Introduction to Tumor Virus

●​ Virus Structure:
○​ composed of genetic material (DNA/RNA) enclosed within a capsid protective (protein coat)
○​ some possess an envelope made of phospholipids & proteins, derived from the host cell's membrane.
○​ Capsid Shapes:
■​ Icosahedral
■​ Prolate (head-tail)
■​ Filamentous

Life Cycle of Viruses

●​ 1. Viral Entry:
○​ Virus surface proteins interact with host cell proteins for attachment/adsorption to the host cell
membrane.
○​ A hole forms in the cell membrane → allows virus particles / genetic material to enter the host cell,
where replication can begin.
●​ 2. Viral Replication:
○​ The virus takes control of the host cell's replication machinery.
○​ distinction between susceptibility & permeability is made
○​ Permissibility of the host cell determines the infection's outcome.
○​ Once control is established, the virus replicates rapidly, producing millions of copies.
●​ 3. Viral Shedding: (final stage)
○​ After exhausting the host cell's resources, the cell often dies.
○​ Newly produced viruses are released (shedding) to find new hosts.
●​ 4. Viral Latency (Lysogenic):
○​ Some viruses can "hide" within a cell to evade host defenses / continuous replication isn't beneficial.
○​ virus is inactive & produces no progeny until an external stimulus (light, stress) prompts activation.

Rous Sarcoma Virus (RSV)

●​ Structure of RSV:
○​ env gene: Encodes glycoprotein spikes for virion adsorption to the cell surface.
○​ gag gene: Encodes core proteins forming the virus's protein shell.
○​ pol gene: Encodes reverse transcriptase & integrase.
○​ Genome: Two identical copies of genomic RNA.
●​ RSV-induced Focus:
○​ Infected cells become rounded, overgrow, pile up, forming a "focus."
○​ Surrounding uninfected cells exhibit contact inhibition & stop proliferating.
●​ Transformed Cells by Virus Forming Foci:
○​ Normal fibroblast cells display contact inhibition (density inhibition).
○​ Transformed cells lose this inhibition and grow on top of each other.
○​ a group of descendant cells is a "cell clone," and the focus is a "clonal growth."
○​ Cell transformation by RSV resembles tumor appearance ; the program is activated upon infection.
●​ Temperature-sensitive Mutant (Berkeley Experiment):
○​ Mutant viruses lose function at high temperatures (denaturing mutant proteins).
○​ Viral genome copies persist in cells (replicating with cell proliferation).
○​ The continued action of the virus is required for both the initiation & maintenance of the
transformed phenotype.
SV40 Virus

●​ Characteristics:
○​ Icosahedral symmetry.
○​ dsDNA genome.
○​ Discovered in 1953 (monkey virus).
○​ Causes cytopathic effect: vacuoles (in permissive cells).
○​ Nonpermissive in rodent hosts (doesn't complete lytic cycle but can transform cells).
○​ Able to seed tumors in vivo (in nonpermissive hosts).
●​ SV40 Genome (circular dsDNA):
○​ Form I (kinked): Closed, circular, super-coiled dsDNA.
○​ Form II (open spread circle): Relaxed, nicked circular dsDNA.
○​ Form III: Linear form.
●​ Property of Transformed Cells:
○​ Anchorage Independence: Transformed cells can form spherical colonies in soft agar gel, unlike
normal cells which require attachment to a solid substrate to grow.
●​ Test of Tumorigenicity:
○​ To check if cells are fully transformed & have neoplastic traits:
■​ Inject in vitro transformed cells into a syngeneic host mouse.
■​ Inject into an immunocompromised host mouse (nude mouse).
●​ Expression of the SV40 T antigen:
○​ The large T antigen (LT) is expressed in the nuclei of transformed cells.
○​ BK virus (an SV40 relative) infection caused transformation & tumorigenesis in the bladder of a
kidney transplant patient.
●​ How is the viral genome replicated/maintained during transformation?
○​ If the viral genome were lost during cell proliferation, transformation might not be maintained
○​ Viral genomes typically lack centromeres for proper allocation during cell division.
○​ In a lytic cycle, the viral genome exists as extrachromosomal molecules, replicating independently
of the host genome, producing many copies.
●​ SV40 Genome is Integrated into Host Genome (in transformed cells):
○​ SV40 DNA from free virus sediments more slowly (lower molecular weight) than SV40 DNA from
transformed cells.
○​ In transformed cells, SV40 DNA is covalently associated with the high molecular weight
chromosomal DNA of the host cells.
○​ Formation of Integrated SV40 Genome:
■​ Occurs with low efficiency.
■​ Involves non homologous (illegitimate) recombination.
■​ Integration occurs at random sites in the host genome.
■​ Results in a covalent linkage of viral and host genomic DNA.
Oncogenes and Proto-oncogenes

●​ src gene (in RSV):

