metastatic melanoma, metastatic NSCLC, advanced or metastatic urothelial carcinoma, advanced relapsed or metastatic esophageal squamous cell carcinoma (ESCC), metastatic CRC (MSI-H or dMMR), advanced renal cell carcinoma and relapsed or refractory (R/R) chronic Hodgkin lymphoma (cHL)²⁰⁶. Pembrolizumab has shown significant anti-tumor activity in HNSCC patients, resulting in improved ORR and moderate toxicity. Pembrolizumab received accelerated approval from the FDA as a monotherapy in 2016 for the treatment of R/M HNSCC patients with disease progression or after platinum-containing chemotherapy based on the KEYNOTE-012 phase Ib study that showed a response rate of 18% at 9 months, a median OS at 8 months and a 6-month PFS of 23%. Further, in this trial grade 3 and 4 toxicities occurred in only 9% of patients²⁰⁷. A confirmatory phase III study was undertaken that compared pembrolizumab to standard therapies in patients with platinum-resistant R/M HNSCC. In this open-label study 247 patients were randomly assigned to receive pembrolizumab and 248 were to receive standard therapy (methotrexate, docetaxel, or cetuximab). In the cohort given pembrolizumab, the MST was significantly higher as compared to the cohort given standard therapy (8.4 months versus 6.9 months, respectively). In addition, significantly fewer patients experienced grade 3 treatment-related AEs, i.e., 13% versus 36%²⁰⁸. In 2019, the FDA approved pembrolizumab, alone or with chemotherapy, as a first-line treatment in patients with unresectable R/M HNSCC. Another study using pembrolizumab monotherapy or a combination of pembrolizumab with a platin-based agent and 5-fluorouracil was compared to cetuximab with platin-based and 5-fluorouracil chemotherapy. The pembrolizumab combination proved to have better OS. Patients survived an average of 13 months compared to 10.7 months on the cetuximab combination²⁰⁹. However, the effectiveness of pembrolizumab treatment was dependent upon the PD-L1 combined positive score (CPS). Further, immune cell PD-L1 expression has been shown to be predictive for PD-1 efficiency in different forms of solid cancers²¹⁰. In the KEYNOTE-048 study, patient survival was highest when the PD-L1 CPS score was greater than 20 (14.9 months for pembrolizumab monotherapy vs. 10.7 for chemotherapy and cetuximab)²⁰⁹. Furthermore, when the PD-L1 CPS score was greater than 1, pembrolizumab therapy was superior to cetuximab-based therapy (12.3 months vs. 10.3). Based on the results of the KEYNOTE-048 study, pembrolizumab as monotherapy or in combination with chemotherapy was approved as first-line therapy for all patients with R/M HNSCC with a CPS²⁰⁹.

In addition to patients with R/M HNSCC, pembrolizumab has been studied as a monotherapy in patients with metastatic melanoma and NSCLC²¹¹, advanced or metastatic urothelial carcinoma²¹², R/R cHL²¹³, metastatic CRC, ESCC²¹⁴, cervical cancer, Merkel cell carcinoma, endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and primary mediastinal large B-cell lymphoma. Cemiplimab has been approved for the treatment of locally advanced (la) or metastatic (m) cutaneous squamous cell carcinoma (cSCC)²¹⁵, basal carcinoma (BCC), and locally advanced or metastatic NSCLC²¹⁶. However, its effectiveness in the treatment of patients with R/M HNSCC has not yet been demonstrated²¹⁷. Table 4 provides an overview of the currently approved mAbs in the field of ICI therapy.

In addition to the anti-PD-1 antibodies, atezolizumab, durvalumab, and avelumab are IgG1 mAbs targeting PD-L1²¹⁸. Atezolizumab, an anti PD-L1 mAb, documented clinical efficacy in patients with previously treated, advanced HNSCC; however, additional studies are ongoing²¹⁸. A recent phase II study (CheckRad-CD8) in patients with HNSCC demonstrated the feasibility of single-cycle induction treatment with cisplatin–docetaxel and durvalumab combined with tremelimumab (anti-CTLA-4 mAb). This trial achieved a high pathologic CR (pCR) rate in patients with locally advanced HNSCC²¹⁹. A phase II study (HAWK) also demonstrated the anti-tumor activity of durvalumab (anti-PD-L1 mAb) monotherapy in patients with R/M HNSCC. However, two phase III studies (EAGLE and KESTREL) investigating the efficacy of durvalumab or durvalumab combined with tremelimumab (anti-CTLA-4 mAb) versus the standard of care or the EXTREME treatment regimen failed to demonstrate an improvement in the OS of patients with R/M HNSCC²²⁰. Further, avelumab (anti-PD-L1 mAb) monotherapy has also shown clinical efficacy in patients with platinum-refractory/ineligible R/M HNSCC in a phase Ib study²²¹.

