Immunotherapy beyond checkpoints inhibitors

Types of immunotherapy
Oncology
cancer vaccines
adoptive T cell transfer
oncolytic viruses
monoclonal antibodies
cytokine
treatment of cancer with immunotherapy

1. Immunotherapy beyond checkpoint inhibitors Presenter: Dr. Gaurav Kumar Postdoctoral trainee Dept. of Medical Oncology BBCI, Guwahati

2. Adoptive T Cell therapy

3. ● The goal of adoptive T cell therapy (ACT) is to generate a robust immune-mediated antitumor response through infusion of ex vivo manipulated T cells. ● ACT strategies: Isolation of naturally occurring tumour -specific T cells from existing tumour masses (TIL based ). (2) Genetic modification of blood-derived T cells to allow for specific recognition of tumor cells.

4. TIL-based ACT ● Isolation of TILs from tumour tissue of the patient, followed by massive expansion of unselected TILs and infusion back into the patient. ● Before infusion, lymphodepleting chemotherapy ( ChT ) is administered to create ‘physical space’ for the high number of TILs. ● Following infusion, the immune-stimulating cytokine interleukin-2 (IL-2) is administered to the patient to support the survival and continued expansion of the TILs in vivo. ● 100×10 ⁹ cells are infused. ● Any patient with a resectable tumour which contains tumour -reactive T cells can benefit from this approach.

5. TIL-based ACT ● Constraint 1) Laborious ex vivo expansion of huge numbers of TILs. 2) Intensive treatment with high-dose conditioning ChT and high-dose IL-2, necessitating hospitalisation for around 3 weeks. 3) All patients experience grade 3 and 4 toxicity.

6. Genetically Modified T Cells Valid in situations in which endogenous antitumour reactivity is lacking. Accomplished by ● Transducing T cells with either tumour antigen-specific T cell receptors (TCRs ) . ● Transducing T cells with chimeric antigen receptors (CARs) .

7. Tumour antigen-specific T cell receptors (TCRs) ● TCRs are naturally occurring surface receptors on T cells that can recognise peptide antigens presented on the surface of host cells by the (MHC)/ (HLA) system. ● Genetically modified TCR gene therapy purpose…. alter T cell specificity through the expression of a new TCR alpha and beta chain pair that is specific for a tumour antigen. ● Steps: 1) TCRs from rare T cells that are able to recognize naturally processed and expressed tumor antigens are isolated and sequencing of these tumor-specific TCRs done . 2 ) Cloned into retro- or lentiviral vectors . 3 ) Vectors used to transduce peripheral blood T cells from patients ex vivo. 4 ) Expansion and re-infusion.

8. Constraints (TCR) 1) High-dose conditioning ChT …toxicity including neutropaenia and risk of sepsis. 2) Safety risks with genetically modified T cell therapies: ● On-target off- tumour toxicity , when infused T cells recognise normal tissue due to expression of the same antigen (l)e gp100 and MART-1 which are expressed by both melanoma cells and normal melanocytes . ● Off-target reactivity , when infused T cells can cross-react against peptides other than the targeted ones ● Cytokine-release syndrome (CRS), when infused T cells induce sudden and dramatic increase of inflammatory cytokines.

9. Genetic modification of T cells with CARs ● Combines antibody-like recognition with T cell-activating function . ● The construction of a CAR relies on the identification of a suitable antibody to a cell surface molecule of interest, and, in contrast with the TCR modification approach, CAR recognition does not rely on peptide processing or presentation by MHC . ● All surface-expressed target molecules represent a potential CAR-triggering epitope .

10. ● First-generation CARs : Composed of an antigen-binding region (a single-chain antibody variable fragment [ scFv ]) derived from an antibody with the desired specificity, which was fused to the CD3ζ T cell signaling domain, allowing T cell activation comparable to triggering of the native TCR. These early CARs provided only activation signal 1 to T cells, and were shown to lead to CAR-T cell anergy upon repeated antigen stimulation. ● Second-generation CARs : Additional co-stimulatory domain , such as CD28 and 4-1BB , which provides activation signal 2 upon scFv engaging the target antigen. ● Third-generation CARs : which incorporate an additional co-stimulatory domain are now in development to further potentiate persistence and activity of infused CAR-T cells.

