The development of new cancer treatments involves recognition of the diverse complexity of tumors, their associated microenvironment, and tumor cell metabolism, posing challenges for researchers in finding treatments broadly applicable enough to make a substantial impact on the disease population of interest, yet specific enough to leave normal cells unharmed.
The therapeutic landscape of cancer continues to evolve over time, presenting researchers with new challenges in developing treatments with high specificity for cancer cells. Tumor cells employ a number of characteristic host-evasion techniques, with many specifically geared toward immunosuppression.
These techniques, when combined with other cancer hallmarks such as uncontrolled proliferation, angiogenesis, resistance to programmed cell death, tissue invasion, and metastases (Weinberg, 2014), allow for tumor cells to grow at their primary location and eventually spread systemically throughout the body. Development of novel bioinformatics and genomic technologies have accelerated the identification of target molecules expressed specifically on tumor cells. By selecting targets that play significant roles in immune regulation, programmed cell death, cell growth, circulatory vessel generation, and membrane integrity maintenance, researchers are developing drugs that bind with high specificity, affinity, and selectivity to tumor-specific targets.
Oncology Treatments Using and Cytokines and Antibodies: what can history teach us?
Cancer immunotherapy is not a novel concept. Immunotherapies based on manufacturing of antibody and cytokine drugs have been used in the treatment of cancer for more than three decades. The first cytokine to receive FDA approval was recombinant IFN-α for the treatment of hairy cell leukemia in 1986, and the next approval in 1992 of a cytokine was recombinant IL-2 for the treatment of metastatic renal cell carcinoma. Approval was subsequently granted for metastatic melanoma in 1998. In general, cytokines as monotherapy treatments for cancer have not fulfilled the promise of efficacy seen in preclinical experiments and are often associated with severe dose-limiting toxicities in the clinic. Disturbing inflammatory responses and/or surprising lack of efficacy has been the hallmark of cytokine therapies such as IL-1, IL-3, IL-4, IL-6, IL-12, IL-15, IL-18 and TNF-α have resulted in failed development programs.
Despite their central role in immune modulation, only a handful of cytokine therapeutics have achieved regulatory approval. Major challenges associated with the therapeutic use of cytokines relate to their short serum half-life and unfavorable bioavailability. High doses are required to overcome these problems, which often result in dose-limiting toxicities.
Monoclonal antibody technology was developed in 1975 and the first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3-specific transplant rejection drug, Orthoclone OKT3, in 1986 - more than 30 years ago. However, its use was limited due to induction of HAMA, human anti-mouse antibodies and it was withdrawn in 2011. The first chimeric monoclonal antibody for the treatment of cancer was Rituxan, approved in 1997 for B-cell non-Hodgkin’s lymphoma. Since 1997, more than 90 additional antibody products have been approved by the FDA for a variety of indications, with 10 approvals to date in 2020, and another 18 currently in regulatory review.
The newest iteration of antibody therapies is Antibody-Drug Conjugates (ADCs), where a cytotoxic payload, such as an auristatin or maytansine derivative, is chemically conjugated to highly specific antibodies which transport the toxic drugs selectively to tumor cells and the cytotoxic payload is released inside the tumor cells, sparing normal cells. There are currently nine approved ADC drugs, all for the treatment of cancer. The monoclonal antibody industry has expanded exponentially and is now valued at >$100B and antibodies are the largest drug class in terms of sales.
However, to reach this pinnacle, much basic research on antibody engineering, manufacturing technologies and understanding of antibody immunogenicity and PK was required over the last 30 years.
Newer Approaches Targeting the Immune System
In the last decade, immune checkpoint inhibitors targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) have provided a major paradigm shift in treatment of solid tumors. These checkpoint inhibitor therapies, currently all antibody-based, improved long term remission and overall survival in several cancers including malignant melanoma, non-small lung cancer, renal cancer and others. However, efficacy remains still limited as up to 70% of cancer patients may not respond to checkpoint inhibitors.