The use of ipilimumab to treat metastatic melanoma resulted in 20% of patients surviving more than 4 years, with a small percentage of patients surviving 10 years or longer²²². Ipilimumab is also widely used to treat patients with lung cancer, kidney cancer, and prostate cancer²²². However, its effectiveness for these other tumors is usually considered unsatisfactory. In general, the antitumor efficiency of PD-1/PD-L1 inhibitors are better than CTLA-4 inhibitors. In patients with advanced melanoma, pembrolizumab has been shown to be more effective than ipilimumab at extending PFS and OS²²³. In addition, the use of Nivolumab in patients with cHL has documented a treatment response rate of more than 80%²²⁴. Despite the antitumor efficacy of ICIs, adverse effects must be considered. The most common irAEs, include rash, colitis, diarrhea, hepatotoxicity, endocrinopathies, and occasionally can include fatal AEs²²⁵. Thus, the clinical utility of ICIs is limited by the relatively low response rates and relatively high treatment-related toxicities. While some biomarkers appear able to predict therapeutic responses and irAEs of ICI therapy, additional studies are required to identify patients’ subsets expected to be responsive. Further, patients with higher mutational and neoantigen levels have been shown to have significantly better clinical benefit to anti-CTLA-4 antibodies²²⁶. Tumor PD-L1 expression has also been associated with significantly improved ORR in patients treated with PD-1/PD-L1 inhibitors. In one clinical study the ORR in the PD-L1 positive cohort was 34.1% while the PD-L1 negative cohort was 19.9%²²⁷. Thus, biomarkers are used to identify cancer patients who will benefit from ICI therapy. In other studies, the expression of CD177 and CEA Cell Adhesion Molecule 1 (CEACAM1) has been related to colitis following ipilimumab treatment²²⁸, i.e., patients treated with ipilimumab, with increased expression of CD177 and CEACAM1, receive prophylactic countermeasures to reduce immune-related colitis. It is expected that additional biomarkers will be identified to predict patients at risk of irAEs or with a higher probability of therapeutic benefit; for instance, mismatch repair (MMR) deficiency and infiltrating T-cell to tumor burden ratio^{229, 230} with potential to further improve outcomes.

Thus, despite its potential, ICI therapy has drawbacks, including an inability to induce a respond in a significant number of patients. This failure is associated with a tumor’s refractoriness, acquired resistance, and deleterious irAEs. The goal of increasing ICI efficacy based on ICI combinations has been extended beyond the use of mAbs to CTLA-4 and PD-1. The rationale is the targeting of differential mechanisms by distinct checkpoint receptors and their corresponding ligands in the TME. This co-targeting is proposed to trigger additive immune responses, increased PFS and reduce the number of unresponsive patients. In March 2022, the US FDA approved another combination immunotherapy, termed Opdualag, which is the combination of the LAG-3 and the PD-1 ICIs, specifically relatlimab and nivolumab for the treatment of adult and some pediatric patients with unresectable or metastatic melanoma²³¹. LAG-3 is expressed by many T-cell subsets, including: CD4 T-helper cells, cytotoxic CD8 T-cells, activated T-cells, NK-T-cells, effector CD4 T-cells, regulatory T-cells, CD8 tumor-infiltrating lymphocytes and tumor-infiltrating antigen-specific CD8 T-cells^{232, 233, 234}. However, LAG-3 expression is not limited to T-cells, and it is also found on B-cells, natural regulatory plasma cells or plasmacytoid DCs ^{231, 234, 235}. Opdualag was clinically evaluated in a multi-institutional, randomized, and blinded phase II/III trial that enrolled 714 patients. The purpose of this study was to determine whether relatlimab in combination with nivolumab was more effective than nivolumab as a monotherapy against unresectable melanoma or metastatic melanoma. In this study Opdualag demonstrated a significant improvement in PFS as compared to nivolumab alone with a PFS median of 10.1 months for the Opdualag cohort versus 4.6 months in the nivolumab cohort. However, Opdualag did not demonstrate a significant improvement in OS as compared to nivolumab alone. To date, the Opdualag cohort has yet to lose half of the patients, so it has not reached a MST versus 34.1 months for the MST in the nivolumab cohort²³⁶.