11. Experience from CD19-specific CAR-T cells : ● Patients should receive lymphodepleting ChT . ● Patients with ALL, in particular, have very high response rates. ● Off-tumor toxicity is primarily limited to B cell aplasia , a condition managed with prophylactic infusions of immunoglobulin. ● Patients often develop severe CRS . ● No clear dose–response relationship between the number of CAR-T cells infused and the likelihood of response.

12. CAR-T cell therapy against solid tumors: Limited success ● Inefficient T cell homing to the tumor site. ● More difficult antigen selection due to high antigen heterogeneity across the same malignancy. ● Physical barriers to tumor infiltration by T cells. ● High risk of on-target, off-tumor toxicity because potential target antigens are more likely to be expressed in other essential organs. ● Potent immunosuppressive factors that render T cells dysfunctional in the tumor microenvironment.

13. Concluding Remarks ● TIL ACT can induce long-term remission in patients with otherwise treatment-resistant widespread MM. ● The use of TIL ACT is, however, still experimental and restricted to reference cancer centers with expertise in TIL production and clinical management. ● The first two commercial gene-modified CD19-targeting CAR-T cell products have been approved by the Food and Drug Administration (FDA) as a standard therapy targeting CD19-positive B cell malignancies with significant clinical efficacy. ● Indications of clinical effect in certain solid cancer types have been reported but a major clinical breakthrough for gene-modified TCR/ CAR-T cells is still awaited.

14. Cancer Vaccines

15. Definition : ● Active immunotherapy with cancer vaccines aims to instruct the host immune system to recognize cancer as a foreign ‘non-self’ tissue and mount specific immune responses that eliminate malignant cells. ● Malignant diseases….evolve by evading anti-tumor immunity, and cancer vaccines aim to (re-)establish immune responses against tumor-associated antigens (TAAs). ● Turn cold tumors (few or no TILs) into hot tumors (many TILs). ● Cancer vaccines: Induce de novo immune responses or boost existing suboptimal responses by providing new/stronger antigenic stimuli.

16. Steps: 1) Selection of TAAs: Immunogenic TAAs for optimal vaccines categories of ‘non-(or distant-from)-self’ antigens: Oncogenic virus antigens & Mutation-based neoantigens . 2) Selection of the vaccine platform that delivers sufficiently concentrated antigens to vaccine-draining lymph nodes (LNs) for dendritic cell (DC) presentation to both CD4+ and CD8+ T cell precursors. Successful vaccine platforms….DNA, RNA and synthetic long peptides (SLPs), consisting respectively of concentrated nucleic acids encoding TAAs or peptides harbouring the T cell epitopes themselves. 3) Vaccines need strong adjuvants : RNA and DNA vaccines…built-in adjuvants , SLP vaccines needs to be supplemented with an appropriate adjuvant. 4) Use in combination with immune- modulatory treatments e.g ….standard chemotherapy or checkpoint blockade… counteract the immune hostile cancer microenvironment.

17. Route of administration of vaccines : ● Crucial to efficiently target antigens to DCs in vivo and/or activate the T cell pool that can be more easily redirected toward the tumor site. ● Subcutaneous ( s.c .) administration or delivery into DC-rich LNs have been preferred for peptides/DC-based vaccines. ● Intramuscular ( i.m .) injection for DNA-based vaccines. Efficiency of vaccination improved by electroporation . ● Liposome-encapsulated RNA vaccines, intravenous ( i.v .) administration has been advocated, because these liposomes can reach numerous LN-DCs following i.v . administration.

18. Oncolytic Viruses ● Oncolytic virotherapy , that selectively infect and kill cancer cells while sparing normal tissues. ● Killing of tumor cells upon virus infection local inflammatory environment resulting in tumor antigen release and recruitment of immune cells contribute to the amplification of antitumor immunity. Technical Procedures: ● Direct anti-tumor cytotoxic effects of viruses replicating within malignant cells. ● Induction of an anti-viral immune response increasing the probability of a reaction against the released TAAs.

19. ● To improve the immunological activity of oncolytic viruses, they can be engineered to express pro-inflammatory molecules as immune adjuvant. Clinical Results ● T-VEC ( talimogene laherparepvec ), a genetically engineered herpes simplex virus to express GM-CSF, was the first oncolytic virus therapy approved by the FDA for the treatment of unresectable MM, based on results of the OPTIM trial. ● Coxsackievirus A21 (CVA21), has been tested for the treatment of different solid cancers and has shown both local and distant clinical responses ( Andtbacka et al, 2015).

