The Challenge of Immunologically Hot vs. Cold Tumors

Cancer immunotherapy has revolutionized the oncology landscape over the last fifteen years, working by restoring the innate immune system’s ability to detect and mount a response to malignant cancer cells that otherwise evade immune surveillance. Today, there are over 30 immunotherapies approved to treat different cancers, with perhaps the most dramatic benefits observed in relapsed/refractory B-cell acute lymphoblastic leukemia using chimeric antigen receptor-T (CAR-T) cell therapy — in which complete remissions have been achieved in over 70% of adult patients.1 Checkpoint inhibitors have similarly delivered remarkable long-term remissions in patient subsets with advanced melanoma (with 50% of patients surviving at 5 years), and advanced non-small cell lung cancer (NSCLC; 27%), effectively rewriting the narrative for these historically hard-to-treat cancers in settings where the disease has already advanced.2 However, for approximately 70% of patients who receive a diagnosis, the promise of cancer immunotherapy remains out of reach.3

The effectiveness of immunotherapy fundamentally relies on the body’s immune system to target and destroy cancer cells. If immune cells are excluded or scarce in or around the tumor, if their immune function is suppressed, or if they are unable to detect tumor antigens as markers of a threat, this means that cancer immunotherapy has no active population of functional immune cells to mobilize against the tumor. Tumors that fit this profile are known as immunologically cold, and usually exhibit the poorest responses to immune checkpoint inhibitors.4

In this blog article, we explore the core challenges that cold tumors present to effective immunotherapy, recent advances aimed at converting them into immunologically active (or “hot”) tumors, and how our lead candidate, Tumor Defense Breaker™ L-DOS47, is designed to overcome one of the most critical obstacles: the suppressive tumor microenvironment (TME).

Immunologically Hot vs. Cold Tumors

Cancer immunotherapies broadly fall into 5 categories that rely to different degrees on a common denominator: the patient’s immune system. These categories are:

(1) Autologous or allogeneic cell-based immunotherapy, which involves replenishing the patient’s immune system with their own modified, functional immune cells or immune cells from a donor (e.g. bone marrow transplantation, BMT);

(2) Immunomodulating drugs or other substances that take the brakes off the patient’s innate immune system, and help it recognize and mount an immune response against malignancies (e.g. the anti-cytotoxic T-lymphocyte-associated protein 4 [anti-CTLA-4], ipilimumab, Yervoy®, for melanoma);

(3) Antibody-based targeted therapies that specifically target proteins or markers on the tumor and its microenvironment to block tumor growth, flag cancer cells for attack by the immune system, and/or deliver potent anti-cancer drugs directly to the tumor (e.g. PD-1 inhibitors, such as pembrolizumab, Keytruda®);

(4) Preventative and therapeutic vaccines that prime and train the patient’s immune system to recognize and target specific cancer-associated antigens when it encounters them (e.g. Sipuleucel-T, for prostate cancer); and

(5) Oncolytic virus therapies, in which modified viruses that selectively infect and cause tumor cells to burst (lyse), in turn causing them to release antigens that help the immune system recognize and attack the remaining cancer cells (e.g. T-VEC for advanced melanoma).5

How well the immune system can mount a cell-mediated attack on cancer cells depends on the activation, infiltration, viability, detection and elimination of tumors by effector T cells — the immune system’s front-line fighters.6

It is estimated that approximately 50% of all human tumors are immunologically hot.7 Hot tumors (or immune-inflamed tumors) are highly infiltrated by T cells and express programmed death-ligand 1 (PD-L1), an immune checkpoint and cell-surface protein that helps tumors evade detection by innate immune cells.8 In cancer, PD-L1 binds to its receptor, programmed cell death protein 1 (PD-1), found on the surface of T cells, which effectively works to silence T cell activation and suppresses their ability to recognize and attack the tumor cells. In this context, antibody-based cancer therapies that bind to and inhibit PD-1 (such as pembrolizumab and nivolumab, Opdivo®), or PD-L1 (e.g. atezolizumab, Tecentriq®), work to block the interaction between the tumor’s PD-L1 and the T cells’ PD-1 immune checkpoints, leaving the tumor vulnerable to immune surveillance and attack. Immunologically hot tumor cells additionally present a high Tumor Mutational Burden (TMB), or multiple mutations in the tumor’s DNA, that in turn produce neoantigens which can be detected as threats by the immune system.