○​ Important for the transformation of infected cells. (RSV also has env,gag,pol genes).
●​ Generating FISH Probes to Detect src DNA:
○​ src DNA was detected in almost all cells, include uninfected chicken cells
○​ src-related sequences were also found in other related birds.
○​ sequences were not solely from retrovirus infection but might be present in ancestral vertebrates.
●​ The origin of oncogene: The viral oncogene (v-src) originated from a cellular gene.
●​ c-src (cellular src):
○​ Similar to the viral src(v-src)
○​ Encodes a non-receptor tyrosine kinase protein (SRC gene in humans). This protein phosphorylates
specific tyrosine residues in other tyrosine kinases.
○​ Elevated c-Src tyrosine kinase activity is linked to cancer progression by promoting other signals.
○​ Mutations in src gene could involved in the malignant progression of colon, breast, prostate cancers.
○​ This proto-oncogene may play a role in regulating embryonic development and cell growth.
○​ c-Src promotes cell proliferation and motility.
●​ Capture of c-src by avian leucosis virus (ALV):
○​ ALV (which normally doesn't carry an oncogene) can acquire the c-src gene from the host cell,
becoming a highly oncogenic virus like RSV. This illustrates how retroviruses can "capture" host
proto-oncogenes.
●​ Proto-oncogene:
○​ A normal cellular gene (like c-src) that, when altered (by mutation or overexpression), can
contribute to cancer.
○​ Activation of a proto-oncogene (src) can lead to oncogenesis.
○​ Other mutational mechanisms can reshape a normal c-src gene with similar oncogenic outcomes.
○​ A single gene activation can be sufficient for cellular transformation – pleiotropy (1 gene
influencing multiple phenotypic traits).
○​ Other genes similar to src with potent transforming capacity may exist in vertebrate genomes.

Mechanisms of Viral Oncogenesis

●​ Viruses Carrying an Oncogene (RSV with v-src):

○​ Rapid transformation (days or weeks).
●​ Insertional Mutagenesis : Provirus without oncogene also induces transformation
○​ Viral transcriptional regulatory elements (strong promoters/enhancers in LTRs) integrate near a host
proto-oncogene.
○​ This integration activates / dysregulates the expression of the host proto-oncogene (Myc).
○​ Cells with this activated proto-oncogene gain a clonal growth advantage.
○​ Transformation is much slower (many weeks or months) than with viruses carrying oncogenes.
●​ A third class: HTLV-I
○​ Infects about 1% of inhabitants of Kyushu, Japan.
○​ Lifelong infection carries a 3-4% risk of developing T cell leukemia.
○​ Transmitted via milk-borne
○​ No insertional mutagenesis is detected as the primary mechanism.
○​ 1 / 2 viral proteins (Tax gene product) are crucial.
■​ Tax activates transcription of the proviral DNA (leading to progeny viral RNA genomes).
■​ Tax activates cellular gene expression, including IL-2 & GM-CSF – important for T cell
growth, maturation, activation. This chronic stimulation contributes to leukemia
development.
Types of Resistance

●​ Intrinsic Resistance: Cancer is resistant from the outset.

●​ Acquired Resistance: Cancer becomes resistant after initially responding to therapy. This is increasingly
common as therapies improve.

Mechanisms of Cancer Drug Resistance

●​ Multi-drug Resistance (MDR)

○​ Affects all classes of drugs
○​ Often results from alterations in drug detoxification mechanisms
■​ uptake, metabolism, sequestration, efflux via pumps like P-glycoprotein
●​ Single-Agent Resistance
○​ Typically arises from alterations in drug targets
■​ mutations in targets / bypass mechanisms
●​ Shared Mechanisms for Single and MDR:
○​ Alterations in growth-promoting pathways.
○​ Altered differentiation pathways (e.g. EMT).
○​ Different cells of origin.

Single drug resistance Multi drug resistance

Drug specific to 1 drug / narrow class of drugs broad range of structurally & mechanistically
spectrum unrelated drugs

Primary cause alterations in drug target / pathway general mechanisms like increased drug efflux,
altered metabolism, enhanced DNA repair,
reduced apoptosis

Eg Gleevec resistance due to Bcr-Abl mutation P-glycoprotein mediated efflux of various

chemotherapy drugs

Implication may still respond to other drugs with different limits treatment options ; high failure rate
mechanisms

Factors Increasing Likelihood of Drug Resistance

●​ Heterogeneity of the original cancer cell population.

●​ Increased mutation rate / epigenetic changes.
●​ Inducibility of resistance mechanisms.
●​ Stage of the cell cycle (quiescent cells are often more resistant).
Detailed Mechanisms of Drug Resistance/Sensitivity