CD8+ cytotoxic T cell (CTL)-mediated immune responses, although pivotal for the maintenance of homeostasis, require a supervisory system or checkpoint mechanism to keep a strict balance between immune cell activation and proliferation. As a way to keep the immune system in check, and to avoid the hyperactivation of T cell responses, blockade checkpoints have evolved which mediate negative feedback control over immune activation and thus serve as a “brake” in immune function (Dong, Li, Zhou, Huang, 2018).
The first identified negative regulator of CTL activation was cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) which is constitutively expressed on regulatory T cells and is up-regulated upon T cell activation. CTLA-4 binds to CD80/CD86 co-stimulatory molecules on the surface of antigen presenting cells with a higher affinity than CD28, its classical ligand. Therefore, when CTLA-4 is upregulated it prevents CD28 from binding CD80/CD86 in a competitive manner, resulting in a net inhibitory signal and inactivation of the T cell (Grosso, Jure-Kunkel, 2013). CTLA-4 can outcompete CD28 depending on the amount of CD28 vs. CTLA-4 that is expressed on the T cell surface.
Consequently, T cells constantly surveil and regulate the amount of pro- or anti-immune behavior that they elicit. Responses that net an inhibitory signal force T cells into an inactive or anergic state (Vajaitu, et al., 2018). Without this blockade, CD8+T cells elicit cytotoxic effects and kill target cells.
Programmed Cell Death Protein 1 (PD-1) plays a key role in blocking immune responses and promoting self-tolerance through modulation of the activity of T-cells, inducing apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells. PD-1 is expressed on T cells, B cells, Natural Killer cells (NKs), and Myeloid-Derived Suppressor Cells (MDSCs). PD-1 has two ligands, PD-L1 and PD-L2, which are expressed on antigen presenting cells and activated T cells, however, PD-L1 is also expressed by tumor cells as an “adaptive immune mechanism” to attenuate the host immune response against tumor cells and lead to cancer immune evasion.
Blocking the inhibitory T cell signaling generated by CTLA-4 or PD-1 binding with their cognate receptors causes formerly activated T cells to transition into an anergic state where tumor cells flourish uninhibited. Tumor cells therefore find creative ways of evading immune detection and regulatory checkpoints via immunoediting. Tumor cells can repress MHC class I antigens that are required for activation and proliferation of CTL or they can down-regulate their own tumor-associated antigens (Weinberg, 2014). This combined action by tumor cells results in a net inhibitory signal in any T cell that could be activated by tumor-specific antigens presented to T cells by dendritic cells.
Although these checkpoint inhibitor drugs have shown remarkable improvements in some tumor types, most patients do not respond to checkpoint inhibitor therapies. The early iterations of checkpoint inhibitors, CTLA-4 for instance, exhibited toxicity issues and led some to question their utility. For example, Pfizer’s development of Tremelimumab anti-CTLA-4 drug progressed to Phase III clinical trials and was designated a failure by Pfizer in 2008 and development discontinued.
With an improved understanding of immune-related toxicities and methods to ameliorate them, the drug was resurrected and is currently in late-stage clinical trials by MedImmune/ AstraZeneca. Interestingly, anti-PD-1 and anti-PD-L1 therapies appear to exhibit lower toxicity levels than anti-CTLA-4 antibodies and therefore have multiple approvals across various tumor histologies within the market landscape.
To circumvent tumors developing resistance to anti-cancer therapies, treatment regimens therefore almost always include a cocktail of therapies. Regimens include cisplatin, for example, which acts as a DNA alkylating agent and causes cells to undergo apoptosis, in combination with Hydroxyurea, that works on ribonucleotide reductase, and an antibody therapy such as a PD-L1 inhibitor such as Keytruda. Recurrence rates in cancers create an additional layer of challenges in finding meaningful ways to promote tumor reduction, progression-free survival, and overall survival rates.
Targeted Cell Therapies
Cell therapy involves the transfer of cells to a patient for the purpose of therapeutic intervention. The most challenging aspect of cell therapy is finding a target that, in order to be a safe treatment option, has to be expressed on cancer cells while not being expressed on normal healthy cells. Lack of discernible biomarkers that have the high level of specificity required has led to substantial discovery efforts for the last 20 to 30 years. However, only a small number of 20-50 tumor-specific targets are actively being investigated for clinical testing. Considering that approximately 15,000 genes are expressed in a normal cell, this number exemplifies the difficulty of generating a novel, safe, and efficacious targeted drug candidates for the treatment of cancer.