In immunologically cold tumors, including cancers of the breast, ovary, pancreas and brain (glioblastoma), the absence or limited infiltration of immune cells within the tumor and its microenvironment pose significant challenges to the efficacy of cancer immunotherapy.9 Cold tumors are either immune-desert, with T cells unable to infiltrate the main body of the tumor and the supporting tissue around the cancer cells (respectively, the tumor parenchyma and tumor stroma), or they are immune-excluded (“altered”), with T cells remaining in the tumor stroma and unable to penetrate the parenchyma.10 In immunologically cold tumors, T cells are unable to effectively infiltrate the tumor and its stroma due to a combination of physical barriers (a stiff extracellular matrix, ECM, and abnormal blood vessel function that prevents immune cell entry, known as endothelial suppression), and an active network of immune-suppressing signals that dampen T cell activation and function.11 Tumors are especially adept at coopting immune cells that normally serve to suppress autoimmunity or prevent overactivation of the immune system (such as regulatory T cells, Tregs, a type of helper T cell, and myeloid-derived suppressor cells, MDSCs), and exploiting them to evade immune detection and promote tumor survival — effectively turning the body’s immune system against itself. In addition, tumors themselves deploy a range of mechanisms to evade immune surveillance, including releasing abnormal levels of cytokines and chemokines that block immune cell signaling, and producing metabolites that weaken immune cell activity. Cold tumors are characterized by almost no PD-L1 expression and minimal expression of neoantigens, making them less likely to be detected by the immune system and to trigger an immune response.12

To overcome treatment challenges presented by immunologically cold tumors, a growing body of research is advancing strategies to turn them immunologically hot, ultimately aiming to render them more responsive to immunotherapy. These strategies include promoting infiltration of T cells into the tumor parenchyma (using CXCR4 and TGFβ inhibitors, and antiangiogenic therapies), increasing availability of tumor-specific T lymphocytes (using cell-based immunotherapy and cancer vaccines), activating dendritic cells (DCs) to promote the presentation of tumor antigens to T cells (by administering local immune adjuvants), and promoting T cell priming and activation by inducing immunogenic cell death (using oncolytic viruses, chemotherapy and radiotherapy).13

In reality, while the classification of tumors as hot or cold has proven utility for treatment planning, tumor biology is heterogeneous and the distinction between the two is not always clear cut. Some tumors exhibit attributes characteristic of hot, cold and altered tumors, and not all patients with hot tumors respond to immunotherapy (some tumors may also develop resistance to treatment later on, essentially transitioning from immunologically hot to cold).14 In this context, the TME plays a key role in the immune trajectory and fate of tumors.15

The Role of the Tumor Microenvironment (TME)

Whereas healthy cells primarily generate energy in their mitochondria using oxygen through oxidative phosphorylation, tumors preferentially generate energy by breaking down glucose through glycolysis, even in the presence of oxygen and functioning mitochondria — a process is known as the Warburg effect.16 This leads to the accumulation of lactate (the ionized form of lactic acid) in the tumor and, subsequently, to acidification of the extracellular pH in the TME, creating an immunosuppressive environment that favors immune escape and cancer cell growth.17

The TME is a dynamic, sophisticated ecosystem of diverse cells embedded in a fibrotic, blood vessel-rich network that plays a crucial role in shaping the development, growth, and immune-escape of solid tumors.18 Far from passive, the TME acts as an active accomplice in immune evasion and therapeutic resistance. Acidic conditions in the TME profoundly limit T cell activation by disrupting receptor signaling pathways and enhancing the activity of Tregs and MDSCs that suppress cytotoxic T cell activity, suppressing T cell motility, and restricting glucose consumption by tumor-infiltrating T cells, leading to T cell exhaustion and tumor progression.19 In this immunologically hostile setting, inhibiting lactate production or alkalizing the physiological pH of the TME has emerged as a promising therapeutic target to take the brakes off anti-cancer immunity, ‘heat up’ cold tumors, and prime them to become more responsive to today’s leading cancer immunotherapies.20

