Immunotherapy Notes

Immunotherapy

Introduction

Immunotherapy involves either stimulating or suppressing the immune system to treat various diseases. Lei Huang, PhD, from the Translational and Clinical Research Institute at Newcastle University, studies immune responses and immunotherapy.

Key Questions in Immunotherapy

1. What is Immunotherapy?
2. What diseases respond to Immunotherapy?
3. How effective is Immunotherapy?
4. Does Immunotherapy cause any toxicities and how?
5. Is Immunotherapy expensive?

Growth in Immunotherapy

There's been significant growth in immunotherapy research and publications, especially in cancer immunotherapy. The growth of immunotherapy publications in PubMed depicts a rise in both cancer immunotherapy and general immunotherapy from 1970 to 2020.

Types of Immunotherapy

1. Vaccines:

   • Prophylactic or therapeutic vaccination
   • Live attenuated pathogens
   • Killed pathogens
   • Artificial vaccines (DNA, mRNA vaccine, viral vectors, protein, VLP)
   • Cancer vaccines

2. Immune Modulatory Drugs:

   • Immunosuppressant drugs
   • Immunostimulatory drugs

3. Biologicals:

   • Monoclonal Antibodies (mAbs) (humanized):
     • To eliminate cancerous cells (cytolytic, depleting mAbs)
     • To block cell interactions & cell signaling (blocking mAbs)
   • Soluble fusion proteins (‘X’-Ig)
   • Bi-specific Antibodies

4. Cell Therapies (adoptive cell transfer):

   • Hematopoietic Stem Cells (HSCs) - to treat cancer (Graft vs Host Disease, GvHD)
   • Chimeric Antigen Receptor (CAR) T cells (CAR-Ts) – to treat cancer
   • Dendritic Cell therapy – to treat cancer or autoimmune syndromes

Cancer Immunotherapy

• Therapeutic effect: Promising yet much to learn.
• Adverse effect: Autoimmunity, Neurological complication
• Approaches:
  • Vaccines
  • Immune checkpoint Blockade (ICB): targeting PD1, CTLA4, TIM-3, LAG-3, IDO, CD73
  • Immunosuppressive Molecules: IL10, TGF$\beta$, PGE2 etc.
  • Adjuvants: pI:C STING activator
  • Adoptive T cell transfer: Peptide, mRNA, Agonistic mAb OX40 CD40 TIL, CAR-T, TCR-T

Inflammation, Immunity & Immunotherapy

• Inputs: Wounds, Malignancies, UV light, Transplants, Allergens, Infections
• Outputs:
  • Immunity (pathogen clearance, tissue injury)
  • Tolerance (tissue healing) (immune checkpoints)
• Related Diseases: Autoimmune diseases, Allergies/Asthma, Transplant rejection, Chronic infections, Cancers
• Immunotherapies:
  • Anti-inflammatory drugs
  • Immune adjuvants
  • Vaccines
  • Immune checkpoint inhibitors
  • Chimeric Antigen Receptors (CARs)
• States: Healthy, Inflammation

Immunotherapy: Stimulating or Suppressing Immune Responses

• Immune Stimulants (to prevent and treat infections):
  • Adjuvants & Antigens (e.g., COVID vaccines)
  • Prophylactic & Therapeutic vaccines (to prevent and treat cancer)
  • Cancer Vaccines
  • Immune checkpoint blockade (mAbs, IDO inhibitors)
  • Adoptive Transfer of Tumour-Specific T cells
  • Chimeric Antigen Receptors (CAR-T cells)
  • Oncolytic Vaccines
• Immune Suppressants (to treat autoimmune/allergy syndromes & prevent transplant rejection):
  • Monoclonal Antibodies (mAbs)
  • Immunomodulators (anti-inflammatory drugs, e.g., kinase inhibitors)
  • Regulatory T cells (Tregs) & DCs
  • Inducing immune suppressive mechanisms (e.g., IDO inducers)
  • Tolerogenic vaccination

Historical Perspective: Edward Jenner's Experiment (1796)

Edward Jenner's experiment demonstrated the principle of vaccination: inoculating with cowpox to protect against smallpox.