●​ Decreased Uptake
○​ Mediated by SLC transporters (>300 membrane-bound proteins)
○​ SLCs transport diverse substrates (nutrients, drugs, xenobiotics) ; About 40 SLC genes are involved
in uptaking specific cancer drugs.
○​ Mutations in SLCs can affect drug efficacy by:
■​ Causing misfolded proteins leading to degradation.
■​ Preventing trafficking of the transporter to the cell surface.
■​ intracellular accumulation of misfolded protein, triggering the unfolded protein response.
●​ Increased Efflux
○​ Mediated by ABC transporters (superfamily of 48 transporters).
○​ transporters use energy from ATP hydrolysis to move substrates across membranes (uptake/export)
○​ P-glycoprotein (P-gp) / MDR1 / ABCB1 / CD243:
■​ An important ATP-dependent efflux pump with broad substrate specificity, pumping many
foreign substances out of cells.
■​ Evolved as a defense mechanism; found in animals, fungi, and bacteria.
■​ Expressed in liver, pancreas, kidney, colon, jejunum, and brain capillary endothelial cells.
■​ Role in Multi-drug Resistance: P-gp and other ABC transporters extrude numerous chemical
compounds, leading to:
■​ Failure of cancer chemotherapy (MDR).
■​ Prevention of carcinogen accumulation in healthy tissues.
■​ Affecting ADME of drugs.
■​ Protection from environmental toxins.
■​ Acquired Resistance: Meta-analysis showed MDR1 mRNA and protein levels increase
significantly after chemotherapy, indicating a role in acquired resistance.
■​ Intrinsic Resistance: Overexpression is frequent in tumors with inherent resistance. MDR1
gene amplification is rare in clinical samples.
■​ P-gp in Clinical Carcinogenesis: Lung carcinomas in smokers often express more P-gp and
are more drug-resistant.
■​ Clinical Significance: ~50% of human cancers express P-gp at levels sufficient for MDR.
P-gp inhibitors have shown limited success.
■​ P-gp is sufficient for MDR but may not be necessary or the sole cause.
●​ Reduced Apoptosis: Cancer cells become less sensitive to drug-induced cell death.
●​ Altered Cell Cycle Checkpoints: Changes allow cells to bypass drug-induced arrest.
●​ Altered Growth Pathways: Activation of alternative survival pathways.
●​ Increased Metabolism of Drugs: Drugs are inactivated more rapidly.
●​ Increased or Altered Targets: More target molecules need to be inhibited, or the target is mutated.
●​ Increased Repair of Damage: Cells become more efficient at repairing drug-induced damage.
●​ Compartmentalization: Drugs are sequestered away from their targets.
Mutations Associated with Drug Target

●​ Increased target expression reduces inhibitor effectiveness.

●​ Example: Gleevec (Imatinib) & Chronic Myeloid Leukemia (CML):
○​ Ph Chromosome: A genetic abnormality t(9;22)(q34;q11) creating the BCR-ABL1 fusion gene.
○​ BCR-ABL1 Protein: A constitutively active tyrosine kinase that drives uncontrolled cell division.
○​ Gleevec Action: Inhibits the BCR-Abl kinase by binding to its catalytic cleft.
○​ Acquisition of Resistance to Gleevec:
■​ Target Mutation: T315I. The bulkier isoleucine residue protrudes into the drug-binding
cavity, sterically hindering Gleevec binding.
■​ Gene Amplification: Increased copy number of the BCR-ABL gene leads to overexpression
of the target protein, overwhelming the therapeutic concentration of Gleevec.
■​ Screening for Resistant Mutants: Cultured cells (BaF3) dependent on Bcr-Abl activity are
mutagenized and selected for Gleevec resistance. Sequencing reveals mutations, many of
which are single amino acid substitutions throughout the Abl kinase domain.
○​ Backup Inhibitors:
■​ AMN107 (Nilotinib): More potent than Gleevec against unmutated Bcr-Abl, but some
Gleevec-resistant mutants (T315I) are also resistant to AMN107.
■​ AP24534 (Ponatinib): A derivative of Gleevec designed to avoid the steric clash with the
T315I mutant and can bind to it.
○​ Gleevec Resistance in Other Cancers: observed in GISTs due to "gatekeeper residue" mutations.

Downstream Pathway and Drug Resistance

●​ Dysregulated Apoptosis: Reduced efficiency of linking drug-induced DNA damage to cell death.
●​ DNA Damage Repair:
○​ Many chemotherapies induce DNA damage (platinum drugs, topoisomerase inhibitors).
○​ Increased DNA damage repair capacity in cancer cells leads to resistance.
○​ MMR: Mutations in MMR genes (MLH1, MSH2) can cause resistance to various cytotoxic
chemotherapies.
○​ Genomic Instability: A hallmark of cancer, contributing to heterogeneity and resistance.
○​ PARP Inhibitors: Target the DNA repair enzyme PARP ; show synthetic lethality in tumors with
BRCA1/BRCA2 mutations (impaired homologous recombination).
■​ Resistance to PARP Inhibitors: occur due to in-frame deletions in BRCA2 that partially
restore its DNA repair function.
●​ Activation of Prosurvival Adaptive Responses:
○​ Cytotoxic drugs can activate prosurvival signals, morphological changes (EMT), autophagy.