CAR-T and CAR-NK Engineered Cells
CAR-T cells use a patients’ own cells which are first isolated, activated and then transfected with a gene construct encoding a chimeric antigen receptor to generate a tumor-targeted artificial T-cell receptor. The receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. The specific CAR programs the T-cells to target an antigen present on the surface of tumors.
For safety, CAR-T cells are engineered to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. The CAR-T cells bind to their cognate antigen on tumor cells and become activated, resulting in their proliferation and killing of tumor cells. Once they are infused into a patient, they act as a "living drug" against cancer cells.
As depicted in Figure 1 below, various hematopoietic cell types can be engineered as cell therapies and are generally targeted using single chain antibodies (scFvs) or TCRs (T cell receptors) to initiate a signal cascade which results in the death of the tumor.
Figure 1: Top targets of cell therapies for blood and solid tumors. CD19 continues to be the top target for blood cancers, whereas the top targets for solid tumors are agents for which the tumor-associated antigen (TAA) has not been disclosed (Yu, et al. 2020).
A target, for example CD19, is identified as being expressed in B Cell Leukemia (Beutler, 2001). CD19 is not expressed in normal cells, therefore it meets the criterion of high specificity for tumor cells. For CAR-T therapy, single chain antibodies against CD19 are developed and introduced via retroviral or lentiviral vectors into patient T cells. The cytoplasmic domain of the CD19 receptor is engineered so that when the anti-CD19 antibody binds to its target, it activates the T cell and kills the tumor cells while rendering CD19-negative normal cells unharmed. Two autologous CAR-T drugs have been approved targeting CD19, Yescarta and Kymirah, and both show with remarkable efficacy, but these patient-specific drugs are complex and very expensive to manufacture likely precluding widespread use.
Yescarta and Kymirah, are patient-specific drugs costing $375,000 and $475,000, respectively and sales of these drugs reflect just 1,000-2,000 patient treatments in 2020. High manufacturing costs are challenges are due to the autologous nature of these cell therapies, i.e., the starting cells come from the patient themselves. Alternative manufacturing and cell engineering methods are being developed using allogenic CAR-T cells or allogeneic CAR-NK cells, where an “off the shelf” approach allows cell therapies to be developed as cell lines instead of patient-specific therapies and thus substantially reduce manufacturing costs to a fraction of autologous therapies.
Similarly, B-cell maturation antigen (BCMA) for the treatment of multiple myeloma using (CAR) T cell therapy has exhibited an 80% response rate in Phase III trials in a combined effort of Bluebird Bio Inc. and Celgene Corp. The results of this trial, including a 30-40% cure rate compared to the current standard chemotherapy regimen for multiple myeloma, which exhibits a 20% response rate, are a substantial improvement for the disease treatment.
One notable example of “on-target, off-tumor” toxicity was observed when HER2, a biomarker expressed primarily in breast cancer, was used to develop HER2-CAR-T cells. One patient treated with a high dose of HER2-CAR-T cells developed respiratory distress within 15 minutes of receiving a single dose of 1010 CAR-T cells, followed by multiple cardiac arrests over the course of the next five days, eventually leading to death.
The investigators attributed toxicity to the recognition of HER2 in lung epithelium by CAR-T cells resulting in inflammatory cytokine release producing pulmonary toxicity and CRS causing multi-organ failure. Alternative cell engineering approaches can be used to target the same receptor and more recent studies indicate that reducing CAR-T dose or modifying components of the vector construct may ameliorate toxicities.
Gene Therapies for Cancer
Gene therapies are a field unto themselves, with many different techniques attempting to solve a similar problem: gene mutation or aberrant protein expression resulting in disease. Gene therapies all involve methods for gene transfer and encompass viral, naked DNA, and non-viral systems. Viral gene therapies leverage the evolutionary history of viruses, which have evolved over millions of years to have the capacity to replicate. In many viral systems, the components required for replication can be isolated and separated from the remainder of the viral genome. Viral DNA specific to replicative abilities are isolated and inserted into a cell line called a producer cell with the therapeutic gene inserted into the edited viral genome.