• Inoculate with cowpox.
• Wait 2 months.
• Inoculate with smallpox.
• The child recovers from cowpox and does not develop smallpox.
• Cowpox = Vaccinia
• Smallpox officially eradicated. Timeline 1965-1980.

Immune Memory after Flu Infection

• Primary infection, Re-challenge
• Protection from B cells, T cells.
• Timeline: Day 0 - Primary infection, Day 28 - Re-challenge with X31: H3N2 PR8: H1N1 and PR8: H1N1

Vaccines: Components and Types

• Vaccines contain Adjuvants (immune stimulants) & Antigens.
• Adjuvants stimulate innate immunity (pro-inflammation), which potentiates adaptive immunity to specific Antigens.
• Prophylactic vaccines are used to stimulate protective immune memory in healthy individuals (Recall response).
  • Edward Jenner used Vaccinia (cow pox virus) to protect against smallpox virus (variolation).
  • Vaccines were developed against epidemic infections such as measles, mumps, rubella (MMR) & polio.
  • Can vaccine stop the COVID pandemic?
• Therapeutic vaccines are designed to stimulate immunity in patients who already have chronic infections or cancer.
  • Therapeutic vaccines are less effective due to inflammatory processes that inhibit natural & vaccine-induced immunity.
  • Improving vaccine design is an active research field.
• Vaccine to induce immune tolerance (Tolerogenic vaccine).

Principles of Vaccine Design

• Louis Pasteur (1822-1895):
  • Germ theory
  • Pasteurization
  • Vaccine principles (mid 19th century):
    1. Isolate
    2. Inactivate
    3. Inject
• COVID vaccine platforms (2014-2020).

mRNA and DNA Vaccines

mRNA Vaccine

• mRNA Vaccine leading to CD4 T cell, B cell (MHC class II) and Antibody production.
• Process: Formulated mRNA vaccine undergoes Endocytosis, mRNA escapes Endosome. Ribosomes then create Proteins/Peptides that bind to MHC class II.

DNA Vaccine

• DNA Vaccine leads to CD8+ T cell and MHC class I interaction.
• Process: Endocytosis, the Proteins/Peptides undergo Proteasome and enter Endoplasmic Reticulum. They then bind to MHC class I through the Golgi apparatus.

Designed T cell vaccine Considering MHC Restriction

• (a) Self MHC-peptide A leads to Killing
• (b) Nonself MHC leads to No killing
• (c) Self MHC-peptide B leads to No killing
• H-2: Mouse MHC.

Adaptive Immunity to Pathogens and Vaccination

• Natural Immunity:
  • Natural infection leads to Morbidity (illness) and Re-infection, potentially Sub-clinical infection. It results in Boosted Immunity to pathogens that lasts many years.
• Vaccines (prophylactic):
  • Infection or Sub-clinical infection leads to Boosted Immunity to pathogens.
• Vaccines (therapeutic) and antiviral lead to Viral Immunity.
• Persistent virus infection (many years) can lead to Viral pathologies (relapse).
• Immune deficiency syndromes lead to Increased risk of cancer & infections.

Cancer and Autoimmune Diseases

• Reciprocal disease processes in cancer & autoimmune syndromes.
• Tumor and Autoimmune Progression over Time.
• Early Markers of Disease Progression.
• Future Goals: Early interventions to impede progression using Prophylactic Tumour Vaccines or Tolerogenic Vaccines and Immune modulators.
• Shared molecular mechanisms (common DAMP?).
• Tumour vaccines (insufficient), Immune activators, Antigen therapy, Immune regulators.
• Inflammation that stimulates immunity, or suppresses immunity leading to self tolerance.