Drug Resistance in Prosurvival Pathways

●​ Inhibition of EGFR: Using monoclonal antibodies / TKIs

●​ Resistance Mechanisms to EGFR Inhibitors:
○​ Activation of alternative RTKs.
○​ Acquired mutations in pathway-relevant kinases (bypass inhibition).
○​ Activation of RTKs by stromal-derived growth factors.
●​ Backup Inhibitors of EGFR: (Iressa/Gefitinib) act by blocking ATP-binding sites of the receptor kinase.
Mutant EGFR can show varied responses.
Tumor Stem Cells (CSCs) and Drug Resistance

●​ Markers for CSCs:

○​ Breast Cancer: CD44 high / CD24 low phenotype.
○​ Brain Tumor: CD133 high.
○​ Lung Cancer: CD24-low / CD44-high
○​ Colon Cancer: ALDH1
○​ Glioma: Nestin-positive cells (normal neural stem cell marker) often near capillaries.
○​ identified in various cancers (pancreatic, colorectal, hepatocellular, hematopoietic malignancies).
●​ Stem Cell Division Models:
○​ Asymmetric Division: One daughter cell remains a stem cell, the other differentiates (may become a
"transit amplifying progenitor cell" which divides rapidly before terminal differentiation). This
allows stem cells to divide periodically.
○​ Symmetric Division: Produces two identical daughter cells.
●​ Location and Characteristics of CSCs:
○​ CSCs share programs with normal stem cells in tissues ; often found in specific niches within
tumors.
●​ Intra-tumor Heterogeneity:
○​ Genetically distinct sub-clones coexist within tumor ; intermingle & contribute to genetic instability.
○​ Metastatic site mutations travel back to the primary tumor.
○​ Epigenetic plasticity also contributes to diversity.
●​ Heterogeneity Complicates Treatment:
○​ tumor composition (subpopulations) could allow for personalized, rational combination therapies
based on drug-genotype matrices.
●​ Role of CSCs in Therapy Response:
○​ If therapy depletes transit-amplifying cells but not CSCs, CSCs can regenerate the tumor.
○​ CSCs are often significantly more resistant to therapy than the bulk of cancer cells.
○​ CSCs are doubly dangerous: they survive treatment ; can generate new tumors
●​ Therapeutic Strategies Targeting CSCs:
○​ Developing agents that preferentially eliminate CSCs.
○​ Eliminating CSCs alone may not be curative, as non-CSCs in some tumors can spontaneously
generate new CSCs.
○​ Targeting both CSC and non-CSC populations is likely required for durable clinical responses.

Causes of Cancer

●​ Age, Chemical Exposure, Diet, Radiation, Infection, chronic inflammation, obesity, smoking
●​ Genetic Factors
○​ Oncogenes and Tumor Suppressors:
■​ Proto-oncogenes: Normal genes that can become oncogenes when mutated (MYC, ErbB2,
PI3KCA, CCND1)
■​ TSG: Normally prevent cancer by inhibiting cell division / causing cell death. Mutations
lead to loss of this protective function. (P53, BRCA1/2, PTEN)
○​ Non-coding Mutations: The majority of somatic variants in cancer genomes. Mechanisms include
changes in transcription factor binding, miRNA binding sites, and higher-order chromatin structure
○​ Epigenetic Mutations: Changes in histone & DNA modifications
■​ Driver Mutations in Histone Modifications: Histone H3.3 mutations in gliomagenesis.
■​ G34R/V: More prevalent in cerebral hemispheres, older patients; inhibits SETD2
function (H3K36me3).
■​ H3.3K27M: Primarily in midline locations, younger patients; inhibits EZH2
function (H3K27me3).
Conventional Cancer Therapy

Surgery Therapy

●​ Oldest and often effective treatment

●​ Removes the tumor and nearby tissue.
●​ Types and Purposes of Cancer Surgery:
○​ Prevention (Prophylactic): Done before cancer develops to lower risk.
■​ removing precancerous polyps, mastectomy/oophorectomy for BRCA1/2 mutation carriers
○​ Diagnostic and Staging:
■​ Diagnostic: Biopsy is the main way to diagnose most cancers (thoracotomy, thoracoscopy,
mediastinoscopy)
■​ Staging: Determines tumor size and spread to guide treatment
○​ Tumor Removal (Curative): Main goal is complete removal of localized, early-stage tumors and
some nearby healthy tissue (margin). Lymph node dissection may be done if nodes are involved.
■​ Difficulties in Removal: Tumor too big, risky location, too small to see, poor patient health
■​ Debulking (Cytoreductive Surgery): Removing part of a tumor when complete removal is
not possible or too damaging. Can make chemo/radiotherapy more effective
■​ Important Considerations: Avoid cutting into the tumor; remove the biopsy track
○​ Relieve Symptoms (Palliation): relieving pain, restoring function (feeding tube), stopping bleeding
○​ Repair Damaged Tissue (Reconstruction): Restores appearance or function, can be done during
tumor removal or later
○​ Support Body Functions: E.g., tracheostomy for breathing, gastrostomy for nutritionSupport
Other Treatments: E.g., placing ports for drug delivery
●​ Issues of Surgery Therapy
○​ Difficult to remove all cancer tissue.
○​ Risks & side effects: bleeding, blood clots, nearby tissues/organs damage, drug reactions, pain,
infections, slow recovery.
●​ Combination with Other Therapies
○​ Neoadjuvant Therapy: Chemo and/or radiotherapy given before surgery to shrink the tumor.
○​ Adjuvant Therapy: Chemo and/or radiotherapy given after surgery to destroy remaining cells &
reduce recurrence risk. Timing is crucial to allow surgical wound healing.