At Helix BioPharma, our lead candidate, Tumor Defense Breaker™ L-DOS47, is designed to deliver exactly on this promise. L-DOS47 is an antibody-enzyme conjugate (AEC) that works to elevate the pH of the acidic TME and deliver a much-needed assist to antibody-based immunotherapies, such as pembrolizumab. The compound consists of a urease enzyme linked to nanobodies that specifically target carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) — a protein minimally present in healthy tissues but over-expressed in solid tumors (such as NSCLC), where it also mediates immune suppression.21 Upon binding to CEACAM6, the urease enzyme reacts with naturally-occurring urea in the TME, converting urea into ammonia and carbon dioxide, and effectively neutralizing the acidity of the TME.

This mechanism restores the conditions necessary for immune cell infiltration and activity and increases response to checkpoint inhibitors, as evidenced by a recent study combining L-DOS47 with the anti-PD1 checkpoint inhibitor, pembrolizumab, in a mouse model of pancreatic adenocarcinoma (PDAC). In this study, the combination of our compound with pembrolizumab resulted in a 70% greater reduction in tumor volume compared to pembrolizumab monotherapy, as well as a 50% greater reduction in tumor weight compared to pembrolizumab alone in 4 weeks.22 These findings are broadly consistent with findings from earlier clinical trials, in which L-DOS47 improved responses to chemotherapy (pemetrexed and carboplatin) in heavily pre-treated patients with advanced NSCLC—suggesting the conversion of cold, treatment-resistant tumors into more immunologically-active and therapeutically-accessible environments.23

A Stronger Immune Response Starts with the Right Conditions

Despite extraordinary advances in cancer immunotherapy, too many people still face cancers that resist even the most sophisticated immune-based treatments. Immunologically cold tumors, defined by immune exclusion, suppression, and/or absence, remain one of the most challenging frontiers in oncology. At Helix BioPharma, we believe that unlocking immunotherapy’s full potential means addressing the TME head-on to level the battlefield.

Our lead candidate, L-DOS47, is engineered to directly address this critical challenge. By directly targeting CEACAM6 and neutralizing the pH of the acidic TME, L-DOS47 helps reverse immune suppression and re-enable immune recognition. In doing so, it lays the foundation for a more potent response to checkpoint inhibitors and other immune-based approaches — not by replacing these therapies, but by amplifying their impact where they are needed most.

We’re not just trying to make cold tumors hot; we’re working to give cancer immunotherapy its best chance to succeed. Our upcoming study combining L-DOS47 with pembrolizumab in NSCLC reflects this commitment, and moves us closer to a future where immunotherapy becomes an option — and a lifeline — for more people, across more types of cancer.

References

1 https://ashpublications.org/blood/article/144/Supplement%201/1421/531486/Meta-Analysis-of-Complete-Remission-Rates

2 https://www.cancerresearch.org/immunotherapy-facts; https://link.springer.com/article/10.1007/s40257-022-00681-4; https://jitc.bmj.com/content/13/2/e010674

3 https://pmc.ncbi.nlm.nih.gov/articles/PMC9442672/

4 https://www.cancer.gov/publications/dictionaries/cancer-terms/def/cold-tumor; https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1142862/full#B18

5 https://www.cancerresearch.org/immunotherapy-facts

6 https://www.nature.com/articles/s41392-024-01979-x

7 https://ehoonline.biomedcentral.com/articles/10.1186/s40164-024-00543-1

8 https://www.nature.com/articles/s41392-024-01979-x

9 https://www.cancer.gov/publications/dictionaries/cancer-terms/def/cold-tumor

10 https://pmc.ncbi.nlm.nih.gov/articles/PMC10965539/

11 https://pmc.ncbi.nlm.nih.gov/articles/PMC10470046/; https://pmc.ncbi.nlm.nih.gov/articles/PMC9475465/