Immune Modulatory Drugs

• Small molecules that target signaling pathways, enzymes, cellular recognition, etc. to stimulate or suppress immunity.
• Immune stimulants: used to treat hypo-immune syndromes (adjuvants).
  • PRR activators: Toll-Like Receptor (TLR) ligands: LPS (TLR4), Imiquimod (TLR7), CpG oligonucleotides (TLR9), DNA (microbial DNA) or RNA.
  • Cytokines: interferon 1 (IFN$\alpha$), GM-CSF, IL-2, IL-12.
  • Chemokines: CCL3, CCL26, CXCL7.
  • IDO inhibitors (1MT = INDOXIMODTM, NLG919, Epacadostat, BMS-986205, etc.).
• Immune suppressants: used to treat hyper-immune syndromes.
  • Steroid hormones: e.g., cortisone, but serious side effects.
  • Cyclosporine: blocks cell proliferation by inhibiting calcineurin.
  • Tacrolimus: as above, but more potent and fewer side effects.
  • Sirolimus (Rapamycin): blocks mToR to prevent T cell proliferation.
  • Interferon 1 (IFN$\beta$): used to treat MS patients (MoA unclear).
  • JAK inhibitors (e.g. Tofacitinib).

Biologicals

• Monoclonal Antibodies (mAbs):
  • Historically produced by B cell hybridomas (B cells fused with myeloma cells).
  • Hybridomas are grown in large quantities and secreted mAbs are harvested and purified on an industrial scale.
  • Most mAbs must be ‘humanized’ (to reduce immunogenicity in patients).
  • Recombinant antibodies (CHO expression system).
  • Cytolytic (depleting) mAbs are used to eliminate cancerous cells.
  • Blocking mAbs are used to inhibit cell interactions & cell signaling (e.g., anti-TNF$\alpha$ & anti- IL6 to treat arthritis patients).
  • Agonistic mAbs eg, aCD40
• Soluble fusion proteins (‘X’-Ig): recombinant immunoglobulins.
  • CTLA4 blockade: (CTLA4-IgG1; Abatacept): inhibits T cell responses by blocking B7/CD28 interactions between Antigen Presenting Cells (APCs) & T cells. Approved for treating arthritis patients.
  • Cytokine blockade: (TNFR-IgG1; Etanercept): inhibit cytokine signaling to suppress autoimmune pathologies. Approved for treating arthritis, psoriasis and other autoimmune syndromes.

Monoclonal Antibodies (mAbs) Generation & Manufacture Applications in Cancer

• ADCC = antibody-dependent cell-mediated cytotoxicity
• CDC = complement-mediated cytotoxicity
• Qu: What information is needed to target mAbs to tumour cells only?

Monoclonal Antibodies (mAbs) used as in Cancer Treatments

Listing of trade names, targets and cancer types for various FDA approved Monoclonal Antibodies.

Abbreviations: HER (human epidermal growth factor receptor), VEGF (vascular endothelial growth factor), CD (cluster of differentiation), CTLA (cytotoxic T-lymphocyte antigen).

Antibody Use for Cancer Therapy

• (A) Signaling pathways blocking:
  • Cetuximab/Panitumab block EGFR, affecting RAS, PI3K, ERK, AKT, thus impacting Proliferation and Survival of cancerous cells.
• (B) ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity):
  • Tafasitamab targets CD19, enhancing Fc region interaction with FcyRIII, leading to Granzyme and perforin release from NK cells, resulting in Lysis.
• (C) CDC (Complement-Dependent Cytotoxicity):
  • Naxitamab targets GD2, initiating C1q binding and MAC formation, resulting in Lysis.
• (D) ADCP (Antibody-Dependent Cell-Mediated Phagocytosis):
  • Tafasitamab targets CD19, enhancing FcyRI interaction, leading to Phagocytosis by Macrophages.