Radiotherapy

●​ Uses high-energy particles/waves (X-rays, gamma rays, electron beams, protons) to destroy/damage cancer
cells by making small breaks in their DNA, preventing growth/division and causing death.
●​ Goals of Radiotherapy
○​ Cure or shrink early-stage cancer.
○​ Prevent recurrence.
○​ Treat symptoms of advanced cancer (palliative).
○​ Treat recurrent cancer (re-assessment needed for same area).
●​ Ways Radiotherapy is Given
○​ External Radiation (External Beam): Machine directs rays from outside the body to the tumor.
Usually given over many weeks as outpatient visits.
○​ Internal Radiation (Brachytherapy): Radioactive source placed inside body, in or near the tumor.
○​ Systemic Radiation: Radioactive drugs given orally / intravenously, traveling throughout the body.
●​ Issues of Ionizing Radiation
○​ Early Side Effects: Fatigue, skin problems, hair loss, nausea.
○​ Late Side Effects: Scar tissue affecting lung/heart function; bladder, bowel, fertility, sexual
problems (if pelvis/belly treated); can cause a second cancer.
○​ Kills all fast-growing cells, including some normal ones.
Chemotherapy

●​ Uses powerful drugs to shrink or kill cancer cells. Works throughout the whole body, so can kill
metastasized cancer cells far from the primary tumor
●​ Main Goals
○​ Cure.
○​ Control.
○​ Ease symptoms (Palliation).
●​ Determining Which Drugs to Use
○​ Type and stage of cancer.
○​ Patient's age and overall health.
○​ Other health problems
○​ Past cancer treatments.
●​ Approaches to Treatment (Delivery Methods)
○​ Intravenous (IV): Most common, infused into a vein.
○​ Oral: Pills
○​ Topical: Cream/lotion for skin cancer.
○​ Direct Placement: Injected into spinal fluid or brain (via a device).
○​ HIPEC: For cancers spread to abdominal cavity lining (appendix, colon, stomach, ovaries).
Delivered directly into the abdomen after surgery in the OR
■​ Prevents remaining cells from growing into new tumors. Higher chemo concentration
delivered more effectively and safely than standard IV chemo for abdominal cancers. Better
at killing microscopic cancer cells. Fewer side effects due to the peritoneal plasma barrier
●​ Different Types of Chemotherapy
○​ Hormone Therapies: Prevent hormone production or interfere with hormone action (e.g.,
Tamoxifen for ER+ breast cancer).
○​ Signal Transduction Inhibitors: Target continuous cell division signals (e.g., Trastuzumab for
HER2+ gastric cancer).
○​ Gene Expression Modulators: Modulate genes controlling cell cycle/apoptosis (e.g., Romidepsin,
a histone deacetylase inhibitor).
○​ Apoptosis Inducers: Cause cancer cell death by bypassing their strategies to avoid apoptosis.
○​ Angiogenesis Inhibitors: Block tumor growth by interfering with the formation of new blood
vessels needed for oxygen and nutrients.
○​ Monoclonal Antibodies that Deliver Toxic Molecules: Antibody binds to target cell, delivering a
linked toxin (radioactive substance, chemical) to kill the cell.
●​ Risks of Chemotherapy
○​ Chemo drugs kill fast-growing cells, affecting normal fast-growing cells too.
○​ Side Effects: Fatigue, hair loss, easy bruising/bleeding, infection, anemia, nausea/vomiting, appetite
changes, fertility problems.
○​ Some chemo/radiation works by producing free radicals. Antioxidant vitamins (A, E, C) can prevent
free radical formation, so should not be taken unless advised by a doctor.
Hormone Therapy

●​ Systemic therapy that adds, blocks, or removes hormones to slow/stop cancer cell growth
●​ Usually involves medications preventing cancer cells from getting hormones needed for growth.
●​ Treatment option for prostate, breast, ovarian, uterine, or thyroid cancers.
●​ Factors influencing choice: age, tumor type/size, presence of hormone receptors on the tumor.
●​ Hormone Therapy for Breast Cancer
○​ Used if tumor growth depends on Estrogen or Progesterone (ER/PR positive).
○​ Tamoxifen: An ER blocker, prevents estrogen uptake by cancer cells.
●​ Side Effects of Hormone Therapy
○​ Breast Cancer Patients: Hot flashes, vaginal issues, decreased sex drive, mood changes.
○​ Prostate Cancer Patients: Reduced erectile/orgasmic ability.
○​ Aromatase Inhibitors: Joint/muscle pain, increased risk of osteoporosis.
Targeted Therapy (2nd Generation)

●​ Targets specific molecular changes in cancer cells

●​ Precision Medicine and Targeted Therapy
○​ Therapy against actionable gene mutations shows higher response rates and longer survival than
conventional chemo.
○​ NGS identifies genetic aberrations to guide therapy.
○​ Patients may be recommended for targeted therapy, immunotherapy, or clinical trials if no approved
drugs exist for their specific mutations.
●​ Development of Targeted Drugs
○​ Small Molecule Compounds: For intracellular targets, can enter cells easily. Developed via
high-throughput screens and chemical modification.
○​ Monoclonal Antibodies: Large, for extracellular or cell surface targets. Developed by immunizing
animals and humanizing the antibody.