ADOPTIVE CELLULAR THERAPY

ACT is the last major form of immunotherapy that we will discuss in this review. The field of immunotherapy, based on ACT started with the isolation and in-vitro expansion/activation of tumor infiltrating T-cells (TILs) from a patient’s primary tumor, followed by infusion back into the cancer patient²³⁷. To date, no cellular TIL therapy has received FDA approval and all clinical studies are regulated by 21 CFR-1271 for the manipulation of these experimental products. These efforts have extended to include the use of NK cells since they can also provide a rapid and potent cytotoxicity to solid tumor metastasis and hematological cancers²³⁸. There are many forms of ACT, including the infusion of cultured TILs obtained directly from the tumor²³⁷; isolating and expanding T-cell clone(s)²³⁹; using T-cells that have been genetically engineered in vitro to recognize and attack tumor cells or infusing allogeneic products containing lymphocytes. The first use of genetically engineered T cells used TCR α and β heterodimers to form TCR–CD3 signaling complexes with specificities for target tumor antigens as reported in 1989^{240, 241}. A second approach to genetically engineer T-cells uses a product termed chimeric antigen T cells (CAR T-cells) that were described for the first time in the mid-1990s but failed in preclinical studies and early clinical trials due to the technical challenges and knowledge gaps that were addressed a few years later²⁴². Most forms of clinical ACT are combined with lymphodepletion chemotherapy to support T-cell proliferation and persistence²⁴³. ACT with CAR-T cells, the only form of ACT that includes therapeutics approved by the FDA to date has resulted in positive outcomes in clinical trials with melanoma and hematologic malignancies, as well as with some solid cancers. However, ACT efficiency is limited due to toxicity and in some instances mortality secondary to cytokine release (“cytokine storm”, or “cytokine release syndrome”) and cerebral edema²⁴⁴. One of the earlier forms of ACT is allogeneic hematopoietic stem cell transplantation (aSCT). The manipulation of stem cells is regulated by the FDA based on 21-CFR-1271. This form of ACT provided three definitive observations supporting the therapeutic role of such T-cell products. The first observation was decreased relapse rate with the use of matched sibling donor compared to syngeneic twin donor²⁴⁵. The second was a lower relapse rate in patients who developed acute or chronic graft versus host disease (GVHD)²⁴⁶. The third observation was an increase in relapses and lower response rate when T-cell depleted stem cell products were used to reduce GVHD²⁴⁷. These observations underscored the role of a graft versus leukemia (GVL) effect, which is associated with the infusion of donor T-cells and their response to minor histocompatibility mismatch²⁴⁸ and have led to the use of donor lymphocyte infusion (DLI) to prevent/treat relapse post-transplant^{249, 250}. Numerous studies using DLI with a set number of total T-cells per Kg²⁵¹, ones depleted of MHC responsive T-cells²⁵², or ones with activated DLI²⁵³ have been undertaken with varying degrees of success. Recent studies using alternate dosing and timing of ATG have been used to regulate T-cell recovery and or T-cell activation for control of GVHD with retention of GVL^{254, 255}.