The producer cells provide the proteins essential for vector genome replication and packaging of the therapeutic vector product. This general approach is widely used for retrovirus, lentivirus, and adenovirus vector production. The viral product is purified and can be used directly for patient treatment or viral vectors used to engineer cells, such as CAR-T or CAR-NK cells.
As in cell therapies, there are myriad approaches to developing or using gene therapies. Gene therapies can be used to target specific genes essential for oncogenesis, such as tumor suppressor genes, that have been mutated in certain cancer types. One such gene, p53, is the most commonly mutated gene in cancer. p53 can suppress the formation of tumor cells by orchestrating the repair of broken or mutated DNA.
When a normal cell undergoes a mutational event (e.g., exposure to toxic chemicals, radiation, etc.), p53 performs the role of stopping the cell division cycle to repair the mutated DNA segment. If a fix cannot be made, p53 activates an apoptotic signal triggering cell death. In almost 50% of all human cancers, p53 is mutated and in the remaining cancers where the p53 gene is not mutated, the p53 signaling pathway is frequently disrupted. Therefore, when this tumor suppressive regulatory mechanism is turned off, cancer cells are able to undergo uncontrolled cell division.
Many gene therapy approaches have been employed clinically targeting an array of genes and mechanisms (e.g., tumor suppressors such as p53 or Rb) designed to detect and eradicate cancer cells. Additional approaches involve gene delivery of cytokines or chemokines, such as IL-12, interferons, or IL-2 to activate the immune system. Prodrug or suicide gene therapy approaches involve delivery of a foreign metabolic enzyme into cancer cells and then systemic delivery of a toxic drug that is selectively activated by this enzyme. The HSV thymidine kinase gene has been widely used along with the antiviral ganciclovir, a chemotherapy used for treatment of HSV. In normal cells, ganciclovir does not have significant effects, but when it encounters the HSV thymidine kinase, it is phosphorylated to a monophosphate derivative.
Cellular enzymes then convert the monophosphate to ganciclovir triphosphate, which is incorporated into the DNA of the cancer cell causing premature DNA chain termination and apoptosis. Oncolytic viruses which replicate selectively in cancer cells, but spare normal cells are moving into late-stage clinical trials and show promise. To date, three gene therapies have been approved by regulatory bodies.
China approved an Adenovirus-p53 called Gendicine and an oncolytic Adenovirus termed Oncorine. In 2015, the US FDA approved Imlygic, an oncolytic Herpes virus encoding the GM-CSF gene for the local treatment of recurrent unrespectable melanoma.
Gene editing is another technique with the theoretical capacity to edit, correct, and alter genes, therefore selectively fixing mutations specifically to treat diseases. It is of this concept that the gene editing story of CRISPR was born. CRISPR, which stands for clustered regularly interspaced short palindromic repeats, is a technique that gives researchers the ability to selectively edit DNA. CRISPR works with the CRISPR associated protein 9 (Cas9), and is derived from the natural gene editing system in bacteria. Bacteria are able to scan the DNA of invading viruses and to capture snippets of viral DNA segments as a way to remember the virus in case of a future attack.
If a secondary attack from the same virus happens in the future, short RNA sequences are replicated by the bacteria to attack the viral DNA, disabling the virus’ invasion. The laboratory technique functions similarly, with researchers isolating specific DNA sequences of interest and using the Cas9 system to cut DNA at the location of interest. The cutting of the DNA sequence at a desired location triggers the cells innate DNA repair mechanisms to repair or replace genetic material in that specific location, thus correcting the genetic defect through endogenous mechanisms. Scientists Jennifer Doudna and Emmanuelle Charpentier, who discovered the gene-editing technique CRISPR, earned the Nobel Prize in Chemistry in 2020. This discovery has made a monumental impact in the potential for the treatment of disease, although this technology also has concerning ethical considerations.