●​ FDA Approved Molecular Targeted Drugs for Different Cancers

○​ LADC: Most common lung cancer type, common in smokers, young women, Asian populations
■​ Approved Drugs for LADC (targeting EGFR): [Lecture 12, p. 43]
■​ TKIs: e.g., erlotinib, gefitinib. Bind to EGFR's tyrosine kinase domain, stopping its
activity.
■​ Monoclonal Antibodies: e.g., cetuximab, necitumumab. Bind to EGFR's
extracellular part, preventing EGF binding and cell division.
○​ Problem for KRAS Target: Mutated KRAS is hard to target directly. Efforts focus on downstream
proteins (e.g., MEK1/2 inhibitors like trametinib, cobimetinib). The NIH has a "RAS Initiative"
■​ Inhibition of MEK1/2 can activate autophagy (LKB1→AMPK→ULK1 axis). Combination
of trametinib (MEK inhibitor) + chloroquine (autophagy inhibitor) showed partial response
in pancreatic cancer
○​ Gastric Cancer (GC): Common digestive system malignancy, high mortality. Surgery is primary
but often ineffective due to advanced disease at diagnosis. Chemo has 20-40% response rate, 6-11
months median OS. Targeted therapy aims to improve specificity and reduce toxicity
■​ Targeted Pathways for GC:
■​ ~20% have too much HER2 (growth-promoting protein).
■​ Targeting VEGF (protein for new blood vessel formation).
■​ Targeted Drugs for GC: (List not detailed, but implied to target HER2 and VEGF
pathways)
○​ CRC: Starts in colon/rectum. Treatments: surgery, radiation, chemo, targeted therapy
■​ CRC Targeted Drugs (FDA approved):
■​ VEGF Pathway Inhibitors (anti-angiogenesis):
■​ Bevacizumab (mAb against VEGF-A).
■​ Ramucirumab (mAb against VEGFR2).
■​ Ziv-aflibercept (VEGF trap).
■​ EGFR Inhibitors:
■​ Cetuximab (chimeric mAb against EGFR).
■​ Panitumumab (fully human mAb against EGFR).
■​ Note: These EGFR inhibitors don't work in CRC with KRAS, NRAS, or
BRAF mutations.
Adoptive Cell Therapy (ACT)

●​ Definition: Uses a patient’s own T lymphocytes with anti-tumor activity, expanded in vitro, and reinfused
into the patient
●​ Highly effective for metastatic melanoma Types of ACT: TILs, TCRs, CARs

TILs

●​ Source: Immune cells found in tumor biopsies, conditioned to the tumor microenvironment. Abundance
varies by tumor type/stage and can relate to prognosis
●​ Process
○​ Collect autologous TILs from patient tumors.
○​ Detect lymphocytes (e.g., using CD3 marker or gene expression methods like Microarray/RNA
Sequencing with deconvolution).
○​ Activate and expand T cells using cytokines (e.g., IL-2).
○​ Select TIL lines with best tumor reactivity for further expansion in a REP using anti-CD3 activation.
○​ Optional: Preliminary chemotherapy regimen (7 days before TIL infusion) to deplete endogenous
lymphocytes, giving transferred TILs better access to tumor sites.
●​ Clinical Success
○​ Induces complete, durable regression of metastatic melanoma
○​ In colorectal cancer, associated with microsatellite instability cancers and effective immune
checkpoint inhibitor therapy in GI cancers.
○​ Associated with better outcomes in epithelial ovarian cancer.
○​ Combination with prior immunotherapy (e.g., IL-2, anti-CTLA4) shows higher, more durable
responses, suggesting synergy.

TCR Therapy

●​ Rationale: For patients whose T cells haven't recognized their tumors or whose T cells can't be sufficiently
activated/expanded . Allows for personalized treatment by choosing optimal targets and T cell types.
●​ T-cell Receptor (TCR) Structure and Function
○​ Antigen receptors on T cells.
○​ Disulfide-linked membrane-bound heterodimer (usually α and β chains, or γ and δ chains)
complexed with invariant CD3 chains (CD3δ, CD3ε, CD3γ).
○​ Two T cell subsets: γδ TCR (minor), αβ TCR (major).
○​ Antigenic diversity from random rearrangement of V, D, J gene segments. TCR α chain: V, J, C
segments. TCR β chain: V, D, J, C segments.
○​ Recognizes peptide fragments of antigen bound to MHC molecules.
●​ Activation of T cells: Requires TCR binding to MHC-presented antigen AND CD28 (on T cell) binding to
B7 (CD80/86 on antigen-presenting cell)
●​ Development of Engineered TCR-T cells
○​ Choose Tumor Antigen: TAA or tumor-specific endogenous protein presented by tumor cell MHC.
○​ High Affinity T cell Clone Isolation: Isolate high affinity T cells clone for the chosen tumor
antigen.
○​ TCR α/β Gene Cloning & Packaging: Clone the TCR α/β genes from these high-affinity T cells.
Use retro- or lenti-viruses to deliver these genes into donor T cells from the patient's peripheral
blood.
○​ Improving TCR Performance:
■​ Enhance TCR affinity (e.g., amino acid substitutions in CDRs).
 ■​ Increase TCR expression (e.g., murine-human hybrid TCR).
■​ Increase stability of CD3ζ/TCR complex.
■​ Reduce mispairing of TCR chains (e.g., add a second disulfide bond between α and β
constant regions, add cysteines, use mouse TCR genes).
■​ Activation with cytokines like IL-2.
○​ Adoptive Transfer: Infuse engineered T cells back into the patient.
●​ Advantages and Disadvantages of TCR-T
○​ Advantages: Function through well-understood T cell signaling pathways; natural way the body
fights foreign elements.
○​ Disadvantages: Restricted to one HLA type; α/β TCRs cannot target non-protein TAAs (e.g.,
carbohydrate or lipid antigens).