In the 1980s the concept of autologous stem cell transplantation (auto SCT) was developed as a rescue tool to enable high dose chemotherapy for higher grade non-Hodgkin’s lymphoma, in 2^nd complete or partial remission²⁵⁶, or multiple myeloma responding to initial chemotherapy^{257, 258}. Later studies by Carl June’s group investigated the benefit of activated T-cells post auto transplant with no clear improvement in transplant efficacy^{259, 260}. However, studies by Rapoport and Stadtmauer et al demonstrated that a combination of pre- and post-transplant vaccine combined with activated donor T-cells may help restore post-transplant immunity against Pneumococcal pneumonia or influenza^{261, 262}. The same group applied the same concept of combination immunotherapy described above to transfer immunity against tumor specific antigens^{263, 264}. They then took the next step of demonstrating the first clinical success of infusing chimeric antigen receptor T-cells (CAR-T) in patients with chronic CLL²⁶⁵. The initial ACT protocols used TILs, which are T-cells moved from the bloodstream, arrested in a primary tumor where they can potentially proliferate. TILs are a heterogeneous population of polyclonal T-cells that have broad antigen recognition that includes tumor cells²⁶⁶. It is believed CD8+ and CD4+ T-cell populations have a critical role in regulating the growth of tumors via a variety of mechanisms^{267, 268}.

The favorable prognostic significance of a high T-cell infiltrate is recognized as a characteristics of melanomas²⁶⁹, as well as other tumors. Unfortunately, these infiltrating TILs are largely therapeutically ineffective due to inflammatory infiltrates in the TME that include suppressive regulatory T cells and myeloid suppressor cells^{270, 271}. While preclinical studies demonstrated the potential of isolated immune cells to mediate tumor regression²³⁷, the first clinical study of TILs was performed in 1988 in patients with metastatic melanoma⁴³. Patients in this study were also given adjuvant IL-2, a strategy discussed above in the cytokine section of this review. This study found that ACT of TILs in combination with adjuvant IL-2 resulted in a 55% ORR.

However, since many of the T-cells within a TIL population are dysfunctional²⁷² (or absent due to low tumor immunogenicity in some cancers)²⁷², this strategy does not work for a significant proportion of patients²⁷³. Further, the isolation and expansion of TILs relies on the creation of a population of T-cells that may be able to overcome this dysfunction either through co-culture with IL-2 and/or selection for tumor antigen specific T-cells. While some of these TIL trials have shown significant success as a treatment modality, widespread adoption has not occurred. The traditional manufacturing process for the clinical use of TILs takes approximately 5–7 weeks from tumor resection to TIL production²⁷⁴, which may be too long for some patients with metastatic disease. Furthermore, isolated TILs are frequently unable to grow or demonstrate strong effector responses²⁷⁵, which may be associated with poor TME immune infiltration. Combination approaches including the addition of checkpoint blockade to TIL products (NCT02621021; NCT02652455; NCT03645928; NCT03215810) are targeting improved TIL efficacy and to overcome these issues. In a recent phase III clinical trial of 186 patients with unresectable stage IIIC-IV melanoma that were predominantly refractory (86%) to anti-PD1 monotherapy, the efficacy of 4 doses of ipilimumab versus one dose of TIL therapy (5×10⁹) was evaluated. The median PFS for ipilimumab was 3.1 months versus 7.2 months in the TIL cohort and the ORR was 21% for ipilimumab versus 49% for the TIL cohort. Consistent with these observations the median OS was 18.9 months in ipilimumab versus 25.8 months in TIL cohort²⁷⁶. This study suggests that TIL therapy might provide an encouraging option for patients unresponsive to ICIs. At present, TIL therapy is being tested mainly as a second-line treatment in melanoma patients²⁷⁷.

TCR-engineered T-cell technologies provide an extension for the TIL therapy rationale. Since TILs have acquired tumor recognition, this suggests the possibility to genetically modify naïve lymphocytes to recognize tumor antigens. In the late 1990s at the NIH, researchers were able to successfully transduce human peripheral blood lymphocytes (PBL) with genes encoding a MART-1-specific TCR, which is a tumor antigen that is expressed by most melanoma tumors²⁷⁸. Based on this approach an anti-MART-1 TCR was tested in an early clinical study of 15 patients with melanoma²⁷⁹ demonstrating clinical responses in 2 of 15 patients with metastatic melanoma supporting TCRs as a feasible therapeutic strategy. Nevertheless, identifying individual neoantigens for a patient is laborious, time consuming and costly, so targeting a shared mutational antigen specific to numerous tumors and clonally conserved is presently considered more practical. It should be noted that genetically engineered TCRs are HLA restricted and thus must be HLA matched for efficiency. Because of this many of the TCRs being studied clinically are restricted to HLA-A*0201, which is under-represented in African and Asian populations but found in nearly half of Caucasian populations²⁸⁰. As TCR studies expand, it is critical that non-HLA-A*02 serotypes be considered to benefit diverse patient populations. Furthermore, since HLA alterations can provide escape mechanisms for some TCRs, the ability to overcome these resistance mechanisms will be critical to future development.