Co-Stimulatory Pathways (Signal 2): Immunotherapy targets in cancer

• Immune Checkpoint Blockade mAbs
• CTLA4 mAb blocks T cell suppression by binding to CTLA4
• PD1/L mAbs block T cell suppression by binding to PD-1 or PD-1 ligands (PD- L1/L2)
• Positive (immunogenic) & negative (tolerogenic) co- stimulatory pathways
• T cell stimulation and Immune checkpoints in cancer

Development of novel cancer treatment: Immune checkpoint blockade

• Anti-CTLA-4 and Anti-PD-1 against cancer.
• Control vs Anti-CD28 vs Anti-CTLA-4
• Control vs anti-PD-1

Cell Therapies

• Tumour cell therapies: Tumour cells can be made more immunogenic.
• T cell therapies:
  • Generate autologous tumour-specific T cells (high cost and cannot be used for large numbers of patients).
  • Chimeric Antigen Receptors (CARs): used to engineer T cells to recognize tumour cells specifically and kill them.
  • Regulatory T cell (Treg) therapies: potential use to treat patients with hyper-immunity such as Type I Diabetes (T1D) and Graft versus Host Disease (GvHD) following bone marrow transplantation to treat leukaemia/lymphoma.
• Dendritic Cell (DC) therapies: (kind of vaccination approach)
  • Provenge (Sipuleucel-T): to treat prostate cancer. Patient blood cells are used to generate mature DCs (APCs) that present prostate antigen (PAP) to T cells. Mature DCs are infused back into patients as a tumour-specific vaccine (but high cost & relatively low survival advantage).
  • Regulatory DC therapies: potential use to treat arthritis patients (in clinical trials). DCs are prepared from blood cells to select DCs with immune regulatory functions. Regulatory DCs are then infused into arthritic joints

Cell Therapies: TILs, TCR-T & CAR-T

• Isolate TILs (Tumor-Infiltrating Lymphocytes) from the tumor tissue.
• T cell isolation from patient peripheral blood.
• Engineering T cells to target tumor cells using artificial genes, leading to TCR-T or CAR-T.
• Re-infusion of modified T cells back into the patient.

SCFV and CAR-T

Diagrammatic representation of scFV and different generations up to the fifth generation of CAR-T cells, illustrating the evolution and complexity of CAR-T cell designs.

Treatment for Autoimmune Diseases: Clinical Immunotherapy to reduce inflammation

Effective treatment for Rheumatoid Arthritis using TNF signaling blockade reagents such as Enbrel, Etanercept. Enbrel (Etanercept) is a TNF receptor IgG1 (Fc).

Applications & targets in multiple diseases

Listing of Monoclonal Antibodies (mAbs), their Application, Mechanism/Target, and Mode (chimeric/humanized) for various diseases.

Treating Rheumatoid Arthritis

• Healthy vs RA Joint composition and factors involved such as: Laminin, Collagens, Hyaluronic acid, Lubricin, Fibronectin and also Bone loss.
• Role of RANKL, Increased angiogenesis, Expanded synovial sublining, Chemotactic factors, Pro-angiogenic factors, IL-6, Chemokines, IL-1, TNF, T cell, B cell, Macrophage, Dendritic cell, Osteoclast, Chondrocyte.

Summary (Immunotherapy)

1. Immunotherapy prevents infections (prophylactic vaccines; MMR).
2. Immunotherapy alleviates the severity of chronic inflammatory diseases (RA, MS, SLE, allergies).
3. Immunotherapy protects transplanted organs & tissues (immunosuppressants; Rapamycin).
4. Immunotherapy shows promise in some cancer patients.
5. Many new immunotherapies to treat chronic inflammatory diseases are under scrutiny.

BUT – major problems still need to be addressed:

• Vaccines are ineffective in many chronic infections & cancers
• Immunotherapy is often required for long periods and rarely leads to stable disease remission
• Immunotherapy has undesirable side effects & can be expensive