CAR-T

●​ Rationale: Overcomes the limitation of TIL and TCR-T therapies which require antigen presentation by
MHC
●​ Patient’s T cells are equipped with a synthetic receptor (CAR).
●​ Workflow of CAR-T: (Diagrammatic representation, general steps include T cell collection, engineering,
expansion, infusion)
●​ Engineered CAR Structure
○​ Antigen Recognition Targeting Moiety: Typically a scFv derived from an antibody.
○​ Hinge/Spacer: Connects scFv to the transmembrane domain.
○​ Transmembrane (TM) Domain: Anchors CAR in the T cell membrane.
○​ Signaling Domains: Commonly CD3ζ (primary activation signal).
○​ Co-stimulatory Domains: (e.g., CD28, 4-1BB) enhance T cell activation, proliferation, persistence.
●​ Alternative CAR-T Strategy (Universal)
○​ Uses CAR T-cells expressing a high-affinity receptor for a molecule like FITC.
○​ A two-part CAR-T adaptor molecule (CAM) is used: FITC conjugated to a tumor-homing ligand.
This allows the CAR-T cell to be directed by the adaptor.
●​ Advantages of CAR-T Therapy
○​ Can bind to cancer cells even antigens aren't presented via MHC (unlike TCR-T which requires
APCs).
○​ However, CAR T cells can only recognize antigens naturally expressed on the cell surface, so the
range of potential targets is smaller than with TCRs.
○​ Being explored for many cancer types. "Off-the-shelf" CAR-T cells from stem cells are in clinical
trials for timelier treatment.
 TILs TCR-T CAR-T

source of T Harvested directly from the Typically T cells isolated from Typically T cells isolated from
cells patient's tumor biopsies; these the patient's peripheral blood the patient's peripheral blood
cells are already "conditioned via leukapheresis
to the tumour
microenvironment"

mechanism Relies on the T cells' naturally Uses T cells genetically Uses T cells genetically
of tumor occurring TCRs that have engineered to express a new engineered to express a
recognition already recognized antigens TCR that has high affinity for synthetic CAR. The CAR's
within the tumor a specific tumor antigen antigen-binding domain
microenvironment. presented by MHC molecules (usually an scFv from an
antibody) directly recognizes
antigens on the cancer cell
surface

genetic Primarily involves selection Requires genetic engineering Requires genetic engineering
engineering and expansion of naturally to introduce the genes for the to introduce the gene encoding
tumor-reactive T cells. new, specific TCR α/β chains the synthetic CAR

MHC MHC-restricted: Natural MHC-restricted: The MHC-independent: CARs

restriction TCRs recognize antigens engineered TCR recognizes can bind to target antigens on
presented by MHC molecules. peptide antigens presented by the cancer cell surface directly,
MHC molecules on cancer without needing MHC
cells. Restricted to one HLA presentation
type.

Target Peptides derived from tumor Peptides derived from Primarily surface antigens.
antigen type antigens presented on MHC intracellular or surface tumor Cannot target IC antigens
molecules antigens presented on MHC unless a fragment is expressed
molecules.. Cannot target on the surface. The range of
non-protein antigens like potential antigen targets is
carbohydrates or lipids described as smaller than with
TCRs because they must be on
the cell surface

Need for Relies on T cells that have Can be used even if the the recognition specificity is
Pre-existing already infiltrated and patient's T cells have not engineered, so pre-existing
Tumor potentially recognized the naturally recognized their recognition by the patient's T
Recognition tumor. tumors, as the recognition cells is not required for the
by Patient's T specificity is engineered CAR to function.
cells

Key Effective for metastatic Can be personalized by Ability to bind cancer cells
advantage melanoma, can induce choosing optimal targets; even if antigens aren't
complete and durable functions through natural T presented via MHC,
regression cell signaling pathways potentially making more
cancer cells vulnerable

Key Process can be lengthy; not all Restricted to one HLA type; Can only recognize antigens
limitation patients have sufficient TILs cannot target non-protein naturally expressed on the cell
or TILs that can be effectively antigens surface; range of potential
expanded. targets smaller than TCRs in
terms of intracellular origin
Side Effects of Adoptive Cell Therapy

●​ Vary by ACT type, target, cancer location/type, and patient health

●​ Often due to an overactive immune response:
○​ CRS / Cytokine Storm: Excessive inflammation.
○​ Neurotoxicity: Inflammation in the brain.
●​ Side effects can range from mild to life-threatening.
●​ Specific examples: acute kidney injury, bleeding, heart arrhythmias, chills, constipation, cough, decreased
appetite, delirium, diarrhea, dizziness, edema, encephalopathy, fatigue, febrile neutropenia, fever, headache,
hypogammaglobulinemia, hypotension, hypoxia, infections, nausea, pyrexia, tachycardia, tremors, vomiting

FDA Approved CAR T Cell Therapies

●​ Axicabtagene ciloleucel (Yescarta®): CD19-targeting; for subsets of lymphoma patients.