A second generation of genetically modified T cells was developed that are used clinically, known as CAR-T cells, have been developed for adoptive transfer and have achieved substantial advances in the treatment of malignant tumors²⁸¹. CAR-T-cell therapies utilize antibody fragments that recognize tumor membrane tumor specific antigens. The first generation of CAR-T cells used genetically modified T-cells with antibody specificity by expressing immunoglobulin-TCR chimeric molecules as functional receptors²⁴⁰. However, these CAR-T cells were unable to survive in the host. In 1998, Maher et al. established a new generation of CAR-T cells by introducing costimulatory molecules such as CD28 into the engineered CARs to allow these modified T cells to survive and remain active in the body of a patient^{282, 283}. They subsequently demonstrated that CD19-specific CD28/CD3-ζ dual-signaling CAR-modified T cells could induce molecular remissions in adult patients with acute lymphoblastic leukemia (ALL)²⁸⁴. In addition, other molecules have been examined for their efficacies when conjugated in CARs, and Porter et al. demonstrated that autologous T-cells genetically modified to target B-cell antigen CD19 by expressing anti-CD19 linked to CD3-ζ and 4-1BB signaling domains resulted in potent CD19-specific immune responses in chronic lymphocytic leukemia (CLL) patients²⁶⁵. These findings support the antitumor efficacy of CAR-T therapies in human cancers. TCR-T therapy with TCR gene transfer to PBLs derived from melanoma patients could generate effector T cells with antitumor reactivity in vitro²⁷⁸. Thus, both CAR-T-cell and TCR-T-cell therapies have achieved substantial advances in cancer treatment and have generated encouraging clinical outcomes.

Table 5 identifies the currently approved CAR-T products including four approved CAR-T products for non-Hodgkin lymphomas (NHL), two for B-cell ALL and two for multiple myeloma (MM)²⁸⁵. The first FDA approved CAR-T product was axicabtagene ciloleucel (Yescarta), which was approved in a phase 2, multicenter trial that included 111 patients with R/R high grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma, or transformed follicular lymphoma (FL). Following lymphodepleting chemotherapy with fludarabine and cyclophosphamide, patients received a single infusion of a personalized cell product. Patient results from this trial demonstrated a high ORRs of 82% with CR rates of 54%. After a median follow-up of 15.4 months, 40% of patients remained in remission and the OS was 52% at 18 months. Common toxicities included CRS and immune effector cell-associated neurotoxicity syndrome (ICANS). Grade 3 and higher CRS and ICANS were reported in 13% and 23% of patients, respectively²⁸⁶. Based on these data, axicabtagene ciloleucel became the first FDA approved CAR-T product in October 2017. Longer-term follow-up of this trial was published by Locke et al. with a median follow-up of 27.1 months²⁸⁷. Tisagenleucleucel (Kymriah) obtained the second FDA approval for a CAR-T cell therapy and was approved in 2017 for pediatric and young adults with R/R B-ALL²⁸⁸. It was also the second CAR-T cells product to be approved by the FDA for the treatment of R/R HGBCL in another study²⁸⁹. These genetic engineered cell products have documented durable activity and manageable safety profiles, relative to conventional salvage chemotherapy in several clinical trials²⁹⁰. In February of 2021, lisocabtagene maraleucel (Breyanzi) became the third CD19-targeted CAR-T product to be FDA approved for R/R HGBCL²⁹¹. Brexucabtagene autoleucel (Tecartus) was the first anti-CD19 CAR-T cell therapy approved for R/R mantle cell lymphoma²⁹². It was also recently approved for adult patients with R/R B-acute lymphocyte leukemia ²⁹³. The first CAR-T cell therapy available for treatment of patients with MM is idecabtagene vicleucel (Abeccma). Its target antigen is the B-cell maturation (BCMA) antigen, and it received accelerated FDA approval in March of 2021 for patients with R/R MM²⁹⁴. The approval of ciltacabtagene autoleucel (Carvyktitm) in February 2022 by the US FDA provided a second CAR T-Cell therapy for R/R MM patients who have failed 4 or more lines of therapy. This CAR-T cell products has BCMA-targeting single-domain antibodies that detect two epitopes of BCMA²⁹⁵.