20. Vaccine results RNA vaccines…. ● A self- adjuvanted RNA-based vaccine (CV9103) encoding the antigens PSA, PSCA, PSMA and STEAP1 was well tolerated and induced immune responses against multiple epitopes in a phase I/ IIa study with advanced prostate cancer patients ( Kübler et al, 2015). ● Personalised neo- epitope -containing liposomal RNA vaccines generated specific immune responses in advanced melanoma patients ( Sahin et al, 2017). DNA vaccines…. ● Delivered by electroporation have induced robust T cell responses to the E6 and E7 proteins of high-risk HPV16 and 18 . ● Randomised phase II trial, more high-grade cervical epithelial neoplasia lesions regressed following HPV DNA vaccination than spontaneously ( Trimble et al, 2015).

21. DC based vaccines…. ● Sipuleucel -T first active cancer immunotherapy to gain regulatory approval from the Food and Drug Administration (FDA) upon demonstration of a median survival improvement of 4.1 months and a death risk reduction of 22.5% in CRPC in a randomised , double-blind phase III trial . ● Autologous DCs pulsed with whole lysate from three allogeneic melanoma cell lines (TRIMEL) increased survival and disease stabilisation in most of the treated metastatic melanoma (MM) patients . ● Autologous DCs modified with a pox vector encoding the TAA’s carcinoembryonic antigen (CEA) and mucin 1 (MUC1) (PANVAC™) reduced recurrence and prolonged survival in tumor- resected disease-free colorectal cancer patients.

22. Antibodies

23. ● Monoclonal Antibodies ( mAbs ) ● Antibody-Drug Conjugates (ADCs) ● Bispecific Antibodies

24. ● Supplement our immune system by creating and customizing antibodies against specific cancer targets in the lab. These are often referred to as monoclonal antibodies due to their identical structure . ● In 1997, the U.S. Food and Drug Administration (FDA) approved the first antibody for the treatment of cancer—the monoclonal antibody rituximab for leukemia—and since then, over a dozen more have received FDA approval. Monoclonal Antibody

25. Monoclonal Antibody

26. Antibody-Drug Conjugates (ADCs) ● Antibody-drug conjugates (ADCs), in which a targeted antibody is equipped with anti-cancer drugs , so that when the antibody targets and binds to cancer cells, it also delivers a toxic drug that can kill the cancer cells .

27. Bispecific Antibodies ● Another new type of antibody-based immunotherapy that has been developed is bispecific antibodies . ● These are made by taking the targeting front end regions of two different antibodies and combining them together to create a product that can bind to two different targets. ● The first bispecific antibody— blinatumomab —was approved by the FDA in 2014 for subsets of patients with leukemia . ● Blinatumomab is known as a  bispecific T cell engager ( BiTE )   because it is designed to bind cancer cells as well as T cells. As a result, blinatumomab can bring T cells into close proximity with cancer cells .

28. Bispecific Antibodies ٭ Radio-immunotherapy

29. Cytokines in Cancer Immunotherapy

30. ● Cytokines are secreted or membrane-bound proteins that act as mediators of intercellular signaling to regulate homeostasis of the immune system . ● They are produced by cells of innate and adaptive immunity in response to microbes and tumor antigens. ● T wo cytokines have achieved FDA approval as single agents for cancer treatment: high-dose, bolus IL-2 for metastatic melanoma and renal cell carcinoma and IFN-α for the adjuvant therapy of Stage III melanoma . ● IL-12 has demonstrated anti-tumor activity in murine models of melanoma, colon carcinoma, mammary carcinoma and sarcoma. ● Recombinant GM-CSF was approved by the FDA to shorten the time to neutrophil recovery and reduce the incidence of infections following induction chemotherapy in patients with acute myelogenous leukemia. ● GM-CSF is also used to mobilize hematopoietic progenitor cells for autologous and allogeneic bone marrow transplantation.

31. ● Single agent GM-CSF has been reported to have antitumor activity in melanoma when injected directly into metastatic lesions.   Cytokine-Antibody Fusion Molecules ● A cytokine-antibody fusion molecule is a genetically engineered fusion protein consisting of an antibody with a functional cytokine and an antigen-binding site designed to deliver cytokines to the tumor microenvironment. ● The prototype fusion molecule has utilized various antigen-binding moieties fused to recombinant human IL-2 . ● Phase I and II clinical trials of this recombinant fusion molecule in both adult melanoma and pediatric neuroblastoma patients have demonstrated its safety in patients at doses and schedules that are able to induce immune activation.