●​ Tisagenlecleucel (Kymriah®): CD19-targeting; for subsets of leukemia and lymphoma patients.
○​ CD19 is a biomarker for B cells.

On-going CAR-T Projects (Clinical Evaluation Targets)

A wide range of targets are being evaluated, including​

CEA, EBV-related antigens, EGFR, GD2, GPC3, HER2, HPV-related antigens, MAGE antigens, Mesothelin,
MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, Claudin 18.2, BCMA, CD19, CD22, CD30, CD33, CD56, CD123
(IL-3R).

Cytokine Treatment

●​ Mechanism: Designed to alter immune homeostasis within tumor & provoke/ disinhibit immune effectors
●​ FDA Approved Examples
○​ IFN-α: For metastatic renal cell carcinoma and melanoma. Works to activate NK cells.
○​ IL-2: For metastatic renal cell carcinoma and melanoma. Is a T cell growth factor.
Immune Checkpoint Therapy

●​ Immune Checkpoints: Regulators of the immune system crucial for self-tolerance, preventing
indiscriminate attacks on normal cells. They need to be activated (or inactivated) to start an immune
response
●​ Cancer cells can use these checkpoints to avoid immune attack. When checkpoint proteins bind their
partners, they send an "off" signal to T cells.
●​ The immune system uses checkpoints to distinguish normal ("self") from "foreign" cells.
●​ Mechanism of Immune Checkpoint Inhibitors: Block checkpoint proteins from binding their partners,
preventing the "off" signal and allowing T cells to kill cancer cells. These inhibitors don't act directly on the
tumor but "take the brakes off" an existing immune response
●​ Known Checkpoint Proteins : CTLA-4 (CD152), PD-1 (CD279), LAG-3 (CD223), TIM-3 (HAVcr2),
KIRs (CD158), 4-1BB (CD137), GITR (CD357).

PD-1 and PD-L1/2 Inhibitors

●​ PD-1: Checkpoint protein on T cells and NK cells. Acts as an "off switch"

●​ PD-L1/PD-L2: Proteins on some normal and cancer cells.
●​ When PD-1 binds PD-L1 or PD-L2, it tells activated immune cells to leave the other cell alone. Some cancer
cells have high PD-L1/L2 levels to evade immune attack.
●​ Inhibitors: Monoclonal antibodies targeting PD-1 or PD-L1/L2 block this binding, boosting the immune
response against cancer
●​ Currently Available Drugs (given by IV):
○​ PD-1 Inhibitors: Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo).
○​ PD-L1 Inhibitors: Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi).

CTLA-4 Checkpoint Protein

●​ Mechanism
○​ T cell activation requires TCR engagement with antigen + CD28 (on T cell) co-stimulation by
binding CD80 (B7-1) / CD86 (B7-2) on APCs.
○​ CTLA-4 is a homolog of CD28.
○​ CTLA-4 on T cells binds B7 proteins with ~20 times greater affinity than CD28, outcompeting
CD28 (Inhibition and Ligand competition).
○​ Excess soluble CTLA-4 can bind B7 on APCs, restricting B7 availability for CD28 binding
(Unresponsiveness).
●​ CTLA-4 Inhibitor
○​ Blocking CTLA-4 has therapeutic benefits.
○​ Ipilimumab: FDA approved for melanoma (2011).
○​ Tremelimumab: (Not yet approved).

Summary of Immune Checkpoint Inhibitors' Targets

●​ PD-L1 and PD-L2 (bind to PD-1): Found on tumor cells and APCs.
●​ B7-1 and B7-2 (bind to CTLA-4): Found on APCs.
Side Effects of Immune Checkpoint Inhibitor Therapy

●​ Serious adverse events due to excessive immune activation – irAEs

●​ Effects vary based on patient health, cancer type/stage, drug type, and dose.
●​ May include: Diarrhea, fatigue, rashes, itchiness, widespread inflammation (rare).

TCV

●​ Strategy: Boost the host’s own immune responses against tumors

●​ 1 method involves growing DCs from individuals, expose them to tumor cells/antigens, using these
"tumor-pulsed" DCs as vaccines → mimics normal cross-presentation and generates CTLs against tumor
cells.
●​ Examples
○​ Sipuleucel-T (Provenge): FDA approved TCV for prostate cancer treatment.
○​ Lapuleucel-T (Neuvenge): Tested for HER2/neu positive breast cancer, scheduled for bladder
cancer trials.
○​ Both developed by Dendreon Corporation for therapeutic (not preventive) purposes.