In the US, the first use of CRISPR technology in the clinic was an ex vivo study from the University of Pennsylvania in 2019 where they used CRISPR to selectively edit three genes, two genes that encode the T cell receptor and a third gene encoding the checkpoint PD-1. They then used a lentivirus to insert a cancer-specific synthetic T cell receptor, which tells the edited T cells to target an antigen called NY-ESO-1. The small study demonstrated proof of concept and safety, but concerns persist about “off-target” effects in which unintended DNA gets modified. Note that related gene editing technologies using TALENs or Zinc Fingers have been evaluated clinically for more than 10 years but have not shown substantial efficacy.
Development of new cancer treatments involves recognition of the diverse complexity of tumors, their associated microenvironment and tumor cell metabolism, posing challenges for researchers in finding treatments broadly applicable enough to make a substantial impact on the disease population of interest, yet specific enough to leave normal cells unharmed. Historic treatments such as chemotherapy, monoclonal antibody therapy, immune therapies, and radiation therapies have all fallen short of providing cures.
We now have new cancer treatment technologies where cures are emerging, albeit with inherent complexities of manufacturing and cost. Combinations of new chemical and biological agents with checkpoint inhibitors are being vigorously pursued. It is becoming clear that we need better rationales when designing trials for combination immunotherapies as there have been several late-stage clinical trial failures. Continued engineering of cell therapies are moving towards universal CAR-T or CAR-NK cell products and enhancing function of CAR-expressing cells by using multiple CARs or incorporating bi-specific antibodies. Novel approaches to prime the tumor microenvironment and enhance the sensitivity of checkpoint antibodies is showing promise as are new approaches to normalize defective tumor microenvironment.
Most cancer treatment is still focused on tumor reduction, improving progression-free survival, and increasing quality of life. Research is now focused on identification of drug targets that provide greater selectivity and are truly tumor-specific. Cell and gene delivery technologies and being used to direct novel targeted therapies to improve the specificity and safety profile of eliminating cancer cells. The integration of genomics, proteomics, and machine learning is being used to develop novel techniques that are evolving in both the cell and gene therapy areas. These integrations may offer substantial improvements when compared to the current standards of care for oncology patients.
Sunil Chada, Ph.D.
Dr. Sunil Chada is President of DNASolve Biopharma Consulting which was founded to help cell and gene therapy companies to advance novel therapies from concept to clinical trials. He has managed or been involved in clinical translation of >20 novel gene and cell therapy drug candidates using multiple virus and cell approaches. For the last 10 years, he has served as Chief Scientific Officer at Multivir Inc. and prior to this, Dr. Chada was the Senior Vice President of Translational Medicine at Intrexon Corporation. Dr. Chada served in several positions at Introgen Therapeutics, Inc. including Vice President of Research. Dr. Chada previously served as adjunct faculty at the M.D. Anderson Cancer Center of the University of Texas and served as Special Advisor to the Chief Scientific Officer of their Moonshots program. Dr. Chada was also involved in discovery and preclinical research at Chiron Corporation and Viagene Inc. Dr. Chada received a B.Sc. from the Department of Cell and Molecular Biology at Kings’ College, University of London, an M.S. in Molecular Biology from the University of California at Los Angeles, and a Ph.D. in Molecular Genetics and Microbiology from the University of Massachusetts Medical School.
- Beutler, E. (2001). The cline affair. Molecular Therapy, 4(5), 396-397. doi:10.1006/mthe.2001.0486.
- Chada, S. (2021, Oct. 21). Personal interview [Personal interview].
- Dong J, Li B, Zhou Q, Huang D (2018) Advances in evidence-based medicine for immunotherapy of non-small cell lung cancer. Journal of Evidence Based Medicine, 1-10.
- Grosso JF, Jure-Kunkel MN (2013) CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immunity 13, 1-14.
- Vajaitu C, Draghici CC, Solomon I, Lisievici CV, Popa AV, Lulp M, Caruntu C, Constantin MM, Voiculescu VM (2018) The Central Role of Inflammation Associated Checkpoint Inhibitor Treatments. Journal of Immunological Research 4625472, 1-10.
- Weinberg RA. (2014). The biology of cancer. Garland Science. New York, NY, US.
- Yu, J. X., Upadhaya, S., Tatake, R., Barkalow, F., Hubbard-Lucey, V. M. (2020). Cancer cell therapies: The clinical trial landscape. Nature Reviews Drug Discovery, 19(9), 583-584.