Table 5:

CAR-T cell therapeutics approved by the FDA and their indications.

Trade name	Generic name	Target antigen	Indication(s)	FDA approval
ABECMA	idecabtagene vicleucel	BCMA	R/R multiple myeloma after ≥ 4 prior lines of therapy	Mar-21
BREYANZI	lisocabtagene maraleucel	CD19	R/R LBCL after ≥ 2 prior lines of therapy	Feb-21
			LBCL, refractory to first line chemoimmunotherapy or relapse within 12 months of first line chemoimmunotherapy
			LBCL, refractory to first line chemoimmunotherapy or relapse after first line chemoimmunotherapy and not eligible for HSCT due to comorbidities or age	Jun-22
CARVYKTI	ciltacabtagene autoleucel	BCMA	R/R multiple myeloma after ≥ 4 prior lines of therapy	Feb-22
KYMRIAH	tisagenlecleucel	CD19	R/R B-cell precursor ALL (patients ≤ 25 years)	Aug-17
			R/R LBCL after ≥ 2 prior lines of therapy	May-18
YESCARTA	axicabtagene ciloleucel	CD19	R/R LBCL after ≥ 2 prior lines of therapy	Oct-17
			R/R FL after ≥ 2 prior lines of therapy	Mar-21
			LBCL, refractory to first line chemoimmunotherapy or relapse within 12 months of first line chemoimmunotherapy	Apr-22
TECARTUS	brexucabtagene autoleucel	CD19	R/R MCL	Jul-20
			R/R B-cell precursor ALL (adult)	Oct-21

ALL, acute lymphocytic leukemia, LBCL, large B-cell leukemia; R/R, Relapsed/Refractory; HSCT, hematopoietic stem cell transplant; MCL, mantel cell lymphoma

THE FUTURE OF IMMUNOTHERAPY

The advancement of immunotherapy is promising and requires unlocking the pharmacokinetic and pharmacodynamic mechanisms required to improve the persistence, durability, and efficacy of these therapeutics. Further it may be possible to engineer additional desired phenotypes into TILs. CAR-T cells and TCR-T cells to address these critical parameters and reduce toxicity. This may include providing supportive cytokines or other immunomodulators either by co-administration with ACT infusion or by gene editing of the effector cells. Using DCs or artificial APCs to present peptide mixtures to the host T-cells to expand the tumor specific T-cell clones to support the expansion of active, specifically primed T-cells of the right phenotype to target tumor cells and limit exhaustion. Combinatorial approach (es) of CAR-T cells, vaccines or TILs and ICIs or other drugs to suppress MDSC expansion or function have shown potential in multiple preclinical models and encouraging results clinically. This area of clinical investigation has potential to rapidly improve outcomes. To date, PD-1 has provided the apparent best monotherapy, but its efficiency has been shown to be improved using multiple inhibitory pathways (e.g., PD-1, CTLA-4, LAG-3). Ultimately, the ideal combination for efficiency will likely include co-infusion of T-cell effectors (CAR-T, TCRs, TILs and or DLI) with concurrent or adjuvant ICI blockade to enhance T cell potency. Further, expansion of bioactivity to achieve improved outcomes will include cytokines or other approaches to limit T-cell exhaustion, facilitate expansion, and limit effector cell apoptosis to achieve maximal anti-tumor responses with minimal off-tumor collateral toxicity. Within this review we have touched on these approaches and have referenced reviews that provide additional insight into these future strategies.

HIGHLIGHTS.

In recent years CAR-T cells, Check point inhibitors have provided a major focus for the treatment of neoplasia.

However, cytokines, vaccines, vaccine adjuvants and biologic response modifiers have also been recognized as effective interventions.

Combination approaches utilizing all of these modalities are expanding anti-tumor efficiency.